CORD-19:3c1f47a07aaf0e393dee9fbd08596a4b9f4f0a03 JSONTXT 9 Projects

Infection control O14 Surgical site infection surveillance in France: the first 1999-2004 trend analysis O21 Evaluation of a rapid molecular dipstick assay for the direct detection of methicillin-resistant Staphylococcus aureus in clinical specimens O23 Impact of hypochlorite disinfection on MRSA rates Resistance surveillance O24 Multi-drug resistant enterococci among Portuguese swine after growth promoter ban O27 European Antimicrobial Susceptibility Surveillance in Animals (EASSA): Results (2002/2003) for enteric bacteria from healthy cattle, pigs and chickens from 8 countries Abstract Background: Surgical site infection (SSI) is one of the most frequent nosocomial infections. Since 1999, a national coordination of regional networks has been implemented to gather SSI incidence data according to standardised method. The aim of the current study was to describe the largest database ever collected in France on SSI and to analyse a 6-year temporal trend. Methods: Surgery patients were enrolled by voluntary participating wards in a yearly 3-month incidence survey. In each ward, 200 consecutive surgery procedures should be included and patients followed up to 30 days after surgery. SSI was defined based on standard CDC criteria. For each patient, risk factors were collected on the day of surgery including age, ASA score, Altemeier wound class, type and duration of procedure, emergency/elective, and when videoscopy surgery was performed. Results: During 6 years, the study included 620,176 operations (17,430,253 operated patient-days follow-up; median post-operative follow-up: 28 days). The overall SSI incidence rate was 1.68%. Organ space and deep incisional SSI accounted for 41.7% although their proportion varied according to the type of surgery. SSI incidence rate increased from 0.91% [0.88-0.94%] for NNIS-0 patients to 13.8% [12.5−15.2%] for NNIS-2, 3 patients. The SSI incidence varied from 1.15% for herniorraphy to 9.2% for colon surgery. In NNIS-0 patients, emergency surgery increased the SSI risk for C-section whereas videoscopy surgery was at lower risk for cholecystectomy. From 1999 to 2004, NNIS-0 SSI incidence decreased from 1.1 to 0.9 for 100 operated patients (relative difference: −18%). According to procedure, the trend remained significant only for herniorraphy. Conclusion: This database provided thorough standardised estimate of SSI incidence according to various surgery procedures. Impact of the national policy on SSI incidence remains to be further evaluated, although encouraging results were evidenced for specific surgery. Objectives: Healthcare associated infection (HAI) incidence rates after delivery range from 0.26% to 20.3% according to the mode of delivery, the maternity activity, women risk factors. Data on HAI surveillance in maternity units are lacking. The Mater Sud-Est Study Group is a HAI continuous surveillance network on maternity units located in south eastern France. We report changes in risk-adjusted HAI rates over a 6-year long surveillance period in this maternity units network. Methods: 161,077 vaginal deliveries and 37,074 cesarean deliveries were included in the surveillance between January 1st 1997 and December 31st 2003. We studied the changes in four HAI: endometritis and Urinary tract infection (UTI) after vaginal deliveries, surgical site infection (SSI) and UTI after cesarean deliveries. We used a logistic regression modeling to estimate risk-adjusted HAI rates. The year of delivery was considered as a risk factor. The trend of risk-adjusted HAI rates over the study period was studied by a linear regression of the year-of-delivery odds ratios for each targeted HAI. Results: The rate of endometritis and UTI after vaginal deliveries was 0.3% (534/161,077) and 0.5% (728/161,077) respectively. Over the study period the decrease in endometritis odd ratios was statistically significant. We found no statistically significant trend in vaginal delivery's UTI. The rate of SSI and UTI after cesarean deliveries was 1.5% (571/37,074) and 1.8% (685/37,074) respectively. Over the study period the decrease in SSI and UTI odd ratios was statistically significant. Conclusion: These findings highlight the positive effect of participating in a surveillance network for infection control and for improvement of care. Infection Alert, Investigation & Surveillance Network (RAISIN) Bordeux, FR Background: National surveillance of occupational blood and body fluids exposures (BBFE) in France is conducted since 2002 through the Nosocomial Infection Early Warning, Investigation and Surveillance Network (Raisin) in collaboration with Geres (Groupe d'Etude sur le Risque d'Exposition des Soignants aux Agents Infectieux). Methods: Participation of healthcare facilities (HCF) is voluntary and anonymous. BBFE occurring from 01/01/04 to 31/12/04 were documented using a standardised questionnaire documenting the nature, circumstances (mechanism, type of device, infectious status of the source) and follow-up of each BBFE. Incidence of BBFE is reported per 100 hospitalisation beds, by type of personnel per 100 full time equivalents (FTE), or by type of material per 100,000 devices. S4 17th ECCMID / 25th ICC, Oral presentations Results: In 2004, 13,041 BBFE were documented in 371 participating HCF, which accounted for 15% of HCF and 29% of hospitalisation beds in France. BBFE overall incidence was 8.9 per 100 beds. Considering that all French hospitals account for 465,494 beds, 41,276 [95% CI: 40,896-41,656] BBFE could have occurred in France in 2004. HCV or HIV status of the source was not known for more than 20% of documented BBFE. Post-exposure prophylaxis (PEP) was prescribed to 4.5% of exposed personnel (vs. 5.8% in 2003 and 6.3% in 2002); this decrease may reflect the impact of April 2003 French recommendations, which reduced PEP indications. For the first time in 2004, sutures were the most frequent cause of BBFE associated with needles (more than subcutaneous injections) and accounted for 1,103 (11%) of all BBFE; one third occurred among residents, and 20% in ICU or emergency rooms (beyond surgery or obstetrics). Prevention through education and use of safety devices (such as blunt suture needles) may thus be a priority. Data from a cohort of 173 HCF which participated in 2003 and 2004 also were compared and demonstrate significant progresses. Compliance to glove use increased from 58.6% in 2003 to 62.3% in 2004, and BBFE incidence among nurse assistants fell from 2.3 in 2003 to 2.1 per 100 FTE in 2004. Last, BBFE incidence fell from 17.2 to 13.7 per 100,000 catheters, and from 71.6 to 43.2 per 100,000 implantable venous access systems. Conclusion: AES-Raisin is one of the biggest BBFE surveillance network and results demonstrate an increase in observance to standard precautions and a significant decrease in the incidence of some types of BBFE. They also point out future priorities for improvement. O18 A randomised trial of 2% chlorhexidine in 70% alcohol compared with 10% povidone-iodine for venipuncture site disinfection: effects on blood culture contamination rates G. Suwanpimolkul, M. Pongkumpai, W. Kulwichit, C. Suankratay (Bangkok, TH) Background: Contaminated blood cultures have been recognized as a bothersome issue for decades, and continue to cause a frustration for clinicians. The contamination rates vary widely between institutions from less than 1% to over 6%. Skin antiseptics can prevent the contamination of blood cultures. To our knowledge, no randomised trial of 2% chlorhexidine in 70% alcohol for venipuncture site disinfection has been conducted. Objective: Our study was aimed to evaluate the efficacy of venipuncture site disinfection with 2% chlorhexidine in 70% alcohol compared with 10% povidone-iodine in preventing blood culture contamination. Patients and Methods: A prospectively randomised investigator-blinded trial was conducted in all patients hospitalised in Internal Medicine wards and attended at emergency department at King Chulalongkorn Memorial Hospital, Bangkok, Thailand from August 15 to October 31, 2006. Antecubital venipuncture sites were randomly disinfected with either 2% chlorhexidine in 70% alcohol or 10% povidone-iodine, and blood cultures were drawn by medical students or residents. The blood culture contamination rate associated with each antiseptic was then determined. Results: Of 2,146 blood culture collected during the study, 108 (5.03%) were contaminated with skin flora. The contamination rate for blood cultures after 2% chlorhexidine in 70% alcohol was 3.2% (34 of 1,068), compared with a rate of 6.9% (74 of 1,078) (P < 0.001) after 10% povidone-iodine. Of the inpatient wards, the contamination rate was 2.6% (18 of 695) and 3.9% (28 of 709) after 2% chlorhexidine in 70% alcohol and 10% povidone-iodine, respectively (P = 0.013). Of emergency department, the contamination rate was 4.3% (16 of 373) and 12.5% (46 of 369) after 2% chlorhexidine in 70% alcohol and 10% povidone-iodine, respectively (P < 0.001). The most common contaminant organism was coagulase-negative staphylococci (81%). Conclusion: 2% chlorhexidine in 70% alcohol is superior to 10% povidone-iodine for venipuncture site disinfection before blood culture sampling. Objectives: The inanimate hospital environment can become contaminated with nosocomial pathogens. Hydrogen peroxide vapour (HPV) decontamination has proven effective for the eradication of persistent environmental contamination but the rate of recontamination following HPV decontamination is largely unknown. We investigated the extent of methicillin-resistant Staphylococcus aureus (MRSA), vancomycinresistant enterococci (VRE) and gentamicin-resistant Gram-negative rod (GNR) contamination in a ward side-room occupied by a patient with a history of MRSA, VRE and GNR infection and colonisation. Methods: Fifteen standardised sites in the room were sampled using a selective broth enrichment protocol to culture for MRSA, VRE and GNR. Sampling was carried out before cleaning, after cleaning, after HPV decontamination and at intervals over the subsequent 19 days on two separate occasions. Results: Environmental contamination was identified before cleaning on 60%, 30% and 6.7% of sites for MRSA, GNR and VRE, respectively and 40%, 10% and 6.7% of sites after cleaning (figure). Only one site (3.3%) was contaminated with MRSA after HPV decontamination (figure). No recontamination with VRE was identified and no recontamination with MRSA and GNR was identified in the two days following HPV decontamination (figure). Substantial recontamination towards pre-cleaning levels was identified by day five and six after HPV decontamination for MRSA. Recontamination with GNR at approximately post-cleaning levels was noted on days 7, 8 and 19 ( Figure 1 ). Infection control S5 infection control measures. Detection of risk groups and infection sources and knowing the transmission ways of infections are important for the prevention from NI. In this study it was aimed to evaluate the knowledge level and behaviour models of hospital cleaning staff about NI in our setting which is a 1788 bedded tertiary care educational hospital. Methods: A questionnaire of with 21 questions was implemented to the hospital cleaning staff, who volunteered to enter the study. The questionnaire was composed of two parts: first part contained parameters for determination of sociodemographic properties and the second contained questions about evaluation of the knowledge about prevention from NI. Questions were prepared by using the references about the subject and by the help of the executives of the cleaning staff firm and statistics unit. Data were evaluated by SPSS 13.0 programme using Chi square and Student's t tests. The questionnaire was completed by one by one interview method. Results: A total of 240 of 290 (82.7% of total, 122 male, 118 female, aged 36.2±8.7) hospital cleaning staff volunteered to enter the study. When evaluated according to the educational status; 55.4% were graduated from primary school and only 54% had been working in the hospital more than three years. Mean knowledge level was 18.15±3.97 (maximum 24). Knowledge level was not associated with gender, educational status and duration of working as cleaning staff (p > 0.05) but mean knowledge level of the staff working in the clinics was found higher than staff working in administrative sections (p < 0.05). 71.3% had received a formal education about prevention from NI before starting working but their mean knowledge level was not different from the others (p = 0.294). Only 48% and 50% knew the true order (x,y), while cleaning the patient rooms. 58.8% thought that they could prevent themselves from NI by hand washing before and after cleaning process, 80.8% stated they obeyed handwashing rules and 90.4% stated that they used gloves. Only 48.3% stated that they dried their hands by paper towels. Conclusion: Measurement of the level of the knowledge of the hospital cleaning staff may be beneficial for determination of the existing problems. Periodical well-established educational programmes should be started to improve the current situation. Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) causes increasing healthcare problems worldwide. Rapid and sensitive screening methods for direct detection of MRSA are essential to limit further spread in the hospital. The purpose of this study was to evaluate the new molecular dipstick assay GenoQuick ® MRSA (Hain Lifescience, Nehren, Germany) for the direct and specific detection of MRSA in clinical specimens. Methods: The analytical specificity of the assay was evaluated by using a subset of 25 MRSA isolates (including SCCmec types I−V); 17 methicillin-susceptible S. aureus (MSSA) and 38 coagulase-negative staphylocococi (CoNS) of different culture collections. The lower detection limit of the assay was determined by serial dilutions of MRSA strains representing SCCmec types I to IV. The test was evaluated for direct detection of MRSA in clinical swab specimens. MRSA carriage was analysed by both the standard culture methods [Chromagar MRSA (Becton Dickinson, Heidelberg, Germany), Columbia blood agar, trypticase soy broth] and two PCR assays [GenoQuick MRSA and GenoType ® MRSA Direct (Hain Lifescience)]. Both PCR assays were performed directly from the swab and after overnight incubation in trypticase soy broth. MRSA isolates were confirmed using a mecA gene and S. aureus specific PCR. Susceptibility testing was performed with an automated system (VITEK 2, bioMérieux, Nürtingen, Germany). Results: The lower detection limit of 25 CFU was determined with serially diluted MRSA strains. For analytical specificity all MRSA strains representing SCCmec types I to V were tested positive by the assay. The MSSA and CoNS were tested negative, respectively. Of 187 patient specimens tested for clinical evaluation, 24 were identified MRSA-positive by culture and by both PCR assays. One specimen was positive only by PCR. Among the 163 culture-negative specimens, 162 were negative with both PCR assays. The GenoQuick assay showed a diagnostic sensitivity of 100%, and a diagnostic specificity of 99.4%, a positive predictive value of 96% and a negative predictive value of 100%. Time-to-result for the direct detection of MRSA from clinial specimens is reduced to 2h 20min with the molecular GenoQuick MRSA dipstick assay (2h 5min for amplification and 15 min for detection). Conclusions: The GenoQuick MRSA dip stick assay proved to be a rapid, sensitive and specific assay for direct detection of MRSA in clinical swab specimens in 2h 20min. O22 Throat swabs are necessary to reliably detect carriers of Staphylococcus aureus Background: The anterior nares are considered to be the primary colonisation site of Staphylococcus aureus (S. aureus) and approximately 30% of healthy people carry the bacteria in their anterior nares. However, recent studies indicate that the throat may be an additional important site of colonisation (Nilsson P. J Clin Microbiol 2006). Most screening programmes for S. aureus including methicillin-resistant S. aureus (MRSA) require a swab from the nose only, and a swab of the throat is not considered as standard. Objectives: To determine the frequency of positive S. aureus cultures with positive samples from the throat and negative from the nares. Methods: Specimens were obtained with a sterile polyester fiber-tipped swab moistened with sterile saline from the anterior nares (5 rotations in each anterior nostril), the posterior wall of the pharynx, and the soft palate. Swabs were transported to the laboratory in a transport tube (M40 Transystem, Copan, Brescia, Italy) and put in selective enrichment broth (Chapman broth containing brain heart infusion broth with 6% NaCl, Biomedics, Madrid, Spain). Results: A total of 905 individuals were screened for S. aureus between 2000 and 2005. Complete data were unavailable from 54 individuals who were excluded. Overall, S. aureus was isolated in 386/851 (45.4%) individuals from any site. Screening results No. of % of overall Nares Throat individuals positive pos pos 196 50.8% pos neg 119 30.8% neg pos 71 18.4% 386 100% neg neg 465 Conclusion: Limiting S. aureus screening to the nares fails to identify 18.4% of carriers. Additional cost can be avoided by pooling the specimens while maintaining the higher sensitivity. Therefore, optimal screening for S. aureus should include swabs from both the nares and the throat. This may be even more important if screening is focused on MRSA carriage. Objectives: MRSA rates in a hospital in the North-East of Scotland were significantly declining due to a series of infection control interventions. S6 17th ECCMID / 25th ICC, Oral presentations These included terminal disinfection of the environment in isolation rooms and cohort areas by application of 1:1000 sodium hypochlorite in place of detergent (ECCMID '06, abstract P1333). We evaluated the effect of replacing sodium hypochlorite with a standard detergent. Methods: From January 1997 to May 2006, monthly percentage, nonduplicate S. aureus clinical cases caused by MRSA were collated. In February 2005 hypochlorite cleaning solution as replaced by a standard detergent. Other infection control measures remained unchanged. Dynamic regression analysis with linear transfer functions and interrupted time-series analyses were used to estimate the effect to the intervention. Results: Previously, MRSA rates wee successfully reduced due to environmental screening (p = 0.03), use of hypochlorite for environmental disinfection (p = 0.002), use of alcohol based hand disinfection (p = 0.03) and patient admission screening (p < 0.01). Stopping the hypochlorite disinfection was associated with a sudden increase in clinical cases of MRSA from 10 to 25% over a 6 month period (p = 0.03), with levels approaching those seen prior to the start of the infection control programme in 2001 (see figure). Conclusions: Stopping hypochlorite environmental disinfection was strongly associated with an increase in clinical MRSA cases. This work adds significantly to the meagre published evidence that environmental contamination is important in the spread of MRSA. Objectives: To determine antibiotic-resistant enterococci in Portuguese piggery samples and to analyse antibiotic resistance genes among these strains after European growth promoter ban. Methods: Samples from waste treatment and dry faeces from 2 pig farms in the South of Portugal were studied during 2006. Samples were plated onto selective Slanetz-Bartley agar with or without antibiotics. Bacterial identification was performed by both standard biochemical profiles and amplification of species specific genes. Antibiotic susceptibility (12 antibiotics) was determined by disk diffusion method (CLSI). Detection of genes coding for resistance [vanA, B, C1, C2, ermA, B, C, tetM, L, O, K, S, aac(6 )-Ie-aph(2 )-Ia, aph(2 )-Ib,Ic,Id, aph(3 )-IIIa, vat E] were searched by PCR. Results: We identified 84 enterococci (9 E. faecalis, 17 E. faecium, 1 E. gallinarum, 2 E. casseliflavus and 55 Enterococcus spp.) . Most isolates showed decreased susceptibility to tetracycline, minocycline, erythromycin, and quinupristin-dalfopristin (95%, 94%, 80%, 54%, respectively), and to a lesser extent to high-level of resistance (HLR) of streptomycin, nitrofurantoin, ciprofloxacin, HLR to gentamicin, chloramphenicol and ampicillin (52%, 33%, 32%, 21%, 11%, 10%). All were susceptible to glycopeptides. Non-susceptible isolates to tetracyclines, aminoglycosides and macrolides contained tetM (55%), tetL (54%), tetM+tetL (32%), tetS (5%), aac(6 )-Ie-aph(2 )-Ia (89%), aac(6 )-Ie-aph(2 )-Ia+aph(3 )-IIIa (47%), ermB (49%). vanC1 was linked to E. gallinarum, and vanC2 to E. casseliflavus as expected. Conclusions: Although all growth promoters were progressively removed from EU in the course of the last 10 years, antibiotic multiresistant enterococci were isolated in Portuguese piggeries. Whether persistence of these antibiotic resistant strains is due to selection by antibiotics or other agents deserves further studies. O25 Experiences and results from the surveillance programme of resistance in feed, food and animals in Norway (NORM-VET) 2000-2005 M. Norström, H. Tharaldsen, M. Sunde (Oslo, NO) Objectives: The monitoring programme for antimicrobial resistance in the veterinary and food production sectors (NORM-VET) was established in 2000. The goals of the programme are to monitor the antimicrobial resistance situation in feed, food and animals over time, in relation to the human situation and to the resistance situation in other countries. Data from NORM-VET could form a basis for risk assessments and be a tool for targeting interventions and further to evaluate the effectiveness of such interventions. This study was performed to summarise the experiences and results obtained during the first six years of the programme. Methods: The zoonotic agents Salmonella (from feed, animals and food) and Campylobacter jejuni (from broiler and broiler meat) were monitored annually. E. coli and Enterococcus spp. (indicator bacteria) were sampled from various animal species and meat products biannually. Specific clinical isolates from the routine diagnostic have been included biannually. The isolates have mainly been tested using a microdilution technique (VetMICTM). The minimum inhibitory concentrations were recorded and analysed in WHONET 5.3. For the categorising of the isolates as resistant or susceptible epidemiological cut-off values were applied. The occurrence of resistance in the monitored species and products is in an international perspective low and the results from the first six years of the programme show that the situation is stable. Conclusion: Evaluation of the first six years of the programme has recognized that the relatively low number of isolates of each species and source included complicates the conclusions possible to draw from the data, especially evaluating trends over time. Even though the run costs of the programme has been limited to a minimum, it is still useful for the purpose of monitoring antimicrobial resistance within a country as Norway, where the resistance problem in the animal and food sectors still is at a very low level. It also consists as valuable source for further research of antimicrobial resistance mechanisms and development. However, the use of this source to perform risk assessments is limited as there still is a lack of even more specific data as for instance data on usage at animal or farm level. O26 Comparison of antibiotic susceptibilities of Staphylococcus aureus and S. intermedius isolates from dog owners and their dogs Objectives: Antibiotic resistance in veterinary isolates has been reported to be higher than in human isolates due to frequent empirical use, and there are concerns about transfer of resistance between staphylococcal species. S. intermedius is the more common colonising species in dogs, but S. aureus, including MRSA, may also be present. Case reports suggest there can be cross-infection between companion animals and man. Increasing concern about MRSA in the community has led to recommendations for surveillance of antibiotic resistance in isolates from companion animals. This study compared antibiotic resistance in isolates of S. aureus and S. intermedius from dogs and their owners. Methods: A cross-sectional study of owners and their dogs was performed using a convenience sample of 800 pairs recruited at six veterinary practices. Nasal swabs were collected from both owner and Resistance surveillance S7 dog, and held at 4ºC in transport medium until culture within 8 h of collection. Subjects completed a questionnaire providing demographic information of owner and dog, and stating if the dog had received antibiotics within the last 3 months. Swabs were inoculated onto blood agar and mannitol salt agar and placed in 5% salt meat broth for enrichment. S. aureus or S. intermedius were identified by means of coagulase, VP, polymyxin susceptibility, and trehalose fermentation. Several colonies of each isolate were tested for susceptibility to methicillin. Antibiotic susceptibilities were determined by disc diffusion and interpreted using CLSI guidelines. Results: S. aureus: 168 owners (25%) and 64 dogs (8.5%) were colonised. 16 owners and their dogs were concurrently colonised. 6 dogs (1.3%) and 4 humans (0.5%) were colonised with MRSA. Resistance to oxacillin, clindamycin, gentamicin, tetracycline and fusidic acid was significantly higher in dog isolates. S. intermedius: 64 dogs (7.9%) and 8 owners (1.1%) carried S. intermedius. Four colonised owners had colonised dogs. Methicillin resistance was not detected. Resistance to chloramphenicol, clindamycin, tetracycline, and cotrimoxazole was higher in dog isolates. Resistance to fluoroquinolones and gentamicin was only displayed by dog isolates. Conclusions: Methicillin resistance was found only in S. aureus, but resistance to other antibiotics was higher in S. intermedius. Dog isolates were more resistant than human for both species. Veterinary use of antibiotics may increase resistance and the risk of transmission of resistant strains. Objectives: Antimicrobial susceptibility to human-use antibiotics was investigated among the commensal bacteria E. coli (Ec) and Enterococcus spp. (Ent) from healthy food animals at slaughter across the EU. Methods: Colon or caecal content was randomly collected at 4 abattoirs per country (n = 5 per host). Each herd/flock was sampled once. Ec and Ent were isolated using standard methods. Antibiotic susceptibility testing was done by agar dilution (CLSI, M31-A2) against 9 (Ec) and 5 (Ent) antibiotics in a central laboratory. Resistance (CLSI, M100-S16) was assessed per drug/organism/country. Results: A total 1465 Ec were recovered (cattle n = 490, pigs n = 494, chickens n = 481). Mean resistance (%) for Ec was: ampicillin (A) 3, 29, 53; cefepime 0, 0, 0; cefotaxime 0, 0, 0.4; ciprofloxacin 1, 0.4, 6; chloramphenicol 2, 16, 15; colistin 0, 0.4, 0; gentamicin (G) 1, 2, 2; tetracycline 8, 66, 65; and trimethoprim-sulfamethoxazole 4, 42, 52 respectively. For Ec, Italy and Spain consistently showed the highest resistance; Denmark showed the lowest. A total of 718 Ent isolates were recovered, comprising 356 E. faecium, 83 E. faecalis and 279 other species including E. durans, E. hirae and E. casseliflavus. All Ent but one bovine isolate, were susceptible to linezolid. For E. faecium resistance to A and G was 0−2%; vancomycin (V) resistance amounted to 1.9 to 3.5%, whereas resistance to quinupristin/dalfopristin (Q/D) combination varied: 8% (cattle), 19% (pigs) and 20% (chickens). Though low prevalence of E. faecalis limited conclusions, particularly in chickens (n = 6), G and V resistance was low in cattle and pigs (0-11%), resistance to Q/D was very high (46 and 83%, respectively). In the other Ent species, resistance among the 3 hosts to G and V was low (0−1.6%). Resistance to A was absent except in chickens: 9.8%. Q/D resistance was among the highest: 5−9% for livestock hosts; 26% in poultry. Striking differences among countries were absent for Ent. Conclusion: This pan-EU survey, with uniform methodology, shows that antimicrobial resistance among enteric commensal bacteria at slaughter was variable. For Ec, prevalence of antimicrobial resistance varied for older antimicrobials and between countries but resistance to newer medically important antimicrobials was absent or very low. With respect to Ent, antimicrobial resistance rates varied for quinupristin/dalfopristin, but resistance was absent or very low for other antimicrobials including linezolid and vancomycin. O28 Demography and antibacterial susceptibility of communityacquired respiratory tract infection pathogens in Year 6 vs Year 5 of the PROTEKT surveillance programme D.J. Farrell, C. Couturier, D. Felmingham (London, UK; Paris, FR) Objectives: Patterns of antibacterial resistance among communityacquired respiratory tract infection (CARTI) pathogens vary among countries. PROTEKT is a global surveillance study monitoring antibacterial resistance among CARTI pathogens. We report here the results of the sixth year of the PROTEKT study (Y6: 2004-2005) and changes compared to Y5 (2003-2004). Methods: Clinical isolates of Streptococcus pneumoniae (SPN) and Haemophilus influenzae (HI) from respiratory samples were submitted from 93 centres from 28 countries. MICs and susceptibilities were determined according to CLSI guidelines. Genotyping was performed to define macrolide resistance mechanisms. Frequencies were compared for Y5-Y6 common sites using c 2 or Fisher's exact tests as appropriate with a = 0.05. Results: In Y6, a total of 5182 SPN and 1609 HI were collected. SPN/HI distribution by site were: sputum & BAL (55%/72%), blood (19%/2%), nasopharynx (10%/13%), ear (10%/7%), sinus (6%/6%). The distribution of specimens by age groups were: 2 y 12%, 3−14 y 14%, 15−64 y 44%, and >64 y 30%. SPN penicillin and erythromycin resistance (PR and ER) prevalences were 19% and 35% respectively [20% erm(B), 9% mef(A), 5% erm(B)+mef(A), and 1% ribosomal mutation]. SPN with both PR and ER was >25% in Far East, South Africa and France and >15% in Hungary, Poland, US and Australia. Significant increases in PR were seen in Poland and China. Falls in PR were seen in Italy, Spain and Japan. Erythromycin resistance was stable in all countries except Germany and Venezuela (decrease). SPN with multiple drug resistance (>2 antibiotic classes) increased in Poland and decreased in Russia. Prevalence of isolates resistant to at least 5 antibiotic classes increased in China. Betalactamase (BL) production in HI was 14% overall, while 2.9% of HI were BL negative and ampicillin resistant (BLNAR). BL production frequencies were stable in all countries except South Africa (increase). Telithromycin showed a sustained activity against both SPN (99.6% susceptible, MIC50/90 0.015/0.25 mg/L) and HI (99.3% susceptible, MIC50/90 1/2 mg/L). Conclusions: Resistance to several first-line antibacterials is a continuing problem worldwide. The last 2 years of PROTEKT indicate changing patterns of resistance in several countries. Telithromycin exhibited significant in vitro activity against the principal CARTI pathogens, including strains resistant to other agents. O29 Community-acquired respiratory tract infections in Europe caused by S. pneumoniae, H. influenzae and Moraxella catarrhalis: report from 10 years of monitoring by the SENTRY Program R. Jones, M. Stilwell, T. Fritsche, H. Sader (North Liberty, US) Objectives: To determine the antimicrobial susceptibility (S) patterns for S. pneumoniae (SPN), H. influenzae (HI) and M. catarrhalis (MCAT) when tested by reference CLSI methods using samples collected from 1997-2006 in 13 European (EUR) nations. Trends in S and the occurrences of well defined resistance (R) mechanisms were assessed. Methods: A collection of community-acquired respiratory tract infections (CA-RTI) pathogens (6,753 SPN; 6,280 HI; and 1,908 MCAT for all years) were annually forwarded to a central reference laboratory for S test processing and confirmation of organism identity. All antimicrobials were tested by CLSI methods, results interpreted by M100-S16 (2006) and quality control rigidly applied to assure accuracy. Analysis of trends used mean S rates by nation for the initial and last 3 years sampled. Oral presentations Mobile resistance elements in Gram-negatives S1 Plasmids in clinically-significant Gram-negative bacteria Bacteria carry extrachromosomal, self-replicating genetic elements, called plasmids. Natural plasmids have systems guaranteeing their autonomous replication but also mechanisms controlling the copy-number and ensuring stable inheritance during cell division. Plasmids have evolved as an integral part of the bacterial genome, providing additional functions to their host. The best example of functions associated to a plasmid is given by the antimicrobial resistance. Resistance genes located on plasmids offer immediate advantage to their host under antimicrobial pressure and they can be easily exchanged among bacteria of different species and genera, through the natural process of conjugation, rapidly spreading in bacteria of diverse origin and source. Plasmids are playing the major role in the diffusions of antimicrobial resistance in Gram-negative bacteria, promoting the spread of resistance genes coding b-lactamases such as CTX-M, SHV, TEM, DHA, CMY, VEB, or aminoglycoside modifying enzymes or proteins mediating quinolone resistance. Plasmid-mediated antimicrobial resistance arises from a complex multifactorial process supported by a panoply of mobile genetic elements that contain and transfer resistance determinants. Bacterial plasmid sequence comparisons provided the historical events through which these genetic determinants are assembled by transposition, homologous recombination, and illegitimate recombinational events. Plasmids also play a very important role in bacterial pathogenesis as carrier of virulence genetic traits. Several resistance plasmids have been described in Enterobacteriaceae to carry virulence factors, such as bacteriocins, siderophores, cytotoxins, or adhesion factors and virulence plasmids have been described to carry resistance genes. Plasmids are consequently a fashionable field of scientific research. Among the most important issues of clinical microbiology, increasing prevalence of bacterial pathogens expressing resistance to multiple antimicrobial agents is of special concern. The observed high frequency of multiresistance suggests mechanisms by which bacterial species can concentrate and efficiently exchange a variety of resistance determinants. Lateral gene transfer, which has been proposed as a fundamental process underlying bacterial diversity, is mainly mediated through plasmids and phages. While integrons are capable of concentrating resistance genes, they are uncapable of self-transposition. The mobility of the resistance genes is mediated by insertions sequences and transposons. Along with the discovery of novel resistance genes, the number of different transposable elements isolated and characterised at the nucleotide sequence level is exploding, and over 1500 different ISs have been identified to date. Bacterial insertion sequences (IS) are small DNA segments (<2.5 kb) with a simple genetic organisation. Most IS elements exhibit short terminal inverted-repeat sequences (IR) and encode a transposase, an enzyme that is required for transposition. These mobile elements play an important role in assembling sets of "accessory" functions in bacteria (such as genes forming parts of degradative or catabolic pathways) and in dissemination of resistance genes. By inserting within a coding sequence they may inactivate the gene, or by inserting upstream of a gene they may modify its expression. ISs may also help integration of plasmids into the chromosome of bacteria. Some ISs, such as ISEcp1, are capable of mobilising nearby DNA, by a mechanism similar to one-ended-transposition, while others may form a composite transposon, being capable of carrying the sequence located between the two ISs. True transposons such as Tn7, and the large group of type II transposons of the Tn3 family are complicated structures and include multiple antibiotic resistance genes carried by integrons. The association of integrons with transferable elements may promote rapid dissemination among clinical strains, and create further opportunities for inclusion of additional resistance determinants. Recently novel genetic structures, Repeated Elements (Re), have been described being capable of mobilising resistance genes through a transposition/recombination process that still needs to be fully understood. The study of novel resistance mechanisms and their underlying genetic background revealed novel mobile DNA elements responsible for their mobilisation. These elements are of interest for clinical microbiology, since they are associated to the emergence of novel antibiotic resistance genes, and for fundamental research, since novel genetic mechanisms for gene mobilisation and dissemination were identified. Synthetic antibacterial drugs such as antifolates, sulfonamides and quinolones have been widely used in human and veterinary practice over the last 60 years. The clinical efficacy of these drugs is undermined by the integration and spreading of resistance borne on transmissible plasmids and genetic islands. In 1977, Pattishall et al. divided the plasmid-borne trimethoprim resistance genes into two distinctive structural classes later referred to as dfrA and dfrB. The number of identified gene types has grown to more than 30 at present. We matched the contexts of all sequenced dfr genes with signatures of two gene capture mechanisms. These are uptake of gene cassettes in integrons by site-specific recombination and one-ended transposition mediated by IS91-like elements referred to as common regions or ISCR. The data we assembled showed that trimethoprim resistance in Gram-negative bacteria is spread, at nearly full coverage, via either of these recombinational paths. Nineteen types of dfr genes in three families, examplified by the gene types A1, A12 and B1, are borne on integron-based cassettes while seven dfr gene types, A3b. A9, A10, A19, A20, A23 and A26 (Grape et al., unpublished) have been inserted via the second recombination path. IS91 family insertion elements use a one-ended transpositional mechanism to produce single-stranded DNA through rolling circle replication. Integron cassettes use recombination sites that are in fact recognized by the cognate tyrosine recombinase in the form of harpins. This suggests that IS91 might enhance excision and integration of cassettes that are situated within replication range of IS91-like elements. If the two gene capture systems are indeed engaged in a functional crosstalk this could explain their frequent co-localisation. The spreading of resistance to other synthetic drugs differs. For instance, sulfonamide resistance in Gram-negative bacteria is ubiquitous but much less diverse with only three gene types in the metagenomic pool. The recombinational spread of sul genes is cryptic and may be diverse. However, one of the genes, sul1, is strongly linked with both integrons and common regions of the IS91 type. Sulfonamides were introduced prior to other modern antibacterial drugs suggesting that it was the key selecting factor for making the dual recombinational platform of integrons and IS91-like elements fixed in most bacterial populations. Quinolone resistance gets contribution from transmissible qnr alleles of three families reported in connection with IS91-like regions (Robicscek et al. 2006 ). 17th ECCMID / 25th ICC, Oral presentations Bacteria are noteworthy for their remarkable genetic plasticity responding and adapting to environmental changes that often as not involves the exchanging of DNA molecules in the same cell and between bacterial cells. Insertions sequence common regions (ISCRs) were first discovered and reported in the early 1990's as a DNA sequence of 2,154 bp that was found in two complex class 1 integrons, In6 and In7. The sequence was initially described as a common region (CR) to distinguish it from the 5 and 3 conserved sequence of class 1 integrons. ISCRs were also interesting in that they are intimately associated with different resistance genes carried on each integron. ISCR elements have been found in many Gram-negative bacteria and have been associated with an array of different antibiotic resistant functions including qnr, ESBLs, AmpC b-lactamases, metallo-b-lactamases, aminoglycoside and chloramphenicol resistance, dhfr and sul genes. Some bacteria contain several copies of these elements that have probably occurred via replication followed by homologous recombination. They can be located on either the chromosome or/and plasmid. There are now at least 15 known types of ISCR elements that when aligned share key amino acid motifs with each other and that of IS91. These motifs have been shown to be involved in DNA recognition and mobility. ISCR elements are very unusual in that, like IS91, they can mobilise large sections of adjacent DNA via a rolling circle replication. Normally, termination of replication occurs at a defined site; misreading of this site leads to replication of large sections of DNA to the left-hand end of the element including antibiotic resistance genes. Many elements are found next to class 1 integrons which we believe to be expressed and active -possibly driven by a hybrid promoter. The only ISCR element shown not to be expressed is ISCR2. They are now commonly found in Enterobacteriaceae, Gram-negative non-fermentors and occasionally Gram-positive bacteria and have been found in clinical isolates from every continent. Their impact on the spread of antibiotic resistance genes is only now beginning to be understood. Diagnosis and treatment of septic shock (Symposium arranged with the International Sepsis Forum) S5 The innate immune response to P. aeruginosa in human monocytes is mediated mainly by TLR-2 and mannose receptor P. Xaplanteri, V. Le Cabec, G. Dimitracopoulos, F. Paliogianni (Patras, GR; Toulouse, FR) P. aeruginosa is a human pathogen that causes life threatening infections in immunocompromised host. We have previously shown that Extracellular Slime Glycoliprotein (GLP) produced by all mucoid and non mucoid strains is a potent activator of NFkb transcriptional activity and TNF-a induction in fresh human monocytes. Objectives: To examine which innate immune receptors are involved in signaling proinflammatory cytokine production induced by P. aeruginosa. Methods: We stimulated for 6−24 h fresh human monocytes either with P. aeruginosa Slime GLP (100 mg/mL) or viable bacteria (MOI 10: 1) in the presence or absence of specific blocking antibodies against TLR4, TLR2 and Mannose receptor (10 mg/mL). Proinflammatory cytokine production (IL-1b, IL-6, TNF-a) was measured in the culture supernatants by ELISA. Phagocytosis was inhibited by the addition of Cytochalasin D (1 mg/mL) 30 min before stimulation. Receptor mediated activation of NFkb was examined by cotransfection experiments, using human embryonic kidney (HEK)-293 cells and plasmids encoding mannose receptor, TLR4 and TLR2−CD14 complex along with NFkb reporter driving the expression of luciferase gene. NFkb activation was detected by measuring luciferase activity. Results: Blocking of phagocytosis or mannose receptor function resulted in 90% inhibition of proinflammatory cytokine production. TLR2 blocking resulted in 70−80% inhibition whereas TLR4 blocking had less prominent effect (30−40%). Challenging of HEK-293 cells expressing only mannose receptor with Slime GLP or viable bacteria resulted in NFkb activation by 6−10 fold and 20−30 fold correspondingly. Co expression of TLR2 in the same cells increased the NFkb activation by 3 fold in both ways of stimulation, whereas coexpession of TLR4 resulted in just detectable increase, 1.2−1.5 fold. Conclusion: Our results suggest that mannose receptor acts not only as phagocytosis receptor for P. aeruginosa, but also as signaling receptor and cooperates with TLR2 for maximum NFkb activation and proinflammatoty cytokine production. These findings provide a new insight for future development of new immunotherapies. Challenges in pharmacokinetics and pharmacodynamics S11 PK/PD in critically ill patients Antimicrobial treatment of infections in critically ill patients remains a significant challenge with persisting high mortality and morbidity. Early and appropriate antibacterial therapy remains an important intervention for such patients. To optimise antibacterial therapy, the clinician should possess not only knowledge of the pharmacokinetic and pharmacodynamic properties of commonly used antimicrobials, but also how these parameters may be affected by the pathophysiological changes occurring during sepsis or septic shock. Pathophysiological changes can be contrasting. Sepsis, and the nonantibiotic treatment, increasing renal preload may result in higher antibacterial clearances. Alternatively, sepsis can induce multiple organ dysfunction, including renal and/or hepatic dysfunction, causing a decrease in antibacterial clearance. Aminoglycosides, b-lactams and carbapenems are distributed in plasma and interstitial space, therefore their concentrations could be dramatically affected during sepsis. These drugs can have a higher Vd during sepsis leading to a reduced Cmax. Some patients with serum creatinine within the normal range can have higher than normal drug clearances, thereby producing low serum concentrations. If a drug needs to have a minimum serum concentration maintained (e.g. b-lactams), a high drug clearance will lead to underdosage for renally excreted drugs. All b-lactams should, in such patients, be administered more frequently than suggested in non-sepsis patients; administration by continuous infusion should be considered. In relation to the aminoglycosides, this means that not only are large doses required to be administered, but because of a high CLCR these antibacterials may also need dosing even more frequently than every 24 hours. Regarding the renal clearance of the fluoroquinolones, in the presence of a high CLCR can be assumed that fluoroquinolone clearance is also high. If this were true these antibacterials would also need to have higher daily doses than proposed in the standard literature. In conclusion the treatment of infections in critical ill patients remains a significant challenge given the persisting high mortality rates. Data suggest that effective antibacterial therapy remains the most important intervention available to the clinician. In treating sepsis, a clinician must be aware of the impact of the various pathophysiological and subsequent pharmacokinetic changes that can occur during sepsis. O16 Bacterial population kinetics on hands during two consecutive surgical hand disinfection procedures G. Kampf, C. Ostermeyer, T. Kohlmann (Hamburg, Greifswald, DE) In Europe, the efficacy of surgical hand antiseptics is investigated only for a single application. In clinical practice, however, consecutive applications are common but the effect on the bacterial density on hands is largely unknown. We therefore studied the effect of different consecutive applications on the resident bacterial flora on hands. A propanol-based hand rub (Sterillium ® , based on 45% propan-2-ol, 30% propan-1-ol and 0.2% mecetronium etilsulfate) and the reference alcohol of EN 12791 (60% n-propanol) were tested in two consecutive applications with 3 h between them. Four variations were tested. The first application of Sterillium was always for 1.5 min, the second application was for 1.5, 1 or 0.5 min. The reference alcohol was applied for 3 min (both applications). The efficacy of the three product variations was compared to the reference treatment of EN 12791. All experiments were performed in a Latin-square design with 20 volunteers. Pre-and post-values (immediately and 3 hr after each applications) were obtained according to EN 12791. The first reference disinfection (3 min) reduced the bacterial density by 2.87±1.00 log 10 -steps (immediate efficacy) and 2.27±1.25 log 10 -steps (after 3 h). Sterillium applied for 1.5 min yielded a similar reduction at both sampling times. Immediately after the second 3 min reference disinfection bacterial density was reduced by 0.45±1.05 log 10 -steps. Application of Sterillium yielded larger reductions with 0.71±1.32 (0.5 min application time), 0.79±1.63 (1 min application time), and 1.12±1.04 log 10 -steps (1.5 min application time). The difference between the four treatments, however, was not significant (p = 0.089). After 3 h under the surgical glove bacterial density further decreased with 1.11±1.04 log 10 -steps for the reference disinfection. A 1 min application of Sterillium yielded the largest reduction (1.89±1.02) followed by a 1.5 min treatment (1.67±0.98) and a 0.5 min treatment (1.08±0.86). There was a significant difference between all four treatments (p = 0.005; Friedman test) but none of the short treatments with Sterillium was significantly less effective than the 3 min reference treatment (p > 0.05; Wilcoxon-Wilcox test). Overall, a simple 1.5 min application of a well-formulated propanolbased hand rub for surgical hand disinfection keeps the bacterial density as low as possible even in two consecutive surgical procedures of 3 h. Conclusions: HPV is more effective than standard terminal cleaning for the eradication of nosocomial pathogens. Recontamination was not immediate for MRSA and GNR but returned towards pre-cleaning levels for MRSA and post-cleaning levels for GNR within a week in a room occupied by a patient colonised with MRSA and GNR. This finding has important implications for the optimal deployment of HPV decontamination in hospitals. . Trends toward greater PEN-and ERY-R were noted in 7 and 12 countries, respectively. FQ-R (0.9% overall) indicated by levofloxacin MIC at 4 mg/L was >1% in Belgium, Italy (7.8%; clonal), Spain, UK, Israel and Ireland. MCAT had a uniform BL-production rate (95%, range 92-100%) across EUR and macrolides (MIC90, 0.25 mg/L), tetracyclines ( 2 mg/L), FQs ( 0.06 mg/L) and enzyme stable b-lactams remained very potent. BL(+) rates in HI ranged from 3.6% (Germany) to 23.9−30.8% (France, Israel). BLNAR HI strains (Table) were found in 5 nations (range, 0.5−1.7%) with an overall average of only 0.3%. A/C-R was rare (0.4%) and noted in 3 nations; highest (5.5%) in Spain. HI-S rates of >90% were documented for A/C, azithromycin, CRO, FQs, RF, chloramphenicol, oral cephems and tetracyclines. a Average of last 3 years of participation. PNSP = PEN-non-S SPN, ENSP = ERY-non-S SPN; BLP = b-lactamase production, BLNAR = b-lactamase-negative AMP-R. The SENTRY Program has consistently monitored EUR CA-RTI pathogens and observed relatively stable R patterns among HI and MCAT but variable rates among nations. SPN R-rates for PEN, macrolides and to a lesser extent FQs continues to evolve at varying velocities among the 13 countries sampled from 1997-2006. These results corroborate data reported by EARRS for 2004. Objectives: To determine the rate, characteristics and risk factors of intestinal carriage of nalidixic acid (NA) resistant E. coli (E. coli NAR) in healthy children who have never received quinolones as antibiotic treatment. Methods: Two consecutive surveys, 10 months apart, were carried out to determine the prevalence of E. coli NAR in a cohort of healthy children aged 3−5 years from 9 Day Care Centers (DCC) in 3 Arab villages of northern Israel. Parents were interviewed on demographic and socioeconomic characteristics and medical history of the children and their families. Stool samples obtained from the cohort of children and from a sub-sample of siblings and mothers were inoculated onto MacConkey agar plates, containing NA (32mg/mL). The NA Minimal Inhibitory Concentration (MIC) was determined by the broth microdilution technique. The E. coli NAR strains were examined for ciprofloxacin resistance. Selected strains were evaluated for efflux pumps activity and for point mutations in the gyrA and parC genes. Uni-and multivariable analyses were used to identify risk factors of E. coli NAR carriage. Results: We found that 17.2% (34/198) of the children carried E. coli NAR in the first survey and 42.3 (85/208) in the second. Among the children examined in both surveys (n = 147), 9.4% harboured E. coli NAR in the first survey but became negative in the second while 38.5% children found negative in the first survey carried E. coli NAR in the second. E. coli NAR were isolated in both screenings among 11 (9.4%) children. 