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Characterization of two Achromobacter xylosoxidans isolates from patients with pertussis-like symptoms
Abstract
Objective: To characterize two Achromobacter xylosoxidans recovered from 2 patients diagnosed with pertussis during a Bordetella pertussis surveillance program. Methods: Nasopharyngeal swabs from 2 children under 1 year of age with clinical suspicion of pertussis were analyzed by culture and PCR. Results: Two Achromobacter xylosoxidans A8, closely related to Bordetella spp. were recovered from 2 patients diagnosed of pertussis, both carrying the ptxA gene and IS418 the pertussis toxin encoding gene. Subsequently, antibiotic susceptibility was evaluated by disk-diffusion method and by PCR. Conclusions: Although more detailed studies are needed, the present data highlight the possibility that Achromobacter xylosoxidans, closely related Bordetella pertusssis microorganisms and not covered under the vaccine umbrella, might also result in cases of whooping cough. Thereby further surveillance is necessary to determine the extension and relevance of their pathogenic role in order to discriminate their real public health implication.
Respiratory tract infections are the leading cause of mortality among children under five years of age worldwide, causing around 1.5 millions of deaths yearly, especially in low and middle income countries (LMC) [1] . Among these infections, whooping cough (pertussis) is particularly severe, with the WHO reporting more than 16 million cases of pertussis and around 195 000 deaths worldwide in 2008, 95% of which were in LMC [1] . Pertussis is caused by Bordetella pertussis (B. pertussis). It is potentially preventable by vaccination and is characterized by a series of early symptoms which may be easily misdiagnosed as a common cold, followed by a series of paroxysmal symptoms at 1 or 2 weeks of evolution, which may include coughing, whooping sounds, vomiting among others that might result in death [2] .
The introduction of the pertussis vaccination resulted in a decrease in disease burden, but since the 80-90's of the last century, the number of cases has risen again [3, 4] . This reemergence of pertussis may be related to different causes, including less effectivity of the current acellular pertussis vaccine with respect to the whole cell vaccine [4, 5] , the selection of variants of B. pertussis not covered by the vaccine [5, 6] , as well as the underestimated role of other Bordetella species, as such as Bordetella holmesii [7] . Following these trends, in the last years an increasing number of cases, mainly in rural areas, has been described in Peru [8] .
The members of the Achromobacter genus are ubiquitous, being detected in different sources, including water environments or different human microbiotas [9, 10] . Achromobacter is a genus closely related to Bordetella and it has been proposed to have a potential virulence from an evolutionary point of view [11] . In this line Achromobacter xylosoxidans (A. xylosoxidans) has been described as a cause of different infections, mainly bacteremia in immunosuppressed patients, but has also been related to respiratory diseases, including infections in patients with cystic fibrosis, bronchiectasis and pleural emphysema, among others [10, [12] [13] [14] [15] . However, to our knowledge it has never been reported as a causal agent of pertussis-like illness.
The aim of this study was to characterize 2 A. xylosoxidans isolated from pediatric patients diagnosed with whooping cough.
Nasopharyngeal swabs from 2 children under 1 year of age with clinical suspicion of pertussis were collected during a surveillance of pertussis and sent to the Instituto de Investigación Nutricional/Universidad Peruana de Ciencias Aplicadas. Relevant clinical data were recorded in a questionnaire. The study was approved by the Ethical Committee of the Hospital Nacional de Salud del Niño, Lima (Peru). The samples were collected after the parents had provided signed informed consent.
Clinical and epidemiological features were registered for each patient, including: age, clinical symptoms (paroxysm of coughing, cyanosis, respiratory distress, difficulty feeding, fever, flush, diarrhea, vomiting, wheezing, runny nose and expectoration), days from the onset of symptoms and time elapsed until the sample was collected and sent to the laboratory.
