CORD-19_Custom_license_subset  

CORD-19:e6e0128e5f23630f1be605cb1a7fb4cc8261f323 JSONTXT 8 Projects

Annnotations TAB TSV DIC JSON TextAE Lectin_function

Id Subject Object Predicate Lexical cue
T1 0-21 Sentence denotes Under the microscope:
T2 22-81 Sentence denotes From pathogens to probiotics and back Under the microscope:
T3 82-119 Sentence denotes From pathogens to probiotics and back
T4 121-129 Sentence denotes Abstract
T5 130-413 Sentence denotes The review centers on the human gastrointestinal tract; focusing first on the bacterial stress responses needed to overcome the physiochemical defenses of the host, specifically how these stress survival strategies can be used as targets for alternative infection control strategies.
T6 414-572 Sentence denotes The concluding section focuses on recent developments in molecular diagnostics; centring on the shifting paradigm from culture to molecular based diagnostics.
T7 574-701 Sentence denotes The unifying theme of my research centers on the microbiology of the gut (more specifically, the human gastrointestinal tract).
T8 702-782 Sentence denotes My interests in this niche map to two main thematic areas (outlined in Fig. 1 ).
T9 783-1018 Sentence denotes The first, deals with the elucidation of bacterial stress responses (i.e. the molecular mechanisms enabling bacterial gastrointestinal persistence), and the exploitation of these mechanisms in the design of novel biocontrol strategies.
T10 1019-1152 Sentence denotes The second, molecular diagnostics, centers on the identification and characterization of new and emerging gastrointestinal pathogens.
T11 1153-1201 Sentence denotes Herein, I review my contributions in this field.
T12 1202-1438 Sentence denotes My research career began with a focus on bacterial stress responses; determining how bacteria overcome the various stresses encountered in food production/storage and subsequently, following ingestion, within the gastrointestinal tract.
T13 1439-1681 Sentence denotes The Gram-positive foodborne bacterium Listeria monocytogenes, a physiologically robust and versatile gastrointestinal pathogen, proved to be the ideal model organism in the early years, which focused on four main stress resistance mechanisms:
T14 1682-1684 Sentence denotes 1.
T15 1685-1800 Sentence denotes Acid tolerance -The ability to resist the low pH of pickled foods and the acidic environment of the stomach (»pH 2)
T16 1801-1803 Sentence denotes 2.
T17 1804-2003 Sentence denotes Osmotolerance -The ability to grow and survive in dried foods and to resist the low a w of the gastrointestinal tract (with an osmolarity equivalent to 0.3 M NaCl -approximately twice that of blood).
T18 2004-2006 Sentence denotes 3.
T19 2007-2139 Sentence denotes Bile tolerance -Resistance to the biological detergent bile, produced in the liver and stored inter-digestively in the gall bladder.
T20 2140-2268 Sentence denotes As well as aiding the emulsification and digestion of fats, bile forms an important component of the innate defenses of the gut.
T21 2269-2271 Sentence denotes 4.
T22 2272-2401 Sentence denotes Barotolerance -the ability to overcome pressures in excess of 300 MPa, associated with high pressure processing of certain foods.
T23 2402-2527 Sentence denotes The first significant challenge encountered by gastrointestinal pathogens, following ingestion, is the low pH of the stomach.
T24 2528-2602 Sentence denotes Gastric transit is thus an essential first step in establishing infection.
T25 2603-2729 Sentence denotes In silico analysis of the L. monocytogenes genome identified lmo0292, a gene predicted to encode an HtrA-like serine protease.
T26 2730-2837 Sentence denotes 1 Mutation analysis revealed a role for htrA in acid tolerance and virulence potential in L. monocytogenes.
T27 2838-3008 Sentence denotes Furthermore, transcriptional analysis revealed that htrA is not only induced by low pH stress, but is also under the control of the two-component regulatory system LisRK.
T28 3009-3156 Sentence denotes 2 Originally identified as an acid tolerance locus and listerial virulence factor, LisRK is also linked to b-lactam resistance in L. monocytogenes.
T29 3157-3230 Sentence denotes 3 Moreover, we identified LisRK and HtrA as important osmotolerance loci.
T30 3231-3427 Sentence denotes 2 Indeed, mutational analysis revealed that in addition to regulating HtrA, LisRK likely controls the transcription of one or more additional systems necessary for optimal listerial osmotolerance.