21% and 34% of the E. coli NAR strains were also resistant to ciprofloxacin in the first and second surveys, respectively. Persistent resistance to NA was associated with an increase in MIC, number of mutations in the gyrA and parC genes and presence of efflux pumps. Acquisition of resistance to NA was significantly higher in two of the DCCs located in one of the 3 villages. The carriage of E. coli NAR was not associated with the child's age or gender, use of antibiotics, or carriage of E. coli NAR among mothers and siblings. Conclusions: The lack of evidence for intra-familiar spread of E. coli NAR and the significant acquisition of E. coli NAR in 2 specific DCCs suggest that other means of transmission such as the food or waterborne routs may explain the high carriage rate of E. coli NAR. Persistent carriage of E. coli NAR is of concern in view of the association with an increase in both phenotypic and genetic markers of resistance to quinolones. rarely cultured enterococci had a MIC90 of 2 mg/L (range, 2 mg/L). Corynebacterium spp. (342) were most susceptible (S) to linezolid (MIC50, 0.25 mg/L) while enterococci and Listeria were 4-to 8-fold less S (MIC50, 2 mg/L). Evaluations of R trends in those species failed to identify any MIC creep over the seven years monitored (data not shown). Table. Linezolid potency against uncommonly isolated Gram-positive organisms (3, 251) . ii. On a given continent, PVL-positive CA-MRSA have spread from country to country. For instance, in Europe, PVL-positive CA-MRSA of ST80 were recently detected in Slovenia, Romania and Croatia. iii. New PVL-positive CA-MRSA clones are emerging on different genetic backgrounds. While most of the clones described in 2003 had an agr3 background, the newly described clones are agr1 or agr2. Clone ST22 (agr1) has been found in Europe only and clone ST377 (agr1 with a type V SCCmec) was reported simultaneously in Europe and Australia. iv. PVL-positive CA-MRSA, which were initially susceptible to most antistaphylococcal antibiotics, have acquired new antibiotic resistance determinants, to gentamicin and ofloxacin for instance. v. The prevalence of PVL-positive CA-MRSA varies considerably from one continent to another (high for instance in the USA (~50%), Greece and Algeria, low in most European countries). vi. PVL-positive CA-MRSA are gradually causing an increasing number of hospital-acquired infections in countries where their prevalence is high. Conclusion: To counter this emerging global threat to public health, systematic surveillance of both hospital and community isolates is required, together with measures designed to limit their spread. In the Nordic countries and the Netherlands, nosocomial MRSA has successfully been contained for more than 25 years keeping the prevalence of MRSA at <1% in these countries. This has been accomplished by enforcing preventive contact isolation of all hospitalised patients suspected or confirmed as MRSA positive. This has been facilitated by the fact that in these countries the predominant risk factor for patients for MRSA acquisition was hospitalisation abroad, making these at-risk patients relatively easy to identify. Additionally, secondary cases seldom arose after hospital discharge. The recent change in epidemiology where transmission of MRSA increasingly occurs outside hospitals has changed the situation, and an increasing number of MRSA has been reported in all the above mentioned countries. Since the mid-1990s the number of new cases of MRSA in Denmark has increased by 10-fold. The majority of these MRSA has been identified in primary healthcare (community onset) and, in approximately 33% of the cases, the patient had had no connection with healthcare institutions for at least one year prior to the diagnosis. These cases have therefore been considered as community acquired (CA-MRSA). CA-MRSA infections have especially been reported from children and younger adults. Skin and soft tissue infections dominate and represent approximately 90% of the cases. Approximately 30% of CA-MRSA cases have been reported in persons with another ethnicity than Danish. This could indicate that there is a continued influx of MRSA from high prevalence areas into Denmark. Strains belonging to the ST80-IV complex have been the most frequent cause of CA-MRSA in Denmark, however, an increasing diversification has recently been observed including almost all strains known as associated with CA-MRSA. During the same time period the number of MRSA diagnosed in hospitals also increased. This has happened despite continued use of contact isolation of MRSA positive patients during. Hospitalisation of MRSA carriers not identified on admission due to lack of risk factors is probably one important factor responsible for the increased number of nosocomial MRSA in Denmark. An increase in the prevalence of MRSA outside hospitals including in otherwise healthy persons, is thus of great concern as this not only gives rise to CA-MRSA infections but also inevitably will increase the risk of nosocomial MRSA infec-tions and outbreaks. In order to keep Denmark as a low prevalence country we believe that MRSA should be contained and eradicated not only in hospitalised patients but also when diagnosed in the community. This has lead to new national guidelines for preven-tion and eradication of MRSA including making both carriage and infection with MRSA mandatory reportable. The first question to ask when considering any screening programme for the identification of CA-MRSA carriage is why? The importance of epidemiological data cannot be underestimated. Data on prevalence can inform empirical therapy requirements or help in the design of intervention strategies. Prevalence can be established for the population in general, or, specific subgroups such as children, military personnel, indigenous or disadvantaged populations can be targeted. Community intervention measures similar to the 'search and destroy' techniques used in hospitals would require large-scale screening. A narrower approach concentrating on high-risk groups to enable programmes such as education on hygiene to reduce transmission would allow a more targeted screening programme. A second more pragmatic area for screening is on admission or during hospitalisation. In some areas in the world there is a blurring of the boundaries of CA-MRSA and HA-MRSA. These organisms have now been reported as a cause of hospital acquired bacteraemia, surgical and prosthetic joint infection and to colonise neonates and adult patients in hospitals. Targeted screening of high-risk patients or all patients on entry into high-risk units are strategies to prevent CA-MRSA from becoming endemic in hospitals. Screening to control recognized outbreaks entails identification of the reservoir to enable appropriate isolation, cohorting and decolonisation measures. The value of screening healthcare workers for carriage of CA-MRSA has yet to be established. How to screen is the next question. Which site(s) give the best yield? Which laboratory method should be used? The ideal test would have high negative and positive predictive values, be rapid, inexpensive, applicable to the routine laboratory, and have high through put capabilities. Currently used tests are traditional microbiological culture and identification methods, use of selective media including various types of CHROMagar and molecular techniques using real time PCR. The requirement for broth amplification increases the sensitivity for some of these methods but also increases the time to identification. There are many different CA-MRSA clones in the world, in Western Australia alone there are over 40 different multi locus sequence (MLST)/SCCmec CA-MRSA clones. Any molecular test must therefore have the ability to identify all clones in diverse regions of the world. The last questions are when and whom to screen. The answer is very much dependent on the purpose of the screening. The range can be from large-scale periodic population screening for epidemiological purposes to continual hospital unit, high-risk patient screening. There are no established guidelines or recommendations for screening for CA-MRSA. Many questions have yet to be answered particularly, whether control of transmission in the community is possible. By looking at what evidence is available we can move a step closer to control of this very significant community pathogen. Rates of infections caused by CA-MRSA continue to increase, causing over 50% of skin and soft tissue infections seen in the emergency department in a recent report. Strategies for control of CA-MRSA in patients and their families include use of infection control techniques in the household as well as decolonisation with mupirocin, chlorhexidine, and antibiotics in some cases. Given that CA-MRSA has been now been implicated in healthcare-associated infections, efforts to reduce the spread of this organism within the healthcare environment are also important and include appropriate hand-hygiene, isolation, and environmental decontamination. Incision and debridement remains critical in the management of most CA-MRSA infections. The use of antibiotics is indicated in many CA-MRSA infections and therapeutic options include older agents, such as clindamycin, trimethoprimsulfamethoxazole, tetracyclines, and rifampin, and newer agents such as linezolid. In addition, new agents are in development. Pneumococcal pneumonia is a pathogen of global significance and represents a leading cause of infection-related mortality at all ages. Recent developments in the field of pneumococcal pathogenesis reflect significant developments in both genomics and proteomics. The breadth of microbial genetic diversity is increasingly being recognized. Our understanding of the importance of traditional virulence factors such as polysaccharide capsule, pneumolysin and generation of hydrogen peroxide is evolving while important roles for factors such as pneumococcal pili are emerging. Genetic screens are identifying increasing roles for previously poorly characterised proteins involved in metabolism and transport of key molecules. The importance of the physical state of the bacteria in different tissue compartments is also becoming evident. An improved range of models of various aspects of infection ranging from colonisation and sub-clinical infection to fulminant pneumonia and invasive disease are allowing assessment of both microbial and host factors central to disease pathogenesis. The range of receptors involved in the innate response to pneumococci in the respiratory tract is starting to be appreciated and includes Tolllike receptors and receptors primarily involved in the phagocytosis of bacteria. The interaction between soluble factors and the resident phagocytes of the lung is being clarified and the complexity of cytokine networks involved in the innate host response is being elucidated. Important roles for T-lymphocytes, Natural Killer cells and dendritic cells in the response to pneumococci are being identified. Furthermore our understanding of the regulation of the inflammatory response and of the significant role of apoptosis in the regulation of the inflammatory response has lead to important insights into how cell survival and outcome of infection are closely linked. These observations are enabling a more scientific interpretation of many of the central clinical features of pneumococcal pneumonia and may encourage novel approaches to therapy to improve disease responses. Streptococcus pneumoniae (the pneumococcus) continues to be a leading cause of morbidity and mortality worldwide particularly among young children, the elderly and the immunocompromised of all ages. Infection is preceded by colonisation of the nasopharynx, which is the ecological niche of pneumococci. In most individuals colonisation is asymptomatic and does not evolve to disease. The carrier state is more frequent in young children and may reach over 70% in some populations such as those attending day-care centres. Indeed, this latter group has been found to be a major reservoir of pneumococci playing a key role on the amplification and transmission of the bacteria to other individuals. Of interest, several drug-resistant clones internationally disseminated have been found to be frequently carried by this population. In recent years, there has been some debate on the relative contribution of serotype and genetic background to the invasive disease potential of pneumococcal strains. We will present a study that addresses this question. The study was conducted in Portugal and results from the detailed comparison of two large collections of pneumococcal isolates: a group of over 450 invasive disease isolates and a group of over 750 colonisation isolates collected during the same time period which were characterised in detail by antibiogram, serotype, PFGE macro-restriction profiles, and multilocus sequence typing. The invasive disease potential of serotypes and clones will be discussed. Pneumococci cause a spectrum of diseases in humans, from reasonably mild diseases like sinusitis and conjunctivitis to potentially lifethreatening diseases like meningitis and bacteraemia. The current conjugate vaccines are ideally aimed at protecting against all pneumococcal disease, but have been very successful at preventing invasive pneumococcal disease. Understanding invasive disease epidemiology, both preceding vaccine implementation and after vaccine introduction, is crucial to the design and development of future vaccines. Several recent studies have shown that pneumococcal serotypes differ in their invasive disease potential, and this has particular relevance for the selection of serotypes to include in future conjugate vaccines. It is also essential to understand the serotype-specific changes that have occurred subsequent to conjugate vaccine implementation in the USA, as a model for what might occur post-vaccine implementation in other countries. The recent history of pneumococcal disease prevention is dominated by the development in the 1990's of conjugate pneumococcal vaccine for administration to infants, and its implementation starting in 2000 in the USA. The vaccine as currently formulated affords protection against invasive pneumococcal disease (IPD) due to the 7 most common serotypes causing IPD among children less than 2 years of age prior to vaccine introduction in the USA. This formulation is not optimal for many developing countries where serotypes such as 1 and 5 are important. Global, second generation vaccines therefore include at least 10 serotypes. The importance of prevention of pneumococcal disease extends beyond IPD to pneumonia, meningitis, otitis media, prevention of antibiotic-resistant infections, and even prevention of mortality in young infants. Each of these presents specific challenges. The vaccine has been shown to prevent between 25% and 37% of all cause X-ray confirmed pneumonias and this implies a level of protection against pneumococcal pneumonia due to vaccine serotypes in excess of 70%. This reduction in pneumonia has translated into a reduction in allcause mortality due to the vaccine of 16% in rural Africa. The major preventable burden of disease in Africa may be among HIV infected children where the vaccine has also been shown to reduce pneumonia burden. An innovative aspect of the vaccine is the fact that it may reduce the pneumococcal super infections that follow viral respiratory infections, such as influenza, and conjugate vaccination of children may thus be a useful adjunct to pandemic influenza planning preparedness. The interruption of carriage of vaccine serotypes has been shown to reduce IPD in adults in the USA due to herd immunity so the population benefit in an influenza pandemic may extend beyond protection just of immunised children. Herd immunity has also recently been shown to protect young un-immunised infants from IPD. Conjugate vaccine has had little impact on all cause otitis media in randomised trials, but post-marketing studies in the USA suggest that healthcare utilisation for otitis episodes has reduced significantly post vaccine introduction suggesting a major role for herd immunity and allowing physicians to feel more comfortable in a "wait and see" attitude to otitis once the prospects of significant complications due to the pneumococcus are likely to be rare. The conjugation of pneumococcal polysaccharides to Haemophilus influenzae protein D has allowed the development of a more efficacious vaccine against otitis media. There has been a dramatic reduction in antibiotic resistance among vaccine serotypes causing IPD in the USA, in both children and adults, but resistance, driven by continuing high levels of antibiotic use, is now selecting resistance in non-vaccine serotypes. In particular, multiresistant global clones of serotype 19A are now dominant in the USA and a 13-valent vaccine formulation including serotype 19A is under development. The implementation of immunisation programmes including conjugate pneumococcal vaccine to infants in European countries affords the opportunity to monitor the impact on all of these health outcomes in both children and adults. Infective endocarditis -time for change? S50 Treatment options for infective endocarditis: new drugs for bad bugs? In the USA infections due to both hospital and community-acquired methicillin resistant Staphylococcus aureus (MRSA) are increasing rapidly in both frequency and severity. As a result new treatment options are badly needed. This presentation will first address the role of traditional therapy focusing on the gradual development of increasing resistance to vancomycin among MRSA. Next it will focus on alternative oral therapies such as clindamycin, cotrimoxazole and minocycline and their role in the treatment of community-acquired MRSA, an organism that has displaced "traditional" MRSA in many venues. The efficacy of the newly approved antibiotics linezolid, daptomycin, and tigecycline will be addressed through the presentation of the results of large phase 3 clinical trials in complicated skin and skin structure infections, hospital-acquired pneumonia, and bacteraemia. Discussion will focus on the efficacy of these agents in the MRSA subgroups. Finally, the efficacy of antibiotics in development will be presented again focusing on the MRSA subgroups from phase 2 and 3 studies. These antibiotics will include dalbavancin, telavancin, oritavancin, iclaprim, ceftobiprole and ceftaroline. As the epidemiology of staphylococcal infections changes new therapeutic agents are becoming available to clinicians. Therapeutic utility will depend on understanding the unique characteristics and efficacy of the new agents in clinical trials in order to clearly understand their role in the new therapeutic paradigms. Antimicrobial resistance is a serious problem with increasing strains of bacteria becoming resistant to many or all available antibiotics. Tetracyclines were already introduced 50 years ago but have undergone a considerable resistance development in nosocomial and communityacquired pathogens. In June 2005, a minocycline derivative, the new glycylcycline antimicrobial tygecycline, was approved by the FDA for treatment of complicated intra-abdominal and complicated skin and skinstructure infections. Tigecycline, a bacteriostatic agent, binds to the 30S ribosome unit, blocks entry of amino-acyl tRNA molecules, and prevents protein synthesis; it has a broad antibacterial spectrum with activity against Gram-positive and Gram-negative pathogens, including multidrug-resistant organisms and anaerobes. The modification of the basic tetracycline molecule has overcome the two resistance mechanisms seen with tetracyclines (efflux, ribosome protection). Tigecycline exhibits linear pharmacokinetics, has a long half-life (24−48 hours), is highly protein bound (71−89%), and has a large volume of distribution (7−9 L/kg). Tigecycline is not extensively metabolised; the primary route of elimination is biliary excretion (59%) and 33% is excreted unchanged in urine. The recommended dosing regimen is an initial dose of 100 mg, followed by 50 mg every 12 hours. Time above MIC and AUC/MIC ratio are the most important pharmacodynamic parameters. In two double-blind studies the safety and efficacy of tigecycline versus aztreonam plus vancomycin were compared in hospitalised adults with complicated skin and skin-structure infections. Over 1000 patients were randomised and the results of the pooled analysis determined that tigecycline monotherapy was as effective and statistically non-inferior to aztreonam plus vancomycin. Two double-blind studies including more than 1000 patients analysed safety and efficacy of tigecycline versus imipenem/cilastatin in adults with complicated intra-abdominal infections. Results of the pooled analysis determined that tigecycline was efficacious and statistically noninferior to imipenem. Nausea (20−30%) and vomiting (15−20%) are the most common adverse effects observed in clinical trials. They usually occur within the first two days of therapy, are more common in women (48%) than in men (24%), and may also be age related. Tigecycline is an important addition to the antimicrobial armentarium. It has a broad spectrum of activity and its ability to evade numerous mechanisms of resistance makes it a promising solution to the treatment of multi-drug resistant organisms. This is very helpful in selected patients in the ICU. The increasing resistance of Gram-negative bacteria and particularly amongst Pseudomonas aeruginosa and Acinetobacter baumannii, raises a major therapeutic problem. The lack of new effective agents in the near future has revived interest in the re-use of some molecules, abandoned because of their toxicities. Colistin (colistimethate sodium) is a polymyxin antibiotic available in parenteral formulation and used for intravenous, aerosolised and intrathecal/intraventricular administration. The bactericidal activity of colistin is due to a detergent effect on the cell membrane and therefore not dependent upon bacterial metabolic activity. Because this drug was developed during the 1950s, only little pharmacokinetic and pharmacodynamic information are available and the optimal daily dose for severely ill patients is still unknown. Colistin, used both intravenously and by inhalation, was used until last decade mainly in cystic fibrosis patients. Recently, colistin has been used in non cystic fibrosis patients as a salvage therapy for the treatment of infections with Gram-negative bacteria resistant to all other antibiotics. Clinical reports on the efficacy of intravenous colistin in these patients are not randomised studies and in most of them, colistin was frequently associated with other antimicrobial agents with a lack of a control group. A good outcome occurred in most infections (52−75%) but with the poorest results observed in pneumonia (25%). In vitro studies have shown synergistic effect between colistin and rifampin but no clinical study has been designed to confirm this synergy. Nephrotoxicity and neurotoxicity are the most common toxicities of colistin. However recent studies demonstrated a reduced toxicity as compared with previous studies, even if a higher dosage is used. This low renal toxicity rate was observed in both critically ill patients and patients treated with prolonged courses of colistin. There are also recent clinical reports on the use of colistin in continuous intravenous infusion to minimise potential toxicity. This reduction of toxicity without impairment of efficacy (concentration-dependent killing in in-vitro timekill studies) should be confirmed by further studies. Neurotoxicity has also been more frequently reported in case of renal dysfunction. Development of resistance to colistin is a major problem, especially in A. baumannii. This highlights the urgent need to preserve this molecule by the best appropriate dose regimen and by an optimal synergistic combination with other antibiotics. Fosfomycin is a phosphonic broad spectrum antibiotic discovered in Spain in 1969, licensed in many countries since then, but used only sporadically in the clinical setting. In fact, It is not mentioned specifically in the current editions of the most prestigious textbooks of internal S13 medicine or infectious diseases. The emergence of infections caused by bacteria resistant to almost all antimicrobial agents may have renewed the interest for addressing the contributions, limitations and future clinical indications of this drug. Fosfomycin exhibits a rapid bactericidal activity against a large number of aerobic Gram positive and Gram negative bacterial species by inhibiting the initial step of cell wall syntesis. Some important multiresistant pathogens such as penicillin-resistant pneumococci, methicillin-resistant staphylococci, vancomycin-resistant enterococci and ESBL-producing enterobacteriaceae are usually susceptible to the drug. To date, no cross-resistance with other antibiotics has been reported. Favourable pharmacokinetic properties include a low molecular weight, a negligible protein binding, a large volume of distribution in human tissues and a good penetration into the inflamed CSF. The disodium salt of fosfomycin can be administered intravenously in high doses due to its very low toxicity, achieving plasmatic peak levels that are several times above the MIC of susceptible microorganisms (breakpoint 64 g/mL). An oral salt of fosfomycin and tromethamine with enhanced bioavailability is also available since 1990. The major drawback of fosfomycin is the frequent development of resistance during therapy. This phenomenon, which has been demonstrated in "in vitro" and "in vivo" studies, precludes its parenteral use as a single agent in the clinical practice. As a counterbalance, fosfomycin can act synergistically with other antibiotics, especially with those that inhibit later points in cell-wall synthesis. Such synergism has been shown repeatedly against different strains of Staphylcoccus aureus, CNS, Streptococcus pneumoniae and Pseudomonas aeruginosa. Moreover, in experimental animal models of MRSA endocarditis and cefalosporin-resistant pneumococcal meningitis, fosfomycin and b-lactam (or vancomycin) combinations have proved to be superior than monotherapies, preventing the emergence of drug-resistant populations as well. However, the reported clinical experience with these combinations is very scarce and, therefore, there are not evidence-based recommendations supporting its use. On the other hand, various clinical trials have indicated that fosfomycin trometamol, as a sole antibiotic, is effective and safe for the treatment of uncomplicated UTI, providing high and long lasting urinary fosfomycin levels, which help to prevent the emergence of resistant strains. Because of the increasing prevalence of CTX-M type ESBL-producing E. coli in the community, the oral administration of a single dose of 3 gr. of fosfomycin trometamol may be considered nowadays as a first line therapy for uncomplicated UTI in young women. However, the possible emergence of fosfomycin-resistant mutants among ESBL-producing enterobacteriaceae should be taken into account. In conclusion, after 40 years of its discovery, only the oral form of fosfomycin -tromethamine -has attained a solid position in the antimicrobial guidelines. Combinations of iv fosfomycin with other antibiotics may be considered in selected cases, but whether these combinations yield improved outcomes in specific infections due to multirresistant pathogens remains to be determined. Logistic reasons and financial limitations will make difficult the realisation of appropriate prospective studies to answer this issue in the near future. Chloramphenicol is still used extensively in non-industrialised countries for the treatment of severe infections like pneumonia and typhoid fever because it is cheap and effective. Chloramphenicol proved to be an effective alternative for the treatment of pneumococcal and meningococcal disease and also in patients with meningitis caused by Haemophilus influenzae. Chloramphenicol's loss of favour began in the 1960s, when it was shown to have two distinct toxic effects on hematopoiesis: a frequently observed, dose-dependent anaemia, reversible on cessation of therapy, and an irreversible, 'idiosyncratic' aplastic anaemia, which had an incidence of approximately 1 case per 30,000 courses of therapy, a high case fatality rate, and no correlation with the or duration of treatment. The use of chloramphenicol has then become restricted to life-threatening infections for which there are no acceptable alternative treatments like brain abscesses. Analogues of chloramphenicol have been developed that lack the aromatic NO 2 group that is thought to cause irreversible marrow aplasia. Although the clinical efficacy of such compounds has not been evaluated, they are effective in vitro against most bacteria, even those that are resistant to chloramphenicol. The data provided in support of a hypothesis that these molecules needed a nitro group in order to cause the blood dyscrasias were insufficient in themselves. Florfenicol has followed chloramphenicol in veterinary medicine. The potential for florfenicol to cause blood dyscrasias, such as aplastic anaemia, in humans was discussed in relation to the chemically related chemicals chloramphenicol and thiamphenicol. Human epidemiological data could not be used to demonstrate the safety of florfenicol as florfenicol has never been used in human medicine. Nevertheless, because there is as yet no way to monitor the potential for irreversible bone marrow toxicity, it is unlikely that such compounds will become available for the treatment of chloramphenicolresistant infections in general -and meningococcal disease in particular. The future of chloramphenicol is not entirely certain. On one hand, it is a well-studied, long-standing treatment for fighting a number of different infections; it is also very inexpensive and consequently has very widespread use. It would, however, be a very good thing if we could find a new drug or group of drugs that produced similar results in fighting infection but did not have as many serious side effects. It would take a long time before any drug could ever replace chloramphenicol, because of both its broad use and its very low cost. Tropical medicine: from basic science to field work O57 Evaluation of malaria status and immune response to Plasmodium falciparum MSP-2 Objectives of study: MSP-2 is a highly polymorphic 45−53 kDa merozoite surface antigen and very immunogenic malaria antigen, which is considered as a promising vaccine candidate. The 3 S and 5 S end regions of the gene are highly conserved, whereas a large central region is variable. Many studies have suggested a protective role for specific IgG antibodies against variable regions of MSP-2. This study was designed to analyse the reactivity of human sera from people living in a malariaendemic area of The Gambia (village of Keneba) against different domains of MSP-2. The association of haemoglobin and parasitaemia as two indicators of clinical malaria with acquired immunity were analysed to elucidate the pattern of protective immunity. The current study was designed and carried out in Liverpool School of Tropical Medicine. 179 human sera were randomly selected from McGregor's Keneba Sera Collection (1966) (1967) (1968) (1969) (1970) (1971) (1972) (1973) (1974) (1975) (1976) (1977) (1978) (1979) (1980) . Different domains of MSP-2 were synthesized using GST gene fusion system and crude schizont extract was prepared from in vitro culture of Plasmodium falciparum. Total IgG and IgG subclass responses were measured by ELISA after a checkerboard study was performed for each antigen to standardise the concentration of both antigens and antibody. Results: Most sera predominantly recognized the immunodominant regions of the molecule. Increasing the age was negatively correlated with parasitaemia and positively with IgG antibody responses and haemoglobin levels indicating that total IgG responses to domains 2, 3 and crude schizont extract coincidental to a decrease in parasitaemia density and frequency. IgG3 response was the main IgG in those who had no parasitaemia at the final time point. IgG2 and IgG3 were increased amongst individuals with no parasitaemia and with higher levels of haemoglobin at higher ages that had exposure to parasite over a long period of time. Conclusion: IgG3 was the main antibody, mainly against domain 3 of MSP-2, that was associated with increase in haemoglobin levels and decrease in parasitaemia suggesting that domain 3 is preferred over other domains and crude schizont extract in presenting a clearer picture of immunity against malaria. These results could be an evidence of protective role of these antibodies against malaria disease and, therefore, domains 3 can be considered as reliable vaccine candidate antigens. Objectives: Our country is hyperendemic for dengue virus infection. Serosurveillance indicates that almost all native adults have been infected, mostly asymptomatically. A long-held mechanism explaining clinical severity involves sequential infections by different serotypes. Even though some of its peer flaviviruses are known to reside persistently within the host and contribute to host illnesses, dengue virus has not been shown to behave in a similar fashion. As dengue is a haematotropic virus, we sought to find evidence of its persistence in the bone marrow of previously-infected persons. Methods: We studied patients clinically suspected or known of haematologic malignancies and indicated to have diagnostic bone marrow initial or follow-up studies. A fraction of cellular marrow was employed for RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) by dengue-specific primers. Serologic assessment by haemagglutunation inhibition test (HI) for dengue and chikungunya viruses and by enzyme-linked immunosorbent assay (ELISA) for dengue was performed on the study of bone marrow study and, in some cases, 14−60 days later, to minimise a chance of including patients with recent dengue infection. Serologic interpretation was made according to standard criteria. Demographic data of all patients were analysed, especially for the history of recent febrile illnesses which could be due to dengue infection. Background: Intermittent preventive antimalarial treatment of infants (IPTi) is considered a promising malaria control strategy. Among the factors that influence the extent of protection provided by IPTi are malaria endemicity, seasonality, drug resistance patterns and the IPTi application schedule. Studies modeling the effect of malaria incidences on IPTi are scarce. The aim of this study was to describe how far protective efficacy of IPTi depends on the incidence rate of clinical malaria. Methods: One-thousand seventy infants were enrolled in a registered controlled trial on the efficacy of sulfadoxine-pyrimethamine based IPTi in the Ashanti Region, Ghana. In an ecological analysis, malaria incidence rates in the first year following IPTi were stratified by the village of residence and month of birth of participants and the spatiotemporal variation of the malaria incidence on the protective efficacy of IPTi was analysed. Findings: The rate of malaria attacks during the first year of followup was highly dependent on the month of birth and on the village of residence of the children. Protective efficacy of the first IPTi administration (IPTi-1) correlated with malaria incidences in children living in a particular village or born in a particular month (r2 0.48, p < 0.04 and r2 0.63, p < 0.003, respectively). A corresponding trend was seen after the second (IPTi-2) and third (IPTi-3) drug administration. Interpretation : Correlations between IPTi efficacy and malaria incidences may have implications on IPTi implementation strategies I will discuss the role mathematical modelling in pandemic planning and response. Recent research examining whether antiviral prophylaxis and social distance measures could be used to contain a nascent pandemic at its point of origin will then be reviewed. Containment is potentially feasible, but requires rapid detection of the initial transmissible case cluster and a rapid and organised response to each new case. These may be demanding criteria for much of SE Asia. If containment fails, slowing spread becomes a policy priority and in that context I will discuss the potential impact of restrictions on international travel. To conclude, I will discuss pandemic mitigation strategies which make best use of limited vaccine and antiviral supplies. Infections are among the most frequent complications occurring in cancer patients undergoing antineoplastic chemo-or immunotherapy. Invasive fungal infections today represent the main causes of fatal outcome. Early diagnosis of probable or proven invasive aspergillosis is therefore one of the most important objectives of supportive care in patients with profound and prolonged neutropenia. With the advent of more effective and well-tolerated antifungals active against aspergillosis, the incidence of other mould infections such as zygomycosis is on the rise. The use of highly aggressive chemotherapy induces severe mucosal damage in many cancer patients. Apart from neutropaenic enterocolitis, a broad spectrum of infections associated with impairment of mucosal barriers may be clinically important, e.g., streptococcal bacteraemia, septic enterococcal infection, or candidaemia. The widely spread use of multi-lumen central venous catheters causes a considerable number of bloodstream infections caused by coagulase-negative staphylococci, S. aureus, Gram-negative bacilli, or Candida spp. Primary removal of foreign material may be important for the successful management of these catheter-related infections. Allogeneic hematopoietic stem cell transplantation with reducedintensity conditioning has become more common also for treatment of aggressive haematologic malignancies in elderly patients as well as in patients with severe co-morbidity, who formerly have not been taken into consideration for myeloablative transplant procedures. Despite a marked reduction of complications related to pancytopenia combined with acute graft-versus-host reaction, the rate of severe and life-threatening fungal infections and cytomegalovirus diseases has turned out to be comparable to conventional allogeneic transplantation. Most importantly, more than half of these infectious complications emerge after more than 90 days post transplant, i.e., in patients already managed on an outpatient basis. Tuberculosis: fast forward to new approaches S17 The broad introduction of monoclonal antibodies to CD20 and CD52 and of nucleoside analogs into the treatment of patients with B-or T-cell lymphomas has lead to long-term depletion of B cells, eventually associated with decreasing levels of serum immunoglobulins, and T-cell deficiency lasting for many years. Particularly in patients being treated with these compounds for relapsed or refractory malignancy, a high number of infections caused by pathogenic viruses such as CMV, invasive fungi or Pneumocystis jiroveci are observed. Targeted prophylaxis is warranted for selected patient groups, whereas high alertness and early pre-emptive antimicrobial intervention is mandatory in the majority of these patients. In the last decade, IS6110 restriction fragment length polymorphism (RFLP) typing has gained recognition as the gold standard in the molecular epidemiology of tuberculosis. The application of RFLP typing has provided new insights in, e.g., the natural history of tuberculosis infection and (inter-)national transmission of multi-drug resistant tuberculosis in Europe (to be presented). However, this method is technically demanding and labour intensive, and requires weeks of culturing to obtain sufficient DNA. The complex RFLP patterns are difficult to interpret and exchange. The new generation genotyping of Mycobacterium tuberculosisbased on the detection of the variable number of tandem repeats (VNTRs) of mycobacterial interspersed repetitive units (MIRUs) -is increasingly used to solve these problems. VNTR typing is based on PCR amplification of multiple genomic loci to determine the number of tandem repeats at these sites. This typing is much easier to perform than IS6110-RFLP typing, and is applicable to crude low-concentration DNA extracts. Current studies are even oriented to apply VNTR typing directly to clinical material. Moreover, the results are expressed as numerical codes and are, therefore, easy to compare and exchange. Recently, in an international collaboration, the resolution, stability and technical applicability of 29 MIRU-VNTR loci was compared. This study comprised the initially explored 12 loci (as applied in the USA) and most of the exploitable loci disclosed in the international context so far. The typing results of 824 M. tuberculosis isolates revealed the 24 most optimal loci. The use of 15 selected loci is recommended as the new international gold standard for typing of M. tuberculosis complex isolates. A recent study pointed out that VNTR typing on basis of 24 loci is useful to study the phylogeny of the complex. As experienced after the standardisation of IS6110 RFLP typing in 1993, it is expected that the recent (December 2006; J. Clin. Microbiol.) international consensus on VNTR typing will facilitate the comparison of molecular epidemiological data from various geographic regions, the spread of multi-drug resistance in Europe, the establishment of an international VNTR database and the meta-analysis of worldwide typing results in order to study the population structure and spread of M. tuberculosis. Many institutes in the world have large databases of IS6110 RFLP patterns of M. tuberculosis isolates from extended time periods. Because in the epidemiology of tuberculosis transmission and manifestation of the disease can be separated in time by multiple years, the switch to VNTR typing can not be done without any overlap of the application of the two typing methods. During the ECCMID meeting the first re-typing results in The Netherlands and considerations underlying the choice of isolates to be re-typed will be discussed. A large proportion of new cases of tuberculosis (TB) in the world arise from a reservoir of people latently infected with Mycobacterium tuberculosis (Mtb). In TB endemic areas of the world, the average time to diagnosis a case of TB after the onset of active disease is 6 months. During these 6 months, such a case of TB is likely to infect, on the average, 10 close contacts. If one of these 10 newly infected contacts then develops TB sometime in his life time, he will infect 10 other persons before diagnosis. The cycle then repeats itself. Thus, if a TB control programme is only focused on the treatment of active cases, TB will never come under control. The fundamental question in TB control, therefore, is "can we predict who among the latently infected people will progress to active disease so that such a person can be given preventive treatment?" To answer this question, the mechanism of Mtb persistence and reactivation needs to be better understood. A variety of models for bacterial persistence has been proposed recently. Most genetic studies that provide evidence for the putative role of a gene in persistence are based on demonstrating that an Mtb strain disrupted in such a gene is attenuated in an animal model of TB. However, several recent gene disruption studies have shown that Mtb mutants can also become hypervirulent in an animal model. This suggests that some Mtb genes may actually help to temper the virulence potential of Mtb so that the organism can establish a stable niche in a host without harming the host. We provide evidence that a cluster of genes in an operon called mce1 in Mtb is involved in lipid transport and metabolism that helps to remodel the bacterial cell envelope. The operon appears to respond to a signal induced by the host's granuloma cell turnover, while the host's proinflammatory cells that comprise the granulomas appear to respond to the changing bacterial cell envelope lipid turnover. An Mtb mutant disrupted in the mce1 operon becomes hypervirulent in a mouse model of TB; it is unable to induce well-organised granulomas in mouse lungs, and the mouse dies from an uncontrolled bacterial proliferation. This mutant accumulates free mycolic acids on its surface. On the other hand, an Mtb strain disrupted in the negative regulator of the operon (mce1R) constitutively expresses the mce1 genes in vivo. This mutant is also hypervirulent but for an opposite reason-it causes rapidly progressive, hyper-proinflammatory response. Thus, the mce1 operon appears to serve as a homeostatic regulator of pro-inflammatory response by the host, helping to establish a balanced relationship favourable to both the host and Mtb. Understanding and exploiting this relationship may lead to new ways to predict a subset of latently infected people who progresses to active disease; it may also lead to the development of a therapeutic vaccine to prevent latently infected people from ever developing TB. Strain Sv29 was sequenced up-and downstream of cpsA. Approximately 25 kb of DNA (9 kb upstream of the cpsA start and 15 kb downstream) were comprised between an upstream dexB and a downstream aliA gene. 14 genes were found between the cpsA start and the aliA stop, comprising genes identical to the pneumococcal capsulation genes wzg (capsular polysaccharide expression regulator), wzh (Wze phosphotyrosine phosphatase), wzd (Wze phosphorylation), wze (autophosphorylating protein-tyrosine kinase), wchA (undecaprenyl phosphate glucose-phosphotransferase), wzy (oligosaccharide repeat unit polymerase), wzx (flippase) and several genes with putative glycosyl, glycerol and acetyl transferase activity and a putative UDPgalactopyranose mutase. Four genes were upstream between the cpsA start and the dexB start, comprising dexB, aliA-like orf1 and 2 genes and an unidentified gene. Objectives: Staphylococcus aureus can be a respiratory pathogen in cystic fibrosis (CF) and its pathogenicity is related to a combination of virulence and antibiotic-resistance genes. Little is known about the agr-group in CF-isolates. This study investigated the antibiotic-resistance, the distribution of 16 virulence determinants in 98 S. aureus strains (single or serial isolatessame strains removed), subdivided in agr-classes, isolated during a period of 18 months from the sputum of 60 CF patients, receiving various courses of antibiotic-therapy. The following were performed: agr-typing by ScaI-RFLP method; investigation of virulence genes by multiplex-PCR; antibioticresistance by the disk diffusion method according to the CLSI guidelines. Results: MRSA was isolated in 8% of cases. All staphylococci showed the following resistance profile: 37% for ERY, 21% for GM, 5% for DA, 14% for CIP, 11% for LVX, 12% for RD, 6% for TE and 3% for C. Among the strains, agrII was the most prevalent (35%) while agrI-III-IV were isolated approximately in 21% of cases. Within the agr-groups the distribution of virulence determinants revealed a common conservedcore background (sarA-rnaIII-spa-icaA-hla-hlg), whereas accessorygenes were always represented, in different combinations, and copossessed significantly. Capsule type-5 was prevalent in agrI while type-8 was prevalent in agrII/III. agrI was associated with the presence of many virulence-genes such as cytotoxins and proteases (sea-lukE-splB) and in one agrI isolate lukS/F-PVL was found; agrII also possessed an adhesin sdrE; agrIII was associated with adhesins and enterotoxin (fnbA-sea-cna), the exfoliatinA was never detected; agrIV possessed the cytotoxin lukE, adhesin cna and exfoliatinA, eta. Protease gene (splB) was always found associated with cytotoxin lukE in agrI-II-III and, mostly, sdrE adhesin was associated with these. A frequent inversecorrelation was found between cna and lukE-splB in all agr-groups and a close correlation was observed between cap8 and cna in agrIII-IV. Conclusion: Our data demonstrated for the first time an increase of MRSA in our centre; furthermore, CF-persistent infections were not associated with a distinct agr-specificity group or a