A direct nucleic acid extraction was performed from the nasopharyngeal samples using the High Pure Template Preparation Kit (Roche Applied Science, Mannheim, Germany) following the manufacturer's instructions, and pertussis status was confirmed by PCR. Thus, both 191 bp of the ptxA gene and 145 bp of the IS418 were amplified using primers described elsewhere [16, 17] . To confirm the correctness of the amplifications the PCR products were recovered, purified (SpinPrepTM Gel DNA Kit, San Diego, USA) and sequenced (Macrogen, Seoul, Korea). Additionally, the presence of adenovirus, influenza A, B and C, syncytial respiratory virus A and B, parainfluenza 1, 2, 3, 4a, enterovirus, rhinovirus and coronavirus was verified as described elsewhere [18] .
The nasopharyngeal samples were cultured in Bordet-Gengou agar, incubated at 35 C in a moist atmosphere and maintained for 5-7 days under aerobic conditions. Colonies were identified based on characteristic morphology, Gram stain appearance and catalase, oxidase testing. Positive cultures were confirmed by PCR of the ptxA gene as mentioned previously. Additionally, a DNA fragment of 663 bp, including the first 517 bp of the parC gene, was also amplified using the primers (5 0 -GTGATCATTCCCAAGCGCC-3 0 and 5 0 -TGAGCAGCAT-CACGGGCA-3 0 ) and the conditions previously described by Ohtsuka et al [19] . These primers are able to amplify the parC gene from both Bordetella spp and A. xylosoxidans, which may be easily differentiated by the presence of several additional nucleotides in the N-terminal region of the parC gene of A. xylosoxidans sequence.
The susceptibility levels to amoxicillin, azithromycin, chloramphenicol, cotrimoxazole, erythromycin, clarithromycin, cefotaxime, ampicillin, moxifloxacin and tetracycline were performed by disk diffusion.
The 23S rRNA gene was amplified from bacterial DNA extractions using the primers and conditions described previously [20] . Additionally, the rplV and rplD genes from both nasopharyngeal swabs and bacterial origins were amplified using Bordetella-specific primers: rplV-F:
Both children presented fever, respiratory distress and flushed, while only one presented cough paroxysm ( Table 1) . One of the children was treated with amoxicillin plus cephalexin (200 mg), while the other received ceftriaxone (100 mg). In both cases the treatment was successful and the children were discharged from hospital. In both cases the PCR of the direct DNA extraction from nasopharyngeal swabs was positive for the presence of the ptxA gene and IS418, which showed a 100% of identity with that of B. pertussis (GenBank accession number NC_002929). Additionally, none of the other pathogens sought was detected, and thus, the patients were diagnosed with pertussis.
The antibiotic susceptibility analysis showed that the isolates present high levels of resistance to tested antibacterial agents, including all analyzed macrolides (Table 2) .
Surprisingly, when the macrolide target encoding genes were amplified and sequenced, the results showed the presence of A. xylosoxidans A8 with 100% (23S rRNA gene) 99% (rplD gene) and 98% (rplV gene) of identity with the respective DNA sequences of A. xylosoxidans A8 (GenBank access CP002287.1), being even higher than the identity levels with the same DNA sequences of B. pertussis (Genbank access NC_002929) or other Bordetella spp. These results were also confirmed on PCR analysis of the grown microorganisms.
For further identification tool, we included the analysis of the parC gene sequence, which also showed that the closest related sequence was that of A. xylosoxidans A8, being 2-6 amino acid codons longer than other Achromobacter spp. and Bordetella spp. ParC sequences present in GenBank. Thus, despite the presence of 27 differences (96% of identity) in the encoding ParC DNA sequence both samples, only showed 3 different amino acid changes: S15P; D20G and Y66F.
Regarding the aforementioned macrolide targets no mutation was found in the 23S rRNA gene and only 5 and 7 silent mutations were found in the rplD genes on comparison with the A. xylosoxidans A8 wild-type sequence, while 6 silent mutations plus the T134A transversion resulting in the amino acid change V45A was observed in both rplV genes.
