QRDR mutations, efflux system & antimicrobial resistance genes in enterotoxigenic Escherichia coli isolated from an outbreak of diarrhoea in Ahmedabad, India

Background & objectives: Diverse mechanisms have been identified in enteric bacteria for their adaptation and survival against multiple classes of antimicrobial agents. Resistance of bacteria to the most effective fluoroquinolones have increasingly been reported in many countries. We have identified that most of the enterotoxigenic Escherichia coli (ETEC) were resistant to several antimicrobials in a diarrhoea outbreak at Ahmedabad during 2000. The present study was done to identify several genes responsible for antimicrobial resistance and mobile genetic elements in the ETEC strains. Methods: Seventeen ETEC strains isolated from diarrhoeal patients were included in this study. The antimicrobial resistance was confirmed by conventional disc diffusion method. PCR and DNA sequencing were performed for the identification of mutation in the quinolone resistance-determining regions (QRDRs). Efflux pump was tested by inhibiting the proton-motive force. DNA hybridization assay was made for the detection of integrase genes and the resistance gene cassettes were identified by direct sequencing of the PCR amplicons. Results: Majority of the ETEC had GyrA mutations at codons 83 and 87 and in ParC at codon 80. Six strains had an additional mutation in ParC at codon 108 and two had at position 84. Plasmid-borne qnr gene alleles that encode quinolone resistance were not detected but the newly described aac(6')-Ib-cr gene encoding a fluoroquinolne-modifying enzyme was detected in 64.7 per cent of the ETEC. Class 1 (intI1) and class 2 (intI2) integrons were detected in six (35.3%) and three (17.6%) strains, respectively. Four strains (23.5%) had both the classes of integrons. Sequence analysis revealed presence of dfrA17, aadA1, aadA5 in class 1, and dfrA1, sat1, aadA1 in class 2 integrons. In addition, the other resistance genes such as tet gene alleles (94.1%), catAI (70.6%), strA (58.8%), bla TEM-1 (35.2%), and aphA1-Ia (29.4%) were detected in most of the strains. Interpretation & Conclusions: Innate gene mutations and acquisition of multidrug resistance genes through mobile genetic elements might have contributed to the emergence of multidrug resistance (MDR) in ETEC. This study reinforces the necessity of utilizing molecular techniques in the epidemiological studies to understand the nature of resistance responsible for antimicrobial resistance in different species of pathogenic bacteria

Development and Evaluation of a PCR Assay for Tracking the Emergence and Dissemination of Haitian Variant ctxB in Vibrio cholerae O1 Strains Isolated from Kolkata, India

A PCR-based assay was developed to discriminate the classical, El Tor, and Haitian types of ctxB alleles. Our retrospective study using this newly developed PCR showed that Haitian ctxB first appeared in Kolkata during April 2006, and 93.3% of strains isolated during 2011 carried the new allele. Dendrogram analysis showed a pulsed-field gel electrophoresis (PFGE) pattern of the new variant strains isolated recently that was distinct from the PFGE pattern of the strains carrying classical ctxB that closely matched the 2006 to 2007 variant strains

Trends in the prevalence of diarrheagenic Escherichia coli among hospitalized diarrheal patients in Kolkata, India