diffused antibioticresistance of the strains, but rather S. aureus isolates have a complex distribution and combination of virulence determinants which contribute to S. aureus pathogenicity in CF patients. Objectives: Proliferation and differentiation of neural progenitor cells in the dentate gyrus is increased after infection in animal models of pneumococcal meningitis. Methods: To evaluate neurogenesis in patients with acute infection of the central nervous system, brain sections of 18 patients dying from bacterial meningitis and 8 patients dying from non-neurological diseases were investigated by immunohistochemistry. Results: In the dentate gyrus of the hippocampal formation, the density of Proliferating Cell Nuclear Antigen (PCNA)-expressing cells was higher in patients with bacterial meningitis compared to the control group (p = 0.02). Furthermore, the number of cells expressing the immature neuronal marker protein TUC-4 was increased in brain sections of persons dying from bacterial meningitis compared to control cases (p = 0.004). In the subventricular zone, no difference of cells expressing TUC-4 was observed between both groups (p = 0.39). Immature neurons expressing TUC-4 had no morphological features of apoptotic cell death and no evidence of DNA fragmentation. The increased proliferation of neural progenitors suggests that endogenous mechanisms may limit consequences of neuronal destruction after meningitis. Stimulation of neurogenesis might help to improve therapy of acute inflammatory diseases of the brain. Methods: C57Bl6-mice (n = 21) were trained to find a hidden underwater platform within less than 90 s (18 trials over 3 days). Swim tracks and the latency to escape from the water were recorded by a video camera. Thereafter, mice received CpG-DNA (0.001 mg/d) or the same volume of sterile saline over 28 days into the right ventricle after stereotactic implantation of a catheter connected to a subcutaneous osmotic pump. Water maze testing was peformed weekly. Results: After initiation of treatment, the latency to reach the hidden platform in the water maze was sigificantly longer in mice receiving CpG-DNA than in control animals (p = 0.006 at day 28 after initiation of treatment). Histology showed profound inflammation with invasion and activation of T-and B-cells and axonal damage. Furthermore, ependymal damage and loss was seen in CpG-treated animals. Additional experiments with TLR9-deficient mice showed no major inflammation after CpG-treatment over 28 days. Background: Early detection of microorganisms in blood is essential for early diagnosis and treatment of bloodstream infections. Calorimetric detection of microbial growth may be more sensitive and rapid than blood culture systems. We compared microbial detection in spiked blood specimens using a commercial system (CO 2 detection by colour change) and an isothermal microcalorimeter. Methods: Escherichia coli (ATCC 25922) or Staphylococcus aureus (ATCC 29213) were added at 3 different concentrations to anticoagulated blood obtained from healthy volunteers. Aliquots of 7.5 ml spiked blood were added into aerobic blood culture bottles containing 40 ml growth medium and incubated in the blood culture instrument (BacT/ALERT, bioMérieux, Durham, NC, USA). Before incubation, 1 ml of the bottle content was inoculated in glass ampoules containing 2 ml sterile trypticase soy broth for calorimetry (TAM III, Thermometric AB, Järfälla, Sweden). Blood cultures and calorimetry ampoules were simultaneously loaded in corresponding instruments and incubated at 37ºC for 72 hours. Time to positivity was defined as the interval from incubation start until positive signal (BacT/ALERT) or until the heat flow rate increased 10 mW above baseline (calorimeter detection limit = 0.3 mW). Results: Time to positivity was considerably shorter using calorimetry (Table) . Blood without added pathogens produced no heat signal above baseline. Spiked specimen heat signals rose to >100 mW as the pathogens multiplied during the incubation period. The objective of the work carried out was to determine the increase rate of MRSA detection using an enrichment step compared to direct culture on chromogenic agar. MRSA screening swabs from neonates in the NICU were compared: each swab was first cultured directly onto chromogenic MRSA ID agar and then incubated in Brain Heart Infusion broth for 24 hours. The broths were subsequently subcultuerd onto MRSA ID agar. All plates were incubated for 48 hours aerobically at 37ºC and checked for the presence of green colonies, which is indicative for MRSA, at 24 and 48 hours. All green colonies had confirmatory tests for MRSA. The limit of detection was determined for both direct and enriched cultures on MRSA ID agar using doubling dilutions up to 1/4096 from an initial 0.5 McFarland suspension. Plates were incubated for 24 hours aerobically at 37ºC after which colony counts were performed. A total of 1181 swabs were tested using direct and enriched culture methods. With direct culture, 16 isolates of MRSA were detected after 24 hours incubation and a further 6 after 48 hours incubation (22 in total). Following enrichment, 34 isolates were detected after 24 hours incubation and one more following 48 hours incubation (35 in total). Enrichment yielded a 59% increase in the number of isolates of MRSA detected compared to direct culture. The limit of detection for direct culture was 69,000 cfu/mL at 1/128 dilution with 84,000 cfu/mL at 1/2048 dilution following enrichment. Results showed that the limit of detection was higher following enrichment indicating a 16-fold increase in sensitivity compared to direct culture. The comparison of patient samples correlated with this by showing an increase of 59% in the rate of detection of MRSA following enrichment. In conclusion enrichment has proven to dramatically increase the sensitivity and rate of detection of MRSA compared to direct culture alone and in order to ensure maximum sensitivity in the detection of MRSA enrichment should be employed as the method of choice in routine laboratories. The limited additional help of the inflammatory markers CRP or PCT in bacteraemia prediction compared to a model of clinical assessment and routine laboratory information required for septic evaluation suggest the need for further justification of the use of these measurements in cost-constrained settings. Objectives: The increasing cross-border transfer of patients and healthcare workers (HCW) within the EU causes enormous problems with regard to control the spread of multi-resistant pathogens. Especially within the Dutch-German border region, there are great differences in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA): the Netherlands <1%, Germany~5−25%. In addition, within the last two years an increasing number of community-acquired (CA)-MRSA could also be noted in the Netherlands, which represent an additional danger. The objective of this study was to elucidate the molecular cross-border epidemiology of MRSA within the EUREGIO Twente/Münsterland. Methods: The first MRSA isolates of all patients and HCWs obtained within the EUREGIO MRSA-net Twente/Münsterland were typed. The epidemiological backbone of the project is a typing network providing a common language based on S. aureus protein A (spa) gene sequencing. Results: Since the beginning of the project in July 2005, more than 1400 isolates from in-and out-patients and nursing home inhabitants were typed. Three spa types (t025, t091, t162) were detected only within the Euregio. The spa types t004, t009, t012, t024, t036, t051 and t084 were determined in the German part of the Euregio, only. However, the majority of spa types on the German side were in concordance to the national epidemiological trend with spa t001, t002, t003, t004 and t032 as the most common spa types. The spa type t044 -often associated with CA-MRSA in central Europe -was detected on both sides; 1% on the German and~15% on the Dutch side of the border, underlining the emerging problem of CA-MRSA. Focusing on hospitalised patients, MRSA in blood cultures -only detected on the German side -were mainly spa type t001, t003, t004, t008 and t032. Conclusion: In summary, the use of spa typing elucidated differences in the regional epidemiology of MRSA. However, cross-border spread of MRSA was also detected. Therefore, new co-ordinated regional and cross-border strategies have to be developed in the MRSA-net to fight against MRSA. Objectives: The success of pandemic hospital-acquired MRSA clones may be due to high adhesive properties. Adhesion is mediated by various different cell wall proteins called adhesins that are tightly regulated. The accessory gene regulator (agr) is one acting regulator. agr dysfunction is associated with overexpression of certain adhesins and seems to be responsible of the success of the CMRSA-3 clone in Canada. In this way, we compared adhesive properties, the adhesin gene content and the functionality of agr of MRSA Lyon clone isolates to those of MSSA isolates. Methods: We studied S. aureus isolates responsible for bloodstream infections in intensive care units in 2003. Nineteen isolates were susceptible to methicillin and 13 MRSA isolates had characteristics of the MRSA Lyon clone (sequence type 8, agr allele type 1 and positive for the sea gene). The binding behaviour onto airway epithelial cells (HAECs) were determined by quantifying the number of adherent bacteria per cell (30 cells per strain were used). The adhesin gene profile (bbp, can, eno, ebpS, fib, clfA, clfB, fnbA and fnbB) was determined with multiplex PCR for each isolates. The dysfunction of agr was detected by the absence of delta-haemolysin production. Results: The number of S. aureus isolates per cell was more homogeneous for MSSA isolates than MRSA isolates, but the adhesion of MRSA isolates was significantly higher than MSSA isolates (68.94 ± 8.483 and 34.31 ± 3.446, respectively, p < 0.001) (Figure) . MRSA isolates were all positive for eno, fib, clfA, clfB, fnbA and fnbB, only. MSSA isolates were all positive for eno, clfA, clfB and fnbB and inconstantly positive for others. The absence of delta-haemolysin production tended to be more frequent among MRSA isolates (7/13 versus 16/19 isolates), but the difference was not significant (p = 0.11). No correlation between the adhesive property and the absence of deltahaemolysin production was observed, both in MRSA and in MSSA isolates. We demonstrated that adhesive properties onto HAECs of MRSA Lyon clone isolates was higher than that MSSA isolates, but was heterogeneous despite similar genetic background and adhesin gene profile. This could be the result of overexpression of adhesins independently of an agr dysfunction. Objectives: Although many distinct genetic lineages of communityassociated MRSA (CA-MRSA) have been reported worldwide, one clone has proved highly successful in the USA. The ST8-SCCmec IVa (or socalled USA-300) clone has been associated with community outbreaks, nosocomial transmission, and identified as the primary cause of skin and soft tissue infections (SSTI) presenting to Emergency Departments across the USA. Following enhanced surveillance for CA-MRSA, and in the light of these observations, we have mapped the epidemiology of this clone in England and Wales. Methods: Over a 2 year period (2004/5), all MRSAs referred to the national Staphylococcus Reference Unit for England and Wales were characterised microbiologically. Isolates identified as CA-MRSA were subjected to detailed characterisation (including MLST, spa typing, toxin gene profiling, PFGE and antibiotic susceptibility testing). Epidemiologic, demographic and clinical data were also collated. Results: Of ca. 300 CA-MRSA identified, 40 belonged to the ST8-SCCmecIVa clonal lineage; the third most common CA-MRSA occurring nationally. All 40 isolates were PVL-positive, spa t008 and belonged to agr group 1. Nine different sub-types were distinguishable by PFGE; almost half belonged to a single pulsotype. All were resistant to b-lactams; resistance to erythromycin, ciprofloxacin, gentamicin, tetracycline and fusidic acid was variable. All were susceptible to clindamycin, trimethoprim, vancomycin, linezolid and mupirocin. The majority of patients (32, 80%) were community-based, including 23 males and 17 females; ages ranged from <1 to 89 y (median 25 y). 60% presented with skin infections (e.g. boils, abscesses or insect bites) and 25% with soft tissue infections. One patient died. Most cases were sporadic, but 4 community-based clusters were identified involving MSMs, household contacts and individuals attending the same GP surgery. Eight patients gave a history of foreign travel (e.g. the USA), the remainder were acquired domestically. The ST8-IVa lineage of CA-MRSA has been occurring in England and Wales since at least 2002. Whilst numbers remain small, the occurrence of community-based clusters and the death of one patient give cause for concern. The emergence of resistance to multiple classes of antimicrobials allied to evidence of clonal expansion highlight the need for continued surveillance to underpin effective therapeutic and infection control strategies. On secondary efficacy endpoints, the three treatment arms were similar in terms of clinical and mycological response, and there were no significant differences in the incidence of emergent fungal infections during the study and in the incidence of relapse post-treatment. There were no clinically significant differences between the three treatment arms in terms of adverse events, including those of special interest, such as hepatic and renal function adverse events. The incidence of death during treatment was similar across treatment groups. Conclusion: MICA100 and MICA150 were equally effective and were non-inferior to CAS on the primary efficacy endpoint of treatment success as defined by the investigator. There were no significant differences between the MICA treatment arms and CAS on all secondary endpoints. All three treatment regimens had similar safety profiles. Objective: To compare the efficacy and safety of micafungin (MICA) with AmBisome ® (L-AmB) in a paediatric subpopulation in a large, randomised, phase III trial in patients with invasive candidiasis or candidaemia. Methods: In this double-blind, multicentre study, non-neutropaenic and neutropaenic paediatric patients aged 15 years with clinical and microbiological evidence of invasive candidiasis or candidaemia were randomised 1:1 to receive intravenous MICA (2 mg/kg/day) or L-AmB (3 mg/kg/day) for a minimum of 14 days. All randomised patients were included in the full analysis set (FAS). Efficacy analyses were also performed for the per protocol set (PPS), which included all patients who had confirmed invasive candidiasis or candidaemia but did not have a further invasive infection caused by a non-Candida fungal pathogen, for whom the investigator's assessment of overall treatment success at the end of therapy (EOT) was available, and who received at least five doses of study drug and did not receive a prohibited antifungal medication. The safety set included all FAS patients. Overall treatment success was based on clinical and mycological response at the end of therapy, as assessed by the investigator. The post-treatment follow-up period was 12 weeks. Results: A total of 106 patients (52 MICA and 54 L-AmB patients) were included in the FAS; 83 patients were included in the PPS. Age groups, including prematurity at birth, were well represented; candidaemia was the primary infection in the majority of patients in both treatment groups. The key efficacy results are shown in the table. The incidence of treatment-related adverse events (AEs) was lower with MICA than with L-AmB (36.5% versus 42.6%, respectively), as was the incidence of serious AEs (3.8% versus 9.3%, respectively). In addition, fewer patients receiving MICA than L-AmB discontinued therapy because of AEs (3.8% versus 16.7%, respectively). One cipro-resistant isolate (MIC > 32) was isolated from a cipro-treated subject. 9 levo subjects were clinical successes, 2 were failures. With cipro, 7 subjects were clinical successes, 2 were failures and for 3, the outcome was unknown. The urine pathogen was eradicated in 10 and was unknown for 1 levo subject compared to eradicated in 7, persisted in 3, and unknown in 2 cipro-treated subjects. A repeat blood culture was obtained for 7 levo and 8 cipro subjects, with the blood pathogen eradicated in all subjects. For one other cipro-treated subject, the blood pathogen was presumed persisted based on clinical failure. The outcome for the cipro-resitant E. coli was unknown. Conclusions: For the overall population (cUTI and AP) and for the cUTI and AP cohorts. 5 doses of levo were non-inferior to 20 doses of cipro. Levo and cipro eradicated blood pathogens in all subjects who had a 2nd blood culture. Approximately two-thirds of patients were from the USA; the remainder were recruited from Canada, Australia, South Africa, Russia and various other countries across Europe, Asia and South America. Patients with infections due to MRSA tended to be younger (<65 years of age) and a greater proportion were of Black race compared with patients with infections not due to MRSA (non-MRSA; Table) . MRSA was most commonly associated with major abscesses, whereas non-MRSA was most commonly associated with deep/extensive cellulitis. Regardless of MRSA status, cSSSI occurred more frequently on the lower extremities. Conclusion: Together, ATLAS 1 and 2 represent the largest prospective, double-blind studies ever conducted in patients with cSSSIs. The MRSA cohort was also the largest ever enrolled in a registrational programme. MRSA infections appeared to be more common in patients who were younger, of Black racial origin, and in those with a major abscess compared with patients with infections not due to MRSA. Objectives: The use of aminoglycosides has decreased due to doubts regarding lack of efficacy and adverse effects. Yet, the prevalence of microbial resistance to aminoglycosides has remained low. This study sought to compare the efficacy and adverse effects of aminoglycosides as a single antibiotic to other antibiotics for the treatment of patients with infection. We searched for randomised trials comparing the efficacy of aminoglycoside antibiotic treatment to non-aminoglycoside antibiotic in patients with infection in the Cochrane Library, MEDLINE, EMBASE, LILACS, databases of ongoing trials and conference proceedings. Two reviewers assessed trial eligibility, quality and extract data. Pooled relative risks (RR) with 95% confidence intervals (CI) were calculated for dichotomous data. We conclude that each of the three major clones can readily be demarcated from other A. baumannii members, and that molecular diagnostics could be developed to identify them rapidly. Analysis of the recombination/mutation rate shows that nucleotides do not change by recombination more frequently than by mutation, showing that recombination is unlikely to disrupt the clonal frame of the clones, which can therefore be expected to be stable over very long periods of time. Objectives: Molecular epidemiology studies of clinically relevant microorganisms frequently focus on a comparison between the assigned types of different typing methods. Therefore, there is a critical need to understand the correspondence between types produced by different methods. This may be useful not only for the comparison of the genetic backgrounds of the particular set of isolates under study but also to produce a broader view of how the results of the different typing methods are related. To achieve this goal, a framework of measures for the quantitative comparison of typing methods results was developed (Carriço et al., Illustration of a common framework for relating multiple typing methods by application to macrolide-resistant Streptococcus pyogenes, J. Clin. Microbiol. 2006) and is now implemented in a free-access web-based interface, on which users can analyse their data anonymously and retrieve the results. Methods: To facilitate the use of the framework developed, a web-based interface was implemented to provide researchers with a user-friendly interface to compare their typing methods results. On the website, we also provide a set of Bionumerics TM scripts to allow the users to implement the proposed analysis on their Bionumerics databases while offline. The web-based interface and the Bionumerics scripts allow for the calculation of several measures: Simpson's index of diversity with confidence intervals for accessing the diversity of typing results for different methods; Adjusted Rand and Wallace coefficients for bidirectional and unidirectional (respectively) comparison between the results of two typing methods results. Based on these measures the users can quantitatively evaluate the discriminatory power of the typing methodologies used and their concordance. The web-based interface and the Bionumerics scripts provide users with the ability for a quantitative assessment of correspondence between typing methods results. This can be used to evaluate the predictive power of a typing methodology when compared to another, which can guide the user in the choice of a 'gold standard' for clone definition. Furthermore, as new microbial typing methods are proposed, this methodology allows for their comparison in terms of type assignments with established methodologies. Despite several case control studies and enhanced surveillance of both human cases and animal environmental isolates, there is still much unknown about the epidemiology of Campylobacter infection. We describe a multi-agency project evaluating the utility of sequence typing of human infection isolates to inform surveillance and distinguish environmental sources of human infection. Multi-locus sequence typing (MLST) developed for C. jejuni and C. coli was applied to all human case isolates from four local authorities in North West England, two largely rural and two largely urban, over a continuous 3-year period. Isolates from drinking water supplies and recreational surface waters were also sequence typed. We describe here an analysis of the three year study data and steps taken to adapt the methodology for use in routine surveillance settings. The study has demonstrated several interesting correlations between specific sequence types and a number of explanatory variables for disease (eg month of infection, locality of residence, and travel abroad). Other studies provide increasing evidence of host-association for specific MLST types of campylobacter and these findings are related to the three year dataset. In addition, many new types have been described in surface waters from this project and there is evidence for a distinct water-borne population that has not so far been described in human infection. In recent years, the use of cell culture and molecular biology deeply changed our knowledge on rickettsiae. As a matter of fact before 1990, 7 pathogenic rickettsial species were identified ( New rickettsial diseases were found under 3 main conditions: -In place where none was identified, typical rickettsial diseases (including fever and a rash) were found (Japan, China). -In some place typical rickettsioses could be caused by different organisms. In such cases, the new Rickettsia was misdiagnosed with a previously identified bacterium (such as R. massiliae with R. conorii). -In some cases atypical clinical findings were found (no rash, no fever) to be caused by Rickettsial organisms such as R. slovaca or R. helvetica. These findings challenge the old dogma postulating that of one tick borne rickettsiosis was prevalent in one geographic area. For many years for example, R. rickettsii, the agent of Rocky Mountain Spotted Fever, was considered the only spotted fever group rickettsia in the USA and the only tick-transmitted rickettsiosis in America. R. felis, a flea-transmitted spotted fever, and R. parkeri, a tick-transmitted spotted fever, have been shown since to infect human beings in the USA and in Urugay. Moreover, R. africae has been found in patients in West Indies. Many Rickettsia have been identified in ticks but have not been currently found in patients. These Rickettsiae should be considered potential pathogens. These new findings should stimulate investigations to identify new rickettsial diseases. Patients with atypical rash or fever after arthropod bite should be targeted. Skin biopsies are the preferred samples in this purpose. Molecular tests used for this purpose will be proposed. Can pharmacokinetic-pharmacodynamic parameters drive dosing regimens that are less vulnerable to resistance? S156 Can pharmacokinetic-pharmacodynamic parameters drive dosing regimens that are less vulnerable to resistance? Pro In recent years, the rules for selection of antimicrobial agents have been undergoing critical revision in terms of optimum dosing for control of infectious diseases, with the goal of potentiating treatment efficacy and reducing the risk of selecting multidrug resistant pathogens. The most important criterion for rational choice of an antimicrobial agent is defined by its pharmacodynamic (PD) characteristics, and thus its antimicrobial activity which can be summarised as the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The second criterion for selecting an antibiotic is due to its pharmacokinetic (PK) characteristics, since it has been demonstrated that antibiotic concentrations at the infection site influence the intensity and duration of the effect, and together with the PD parameters, provide a general inter-relationship and contribute to defining the potential clinical efficacy of a drug. Pharmacodynamics have been upgraded in the last 10 years with in vitro and animal models which have helped to identify fairly precise correlations with therapeutic efficacy of antibiotics. Moreover, the principles that link concentrations of antibiotics in humans and their effects have been outlined in order to determine the optimal dosing interval. Antimicrobial drugs with a concentration-dependent activity, i.e. the newer quinolones and aminoglycosides, should be administered as a single daily dose to maximise the peak serum concentration/MIC ratio. AUC/MIC ratio is also an important parameter for PK/PD correlations. Existing data on fluoroquinolones suggest that a ratio of 100-125 correlates with high bacterial eradication and optimal clinical outcome in infections due to Gram-negative pathogens, while a ratio of 50 is associated with a high probability of eradication of S. pneumoniae strains. For this group of antibiotics the new concept of the so called mutant prevention concentration (MPC) and mutant selection window (MSW) may be helpful in restricting the enrichment of mutant subpopulations and consequently, at least partially, in controlling the spread of resistance. Beta-lactams, having time-dependent efficacy, usually do not have great post antibiotic effects (PAEs), and the parameter which seems to better correlate PD with PK is the time duration with concentrations higher than the MIC (T > MIC). These antibiotics need to be given with short dosing intervals or even by continuous infusion to maintain plasma levels exceeding the MIC for a sufficiently long period. In summary, we can theoretically optimise the dosage regimen of antibiotics (dose, administration route and interval between doses) in clinical practice by correlating the PK and PD pertaining to each antibiotic class, based on experimental animal studies and these PK/PD parameters may contribute to the containment of resistance for all drug classes and especially the most important ones used in serious infections in intensive care patients. A. Bartoloni (Florence, IT) Antibiotics are the most commonly purchased class of drugs in lowresource countries, where the infectious diseases are extremely frequent and the bacterial infections are the major cause of death, especially in childhood. The phenomenon of microbial drug resistance, which represents a global public health problem, is particularly serious in these countries, as resistance rates are even higher than in industrialised countries and the therapeutic options are often unavailable or too expensive. Surveillance of antibiotic susceptibility is a key element to provide updated information on the magnitude and trends in resistance, and to plan and monitor intervention strategies aimed at preserving the therapeutic efficacy of antibiotics. In low-resource countries, effective surveillance programmes are difficult to implement for a number of reasons, including scarce financial resources, lack of laboratory facilities and, where laboratories do exist, lack of quality control, reliable reagents and adequate supervision. In these settings, the development of reliable and low-cost alternative methods could facilitate the implementation of large-scale surveillance. There is an increasing agreement about the importance of extending the surveillance of antibiotic resistance to the commensal microbiota of humans and animals. This bacterial population, although not being a specific target, is continuously exposed to the selective pressure generated by antimicrobial chemotherapy and may become a potential reservoir of resistant strains that can cause infections, and of resistance determinants that can be transferred to pathogenic bacteria. Therefore, surveillance of antibiotic-resistant bacteria carried by healthy individuals is considered an indicator of the spread of antibiotic resistance that could also be useful to predict the emergence of resistance in pathogenic bacteria. In this perspective, resistance patterns of some members of the commensal microbiota, such as the faecal Escherichia coli, have been evaluated in various epidemiological settings. For this purpose, different microbiological approaches have been implemented and evaluated as useful tools to conduct large scale resistance surveillance studies and to monitor resistance control programmes in a cost-effective manner. The emergence of extended-spectrum b-lactamases (ESBLs) in Enterobacteriaceae in the 1980s marked an important turning point in routine laboratory diagnostics. Soon after their discovery, phenotypic methods were developed for their detection and confirmation. Confirmation depended largely on the ability of clavulanate to inhibit the main culprit enzymes at the time, the TEM and SHV variants. Later this technique proved reliable for CTX-M type enzymes. Many susceptibility testing methods recommended the use of ESBL screening and confirmation tests on a routine basis, including CLSI, BSAC, CA-SFM, and SRGA. However, a number of issues have emerged with routine use over the years: 1. The problem of defining an adequate number of substrates to ensure sufficiently sensitive screening. Ideally, one should include a minimum of 4, namely cefpodoxime, ceftazidime, ceftriaxone or cefotaxime, and aztreonam, and at concentrations that often differ from those used for susceptibility breakpoints. 2. The lack of reliable phenotypic methods to detect ESBLs in species with inducible AmpC b-lactamases. Some of these species have been shown to be important reservoirs for ESBLs, and resistance to extended-spectrum cephalosporins cannot solely attributed to stable de-repression of AmpC. 3. The failure of current methods to provide advice on the interpretation of a positive screening test but a negative confirmation test, especially if the isolates are "susceptible" to extended-spectrum cephalosporins using method-recommended breakpoints. Such strains have been shown to harbour OXA enzymes, inhibitor-resistant TEM enzymes, or particularly plasmid-borne AmpC enzymes with significant frequency. Thus there is no current phenotypic or genotypic test that can be practically and effectively applied in the routine laboratory with sufficient sensitivity to detect the emerging range of transmissible enzymes. However, we can rely to a great extent on the selection of or change to appropriate susceptibility breakpoints. A range of recent studies has suggested that failures of treatment with extendedspectrum cephalosporins are likely when strains of Enterobacteriaceae have MICs elevated above the wild-type. Further, application of pharmacokinetic/pharmacodynamic (PK/PD) principles to the most widely recommended dosing schedules of cephalosporins suggest that the susceptibility breakpoints recommended by many methods are too high, and should to lowered to values that fortunately coincide wild-type cutoff values. Hence, lowering of susceptibility breakpoints for extendedspectrum cephalosporins to those defined by PK/PD will indicate the presence of an ESBL or plasmid-borne AmpC enzyme with sufficiently high likelihood to allow laboratories to report both resistance to these agents and provide advice about appropriate infection control procedures. Objectives: Since antibiotic resistance often is associated with a biological fitness cost for bacteria it is assumed that a reduction of antibiotic use is followed by a reduction in resistance rates. Over the last 10 years trimethoprim (TRI) resistance in Escherichia coli has increased in Sweden. In Kronoberg county the TRI resistance has increased from Antibiotic usage S33 7% (1990) to 11% (2004). So far no prospective intervention in the community has been carried out to investigate whether a substantial decrease of the use of a single antibiotic will result in a corresponding decrease in antibiotic resistance. Method and Material: Physicians (n = 564) in county Kronoberg (approx. 180,000 population), Sweden, were convinced, by personal visits and mail, to cease their use of TRI and cotrimoxazole during two years starting October 1st 2004. Monthly sales data for oral antibiotics were retrieved from the National Corporation of Swedish Pharmacies and made known to the physicians. All E. coli isolated from urinary tract specimens at the Dept of Clin Microb, Växjö, were included in the analysis. The susceptibility testing methodology was stable since 1990 and the baseline consisted of quantitative data from 1990-2004. In order to study the diversity among the isolates 2900 E. coli from the period preceding and 1200 isolates from the end of the intervention were phenotyped using the PhenePlateTM system. Results: An immediate and sustained decrease of 85% in total use of TRI was achieved. TRI use was in most cases replaced by mecillinam (MEC), nitrofurantoin (NIT) and ciprofloxacin. The total decrease in antibiotics used for UTI treatment was 4%. Resistance to TRI did not decrease (10% and 12% in 2005 and 2006, respectively). Resistance to NIT and MEC did not increase despite the substantial increase in the use of these drugs, 31% and 69%, respectively. Resistance to fluoroquinolones (FQ) increased from 4% to 10% between 2000 and 2006. The phenotypic diversity index was 0.954 in the period prior to the intervention, 0.964 during the first 4 months and 0.949 during the last 4 months of the intervention. Conclusion: A substantial and sustained decrease in the use of TRI in a 180,000 population did not result in any drastic changes in resistance rates. Whether the increase in FQ-resistance was related to the increase in use remains to be evaluated. The diversity index, based on E. coli phenotypes, was stable indicating that the intervention did not favour any certain clone to expand in the community. Objectives: Increasing use of antibiotics and spread of resistant pneumococcal clones in the beginning of the 90ies alarmed the medical profession and medical authorities in Sweden. A coordinated effort to prevent further spread and preserve the effectiveness of antimicrobial agents was therefore initiated Methods: Strama (The Swedish strategic programme for the rational use of antimicrobial agents and surveillance of resistance) was initiated in 1994. The Strama network includes a broad representation of medical disciplines, professional organisations and relevant authorities. The programme includes surveillance of antibiotic use and resistance, implementation of rational use of antibiotics and development of new knowledge. The main goal is to preserve the effectiveness of antimicrobial agents. Results: Yearly validated data on antibiotic use and resistance were made publicly available on a website. Multidisciplinary Strama-groups were formed in each county to disseminate knowledge and implement rational antibiotic use. Studies were performed on indications for antibiotic use in out-patient care, hospital care and nursing homes. Between 1995 and 2004 antibiotic use for out-patients decreased from 15.7 to 12.6 DDD/TID and from 536 to 410 prescriptions per 1000 inhabitants and year. The reduction was most prominent for children 5−14 years old (52%) and for macrolides (65%). During the period, the number of hospital admissions for acute mastoiditis, rhinosinusitis and quinsy was stable or declining. Although the epidemic spread in southern Sweden of penicillin-resistant S. pneumoniae was curbed, the national frequency increased from 4% to 6%. A hospital outbreak of MRSA could be terminated using aggressive infection-control measures. Resistance remained low in most other bacterial species during the period. Conclusions: This multidisciplinary, coordinated programme has contributed to the reduction of antibiotic use without measurable negative consequences. Despite this, antibiotic resistance in several bacterial species is slowly increasing which calls for continued sustained efforts to preserve effectiveness of available antibiotics. Such efforts will include interventions to further improve antibiotic use and to improve compliance to basic hygiene precautions. The protocol was designed to present demographic data as well as the amounts and indications for antimicrobial agents against bacteria. Treatments were recorded in relation to diagnoses, prophylactic use, community acquired (CAI) and hospital acquired infection (HAI). 19 pre-defined diagnosis groups were used. The previously presented STRAMA protocol and web-based reporting system was used. Results: 19 hospitals participated in the study. 3,398 patients treated with antimicrobial agents were included out of 11,224 admitted. 30% of the patients were treated with antimicrobials. 3,554 treatments were recorded. 377 (10.6%) were given to children (<17 years) and 47.6% to women. The indication for treatment was CAI in 15%, HAI in 9.2% and prophylaxis in 7.6%. For adults cultures were taken before parenteral treatment in 57% and oral treatment in 51%. The most commonly used antimicrobials for adults, in DDD, in treatment and prophylaxis were penicillins with betalactamase inhibitors (23%, 0−75, and 26%, 0−66), cephalosporins (14%, 4−38, and 30%, 0−60), fluoroquinolones (14%, 8−31, and 11%, 0−52). The total amount of antimicrobials used for adults was 52 DDD/100 admitted patients (33−88). Two diagnosis groups were predominating; pneumonia (19% of all therapies) and skin and soft tissue infections (13%). Analysis of antibiotic usage in different countries shows countries having mainly penicillins with betalactamase inhibitor or cephalosporins as the predominating drug. Seven countries showed a more varied use of drugs. The patterns of treatment and prophylactic usage were similar in countries with heavy use of one ATC-class. Length of peri-operative prophylaxis was dominated by >1 day in all surgical specialities; in general surgery 59%, in orthopaedic surgery 52%, in urology 76% and in ENT 89%. One-dose peri-operative prophylaxis ranged from 2% to 27%. One-day prophylaxis was 34% in orthopaedic surgery. The study describes wide differences (three fold) in consumption between European hospitals. Peri-operative prophylaxis is too long in all surgical specialities. There is limited variation in the use of different antibiotic groups in most hospitals -a risk factor for emergence of antibiotic resistance. Our web-based PPS provides a tool for quality assessment of antibiotic prescribing in European hospitals. Methods: Face to face structured interviews were conducted in 11 countries (Austria, The Netherlands, Sweden, UK, Belgium, Italy, Malta, Israel, Czech Republic, Lithuania and Croatia). The study population included respondents of our previous survey who agreed to be interviewed. We aimed to recruit at least 100 respondents in each country, including a group of users of self-medication and non-users. Scales were grouped based on theoretical concepts and confirmed by factor analysis. The four scales were: attitudes towards appropriateness of self-medication with antibiotics for bronchitis, beliefs about antibiotics for minor ailments, attitudes towards situational use of antibiotics and knowledge about antibiotics and viruses or bacteria. Knowledge of antibiotic resistance was measured by an open-ended question. Analysis: To deal with the possible confounding effect of both use of selfmedication and education, we performed stratified analyses, i.e., we studied the differences between countries separately for users and non-users of self-medication, and for respondents with high and low education. The differences between countries were considered relevant when regression coefficients were significant in all stratum-specific analyses. Objective: Antimicrobial resistance to ciprofloxacin, a valuable secondline antibiotic, is increasing. To limit this increase the appropriate use of ciprofloxacin should be encouraged. The objective of this study was to reduce the number of inappropriate prescriptions and improve the quality of ciprofloxacin prescriptions by way of educational intervention. Methods: Five units (197 beds) of the departments of Internal Medicine, Gastro-Enterology, Surgery, Urology and Pulmonary Diseases, selected because of a high rate of ciprofloxacin prescription, participated in a prospective intervention study. The study comprised three periods of three months: 2 observation periods (phase 1 and 3) and an intervention period (phase 2). A follow up of 3 months was done 9 months after start of intervention. During the two observation periods all ciprofloxacin prescriptions were registered and the quality of each ciprofloxacin prescription was evaluated in a standardised manner by two experts in infectious diseases independently. During the intervention period physicians prescribing ciprofloxacin were interviewed by a medical microbiologist, and educational presentations were given to physicians of the participating units. Results: During phase 1 491 prescriptions per 1000 admissions of ciprofloxacin were written, declining to 184 prescriptions per 1000 admissions in phase 3, a reduction of 62.5%. The greatest reduction was observed in units of the Departments of Surgery and Urology (83.9% and 75.6% respectively), mainly due to a reduction of erratic prophylactic use. Unjustified prescriptions (no use of antibiotics indicated) decreased with 25.9%. Inappropriate prescriptions (wrong choice of antibiotic or duration of prescription) declined from 69.5% to 57.7%, mainly due to the decrease of ciprofloxacin courses of too long duration. Appropriate prescriptions increased with 33.5%. Nine months after intervention 136 prescriptions per 1000 admissions of ciprofloxacin were prescribed, a total reduction of 72.3%. Intervention by direct consultation of a medical microbiologist and educational presentations led to significant reduction of the use of ciprofloxacin and improvement of the quality of ciprofloxacin prescription. Nine months after the intervention the use of ciprofloxacin had declined even further, indicating a sustained effect of the intervention measures. There was a trend towards a greater rise in creatinine in Group 2 versus Group 1: final creatinine increased by 88% vs 52% from baseline (p = 0.08), which was reversible on stopping study drug treatment. Mortality was 23% (15/64) at 10 weeks with no difference between the two groups. Conclusions: Amphotericin B at 1mg/kg/d with flucytosine is more rapidly fungicidal in the treatment of HIV-associated cryptococcal meningitis compared to standard dose amphotericin B. Increasing the dose of amphotericin B did not result in a clinically significant increase in nephrotoxicity. Cases diagnosed by culture and confirmed by restriction fragment length polymorphism as C. gattii and HIV negative cases diagnosed by serology and histopathology with no exposure to international endemic areas were included. Cases reported to provincial authorities between 1999 and 2006 were interviewed to determine clinical presentation and epidemiological characteristics. Frequencies were calculated based on denominators available using SPSS ® v14.0. As of November 16 2006, 162 cases of locally-acquired cases of C. gattii infection occurred in BC with an annual provincial average of 6.3 cases/million population. Mean age was 59 years (2−92 years) and 56% were male. 16 cases (10%) were asymptomatic. 2 (1%) had dermatological findings. Most common symptoms included cough (46%), shortness of breath (44%), fever and night sweats (both 35%). Chest X-rays revealed nodules in 65 cases (66%) and infiltrates in 14 cases (14%). 19 cases (30%) were on oral steroids in the year prior to diagnosis and 50 (49%) were current smokers. Only 5 (4%) were HIV positive, 3 (3%) were organ transplant recipients, 30 (31%) had a chronic lung disease and 26 (26%) had a history of cancer. 103 cases (64%) were culture-confirmed as C. gattii and 81 isolates (79%) were genotyped as VGIIa. 