Achromobacter spp. are frequent environmental microorganisms, causing a series of opportunistic illnesses, including respiratory diseases [15, 21] and possess a high evolutionary virulence potential, because of their phylogenetical proximity to Bordetella. In this line, the description of A. xylosoxidans in pediatric whooping cough patients is strongly suggestive of its implication as an etiological agent, especially as the isolate analysis showed the presence of the ptxA gene as well as IS418. A DNA fragment including the N-terminal of parC gene was also amplified to confirm these data. This gene was selected because the N-terminal section in A. xylosoxidans A8 has 5 to 6 more amino acid than the described ParC of Bordetella spp. Moreover, this sequence is also 2 to 5 amino acids longer than ParC of other Achromobacter.
The presence of non-B. pertussis microorganisms causing whooping cough has a direct implication in the lack or diminishing protection of vaccines and having a direct public health impact. Nonetheless, in this case only one child was partially vaccinated since the first vaccine dosage is provided at 2 months of life.
While IS418 is present with a variable copy number in different Bordetella genus members, the so-called pertussis toxin is exclusively produced by B. pertussis [22] . However, different members of the genus Bordetella, such as Bordetella bronchiseptica and Bordetella parapertussis, which have been involved as etiological causes of this illness, possess closely related non-functional genes [22] . Other Bordetella genus members, such as B. holmesii, which account for 0.4%-29% of all diagnosed pertussis cases [23, 24] , do not present the either pertussis toxin gene or close-related genes, a characteristic which has been used in different studies to facilitate the differentiation between B. pertussis and B. holmesii [25] . Thus, pertussis toxin functionality, although relevant, is not essential for the presence of pertussis-like symptoms. In our case no data about gene functionality was obtained, remaining to be established (studies ongoing).
The antimicrobial susceptibility analysis, showed high levels of resistance to macrolides. This is a matter of concern, since macrolides are considered as the treatment of choice for pertussis [26] , and again highlight the need to correctly determine the etiologic agent, causing the illness. Along this line, it has been reported that different Bordetella species present different levels of macrolide resistance, which is especially high in B. holmesii [7] .
Regarding macrolide targets, the only amino acid codon alteration with respect to A. xylosoxidans A8 wild-type sequences was observed in the rplV gene. Thus, the V45A amino acid codon substitution was observed in the protein L22 in both isolates. However, to the best of our knowledge, substitutions at this amino acid codon, or at equivalent positions in other microorganisms, have not been related to macrolide resistance. Nonetheless, the presence of this specific amino acid change may be a polymorphism without involvement in the development of macrolide resistance. Obviously, the presence of transferable mechanisms of macrolide resistance can not be ruled out. Thus, a GenBank search has showed the presence of the ere(A) gene in other members of the Achromobacter genus (GenBank access: DQ112222.1).
Interestingly, some silent DNA mutations were found in both strains in the rplV, rplD and parC genes. These close similarities between the two isolates, together with the short isolation frame time between the two isolates suggest the presence of a specific A. xylosoxidans A8 clone or a close related microorganism, with the ability to cause pertussis-like illness in the area. Taking this into account the recent report by Spilker et al [27] is of special interest. These authors reported the presence of several new putative Bordetella-like species, suggesting the presence of an undescribed genus closely related to both A. xylosoxidans and Bordetella spp.
In summary, the present results show the presence of A. xylosoxidans A8-like microorganism possessing the ptxA gene and IS418. Due the proximity of the Achromobacter genome to that of Bordetella spp. causing whooping cough, in depth characterization of the isolates recovered is need. The present data highlight the possibility that closely related B. pertusssis microorganisms, not covered under the vaccine umbrella, might also result in cases of whooping cough, thereby requiring further surveillance to determine the extension and relevance of their pathogenic role in order to discriminate their real public health implication.
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