T31 3428-3686 Sentence denotes Furthermore, it appears that LisRK may act at the initial stages of the listerial osmotic stress-response, with LisK possibly functioning as the primary sensor and LisR (and its genetic targets, e.g. htrA) as the primary respondent(s) to elevated osmolarity.
T32 3687-3894 Sentence denotes LisRK may thus represent a new dimension in the osmosensing and osmoregulatory capabilities of L. monocytogenes, functioning independently of, but in tandem with, osmolyte uptake synthesis (described below).
T33 3895-3926 Sentence denotes Osmotolerance L. monocytogenes.
T34 3927-4057 Sentence denotes In the late 1990s, knowledge of the listerial osmotolerance response was limited; confined mainly to physiological investigations.
T35 4058-4173 Sentence denotes My strategy was to apply genetic approaches to gain fresh insights into the mechanisms of bacterial osmoadaptation.
T36 4174-4389 Sentence denotes Indeed, my first published paper 4 described the identification and characterization of BetL, a membrane protein which protects Listeria at elevated osmolarities, by transporting the osmoprotective compound betaine.
T37 4390-4664 Sentence denotes Representing the first genetic analysis of osmotolerance in Listeria, this study also identified a putative s B -dependent promoter binding site upstream of betL, suggesting that betaine uptake in Listeria might be regulated, at least in part, at the level of transcription.
T38 4665-4803 Sentence denotes Indeed, we later proved the hypothesis, recording a 1.6-fold increase in betL transcript levels following exposure to elevated osmolarity.
T39 4804-5129 Sentence denotes 5 Later, while on sabbatical at the laboratory of Prof. Janet Wood at the University of Guelph, Ontario, Canada, I employed a nisin-controlled expression (NICE) system to decouple transcriptional control of betL, revealing that the BetL protein is itself activated (post-translationally) in response to changes in osmolarity.
T40 5130-5311 Sentence denotes Rapid activation of pre-existing BetL, in response to relatively low NaCl concentrations, suggested that the protein is one of the primary respondents to rapid fluxes in osmolarity.
T41 5312-5521 Sentence denotes 6 Furthermore, in addition to transcriptional and post-translational control, in silico analysis strongly suggests a role for translational control, not only of betL but of listerial osmoregulation in general.
T42 5522-5737 Sentence denotes 7 However, despite its obvious role in listerial osmotolerance, deleting betL did not significantly impair growth of the pathogen at elevated osmolarity; suggesting the existence of multiple osmolyte uptake systems.
T43 5738-5939 Sentence denotes In support of this, we described the identification and characterization of OpuC, a carnitine uptake system which contributes to osmotolerance, chill stress and virulence potential of L. monocytogenes.
T44 5940-6211 Sentence denotes 8, 9 Indeed, Sleator et al., 8 provided the first direct link between osmotolerance and virulence in Listeria, a link which we ascribed to the accumulation of carnitine, a protective compound similar to betaine but present at much higher concentrations in animal tissues.
T45 6212-6488 Sentence denotes Following the discovery of Gbu, a third osmolyte uptake system, by Gary Smith's group at the University of California, I undertook a systematic approach to knocking out each of the three uptake systems; BetL, OpuC and Gbu, creating a bank of single, double and triple mutants.
T46 6489-6867 Sentence denotes This bank allowed me to elucidate, for the first time, the exact role of each transporter against its native background; plotting a hierarchy of osmolyte and transporter importance under osmotic 10 chill stress 11 and desiccation conditions 12 We subsequently pushed the analysis further; 13 establishing the relative importance of each transporter in food and during infection.
T47 6868-7016 Sentence denotes Interestingly, while betaine appeared to confer most protection in food, the hierarchy of transporter importance differs depending on the food type.
T48 7017-7156 Sentence denotes While in the animal model, OpuC appears to play the dominant role, with the remaining systems contributing little to the infection process.
T49 7157-7304 Sentence denotes These findings have had a significant impact on the development of effective control strategies for L. monocytogenes in foods and during infection.
T50 7305-7592 Sentence denotes In addition to osmolyte uptake, we also identified the first osmolyte synthesis system in L. monocytogenes, proBA 14 While inactivation of proBA had no appreciable effect on virulence, growth at elevated osmolarity was significantly reduced in the absence of efficient proline synthesis.