4 cases (3%) died due to their C. gattii infection. Most cases were treated with fluconazole and amphotericin B for several months. Most cases were exposed on Vancouver Island but the range of the fungus has spread over the years and 4 recent cases were exposed on the BC mainland. Clinicians should be aware that C. gattii can be acquired in Canada and that the epidemiological and clinical characteristics of this disease differ from C. neoformans. Objectives: The incidence of invasive zygomycosis (IZ) appears to be increasing, especially in patients treated for haematological malignancy. Presently, no biological markers exsist that facilitate early diagnosis, and one has to rely on conventional diagnostic methods, such as culture, which lack sensitivity. We investigated the presence of zygomyceteantigen (zAg) and zygomycete DNA in bronchoalveolar lavage fluid (BALF) of patients with documented IZ and of those at risk for this disease. Methods: BALF of 18 neutropaenic patients, who underwent a bronchoscopy on suspicion of invasive fungal infection (IFI), was investigated for the presence of water-soluble somatic zAg by immunoblotting with a commercially available monoclonal antibody (anti-Rhizomucor, Dakocytomation, Denmark). Five of 18 patients had proven IZ. Of the remaining 13 patients without documented IZ, 8 had proven or probable invasive aspergillosis (IA). BALF from 10 non-haematology patients was used as control. BALF samples were also investigated for presence of zygomycete DNA by PCR, using 18S primers. Results: The BALF of the 5 patients with proven IZ were positive for zAg. In the remaining 13 neutropaenic patients, zAg was detected in BALF of 7. Four of these had probable or proven IA The number of zAg positive BALF from neutropaenic patients with suspected IFI was higher than in the control group (7 of 13 versus 1 of 10, P < 0.002). Zygomycete DNA was detected in 11 of 12 BALF samples positive for zAG, and all BALF samples negative for zAg were also negative for zygomycete DNA by PCR. Conclusion: zAg and zygomycete DNA are present in BALF of patients with IZ, and might be a useful tool for early diagnosis. The presence of both markers in BALF samples from high-risk patients without a clinically manifest disease might indicate the presence of colonisation or subclinical infection. Results: All the strains tested were able to form biofilm in all the media tested. However, differences were detected between the different media. The isolates were able to form biofilm faster in Middlebrook 7H9 (100% of surface covered in day 28 for all the strains) than in tap water (100% of surface covered in day 63 for all the strains). The day 70 none of strains covered all the surface when using PBS-5% glucose. No difference was found between species in biofilm development. Conclusions: All the NPRGM tested were able to develop biofilm in all tested media. The biofilm development is dependent on the type of the culture media, being faster when a rich media is used. No differences were detected in biofilm development among the different species. Acknowledgments: T.J. Kinnari was funded by a grant from the Academy of Finland. N. Zamora was funded by a grant from the Fundación Conchita Rábago de Jiménez Díaz (Madrid, Spain). In vitro, in sessile bacteria, gapB expression was high in a medium with a low Fe content, irrespective of the glucose content. Simultaneously with the increased expression of gapB, PIA production could be visualised through CLSM in sessile bacteria in all media. In vivo expression of gapA was high and remained constant over a period of two weeks. The expression of gapB decreased during the initial phase of implantation, but reached the expression level of gapA after two weeks of implantation. The persistent expression of gapA in sessile bacteria could indicate a role in biofilm formation, especially in the early stages, while expression levels of gapB could indicate a role in the later phases of biofilm formation. Results of gene expression and CLSM indicate a link between gapB and PIA production that is influenced by both Fe and glucose. example Ssp, a surface-associated lipase, Aas, an autolysin adhesin, SdrI, a collagen binding protein, and urease activity. Purportedly, the uropathogenicity of S. saprophyticus can be attributed to its ability to cope with the high range of variation in salt-and urea-concentration in human urine. The aim of this study was to elucidate virulence factors of uropathogenicity. Methods: Bacterial growth was examined under different conditions using a modified model of artificial urine as previously described. Bacterial aggregation was observed in bright light and electron micrographs. Biofilm formation was tested using native polystyrene and crystalline pre-coated microtiter plates. Results: In S. saprophyticus strain CCM 883, in contrast to wild-type strain 7108, generation-time was increased. In S. saprophyticus wild-type strains 7108 and 9325, bacterial aggregation appeared near large crystal structures in contrast to its urease negative derivative strain GJ1187 and the wild type strain CCM 883. In native polystyrene and crystalline precoated microtiter plates, biofilm formation was observed in strain 7108 in contrast to strain CCM 883. The biofilm formation in S. saprophyticus seems independent of agr-, Ssp-, SdrI, since no difference between knockout mutants to wild type strain was observed. Conclusion: Bacterial aggregation could be due to increased adhesion. The lipase activity may modulate hydrophobicity and Ca 2+ binding. Higher local Ca 2+ concentration, as well as strengthened bacterial aggregation, may lead to increased crystal formation. Bacteria may adhere to crystals, additionally to the later mechanism due to biofilm formation. In crystallisation process, bacteria were embedded into the crystal structure. This may be a new model of infectious stone genesis. This study strongly suggest that S. saprophyticus wild-type strain CCM 883 lacks important virulence factors in contrast to wild-type strain 7108 and biofilm formation may play an important role in S. saprophyticus urinary tract infection. Objectives: The influence of biofilm formation in catheter-related candidiasis has been established and it has been shown that the development of biofilm by the colonising yeasts confers resistance to antifungals. The purpose of this work was to demonstrate the production, by the Candida albicans yeasts growing as a biofilm, of a molecule able to reduce biofilm growth. Methods: An in vitro model of C. albicans biofilm associated with 100% silicone catheters was used. The supernatant medium recovered from C. albicans (ATCC 3153) biofilm aged of 24 hours was added during the course of a new biofilm formation made with 4 C. albicans strains (182, 444, 2091 and ATCC 3153), and its influence was subsequently evaluated with XTT method. Ultrafiltration and purification assays to characterise the modulating fungal molecule responsible for the biofilm supernatant activity are presented in this study. Results: The addition of the supernatant to adherent C. albicans cells induced a significant (p < 0.001) inhibition of the biofilm growth. This activity was not observed when the supernatant was added to preformed biofilms. Ultrafiltration and purification assays demonstrated that this molecule is small (<3000 Da) and hydrophilic. Conclusions: These preliminary results suggest that this new molecule, naturally produced within yeasts communities, could be a good candidate for the prevention of biofilms associated with indwelling medical devices. In 1994 the first article describing an 'early secreted' antigen (esat-6) from the culture filtrate of M. tuberculosis was published. Three key properties of this antigen are that it is highly immunogenic, it is not found in BCG or many environmental mycobacteria, and it is a key virulence factor. Its immunogenicity and specificity led to a series of studies to assess its potential as a major component of new diagnostic tests. Such tests have evolved into two major forms-a whole blood assay marketed under the name Quantiferon and an ELISPOT assay marketed under the name T-spot. New antigens have been assessed for improved test performance, most notably CFP-10. Studies of in-house versions of these assays, and more recently the commercial kits, have shown encouraging results in TB cases for the diagnosis of disease and in their close contacts for the diagnosis of M. tuberculosis infection. It is clear that these tests are not confounded by prior BCG vaccination and are probably not subject to boosting by injected tuberculin. Their quantitative readouts may also provide important information with respect to infectious load. However, some findings have caused concern: disparity in test performance between temperate non-TB endemic and tropical TB-endemic settings, evidence of relatively poor sensitivity to the presence of certain M. tuberculosis strains (eg. M. africanum) and in certain sub-groups (eg. Pulmonary TB, certain TB contacts), and rapid unexplained test reversion. It is therefore becoming clear that T cell assays may have a niche in the diagnosis of certain TB cases and in certain individuals suspected of having M. tuberculosis infection, but this niche has not yet been fully defined. More longitudinal studies, especially following TB case contacts for the development of disease, are indicated. Also more studies should be undertaken in TB endemic areas. Finally, if these tests are to have any role in TB control globally they would need to be much cheaper and more easy to use. Some advances with respect to these two issues have already taken place. Over the last ten years unprecedented progress has been made in the treatment of HIV infection by the application of combination antiretroviral therapy (cART). Three classes of drugs have been contributed to this success: the nucleoside (NRTI) and the nonnucleoside (NNRTI) reverse transcriptase inhibitors, and the protease inhibitors (PI). With the advent of the fusion inhibitor enfuvirtide (T20) the field has been opened for new classes of drugs. During the last year a whole bundle of drugs with new targets has been studied in humans. Among the most developed compounds are CCR5 antagonists and integrase inhibitors. Other classes in earlier phases of clinical development include attachment inhibitors and maturation inhibitors. It is expected, that integrase inhibitors and CCR5-antagonists will be introduced into clinical practice in 2007/2008. Both of these drug classes have been first studied in heavily treatment experienced patients having failed NRTI, NNRTI and PI therapy. In preliminary analyses the integrase inhibitor MK-0518 combined with optimised background treatment (OBT) has shown clinical superiority over OBT alone. Up to 72% of patients treated with MK-0518 have reached complete viral suppression (HIV-RNA <50 c/mL) after 16 weeks. Preliminary data also suggest a high virological potency of the drug in treatment naïve patients. MK-0518 was applied bid and was well tolerated in both pretreated an treatment naïve patients. Two CCR5 antagonists are currently studied in clinical trials, maraviroc and vicriviroc. Vicriviroc together with OBT has shown superiority over OBT alone in treatment experienced patients, but has failed in a phase II study in treatment naïve patients presumably due to matters of dosing. Further studies to define the optimal dose (together with ritonavir boosting) are underway. With maraviroc results of a study in treatment experienced patients exhibiting dual tropic virus (R5/X4) are available. In this population, maraviroc did not show additional virological efficacy over placebo, but lead to a greater increase of CD4 cells. In conclusion, new drugs in new classes improve the treatment options for patients with multi-resistant HIV considerably. Since these drugs seem to be very well tolerated there is also a potential for their use in treatment naïve patients in the future. Antiretroviral therapy is highly effective in controlling HIV replication, inducing immune reconstitution and preventing AIDS-related morbidity and mortality. Current antiretroviral regimens however cannot achieve virus eradication and virological rebound virtually always occurs after therapy discontinuation. Long-term highly active antiretroviral therapy (HAART) is life saving, but poses several challenges. Potential adverse events can be categorised as problems related to adherence, pharmacokinetics, toxicity and drug resistance. Various interventions have been explored to improve and maintain good adherence among treated individuals, ranging from treatment simplification strategies to addressing mental health problems, and a major challenge is related to the need for integrated biomedical, social and behavioural interventions. Inadequate adherence however accounts for only a subset of subtherapeutic drug levels in clinical practice. There is a significant degree of inter-individual variability in absorption, metabolism and cellular transport of antiretroviral drugs, with potential impact on both efficacy and toxicity of therapy. Important determinants include gender, age, ethnic origin, and genetic polymorphisms. Although evidence of the influence of pharmacogenomics is growing, there remain limited data to guide clinical practice. Proton pump inhibitors are an example of commonly prescribed medications that can affect antiretroviral drug levels by reducing the absorption of certain protease inhibitors (PIs) and increasing exposure to others. Herbal medicines such as St. John's wort, widely used by HIV patients, also have the potential to interact with antiretroviral drugs. Thus, patient education and good communication across care providers are paramount to prevent unfavourable drug-drug interactions. The risk of toxicity associated with long-term antiretroviral therapy is difficult to predict reliably in an aging population of HIV-infected persons. Major examples of HAART-related toxicity include mitochondrial damage and lipoatrophy associated with the use of nucleos(t)ide reverse transcriptase inhibitors (NRTIs), and fat accumulation, dyslipidaemia and insulin resistance associated with the protease inhibitors (PIs). The risk of cardiovascular disease is a particular concern, due to the combined effects on the cardiovascular system of HIV infection, HAART and lifestyle related risk factors such as smoking which are common among HIVinfected persons. In addition, renal, hepatic and bone toxicity represent emerging side effects of long-term treatment. Patients starting therapy on recommended first-line regimens achieve a high degree of virological and immunological success. However, drug resistance continues to emerge as a result of problems with adherence and tolerability. Furthermore, there remain a large number of HIV-infected persons who have accumulated drug resistance through mono and dual therapy in the pre-HAART era and the suboptimal use of therapy in the early HAART era. In addition, a substantial proportion of newly infected persons acquire drug-resistant virus. New drugs and drug classes are required to optimise treatment among drug-experienced persons and reduce the risk of side effects. Several new compounds have recently entered clinical use that show improved tolerability profile or activity against drug resistant virus. Improved methods for detecting and interpreting drug resistance are also being developed to adequately guide treatment. In spite of these concerns however, the benefits of HAART far outweigh the risk of adverse events. The results strongly suggest that keratinocytes are able to produce cytokines in response to L. major stimulation through in part-TLR-2. Aspergillus fumigatus, a termotolerant saprophyte, is associated with a wide spectrum of diseases in humans, that includes saprophytic colonisation of preexisting cavities, allergic asthma, allergic bronchopulmonary aspergillosis occurring as a complication of bronchial asthma or cystic fibrosis, and invasive aspergillosis in immunocompromised patients. Immunocompetent and non-atopic subjects are relatively resistant to A. fumigatus diseases and disease occurs in the setting of host damage. The inherent resistance to diseases caused by A. fumigatus suggests the occurrence of regulatory mechanisms that provide the host with adequate defence without necessarily eliminating the fungus or causing unacceptable levels of host damage. Respiratory tolerance is mediated by plasmacytoid dendritic cells producing IL-10 and inducing the development of CD4+ T regulatory cells expressing FoxP3 and regulating inflammation and allergy through the indoleamine 2,3-dioxygenase (IDO)-dependent pathway. Thus, the IDO-dependent pathway has a unique e central role in this process as it participates in the effector and inductive phases of immunity and tolerance to the fungus. Vaccinia virus (VACV) is the vaccine that was used to eradicate smallpox. Like other poxviruses, VACV is a large DNA virus that replicates in the cytoplasm, produces multiple infectious forms of virus and encodes many virulence factors that are non-essential for virus replication in cell culture. Many of these virulence factors are encoded in the terminal regions of the virus genome and are called immunomodulators because they interfere with the host response to infection. Some of these immunomodulators are present only within infected cells and inhibit signalling pathways induced by ligand engagement of cytokine, interferon or toll like receptors. Other molecules inhibit pathways leading to the induction of apoptosis. This talk will describe proteins from VACV that inhibit apoptosis or intracellular signalling pathways leading to the induction of interferon or proinflammatory cytokines. These studies will illustrate how studying the structure and function of these molecules is increasing our understanding of virus pathogenesis and cell biology. Current trends in parasitology S191 European consensus on malaria chemoprophylaxis Every year several thousand malaria cases with a mortality around 1% are reported in Europe. Visiting friends and Relatives (VFRs) are at particular risk. The complex situation of malaria transmission in various endemic areas requires differentiated recommendations for malaria prophylaxis. Malaria prophylaxis comprises of multiple components. Malaria risk can be reduced by compliant exposure prevention, and with chemoprophylactic drugs for travel to high risk areas are available. Key points for malaria prevention and management include awareness in endemic regions and after return, avoid mosquito bites, compliance with chemoprophylaxis, and seeking immediate diagnosis and therapy in case of fever. The importance of exposure prophylaxis should be emphasized. It is recommended to use mosquito repellents after dusk, especially if outdoor activities are performed. Light-coloured, loose-fitting insecticide-treated clothing with long trousers and long sleeves are suggested. Sleeping under insecticide treated bed nets or in airconditioned rooms which are pre-treated with insecticides (knockdown spray) is recommended. Chemoprophylaxis is recommended in high risk areas. In most settings, either mefloquine (Lariam ® ), atovaquone/proguanil (Malarone ® ) or doxycycline (monohydrate) are used. In German speaking countries, emergency treatment is recommended for trips to regions with low or intermediate malaria risk. This strategy is recommended when the infection risk is lower than the risk of severe drug side effects. Good information by the consulting doctor and personal responsibility of the traveller are essential for the correct handling of emergencyself-treatment. Drugs used include artemether/lumefantrine (Riamet ® ), atovaquone/proguanil (Malarone ® ) or mefloquine (Lariam ® ). The guidelines for the application of the emergency-self-treatment should be discussed thoroughly with the traveller, to make sure that in case of fever the correct action will be taken: 1. In case of fever (sudden onset or rapidly progressive) -axillary temperature >37.5ºC (oral, tympanal or rectal >38ºC) -a doctor should be seen and a malaria blood test should be performed. A working thermometer is essential in the tropics. 2. If no doctor can be seen within 24h and the traveller is in an endemic region for at least 6 days, the fever should be lowered. 3. The malaria emergency medication should be taken with adequate amounts of fluid. 4. In every case, also after the intake of the malaria drug a doctor must be consulted at the earliest possible time. The term echinococcosis describes two diseases which markedly differ in their presentation, behaviour and management: Alveolar echinococcosis (AE) is caused by the metacestode Echinococcus multilocularis and cystic echinococcosis (CE) by E. granulosus. The larval growth of the two bugs is very different and separates the "malignant" AE from the "benign" CE. Thus, current trends are discussed separately. AE: A recent European initiative (EchinoRisk) discovered a continuous increase and spread of the parasite in the final host (fox). In parallel, human cases were observed in regions outside the known endemic areas, such as in the Baltic States, in Hungary and Slowakia. Risk factors for humans are farming and a long-standing, close contact to dogs. The initially asymptomatic liver disease is still rare in Central Europe, and diagnosis is often made at a late stage of the disease. An anatomical classification system has been used (PNM adapted from the TNM system of tumours), allowing now a comparison of the disorder at the time of first diagnosis in different clinical settings. New foci have been discovered in China/Tibet with a prevalence rate of up to 4% of the affected population. Imaging techniques, such as Ultrasound, CT, MRI and PET/CT contributed to a much better description of the lesions. However, the expertise of the radiologist is often not available due to the low incidence of AE. Serology is helpful, but may be misleading due to non-standardised tests. Surgery is not anymore the treatment of choice. Instead many centres apply short term cycles of benzimidazoles which act parasitocidal and accelerate the degeneration of the cyst. Others favour the Watch and Wait concept, and carefully observe the natural degeneration of the cyst(s). Filarial worm infections of humans cause morbidity and even death in developing countries of the tropics. Current antifilarial drug therapies target only the first stage larvae, requiring many years of annual/biannual treatment. Another problem with controlling filarial infections is the lack of any alternative drugs that can be used in the current mass drug administration programmes should resistance develop. Wolbachia, endosymbiotic bacteria that are found in most of the human filarial worms are excellent targets for the discovery of new antifilarial drugs because of their requirement for worm embryogenesis, development and adult survival. In both lymphatic filariasis and onchocerciasis, administration of 6 weeks of doxycline at a dose of 200 mg/day results in a high macrofilaricidal effect of over 80% and 70%, respectively. Targeting of Wolbachia with antirickettsial drugs has lead to the recommendation of doxycycline for use on an individual basis and may be recommended in areas where resistance to current drugs may develop. Evidence that eliminating the endobacteria reduces adverse reactions to current drug therapies and even reduces early stages of lymphatic pathology is also accruing. Research is underway to discover new drugs, preferably those already approved for use in humans, that have antiwolbachial activity and work in a shorter time and are widely applicable. vancomycin MIC: 2 and 16 mg/l, respectively) to study their ability to (i) adhere to fibrinogen (Fg) and fibronectin (Fn) in vitro, (ii) persist in the bloodstream after intravenous inoculation, (iii) colonise aortic vegetations in rats, (iv) multiply in situ thereafter, and (v) compete for valve colonisation when inoculated in mixed cultures. Methods: GISA M1V16 was selected by vancomycin exposure of the glycopeptide-susceptible, methicillin-resistant S. aureus M1V2 (agr type II) in the laboratory. The phenotypic expression of resistance to vancomycin and the stability of the phenotype was analysed by population analysis. Adherence was tested on immobilised Fg and Fn. Rats with catheter-induced aortic vegetations were inoculated with 10 3 −10 6 CFU of the test strains. Rats were killed after 24 h. Vegetations and spleens were cultured and the lower inoculum infecting 90% of vegetations (ID90) was recorded. Blood cultures were drawn within the first 60 min. Infectivity was also compared by co-inoculation with 10× the ID90 of M1V16 and a rifampin-resistant variant of the parent M1V2, to discriminate them in organ cultures. Results: The GISA M1V16 mutant grew on plates containing 16 mg/l of vancomycin and its resistance phenotype remained stable after 5 passages on drug-free medium. Both GISA and parent strains adhered similarly to Fg and Fn in vitro. In rats, the GISA M1V16 was cleared faster from the blood (P < 0.005) and required 100× more bacteria than the parent (10 6 versus 10 4 CFU) to infect 90% of animals. GISA M1V16 had also 100-1000× lower bacterial densities in vegetations and spleen. After co-inoculation, M1V2 and GISA M1V16 produce similar vegetation densities early after injection. However, while the vegetation counts of the parent increased at 48, 72 and 120 h, the counts of GISA M1V16 remained stable. Conclusions: GISA showed attenuated pathogenicity (ID90) and fitness (intra-vegetation growth) in rats with experimental endocarditis. Thus, the GISA phenotype is globally detrimental to infectivity. This might explain its clustering in immuno-compromised patients. Whether a very prolonged exposure to glycopeptides might restore pathogenicity and fitness defects remains to be determined. Most adult cystic fibrosis (CF) patients are chronically infected with P. aeruginosa in their lungs. Two major features of P. aeruginosa are the abillities to form biofilms and become mucoid. Recently, we reported a time-dependent reduction in the ability to form biofilm by non-mucoid P. aeruginosa isolates from the same CF patient obtained over a period of 23 years. In order to investigate whether the declined ability of the PFGEidentical isolates to form biofilm in vitro correlated to pathogenicity in vivo, 5 non-mucoid clones from 1980 and 1988 (early clones), and 1997, 1999 and 2003 (late clones) embedded in alginate were installed in the lungs of five groups of BALB/c mice. Mice were sacrificed after 5 days of infection. The experiment was evaluated by macroscopic lung pathology, histopathology, quantitative bacteriology (CFU/lung) and pulmonary cytokine production. Survival was significantly higher in mice infected with the late clones, as compared to mice infected with the early clones (p < 0.025). Macroscopic pathology was more disseminated in mice infected with the early clones as compared to mice infected with the late clones (p < 0.0015). Inflammation involving polymorphonuclear neutrophils (PMN's) was present more often in mice infected with the early clones as compared to mice infected with the late clones (p < 0.05). In addition, a higher degree of inflammation as well as more mice with athelectasis was observed after infection with the early clones (p < 0.05). CFUs were higher in mice infected with the early clones as compared to mice infected with the later clones (p < 0.04 Results: Animals treated with probiotic displayed reduced incidence, severity and duration of diarrhoea. These animals also gained weight at a greater rate than control animals and mean faecal numbers of Salmonella were significantly reduced in probiotic-treated animals at 15 days post infection. The probiotic mixtures used lead to an amelioration of clinical signs in Salmonella Typhimurium-infected pigs early in the course of infection, and significantly reduced pathogen shedding over a longer timeframe. While the exact mechanism remains to be fully elucidated, this demonstrates the validity of using commensal lactic acid bacteria strains in the prevention of gastrointestinal infection. The in vitro and in vivo procedures used to isolate and select the bacteria are also validated. While the probiotics examined in this study are of obvious interest to those involved in the pig production industry, the similarities between the pig and human gastrointestinal tracts suggest that the probiotics offer potential in cases of human salmonellosis, given that the methodology used is also likely to be applicable to human disease control. Objectives: Plasmid-mediated quinolone resistance (qnr) genes have been identified in enterobacteria from several countries. Such resistances have frequently been associated with strains that produce extendedspectrum b-lactamases (ESBLs). As Salmonella with resistance to quinolone antimicrobials have become increasingly common in isolates from humans in England and Wales, a study has been initiated to investigate the occurrence of such genes in isolates from humans and food, and to assess their importance for public health. Methods: A panel of 118 isolates of S. enterica resistant to nalidixic acid and with concomitant decreased susceptibility to ciprofloxacin (MIC: 0.25−1.0 mg/L), from cases of human infection and from foods in England and Wales, were screened for the presence of qnrA, qnrB and qnrS by PCR. Isolated qnr plasmid was tested by PCR genes and for ESBL genes, and for 18 commonly occurring replicons. All strains positive for qnr genes were further characterised by PFGE. Results: All isolates were negative for qnrA and qnrB; 6 isolates belonging to four serovars (S. Stanley, 2; S. Typhimurium, 1; S. Virginia, 1; S. Virchow, 2) were positive for qnrS. Of these the isolates of Stanley and Typhimurium were from patients infected abroad, the isolate of Virginia was from a patient who had not travelled abroad, and the two isolates of Virchow, one from imported frozen chicken and one from a patient were associated with a series of infections in 2003/2004. qnr plasmids were transferable either by conjugation or transformation; their molecular masses ranged from 13.5 kb (S. Virginia) to >148 kb (S. Stanley). Four of the six plasmids coded for additional resistance, in two cases for resistance to 10 additional antimicrobials. The qnr plasmids from the S. Virchow isolates were of similar size and conferred a similar resistance phenotype as the first qnr plasmid, identified in Shigella flexneri in Japan. These results indicate that plasmidic quinolone resistance, often linked to other resistances, has now appeared in S. enterica in Britain. This is of particular concern for the treatment of infections with invasive serovars such as S. Virchow. Objective: The rapid characterisation of (drug-resistant) Mycobacterium tuberculosis (MTB) would be useful for the research and treatment of tuberculosis (TB). Many important genetic markers of MTB have been identified, notably for drug resistance, but also genotype, bacterial lineage and adaptive potential. A single assay allowing identification of these markers would aid control efforts. Multiplex PCR is unsuitable for simultaneous amplification of several genetic loci in one reaction. With Multiplex Ligation-dependent Probe Amplification (MLPA) only one primer-pair is used for the amplification of multiple genetic targets. We explored the utility of MLPA as a screening tool to rapidly characterise MTB-strains Method: MLPA can identify multiple single nucleotide polymorphisms (SNPs) by amplification of sequence-specific MLPA-probes, rather than target DNA. A sensitive ligation-step ensures specificity of the assay. We designed MLPA-probes specific for a range of discriminatory SNPs in MTB. In addition, we selected three sequences of conserved regions to use as internal controls and for quantitation. The size of MLPA-products corresponds to the specific SNP they targeted. Amplified MLPA-products were identified on an agarose-gel or by fragment analysis, using capillary electrophoresis. Results: Probes were initially validated on DNA from reference strains. All probes were then combined in a single mixture and used to characterise laboratory strains and a panel of clinical isolates from Brazil, Peru and the Netherlands. To determine the predictive value of MTB-specific MLPA, we screened DNA of MTB-strains with unknown genotypes. Results were confirmed by sequencing. There was a very high correlation between sequencing results and results obtained via MLPA. In addition, both the positive and the negative predictive value were high. MLPA-products were clearly visible on an agarose gel. We have shown that with MTB-specific MLPA it is possible to identify drug resistance associated mutations and MTB lineage specific SNPs in a single assay. Depending on the application, probes can be added to or removed from the probe-mix, making it a flexible, multi-purpose method. MLPA products can be easily identified on an agarose-gel, making it especially suitable for screening of TB in the less-developed world. Based on our results we feel that in addition to using MLPA on DNA extracted from culture, performing MLPA directly on DNA from sputum samples is also feasible. Objectives: The species Escherichia coli comprises four different phylogenetic groups (A, B1, B2 and D). In general the majority of infectious as well as colonising strains isolated from patients belong to phylogenetic group B2. Yet, there is a known disparity between fluoroquinolone-susceptible and -resistant isolates, which it is assumed has arisen from the susceptible population: Only a small proportion of resistant isolates belong to group B2, while the majority belong to group D. For a possible explanation of the disparate distribution among phylogenetic groups this work analysed ciprofloxacin-susceptible and -resistant ICU isolates in all phylogenetic groups for differences in virulence factors (VF) and mutation frequencies. Methods: Hundred and forty-six susceptible and 135 resistant isolates were collected from patients in 27 ICUs. All strains were assigned to phylogenetic groups by means of a triplex PCR protocol. In order to compare isolates from different groups and susceptibilities, a similar and as large a number as possible of susceptible and resistant strains were chosen randomly from groups A (23; 27), B1 (7; 11), B2 (17; 9), and D (15; 25). The occurrence of different VF was determined by use of PCR and summarised for each isolate as a VF score. Moreover, the mutation frequency was determined by plating on 100 mg/L rifampicin containing agar. Results: Assignation to phylogenetic groups confirmed differences between susceptible and resistant isolates in the distribution among phylogenetic groups: 82 out of 146 susceptible isolates belonged to group B2, in contrast to just 9 out of 135 resistant isolates, with a majority of 61 isolates belonging to group D. The VF score was highest both in strains belonging to groups B2 and D without significant differences between susceptible and resistant isolates. However, there was a small, but significant difference in the median of the mutation rates between the isolates of different groups, increasing in the order B2 < D < B1 < A. The median mutation rate of B2 isolates (1.3×10 −8 ) differed significantly (P < 0.05) from the remaining isolates as did, conversely, that of the group A isolates (2.4×10 −8 ). Conclusion: Slightly, but significantly reduced mutation rates in strains of the deep rooted phylogenetic group B2 might be an explanation for their rare occurrence among fluoroquinolone-resistant isolates. Objectives: Resistance to ceftazidime (CAZ) has mostly emerged among enzymes of CTX-M-1 lineage. The aim of this study was to understand evolution of CAZ resistance among CAZ susceptible ESBL belonging to this cluster by step-wise in vitro selection experiments. Methods: blaCTX-M-1, blaCTX-M-3 and blaCTX-M-10 genes were cloned into pBGS18 plasmid vector (KanR) using EcoRI and PstI restriction enzymes. Recombinant plasmids were further transformed into E. coli strain MI1443 (del-ampC, plasmid free) and its hypermutable isogenic strain GB20 (mutS::Tn10 MI1443). GB20 transformants corresponding to each ESBL were submitted to daily serial passages with increased concentrations of CAZ (0.5−256 fold respect to MIC). Plasmids containing mutants obtained at distinct concentrations were transformed in MI1443 cells, selecting with CAZ (at 4× their corresponding MIC). MICs to CAZ, cefotaxime (CTX), cefuroxime (CXM), cefepime (FEP), and amoxicillin-clavulanate (AMX) were determined in MI1443 carrying the different evolved and nonevolved recombinant plasmids. blaCTX-M genes from strains containing recombinant plasmids displaying reduced susceptibility to CAZ were sequenced. Results: A high frequency of CAZ mutants was detected (73%; 8/11 transformants tested) with significant increments in CAZ resistance. Interestingly, and with exception of two variants and CXM, a concomitant loss of resistance to all other antibiotics tested (antagonistic pleiotropy) was observed. All mutants contained new blaCTX-M variants except blaCTX-M-52, which evolved from blaCTX-M-3. The most frequent mutations found were D240G and P167S/T, the later conferring higher resistance to CAZ than the former. However, D240G change determined a lower decrease to FEP and CXM than P167S/T. Additionally, two variants were recovered from the evolved blaCTX-M-3 gene: P167S and P167S+A77V, found at 16 and 128 mg/L of CAZ respectively. The double mutant yielded higher resistance level to CAZ, CTX and to a lesser extent to FEP than P167S variant. The A77V change is one of the three polymorphisms between CTX-M-3 and CTX-M-1, whose mutants obtained revealed the highest resistance levels to CAZ and CTX in our study. Conclusions: This work demonstrates an antagonistic relationship between the susceptibility to CAZ and to other b-lactam antibiotics tested. A77V could be a secondary mutation site affecting CAZ or could be involved in re-equilibrium of CTXR/CAZR co-resistance. Objectives: Fluoroquinolone (FQ)-R group A Streptococcus (GAS) worldwide are predominantly of the emm6 serotype. Emm6 GAS are intrinsically low-level FQ-R due to a polymorphism in the FQbinding region in parC, which confers resistance to the older FQs like ciprofloxacin (CIP). The new respiratory's FQs, levofloxacin (LEV) and moxifloxacin (MOX), retain their activity against these first-step parC mutants, although there are concerns that their widespread use might select for second-step, high-level FQ-R mutants. LEV and MOX were introduced into clinical use in 1999 and 2002, respectively, in Belgium. We studied the prevalence and evolution of FQ-R GAS recovered from tonsillopharyngitis and invasive infections in Belgium during 2003-2005. Methods: From a total of 3765 isolates collected from 10 provinces during 2003−05, 261 (7%) isolates were resistant to CIP (MIC 2 mg/mL) and corroborated with MICs of 4 FQs in the presence or absence of reserpine. Clonality was studied by pulsed-field gel electrophoresis (PFGE) and emm typing. Clonal isolates with CIP MIC 2 mg/mL were sequenced for mutations in the FQ binding regions of parC, gyrA, gyrB, and parE. Results: During 2003−05, FQ-R GAS increased from 2%, 5% to 16%. The predominant emm type was emm6, which formed 76% of the total FQ-R GAS during these three years. Emm6 and emm75 isolates together constituted 92% of the FQ-R GAS. Isolates with CIP MICs 2−8 mg/mL showed previously identified mutations in parC leading to amino acid substitutions as S79A (emm6) and S79Yor S79F (emm75), and no changes in gyrA, gyrB or parE. However, an emm6 throat isolate, recovered from a 36-year old female in 2005, exhibited highlevel resistance to CIP (MIC 32 mg/mL), full resistance to LEV (MIC 8 mg/mL) and decreased susceptibility to MOX (MIC 1 mg/mL). This isolate also showed a second-step mutation in the FQ-binding region in gyrA (predicted substitution S81Y). Reserpine-sensitive efflux was not observed for any FQ-R GAS. Conclusions: We report a dramatic increase in FQ-R GAS in 2005 in Belgium probably related to natural fluctuations of the low-level FQ-R emm6 clone. More importantly, we describe, for the first time, an emergence of high-level FQ resistance in a clinical emm6 isolate. Although respiratory FQs are usually not a therapeutic option for GAS infections, dissemination of this parC/gyrA double-mutant emm6 strain needs close monitoring. This method replaced phage typing in combination with SmaImacrorestriction analysis for routine S. aureus strain typing. This study reviews typing results for six month with respect to typeability, reproducibility, discriminatory power and concordance with alternative typing methods. Methods: A total of 1464 S. aureus isolates were characterised. The polymorphic X-region of the protein A gene was sequenced and a spa type was assigned using the Ridom StaphType software. The algorithm BURP (based upon repeat patterns) was used to cluster resulting spa types into different groups. To verify the results a subset of isolates was subjected to SmaI-macrorestriction analysis and MLST. Results: Among 1464 S. aureus isolates more than 200 different spa-types were defined. The twenty spa-types most frequently found comprise more than 60% of all isolates and include most types linked to predominant clonal lineages of MRSA in Central Europe, previously defined by phage typing and SmaI-macrorestriction analysis. Among those clonal lineages distinct differences in spa-type variability were seen; while some lineages (e. g. the Barnim epidemic MRSA, t032, ST22) encompassed a variety of closely related spa-types, others (e. g. a subclone of Rhine-Hesse epidemic MRSA, t003, ST225) are represented only by a single type. For assessment of widely disseminated lineages exhibiting only a single spa-type, additional markers must be applied. More than one hundred spa-types were determined only once. For these isolates BURP proved to be a valuable tool to assign them to particular clonal groups. Thereby, results of BURP grouping were overall concordant with those of SmaI-macrorestriction analysis and MLST. Conclusion: spa-typing in combination with BURP based grouping of isolates proved to be a valuable tool for strain typing with regard to the demands of a national reference centre. The BURP algorithm revealed extremely useful for grouping new and uncommon spa-types. The increasing amount of typing data obtained from outbreak situations enables comparison of closely related spa-types within BURP groups, thus facilitating the definition of criteria for strain relatedness according to those already defined for SmaI-macrorestriction analysis. This will be an important prerequisite for definition of isolates as identical, related of different, which is essential for successful epidemiological outbreak investigation. Although bacterial colonisation is common and mostly asymptomatic in children, it frequently results in a wide spectrum of respiratory tract infections (RTIs): acute otitis media (AOM), sinusitis, conjunctivitis, pneumonia and other lower respiratory infections (LRIs). In children, the majority of bacterial RTIs are caused by Streptococcus pneumoniae (Pnc), Haemophilus influenzae (mostly nontypeable, NTHi) or both. In AOM, NTHi and Pnc are equally common. Pnc is slightly more 'aggressive' than NTHi (i.e. higher temperature, higher polymorphonuclear counts and complications are more frequent), but NTHi can still cause significant disease. The role of NTHi in recurrent and non-responsive AOM in both infants and in older children is recognized, suggesting different biological pathways compared with Pnc. In both acute and subacute sinusitis, NTHi is found as commonly as Pnc. NTHi is the most common causative agent of conjunctivitis and the most significant in relation to the co-presence of AOM and systemic symptoms and signs. The role of NTHi in LRIs in general, and in pneumonia in particular, is not yet clear. In cystic fibrosis, however, NTHi is an important pathogen. Although H. influenzae type b disease has practically been eliminated in vaccinated populations, the picture with S. pneumoniae in the 7-valent Pneumococcal Conjugate Vaccine (PCV7) era is much more complex owing to its limited serotype coverage and some replacement with nonvaccine serotypes. A vaccine with additional pneumococcal serotypes and activity against NTHi would be a very welcome addition to our vaccine armamentarium. Otitis media (OM) is a highly prevalent paediatric disease that is associated with significant morbidity and socioeconomic cost. OM is caused by the synergistic interaction between upper respiratory tract viruses and three predominant bacterial species, all of which are members of the commensal flora of the paediatric nasopharynx. Despite their similar roles as opportunistic pathogens of the middle ear, the pathogenic mechanisms utilised by these bacteria (nontypeable Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis) are unique. Thus, to date, most laboratories have investigated the pathogenesis and prevention of disease caused by these microbes individually. Recently, it has become appreciated that OM, and particularly chronic and recurrent OM, is a disease that includes a biofilm component. By definition, bacteria resident within a biofilm community are inherently resistant to eradication due to their significantly enhanced ability to resist the action of antibodies as well as antibiotics. Moreover, residence within a biofilm promotes the bacterial exchange of DNA, which confers resistance to antibiotics, and induces a markedly reduced bacterial growth rate that commonly results in false-negative bacterial cultures. In the past few years it has been demonstrated that each of the three predominant bacteria involved in OM can form a biofilm in in vitro assay systems and in animal models, and that they participate in biofilms formed on middle ear mucosa specimens recovered from children with recurrent or chronic OM. This enhanced understanding of the biofilm component of middle ear infections is greatly influencing the current approaches to both the management of OM and the design of novel strategies to treat, or better to prevent, OM. It is particularly intriguing that some of the paediatric middle ear mucosa-derived biofilms characterised were of mixed bacterial aetiology, suggesting that progress made on single-microbe directed strategies for the treatment and/or prevention of OM, while highly encouraging, are also likely to be inadequate. Therefore, a significantly greater understanding of how microbial physiology relates to the involvement of biofilms in OM is required to identify points in the disease course that are perhaps more amenable to treatment strategies, and also biofilm-relevant antigenic targets that would be helpful in the rational design of vaccine candidates to prevent OM. Protein D (PD) is a highly conserved surface exposed 42 kDa lipoprotein found in 100% of all NTHi and encapsulated Hi, and is highly immunogenic in humans. Evidence suggests that PD is involved in the pathogenesis of respiratory tract infections, and has been implicated in NTHi adhesion and invasion of host cells. An isogenic PD-deficient NTHi-mutant, compared with the wild type strain, had an approximately 100-fold lower capacity to cause acute otitis media (OM) in rats and induced significantly less impairment of ciliary function in a human nasopharyngeal tissue culture model. The likely mechanism of pathogenicity is that PD is an enzyme (glycerophosphodiester phosphodiesterase, GlpQ) that releases phosphorylcholine (ChoP) from host epithelial cells to the lipooligosaccharide (LOS) on NTHi. LOS-ChoP interacts with platelet-activating factor receptors, inhibiting ciliary beat frequency of human bronchial epithelial cells and activating G protein-related pathways. PD is also capable of inducing bactericidal anti-protein D antibodies that protect against NTHi disease. PD-immunised rats cleared NTHi significantly better than non-immunised animals after middle ear and pulmonary bacterial challenge. In addition, chinchillas, PD-immunised or passively given anti-PD sera, showed protection against NTHidependent OM. PD was recently introduced as an antigenically active carrier protein in an experimental 11-valent pneumococcal conjugate vaccine. In a recent Phase III clinical trial with this experimental vaccine, significant protection was achieved against OM caused by pneumococci and/or NTHi. The currently licensed pneumococcal conjugate vaccine (PCV) has been integrated into infant immunisation programmes of several industrialised countries. New PCV candidates in development with wider serotype coverage or new PCV formulations for both infants and adults will be evaluated for efficacy. Large efficacy trials are not feasible any more, and the acceptance of the new PCVs will rely on immunogenicity studies. In the WHO guidelines (http://www.who.int/biologicals/publications/trs/ areas/vaccines/pneumo/en/index.html) the primary threshold for such trials is the proportion of subjects with antibody concentration of 0.35 mg/mL for all serotypes when measured via the WHO non-22F ELISA. The protective functional mechanism of antibodies to pneumococcal capsular polysaccharides resides in opsonophagocytosis and the demonstration of the opsonophagocytic activity (OPA) has been regarded as an important secondary threshold. Thus far there are only few published PCV efficacy trials that connect with OPA data and their good correlation. Considering those data it is becoming evident that measuring OPA has an added value when evaluating the vaccine efficacy based on immunogenicity studies. In general, the OPA titers and the antibody concentrations correlate well, but different serotypes can show various OPA/EIA ratios meaning that for certain serotypes (like 19F or 1) more antibodies are needed for killing the bacteria than for the rest. Importantly, the elderly and HIV positive patients seem to have antibodies of low functional activity, and measuring OPA in studies in the elderly and HIV patients has been considered important. Various assays for determining OPA have been developed and some laboratories have already performed extensive validation steps. A multilaboratory standardisation of the OPA assay is imminent. Measuring OPA with the classical killing assay consumes serum and time and thus measuring OPA for large materials is not always feasible. However, the development of multiplex and/or high throughput assays will end up in wider use of this technology in the future clinical trials. This would be in concordance with the guidelines for evaluation meningococcal conjugate vaccines by serum bactericidal activity assay. a case study (POET) Bacterial respiratory tract infections are a leading cause of morbidity and mortality in young children. Effective immunisation strategies against these infections have to contend with both the multiplicity of pathogens and a host of disease syndromes. Streptococcus pneumoniae (pneumococcus), and both encapsulated and non-encapsulated forms of Haemophilus influenzae (Hi), are the leading pathogens. More than 90 serotypes of pneumococci exist, but only 10 serogroups appear to be responsible for 80-85% of invasive pneumococcal disease and pneumococcal otitis media (OM) worldwide, ranging from the relatively rare but clinically severe meningitis, to the more frequent infections of the bloodstream and the lungs, such as pneumonia and finally to the extremely common middle ear infections among young children. Middle ear infections are often associated with diagnostic uncertainty, frequent use and misuse of antibiotics, and recurrent and persistent disease. Recurrent infections occur in as many as 10% of children <3 years of age. Recurrent and persistence disease can lead to temporary or even permanent hearing loss and surgery (tube placement). In order to address these issues, preventive acute OM (AOM) strategies should include a vaccine with broader pneumococcal serotype coverage as well as protection against additional otopathogens. This presentation describes a novel vaccine approach in which polysaccharides from the 11 leading streptococcal paediatric serotypes are conjugated to a carrier protein (protein D) derived from Hi. Resistance to erythromycin, clindamycin, moxifloxacin and tetracycline was found in 45.8%, 50.6%, 9.1% and 33.7%, respectively. All the strains were fully susceptible to metronidazole and vancomycin. Resistance to moxifloxacin and erythromycin was tightly associated and MICs of moxifloxacin were significantly higher in patients previously treated with fluoroquinolones. Prevalence of the 027 epidemic strain was 5.7%. It was found in Ireland (n = 1), Netherlands (n = 8) and Belgium (n = 11). This strain exhibits the same phenotypic and genotypic features as the NAP1 clone described in North America: it is positive for binary toxin genes, it has an18-bp deletion in tcdC gene and it is resistant to erythromycin and moxifloxacin. Mean incidence of CDAD was 2.45 cases per 10,000 patient-days but it varied widely from one hospital to another. Patients infected with 027 strain were likely to have a more severe disease (RR = 1.72, 95% CI: 1.07−2.75, p = 0.048), to have received less antimicrobials in the month preceding diarrhoea (RR = 0.76, 95% CI: 0.53−1.10, p = 0.05) and to have been more specifically treated by metronidazole or vancomycin (RR = 1.32, 1.17−1.49, p = 0.02). On-going epidemiologic surveillance of CDAD cases with periodic characterisation of the strains is needed to timely detect clustering of cases and to monitor the emergence of a specific hypervirulent clone. The incidence and severity of nosocomial C. difficile disease seems to be increasing. Additionally, disease with community onset is becoming more common than previously recognized and cases in groups with Acinetobacter -the Gram-negative MRSA? S47 previously low risk have been reported. C. difficile is also emerging as an important animal pathogen in horses, calves and piglets. Described changes correlate with changed populations of C. difficile present in humans. New type BI/NAP1/027 with increased virulence is spreading since 2003, but only part of the recent increase in mortality and morbidity caused by C. difficile infections can be accounted for by this new highly virulent type. The proportion of other binary toxin positive types is also rising among human isolates. Such binary toxin positive strains used to be more often associated with animal than human host and early typing comparisons did not show many similarities between human and animal strains. However, recent studies found a marked overlap between isolates from calves and humans, including two of the predominant outbreak types, 027 and 017. C. difficile has also been found in retail meat samples, suggesting that food could be involved in the transmission of C. difficile from animals to humans. As a response to the new developments in C. difficile epidemiology, new diagnostic and typing methods are being developed aiming in quick and sensitive detection of C. difficile but also in quick recognition of strains with higher virulence. Early diagnosis of CDAD, prompt isolation of symptomatic patients and reducing antimicrobial treatment are essential first steps. The infection control measures include recommendations to isolate infected patient on a single room with designated toilet, to apply proper hand hygiene with soap and water, to use appropriate protective clothing (gloves and aprons or gowns), to intensify environmental cleaning with a chlorine containing disinfectant and to take specific precautions for the use of devices (disposable or dedicated to individual patient). Patient isolation must continue at least until diarrhoea has ceased. Each hospital should have an appropriate surveillance system to recognize an increase of the incidence of CDAD in an early stage. All infection control measures should be written in a local protocol so that additional measures can be carried out as soon as a problem with CDAD arises. When outbreaks occur, additional recommendations include a reinforcement of general and hand washing measures, intensifying of testing patients with diarrhoea for C. difficile, reinforcement of environmental cleaning, information and education of healthcare workers, cleaning department and visitors, cohorting of infected patients, and eventually closure of the unit followed by intensive environmental cleaning. Restricted antibiotic prescribing is also highly recommended to reduce polypharmacy and duration of administration. Second and third generations cephalosporins, clindamycin and more recently fluoroquinolones have been identified as potential risk factors. Although some hospitals report successes for enhanced environmental cleaning with potentially effective agents such as hydrogen peroxide vapour, the evidence is too scarce to consider this as an evidence-based approach. Acinetobacter -the Gram-negative MRSA? S242 Global epidemiology of Acinetobacter Members of the genus Acinetobacter, particularly Acinetobacter baumannii, are now recognized as significant nosocomial pathogens, particularly for the subset of critically-ill patients requiring mechanical ventilation in hospital intensive care units. However, Acinetobacter infections can also be acquired outside the healthcare setting. Wound infections caused by Acinetobacter are now common among trauma patients in conflict zones or following natural disasters, and there have also been reports of other community-acquired infections, mainly in patients with some type of co-morbidity. Most of the latter reports originate from tropical or sub-tropical areas, and may become more common following ongoing changes in the global climate. Strains of A. baumannii harbour inherent or acquired resistance mechanisms against the vast majority of available antimicrobial agents, including the carbapenems. These organisms also have a remarkable capacity to persist in the hospital environment and to cause epidemic outbreaks of infection in hospitals. A recent increase in the number of carbapenem-resistant strains, particularly in eastern Europe, is currently of great concern. The recent EU-funded ARPAC study revealed that 130 of 169 participating European hospitals in 32 European countries had encountered carbapenem-resistant isolates of Acinetobacter, ranging from very rare sporadic resistant isolates to an endemic/epidemic situation. Certain multiresistant epidemic clones are currently spreading worldwide, and epidemics of multiresistant A. baumannii infections have occurred following the repatriation of casualties from the recent wars in Kuwait, Iraq and Afghanistan to European and American hospitals. In general, multiple isolates from a single hospital usually belong to the same clone, but some hospitals yield isolates belonging to more than one clone. Three 'European Clones' (lineages) have been identified in hospitals throughout Europe, but numerous other, more localised, clones have also been characterised, showing that the problem of A. baumannii is not confined solely to the widespread 'European clones I, II and III'. The reasons why certain A. baumannii lineages are more successful than others are not understood at present. Overall, the available epidemiological evidence suggests that A. baumannii is becoming one of the most significant microbial challenges of the current era. Strains in some hospitals are already effectively untreatable, and more scientific efforts and resources are urgently needed to further elucidate the epidemiological and infection control issues related to these infections. Acinetobacter baumannii is an opportunistic pathogen that is mostly involved in nosocomial infections in immunocompromised patients. Whereas A. baumannii has itself a quite high level of naturallyoccurring antibiotic resistance, it may acquire additional resistance traits as a source of multidrug resistance. These resistance mechanisms may involve most of the antibiotic molecules, i.e. aminoglycosides, b-lactams and fluoroquinolones. Rate and spread of specific mechanisms of resistance depends on each country. A similar trend between the number of A. baumannii infections and antibiotic resistance may be observed worldwide. Acquired resistance genes are located on chromosome, plasmids, insertions sequences, transposons, integrons, and the recently described resistance island. One of the most worrying antibiotic resistance problems in A. baumannii is the increasing trend of carbapenem resistance since carbapenems are often used as antibiotics of the last resort. Carbapenem resistance results from metallo-b-lactamases, carbapenem-hydrolyzing oxacillinases and often combined mechanisms of resistance. Methods: During the two flights the patient had no cough but a urinary tract catheter which was disconnected from the reservoir and released relevant amounts of urine on the passenger's seat. The patient's urine was tested positive for Lassa virus, which was taken into account for the risk assessment and contributed to the decision to trace back copassengers potentially exposed. The passengers at risk were defined as those sitting in maximum three rows distance from the patient. 