Validation of the T86I mutation in the gyrA gene as a highly reliable real time PCR target to detect Fluoroquinolone-resistant Campylobacter jejuni.

BACKGROUND: Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide, and increasing rates of fluoroquinolone (FQ) resistance in C. jejuni are a major public health concern. The rapid detection and tracking of FQ resistance are critical needs in developing countries, as these antimicrobials are widely used against C. jejuni infections. Detection of point mutations at T86I in the gyrA gene by real-time polymerase chain reaction (RT-PCR) is a rapid detection tool that may improve FQ resistance tracking. METHODS: C. jejuni isolates obtained from children with diarrhea in Peru were tested by RT-PCR to detect point mutations at T86I in gyrA. Further confirmation was performed by sequencing of the gyrA gene. RESULTS: We detected point mutations at T86I in the gyrA gene in 100% (141/141) of C. jejuni clinical isolates that were previously confirmed as ciprofloxacin-resistant by E-test. No mutations were detected at T86I in gyrA in any ciprofloxacin-sensitive isolates. CONCLUSIONS: Detection of T86I mutations in C. jejuni is a rapid, sensitive, and specific method to identify fluoroquinolone resistance in Peru. This detection approach could be broadly employed in epidemiologic surveillance, therefore reducing time and cost in regions with limited resources.

Fecal Contamination of Drinking Water Was Associated with Diarrheal Pathogen Carriage among Children Younger than 5 Years in Three Peruvian Rural Communities.

Drinking water contamination is a frequent problem in developing countries and could be associated with bacterial pathogen carriage in feces. We evaluated the association between the risk of drinking water and bacterial carrier status in children younger than 5 years in a cross-sectional study conducted in 199 households from three Peruvian rural communities. Fecal samples from children were screened for pathogenic Aeromonas, Campylobacter, and Vibrio species, as well as for Enterobacteriaceae, including pathogenic Escherichia coli. The drinking water risk was determined using E. coli as an indicator of contamination. Nineteen (9.5%) children were colonized with pathogens and classified as carriers, all without diarrhea symptoms. Of 199 drinking water samples, 38 (19.1%) were classified as very high risk because of high fecal contamination (> 100 E. coli/100 mL). Shared-use water sources, daily washing of containers, and washing using only water were associated with higher prevalence of bacterial carriage, whereas there was no association between households reporting boiling and chlorination of water and carrier status. The prevalence of carriage in children exposed to very high-risk water was 2.82 (95% CI: 1.21-6.59) times the prevalence of those who consumed less contaminated water, adjusted by the water source and daily washing. Our results suggest that household drinking water plays an important role in the generation of carriers with diarrheal pathogens. Our findings also highlight the importance of interventions to ensure the safety of drinking water. Further studies are needed to validate the observed association and determine its significance with respect to diarrhea in the community.

A comparison of methods for DNA preparation prior to microarray analysis.

Microarrays are a valuable tool for analysis of both bacterial and eukaryotic nucleic acids. As many of these applications use non-specific amplification to increase sample concentration prior to analysis, the methods used to fragment and label large amplicons are important to achieve the desired analytical selectivity and specificity. Here, we used eight sequenced ESKAPE pathogens to determine the effect of two methods of whole genome amplicon fragmentation and three methods of subsequent labeling on microarray performance; nick translation was also assessed. End labeling of both initial DNase I-treated and sonication-fragmented amplicons failed to provide detectable material for a significant number of sequence-confirmed genes. However, processing of amplicons by nick translation, or by sequential fragmentation and labeling by Universal Labeling System or Klenow fragment/random primer provided good sensitivity and selectivity, with marginally better results obtained by Klenow fragment labeling. Nick-translation provided 91-100% sensitivity and 100% specificity in the tested strains, requiring half as many manipulations and less than 4h to process samples for hybridization; full sample processing from whole genome amplification to final data analysis could be performed in less than 10h. The method of template denaturation before amplification did affect detection sensitivity/selectivity of nick-labeled amplicons, however.

Distribution of Capsular Types of Campylobacter jejuni Isolates from Symptomatic and Asymptomatic Children in Peru.