T51 7593-7813 Sentence denotes Thus, while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of L. monocytogenes, it is required for survival in osmolyte-depleted environments of elevated osmolarity.
T52 7814-8009 Sentence denotes Furthermore, we showed that proline synthesis in Listeria is regulated by feedback inhibition of g-glutamyl kinase, the first enzyme of the proline biosynthesis pathway, encoded by the proB gene.
T53 8010-8137 Sentence denotes Random saturation mutagenesis of the proBA operon generated 3 independent mutations in proB, leading to proline overproduction.
T54 8138-8290 Sentence denotes 15 However, as observed for proline auxotrophy, proline hyperproduction has no apparent impact on listerial osmotolerance or indeed virulence potential.
T55 8291-8594 Sentence denotes Later, using the same random saturation mutagenesis strategy to target betL, we identified a single point mutation in a putative promoter region upstream of betL which led to a dramatic increase in transcript levels under osmo-and chill-stress conditions, and a concomitant increase in stress tolerance.
T56 8595-8752 Sentence denotes 16 Furthermore, the mutation appears to counter a previously unreported "twisted" cell morphology observed for L. monocytogenes grown at elevated osmolarity.
T57 8753-8849 Sentence denotes By 2001, my work, and that of others, had fully elucidated the listerial osmotolerance response.
T58 8850-8975 Sentence denotes 17 In less than 4 years, all of the principal osmolyte uptake and synthesis mechanisms had been identified and characterized.
T59 8976-9136 Sentence denotes This culminated with the publication of the L. monocyogenes genome sequence which we used to perform a complete in silico analysis of osmotolerance in Listeria.
T60 9137-9162 Sentence denotes 18 Cronobacter sakazakii.
T61 9163-9262 Sentence denotes My interest in osmotolerance did not end however with the conclusion of the Listeria story in 2001.
T62 9263-9472 Sentence denotes Motivated by a 2008 joint United Nations-World Health Organization call, I returned to the subject of osmotolerance, almost a decade later, this time targeting the Gram-negative pathogen Cronobacter sakazakii.
T63 9473-9541 Sentence denotes C. sakazakii is an extremely osmotolerant gastrointestinal pathogen.
T64 9542-9664 Sentence denotes 19 The most common route of infection is via powdered infant formula (PIF), resulting in an infant mortality rate of »80%.
T65 9665-9771 Sentence denotes Unravelling C. sakazakii' s osmotolerance mechanisms is thus a key first step in controlling the pathogen.
T66 9772-9899 Sentence denotes Unlike the situation with L. monocytogenes a decade earlier, the genome sequence of C. sakazakii was available from the outset.
T67 9900-10054 Sentence denotes An exhaustive homology transfer based screening strategy, using the Escherichia coli K12 genome as the template, identified 53 putative osmotoleance loci.
T68 10055-10387 Sentence denotes 20 Interestingly, while C. sakazakii contains homologs of all the principal E. coli osmotolerance loci; a key distinguishing feature is that C. sakazakii possesses multiple copies of certain osmotolerance genes; including seven copies of the E. coli proP homolog, each of which potentially encodes a separate osmolyte uptake system.
T69 10388-10527 Sentence denotes This level of degeneracy is apparently unique to C. sakazakii (offering a plausible explanation for its distinctive osmotolerance profile).
T70 10528-10667 Sentence denotes Indeed, gene expression analysis of each of the proP homologs demonstrated an increase in expression levels in response to osmotic upshift.
T71 10668-10859 Sentence denotes 21 Furthermore, heterologous expression against the osmotically sensitive E. coli MKH13 host revealed that 6 of the 7 homologs conferred an osmotolerance phenotype, albeit to varying degrees.
T72 10860-11093 Sentence denotes Further structural analysis of the C. sakazakii ProP homologs revealed that all but one (ProP1, which offers the greatest osmoprotective effect) are 60-70 amino acids shorter than the E. coli ProP; lacking the extended carboxyl tail.
T73 11094-11206 Sentence denotes This immediately suggested a role for the C-terminal domain in modulating the protein's osmoprotective function.
T74 11207-11476 Sentence denotes This hypothesis was proved by spicing the extended C-terminal domain of ProP1 (encoded by ESA_02131) onto the truncated C-terminal end of ProP2 (encoded by ESA_01706); creating a chimeric protein (ProPc) which exhibits increased osmotolerance relative to the wild type.