57 additional persons from the airline and the airport were exposed. With the help of the airline the passenger's lists of both flights were available within two days. 92 passengers from nine countries were identified. Every country received a list with names and contact telephone numbers of all passengers at risk who were allocated to the country as well as a questionnaire about symptoms to be filled for traced passengers. The trace back of the concerned staff was done by the airline and the Belgian public health authorities. 7) . In all cases, the magnitude of the reproduction number was comparable between the two waves. Our best estimate for the initial reproduction number (R 0 ) was 3.7, although it could range from 2 to 11 depending on assumptions regarding incubation and lifespan in mosquitoes. In the best fitting case, each infected individual may have contaminated 3 mosquitoes at most, and each mosquito contaminated 1.4 persons on average. Using data from the first season alone, modelbased extrapolations suggested that epidemic outbreaks were possible as long as more than one third of the population remained susceptible. Conclusion: Despite a thousand-fold change in incidence between the two seasons, the transmission characteristics of Chikungunya were similar; therefore there is no epidemiological evidence for an increase in virulence between seasons. At a time when information systems make it easier to monitor the course of emerging diseases, methods for timely and efficient analysis of the data must also be developed. Results: During the study period, a total of 138 cases of invasive Hi disease were reported from AK (76) and N Can (62). Among the 88 (67%) invasive Hi cases with serotype information available, 42 (48%) were serotype a, 27 (31%) were serotype b, 12 (14%) were serotype f. Among Hia isolates, 35 (83%) occurred in aboriginal peoples; median age was 1.1 years (range 3 mo to 74 years); 62% were male. Two Hia cases (1 adult/1 child) were fatal. Common clinical presentations included: meningitis (33%), pneumonia (29%), and septic arthritis (12%). There were no cases of epiglotittis. Overall annual Hia incidence was 0.9 cases per 100,000 population. Annual incidence rates in aboriginals in AK and N Can were 1.1 and 4.6 per 100,000 persons, respectively; rates in aboriginal children <2 years of age were 22 and 101 cases per 100,000 persons, respectively. PFGE analysis revealed genetically similar Hia strains in both AK and N Can. Conclusions: Serotype a is now the most common Hi serotype seen in the North American Arctic, with the highest rates among indigenous children. Further research is needed to determine sequelae, risk factors, outbreak potential, and the utility of chemoprophylaxis for this disease. Objectives: Telavancin is a novel, multivalent, lipoglycopeptide antibiotic that exerts rapid bactericidal activity against a broad range of Grampositive pathogens. Telavancin has a unique multifunctional mechanism of action that includes both inhibition of bacterial cell wall synthesis and disruption of bacterial membrane function. Previous studies with methicillin-resistant Staphylococcus aureus (MRSA) revealed that telavancin increases cell membrane permeability and causes rapid dissipation of the bacterial membrane potential. This study aimed to further characterise the effects of telavancin on the functional integrity of the MRSA cell membrane using flow cytometry. Methods: Cell membrane potential and membrane permeability were studied in telavancin-treated S. aureus ATCC 33591 (MRSA) cells. The fluorescent dyes, DiOC2(3) and propidium iodide, were used to assess membrane potential and permeability, respectively. Flow cytometry was used to analyse uptake of these dyes in telavancin-treated cells. Results: Telavancin (MIC, 0.5 mg/mL) caused a concentration-and time-dependent dissipation of the membrane potential in MRSA cells. After 60 minutes' exposure to telavancin, 67% of the bacterial population was depolarised at 8 mg/mL (approximate human plasma trough concentration), while 32 mg/mL resulted in 95% depolarisation. Membrane permeability was also concentration-and time-dependent, as seen when cells were exposed to telavancin concentrations ranging from 2 to 64 mg/mL, with 32 and 64 mg/mL yielding rapid, >90% permeabilisation of the cell population. Conclusion: Exposure of MRSA cells to telavancin initiated concentration-and time-dependent membrane depolarisation and increases in permeability, further demonstrating that telavancin interferes with bacterial cell membrane function. These effects were observed at telavancin concentrations that are achieved clinically with 10 mg/kg intravenous dosing, and further highlight the therapeutic relevance of telavancin's multifunctional mechanism of action. Objectives: Telavancin (TLV) is a novel, rapidly bactericidal lipoglycopeptide with activity against methicillin-resistant Staphylococcus aureus (MRSA). TLV possesses a unique, multivalent, multifunctional mechanism that contributes to enhanced activity and includes inhibition of cell wall peptidoglycan (PG) biosynthesis and disruption of bacterial membrane barrier function. In vitro studies suggest that TLV exhibits superior antibacterial potency relative to vancomycin (VAN); this may be due to the lipophilic decylaminoethyl side chain that targets TLV to the bacterial cell membrane. All isolates with the erm(A) gene were susceptible to oleandomycin whereas no inhibition zone was observed for the isolates with the erm(C) gene. Conclusion: S. aureus with the erm(C) gene are more readily susceptible to develop constitutive resistance to clindamycin than those with the erm(A) gene, regardless methicillin resistance. Susceptibility or inducible resistance to oleandomycin was associated with the presence of an inducible erm(A) or erm(C) gene, respectively and might constitute markers of these genes. The risk analysis for the clindamycin use in therapy of infections due to S. aureus inducibly resistant to erythromycin might include the nature of the resistance gene. Objectives: Multiple resistance of Escherichia coli to distinct classes of antimicrobials is usually attributed to simultaneous increased expression of AcrAB-TolC efflux system and loss of porin F generated by transcriptional activators marA, soxS or rob. AcrAB-TolC is also upregulated by SdiA, an E. coli protein that regulates cell division. In this study, multidrug resistant mutants selected in vitro with diazepam (DZ) and lomefloxacin (LOM) from two E. coli susceptible clinical isolates were characterised. Methods: Mutants were selected with DZ or LOM at 2, 3, 4 or 6 × the MIC. Tolerance of strains to 10% cyclohexane was measured in liquid medium. Inner and outer membrane proteins (IMP and OMP) were analysed by electrophoresis on polyacrylamide gels. Penicillin-Binding-Proteins (PBPs) were detected with Bocillin FL and Imperial Protein Stain. Active efflux was evidenced by 0.05 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The expression of the acrA, acrB, marA, soxS, rob and sdiA genes was studied by reverse transcription of total RNA and PCR of cDNA (RT-PCR), using gapA gene as internal control of expression. PCR products were separated on polyacrylamide gels and detected using silver staining. Ag100 strain (induced or noninduced with 5 mM salicylate or 0.2 mM paraquat) was the control strain. Results: Nine different clones were selected with DZ or LOM. Selection frequencies were around 10 −9 to 10 −8 . The nine mutants increased resistance to quinolones, chloramphenicol and b-lactams (2−8 fold ceftazidime, cefpirome and aztreonam MICs). The presence of CCCP increased the susceptibility of mutants to antimicrobial agents. The OMP TolC increased in mutants, coinciding with their increased cyclohexane tolerance. Mutants and parent strains showed a high expression of acrA and acrB genes. Nevertheless, only mutants showed overexpression of sdiA and soxS and OmpF decrease. Neither parent strains nor mutants showed marA or rob overexpression. Increased PBP3 expression was detected in only the mutants selected with 6 × MIC of DZ or LOM. i. DZ (a non-antimicrobial drug) and LOM selected multiple antibiotic resistant mutants that overexpressed the same transcriptional regulators, soxS and sdiA, resulting in TolC increased expression in mutants. ii. The OmpF decrease, which was generated by soxS overexpression, and increased TolC and PBP3 expression contributed to 2−8-fold increment in ceftazidime, aztreonam and cefpirome MICs in mutants. Objectives: To study the role of Qnr-like pentapeptidic proteins of several Gram-positive species in resistance to quinolones in vitro. Methods: Enterococcus faecalis V583 homologue QnrA protein sequence was used to search pentapeptidic proteins with different levels of aminoacidic identity in the NCBI database. A PCR-based strategy was used to clone and express the genes coding for Qnr-like pentapeptidic proteins of E. faecalis ATCC 29212, E. faecalis V583, E. faecium BM4147, Listeria monocytogenes 868 (clinical isolate), Clostridium perfringens 455 (clinical isolate) and Bacillus cereus ATCC 11778 in pPCR-Script vector (Stratagene) in Escherichia coli DH10B. According to CLSI guidelines, MICs of nalidixic acid, ofloxacin, norfloxacin, ciprofloxacin, levofloxacin and moxifloxacin were determined for clinical strains, reference strains and Escherichia coli DH10B harbouring recombinant plasmids containing genes coding for the pentapeptidic proteins. Results: The aminoacidic identity of Qnr-like pentapeptidic proteins of the Gram-positive strains compared to those of plasmid-mediated QnrA1, QnrB1 or QnrS1 quinolone resistance proteins ranged from 20% to 27%. When compared to the homologue of QnrA in E. faecalis V583, the aminoacidic identity ranged from 30% to 35%. All the clones obtained in E. coli DH10B expressing a pentapentidic protein from the Gram positives strains conferred reduced susceptibility to quinolones. The recombinant plasmids expressing pentapentidic proteins in E. coli DH10B increased the MICs from 4 to 12 folds for nalidixic acid, from 6 to 24 folds for ofloxacin, from 2 to 47 folds for norfloxacin, from 12 to 32 folds for ciprofloxacin, from 4 to 21 folds for levofloxacin, and from 4 to 32 folds for moxifloxacin. The pentapeptidic proteins analysed confer reduced susceptibility phenotype in E. coli. These data could provide further evidence about the possible role of pentapeptidic proteins of different Gram-positive species in their natural resistance to quinolones. These Gram positives species could constitute a reservoir of Qnr-like quinolone resistance proteins. Objectives: The Silverman model for the mode of action of daptomycin (DAP) is based on the leakage of potassium ions (K + ), membrane deenergisation and subsequent cell death without lysis. However, it is unknown whether loss of K + is sufficient to cause cell death. In this study we determined the kinetics of both cell death and lysis of S. aureus treated with DAP to establish any relationships between the two. Methods: Time-kill experiments were performed at various concentrations of DAP to determine the kinetics of cell death and samples were taken and prepared for scanning and transmission electron microscopy (EM). Culture samples were taken at intervals and the extracellular and intracellular amounts of ATP determined. The extracellular and intracellular levels of b-galactosidase (b-gal) in a S. aureus strain expressing b-gal in its cytoplasm were also measured. The time-kill experiments show DAP to be rapidly bactericidal at 32 mg/L. DAP at 4 mg/L shows a bacteriostatic effect for the first 60 min and then becomes slowly bactericidal. There is evidence for dosedependent leakage of ATP and b-gal from cells treated with DAP, but this leakage occurs at very different rates. After 30 min of treatment with DAP at 32 mg/L, 90% of the total ATP has been released but only a negligible amount of b-gal. The percentage of b-gal released increases steadily over a period of hours, reaching 40% after 6 h and 90% after 24 h. After 30 min of treatment with DAP at 4 mg/L, 50% of the total ATP has been released and this amount continues to increase steadily. The leakage of b-gal is less apparent and after 24 h the cells have released less than 30%. The EM images show some cellular disruption in the first hour after DAP treatment, with this damage becoming more apparent and widespread over the following hours. The images clearly show that the rate and extent of cellular lysis is dose-dependent. Conclusion: Our findings support the Silverman model for the mode of action of DAP and the hypothesis that it can cause rapid cell death without whole cell lysis. We have, however, shown that cell lysis does occur over time, through both EM images and the leakage of large amounts of b-gal. Our findings, while upholding the Silverman model, suggest that it is too simple to conclude that cell death is due only to loss of K + and membrane de-energisation. The rapid loss of ATP would be an equally likely cause of cell death. Objective: To study the effects of efflux pump inhibitors (CCCP and PAbN) on carbapenems in P. aeruginosa (Pa); to investigate the correlation between the resistance phenotypes and expression levels of efflux pumps of Pa and discuss the mechanism of different phenotypes in resistant Pa. By contrast, all the qnrA1-positive but blaVEB-1-negative isolates were negative for the A/C2 replicon. These results clearly indicated that the genes encoding QnrA1 and VEB-1, when identified concomitantly in a given isolate, were always located on plasmids belonging to the same IncA/C2-incompatibility group that may vary in size and digestion pattern. In addition, plasmids carrying the blaVEB-1 gene but lacking qnrA1 were also of the A/C2 type. On the opposite, plasmids that were qnrA1-positive but blaVEB-1-negative were of distinct replicon types, suggesting independent acquisition of the qnrA gene on different plasmids. Restriction pattern analysis of plasmid DNAs performed using the PstI endonuclease revealed that the blaVEB-1-positive plasmids exhibited different restriction profiles but also sharing common bands, likely corresponding to a common plasmid backbone. Hybridisation performed with an A/C2-specific probe showed an identical signal revealing that the bands carrying the replication control region of the plasmids were of identical size, as expected with plasmids from the same lineage. Conclusion: It is shown here that the IncA/C2 plasmid may be the main vehicle of the blaVEB-1 gene on which the QnrA1 determinant may be added. Understanding these patterns will help to identify potential targets for public health intervention. Objectives: Controversy still surrounds the indications for intrapartum antibiotic prophylaxis (IAP) against group B streptococci (GBS) for women in labour, with arguments for and against the two recognized care pathways; antenatal screening and risk factor assessment. Using data obtained from a large test accuracy study into rapid detection of intrapartum GBS colonisation, the aims were to determine (1) the prevalence of vaginal and rectal GBS in labouring women, comparing standard culture to enrichment culture techniques; (2) what proportion of women present with different risk factors; (3) how both culture positivity and risk factors correlate with the frequency of IAP administration; (4) baby colonisation rates with regard to maternal colonisation site and whether or not IAP was given. Methods: Vaginal and rectal swabs collected from 1185 women in labour for direct culture on streptococcal selective agar (CNA) and enrichment culture (LIM broth). 1125 ear swabs from neonates were collected within 1 hour of birth for enrichment culture. Data on risk factors and antibiotic therapy collected prospectively. 1. Enriched culture gave an overall (vaginal and/or rectal) colonisation rate of 21% compared to 10% with standard culture. 2. 32% of women studied had at least one risk factor, most commonly prolonged rupture of membranes (49%). 3. 50% of women with any risk factor did not receive IAP (Table 1) . 4. Only 21% of GBS carriers received IAP, whilst 67% of women who received IAP were GBS-negative (Table 2 ). 5. The baby colonisation rate was 8% (90/1125). Positive vaginal culture identified 78% of the positive babies; positive rectal culture 89% and both rectal and vaginal positive culture combined 93%. Objectives: Staphylococcus aureus remains the prevalent bacterium causing acute osteomyelitis in children. The involvement of methicillinresistant S. aureus (MRSA) as a cause of acute childhood osteomyelitis was investigated. Methods: Included in the study were all children treated for acute osteomyelitis due to S. aureus in Western Greece from January 2005 until August 2006. Bone scan, MRI and X-rays were performed in order to ascertain the diagnosis and to assess the severity of the disease. S. aureus was isolated from blood and/or bone tissue when surgical drainage was necessary. The Staphylococcal Cassette Chromosome (SCCmec) type and Panton-Valentine leukocidin (PVL) genes (lukS-PV and lukF-PV) were detected by PCRs. MRSA clones were defined by PFGE of SmaI DNA digests. Signs, symptoms and laboratory findings were prospectively registered and statistically evaluated (Mann-Whitney-U test) in all patients until cessation of activity of the disease. Results: Nineteen patients, 12 males and 7 females, median age 11 years, range 2−13 years, were diagnosed as having acute staphylococcal osteomyelitis. Three children had a suspected site of bacterial entrance (injury, burn, insect bite). S. aureus was isolated from the blood of nine patients, from the tissue of nine more patients and from both clinical specimens in one patient. In five children community-acquired MRSA (CA-MRSA) carrying SCCmec type IV and PVL was identified. Among the 14 MSSA isolated from the remaining patients, two were PVLpositive. The maximal ESR and CRP values as well as the time necessary for normalisation of ESR and CRP differed statistically significantly in patients with PVL positive stains (MRSA and MSSA) compared to PVLnegative MSSA (p < 0.05). Surgical drainage was more often necessary among patients with PVL-positive stains. Conclusions: PVL-positive CA-MRSA are recovered not only from patients with superficial but also with invasive musculoskeletal infections. The production of PVL seems to be the main factor that contributes to the course of acute osteomyelitis. Here, we describe the first report of family transmission of C-MRSA PVL-, tsst1+, in a patient (pt) with osteomyelitis. Case report: In October 2006, a 29-year-old alcoholic man with no medical history was admitted in our department of infectious diseases for acromioclavicular osteoarthritis. He was married and had two children (6 and 8 year-old). In 2005 he had a traumatic dislocation of the right acromioclavicular joint and was operated one year later for tendons refection using homogenic graft. Two weeks after surgery, a purulent discharge from the post-operative wound was noted, and MRSA was cultured from swabs. The strain was resistant to oxacillin, kanamycin, tobramycin, and susceptible to ofloxacin, trimethoprimsulfamethoxazole, pristinamycin, clindamycin, vancomycin, and was intermediate to fusidic acid. He was given oral pristinamycin (1 g tid for 8 weeks). One week after the end of treatment, in October 2006, he was admitted for a right acromioclavicular osteoarthritis which required surgical debridement and intravenous vancomycin treatment. A C-MRSA strain PVL− tsst1+ grew from intra-operative samples. The family carriage screening (including the pt) showed that the pt's children were colonised with the same MRSA strain according to the antibiotic susceptibility profile (PVL and tsst 1 detection still in process). The members of the pt's family had no healthcare associated risk factors for C-MRSA. Vancomycin was switched to oral trimethoprimsulfamethoxazole and rifampicin combination for 3 months. Discussion: In 2002, a new type of MRSA producing tsst1 has been described in France and Switzerland. To the best of our knowledge, family transmission of C-MRSA PVL-, tsst1+ strain has never been reported. In the present case report, a cross contamination between the pt and his family is highly suspected. Conclusion: the present case shows that C-MRSA PVL-, tsst 1 might be associated with family transmission. The prevalence of pathogenic Y. enterocolitica in Swiss pigs was high using PCR, however, the isolation rate was clearly lower than in German pigs. Most of the Swiss pigs were infected with bioserotype 4/O:3, which is the most common type found among human Y. enterolitica gastroenteritis in German and Switzerland and is the only type isolated from German slaughter pigs. Pig was also shown to be a reservoir for bioserotype 2/O:9 in Switzerland. The genetic diversity of ail-positive Y. enterocolitica in the pig tonsils collected from one Swiss slaughterhouse was limited. T. van were significantly associated with the CDAD incidence. Conclusion: Hospital-wide use of most antibiotics known to be a risk factor for CDAD at patient level, such as fluoroquinolones, are not associated at institution level with higher incidences of type 027associated CDAD. Possibly, investigation at ward-level might correlate better. Wearing an apron and giving special instructions to visitors in case of diarrhoea appeared to be protective. Objectives: Acanthamoebae have been reported to represent the environmental host of several human pathogens. Vibrio cholerae requires 10 8 to 10 9 cells to cause cholera, and accordingly it needs an environmental host to enhance its growth. Our studies have shown that extracellular V. cholerae O1 and O139 have grown and survived in Acanthamoeba castellanii. The aim of this study is to examine the ability of A. castellanii to protect V. cholerae and to enhance its growth to be able to infect humans. Methods: V. cholerae producing green fluorescent protein was cocultivated with A. castellanii. An antibiotic assay used to examine ability of amoeba to protect intracellular bacteria and to differentiate between extracellular and intracellular bacteria. A. castellanii cultivated with V. cholerae for one week. After gentamicin killing and washing of extracellular V. cholerae, the number of intracellular bacteria was found to be 2×10 5 cells/mL. The amoebae harbouring V. cholerae were re-cultivated and growth of the microorganisms examined by viable counts during 2 weeks. Results: Gentamicin killed only the extracellular bacteria, while ciprofloxacin killed both, thus, the amoeba protected the intracellular V. cholerae from gentamicin. The antibiotic assay differentiated between extracellular bacteria that killed by gentamicin and intracellular bacteria that killed only by ciprofloxacin, which could diffuse into amoeba cells. Re-cultivation of A. castellanii harbouring intracellular V. cholerae resulted in enhanced growth of V. cholerae to 2×10 11 CFU/mL and the bacteria survived for more than two weeks. Thus, amoeba acted as a biological incubator in which intracellular growth of the bacteria occurred to increase their number a million fold and maintained their overall viability. Existence of V. cholerae extracellularly as well as intracellularly in culture medium confirmed its facultative intracellular behaviour, which enhanced its growth and survival. The utilised antibiotic assay differentiates between extracellular and intracellular bacteria. Free-living amoeba is a possible biological factor, which protects and enhances growth of V. cholerae to exceed its infections dose and to supports the idea of amoeba as a second host to the bacterium in nature besides man. Common oral conditions, such as gingivitis and chronic periodontitis, are found world-wide and are among the most widely prevalent microbial diseases of mankind. A primary trigger for these conditions is the complex microbiota found as dental plaque, a complex microbial biofilm. Despite 3000 years of history demonstrating the influence of oral status on general health, recent decades have seen an accelerated effort for the prevention and management of these conditions through groundbreaking advances. One outcome of these advances is the realisation that periodontal diseases are associated with systemic conditions such as coronary heart disease and stroke; higher risk for preterm, low birth weight babies and pose threats to those with chronic disease: diabetes, respiratory diseases and osteoporosis. This presentation will highlight recent research associating periodontal disease with systemic diseases. The relationship between oral infections and systemic disease represents a paradigm shift in current research. A portion of this lecture will be devoted to examining the role of chronic infections and their association with cardiovascular diseases (CVD). These infections include Helicobacter pylori, Chlamydophila pneumoniae, Cytomegalovirus (CMV), and, more recently, periodontopathic bacteria including Porphyromonas gingivalis. Although a number of potential mechanisms have been postulated, the mechanism by which these infections are associated with CVD is still unclear. The various hypotheses concerning this relationship include common susceptibility, inflammation via increased circulating cytokines and inflammatory mediators, direct infection of the blood vessels and finally the possibility of cross-reactivity or molecular mimicry between bacterial and self antigens. The evidence for each of these mechanisms will be presented and the clinical implications for medical and dental practitioners discussed. This introductory lecture will set the stage for subsequent speakers highlighting the medico-dental interface as a required shift in health practices for the future. While both medicine and dentistry are healthcare professions devoted to patient care, the interaction between the two disciplines remains limited. It is rare for dental professionals to be integral members of medical teams or hospital/healthcare settings. While referrals between the two professions occur, improved communications between professionals are required to optimise treatment options. This is increasingly required to capitalise on recent advances in health sciences for the management of chronic diseases that have reduced morbidity. Improvements in longevity have resulted in unique populations with special oral care needs and include those with cancer of the head and neck, immunocompromised, HIV/AIDS, geriatrics, residents of long-term care facilities, patients with lifelong conditions, after organ transplantation and those on prescription medications like those for blood-pressure control or anticoagulation therapy. This session will highlight the unique oral care needs for our changing patient population, and how oral health can be improved when dentists and physicians work together. The role of prescription medications to treat medical conditions and their adverse reactions in the oral cavity will form a separate another area for discussion. Many medications, for instance, are xerostomic. Patients with xerostomia are at increased risk for tooth decay and periodontal disease. The need for increased collaboration between dentists and physicians to prevent or reduce complications by maintaining better oral and medical health will form a core theme for this session. It cannot be any longer ignored that oral health and medical health professionals must unite for optimal management of patient health. A primary step to improve oral health is the routine control of dental plaque, a natural biofilm. Oral hygiene formulations with significant effects on the dental biofilm augment ineffective practices of routine prophylaxis. Chlorhexidine and triclosan formulated in oral hygiene formulations have an extensive history of clinically proven efficacy and safety. An overview of clinically effective oral hygiene formulations and their role as therapeutic strategies will commence this presentation. With triclosan serving as a case-study, the talk will bring together recent advances in the assessment of oral biofilms, analyses of clinical strains including susceptibility to demonstrate the microbiological safety and efficacy of oral hygiene formulations. The past three decades have witnessed no general reduction in the effectiveness of triclosan for its target bacteria including periodontal bacteria. Whilst triclosan is not noted for its activity against enteric bacteria and pseudomonads, laboratory studies demonstrated that chronic sub-lethal exposure of Escherichia coli can select mutant clones with significantly reduced susceptibility. These mutants are either mutated in an enzyme (FabI) associated with fatty-acid biosynthesis or overexpress multi-drug efflux pumps. Initial concern that mutations in FabI might be capable of horizontal transfer between environmental bacteria and noscomial pathogens or that parallel processes might occur directly in Gram-positive pathogens have subsided. Similarly, concern about possible selection of resistance towards third-party agents (antibiotics) that might share the FabI gene target has proven unfounded. Selection of efflux-on mutants, whilst demonstrated in the laboratory is not supported by analyses of isolates from the hospital or domestic environment where agents such as triclosan have been widely deployed. Evidence suggests that efflux-on mutants are unable to compete in natural microbial communities. Whilst there is insufficient evidence to suggest that the uncontrolled use of triclosan in domestic products is totally free of risk, its deployment for oral hygiene, especially where such use might limit the number of refractory infections and their possible complications contributes ultimately to a reduction in antibiotic use is encouraged. Collectively, there is no evidence that the long-term application of triclosan formulations to the oral cavity or skin selects for triclosanresistant bacterial populations. As the increasing elderly population retain more of their natural teeth into later life periodontal disease will pose an increasing problem both for the dentition and potentially general health. The maintenance of an effective level of oral hygiene is the cornerstone of all attempts to prevent and control periodontal diseases and yet the widespread prevalence of these diseases indicates the inability of most people to achieve a level of plaque control commensurate with periodontal health. The inclusion of antibacterial agents, such as chlorhexidine and triclosan/copolymer, into oral care products has provided the consumer with the means to improve their oral health. Randomised controlled clinical trials have demonstrated that a dentifrice containing triclosan/copolymer significantly improves gingival health, prevents the onset of periodontitis and furthermore reduces the progression of the disease. These benefits were initially attributed to the antibacterial action of triclosan but evidence now suggests that it is also an anti-inflammatory agent. The delivery of such benefits is considerable with implications for the maintenance of oral and general health and the costs of dental care. The emergence of multidrug resistance (MDR) in pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae that produce extended-spectrum beta-lactamases (ESBLs) is alarming. Increasing MDR in patients treated for nosocomial infections is causing an increase in costs, morbidity, and mortality. Traditionally, a narrow-spectrum drug was used first, and the most potent drugs were reserved for subsequent use. Unfortunately, the traditional approach increases the chance of treatment failure and the emergence of multidrugresistant strains. In fact, the inappropriate choice of antibiotics is the most important independent risk factor for death. ESBL producers are resistant to most penicillins and cephalosporins and are often cross-resistant to aminoglycosides and fluoroquinolones as well. Carbapenems have been shown to provide superior outcomes compared with other agents for infections caused by ESBL producers. Consequently, the first-line use of carbapenems is optimal for serious infections in which ESBL pathogens are suspected. The major non-fermentative Gram-negative pathogen of concern is P. aeruginosa. P. aeruginosa is a common cause of nosocomial infections and the leading cause of ventilator-associated pneumonia. Initial therapy should include an antipseudomonal b-lactam, such as piperacillintazobactam, an antipseudomonal carbapenem, or an antipseudomonal cephalosporin, plus an aminoglycoside or a fluoroquinolone. A. baumannii generally is susceptible to carbapenems, aminoglycosides, sulbactam, colistin, or tigecycline. Carbapenems are one of the few classes that remain an excellent choice for both nonfermenters. Doripenem is a new broad-spectrum carbapenem with activity superior to that of other carbapenems against P. aeruginosa and similar to other carbapenems against A. baumannii. As antibiotic resistance increases, empiric therapy for severe infections should be based on local pathogen etiology and resistance patterns. A new approach is to start with a high-dose, broad-spectrum antibiotic and then tailor the individual therapy based on microbiological results. Carbapenems are likely to remain an important option for serious Gramnegative infections for the foreseeable future. Since the number of new intravenous agents with activity against Gram-negative organisms is extremely limited, the development of a new carbapenem may help to reduce the mortality, morbidity, and cost of serious nosocomial infections. The widely marketed carbapenems long comprised imipenem (launched 1985) and meropenem (1996) , but the family is now growing, with ertapenem (2002), doripenem (anticipated 2007/2008), and CS-023 (in development). Use is also growing, driven by the spread of extendedspectrum b-lactamases (ESBLs). The carbapenems have much in common with each other, with broader activity and better stability to ESBLs and AmpC enzymes than other b-lactams, but with lability to metallo-and KPC b-lactamases and to the OXA carbapenemases of Acinetobacter clones. Three carbapenem groups nevertheless can be defined. Group 1 agents lack activity versus non-fermenters; they are represented by ertapenem and perhaps in the future also by oral analogues. Ertapenem's utility lies in the treatment of community-acquired infections, where ESBL producers are likely or proven; it has convenient, oncedaily, dosing but is the most vulnerable carbapenem to combinations of ESBL or AmpC plus impermeability. Group 2 comprises imipenem, meropenem, and doripenem, plus analogues available only in East Asia; they are active versus most non-fermenters and are appropriate in nosocomial settings where these pathogens are likely. Doripenem and meropenem are very similar, but with doripenem circa two-fold more active versus most species; imipenem is slightly less active against Gram-negative bacteria except Acinetobacter but is the sole carbapenem active versus Enterococcus faecalis. Doripenem and meropenem need two mutations -loss of OprD and up-regulated efflux -for resistance in Pseudomonas aeruginosa. Imipenem resistance arises by loss of OprD alone and can be selected in therapy; nevertheless, imipenem is unique in evading pseudomonal efflux. Meropenem and doripenem are relatively stable to renal dehydropeptidase, but imipenem needs protection with cilastatin; doripenem and meropenem are chemically stable, allowing prolonged infusion, whereas imipenem is not; doripenem and meropenem have little seizure potential (meropenem is licensed for meningitis), but CNS side-effects limit imipenem dosage. Group 3, comprising analogues active versus MRSA, is represented by the developmental agent CS-023; its minimum inhibitory concentrations for MRSA are more widely distributed than those of anti-MRSA cephalosporins, but it has the wider spectrum typical of a carbapenem. As carbapenem usage expands, it is critical to understand these differences, so that the most appropriate analogues can be selected. Increasing antibiotic resistance and a lack of new drug classes in development are driving the need to optimise use of currently available drugs. The goal of antimicrobial chemotherapy is to optimise the combined pharmacokinetic (PK) and pharmacodynamic (PD) profile of a drug so that the greatest percentage of patients achieves the PD target associated with a favourable outcome, while minimising the development of resistant organisms. Integration of population PK, a PD target, and microbiologic surveillance data by Monte Carlo simulations can generate an empirical dosing strategy that maximises the likelihood that an antibiotic regimen achieves the desired PD end point (exposure target). For b-lactams, the exposure target is the percentage of time that free drug levels are above the minimum inhibitory concentration (T > MIC). For carbapenems, the T > MIC requirement is lower than for other b-lactams. By extending the infusion period to achieve the necessary T > MIC, a lower dose can achieve the same efficacy as a higher dose, while lowering cost and potential toxicity. For example, a recent publication showed that a 1-g 0.5-h infusion of meropenem has a 77.1% rate of target attainment against Pseudomonas aeruginosa isolated from hospitals in Hungary, whereas a 3-h infusion of 0.5 g meropenem would have an 83.8% rate of target attainment. In a retrospective study that compared 0.5 h piperacillin/tazobactam infusions (3.375 g, q6h) with 4-h infusions (3.375 g, q8h) in patients infected with P. aeruginosa, the 4-h infusions reduced 14-day mortality from 31.6% to 12.2% (P = 0.02) and median length of stay from 38 to 21 days in patients with APACHE II scores 17 (P = 0.02). Doripenem is more stable upon reconstitution than other carbapenems, potentially making it more convenient and easier to use as an extended infusion. Furthermore, the study design for the doripenem ventilatorassociated pneumonia phase 3 trial includes an extended infusion regimen (0.5 g over 4 h). In conclusion, the same dose of an antibiotic has the capacity to increase efficacy for more resistant infections if infused over an extended period. Extended infusion of a lower dose of antibiotic may produce efficacy equivalent to shorter, higher-dose infusions while reducing both toxicity and the emergence of resistance. Extended infusion of a carbapenem, such as doripenem, that has neither seizure potential nor high resistance selection is a particularly promising strategy for serious infections. Doripenem is an investigational carbapenem that has completed phase 3 multi-national trials for complicated urinary tract infections (cUTIs), complicated intra-abdominal infections (cIAIs), hospitalacquired pneumonia (HAP), and ventilator-associated pneumonia (VAP). Doripenem's advantages over other carbapenems include: enhanced activity against Pseudomonas aeruginosa (with a low propensity for resistance), the lowest potential for seizures in the carbapenem class, and the greatest stability after reconstitution. Two randomised, double-blind phase 3 cIAI trials compared intravenous (IV) doripenem (500 mg q8h) with IV meropenem (500 mg q8h), with an option to switch to amoxicillin/clavulanate for both treatment arms after 9 doses of IV study drug therapy. Results from the cIAI trials showed that doripenem (500 mg q8h) is welltolerated and non-inferior to meropenem (1 g q8h). Doripenem was microbiologically effective against major causative organisms of cIAIs, including Enterobacteriaceae and Bacteroides spp. Adverse events (AEs) occurring in 3% of subjects in either treatment arm were nausea and vomiting. No seizures were reported. A randomised, double-blind phase 3 cUTI trial compared IV doripenem (500 mg q8h) with IV levofloxacin (250 mg q24h), with an option to switch to oral levofloxacin for both treatment arms. Results from this trial showed that doripenem is well-tolerated and non-inferior to levofloxacin. Doripenem was effective against major causative organisms of cUTIs, including Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae. Doripenem had greater efficacy in patients who remained on intravenous therapy and in patients with levofloxacin-resistant E. coli. Fewer pathogens had a 4-fold increase in minimum inhibitory concentration during therapy with doripenem than with levofloxacin. AEs occurring in 3% of subjects in either treatment arm were headache, diarrhoea, and phlebitis. No serious study drug-related AEs were reported. Urine from patients was obtained and evaluated to assess urine bactericidal titers. Serum and urine drug concentration data collected in Phase 1, 2, and 3 studies will also be discussed. Doripenem also completed a phase 3 trial versus piperacillin/tazobactam for HAP (including early-onset VAP), and a phase 3 trial versus imipenem for VAP (early and late onset). Doripenem's superior stability allowed the VAP trial to utilise an extended infusion (4-h) regimen for doripenem that may maximise for these difficult infections. Tuberculosis is a major health threat with 9 million new cases and 2 million deaths annually. The available vaccine, BCG, protects newborns from miliary tuberculosis, but fails to prevent the most prevalent form of disease, pulmonary tuberculosis in adults. One third of the world population is infected with the etiologic agent, Mycobacterium tuberculosis. Hence: (i) M. tuberculosis can be controlled (though not eradicated) by the immune response induced by natural infection; (ii) BCG fails to induce a protective immune response at least in those individuals who are susceptible to tuberculosis. Current vaccination strategies have to consider both pre-exposure and post-exposure vaccines. Acquired immunity against tuberculosis is a T cell-dependent phenomenon. The T cell system comprises distinct populations. CD4 T cells are undoubtedly of central importance for acquired resistance against tuberculosis. Antigens of M. tuberculosis also stimulate CD8 T cells, probably through crosspriming. In addition, unconventional T cells also seem to participate in immunity against tuberculosis. What can we learn for rational vaccine design? Novel vaccination strategies either focus on subunit vaccines or viable attenuated vaccines. Subunit vaccination strategies are based on the assumption that one or few antigens suffice for an efficient immune response. Hence, the identification of protective antigens represents an essential prerequisite for the success of this type of vaccines. Subunit vaccines come in three forms: Protein/adjuvant formulations, recombinant heterologous carriers or naked DNA constructs. Viable attenuated vaccines are based on the assumption that multiple antigens are required for efficacious protection. Two major strategies are being pursued: Knockout mutants of M. tuberculosis and improved recombinant (r)-BCG vaccines. Improved r-BCG should first be endowed with a higher immunogenicity and second may need a broader antigenic repertoire. Rational vaccination strategies performed in the laboratory of the author focus on improved BCG. A r-BCG strain which expresses listeriolysin induces better protection than wild-type BCG. It is tempting to speculate that a prime/boost scheme comprising prime with improved r-BCG and boost with the most efficacious subunit vaccine candidate will provide optimal protection. Identification of a biosignature that allows distinction between infection/protection and infection/disease in tuberculosis could speed up efficacy testing of vaccines in clinical trials. The Grand Challenge 6 of the Bill & Melinda Gates Foundation aims at identifying such biosignatures. Taking advantage of our increasing knowledge about the immune response to M. tuberculosis will facilitate rational design of novel vaccines against one of the most frightening threats in the world, tuberculosis. The following years will witness the transformation of vaccine candidates from preclinical to clinical trials requiring intensive coordination in order to identify the most appropriate candidate(s). One of the most important characteristics that separate antimicrobials from other drugs used in clinical medicine is that their target (the receptor of the drug) is part of the microbe, and not of men. The activity of the antimicrobial can therefore be studied both in vitro and in model systems, such as animal models, and a relationship between exposure and effect established. The exposure is primarily determined by the pharmacokinetic characteristics of the drug, while the effect is determined by the concentration-effect relationship of the antimicrobial and the micro-organism. However, in most clinical settings, it is the potency of the antimicrobial