Campylobacter jejuni is the leading bacterial cause of diarrhea worldwide. A capsular polysaccharide (CPS) conjugate vaccine is under development and requires determination of the valency. However, distribution of CPS types circulating globally is presently poorly described. We aimed to determine whether CPS type distribution in Peru differs from that in other endemic regions. We used a multiplex polymerase chain reaction (PCR) assay for the detection of CPS encoding genes capable of distinguishing all 35 CPS types on Campylobacter isolates in two prospective communities based studies conducted in cohorts of children less than 59 months of age in Peru. Results showed that CPS type HS4 complex was the most prevalent, followed by HS3 complex and HS15. Differences in CPS type for symptomatology were not statistically significant. Most subjects demonstrated repeated infections over time with different CPS types, suggesting that CPS types may confer of a level of homologous protective immunity. In this dataset, some differences in CPS type distribution were observed in comparison to other low-middle income countries. Further studies need to be conducted in endemic areas to increase our knowledge of CPS type distribution and guide vaccine development.

First Report of New Delhi Metallo-ß-Lactamase Carbapenemase-Producing Acinetobacter baumannii in Peru.

Here we report the first incidence of New Delhi metallo-ß-lactamase (NDM-1)-producing Acinetobacter baumannii in Peru, identified via a strain-based nosocomial surveillance project carried out in Lima and Iquitos. The bla NDM-1 gene was detected by multiplex polymerase chain reaction (PCR) and confirmed by loci sequencing. Acinetobacter baumannii is a nearly ubiquitous and promiscuous nosocomial pathogen, and the acquisition of bla NDM-1 by A. baumannii may facilitate an increase in the prevalence of this important resistance marker in other nosocomial pathogens.

Extensively drug-resistant (XDR) Pseudomonas aeruginosa identified in Lima, Peru co-expressing a VIM-2 metallo-ß-lactamase, OXA-1 ß-lactamase and GES-1 extended-spectrum ß-lactamase.

INTRODUCTION: Pseudomonas aeruginosa has the ability to acquire plasmids and other mobile genetic elements that confer resistance to antibiotics. Bacterial genes encoding different ß-lactamases (bla), such as metallo-ß-lactamases (MBLs) and extended-spectrum ß-lactamases (ESBL), can confer resistance to multiple classes of ß-lactam antibiotics. CASE PRESENTATION: An 83 year old female was admitted in 2012 to the Peruvian Naval Hospital, Centro Médico Naval 'Cirujano Mayor Santiago Távara' (CEMENA), in Lima, Peru. A midstream urine sample was collected and sent to the local CEMENA laboratory for routine urine culture. P. aeruginosa was isolated and initial antibiotic susceptibility testing showed it to be sensitive to imipenem. The clinicians started a course of meropenem, but the patient did not improve. After 5 days, a second urine culture was performed and a P. aeruginosa was isolated again, but this time the strain showed resistance to imipenem. The treatment course was changed to fosfomycin and the patient improved. Phenotypic and molecular laboratory testing to characterize the antibiotic resistance were performed, demonstrating the presence of both MBL and ESBL genes. CONCLUSION: To our knowledge, this is the first report of a P. aeruginosa XDR clinical isolate that co-expresses an MBL (VIM-2), OXA-1 beta-lactamase and the ESBL (GES-1) in Peru. It is also the first report of a VIM carbapenemase in Peru.

Outbreak of gastroenteritis at a Peruvian naval base.

In April of 2003, an outbreak of gastroenteritis was reported in a training command (Centro de Instrucción Técnica y Entrenamiento Naval (CITEN)) at a Peruvian naval base located near Lima, Peru. The Naval Medical Research Center Detachment, in collaboration with the National Peruvian Naval Hospital, conducted an investigation to determine the causative agent and potential source of the outbreak. Between April 3 and 5, 172 (16%) of 1,092 military trainees reported to the CITEN clinic with diarrhea. Of 74 trainees for whom bacterial cultures were performed, Shigella spp. were isolated from 5 (6.8%), Campylobacter spp. from 5 (6.8%), and enterotoxigenic Escherichia coli from 2 (2.7%). Pathogenic parasites were identified in 22 of 64 (34%) trainees for whom microscopic observation for ova and parasites was performed. Stool samples from asymptomatic controls could not be collected, thus we were unable to confirm that the enteropathogens isolated were the etiologic agent(s). Several food items and the hands of food handlers were contaminated with coliform bacteria and drinking water was not adequately chlorinated. Preventative measures have since reduced the number of diarrhea cases at the CITEN.