T75 11477-11713 Sentence denotes 22 In tandem with the sequence based approach, functional screening of a C. sakazakii genomic bank, heterologously expressed against the E. coli MKH13 background, revealed a novel role for ProP1 (ESA_02131) as a carnitine uptake system.
T76 11714-11975 Sentence denotes 23 Indeed, carnitine uptake via ProP1 allows growth at salt concentrations far in excess of that afforded by proline; a finding which has significant food safety implications, given that carnitine is often added to infant formula to boost its nutritional value.
T77 11976-12113 Sentence denotes Furthermore, we showed that carnitine also promoted the growth of L. monocytoenes in infant formula stored at refrigeration temperatures.
T78 12114-12322 Sentence denotes 9 Work in my lab has thus not only elucidated the C. sakazakii osmotoleance response, it has also helped to inform food safety policy -calling for a halt to the practice of adding carnitine to infant formula.
T79 12323-12434 Sentence denotes Furthermore, the osmotolerance mechanisms identified now provide ideal targets for the control of the pathogen.
T80 12435-12604 Sentence denotes One such approach, involves smugglin technology; 17,18 the use of antimicrobial compounds which structurally mimic carnitine, killing the cell rather than protecting it.
T81 12605-12745 Sentence denotes This represents a viable alternative to antibiotic therapy and may lead to effective control of the pathogen in high risk foods such as PIF.
T82 12746-12759 Sentence denotes Metagenomics.
T83 12760-12973 Sentence denotes In addition to focusing on individual gastrointestinal pathogens, my lab also employs a much broader metagenomics approach to identifying novel osmotolerance loci in the human gut microbiome (reviewed in 24, 25 ).
T84 12974-13257 Sentence denotes The human distal colon is one of the most complex and densely populated microbial ecosystems on Earth, with cell densities of up to 10 12 per gram of faeces. [26] [27] [28] A functional screen of over 20,000 clones from a human gut microbiota library revealed 53 osmotolerant clones.
T85 13258-13467 Sentence denotes A combined transposon mutagenesis and bioinformatic strategy revealed three genes (encoding GalE, MurB and MazG) from a single clone, exhibiting high levels of identity to a species from the genus Collinsella.
T86 13468-13646 Sentence denotes 29 Additionally, in silico analysis of two other clones revealed the presence of an additional galE and mazG gene, from Akkermansia muciniphila and Eggerthella sp., respectively.
T87 13647-13858 Sentence denotes Each of the three genes identified was found to be over-represented in the human gut metagenome and abundant among healthy subjects from the MetaHit data set, 24 suggesting an important role in gut colonisation.
T88 13859-14008 Sentence denotes Indeed, as expected, cloning and heterologous expression of the genes in E. coli MKH13 resulted in increased salt tolerance of the transformed cells.
T89 14009-14248 Sentence denotes A phenotype based microarray analysis identified one of the 53 clones as being positive for utilization/transport of carnitine, 30 while another of the loci was shown to be homologous to a brp/ blh-family b-carotene 15,15 0 -monooxygenase.
T90 14249-14494 Sentence denotes 31 In addition to the obvious osmotolerance phenotype, E. coli cell pellets expressing brpA adopt a red/orange pigmentation, when cultured in the presence of exogenous b-carotene, indicating the incorporation of carotenoids in the cell membrane.
T91 14495-14732 Sentence denotes Finally, a significant advantage of functional screens over sequence based approaches is that they provide a powerful means to identify and assign function to novel genes, and their encoded proteins, without any prior sequence knowledge.
T92 14733-14914 Sentence denotes In support of this, we describe the identification and subsequent analysis of an unknown gene (stlA, for "salt tolerance locus A") with no currently known homologs in the databases.
T93 14915-15155 Sentence denotes 32 The stlA gene is rare when searched against the human metagenome datasets, MetaHit and the Human Microbiome Project, and represents a novel and unique salt tolerance determinant which is apparently exclusive to the human gut environment.
T94 15156-15362 Sentence denotes In silico analysis of the L. monocytogenes genome revealed a two-gene operon (formerly opuB), preceded by binding sites for PrfA (positive regulatory factor A) and the alternative stress sigma factor, s B .