that is measured in terms of the MIC and not the concentration-effect relationship itself. Therefore, a relationship needs to be ascertained between the MIC, exposure of the drug and effect. Over the last decade, these pharmacokinetic/pharmacodynamic (PK/PD) relationships of antimicrobials have been established for most classes of drugs. The two major indices describing these relationships are the time the concentration of the antimicrobial remains above the MIC (T >MIC) as a fraction of the dosing interval and the Area under the Time Concentration curve over 24 hours divided by the MIC (AUC/MIC ratio). The Peak/MIC ratio correlates with effect for a number of drugs, but it in many, if not all, cases the effect can not be clearly distinguished from the AUC/MIC effect. Importantly, for virtually all antimicrobials, it is the exposure of the free non-protein bound fraction of the drug that best correlates with efficacy, and is of particular significance when drugs are compared with each other within a class and/or across species, including men. From these PK/PD relationships, a number of issues have come forward. Importantly, it has been shown in a number of clinical trials -now exceeding 10 -including several classes of drugs, that the PK/PD relationships that do exist in animals are, as expected, similar to those in men. Moreover, it has been shown that the quantitative relationships are remarkably similar. In other words, exposures in infected animals result in the same effect as exposures in infected humans under similar conditions. A second, perhaps even more important topic is that these relationships -once well established -can be used to optimise therapy in patients by optimising exposure of the drug. In a relatively simple approach, these relationships are used in daily clinical practice in the use of clinical breakpoints. These MIC values are used to discriminate between high and low probabilities of successful outcome of treatment (S and R, respectively) based on the PK/PD relationships of the antimicrobial together with the clinical setting. For instance, lower exposures to obtain the same effect are needed in the immunocompetent host because of the contribution of the immune system. More sophisticated, these relationships are used to adjust dosing regimens once the MICs of the infectious micro-organisms are known, and thus provide an integrated approach to the individual patient. Finally, PK/PD relationships are used in the development phase of the drug in determining the optimal dose for the indication sought. Alternatively, once dosing regimens are established, they can be used to form an opinion on the possible indications for various clinical treatment modalities. Dissemination of microbial drug resistance observed in the antibiotic era is clearly related to the selective pressure generated by the use of antibiotics in clinical, veterinary and agricultural practices. This notion is universally acknowledged and supported by the several studies that have correlated the emergence and dissemination of resistance with the use of new antibiotics, and by documentation of the absence of acquired resistance in clinical isolates from the pre-antibiotic era. However, the presence of antibiotic-resistant bacteria has recently been reported also in humans and in wild animals living in remote areas where antibiotic exposure has been absent or minimal [1−3] . This unexpected finding raises a question on the mechanisms responsible for spreading and maintenance of antibiotic resistance in similar settings, and could have important implications for the design of strategies addressed at controlling bacterial resistance based on antibiotic restriction policies. Results of studies on antibiotic resistance in settings of minimal antibiotic exposure (including investigations recently carried out in very remote human communities living in the Bolivian Chaco and in the Alto Amazonas jungle of South America, and among wild reptiles (land iguanas) from a remote and protected island of the Galápagos archipelago with no documented sources of antibiotic exposure and minimum human contacts) will be critically reviewed in this presentation, and the mechanisms potentially involved in the dissemination of resistant strains and resistance genes unrelated to antimicrobial consumption will be discussed. Results from the characterisation of resistance determinants carried by bacterial isolates from these settings pointed to their likely origin from antibiotic-exposed areas rather than to a local and independent resistance selection process. However, the mechanisms responsible for this flow of resistance genes and for their maintenance and spread in absence of sustained antibiotic use remain elusive. Objective: The objective of this study was to identify the 43-kDa ChoP containing protein and characterise its function in the virulence of P. aeruginosa. Methods: To identify the ChoP containing protein, bacterial cell fractions were separated by FPLC. Fractions were analysed by Western blot using specific monoclonal anti-ChoP antibodies and those that contained the ChoP epitope were further purified by SDS-PAGE. A 43-kDa protein containing the ChoP epitope was cut out of the gel, trypsinised, and subjected to capillary LC-MS and MS/MS. ChoP epitope expression of whole cell extracts of 92 genetic unrelated P. aeruginosa isolates (46 from chronic infections and 46 from acute infections) was analysed by Western blot analysis using specific monoclonal anti-ChoP antibody. ChoP epitope expression was also analysed by flow cytometry and immunofluorescent microscopy on intact cells of P. aeruginosa. To determine whether the ChoP epitope was involved in the interaction of the P. aeruginosa isolates with the respiratory epithelial cells, standard invasion assays were performed using 16HBE14-bronchoepithelial cells, either treated or untreated with a PAFr antagonist. The 43-kDa ChoP containing protein was shown to be the elongation factor Tu (EF-Tu). The expression of the ChoP epitope at 37ºC was significantly more frequent in P. aeruginosa strains isolated from chronic infections (70%) than from acute infections (28%). The ChoP epitope was shown to be expressed on the outer surface of the bacterial cell and bacterial invasion was significantly inhibited by treatment with the PAFr antagonist compared to controls. We have demonstrated that in P. aeruginosa, the ChoP epitope is associated with EF-Tu. A high percentage of the isolates from chronic infections, in comparison with those from acute infections, express this epitope at 37ºC. This epitope is present on the cell surface and mediates the invasion of the airway epithelial cells via PAFr. Objectives: We aim to develop novel therapies for the treatment of Gram-negative bacterial infections, by isolating monoclonal antibodies that are capable of binding to signal molecules involved in bacterial quorum sensing. Signalling blockade was hypothesised to prevent the production of virulence factors and so allow the host immune system to eliminate the infection. Methods: As Gram-negative bacterial signaling molecules are typically of low molecular weight (less than 500 Da) they have been considered intractable to standard antibody discovery methodologies. Therefore we have used a novel suite of antibody discovery processes termed Haptomics ® to isolate a panel of human antibodies capable of binding to signal molecules produced by Gram-negative bacteria. Results: We have demonstrated the abilities of the antibodies to bind bacterial signaling molecules using a range of in vitro experiments, and we have also evaluated the antibodies using in vivo models of Pseudomonas aeruginosa infection. Results will be presented demonstrating the efficacy of our fully human recombinant antibody fragments and whole IgG molecules in the treatment of Gram-negative bacterial infections. Conclusion: Our approach offers the exciting prospect of using powerful immunological reagents to combat bacterial infection. By targeting the signal molecules, rather than the bacteria themselves, we believe that resistance is considerably less likely to develop to these antibody-based therapeutics. We are currently broadening our approach to develop antibodies capable of disrupting quorum sensing in Gram-positive organisms. Objectives: Pseudomonas aeruginosa is a free-living, extracellular and a common environmental bacterium. It is an opportunistic and nosocomial pathogen causing serious health problems. To compete its predators such as macrophages and environmental phagocytes it utilises different survival strategies such as biofilm and formation of micro-colonies. It also develops resistance mechanisms and produces virulence factors such as lipopolysaccharide, alginate as well as extracellular, quorum sensing regulated enzymes and toxins and type III secretion system (TTSS). The aim of this study is to examine the interaction between P. aeruginosa PA103 and Acanthamoeba castellanii. Methods: Bacteria and amoebae were co-culture and the interaction was examined between P. aeruginosa PA103 and A. castellanii by cocultivation, viable count, eosin staining, electron microscopy, apoptosis assay and statistical analysis. The results showed that P. aeruginosa PA103 induced necrosis and apoptosis to kill A. castellanii by the effects of TTSS proteins ExoU, ExoS, ExoT, and ExoY. In comparison, growth of Acanthamoeba cultured alone and co-cultured with TTSS mutant strain was not affected. The results confirm the nature of P. aeruginosa as a strict extracellular bacterium that needs TTSS to survive in the environment since this system is able to kill its eukaryotic predators such as Acanthamoebae. In all instances but one, the three methods agreed in the susceptibility testing for linezolid. The only strain carrying 23S rRNA mutation showed MIC values for linezolid 4 ug/mL when tested with the Phoenix and with the E-test, while with the VITEK 2 displayed a MIC > 8 ug/mL. Conclusions: Glycopeptide resistance of Enterococci poses serious therapeutic problems and an accurate microbiological diagnosis is fundamental for the treatment. Our results evidenced a high rate of discrepancies in the detection of glycopeptide resistance by three widely diffused susceptibility testing methods. Furthermore, some limits were observed in the detection of linezolid resistance that prospectively could reduce the therapeutic options. Increasing b-lactam resistance in E. coli has become a worrying threat worldwide. In that species, most of the mechanisms involved in the b-lactam resistance are linked to the production of b-lactamases, including clavulanic-acid inhibited expanded-spectrum b-lactamases (ESBLs) and carbapenemases which are the most powerful enzymes able to degradate most b-lactams. Besides the most common ESBLs circulating in Enterobacteriaceae (TEM-, SHV, and CTX-M-types), there are other emerging enzymes which are mostly plasmid-encoded and are distributed in a large variety of species. The ESBL VEB-1 is known to be largerly distributed in South-East Asia, PER-1 in Turkey, Italy, Korea, and also South America, and GES/IBC-type enzymes in Greece and Japan mostly. All these ESBLs are known to efficiently hydrolyze expanded-spectrum b-lactams, and some variants of the GES family are also hydrolysing imipenem at a low level. Other enzymes of the KPC family have been also reported in E. coli. KPC-2 has been shown to be prevalent in E. coli in Israel and KPC-3 has been reportyed sporadically in the USA. These latter enzymes are considered as ESBLs since they are inhibited by clavulanic acid and their hydrolytic efficiencies toward imipenem are very high, thus giving rise to high level resistance to carbapenems in those E. coli isolates. In addition to these ESBL determinants, metallo-b-lactamases (MBL) have been identified in E. coli. These enzymes which are not inhibited by clavulanic acid and hydrolyse carbapenems very efficiently are of the IMP and VIM groups. In E. coli, IMP-1 has been identified in Japan and VIM-2 in Greece, but the most worrying observation is that related to the spread of VIM-1 in Greece. Indeed, this determinant has been identified in clinical isolates also harbouring ESBL encoding genes, thus leading to panresistance in those strains. Noteworthy, most of the ESBL and MBL encoding genes encountered in E. coli are vehiculed by plasmids and located into class 1 integron structures, thus facilitating the occurrence of multidrug resistance via the co-localisation of other antibiotic resistance genes. A particular attention will be needed in the next future to trace the isolates harbouring these worrying determinants, and also to trace the corresponding plasmids which are powerful tools for such dissemination. Severe sepsis is a serious syndrome with organ failure that may affect a large proportion of the patients admitted to the intensive care unit and whose prediction is often difficult [1] . The Surviving Sepsis Campaign (SSC) is a global programme to reduce mortality rates in severe sepsis, endorsed by 11 International Medical Societies. This Campaign produced the guidelines for management of severe sepsis and septic shock [2] . These recommendations were reviewed during the annual meeting of the Society of Critical Care Medicine held in January 2006. The Campaign has taken an action to create National and International networks to collect data and monitor interventions. In Italy the network was settled in September 2006. But, can we monitor therapy for severe sepsis if concepts, tools, ideas and attitudes differ largely between centres? With the aim of answering this crucial question, we conducted a survey on the perception of Severe Sepsis, its diagnosis and monitoring through the national network for the Italian Chapter of the SSC. Twenty-three participating centres (10 community and 13 teaching hospitals) responded to our survey, for a total number of 261 ICU/critical care beds. When the participants were asked to describe the methodology currently used for implementation of data in their institution, 70% of the respondents adopted a continuous data collection methodology and the other 30% a "before and after event" design. On the question whether an educational tool was used to sensitise people to the campaign, 13% of the respondents declared that no specific tool was used and the other 87% (20 centres) stated that they used different educative means (mostly chart documentations and department conferences). When asked when data collection for the diagnosis of sepsis usually takes place, thirteen respondents said that their collection occurred within 24 hours from the diagnosis, only 2 contemporary, and 8 retrospectively. On the question how data were collected 9 institutions out of 23 (39%) answered that the SSC paper tool or database were used, 11 (47%) a combination of the two, and 3 (23%) other systems. In 17 of the 23 centres the responsibility of data collection is assigned to the attending physician; in the remaining 5 cases residents, nurses or students collect the data, but same centres allow the simultaneous collaboration of more figures. Partnership between ICUs and other departments to approach Sepsis in the spirit of the SSC is unfortunately scarce: only 4 (17%) of the respondents have a form of collaboration with other units. The effect of this mentality is that only 6 (26%) of the interviewed hospitals currently apply a screening for severe sepsis in the ward. One main concept that illustrates the spirit of the SSC is the idea of an early intervention to apply the different recommendations. Again, in checking how the time of presentation of sepsis is conceived in the emergency department, in the ward and in the ICU we obtained answers that were quite diverse. Of the 23 participating institutions, 16 responded. Only 50% of physicians in the Emergency Department identified the "presentation time" of severe sepsis with the moment of triage or hospital admission. The remaining 50% had a sort of "a posteriori" diagnosis, losing the possibility of an early approach. Things are much worse looking at the ward, where in 80% of cases the moment of severe sepsis presentation corresponded to the identification of symptoms from the "a posteriori" reviews of the medical notes, the arrival of a critical care consultant or ICU admission. Surprisingly, in the ICU still 30% of the cases is usually diagnosed through the review of the medical reports. The EUCAST Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has recently published a standard for determining the susceptibility of fermentative yeasts to antifungals. Besides, another standard related with the susceptibility of filamentous fungi to antifungals will be published very soon. The EUCAST General Committee has recently published guidelines for harmonisation of breakpoints for antimicrobials and therefore the Antifungal Susceptibility Testing Subcommittee of EUCAST has adopted it. In order, to prove that breakpoints for antifungals can be obtained, EUCAST-AFST decided to make a proof of concept with fluconazole. All the requirements raised for EUCAST committee to obtain break points for antimicrobials have been addressed and fluconazole break points have been attained. The data obtained through the revision system to obtain break points for fluconazole will be discussed. Also the discrepancies of EUCAST and CLSI break points will be examined and analysed. In the near future, it is intention of EUCAST AFST develop break points to all licensed antifungals. Objectives: In recent years K. pneumoniae has been shown to have increasing resistance, due to production of extended-spectrum b-lactamases (ESBLs) or metallo-b-lactamases (MBLs). The aim of this study was to record the bacteraemias due to K. pneumoniae in our ICU, to analyse the patterns of resistance, as well as the clinical characteristics of the ensuing infections. Methods: Prospective observational study in patients who were hospitalised in the ICU, from September 2004 to October 2006. The demographic characteristics of all patients admitted were recorded, as well as the underlying diseases, disease severity as estimated by the APACHE II score and possible factors predisposing to infections, such as previous consumption of antibiotics. Susceptibility testing of culture isolates was performed by MicroScan autoSCAN4. The outcome of all bacteraemias was recorded. Statistical analysis was done with ESPSS v.12. Results: During the study period 290 patients were admitted to the ICU and 160 episodes of bacteraemia were recorded. Of these, 29 (18%) were due to K. pneumoniae. The isolates were resistant as follows: 100% to ampicillin, 72% to b-lactamase inhibitors, 90% to 3rd-generation cephalosporins, 41% to imipenem (MIC > 4 mg/L), 41% to aztreonam, 86% to quinolones and 31% to aminoglycosides. All isolates were susceptible to colistin (MIC 2 mg/L). The carbapenemresistant isolates (CRKP) were also resistant to all other antibiotics tested, except aztreonam and gentamicin (75% and 58% sensitive respectively). The bacteria were isolated in 23 patients (mean age 67 years, 69% male) of whom 56% were surgical. Their mean APACHE II score was 19. Ten bacteraemias were primary, 5 secondary to ventilator-associated pneumonia and 14 catheter-related. The 14days mortality of patients with bacteraemia due to CRKP was 53%, while in those with sensitive strains it was 28% (p = 0.2). Outcome was significantly related to the APACHE II score (p = 0.02). Of the patients who had bacteraemia due to CRKP, 77% had been previously receiving a carbapenem. Of those who had a carbapenem-sensitive isolate, 35% had been receiving a carbapenem (p = 0.4) Conclusions: Infections due to K. pneumoniae resistant to carbapenems represent a serious clinical problem in our ICU and are associated with a high mortality rate. It seems that previous use of carbapenems leads to increased resistance. Larger studies are needed in order to obtain statistically significant results. Objectives: Nosocomial infections surveillance was performed in an Italian ICU in order to pretest the HELICS protocol before its nationwide implementation, and to evaluate the impact and the routes of acquisition of three emerging multiresistant pathogens, Pseudomonas aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia by determining: (i) the occurrence of carriage on admission; (ii) the microrganism-specific ICU-acquired infection rates, by site; (iii) the impact of cross-transmission using molecular typing data, and (iv) the individual risk factors for infection. Methods: A six-months surveillance survey was performed at the ICU of an Italian Hospital, in accordance with the HELICS protocol, excluding patients staying less than three days. An electronic data form, was designed using the SPSS Data Entry Enterprise Server (SPSS Inc.). Clonality was determined by macrorestriction analysis using well established criteria, and the presence of two indistinguishable strains in two patients was considered one episode of cross-transmission. Results: During the study period, 121 patients were enrolled into the survey for a total of 2165 days. The ICU-acquired infection rate was 82.6% patients and the incidence density was 46.2‰ patient-days. The occurrence of P. aeruginosa carriage on admission was 1.6% patients. No episode of A. baumannii or S. maltophilia carriage was identified. The ICU-acquired P. aeruginosa, A. baumannii and S. maltophilia associated infection rates were respectively: 35.9%, 13.0% and 12.4% patients. The incidence density were respectively 19.4‰, 7.4‰, 6.9‰ patientdays. ICU-acquired pneumonia was confirmed to be the first infection type (30.0%), followed by bloodstream infections (BSIs) (25.0%), local CVC-related infections (23.0%), urinary tract infections (13.0%), CVC-related BSI (8.0%), and surgical site infections (1.0%). Eighteen P. aeruginosa, one A. baumannii and four S. maltophilia distinct clones were identified by macrorestriction analysis over a total of 162 isolates. The impact of cross-transmission was estimated to be at least 52.4%, thus defining the preventable proportion of all cross-transmission episodes. Two major risk factors were identified: inappropriate management of invasive devices and of antimicrobial usage. Conclusion: Our study confirms the essential role of epidemiologic surveillance to provide advanced risk-adjusted infection rates as a measure of quality of care. Objectives: The role of the multi-gene PE family of proteins unique to mycobacteria, in the pathogenesis of tuberculosis is still poorly understood. Tumour necrosis factor-alpha (TNF-a) is essential for successfully combatting tuberculosis. Our objective was to understand how PE_PGRS33, a surface-exposed protein known to undergo variation among strains, influences TNF-a release from macrophages, and how this is linked to apoptosis of macrophages. Methods: Recombinant PE_PGRS33 was tested for its ability to release TNF-a from the macrophage cell line RAW264.7 as well as from human monocyte-derived macrophages. Its binding to cell surface TLR2 was analysed by flow cytometry and the role of TLR2 was studied by transfecting cells with dominant-negative TLR2 prior to analysing PE_PGRS33-induced TNF-a release. The signaling pathways activated were studied by assaying for the mitogen-activated protein kinase (MAPK) kinase kinase ASK1 and downstream MAPKs. Cell death was analysed using histone ELISA and also by assaying surface-associated annexin-FITC. Results: PE_PGRS33, a surface exposed protein, elicits TNF-a release from macrophages in a Toll-like receptor 2 (TLR2)-dependent manner. Apoptosis signal-regulating kinase 1 (ASK1) is activated downstream of TLR2. ASK1 activates the mitogen-activated protein kinases (MAPKs) p38 and JNK. PE_PGRS33-induced signaling leads to enhanced expression of TNF-a and TNF receptor I (TNFRI) genes. Mycobacterium smegmatis expressing PE_PGRS33 elicits the same effects as purified PE_PGRS33. Neutralising TNF-a antibodies showed that release of TNF-a is required for triggering apoptosis in macrophages challenged with PE_PGRS33. The death receptor-dependent signals are amplified through classical caspase 8-dependent mitochondrial release of cytochrome c, leading to the activation of caspases 9 and 3. The important aspect of our findings is that deletions within the PGRS domain (simulating those occurring in clinical strains) attenuate the TNF-a-inducing ability of PE_PGRS33. These results provide the first evidence that variations in the polymorphic repeats of the PGRS domain modulate the innate immune response. Our objective was to understand the role of PPK1 in bacterial survival under stress. Methods: To evaluate the role of PPK1 in mycobacterial survival, the gene encoding PPK1 was disrupted in M. smegmatis through homologous recombination. The effect of stress on the wild type and the mutant were then analysed. ppk1 was also silenced by antisensing in M. tuberculosis. Results: The PPK1-deleted strain of M. smegmatis exhibited significantly reduced intracellular survival in murine RAW 264.7 macrophages compared to the survival of its wild type counterpart. It exhibited an extended lag phase of growth when shifted to a low phosphate-containing medium, was unable to grow in nitrogen-depleted medium and impaired in its ability to survive in anaerobic conditions. It showed decreased transcription of the stringent response regulator relA. M. tuberculosis ppk1 could complement the mutant. Antisensing of the ppk1 gene in M. tuberculosis also showed reduced levels of relA transcription. The ppk1 mutant was defective in its ability to form biofilms. Conclusion: ppk1 likely plays a role in mycobacterial persistence and is a potential target for drug development. Objectives: The objective of this study was to evaluate cholesterol oxidase (ChoD) as a putative virulence factor of Mycobacterium tuberculosis. Methods: A two-step recombination protocol was used to replace the cholesterol oxidase gene with a non-functional copy. The pathogenicity of resultant strains was analysed in vitro using peritoneal macrophages and in vivo by mice infection. Results: Cholesterol oxidase is known to be a key enzyme initiating cholesterol degradation processes in many soil bacteria, including fast growing mycobacteria. Pathogenic mycobacteria accumulate cholesterol without its utilisation and express cholesterol oxidase as an extracellular enzyme. A homologous recombination was used to construct an M. tuberculosis strain with an unmarked deletion within the choD gene. The wild type M. tuberculosis strain (Mt-wt), choD-mutant (mut-choD) and mutant complemented with choD gene controlled by a heat shock protein promoter (mut-choD-PhspchoD) were used to study the function of cholesterol oxidase in mycobacterial pathogenesis. Peritoneal macrophages (10 6 /well) were infected with Mt-wt, mut-choD and mut-choD-PhspchoD M. tuberculosis strains (10 5 /well) and incubated for four and six days. The numbers of Mt-wt and mut-choD-PhspchoD viable bacteria recovered from macrophages were at least an order of magnitude higher compared to mut-cho strain as revealed by cfu analysis. The same strains were used for infection of mice. Ten weeks post infection, lungs and spleens were isolated from euthanised mice. The numbers of viable bacteria in each organ were detected by cfu. Analysing at least 10 mice of each group, the strong attenuation of choD mutants was observed compared to wild type and complementation strains. We studied the cytokine/chemokine profiles associated with LTBI and TB in order to gain insight into ongoing immune activation events in Mtb-infection. Methods: Blood was obtained from patients with culture-confirmed TB (n = 4), with recently acquired LTBI (n = 3), and from healthcare workers without Mtb exposure (n = 6). Interferon-gamma (IFN-g) that was released from sensitised lymphocytes upon ex vivo stimulation with Mtb-specific antigens was determined using the QuantiFERON-TB ® Gold In Tube assay as recommended by the manufacturer (Cellestis Ltd., Carnegie, Australia). Microsphere-based multiplex assays (Lincoplex ® , Linco Research Inc., St Charles, MA, USA) were used to assess the plasma concentrations of IFN-g, TNF-a, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL-12p70 IL-13, IL-15, Fraktalkine, GM-CSF, MCP-1, and IP-10 before (NIL) and after (TbAg) ex vivo stimulation. Values were determined using Bio-Plex Manager (Bio-Rad Laboratories, Hercules, CA, USA). The limit of detection was 3.2 pg/mL. Statistical analysis was performed using Prism 4.1 (GraphPad Software Inc., San Diego, CA, USA). The Kruskal-Wallis test was used for multiple group comparison, and the Mann-Whitney test for group to group comparison. Results: IFN-g, IP-10, IL-2, GM-CSF, IL-13 concentrations were significantly higher in patients with TB and LTBI as compared to controls (Table) . Conclusions: Mean plasma concentrations of IP-10 were 50 to 90-fold higher than those of IFN-g, both in patients with active TB and LTBI. Assessment of IP-10 might substantially increase the sensitivity of IFN-g release assays (IGRA) for detecting Mtb infection. Ex vivo profiling of cytokines/chemokines using IGRA in combination with flow-cytometer multiplex assays identified additional candidate parameters to confirm Mtb infection and will improve our understanding of TB pathogenesis. results suggested that all isolates from patients of hospitals I and II were identical. No transmissions of patients from hospital I to hospital II had been found and vice versa indicating prevalence of one prevailing C. difficile strain in the local region. Binary toxin was not found in these isolates indicating the strain neither NAP 1 nor 027. Conclusion: One C. difficile strain seems to be predominating in Northern Bavaria CDAD patients. The increased number of CDAD cases was probably promoted by common events, e.g. increased antibiotic use due respiratory infects in winter. Therefore, infection control programmes to restrict spread of C. difficile should not be restricted to hospitals but also should imply other healthcare facilities (nursing homes, house doctors). Giardia lamblia and Yersinia enterocolitica were isolated in 2 and 1 patients, respectively. C. difficile was detected in 66 (9.3%) of all faeces samples; 13 (26%) patients had moderate-severe diarrhoea and 53 (74%) suffered from mild diarrhoea. Of 66 patients with community-onset CDAD, 31 (47%) were healthcare-associated (onset of symptoms within 48 h following admission or within 4 weeks after discharge) and 53% were community-associated. The mean age of 66 patients (24 males and 41 females) with CDAD was 66 years. The most frequent underlying diseases were cardiovascular disease (41%), urogenital disease (20%), lung disease (14%), diabetes mellitus (11%) and malignancy (9%). Recent antibiotic use was reported by 34 (52%) patients with cephalosporins (33%) and fluoroquinolones (33%) as most frequently. Of 66 patients, 19 (29%) received a PPI and 17 (26%) NSAID. Preliminary results of typing of 14 strains revealed 8 different types with type 001 (21%) and type 015 (14%) as the predominant types. Conclusion: Clostridium difficile was the most frequent cause of diarrhoea in a population of patients who visited the GP because of diarrhoea, but 47% was healthcare-associated. Objectives: Clostridium difficile infection is implicated in 20−30% of cases of antibiotic-associated diarrhoea. Few studies have evaluated risk factors for the development of C. difficile-associated diarrhoea (CDAD) in patients with antibiotic-associated diarrhoea. Our objective was to compare clinical characteristics of hospitalised patients who received antibiotics and developed CDAD with hospitalised patients who received antibiotics and developed diarrhoea with negative C. difficile toxin assay in their stool. Methods: A prospective cohort study of hospitalised patients who had received antibiotics and developed diarrhoea were included. CDAD patients were defined as patients who had diarrhoea and was associated with a positive stool toxin A/B enzyme immunoassay test. Univariate and stepwise logistic regression analyses were used for prediction of C. difficile toxin. The predictive capability of the model was demonstrated by a receiver operator characteristic (ROC) curve. Results: Fifty-two (24%) out of 217 patients were found to be positive for C. difficile toxin A/B in their stool. The logistic regression model included impaired functional capacity, watery diarrhoea, use of a proton pump inhibitor, use of a histamine receptor blocker, leukocytosis, and hypoalbuminaemia. The area under the receiver operator characteristic curve for the model as predictor to positive stool toxin assay was 0.896 [95% CI: 0.661-1.00; p < 0.001], with a 95% specificity and 68% sensitivity. Conclusions: Our results may help clinicians predict the risk of CDAD in hospitalised patients with antibiotic-associated diarrhoea, guide careful antibiotic prescription and early attention to infection control issues. Inpatient cases receiving oral MET or VANC were analysed, and initial dual therapy cases were excluded. Groups were compared on demographics, prior CDAD admissions, and comorbidity proxy measures, the APR-DRG Severity of Illness (SOI) and Risk of Mortality (ROM). Length of stay (LOS), in-hospital mortality, ICU stay, colon resection defined case outcomes. Chi-square and t-tests produced p-values. Results: We identified 32,325 CDAD cases treated first with MET (89%) or VANC (11%). Cases were similar in age (mean 70 years, p = 0.38). Fewer (p < 0.0001) MET than VANC cases were female (58% vs 64%), had a principal CDAD diagnosis (19% vs 31%), prior CDAD admission (10% vs 31%) or prior acid suppressive therapy (42% vs 53%). Higher SOI and ROM were observed in MET vs VANC (p < 0.0001), with 30% MET and 24% VANC rated extreme SOI. During hospitalisation, 15% VANC cases subsequently received MET, and 12% MET received VANC; 9% MET and 11% VANC received IV MET (unknown if specifically for CDAD), and 1.5% MET and 3.1% VANC received rifampin. MET vs VANC had increased LOS (12.8 vs 11.5 days, p < 0.0001), in-hospital death (7.9% vs 6.8%, p < 0.02), and ICU stay (23.2 vs 17.7 days, p < 0.0001). There was no difference in colectomy (1.0% MET vs 0.8% VANC, p = 0.37). Despite lower costs of MET vs VANC therapy, total pharmacy costs were similar ($2,439 vs $2,492, p = 0.52), while total hospitalisation costs were higher ($16,953 vs $14,718, p < 0.0001). Conclusion: Most CDAD patients received MET, and modification to initial therapy occurred in similar proportions of MET and VANC cases. Compared to initial VANC therapy, those treated with MET had higher rates of poor discharge outcomes and greater resource use, however comparisons do not adjust for acute comorbidities. Background: Since 2003 severe cases of Clostridium difficile (CD) associated diarrhoea (CDAD) have been reported in hospitals in North America and Europe caused by a hypervirulent strain CD ribotype 027, toxinotype III. This ribotype is also detected in The Netherlands. Objective: A descriptive study to the incidence of CDAD after the introduction of specific infection control measures in a region with outbreaks due to type 027. Methods: A case of CDAD was defined as a patient with diarrhoea and a positive toxin test on a faeces sample (ICTAB, Meridian). Strains isolated from positive faeces samples were further investigated by PCR ribotyping. The Public Health Laboratory Haarlem, The Netherlands, services an area with 440,000 inhabitants including nursing homes and three hospitals (A, B and C). Monthly admission rates of the latter are 1,050, 1,300 and 1,600, respectively; overall admission rate 47,000 yearly. Retrospective evaluation of laboratory data showed short episodes of CDAD outbreaks in hospital A in October 2002 (incidence 62/10,000 admissions) and June 2003 (121/10,000); in hospital C in December 2003 (50/10,000) and April 2004 (44/10,000). The highest CDAD increase was found in hospital B in August 2004 (>130/10,000). The latter episode was found to be the start of a long-term increase of CDAD with incidence peaks of >100/10,000 admissions per month in all hospitals as well as one nursing home to which hospital patients were transferred. In the 3rd quarter of 2005, 67% of 30 CD strains from hospitalised patients belonged to the hypervirulent ribotype 027. Specific measures against CDAD were introduced in all hospitals and nursing homes: strict barrier precautions as private room, glove use, gowns and hand washing, environmental cleaning with bleach and restrictive use of fluoroquinolones. Also a laboratory algorithm was introduced to investigate all faecal samples from hospitalised patients with diarrhoea. Subsequently, during the 4th part of 2005 until the 3rd part of 2006, CDAD incidence decreased to a mean of 21/10,000 (range 30−13/ /10,000) admissions, corresponding with the national incidence. The percentage type 027 strains decreased from 67% to 36% and 8% in the 3rd 05, 1st 06 and 3rd 06 period, respectively. Conclusion: Specific hygienic measures in combination with restrictions of antibiotic prescriptions and intensified laboratory surveillance are successful to overcome CD 027 outbreaks. Whole-genome multi-strain bacterial microarrays can be used to take a rapid snapshot of gene presence or absence in unsequenced bacterial genomes, and allow large bacterial populations to be interogated for genetic markers of phenotypic, clinical or evolutionary behaviour. We have designed and built a seven-strain Staphylococcus aureus microarray, and used it to compare S. aureus isolates from nasal carriage, community-and hospital-acquired disease. We found the S. aureus genome consists of three parts, core, core variable (CV) and mobile genetic elements (MGE). About 70% of the genes in a S. aureus genome are common to all strains and called core. Surprisingly, about 12% of genes are CV, their presence, absence or variabity can be used to classify an isolate into one of about ten human lineages. This classification matches well with multilocus sequence typing clonal complexes. Many CV genes are regulators of virulence genes, or known or predicted to be expressed on the bacterial surface and to interact with the host during nasal colonisation and infection. Since each lineage carries a unique combination of CV genes scattered throughout the chromosome, there was likely to be a common S. aureus ancestor but early evolutionary divergence of lineages, with possible selection in the nose. MGE often carry key resistance and virulence genes, and show substantial variation within and between lineages, indicating frequent horizontal transfer. Interestingly, we have been unable to identify any markers that differ between carriage and typical invasive community isolates (not CA-MRSA), suggesting community-acquired invasive disease is strongly dependent on host factors. We have also discovered the likely mechanism controlling the independent evolution of lineages. A restriction modification pathway found in all S. aureus called Sau1 can block horizontal transfer of DNA between isolates, and the specificity of the system varies according to lineage. Thus DNA from one lineage is recognized as foreign and digested by other lineages, but as self by isolates of the same lineage. Not only does this explain independent lineage evolution, but has important implications for how S. aureus continues to evolve, and in particular to acquire MGE leading to increasingly antibiotic resistant and virulent strains. S337 Baseline laboratory facilities on remote sites in Africa: too much/too little? The provision of medical care to privately funded, isolated populations in geographically and socio-economically remote areas of the world pose the challenge of providing maximum laboratory support to medical and para-medical personnel in the most cost effective way. This requires a pragmatic approach based on a knowledge of the clinically most significant diseases in the area, either due to a high and clinically significant incidence or a high risk of significant morbidity and or mortality due to disease, the outcome of which may require or may benefit from reliable, affordable and readily available laboratory diagnostics. The author provides a perspective on this challenge based on the provision of medical services to multinational companies operating in remote areas of Africa on a limited budget over a ten year period. In a setting in which a relatively small number of expatriate and national employees are taken care of with limited funding, the healthcare provider needs to be aware of the health risk profile of the region in which the service is rendered, the likely exposure of the population served to the prevalent diseases and the availability of laboratory tests that could or would make a significant difference to clinical disease management if available The main limiting factor in the provision of laboratory support to a medical service is funding. Laboratory equipment may vary from a number of simple dry chemistry based kits to sophisticated spectrophotometer and other technology based analysers. A number of factors impact on this including ease of operation of the provided tests, sensitivity and specificity, quality assurance and sustainability. The absence of trained laboratory personnel is most often a significant determinant in the decision making process regarding the level of sophistication that can be achieved. On the African continent the advent of rapid malaria antigen test kits have had a major impact on suspected malaria case management in the absence of trained miscroscopists. Long standing main stays of side-room diagnostics such as blood and urine test kits capable of detecting a number of metabolic and other diseases prove to be cost effective, regularly useful, affordable and sustainable whilst sophisticated dry chemistry analysis machines find a very limited application on a remote site with limited medical and other infra-structure. Dry chemistry tests that may be useful in this setting are superfluous in a setting in which a patient who requires, e.g., regular kidney function monitoring needs to be evacuated due to the overall limitations beset by the area. Migrants and other mobile individuals, such as migrant workers and asylum seekers, are an expanding global population of growing social, demographic and political importance. Often, grave disparities exist between place of origin and destination, in particular with relation to health determinants. The effects of those disparities can be observed at both individual and population levels. Migration influences the epidemiology of many diseases globally and in nations receiving migrants. Specific disease-based outcomes may vary between migrant group and location. Traditionally, migration health activities have been designed for national application and lack an integrated international perspective. Present and future health challenges related to migration may be more effectively addressed through collaborative global undertakings. The epidemiological relationships resulting from health disparities in migration are reviewed and the growing role of migration and population mobility in global disease epidemiology is highlighted. Implications for national and international health policy and programme planning are presented. In the last decade, Southeast Asia has become the epicentre of a number of new disease outbreaks with the potential for global spread. Nipah virus encephalitis first occurred in Malaysia in 1998 and there is serological evidence that the virus has spread to Cambodia, Thailand, China and India. In Bangladesh, Nipah virus or one closely related to it has caused several outbreaks in Bangladesh with a high mortality rate. The outbreak of severe acute respiratory syndrome (SARS) followed by avian influenza H5N1 in China and their subsequent spread led to fear of global pandemics. Many factors accounting for the emergence of new infections must be in place prior to such outbreaks. The convergence of a number of these factors in Southeast Asia makes this region a hotspot for their emergence. In order to mount a programme to fight emerging viral diseases, it is important to have adequate funds and political commitments. The key to a comprehensive programme includes good surveillance, clinical hospital management, preventing disease spread, strong laboratory support, research and evaluation, and a well-tested national preparedness plan. Surveillance is complicated in the case of a zoonotic disease such as avian influenza H5N1. Hospitals are ill-equipped to cater to large numbers of severely ill patients as in the Nipah virus outbreak in Malaysia. Preventing disease spread in an era of globalisation poses tremendous challenges in developing countries that depend on international trade and tourism. Research and evaluation are greatly hampered by lack of manpower and infrastructure, including the lack of high security laboratories. Although there are guidelines available for pandemic preparedness as in the case of avian influenza, national plans are totally inadequate and do not measure up to expectation. Despite these inadequacies, developing countries in Southeast Asia are making contingency plans in the event of unexpected viral emergence. There is no doubt that this will be an uphill battle but certain measures initiated in the region will hopefully reduce the spread of emerging viral diseases. Antimicrobial drug discovery: the road is long . . . (Symposium arranged with the ICAAC Program Committee) Cell wall inhibitors have traditionally provided the infectious disease community with safe and efficacious agents to treat serious bacterial infections. In addition, metabolism inhibitors, singly or in combination, have continued to provide coverage for some of the more resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Of the cell wall inhibitors in the pipeline, b-lactams have been a major component of antibiotic discovery programmes, but lagged in development as multidrug-resistant Gram-positive bacteria, such as MRSA, and cephalosporin-resistant Gram-negative organisms emerged as prevalent nosocomial pathogens. Compounds in late stage development include anti-MRSA b-lactams such as ceftobiprole, the broad-spectrum cephalosporin that has successfully completed two Phase III trials in complicated skin and skin structure infections (cSSSI). Following are two investigational anti-MRSA b-lactams, the anti-MRSA cephalosporin, ceftaroline, that has completed a Phase II cSSSI trial and the carbapenem CS-023/RO-4908463 with in vitro activity against MRSA and cephalosporin-resistant Enterobacteriaceae. Also in development are two additional carbapenems, ME1036, in Phase I studies, with activity against MRSA as well as against most Enterobacteriaceae, and SMP-601/PZ-601 that has demonstrated in vitro activity against many multidrug-resistant Gram-positive and Gramnegative bacteria in preclinical studies. Other b-lactam agents in development include doripenem, a carbapenem with enhanced activity against Pseudomonas aeruginosa; doripenem has recently completed clinical trials for complicated urinary tract infections and intra-abdominal infections. FR264205 is an unusual preclinical cephalosporin with promising in vitro activity against Helicobacter pylori and AmpC-hyperproducing P. aeruginosa. Additional cell wall active agents include lipoglycopeptide inhibitors that are active against most Gram-positive bacteria. Telavancin and dalbavancin are in late-stage regulatory reviews, and are characterised by long half lives in humans, with a proposed once-weekly dosing regimen for dalbavancin. Iclaprim, a dihydrofolate reductase inhibitor that is the most advanced metabolism inhibitor with completion of a Phase III cSSSI clinical trial, demonstrates in vitro antibacterial activity against MRSA, penicillin-resistant pneumococci and other respiratory pathogens. Other metabolism inhibitors that have been described in early development include peptide deformylase inhibitors and inhibitors of fatty acid biosynthesis. However, none of these early metabolism inhibitors have proceeded to proof-of-concept therapeutic studies at this time. The compound bank approach: complementing to increase the chance of success Since the publication of the first complete bacterial genome in 1995, the genome-derived target based approach to discover new classes of antibacterial drugs with novel mechanisms of action has been employed widely. The literature shows that the success rate of both biochemical screening of individual antibacterial targets, and whole-cell antibacterial screening has been disappointing. In retrospect, this is not surprising given the historical content of the chemical collections that companies employed in their early high throughput screening campaigns. Most companies have engaged in "cleaning-up" their compound libraries in recent years and have increased the molecular diversity of their collection in efforts to increase the chance of success, regardless of therapeutic area. Efforts have also been made to create focussed chemical libraries that are better suited to finding antibacterial compounds. By complementing the screening of diverse, drug-like, infection-friendly compound collections against drugable targets, and early application of structure-based approaches, we should see breakthroughs in the discovery of antibacterial drugs with novel mechanisms of action. The natural product approach: advances over the last 10 years that make it an attractive approach again There is an urgent medical need to discover and develop new antibiotics, since resistance to antibiotics is becoming an increasingly frequent problem and current development pipelines are lacking innovative, novel antibiotic classes. In the past natural products dominated the field of antibiotics and have had a significant role in the discovery and development of commercially successful drugs of all classes. Over 75% of the antibacterial new chemical entities (NCEs) introduced worldwide between 1981 and 2002 were based on natural products. The unmatched chemical diversity and complexity of natural products is one reason for their success over those obtained by pure chemical synthesis. Another reason for their superiority can be explained by the fact that their synthesis evolved naturally in response to needs and challenges of the natural environment generating compounds which are pre-selected for activity. Despite these facts, natural product research has