T95 15363-15681 Sentence denotes 18 Despite exhibiting significant sequence similarity to the betaine carnitine choline transporter (BCCT) family, further analysis suggested that the protein most likely functions as a bile efflux pump (actively extruding toxic bile out of the cell), a conclusion substantiated by radiolabelled bile exclusion studies.
T96 15682-15918 Sentence denotes In addition, functionally inactivating BilE resulted in a significantly increased sensitivity to physiological concentrations of human bile, and a reduction in virulence potential when administered orally to a murine model of infection.
T97 15919-16172 Sentence denotes 33 Renamed BilE (for Bile Exclusion), this protein not only represented a new and important virulence factor in L. monocytogenes, but also stimulated in me a new interest in bile tolerance as an important gastrointestinal associated virulence mechanism.
T98 16173-16320 Sentence denotes This was followed by the functional analysis of bsh, pva, and btlBthree genes previously annotated as bile-associated loci in the public databases.
T99 16321-16559 Sentence denotes 34 While deletion mutants revealed a role for all three genes in resisting the acute toxicity of bile, hydrolysis assays indicated that BSH is likely the only listerial bile salt hydrolase (leading to bile detoxification inside the cell).
T100 16560-16681 Sentence denotes Transcriptional analyses and activity assays further revealed that, as with bilE, bsh is regulated by both PrfA and s B .
T101 16682-16847 Sentence denotes The fact that both bilE and bsh are controlled by s B , along with betL, gbu and opuC (which also boasts a PrfA box), suggested a common function for all 4 proteins.
T102 16848-17046 Sentence denotes 35 In support of this hypothesis, a systematic analysis of strains with mutations in BetL, OpuC and Gbu revealed roles for OpuC, and to a lesser extent BetL, in resisting the acute toxicity of bile.
T103 17047-17208 Sentence denotes Furthermore, real-time gene expression profiling in the presence of bile, using a lux gene reporter system, revealed that both betL and opuC are induced by bile.
T104 17209-17459 Sentence denotes 36 Following ingestion, the first physical stress encountered by the bacterium is the low pH of the stomach (»pH 2), followed by elevated osmolarity (equivalent to 0.3M NaCl) and activity of the biological detergent bile in the upper small intestine.
T105 17460-17710 Sentence denotes Significantly, pre-exposure to elevated osmolarity (0.3 M NaCl for 1 h) resulted in a dramatic increase in the ability of L. monocytogenes to deal with lethal concentrations of bile (adapted cells surviving 1,000 times better than na€ ıve cells 37 ).
T106 17711-17848 Sentence denotes However, a low-pH challenge (as experienced during gastric transit) fails to similarly protect against subsequent osmotic or bile stress.
T107 17849-17916 Sentence denotes Equally, preexposure to bile fails to protect against acid or salt.
T108 17917-18032 Sentence denotes Thus, osmotic stress appears to be at the top of the hierarchy of stress responses during gastrointestinal transit.
T109 18033-18302 Sentence denotes 38, 39 Given that sigB is transcriptionally upregulated at elevated osmolarity, it is likely that the increased osmolarity of the gastrointestinal lumen may be interpreted by L. monocytogenes as an environmental cue, signaling gut entry (as is the case for Salmonella).
T110 18303-18801 Sentence denotes 40 Furthermore, given that s B is known to modulate expression of prfA (the master regulator of the virulence gene cluster which coordinates the intracellular phase of L. monocytogenes infection) it is possible that osmotically induced stimulation of the s B regulon in the upper small intestine may not only facilitate successful gastrointestinal transit, but also prime the pathogen for the next phase of infection, which, in susceptible individuals, is the systemic invasive disease listeriosis.
T111 18802-18974 Sentence denotes 41 Barotolerance Prior to ingestion, gastrointestinal pathogens are often subject to a variety of hostile conditions; particularly stresses associated with food processing.
T112 18975-19139 Sentence denotes One such stress is high-pressure processing (HPP); a non-thermal food processing method that subjects foods (liquid or solid) to pressures between 50 and 1,000 MPa.
T113 19140-19344 Sentence denotes Indeed, in Considine et al., 42 we outline how HPP can inactivate microorganisms and enzymes and modify macromolecular structures, with little or no impact on the nutritional and sensory quality of foods.
T114 19345-19571 Sentence denotes As before, our goal was to identify the genetic loci which allow bacteria to survive HPP; thereby permitting a more in-depth assessment of the implications of HPP in the food industry, both in terms of food quality and safety.