recently gone through a phase of reduced interest. Big pharmaceutical companies in particular have downgraded or even stopped this kind of research. The reasons for this development may be that natural products are often produced in low quantities and as mixtures of similar compounds, the rediscovery of known compounds and the challenge of natural product derivatisation using classical chemical means. The past few years have witnessed major developments in the use of innovative natural product related technologies, such as fermentation optimisation, separation, structure elucidation and dereplication allowing much faster access to sufficient quantities of pure natural compounds. The application of modern medicinal chemistry adapted to the special needs of natural products is also an efficient way to revisit and recycle old antibiotic classes. The use of modern Genome-based technologies, established in the past few years, offers the opportunity to increase the attractiveness of natural products. Genome-based screening technologies provide fast access to the enormous genetic potential of Actinomycetes, soil bacteria known to represent one of the most important sources for bioactive metabolites. Additionally, genetic engineering technologies will help to overcome two of the main hurdles connected to natural products, the difficulty in derivatising complex structures and the quantitative improvement of the production yield. By focussing on examples in the field of genome based screening and genetic engineering this presentation will give an overview of major improvements that may lead to a rediscovery of natural product based drugs to meet the urgent need for new antibiotics. Objectives: Recently, we described the discovery of a novel group 2 coronavirus, coronavirus HKU1 (CoV-HKU1), from a patient with pneumonia. In this study, we examined the molecular epidemiology, genotype distribution and recombination in CoV-HKU1. Methods: We collected nasopharyngeal aspirates of patients with respiratory tract infections over a two-year period. The complete genomes of a total of 22 strains of CoV-HKU1 were sequenced and compared. Genotypes and possible sites of recombination were determined. Results: Phylogenetic analysis of 24 putative proteins and polypeptides showed that the 22 CoV-HKU1 strains fell into three clusters (genotype A, 13 strains; genotype B, three strains and genotype C, six strains). However, different phylogenetic relationships among the three clusters were observed in different regions of their genomes. From nsp4 to nsp6, the genotype A strains were clustered with the genotype B strains. For nsp7 and nsp8, and from nsp10 to nsp16, the genotype A strains were clustered with the genotype C strains. From hemagglutinin esterase (HE) to nucleocapsid (N), the genotype B strains were clustered closely with the genotype C strains. Bootscan analysis showed possible recombination between genotypes B and C from nucleotide positions 11500 to 13000, corresponding to the nsp6/nsp7 junction, giving rise to genotype A; and between genotypes A and B from nucleotide positions 21500 to 22500, corresponding to the nsp16/HE junction, giving rise to genotype C. Multiple alignments further narrowed the sites of cross-over to a 143-bp region between nucleotide positions 11750 and 11892, and a 29-bp region between nucleotide positions 21502 and 21530. Genome analysis also revealed variable numbers of tandem copies of a perfect 30-base acidic tandem repeat which encodes NDDEDVVTGD and variable numbers and sequences of imperfect repeats in the N-terminal of nsp3 inside the acidic domain upstream of papain-like protease 1 among the 22 genomes. All 10 CoV-HKU1 with incomplete imperfect repeats (1.4 and 4.4) belonged to genotype A. Conclusions: Three genotypes, genotype A, genotype B and genotype C, exist in CoV-HKU1. Analysis of a single gene is not sufficient for genotyping of CoV-HKU1, but would require amplification and sequencing of at least two gene loci, one from nsp10 to nsp16 (e.g. pol or helicase) and another from HE to N (e.g. spike or N). Influenza activity in temperate regions of Australia typically occurs between May and September, whereas in tropical regions influenza can occur any time throughout the year. The main purpose of this presentation is to outline the epidemiology of human influenza during 2001-2006 in NSW. The second purpose is to report a quality assurance programme for laboratory diagnosis of influenza throughout Australia, including avian influenza. Epidemiological data from NSW, Prince of Wales Hospital (Sydney) shows that Respiratory syncytial virus (RSV) is the first respiratory virus emerging in the winter season. Influenza A infections typically last for 4−5 weeks during August and September. Infections with other respiratory viruses such as Parainfluenza virus 3 and human metapneumovirus occur from June to late November and December. Summary data from the last 5 years will be presented. Influenza data from 2001 to 2006 indicate that the first ones affected early in the season are children and young adults, whereas during peak season the majority are between 20 and 50 years of age. There is an even distribution between the number of influenza attacks among males and females (51% and 49% respectively). From a total of 500 specimens tested for respiratory viruses during 2001-2006, 51% were true influenza cases, 10% RSV and 17% rhinovirus. Data collected from the Prince of Wales Hospital is submitted on a weekly basis to the New South Wales Department of Health as part of the NSW Influenza Surveillance Program. During 2006 we have undertaken a Quality Assurance Program aiming to improve detection and diagnosis of HPAI H5N1 among Australian and Asian Laboratories in collaboration with the Royal College of Pathologists Australia (RCPA) and the Elizabeth MacArthur Agricultural Institute, integrating human and veterinary health laboratories. The project assesses H5 diagnosis accuracy using serological and molecular techniques, utilising a new avian influenza module within the Serology Quality Assurance Program (SQAP). The report from the first Nucleic acid testing panel is already available to the 30 participant laboratories. The results suggest that testing laboratories around Australia and Asia generally utilise accurate and sensitive methods for detection of H5N1, although some laboratories are only testing for Influenza A and are currently not performing specific H5N1 tests. Objectives: Influenza pandemic preparedness plans are currently being developed on national and international levels. Planning public health responses rely on predictive models by which the impact of different intervention strategies can be evaluated. Previous research has rather focused on producing predictions for certain localities or under specific conditions and on the administration of antiviral drugs. The effectiveness of these interventions depends on various factors which must be explored by sensitivity analyses, based on mathematical models. We investigate how pharmaceutical and non-pharmaceutical interventions can mitigate an influenza pandemic and examine how intervention schedules, restricted stockpiles and contact reduction (social distancing measures and partial isolation of cases) determine the course of a pandemic wave and the success of interventions. Methods: We provide the freely available planning tool InfluSim ( figure) , a deterministic compartment model, which is based on a system of over 1,000 differential equations and designed to operate with an optimal combination of the competing requirements of precision, realism and generality. It allows for producing time courses and cumulative numbers of influenza cases, outpatient visits, applied antiviral treatment doses, hospitalisations, deaths and work days lost due to sickness, all of which may be associated with economic aspects. Results: The model shows that a timely application of antiviral drugs combined with a quick implementation of contact reduction measures is required to substantially protract the peak of an influenza epidemic and reduce its height. Delays in the initiation of antiviral treatment (e.g. because of parsimonious use of a limited stockpile) result in much more pessimistic outcomes and can even lead to the paradoxical effect that the stockpile is depleted earlier compared to early distribution of antiviral drugs. Conclusion: InfluSim efficiently assists public health planners in designing optimal interventions against pandemic influenza. It reproduces the infection dynamics of pandemic influenza like complex computer simulations while offering at the same time reproducibility, higher computational performance and better operability. When controlling pandemic influenza, pharmaceutical and non-pharmaceutical measures should not be used exclusively. Contact reduction measures must be part of mitigation strategies and have the advantage to be not limited per se. Objectives: Although influenza (Flu) is an old infectious disease, it will cause pandemics and requires global cooperation for control and prevention. In addition to the avoidance of exposure during the influenza season vaccination has been the most effective modality for preventing influenza epidemics. However, due to the genetic variability of the influenza it requires vaccination every year. Our Department of Health supported free influenza vaccination for aged people (>65 year-old) for years and the host antibody responses are evaluated. Methods: A total of 89 paired sera was collected before and 3 weeks after the vaccination with one dose of a commercial trivalent influenza vaccine for 2006-2007. The paired samples were tested for influenza A and B antibodies by complement fixation (CF) and quantitative enzyme-linked immunosorbent assay (ELISA). In addition, 83 paired sera were tested by haemagglutination inhibition (HI) using an A/New Caledonia/20/99-like strain. There were 49 males and 34 females, and 47 people were older than 65. Results: Our results indicated that the CF response rates (defined as 4-fold titer increase of the paired sera) for both influenza A and B were low (33.7% and 15.7%, respectively). However, the pre-immunised ELISA titers for both influenza A and B were much higher than normal (average 98.3±45.6 RU/mL for Flu A and 176±57.5 RU/mL for Flu B; normal <22 RU/mL). The overall response rate for HI was 32.5% (27/83), but the response rate of the aged (>65 year-old) was lower (27.7%, 13/47) than people <65 year-old (38.9%, 14/36). However, the rate of pre-immunised sera with protective HI titer (HI 40) was 80.7% (67/83). Conclusion: The antibody responses induced by the 2006-2007 influenza vaccine were low, but the pre-immunised antibody positive rates and titers were high. The phenomenon might reflect the quality of the vaccine and the consequences of previous vaccination. , among nurses with above-average knowledge about the influenza vaccine (OR 2.1) and an underestimation of non-severe AE rates (OR 3.6). Conclusion: HP overestimated the severe AE rates and underestimated the non-severe AE rates of influenza vaccination. The appraisal of these AE rates appears to be very important in the decision of HP to get vaccinated. A better mediation of the actual very low rates of severe adverse effects may improve the influenza-vaccination rates among HP. Objectives: Avian H5N1 influenza viruses could be transmitted to humans, resulting in severe or fatal disease. Aim of this study was to evaluate the immune cross-reactivity between human and avian (H5N1) influenza strains in healthy donors vaccinated for seasonal H1N1/H3N2 influenza A. Methods: Healthcare workers wishing to receive seasonal influenza vaccination at the Spallanzani Institute were enrolled. Blood samples for the assessment of humoral and cell-mediated responses were obtained before and 30 days after vaccination. The frequency of circulating antigen-specific CD4 and/or CD8 T-cells in healthy donors enrolled in the study were analysed by flow cytometry, using intracellular cytokine staining assay after the expansion of effector-cells in vitro. Human sera from the same donors were tested for their HA-inhibition activity against vaccine preparation and neutralisation activity against H5N1 virus. Our data indicate that vaccination may boost cross-subtype cellular and/or humoral immunity against H5N1 influenza. Specifically, H5N1-specific CD4 T-cell frequency was significantly higher in plurivaccinated versus first flu-vaccinated donors. The main target for cross-reactivity between H5N1 and H3N2/H1N1 strains was N1. No correlation between influenza-specific CD4 T-cells and humoral response was observed, suggesting that this response was mainly CD4 T-cellindependent. Differently, CD4 T-cells may help for anti-influenza CD8 T-cell response. Furthermore, human sera from the same donors, tested for their HA-inhibition activity against vaccine preparation, showed a significant rise (73.7%) after vaccination. The same sera were tested for their H5N1 neutralisation activity and 34.2% of subjects was able to show anti-H5N1 antibody rise after seasonal influenza vaccination, showing the existence of an antibody-dependent cross-type immunity. No correlation between influenza specific CD4 T-cells and humoral responses were observed, suggesting that this type of antibody response was mainly CD4 T-cell independent. In this study, we demonstrated that vaccination against seasonal influenza might induce both cellular and humoral cross-reactive immunity against H5N1 avian influenza. This cross-type immunity may represent an important component of the immune response against novel influenza A infections. Considering the local serotype distribution, the direct effect of PCV-7, -10, and -13 was estimated in children under 5 years (<5 y), assuming a 90% vaccination coverage and a 97% effectiveness in reducing IPD caused by vaccine-serotypes. Herd immunity effect was estimated assuming a 40% reduction of IPD caused by vaccine-serotypes. Results: (1) Incidence: The total incidence remained stable throughout the period,~20/100,000 population, whereas the incidence in <5 y increased from 25 to 34. The highest incidence was observed among adults >65 years,~69 per 100,000. 92% of cases presented as bacteraemia. Approx. 100 cases of pneumococcal meningitis were registered annually, with an overall mortality of 20%. Methods: Twenty-seven and 20 healthy adults were immunised with either a PCV or a PPV intramuscularly. Sera were obtained before and at 4 weeks after immunisation to determine IgG pneumococcal antibody titers by enzyme-linked immunosorbent assay and opsonisation tier by opsonophagocytic killing assay to the 7 serotypes represented in the PCV. Results: Antibody titers and opsonisation titers to 7 pneumococcal serotypes were significantly increased after immunisation in PCV and PPV groups. Post-immune antibody titers to serotype 4, 18C and 23F were significantly higher in PCV groups than in those in PPV group. Also PCV elicited significantly higher fold rise of opsonisation titer compared with PPV in serotype 4, 18C and 23F. Antibody titers and opsonisation titers to 7 serotypes have good correlation in pre-immune sera as well as post-immuue sera in both groups. More than 95% of subjects in both groups have antibody titer 1 mg/mL to 7 serotypes after immunisation. Quantitative and qualitative antibody responses to PCV and PPV are good in Korean adults. PCV is more immunogenic than PPV to serotype 4, 18C and 23F. The study included 704 isolates: 301 from years before the introduction of the vaccine (1998-1999) and 298 post-release (2003) (2004) . Key demographic data, serotype and resistance profiles for 5 antimicrobial classes were analysed. Strains displaying resistance (defined as non-susceptibility based on CLSI breakpoints) to >2 classes were considered MR. Statistical analysis was carried out using linear mixed effects models for repeated measures. Results: After adjusting for age, invasive status and region, a multivariate logistic regression model showed that non-vaccine (NV) isolates are 1.9 times (1.3−2.7, p = 0.03) more likely to be MR in the post-vaccine release period. When serotypes are divided into groups according to the prevalence of multiple resistance and their propensity to expand (i.e. become more prevalent after introduction of the vaccine), we found that the serotype 19 not F was 4.4 times more likely to expand (2.1−9.1) than other NV serotypes (p < 0.0001). A group formed by serotypes [11, 15, 33, 35] is also more likely to increase in prevalence, OR 2.4, 1.3−4.5, p = 0.01) than other NV serotypes. Details in resistance prevalence and proportions at each point in time are shown in the table. Conclusion: Groups of both vaccine and non-vaccine serotypes can be categorised according to their propensity to expand and to acquire MR. Serotype 19F slightly decreases in prevalence but increases in MR rate, while serotype 19 not F both increases in prevalence and acquires resistance at a rate that is proportional to the expansion process. About half of the NV serotypes rapidly increased both in prevalence and resistance. Vaccine serotypes (excluding 19F) decreased in prevalence but not in resistance. A. Bartoloni Objectives: ANTRES is a research project aimed at describing antimicrobial use and resistance in low-resource countries of Latin America. In this project, dissemination of microbial drug resistance is monitored in commensal Escherichia coli, which has largely been exploited as an indicator for similar studies. In the baseline study, carried out in 2002, we observed a high rate of faecal carriage of E. coli with acquired resistance to several antimicrobial agents. Here we report the results of a second cross-sectional study conducted in 2005 to evaluate the evolution of antimicrobial resistance rates in the studied areas. Methods: Rectal swabs were collected from 3,193 healthy children aged 6−72 months, living in 4 urban areas (2 in Bolivia and 2 in Peru). Samples were processed by a rapid screening method which allowed detecting of E. coli resistant to ampicillin (AMP), ceftriaxone (CRO), tetracycline (TET), chloramphenicol (CHL), kanamycin (KAN), gentamicin (GEN), amikacin (AMK), streptomycin (STR), trimethoprimsulphamethoxazole (SXT), nalidixic acid (NAL), and ciprofloxacin (CIP). Sampling strategy and microbiological processes were the same as in the baseline study. The data from the 2005 survey confirmed the high resistance rates for AMP (96% vs. 95%), SXT (94% vs. 94%), TET (93% vs. 93%) and CHL (69% vs. 70%) observed in the baseline study, and showed a remarkably increase in the resistance rates to quinolones (57% vs. 35% for NAL and 33% vs. 18% for CIP) and CRO (1.7% vs. 0.1%). A significant increasing trend was also observed for STR and GEN (92% vs. 82% and 27% vs. 21%, respectively). The results of this study confirmed the magnitude of the problem of antimicrobial resistance in these low-resource countries and evidenced an alarming increase in the resistance rates to quinolones and to expanded-spectrum cephalosporins. Results: Overall, most if not all resistance to a drug was in the presence of resistance to one or more of the other drugs. More than half of the isolates resistant to 3CP were resistant to FQ. Whereas only 3−10% of all isolates were resistant to AG, similarly almost all isolates resistant to AG were also resistant to FQ (82−91%) as compared to the much lower rates in those sensitive to AG (6−22%). Large differences were observed between countries in antimicrobial resistance rates, whereas the levels of associated resistance among the group of resistant isolates did not vary to the same extent (Table) . Conclusions: (i) The level of associated resistance for the isolates resistant to the different antimicrobial groups under EARSS study seemed comparable between countries, irrespective of their differences in overall resistance proportions. (ii) The group of isolates resistant to one group of antibiotics was more likely to be resistant to other antimicrobial groups (under EARSS surveillance) compared to their susceptible counterparts. The observation that most resistance is associated with other types of resistance and that the level of associated resistance seems comparable between countries would suggest that the overall reduction of the antibiotic use is probably more important in the society as a whole than targeted reduction of single drugs. Our analysis suggest that efforts should be focused on these 'multi' resistant strains. Asia, and Europe. Following the identification of the first imipenemresistant, KPC-positive Klebsiella pneumoniae isolate in our institution, we initiated a prospective study from August 2006 to November 2006 of 108 consecutive isolates of this organism to determine the prevalence and genetic relatedness of KPC-positive strains. Methods: KPC-specific polymerase chain reaction was performed on total DNA and plasmid DNA from 108 consecutive Klebsiella pneumoniae isolates. Repetitive sequence PCR was performed on all KPC-positive isolates to determine genetic relatedness Results: Of the 108 isolates collected over the four-month period, 13 (12.0%) were shown to harbour the KPC ORF by PCR. Six of the 13 isolates were identified from three patients, while each of the remaining seven came from individual patients. Eight of the eleven isolates were resistant to imipenem by disk diffusion testing while three of the isolates were fully susceptible. Plasmids containing the KPC gene were isolated and verified by PCR from the 8 imipenem-resistant isolates. In contrast, we were unable to isolate any plasmids from the 3 imipenem-sensitive strains; however, they were positive for the KPC ORF using PCR on total genomic DNA. Two distinct clusters of Klebsiella pneumoniae were seen following repetitive sequence PCR. The first cluster of 7 isolates was 86.0% identical suggesting a clonal expansion while the other 5 isolates were unrelated. As expected, multiple isolates from single patients were between 94.8% and 97.4% identical. The prevalence of KPC-possessing Klebsiella pneumoniae in our institution has risen to 9.2% in 4 months thereby compromising empiric treatment options for patients infected with Klebsiella pneumoniae. Both a clonal expansion and horizontal plasmid transfer appear to be involved in KPC dissemination. In addition, these data suggest that the KPC ORF may reside in the chromosome and is not expressed until the proper inducing agent is present or that the KPC ORF may reside on a transposable element that is not expressed until transferred into a plasmid. O362 Pseudomonas aeruginosa resistance rates in association with level of hospital care: data from the EARSS Factors independently associated with increased risk of non-susceptibility to 1 of these agents in P. aeruginosa were intensive care, young age, >48 hours prior hospitalisation, and infections arising from skin/soft tissue and lines. Among Acinetobacter, resistance was concentrated in A. baumannii: 2/110 (2%) of A. baumannii were non-susceptible to all 5 agents, and 36% to 2 classes of agents. Overall, 5% of Acinetobacter were nonsusceptible to GEN and IPM, and 17%, 27% and 63% to TZP, CIP and CAZ respectively. Numbers were too small to assess trend, predictors or agreement with LabBase2. MIN and TGC largely overcame TET resistance in Acinetobacter, with maximum MICs of 8 and 4 mg/L respectively. DOR MICs were closely related to IPM, on average 2.8 dilutions lower for P. aeruginosa and 0.5 dilutions higher for Acinetobacter. BPR MICs were on average 1.2 dilutions higher than CAZ for P. aeruginosa and 2 dilutions lower for Acinetobacter. The plasmid mediated quinolone resistance determinants qnrB (1) and qnrS (9) were identified in 10 of 81 Salmonella enterica isolates with raised ciprofloxacin MICs. S. Corvallis is an uncommon serovar in our experience (0.2% of all isolates) and is remarkable that this serovar represents a high proportion of all qnrS positive isolates detected. There is no evidence of an epidemiological link between the S. Corvallis isolates. Orthopaedic and Trauma surgery is an implant specialty where surgical success is related to the insertion of prosthetic joints (e.g. knee, hip and shoulder replacements) and reconstructive metalwork following trauma (e.g. intra-medullary nails, plates and screws). The worst complication is infection which results in multiple additional operations, extended hospitalisation, prolonged morbidity and poor functional outcomes. Infection rates are low, ideally between 0.5% and 2%. The most common infective organism is Staphylococcus aureus which accounts for 50−70% of cases. Methicillin-resistant coagulase-negative staphylococci are of growing importance but numerically less significant. The clinical presentation, surgical techniques and pre/post operative morbidity of chronic osteomyelitis and infected joint replacement will be discussed to project the patients' perspective of S. aureus. Emerging infectious diseases have a major impact on public and animal health, the economy, and the environment (Science 309: 1680). Human mortality from recently emerged diseases varies, ranging from less than 200 people thus far for H5N1 avian influenza to about 20 million for AIDS. Livestock production has been negatively affected by the direct mortality of animals from emerging infections and depopulation policies to protect the safety of international trade and to control the spread of pathogens. The environmental impact of emerging infections is of special concern for endangered wild animal populations, which can be pushed to the brink of extinction by such events. Animals, and particularly wild animals, are thought to be the source of more than 70% of all emerging infections ( August to December and also rises in the spring. These findings are of importance for the understanding of how influenza virus is perpetuated in nature. Of concern is the presence of H5 and H7 subtypes that are prone to change into highly pathogenic variants in poultry. Many of the strains isolated in our study are prototypes that have caused influenza outbreaks in poultry in Europe during recent years. This indicates that wild bird surveillance for influenza A virus can be of value as a sentinel system to prevent outbreaks in domestic poultry. A result of the domestic animal revolution is that there has never before in history been so much poultry as today. This fact, in combination with a growing number of humans, creates an arena where domestic animals, humans and finally zoonotic pathogens can interact. Influenza A virus is the ultimate actor in this play where some subtypes may change into highly pathogenic forms. These may be transmitted directly to man. Another worrying scenario is that an avian influenza virus will reassort with a circulating human influenza. A genetic trait might then readily go from the human to the bird virus so that avian influenza acquires the capacity to pass from person to person. With this risk in mind, it would advisable to consider any method to reduce the probability of this happening. Contingency planning should take into account the known evolutionary potential of animal viruses and other pathogens to adapt to the environment of humans and domestic animals. Candida. The use of fluconazole susceptibility profile as a surrogate marker for prediction of voriconazole susceptibility has also been proposed recently. In addition, based on the modifications of the CLSI M38-A reference method, new guidelines for in vitro susceptibility testing of antifungal agents against Aspergillus spp. have been developed by AFST-EUCAST. A standard assay for testing antifungal agents against dermatophytes is also under development. Further investigations address the applicability of other methods, which are more practical and/or require shorter incubation periods. Among these are Etest, colorimetric methods, flow cytometry, and ergosterol quantitation. Utility of flow cytometry for AFST of yeasts and moulds is being currently studied and appears promising. Assessment of metabolic activity by using XTT colorimetric assay is also being investigated as a novel approach, particularly for determination of quantitative endpoints for testing caspofungin against Aspergillus, and for susceptibility testing of Zygomycetes. Despite these progressions, utility of AFST in direction of antifungal therapy and prediction of clinical outcome is still limited. While in vitro triazole (particularly fluconazole) susceptibility results for Candida appear to be optimally correlated with clinical outcome, data are either limited and investigational or fail to demonstrate any significant in vitro-in vivo correlation for most of the remaining fungus-antifungal drug combinations. Importantly and conclusively, the results obtained by AFST constitute one of the several factors that influence clinical outcome. Optimisation of test methodologies and parameters for routine use and expanded in vitro-in vivo correlation studies may further enhance the role of AFST as an adjunct in direction of antifungal therapy. In recent years, there has been important progress in our knowledge of bacterial resistance to antibiotics. The availability of a large number of antibiotics has allowed precise individualisation of resistance phenotypes, and enzyme inhibitors have provided clues concerning certain mechanisms of resistance. Detailed analysis of the bacteriostatic and bactericidal activity of antibiotics, alone or in combination, has indicated the limits of in vitro antimicrobial susceptibility tests in the detection of resistance resulting in clinical failure. The goal of combined molecular and therapeutic interpretation of susceptibility tests is to provide an improved logical basis for decision-making in antibiotic therapy by taking into account the recent progress in the understanding of bacterial resistance. The molecular analysis and therapeutic interpretation, designated "interpretative reading", of the in vitro antibiotic susceptibility tests consists of three steps: (1) characterisation of the resistance phenotype with a judicious assortment of antibiotics belonging to the same class; (2) deduction from the observed phenotype of the corresponding biochemical mechanism of resistance; and (3) inference from the deduced mechanism of the predicted resistance phenotype. In vitro antibiotic susceptibility tests, like other tests in biology, should provide objective quantitative data, e.g. MICs. For various historical reasons, they also provide subjective interpretation of the data such as clinical categories. Interpretative reading of antimicrobial susceptibility tests is an attempt to reconcile these two notions by basing interpretation on the most recent knowledge in the field of antibiotic study, in particular that of mechanisms of resistance. The best that one can ask of antibiotic susceptibility testing is detection of resistance, in particular of low-level resistance. This can be achieved by improved interpretation of the results of in vitro sensitivity tests or by the design of certain genotypic approaches. The goal of the proposed approach is to provide the clinician with the necessary results for judicious decision-making in antibiotic therapy utilising available information and to draw his or her attention to the combinations of bacterium and antibiotic for which there is a therapeutic risk. Resistance to aminoglycosides is mostly related to production of modifying enzymes, phosphorylases, nucleotidyltransferases or acetyltransferases. The enzymes vary in their substrate ranges which are often broad and each enzyme is characterised by a particular profile of resistance which allows its putative identification from the conferred phenotype. The enzymes from Gram-positive cocci generally differ from the enzymes detected in Gram-negative bacteria. Generally, the presence of an enzyme confers to the host frank resistance to the antibiotics modified in vitro. However, there are some exceptions, both in Gram-negative and in Gram-positive bacteria. For instance, resistance of Gram-positive organisms to the kanamycin-neomycin group of antibiotics is due to the synthesis of an APH(3 )-III enzyme. The enzyme catalyzes efficient phosphorylation of amikacin in cell-free extracts but does not always determine resistance to this antibiotic. However, bactericidal synergism of amikacin with b-lactams or vancomycin is always abolished. Therefore, the test of kanamycin better predicts the activity of amikacin against Gram-positive organisms. In this case and several others, interpretive reading of susceptibility tests based on identification of resistance phenotypes may help to identify impaired activity of aminoglycosides. Interpretive reading may also allow to identify some pitfalls in the detection of resistance to macrolide and lincosamide antibiotics. Staphylococci may be resistant to macrolides by production of a ribosomal methylase encoded by erm genes conferring the MLSB phenotype or by production of an efflux pump encoded by the msr(A) gene. In case of inducible MLSB resistance, clindamycin that is not an inducer remains active. However, constitutively resistant mutants can be selected by clindamycin and clinical failure during treatment by clindamycin have been reported in a few occasions. The use of clindamycin is probably best avoided in severe infections or infections with heavy bacterial inocula. In case of resistance by efflux, clindamycin is not substrate for the pump and the risk for selection of resistant mutants is not greater than that for erythromycin-susceptible isolates. By a disk diffusion test, the inducible MLSB phenotype can be identified by the flattening of the clindamycin zone facing the erythromycin disk. The relative importance of the contaminated inanimate environment is discussed controversially: Whereas outbreaks due to environmental sources have been described repeatedly, its role as a source for endemic colonisation (and, as a consequence, infection) in intensive care patients has not been firmly established. More recent data show that in an ICU, a high figure of 35% of all cases of P. aeruginosa acquisition may originate from contaminated tap water (clonal relationship between isolates) and that retrograde contamination of faucets by patients may occur in 15%. However, surveillance of intestinal colonisation (which plays a relevant role in the epidemiology of P. aeruginosa) was not undertaken. The majority of colonised patients is already colonised on admission, with preceding antibiotic exposure being responsible for the majority of cases of acquired respiratory tract colonisation. With regard to infection control, adequate hand hygiene is essential, whereas 'sterilisation' of sinks or routine use of water filters seems impractical. Alcohol-based handrubs, via dispensers near the bedside or in coat pockets, should be preferred in most situations over hand washing with soap and water. All open water sources including sinks may be a potential habitat of pathogens like P. aeruginosa, and the use of tap water for critically ill or immuno-compromised patients must be restricted. A second pathogen was reported in 65% of patients and Candida sp. was seen in 14%. There were significant regional variations in the distribution of pathogens and the number of culture tests taken (0−5.9 cultures per patient admission). The principal pathogen was resistant to the most commonly used antibiotics in 60−90% of cases. The prevalence of asymptomatic bacteriuria was about 30% in most regions, while the prevalence of urosepsis varied between 2−27% in the regions studied. Conclusions: Urology sections should be encouraged to monitor the susceptibility of pathogens causing NAUTI in order to tailor a better empirical antibiotic treatment. Urologists need to work out guidelines on when to take blood cultures after urological surgery to obtain a more uniform reporting of urosepsis between regions. The high prevalence of urosepsis after urological surgery is a cause of concern. Escherichia coli accounted for about 80% of organisms in uncomplicated UTIs. In contrast, in complicated UTI many kinds of enterobacteriaceae isolates, staphylococci, enterococci, Pseudomonas aeruginosa, and the other glucose-nonfermentable Gram-negative rod (NFGNR) are isolated. Almost nosocomial UTI (NUTI) is complicated UTI. In the 2003 GPIU study (formerly PEP study), NUTI prevalence rate was 9.4% (326/3350). In the 2003 GPIU study, E. coli was the most frequent pathogen, accounting for about 30% of all pathogens isolated. Pseudomonas (13%), Klebsiella (10%), Enterococcus (9%), and Proteus (7%) were isolated frequently. In our hospital, Enterococcus faecalis was the most frequent of all isolated organisms, accounting for 24%. The other enterococci (12%), S. marcescens (12%), P. aeruginosa (9%), the other NFGNR (9%), E. coli (6%), Staphylococcus epidermidis (6%) were isolated frequently. It is considered that these differences occurred by the difference of patients' background. The fluoroquinolone-and cephem-resistant enterobacteriaceae isolates from patients with NUTI are increasing. Most of the cephem-resistant isolates of E. coli, K. pneumoniae and P. mirabilis are producing Extended-Spectrum b-lactamases (ESBLs). ESBLs are plasmid-mediated broad-spectrum b-lactamases. The ratios of ESBL producers in E. coli and K. pneumoniae are quite different in each country (less than 5% to more than 80%). Most of the nosocomial ESBL producers have acquired resistance to non-b-lactams, such as fluoroquinolones, phosphomycin, co-trimoxazole. Some of the multi-drug resistant isolates have no effective oral antibiotics. According to the 2003-2004 GISP study, the ratios of fluoroquinolone resistance of E. coli and K. pneumoniae accounted for about 20%, respectively, that of enterococci accounted for more than 60%. Mechanisms of resistance to quinolones are mainly target mutations, especially GyrA and ParC. Qnr that is plasmid encoded quinolone resistance reported in 2002. The emergence of qnr also alerts us to the potential rapid dissemination of quinolone-resistant determinants. Qnr shows quinolone specific resistance, but the qnrplasmids reported are integron-associated and carry multiple resistance determinants providing resistance to several classes of antimicrobials including b-lactams and aminoglycosides. Multi-drug resistant isolates cause nosocomial spread easily. There are many reports about nosocomial spread of multi-drug resistant ESBL producers. To prevent prevalence of antimicrobial-resistant isolates appropriate antimicrobial selection is needed. Geographic variations in pathogen occurrence and susceptibility profiles require monitoring continuously, and it is important to update treatment guidelines based on susceptibility profile and the results of clinical trials. Urosepsis accounts for approximately 25% of all sepsis cases and may develop from a community or nosocomial acquired urinary tract infection (UTI). The underlying UTI is almost exclusively a complicated one with involvement of the parenchymatous urogenital organs (e.g. kidneys, prostate) and mostly associated with any kind of obstructive uropathy. If urosepsis originates from a nosocomial infection, a broad spectrum of Gram-negative and Gram-positive pathogens have to be expected which are often multiresistant. In urosepsis, as in other types of sepsis, the severity of sepsis depends mostly upon the host response. The treatment of urosepsis follows the generally accepted rules of the Surviving Sepsis Campaign Guidelines. Early normalisation of blood pressure and early adequate empiric antibiotic therapy with optimised dosing are equally important to meet the requirements of early goal directed therapy. In most cases of urosepsis an early control of the infectious focus is possible and as important. Optimal supportive measures need to follow the early phase of resuscitation. Although most antibiotics achieve high urinary concentrations there are several unique properties in complicated UTI, and thus in urosepsis, that influence the activity of the antibiotic substances: (i) The renal pharmacokinetics in unilateral and bilateral renal impairment and in unilateral and bilateral renal obstruction differ. (ii) Varations in pH may influence the activity of certain antibiotics. (iii) Biofilm infection is frequently found under these conditions, which may increase the minimal inhibitory concentrations (MIC) of the antimicrobials at the site of infection by several 100-folds. In order to assess the antibiotic pharmacodynamic properties in such situations not only the MIC as determined in vitro and the plasma concentrations of the free (unbound) drug, which are the guiding principle for many infections, but also the actual renal excretion and the urinary bactericidal activity of an antibiotic substance should be taken into account. In the treatment of urosepsis it is important to achieve optimal exposure to antimicrobials both in plasma and in the urinary tract. The role of drugs with low renal excretion rate is therefore limited. Since urosepsis originates quite often from catheter associated UTI and after urological interventions, optimal catheter care and optimal strategies to prevent nosocomial UTI may be able to reduce the frequency of urosepsis. 28.5%; p < 0.001) and were more commonly classified into Pneumonia Severity Index high-risk class (group V) (48% vs. 16%; p = 0.001). They also had received more frequently prior corticosteroid (30% vs. 6%; p = 0.001) and antibiotic (44% vs. 17%; p = 0.011) therapy. Multivariate analysis identified COPD (OR = 6.83) and prior corticosteroid therapy (OR = 4.22) as independent risk factors. Patients with CAP due to P. aeruginosa presented more frequently with septic shock at entry (25% vs. 3.8%; p = 0.001) and multilobar pneumonia (50% vs. 25.4%; p = 0.019) than the remaining patients. They were also given more frequently an inappropriate initial empirical antibiotic therapy (68% vs. 9%; p < 0.001). Early (29% vs. 2%; p < 0.001) and overall (55% vs. 8.5%; p < 0.001) case-fatality rates were higher among patients with CAP due to P. aeruginosa. Conclusions: CAP caused by P. aeruginosa is uncommon and occurs mainly in patients with COPD treated with corticosteroids. It frequently presents with shock and causes high case-fatality rates. The risk factors delineated in this study should be considered when selecting initial empirical antibiotic therapy for patients with CAP. Objective: In prospective aetiological studies, a microbial cause is not found in 30−65% of subjects with community-acquired pneumonia (CAP). The objective of this analysis was to identify predictors of microbiological documentation in a large CAP trial. Persistence or deterioration of CXR abnormalities were not correlated with a poor prognosis. Therefore, routine follow-up CXR seems not to be appropriate for patients that respond to therapy and CXR follow-up to exclude a non-infectious cause should not be performed within 4 weeks after initial diagnosis. O414 Is empiric broad-spectrum therapy always necessary in severe community-acquired pneumonia? G. Barlow, D. Nathwani, P. Davey (Hull, Dundee, UK) Objectives: UK guidelines recommend co-amoxiclav/macrolide or a second/third-generation cephalosporin/macrolide for severe (CURB65 3) community-acquired pneumonia (CAP). Observational studies suggest that adherence to guidelines and atypical pathogen cover results in better outcomes. There is concern about the ecological impact, however, of broad-spectrum agents. This study compared mortality in hospitalised patients who had received amoxicillin/macrolide (narrow spectrum, NS) with those who had received either co-amoxiclav/macrolide or a second/ third-generation cephalosporin/macrolide (broad spectrum, BS Objective: Bacteraemia is an important cause of mortality in hospitalised patients. CURB65 has been validated as a predictor of mortality in community-acquired pneumonia (CAP) (Lim et al. Thorax 2003), but not in other causes of sepsis. The aim of this study was to assess the performance of CURB65 in patients with bacteraemia. Methods: A retrospective cohort study was performed using data routinely collected in the delivery of the Hull Bacteraemia Service. As part of this service, patients with confirmed bacteraemia are seen at the bedside by an infectious diseases physician. A typed report, which includes physiological data, is then sent to the patient's physician. Patients were included if they had all CURB65 criteria recorded at the point of bacteraemia. 30-day mortality was established by hospital database and stratified by CURB65 score. A receiver operating curve (ROC) was produced and area under the curve and 95% confidence intervals (CI) calculated. Results: Of 151 patient reports, 61 patients (62% male) had a full set of CURB65 criteria. 49% of patients were over 65 years old and 34% were being managed on a renal ward. The most common bacteria were: MSSA (41% of patients), MRSA (23%); various Gram-negatives (16%); thought to be significant coagulasenegative staphylococci (15%); Group A−G streptococci (10%); and enterococci (8%). The most common sources of bacteraemia were: central venous line (31% of patients); intravenous drug use (15%); urinary tract (13%); contamination (10%); and skin/soft tissue (8%). 