T115 19572-19696 Sentence denotes A L. monocytogenes genome bank was used to screen for barotolerance loci (i.e., resistance to 400 or 450 MPa for 5 minutes).
T116 19697-20009 Sentence denotes The study, which represents the first use of agar plates as a novel methodology for screening genome libraries using HPP, identified systems involved in quorum sensing (Agr), flagellar machinery (Mot) and cytokinesis (EzrA) as conferring enhanced pressure resistance when expressed against an E. coli background.
T117 20010-20156 Sentence denotes 43 In a follow-on study, 44 failure to disrupt EzrA (encoded by lmo1594) revealed that the protein is essential for the growth of L. monocytoenes.
T118 20157-20352 Sentence denotes Thus, while gene deletion proved impossible; over-expression of lmo1594 confirmed a role for the protein in listerial barotolerance, resulting in significantly improved survival rates at 300 MPa.
T119 20353-20574 Sentence denotes In an interesting link with previous sections, and substantiating a role for osmolytes as a listerial passe-partout, 45 both osmolyte uptake and synthesis systems have been shown to play a role in listerial barotolerance.
T120 20575-20731 Sentence denotes Indeed, listerial survival, following exposure to 400 MPa for 5 minutes, increased from 0.008 to 0.02% with added carnitine and to 0.05% with added betaine.
T121 20732-20842 Sentence denotes 46 While proline synthesis is required for optimal survival when betaine and carnitine are absent or limiting.
T122 20843-20845 Sentence denotes 47
T123 20847-20953 Sentence denotes In 2005 I was appointed as a principal investigator (PI) at the Alimentary Pharmabiotic Center (APC), UCC.
T124 20954-21190 Sentence denotes With a mission statement "linking Irish science with industry and society through excellence in research, education and outreach in gastrointestinal health," the APC is arguably one of the world's leading centers of gut health research.
T125 21191-21291 Sentence denotes Indeed, Thomson Reuters Science Watch ranks the APC at number 2 in the world for probiotic research.
T126 21292-21566 Sentence denotes As an APC PI, I developed an interest in probiotics, 48-50 specifically in applying the tools and techniques developed during my research on gastrointestinal pathogen stress responses, to develop improved probiotics 51 specifically with applications in the developing world.
T127 21567-21802 Sentence denotes 52, 53 Given the obvious commercial and clinical relevance of probiotic cultures; improving their stress tolerance profile and ability to overcome the physiochemical defenses of the host is, in my opinion, an important biological goal.
T128 21803-22256 Sentence denotes 54, 55 Patho-biotechnology In 2006 Colin Hill and I coined the term 'patho-biotechnology' to describe the exploitation of pathogen derived stress survival strategies to engineer improved probiotic strains. [56] [57] [58] [59] [60] This approach showed promise not only for the design of more technologically robust probiotic cultures, with improved biotechnological applications, but also in the development of novel vaccine and drug delivery platforms.
T129 22257-22650 Sentence denotes In support of this, Sheehan et al., 61 describes how heterologous expression of the listerial betL gene in Lactobacillus salivarius significantly increased resistance of the probiotic to several ex vivo stresses, including elevated osmo-, cryo-, baro-, and chill tolerance, as well as increased resistance to spray-and freeze-drying (stresses associated with food processing and preservation).
T130 22651-22851 Sentence denotes Furthermore, betL also improved in vivo stress survival in Bifidobacterium breve resulting in significantly improved tolerance to gastric juice and osmolarity conditions mimicking the gut environment.
T131 22852-23137 Sentence denotes Indeed, B. breve strains expressing BetL were recovered at considerably higher levels in the faeces, intestines and cecum of inoculated animals and exhibited significantly lower levels of systemic infection compared to the control strain following oral challenge with L. monocytogenes.
T132 23138-23274 Sentence denotes 57 Similar results were obtained for both B. breve and Lactococcus lactis when betL was replaced with bilE as the transgene of interest.
T133 23275-23693 Sentence denotes 62 These physiologically enhanced probiotics function as ideal drug and vaccine delivery platforms; 63 targeting difficult to treat pathogens such as Clostridium difficile, 64 Acinetobacter baumannii 65 and Mycobacteria. [66] [67] [68] In addition to the obvious clinical applications, described above, genetic manipulation of probiotics has provided fundamental insights into their natural life cycle within the host.
T134 23694-23875 Sentence denotes In Cronin et al., 69 for example, we describe the development of a non-invasive luciferase-based reporter system (pLuxMC1) for real-time tracking of Bifidobacterium species in vivo.
T135 23876-24027 Sentence denotes This construct allowed us, for the first time, to track the colonisation potential and persistence of the probiotic in real time in live animal models.
T136 24028-24262 Sentence denotes Indeed, a significant outcome of the study was the identification of the cecum as a niche environment for B. breve; a finding which may explain why appendectomies may lead to an increased risk of functional gastrointestinal disorders.
T137 24263-24265 Sentence denotes 70
T138 24267-24363 Sentence denotes Molecular diagnostics is arguably one of the fastest growing areas in gastrointestinal research.
T139 24364-24698 Sentence denotes Growth of the so called 'omics' technologies has, over the last decade, led to a gradual migration away from the culture based 'one test, one pathogen' paradigm, toward DNA based multiplex approaches to infectious disease diagnosis, which have in turn led to significant improvements in clinical diagnostics and improved patient care.
T140 24699-24940 Sentence denotes In 2009 our group established a partnership with Serosep (an Irish molecular diagnostics company) and the Clinical Microbiology Department of Cork University Hospital (CUH), focused on the changing culture of medical microbiology in Ireland.
T141 24941-25150 Sentence denotes 71 A retrospective analysis of faecal samples submitted to CUH in 2009 was performed to identify all Campylobacter species detected using Serosep's EntericBio Ò multiplex PCR system, in a single calendar year.
T142 25151-25257 Sentence denotes The results confirmed that routine culture fails to detect over a third of Campylobacter positive samples.
T143 25258-25386 Sentence denotes From a total of 7,194 diarrheal specimens, 349 Campylobacterpositive samples (23.8%) were shown to be Campylobacter ureolyticus.
T144 25387-25749 Sentence denotes This represents the first report of C. ureolyticus in the faeces of patients presenting with gastroenteritis, suggesting a heretofore unreported role for this organism as an enteric pathogen. [72] [73] [74] Indeed, we showed that C. ureolyticus is second only to C. jejuni as the most common cause of Campylobacter associated gastroenteritis in Southern Ireland.
T145 25750-26018 Sentence denotes 75 Furthermore, we reported a prominent seasonal distribution for campylobacteriosis (Spring), with C. ureolyticus (March) preceding C. jejuni/C. coli (April/May) and exhibiting a bimodal age and gender profile; being more commonly detected in elderly female patients.
T146 26019-26319 Sentence denotes Given that cultural isolation of C. ureolyticus was not possible using the established selective methods for the isolation of Campylobacter spp., we were forced to develop a new selective medium, nalidixic acid amphotericin B vancomycin (NAV), capable of isolating C. ureolyticus from faecal samples.
T147 26320-26478 Sentence denotes 76 This medium made it possible, for the first time, to isolate pure cultures of C. ureolyticus and to determine the potential source of infections in humans.
T148 26479-26626 Sentence denotes Building on this, Koziel et al., 77 describes the collection and processing of 164 samples from various domestic animals over a period of 6 months.
T149 26627-26865 Sentence denotes Significantly, Random Amplified Polymorphic DNA (RAPD) analysis identified a feline derived C. ureolyticus isolate as being genetically similar to a strain (CIT 007) isolated from an elderly female patient presenting with gastroenteritis.
T150 26866-27053 Sentence denotes Together with the findings of Koziel et al.,, 75 which reported the detection of C. ureolyticus in bovine samples, it appeared likely that this emerging pathogen has a zoonotic potential.
T151 27054-27226 Sentence denotes Whole genome sequence analysis of CIT 007 appeared to confirm this hypothesis; identifying a number of genetic loci necessary to cause gastrointestinal infection in humans.
T152 27227-27421 Sentence denotes 78 Indeed, whole genome analysis of two further C. ureolyticus isolates, including the type strain, revealing 106 putative virulence associated factors, 52 of which are predicted to be secreted.
T153 27422-27641 Sentence denotes Furthermore, this analysis also suggested that C. ureolyticus is likely to exist as a taxonomic continuum comprised of several species (genomospecies) that are likely to have varying impacts on human health and disease.
T154 27642-27812 Sentence denotes 79 Interestingly, a C. ureolyticus screen in exotic animals led to the identification of strain CIT 045(T) in the faeces of captive lion-tailed macaques (Macaca silenus).