9% of patients were receiving discordant therapy prior to review. Overall, 30-day mortality was 25%. Recrudescence within 90 days occurred in 5.5% of patients. The table shows 30-day mortality stratified by CURB65 score. The area under the ROC was 0.73 (95% CI: 0.6−0.86). Conclusions: This is the first study to assess the performance of CURB65 in a non-respiratory infection. Although preliminary, CURB65 appeared to stratify 30-day mortality in patients with bacteraemia. The cut-off for severe illness may need to be lower, however, than in CAP. There is the potential, therefore, to identify a low-risk cohort of patients (i.e. CURB65 = 0) who may be appropriate for an early switch to oral therapy and discharge from hospital. Likewise, patients at high risk (i.e. CURB65 2) are more likely to need aggressive therapy according to the principals of the surviving sepsis campaign. A large prospective study is warranted. . Mortality occurred rapidly after the onset of symptoms (median 3 days; range 1−17 days) in seven patients with non-menstrual STSS (7/32, 22%). Deceased patients were more aggressively treated in comparison to survivors but they never received clindamycin, linezolid or intravenous immunoglobulins (IVIg). We observed that non-menstrual STSS was a more severe disease as expected. Comparing with menstrual STSS, non-menstrual STSS patients have a high positive blood culture and mortality rate (50% and 22%, respectively). This could reflect an evolution of the S. aureus isolates towards a higher virulence and expression of various superantigenic toxins. Bacteraemia could partially explain the severity of non-menstrual cases and may be a composite form of shock, frontier between septic shock and STSS, potentially more severe than STSS itself. Specific therapeutic intervention has to be implemented for patients with non-menstrual STSS and we proposed the following strategy: rapid wound debridement whatever the importance of local inflammation; rapid addition of anti-toxinic antibiotic such as clindamycin or linezolid; and prescription of IVIg. To determine the best model, Model Selection Criterion (MSC) and coefficient of determination (R2) were taken into account as well as a visual inspection for goodness of fit. Results: Two susceptibility stages were defined for MRSAstage 1: self-replicating and susceptible to linezolid, and stage 2: unsusceptible and metabolic inactive. In the log-growth phase, susceptible bacteria grow with a growth rate constant k s that is greater than their natural death rate constant k d in a certain ratio. Converging to the stationary phase this ratio changes in a non-linear manner, described by an additional N max term. In the presence of linezolid a concentration C dependent kill has to be taken into account. From certain drug specific concentrations on, a maximum effect is reached, described by the maximum kill rate constant k max . However, both the onset of growth and kill can be delayed and modeled by exponential terms, characterised by time t and dg or dk, respectively. The final shape of the curve is smoothed out by a Hill factor/shape factor h. The modified sigmoid Emax-model shown below described the data best, resulting in MSC and R2 values of 4.04 and 0.82. Conclusion: The proposed model incorporating five additional terms could account much better for the in vitro situation than a simple Emax-model. A simultaneous fit describes the actual time-kill curve data sufficiently well. We have recently evaluated a number of antistaphylococcal drugs in vivo in a murine model of peritonitis, with respect to their intracellular antistaphylococcal activity. Objectives: (1) A PK description of azithromycin (AZM), cefuroxime (CXM), dicloxacillin (DCX), gentamicin (GEN) and rifampicin (RIF) in plasma and peritoneal exudate following SC injection. (2) A description of the intracellular accumulation of antibiotics in peritoneal exudate. Methods: Female NMRI mice were inoculated IP with Staphylococcus aureus E19977 in 5% mucin. Two hours later, mice were treated SC with one of the five antibiotics. For each individual mouse an aliquot of peritoneal exudate was saved for differential somatic cell count and measurement of antibiotic concentration. Pelleted cells (300 g, 10 min) from the rest of the peritoneal exudate were lysed in a small volume of dH 2 O. Antibiotic concentrations were measured by bioassay. Intracellular accumulation was calculated by dividing the intracellular with the extracellular concentration. The concentrations found for CXM and DCX were higher than the concentrations found for AZM, GEN and RIF. This corresponds well with the higher dose injected and the relatively high antistaphylococcal activity observed in PD studies. The intracellular accumulation of AZM and RIF found here corresponds well with what has previously been reported. The intracellular accumulation for CXM, DCX and GEN found here, are surprisingly high, since b-lactams are notoriously considered not to accumulate intracellularly, and the 2−4 fold intracellular accumulation of aminoglycosides take several days, but the influx correlates well with the good effect seen in the in vivo model. An inflammatory reaction of the venous vessel is a common clinical problem that is observed after intravenous application of antibiotics and other drugs. The local irritation of the endothelium at the site of infusion leads to an inflammatory response with an increased expression of various cell surface antigens. Among these are CD 34, E-Selectin (CD 62E), ICAM-1 (CD 54) and VCAM-1 (CD 106). We studied the effects of three closely related antibiotics on human endothelial cells in vitro. We used the endothelial cell line EA.hy 926 and analysed the reaction by means of flow cytometry (FACScan, Becton Dickinson). Cells were incubated with clarithromycin or azithromycin at concentrations ranging from 100 mg/l to 800 mg/l and at concentrations ranging from 200 mg/l to 1400 mg/l for erythromycin. Such concentrations occur under therapeutic conditions at the site of infusion. Subsequently, they were stained with fluoresceinisothiocyanate-or phycoerythrin-conjugated monoclonal IgG mouse-antibodies for the four antigens mentioned above. Cells were incubated with the drugs for 2 h and analysis was carried out after an additional time period of 22 h. In control cells, we found positively stained cells at the following levels: CD 34 (2%), E-Selectin (4%), ICAM-1 (14%) and VCAM-1 (2%). The most pronounced changes were observed at 800 mg/l (erythromycin), 600 mg/l (azithroymcin), and 400 mg/l (clarithromycin). Erythromycin (800 mg/l) caused significantly increased expressions of all epitopes [CD 34 (+6%), E-Selectin (+5%), ICAM-1 (+15%) and VCAM-1 (+5%)]. At 600 mg/l the azalide azithromycin provokes a stronger upregulation of the proinflammatory antigens: CD 34 (+17%), E-Selectin (+16%), ICAM-1 (+27%) and VCAM-1 (+17%). Clarithromycin at a concentration of 400 mg/l causes a similar effect as erythromycin at twice this concentration [CD 34 (+6%), E-Selectin (+7%), ICAM-1 (+23%) and VCAM-1 (+4%)]. Analysis of the cell surface markers involved in cell-cell-interactions proved to be a useful approach to further study the mechanism of infusion phlebitis and to compare the proinflammatory effects of related compounds in vitro. Conclusions: This study is the first report of the integron-associated IMP resistance, high prevalence and co-existence of blaIPM-1, blaVIM-2 and blaOXA-23-type among multiple clones of blaOXA-8-type-bearing multiresistant clinical isolates of A. baumannii in Romania. Taking into account the very narrow antibiotherapy choices in these infections, the horizontal transfer of these genes and also the increasing resistance to colistin, the possibility of a further spreading of carbapenemhydrolyzing oxacillinases is of a considerable concern for antimicrobial chemotherapy. (15). The strains were investigated for the presence of metallo-b-lactamase genes by class-1 integron PCR followed by digestion with restriction enzymes, Southern hybridisation and probing with blaVIM probes. Positive isolates were further investigated by PCR and sequencing with primers designed against class-1 integron, blaVIM genes and Tn5090 transposase genes. Results: All isolates were positive by hybridisation with blaVIM-1 and blaVIM-2 radio-labelled probes. Digestion of the class-1 integron PCR products with HincII produced ten different integron RFLP types (Types A−J). Type A was found in six PSA, 3 PPU and one PST strains and consisted of a class-1 integron harbouring aacA4 and blaVIM-4 gene cassettes. Type B was found in 10 PSA strains and the individual EC strain, harbouring an integron with aacC4, blaVIM-4 gene cassettes and the insertion sequence ISPpu17. Eight strains harboured the blaVIM-2 gene cassette in several different gene arrays and only one strain of PSA harboured the blaVIM-1 gene cassette. All class-1 integrons were of the more common form including the 3 Conserved sequence. No class-1 integrons were found that did not have the 3 CS as has been found in several recent blaVIM-2 isolates from diverse geographic regions. Objectives: We sought to evaluate the bactericidal effect of human b-defensin 3 against metallo-b-lactamase (MBL)-producing P. aeruginosa clinical isolates. Materials and Methods: A total of 15 unique multidrug-resistant P. aeruginosa strains were tested that originated from relevant specimens of patients hospitalised at our hospital. Bacterial identification and antimicrobial susceptibility testing was performed with the VITEK 2 automated system. According to their drug susceptibilities all clinical isolates were categorised into 3 distinct resistance phenotypes. Metallob-lactamase (MBL) production was detected with the use of Etest MBL strips, according to the manufacturers' instructions. To determine the MBL types all isolates were subjected to PCR analysis to confirm the presence of blaVIM and blaIMP genes. The bactericidal activity of recombinant hbD-3 was assessed by the liquid microdilution assay in the presence of 10mM sodium phosphate buffer, as previously described. Briefly, exponentially growing bacteria (inoculum density of 1×10 4 cfu/mL) were exposed to different concentrations of hbD3 (0.5 ug/mL and 0.25 ug/mL). Following incubation for 20 min at 37ºC, 0.1 ml of each sample was plated onto McConkey agar plates. Bactericidal effect was expressed as percentage of reduction in numbers of viable bacteria after 18 to 24 hr incubation at 37ºC in ambient air. Results: At a concentration of 0.25 ug/mL, the peptide produced a reduction rate ranging from 55% to 95.4% whereas the use of 0.5 ug/mL of hbD-3 resulted in 68% to 100% killing. The significant variability in the bactericidal effect observed, appears to be independent of the isolates' resistance phenotypes. Conclusion: Despite the relatively low concentrations used, hbD-3 exhibited bactericidal activity against MBL-producing Pseudomonas aeruginosa nosocomial strains and therefore it would be an attractive alternative to current theurapeutic agents. Systems biology is a fast developing research area in life sciences which combines both experimental and theoretical disciplines and which investigates all components of complex biological systems (for example all proteins of a cell). It collects large biological datasets by novel, high-throughput-based technologies and develops computational tools for its bioinformatic analysis. It thus allows a holistic view on complex biological systems and to set up improved biological models, particularly if several different experimental parameters are integrated (for example protein interaction and expression profiling data). Although Systems biology is usually based on screening assays and thus starts without bias, in contrast to hypothesis-driven research, in most cases it eventually leads to a multitude of novel working hypotheses. Particularly interesting are hypotheses derived from bioinformatic concepts like network emergence, robustness and modularity, since it is currently completely unclear if and how they translate into biology. Systems biology-based approaches in infectious diseases are even more complex, as they investigate the interactions between the components of two distinct biological systems, pathogen and host. On the host side, cellular components which directly deal with the control of the infection, i.e. the innate and adaptive immune system, are of particular interest. For the understanding of the pathogenesis, pathogen products counteracting these cellular components are similarly important. By integrating all components of the immune system, Systems biology-based approaches might be able to generate better disease models than previous reductionist approaches focusing on only one or few pathogenic aspects. For example, in herpesviruses, which cause persistent latent infections, both the innate as well as the adaptive immune system and their components play a role at different time points after infection and thus have to be considered to fully understand their pathogenesis. Recently developed technologies in molecular biology allowing genomewide analyses are one of the prerequisites for Systems biology-based approaches. For example, novel sequencing technologies which are powerful enough to evaluate whole genomes within short time are able to detect inter-individual polymorphisms. There is growing evidence that genetic variations on both the host and the pathogen side are often crucial for the outcome of infectious diseases. A variety of other experimental systems have been developed during the last decade and are meanwhile sufficiently potent to allow genome-wide analyses, including microarrays for transcriptional or protein expression profiling, genetic screening systems like the yeast-two-hybrid system used to identify pairwise protein interactions and novel mass spectrometry approaches used for proteomics and metabolomics. In medicine, Systems biology is currently still largely considered as a basic research area with little practical relevance. However, it will lead to substantial changes in medicine within only a few years, not just because Systems biology-based disease models will considerabley improve our understanding of the pathogenesis and will accelerate and rationalise drug discovery, but also since the ability to determinate individual genetic traits and availability of multi-parameter diagnostics will eventually lead to a much more personalised medicine, in which therapeutical interventions are tailored to single individuals. Human immunodeficiency virus represents the best model of evolution on earth. Due to the high rate of replicative cycles, and the errorprone polymerase (able to make about 1 error out of 10,000-30,000 nucleotides), the entire genome of HIV can be changed every day. Based on these factors, it has been calculated that HIV genome varies in one day more than the variability of influenza virus (another highly variable virus) in 10 years, and more than the variability of the human genome during the whole history of mankind. Despite this immense potentiality, only few mutations that are randomly produced during the daily replicative cycles are fixed within the viral genome. This is because of a bottleneck phenomenon related to the constraint of target cells (unable to support the replication of all vira strains daily generated) and because the large majority of the mutations generate variants whose replicative capacity is very low/ absent, or in any case lower than the one that characterised the wild type strain. For this reason, the number of quasispecies is limited if the environment (that is immune and pharmacological pressure) does not change. Of course the situation changes unpo environment change. In case of therapeutic pressure, the ability of the virus to rapidly deselect the wild-type strain in favour of a resistant mutant is a function of the strength of drug regimen. In case of fully suppressive therapy, the virus is unable to generate and/or select new variants, the wild-type strain remains prevalent and the therapy is highly successful. By contrast, if therapeutic pressure is inconsistent, erratic, or not sufficiently potent, the virus continues its replicative cycles under drug presence, and this represents the best environment to select mutant-resistant strains. The rapidity with which this selection occurs is a function of the genetic barrier of each drug, that in turn is related to the number of mutations occurring to generate a new variant fully resistant to each drug. A low genetic barrier means few (1 or 2) mutations are required to generate resistant variants, that will occur rapidly. A high genetic barrier (5−6 or more mutations occurring in sequence) makes more difficult and slower the process of selection of a resistant strain. These data have a strong clinical implication, in view of the impossibility (at least today) to eradicate virus infection from the body. Thus, anti-HIV therapy has to be started before the damage of the immune system is too advanced, and must be strong and consistent. For this reason, until today, at least three drugs rationally combined are needed to achieve this result. A great effort has to be dedicated by physicians and nurses to the achievement of a very high level of adherence, that represents a key factor to maintain a long-lasting protection of HIV-disease progression and a good quality of life. Objectives: API-1252 is a novel antimicrobial, with a mechanism of action targeting fatty acid biosynthesis, currently in development as a new class of oral and intravenous anti-staphylococcal agents. In this study, comparative single-dose response experiments were performed with API-1252 and linezolid to assess their quantitative oral efficacy against hospital-acquired (HA)-MRSA and community-acquired (CA)-MRSA. Methods: MICs were determined by CLSI guidelines. Single dose PK studies were conducted. The thighs of neutropaenic mice were inoculated (~10 6 ) with a single isolate of HA-MRSA or CA-MRSA. API-1252 and linezolid were administered orally in single doses ranging from 1 to 300 mg/kg. CFU were determined in infected thighs 24 hours postdose. Efficacy was determined as the change in CFU/thigh at 24 hours versus the 0 hour controls. Results: MICs of API-1252 and linezolid against both isolates were 0.004 and 2 mg/L, respectively. For the HA-MRSA, CFU reduction was seen with API-1252 doses of 10 mg/kg. While the 10 mg/kg dose displayed a variable effect for the CA-MRSA, doses of 40 mg/kg of API-1252 resulted in sustained antibacterial activity. Linezolid doses of 100 mg/kg were required to maintain sustained antibacterial activity over the 24 hour exposure period for both isolates. With both MRSA isolates, the 80% maximally effective dose (ED80, mg/kg), ED50, and ED5 were 4−20× lower for API-1252 when compared to linezolid. Pharmacodynamic index correlations showed that the fAUC/MIC of API-1252 was the best predictor of efficacy. For both MRSA strains the API-1252 fAUC/MIC ranges resulting in the ED80, ED50, and ED5 were 32−69, 29−32, and 6−25, respectively. These ED values were similar to that observed with MSSA for API-1252 (ICAAC 2006, Abstract F1-759). Conclusion: These data demonstrate the superior dose-response of API-1252 as compared to linezolid against HA-MRSA and CA-MRSA in the mouse thigh model. fAUC/MIC appears to best characterise the PD profile of this novel agent. The low efficacious fAUC/MIC values support the further development of API-1252 as a novel oral and intravenous agent for challenging staphylococcal infections. The emergence of multi-drug-resistant pathogens, including methicillinresistant Staphylococcus aureus (MRSA), combined with the absence of new antibiotics from the pharmaceutical sector demands that alternative anti-MRSA agents are scientifically evaluated and developed as a matter of urgency. Recent studies have shown the enormous potential of the use of phage endolysins as potential therapeutics. Objectives: To characterise the staphylococcal phage K-derived protein, LysK, a bifunctional endolysin with antimicrobial activity against MRSA, with a view to identifying the domain or domains responsible for lytic activity. Methods: The lysK gene was PCR-amplified from phage K cDNA and cloned into the pTOPO (Invitrogen) vector. The domain architecture of LysK was studied by performing deletion analysis on the intact LysK protein using PCR and/or slicing by overlap extension (SOEing) PCR on this pTOPO clone. To assess the activity of LysK deletion derivatives, constructs were recreated in the pQE60 (Qiagen) expression system. SDS-PAGE and zymogram assays were used to visualise the activity of LysK and its deletion derivatives in pQE60 and to assess which domain(s) the lytic activity of LysK could be ascribed to. Results: Bioinformatic analysis of LysK (495 amino acids) suggests that it has a modular structure, containing two peptidoglycan hydrolase domains, CHAP (endopeptidase activity) and Amidase_2 (N-acetylmuramoyl-L-alanine amidase activity), at the N-terminus and a cellwall binding domain at the C-terminus (SH3b). Analysis of deletion derivatives of LysK confirmed that while Amidase_2 and SH3b domains had no significant activity alone, the CHAP domain was as active as the intact LysK, displaying an identical lytic spectra when examined by zymographic assays. While attempting to define the smallest possible functional CHAP domain with antimicrobial activity, we found that the endopeptidase activity associated with the CHAP domain is contained within the first 161 amino acids of LysK. Further deletions resulted in a complete loss of antimicrobial activity. The 161 amino acid truncated CHAP has been found to be active against the main MRSA strains emerging in hospitals in the local area. Conclusion: NXL101 retained activity against highly FQR SA exhibiting a range of QRDR mutations and CFX efflux. NXL101 must target topoisomerases in a manner distinct to that of FQs. Harnessing the host response for antiinfective therapy S459 Controlling pathogenic bacteria with phage lytic enzymes Bacteriophage lytic enzymes are highly evolved molecules used by the phage to quickly destroy the bacterial cell wall to release phage progeny. We have exploited the rapid and lethal action of these enzymes to destroy pathogenic and biological warfare bacteria on mucous membranes and in blood. These enzymes in general are specific for the species or strain from which they were produced, thus avoiding destruction of the surrounding normal commensal organisms found on mucosal surfaces. We now have enzymes that are specific for Streptococcus pyogenes, S. pneumoniae, Bacillus anthracis, Staphylococcus aureus, Enterobacter faecalis/E. faecium and group B streptococci. Our results show that in vitro 10 7 bacteria can be reduced to sterility seconds after enzyme contact. In animal model experiments, we were able to colonise mice with either streptococcal or pneumococcal species (orally or nasally) and remove these completely with phage enzymes delivered to these sites using a single enzyme dose. In a septicaemia model with S. pneumoniae, bacteria are reduced by >2-logs from the blood of infected animals with a single intravenous dose of enzyme. A lytic enzyme called PlyG from the gamma-phage of B. anthracis was specific for all worldwide isolates of B. anthracis. The enzyme specifically killed B. anthracis with no effect on other bacilli or other organisms. When >1 LD100 of B. anthracis bacilli were delivered i.v. to mice we observed a progression of symptoms, leading to survival of only 10% of animals followed for 12 days. When PlyG was injected i.v. 15 min after infection, a significant therapeutic effect was observed in which 90% of the mice recovered fully. Resistance to the enzymes has not been found nor do antibodies neutralise their activity. Furthermore, a combination of antibiotic and enzyme has been shown to work synergistically resulting in efficient lethal activity in cases of antibiotic resistant bacteria. Thus, phage lytic enzymes are a new reagent that may be used in hospitals, nursing homes and the general population to control antibiotic resistant pathogenic bacteria in blood and on mucosal surfaces, offering a capability previously unavailable. Two-component signal transduction systems are ubiquitous in bacteria and are woven within the fabric of the regulatory processes that are used to sense environmental change and respond accordingly. These systems allow the bacterial cell to continually re-programme patterns of gene expression rapidly and in defined ways to bring about cellular adaptation as a direct consequence of specific environmental signals. To achieve this function two-component systems are composed of a signal ligandresponsive sensor histidine kinase that is usually an integral membrane protein, and a response regulator that is often a transcriptional regulator. Signal ligands are sensed by the sensor histidine kinase resulting in ATP-dependent autophosphorylation and the subsequent transfer of the phosphoryl moiety to the cognate response regulator influences its DNA binding and transcriptional activity, and therefore gene expression. For bacterial pathogens these signal transduction pathways are frequently key elements in the regulation of a myriad of virulence responses that facilitate colonisation, survival and persistence within the host environment and at specific host sites. Examples of the types of virulence attributes modulated by two-component systems include biofilm formation, quorum sensing, resistance to host defence peptides, and secreted toxin production. In addition, two-component systems that are essential for bacterial viability are known to exist. For these and other reasons two-component signalling systems and their variants have been recognized as targets for the development of anti-infective drugs. In this presentation the features of two-component systems that make them potentially attractive targets for the development of novel antiinfectives will be discussed. The mechanisms of action and limitations of some classes of previously developed two-component system inhibitors will also be discussed, as will the current and future perspectives for the development of selective inhibitors of these bacterial signalling systems. The global increase of methicillin-resistant Staphylococcus aureus (MRSA) has generated much attention over the last decade. The public have linked the so-called 'superbug' with their experience of dirty hospitals, but the precise role of cleaning in the control of this organism is unknown [1] . There is some support for a link between poor hygiene in hospitals and MRSA, since its epidemiological characteristics permit survival in the clinical environment as well as make it potentially vulnerable to the cleaning process [2] . Unfortunately, we cannot assess the risk of acquiring MRSA from the clinical environment because there is no way of measuring the effect of cleaning. Perhaps it is time to introduce microbiological standards for surface levels in hospitals [3] . This would not only allay concerns over the grading of hygiene by visual assessment, but would provide a means whereby the removal of dirt becomes an evidence-based science [3, 4] . Without such evidence, the importance of a clean hospital will continue to remain speculative. Even if the benefits of cleaning are well established, buffing the floors to a shine will not reduce the number of patients acquiring MRSA. Floors may form a repository for a variety of organisms but they do not play a major role in HAI [5] Pathogens are delivered to patients on hands, and it is more likely that contaminated hand-touch sites are a greater risk for MRSA acquisition [3, 6] . Such areas should be prioritised when managing cleaning schedules, since a targeted approach to hospital cleaning might be a useful control factor for MRSA [7] . We will never be able to guarantee consistent hand hygiene nor a sustained reduction in antibiotic consumption. Improvements in cleaning, however, are not insurmountable [1] . More research on the association between MRSA and environmental hygiene is urgently required. Basic cleaning could deliver significant cost benefits in MRSA control and could ultimately be our only defence against this organism. Carriage of MRSA is most often transient but can be persistent. In patients carriage is associated with an increased risk for the development of infection. Healthcare workers who carry MRSA may transmit the micro-organism to patients. To control transmission and prevent the development of infection, eradication of carriage may be indicated. The decision to start decolonisation therapy should be carefully balanced with the risk for the development of resistance. Factors that are associated with increased failure rates are: the presence of wounds or other skin lesions, the presence of indwelling devices, multisite carriage and the presence of reservoirs at home. In individuals without these risk factors treatment with mupirocin nasal ointment is succesfull in the majority of subjects. It is often combined with the application of antiseptic showering. In individuals who fail on this topical treatment or with the presence of risk factors for failure, systemic antimicrobial treatment is indicated. Well-designed trials are not available so at present an evidencebased recommendation can not be made. The existing data indicate that a combination of two agents including rifampicin is preferred. The choice of the agents should be based on the susceptibility testing results. Treatment failure may be based on recolonisation from a persisting reservoir at home. Therefore, in patients who (repeatedly) fail on decolonisation therapy these sources should be sought for. The European Food Safety Authority (EFSA) was set up in 2002 following a decade of food scares and a loss of confidence by the European public which led to a complete overhaul of the European Union food safety system and policies. EFSA's mission is to provide Risk Assessment on matters related to Food and Feed Safety and to communicate on these risks. In 2005, twenty-four Member States, Iceland, Norway, and Switzerland submitted information on the occurrence of zoonoses, zoonotic agents, antimicrobial resistance and foodborne outbreaks to the European Commission and EFSA. Campylobacteriosis was found to be the most frequently reported zoonotic disease in humans within EU. Reported Campylobacter cases increased by 7.8% compared to the previous year rising to an incidence rate of 51.6 cases per 100,000 people and to a total of 197,363 recorded cases. Salmonellosis remained the second most frequent zoonosis with 171,775 reported human cases, despite the fall by 9.5% to an incidence rate of 38.2 compared to 2004. Salmonella was most often reported from fresh poultry and pig meat where proportions of positive samples up to 18% were detected. In table eggs, findings of positive samples ranged from 0% to 6%, but over the past 5 years, an overall decreasing trend in occurrence of Salmonella in the eggs was observed. In animal populations, Salmonella was most frequently detected from poultry flocks. Salmonella, Campylobacter and viruses were the most important causes of reported foodborne outbreaks in 2005. Egg and bakery products were the most common reported sources of Salmonella outbreaks, whereas broiler meat was an important source for both Salmonella and Campylobacter outbreaks. Foodborne virus outbreaks were most often caused by drinking water, fruits and vegetables. Relatively high proportions of Campylobacter and Salmonella isolates from animals and food were resistant to antimicrobials commonly used in treatment of human diseases. This is especially the case for resistance to fluoroquinolones in Campylobacter isolates from poultry. Foodborne infections caused by these resistant bacteria pose a particular risk to humans due to possible treatment failure. When the results of the routine monitoring of laying-hen flocks are compared to the results from an EU-wide, fully harmonised Salmonella baseline study in laying-hen holdings, the prevalences in the baseline study are remarkably higher than those in routine monitoring. This reflects the different sensitivities of sampling scheme and sample types used and demonstrates that a harmonised protocol should be used when comparing the situation in one Member State with another. Yersinia enterocolitica and Y. pseudotuberculosis infections (yersiniosis) occur predominantly in the northern hemisphere. The clinical manifestations range from acute gastroenteritis to divers postinfectious sequelae, most notably reactive arthritis. The pathogenicity of these Gram-negative bacteria has been studied intensively during the last decades, resulting in identification and molecular characterisation of a set of chromosomally and extrachromosomally (plasmid pYV) encoded pathogenicity factors (PF). Here, the function of the PFs in the context of cell culture and animal infection models will be discussed. The most intriguing PFs are the yersinia outer proteins (Yops) which are "microinjected" into host cells via a type 3 secretion system (T3SS) and function as "tranquilliser" towards the innate host defence. Several PFs of yersiniae have become prototypic members of growing families of PFs: such as the invasin, the trimeric autotransporter (TATA) Yersinia adhesin (YadA) and the siderophore yersiniabactin encoded by high pathogenicity island (HPI). The Yersinia invasin (Inv) is closely related to the intimins (EaeA) of enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC). The Yersinia adhesin (YadA) is a trimeric autotransporter and member of the oligomer coiledcoil adhesin (Oca) family which comprises adhesins of Haemophilus influenzae (Hia), Moraxella catarrhalis (UspA) and other species. The yersiniabactin biosynthetic gene cluster of the HPI is widely spread among members of the family of Enterobacteriaceae and has become a marker for extraintestinal pathogenicity, in particular in E. coli. The molecular analysis of the pathogenicity of Yersinia has not only improved our understanding of bacterial mechanism of invasion and persistence but has also provided us with new tools for laboratory diagnosis and prevention. Several PFs turned out to be useful antigens for (i) differentiation between virulent and avirulent Y. enterocolitica, (ii) detection of a class-specific serum antibody response (serological diagnosis) and (iii) vaccination. Moreover, the T3SS of yersiniae is suitable for delivery of antigens to antigen presenting cells and, thus, opens a new strategy for the design of oral live vaccine carrier strains. Heesemann J, Sing A and Trülzsch K. 2006. Yersinia's stratagem: targeting innate and adaptive immune defence. Curr Opin Microbiol 9: 55−61. S468 Diarrhoea-causing Escherichia coli pathotypes -which genes to target for identification? In the past decades various groups of Escherichia coli associated with diarrhoeal diseases in man have been recognized. Currently, the most frequently identified pathotypes are the enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), enterohaemorrhagic (EHEC), enteroaggregative (EAEC) and diffusely adherent (DAEC) E. coli strains. Individual classes are defined on the basis of more or less characterised interactions between the bacterial cell and the host when inducing diarrhoea. There are several problems associated with the recognition of these strains in the laboratory. Easily detectable phenotypic markers sufficiently specific and sensitive to distinguish these strains from their non-pathogenic counterparts are not known. Although certain virulence factors (toxins, adhesins, invasins) are always, or often present in particular groups, the detection of their expression frequently goes beyond the capabilities of diagnostic laboratories. The use of molecular methods specific to genes of these factors, often in multiplexed PCR systems, is currently the most straightforward diagnostic approach. The detection of some of these genes (e.g. Shiga toxin, stx, or intimin, eae) can serve screening purposes but their identification may not prove the actual virulence of the isolate. Virulence factors can be shared by members of different pathotypes, and can be present in isolates recovered from healthy individuals or in non-pathogenic strains. For some classes it is still not clear which particular combination(s) of these genes are necessary to cause disease (e.g. EAEC), while in others the existence of subgroups with various pathogenic potential are already known to exist (e.g. EPEC, or Shiga toxin producing strains). Currently, the lack of straightforward diagnostic algorithms limits our knowledge on the epidemiology of diarrhoea-causing E. coli. Furthermore, more data on the incidence of these pathotypes should help to understand the role of the individual host's susceptibility in the outcome when encountering these strains. Since their discovery antimicrobials have been extensively used in livestock and poultry, with significant beneficial effects on food animal health and production efficiency. Most classes of antimicrobials used in animals, including fluoroquinolones, have human analogues. Fluoroquinolones (FQ) are used in animal production primarily for: (1) therapy; (2) prophylaxis; (3) infection control (metaphylaxis); and (4) growth promotion in healthy animals. In contrast to human medicine in which treatment is customarily directed at the patient, entire groups of animals may be treated with the use of medicated feed and/or water. Moreover, growth-promoting dosages are usually at low concentrations for extended time periods, therefore both practices are a potentially significant driving force in accelerating the emergence of resistant bacteria in these animals that can be transferred through contact or food to infect humans. The acquisition of quinolone resistance in Gramnegative bacteria is mainly due to chromosomal mutations either in topoisomerase genes (mainly gyrA and parC) or in genes associated with a decreased uptake or by increased efflux of quinolones. In addition, a plasmid conferring low levels of quinolone resistance linked to the presence of the qnr gene, which encodes a protein protecting DNA gyrase and topoisomerase IV from quinolones, has been reported. The above mentioned mechanisms have been described in both Escherichia coli and Salmonella spp. and generate an increased level of FQ resistance in a step-wise model, with the first mutation found either in the gyrA gene or in genes involved in efflux pump(s), such these producing an overexpression of acrAB. However, Campylobacter spp. easily acquire high levels of FQ resistance associated with a mutation in the gyrA gene, likely because this microorganism does not have topoisomerase IV. Several studies have demonstrated an association between FQ use in animals and the subsequent isolation of FQ-resistant bacteria from the same animal. Antimicrobial-resistant enteric pathogens can reach humans through direct animal contact, or more commonly, through ingestion of contaminated water or foods. Three scenarios may be proposed by which the use of FQ in food animals could affect the treatment of diseases in humans: (1) FQ-resistant bacterial pathogens are selected, and food is contaminated during slaughter and/or preparation. After consumption of the food, these pathogens cause an infection that requires antibiotic treatment and therapy is compromised; (2) FQ-resistant bacteria non-pathogenic to humans are selected in the animal. When the contaminated food is ingested, the bacteria transfer FQ-resistance determinants, such as plasmid carrying the qnr gene, to other bacteria in the human gut, commensal and potential pathogens; and (3) FQs remain as residue in food products, which allow the selection of antibiotic-resistant bacteria after the food is consumed. In conclusion, ongoing surveillance of the antimicrobial susceptibility profiles of foodborne pathogens is needed to identify emerging antimicrobial-resistant phenotypes within the food production continuum. Moreover, barriers to stop the dissemination of FQ or other antimicrobial-resistant bacteria from animals to humans should be improved. Objectives: It has been suggested that tuberculosis (TB) in the elderly is often atypical and difficult to diagnose. There is a lack of information about TB in people over 80 years of age. The aim of this study was to examine current clinical manifestations, time to diagnosis and outcomes in old ( 65 years) and in very old ( 80 years) patients with TB. Diagnosis of TB infection was based on anamnesis for TB risk, two step tuberculin skin testing (TST) and chest X-ray. Patients were followed up throughout the period of chemoprophylaxis and adherence was monitored by determining isoniazid metabolites in urine (or urine colour when appropriate). A positive TST was defined as an induration 5 mm. Results: 210 patients, 64% women, mean age 52 years, were evaluated. Baseline illness: 60.5% rheumatoid arthritis, 13.8% cutaneous psoriasis, 13.3% psoriatic arthritis, 11% ankylosing spondylitis, and 1.4% Crohn's disease. Thirty-eight (80.2%) patients were on immunosuppressive treatment. Thirty-one (14.9%) patients had BCG-vaccination, 6 had been treated for TB, and 2 had prior positive TST. Of 201 patients who underwent TST, 84 (41.8%) resulted positive (59 and 25 in the first and second test respectively), and 117 (58.2%) resulted negative. Chemoprophylaxis was given to 79 (39.3%) patients: 78, isoniazid for 9 months and 1 rifampin for 4 months. Three patients (3.8%) experienced a 5-fold increase of transaminase level above the ULN. After a 237 patient-years follow up, one of the 130 patients who finally received anti-TNF treatment developed TB (0.42%; 95% CI: 0.01−1.71). His twostep TST resulted negative and he began on adalimumab. Five months later, TB developed. Conclusion: Systematic and protocolised assessment for TB infection and its treatment when indicated is a reliable and useful method to prevent anti-TNF-associated TB. Our data show that two-step TST is helpful to detect TB infection in a significant number of patients. Prolonged treatment with isoniazid seems to be safe in anti-TNF treated patients. Objectives: Interferon-gamma release assays (IGRA), such as the QuantiFERON ® -TB Gold In-Tube (QFT-GIT; Cellestis Ltd., Carnegie, Australia), are increasingly used for the diagnosis of tuberculosis (TB). These tests exploit immunological mechanisms that normally contain Mycobacterium tuberculosis (Mtb) infecting the human host. Failed containment results in clinical TB and is associated with proliferating Mtb. We thus wondered if in this situation indeterminate or negative QFT-GIT results were associated with positive microscopy, thus serving as a surrogate for the bacterial burden. Methods: Between November 2004 and October 2006, 67 consecutive patients with culture-confirmed TB were prospectively enrolled. Patients had received no or less than 14 days of antituberculous therapy (n = 58) or were recruited within two months since begining treatment (n = 9). Severe immunosuppressive conditions were present in 14 (21%). QFT-GIT was performed according to the manufacturers' instructions. Laboratory investigations included fluorescence microscopy for acidfast bacilli, and culture on both liquid and solid media. Univariate and multivariate analysis were done in StatView ® version 5.0 (SAS Institute Inc., Cary, NC). Results: Overall, 52%, 36%, and 12% of the patients had pulmonary, extrapulmonary, or combined pulmonary and extrapulmonary TB, respectively. In one patient, no microscopy result was available as Mtb was only detected in blood culture. Microscopy was positive in 40/66 patients (61%; 95% CI: 49−72%); respiratory and extrapulmonary specimens were smear-positive in 30/39 (77%) and 10/27 (35%) of TB patients, respectively. Overall, QFT-GIT was positive in 51 (76%; 95% CI: 65−85%) TB patients; indeterminate or negative QFT-GIT results occurred in 6 (9%) and 10 (15%) TB patients, respectively. Age and gender did not influence the QFT-GIT test result. However, the likelihood of having a negative or indeterminate QFT-GIT test result was significantly and independently associated with a positive microscopy (odds ratio [OR] 5.8, 95% CI: 1.2−28.2%; p = 0.03) and with immunesuppression (OR 3.2, 95% CI: 0.91−11.4, p = 0.08). Conclusions: To our knowledge this is the first study that demonstrates a significant association of smear-positive TB and negative or indeterminate QFT-GIT results. This association was independent of, and stronger than, the effect of concomitant immune-suppression. The finding has implications for the use of QFT-GIT in clinical practice. Objective: Duration of exposure to tuberculosis (TB) is a major risk factor for transmission of the disease. Current WHO guidelines, based on transmission of TB on aeroplane flights, recommend an 8 hour period as a cut off for screening. However this was before the routine use of IFN-g assays. We compared Mantoux skin testing with QUANTIFERON-TB GOLD (QFN) in a hospital associated outbreak, stratifying patients by duration of contact. Methods: Hospital contacts of the index case were screened in a sequential manner (close contacts sharing the same room for 8 hours, contacts on the same ward for 8 hours and contacts on ward <8 hours Objectives: Nation-wide surveillance on transmission and resistance of Mycobacterium tuberculosis in the Netherlands has functioned since 1993. Presumably due to a lower transmissibility and a well-organised tuberculosis control, transmission of multi-drug resistant tuberculosis (MDR-TB) was so far limited to single secondary cases. However, a MDR strain with an unusually low level of rifampicin resistance (MIC 1−2 mg/l) was transmitted from a single source to nine persons, of whom three developed active disease so far. In the current study we assessed the molecular basis of the INH and rifampicin resistance of the outbreak strain and its implications for transmissibility and therapy. Methods: The katG and rpoB gene of the MDR strain were sequenced and the minimum inhibition concentration (MIC) to INH and rifampicin was determined. DNA fingerprinting of M. tuberculosis isolates and conventional contact tracing was performed to investigate the spread of the MDR strain. Results: The respective outbreak strain had a Ser315Thr mutation in the katG gene (k315). INH resistant strains with k315 mutation were previously found to maintain a higher transmissibility than other INH resistant strains. An Asp516Tyr mutation was found in the rpoB gene (r516). Phenotypically, most of the outbreak isolates were interpreted resistant, but a part of the isolates were read as susceptible, which confused the therapy guidance and the surveillance of MDR-TB. Conclusion: The relatively high rate of transmission of this k315/r516 variant may be related to evolutionary development of M. tuberculosis to maintain its transmissibility despite the adaptation to withstand the therapy by our most important anti-mycobacterial drugs; INH and rifampicin. More studies are needed to investigate the contribution of k315/r516 MDR strains to transmission of MDR-TB. The consequences of the unusual low level of rifampicin resistance for therapy guidance and surveillance will be determined. It is considered to introduce the term "intermediate susceptibility" to indicate this level of rifampicin resistance. Objectives: The "Real Time TB" assay (RT-TB) is a new multiplex real time PCR assay that allows the rapid detection and differentiation of the M. tuberculosis complex from other mycobacteria. The assay targets the IS6110 element and the RD9 specific region that differentiate M. tuberculosis (IS6110 positive -RD9 positive) from the other mycobacterial species included in the M. tuberculosis complex (IS6110 positive -RD9 negative). We recently evaluated the first version of the RT-TB kit, demonstrating that the assay is sensitive, particularly in smear negative-culture positive clinical samples. Here, we report on the evaluation of a new version of the RT-TB assay in which the labelling of the IS6110 probe has been modified in order to improve its stability and detection level. The new RT-TB BioRad kit includes an IS6110-BRD04 probe (instead of IS6110-Tamra in the previous version) that increases by 10-fold the sensitivity of detection of the IS6110 amplicons. Fifty-two clinical samples collected in routine (sputa, bronchoalveolar and gastric lavages) were included. DNA preparations and RT PCR reactions were carried out according to the manufacturer's instructions. A new specific software developed by the manufacturer was used for the automated analysis of the RT-TB amplification results. Results: In the present study, 2 samples were found to contain inhibitors (as indicated by the negative amplification signals of the corresponding internal controls). The 8 smear positive samples included in the study were all found to be IS6110-POS and RD9-POS. Interestingly, 4 of them showed <1 acid fast bacilli per field on microscopic examination. The good sensitivity of the test was confirmed by 2 smear negative samples which were both found to be IS6110-POS and RD9-POS by the RT-TB assay. These two samples were confirmed to be positive for M. tuberculosis by the Roche Amplicor assay. Finally, the 40 remaining smear-negative samples were all found to be negative for IS6110 and RD9, suggesting that the increased sensitivity of the new IS6110-BRD04 probe does not impair the specificity of the test. The results obtained for the first evaluation of the new version of the RT-TB kit suggest that the assay is sensitive and specific and may be very promising for the detection of M. tuberculosis in clinical samples containing few bacilli. Further experiments are in progress to confirm these preliminary data. Objectives: A Dot-Blot hybridisation assay that detects all mutations occurring in the M. tuberculosis rpoB hot-spot region is being developed. The assay uses five probes, capable of binding to a different target segment within the rpoB hot-spot region of the wild-type M. tuberculosis genome. The present study is a preliminary investigation to assess the suitability of the assay for detection of resistance mutations in rpoB in clinical isolates of M. tuberculosis from Delhi, India. Methods: Susceptibility testing of 142 isolates of M. tuberculosis was carried out by proportion method and confirmed by BACTEC 460TB system. Dot-Blot assay was performed on 106 isolates with two of the probes hybridising to the wild-type sequence of M. tuberculosis, from codons 522 to 527 (Probe D) and 528 to 533 (Probe E). Absence of hybridisation with any of the probes in the assay when a mutation was present indicated Rifampicin resistance, a surrogate marker for Multidrug-resistant M. tuberculosis. Results: Susceptibility testing of 142 isolates revealed isoniazid resistance in 53.5%, rifampicin resistance in 41%, streptomycin resistance in 53% and ethambutol resistance in 37% of strains. Fortyfive strains (32%) were multidrug resistant. Further analysis of the data showed that 96.4% of rifampicin-resistant strains and 100% of ethambutol-resistant strains had co resistance to one or the other antituberculous drug. It was also interesting to note that 77% of the rifampicin-resistant strains were multidrug resistant, while the corresponding figure for ethambutol-resistant strains was 87%. Dot-Blot hybridisation assay with probes D and E was carried out on 106 isolates of M. tuberculosis, of which 43 were resistant to rifampicin. Of the rifampicin-resistant isolates tested, 15 did not hybridise with probe E, indicating a mutation at this site. The results of 10 strains were confirmed by sequencing when a mutation was detected in 9 strains at codon 531 and in one of them at codon 533. The remaining 28 rifampicin-resistant strains, which hybridised with probe E, may have a mutation at a site other than that complimentary to probe E. Of these, 10 hybridised with probe D, indicating a mutation at the site complimentary to probe D (codons 522 to 527). Of the 63 rifampicin-susceptible isolates, 62 (98%) hybridised with probes D and E, indicating a wild-type sequence. The Dot-Blot assay was found to be a sensitive and specific assay. , and methicillin-susceptible S. aureus (MSSA), respectively, by the MALDI-TOF with correct identification rates of 100%, 97.7% and 93.3%, in comparison with those identified by phenotypic methods. The agreement rate was 75.6% with those determined by the API STAPH when these CoNS isolates were identified into species level by the MALDI-TOF. Seven PVL-positive MRSA isolates were correctly recognized by the MALDI-TOF with 100% accordance with the results determined by real-time PCR. The whole process, from the sample preparation to result analysis, can be completed between 1.5 and 2.5 hours, which greatly shortens the time usually needed for current phenotypic identification. Conclusion: MALDI-TOF mass spectrometry provides a rapid and relevant system for clinical identification of staphylococci. Detecting PVL protein directly from clinical isolates provides a bacterial identification system that is desirable in clinical diagnostic services. Objectives: Bacteroides fragilis is the Gram-negative anaerobe isolated most often from human infections. Some B. fragilis strains produce a 20 kDa enterotoxin (BFT). Enterotoxigenic B. fragilis (ETBF) have been isolated from various diarrhoeic animal species and epidemiological studies worldwide note a significant correlation between ETBF and human diarrhoeal disease, especially in children. The prevalence of ETBF in gastrointestinal diseases in the UK, however, has not been investigated, partly due to the lack of a simple identification test that can be used in diagnostic laboratories. The aim of this study was to develop a sensitive and specific multiplex PCR assay for the detection of ETBF directly from faeces, and to investigate if ETBF are associated with cases of community-acquired diarrhoea in the UK. Methods: Primers were designed from published enterotoxin sequences to amplify 416 bp of bft, and multiplexed with primers amplifying 293 bp of nanH (+ve control for presence of B. fragilis). The assay was validated against 136 strains from 52 species. DNA was extracted from stools using the QIAamp DNA stool mini kit (Qiagen). Spiking experiments were used to determine sensitivity of the assay. Stool was obtained from 193 cases of community-acquired diarrhoea, selected on the basis that no other bacterial pathogen had been identified. Where bft was detected in DNA extracts, a second novel multiplex PCR assay was used to determine which of the 3 bft isoforms was present. Results: The PCR assay for ETBF was found to be 100% specific and had a detection limit of 10 4 cfu/g stool. 13% (25/193) of the diarrhoea samples gave a positive result for bft and nanH. The predominant isoform in the ETBF +ve samples was bft-1 (n = 20, 80%). Three samples had bft-2 and 1 had bft-3. A diarrhoeal sample from a 1-yr old male yielded PCR amplimers for both bft-1 and bft-2 suggesting carriage of at least 2 different ETBF strains. Conclusion: The multiplex PCR assay was highly specific for ETBF and allowed detection without the need for culture. This is the first report of ETBF in community-acquired diarrhoea in the UK. The distribution of isoforms in the clinical samples was similar to earlier reports from Europe, but the occurrence of 2 different bft isoforms in a faecal sample has not been described before. The finding of ETBF in a high proportion (13%) of samples for which there was no other bacterial explanation for the diarrhoea merits further investigation of this pathogen.