T155 27813-28118 Sentence denotes Originally believed to be C. ureolyticus on the basis of colony morphology; 16S rRNA analysis showed it to be a completely new Campylobacter species; 80 a finding which we confirmed using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), together with whole genome sequence analysis.
T156 28119-28235 Sentence denotes 81 We named this new species Campylobacter corcagiensis sp nov. (The Corkonian!), in honor of its home town of Cork.
T157 28236-28441 Sentence denotes In parallel with the identification and characterization of C. ureolyticus and C. corcagiensis, our lab has continued to work with Serosep; validating the next two iterations of the Enter-icBio Ò platform.
T158 28442-28799 Sentence denotes The EntericBio Panel II Ò system, developed to detect Shiga toxin-producing Escherichia coli (STEC), differentiating Shiga-like toxins 1 and 2 (SLT-1 and SLT-2) 82 and the EntericBio real-time Gastro Panel I, which allows real-time detection of C. jejuni, coli, and lari, STEC, Shigella spp, and Salmonella spp. directly from faeces, without pre-enrichment.
T159 28800-28802 Sentence denotes 83
T160 28804-28963 Sentence denotes In addition to gastrointestinal specific bacterial diagnostics, we have also targeted gastrointestinal viruses; namely rotavirus, parvovirus and coronaviruses.
T161 28964-29146 Sentence denotes Collins et al., 84 86 described a nationwide screen, between 2008 and 2009, of canine parvovirus (CPV) and coronavirus (CCoV); the most common cause of acute gastroenteritis in dogs.
T162 29147-29318 Sentence denotes A total of 250 faecal samples were collected from both symptomatic and asymptomatic dogs, from a diverse array of sources, representing the major canine groups in Ireland.
T163 29319-29454 Sentence denotes Seven CPV strains and three CCoV strains were identified in symptomatic dogs, with one animal presenting with mixed CPV/CCoV infection.
T164 29455-29632 Sentence denotes Significantly, from a public health perspective, each of these animal viruses represents a vast reservoir of genetic material for the diversification of their human equivalents.
T165 29633-29731 Sentence denotes Thus, a key outcome of these studies is the need for constant genotype surveillance and vigilance.
T166 29732-29932 Sentence denotes This is particularly relevant given that shifting genotype prevalence, the emergence of novel combinations and mixed infections, are all likely to impact negatively on existing vaccination programmes.
T167 29933-29991 Sentence denotes In extreme cases this can lead to breakthrough infections.
T168 29992-30193 Sentence denotes The development of ever faster and cheaper next generation sequencing strategies has resulted in a paradigm shift in molecular diagnostics; changing the focus from single target genes to whole genomes.
T169 30194-30390 Sentence denotes Since 2008, genomic data has outpaced Moore's Law by a factor of 4, with a global annual sequencing capacity estimated to be 13 quadrillion bases; enough data to fill a stack of DVDs 2 miles high!
T170 30391-30862 Sentence denotes Recognizing the potential of biology's big data sets in developing improved diagnostic techniques, our lab began to focus on phylogenetics, 87,88 genomics, [89] [90] [91] [92] protein structure/function prediction [93] [94] [95] [96] [97] [98] and the emerging field of synthetic biology. [99] [100] [101] [102] [103] [104] [105] [106] [107] Indeed, this changing focus allowed me to establish links with the Department of Computing at Cork Institute of Technology (CIT).
T171 30863-30968 Sentence denotes 108, 109 This union produced CIT's first commercial spinout company, nSilico, which I co-founded in 2012.
T172 30969-31140 Sentence denotes Combining all of the above elements, I wrote and coordinated ClouDx-i, a €1.3 million EU FP7 funded project, with CIT, nSilico and the University of Edinburgh as partners.
T173 31141-31370 Sentence denotes The principal scientific and technological objective of ClouDx-i was to develop computational approaches to support rapid molecular diagnosis of infection, embedding these techniques in an efficient end-to-end diagnostic process.
T174 31371-31570 Sentence denotes 110, 111 The project has sequenced and analyzed the genomes of 12 bacterial pathogens, identifying biomarkers for the rapid diagnosis of neonatal infections. [112] [113] [114] [115] [116] [117] [118]
T175 31572-31622 Sentence denotes No potential conflicts of interest were disclosed.