BACKGROUND:　 To analyse the trends in the prevalence of different pathogroups of diarrheagenic Escherichia coli (DEC) among hospitalized acute diarrheal patients.　 METHODOLOGY/PRINCIPAL FINDINGS:　 From the active surveillance of diarrheal disease at the Infectious Diseases Hospital, Kolkata, 3826 stool specimens collected during 2008-2011 were screened for DEC and other enteric pathogens. PCR was used in the detection of enterotoxigenic, enteropathogenic and enteroaggregative E. coli and 10 major colonization factor antigens (CFs) of enterotoxigenic E. coli. The relationship between DEC infected patient's age group and clinical symptoms were also investigated. Multiplex PCR assay showed that the prevalence of EAEC was most common (5.7%) followed by ETEC (4.2%) and EPEC (1.8%). In diarrheal children >2 year of age, EAEC and EPEC were detected significantly (p = 0.000 and 0.007, respectively). In children >2 to 5 and >5 to 14 years, ETEC was significantly associated with diarrhea (p = 0.000 each). EAEC was significantly associated with diarrheal patients with age groups >14 to 30 and >30 to 50 years (p = 0.001, and p = 0.009, respectively). Clinical symptoms such as vomiting, abdominal pain, watery diarrhea, were recorded in patients infected with ETEC. Dehydration status was severe among patients infected by ST-ETEC (19%) and EPEC (15%). CS6 was frequently detected (37%) among ETEC.　 CONCLUSIONS/SIGNIFICANCE:　 Hospital based surveillance reviled that specific pathogroups of DEC are important to certain age groups and among ETEC, CS6 was predominant

A unified approach to molecular epidemiology investigations: tools and patterns in California as a case study for endemic shigellosis

<p>Abstract</p> <p>Background</p> <p>Shigellosis causes diarrheal disease in humans from both developed and developing countries, and multi-drug resistance is an emerging problem. The objective of this study is to present a unified approach that can be used to characterize endemic and outbreak patterns of shigellosis using use a suite of epidemiologic and molecular techniques. The approach is applied to a California case study example of endemic shigellosis at the population level.</p> <p>Methods</p> <p>Epidemiologic patterns were evaluated with respect to demographics, multi-drug resistance, antimicrobial resistance genes, plasmid profiles, and pulsed-field gel electrophoresis (PFGE) fingerprints for the 43 <it>Shigella </it>isolates obtained by the Monterey region health departments over the two year period from 2004-2005.</p> <p>Results</p> <p>The traditional epidemiologic as well as molecular epidemiologic findings were consistent with endemic as compared to outbreak shigellosis in this population. A steady low level of cases was observed throughout the study period and high diversity was observed among strains. In contrast to most studies in developed countries, the predominant species was <it>Shigella flexneri </it>(51%) followed closely by <it>S. sonnei </it>(49%). Over 95% of <it>Shigella </it>isolates were fully resistant to three or more antimicrobial drug subclasses, and 38% of isolates were resistant to five or more subclasses. More than half of <it>Shigella </it>strains tested carried the <it>tetB</it>, <it>catA</it>, or <it>bla</it>_TEMgenes for antimicrobial resistance to tetracycline, chloramphenicol, and ampicillin, respectively.</p> <p>Conclusion</p> <p>This study shows how epidemiologic patterns at the host and bacterial population levels can be used to investigate endemic as compared to outbreak patterns of shigellosis in a community. Information gathered as part of such investigations will be instrumental in identifying emerging antimicrobial resistance, for developing treatment guidelines appropriate for that community, and to provide baseline data with which to compare outbreak strains in the future.</p

Phenotypic and Genetic Heterogeneity in Vibrio cholerae O139 Isolated from Cholera Cases in Delhi, India during 2001-2006

Incidence of epidemic Vibrio cholerae serogroup O139 has declined in cholera endemic countries. However, sporadic cholera caused by V. cholerae O139 with notable genetic changes is still reported from many regions. In the present study, 42 V. cholerae O139 strains isolated from 2001 to 2006 in Delhi, India, were retrospectively analyzed to understand their phenotype and molecular characteristics. The majority of isolates were resistant to ampicillin, furazolidone and nalidixic acid. Though the integrative conjugative element was detected in all the O139 isolates, the 2004–2006 isolates remained susceptible to co-trimoxazole, chloramphenicol, and streptomycin. Cholera toxin genotype 1 was present in the majority of the O139 isolates while few had type 3 or a novel type 4. In the cholera toxin encoding gene (ctx) restriction fragment length polymorphism, the majority of the isolates harbored three copies of CTX element, of which one was truncated. In this study, the ctx was detected for the first time in the small chromosome of V. cholerae O139 and one isolate harbored 5 copies of CTX element, of which 3 were truncated. The ribotype BII pattern was found in most of the O139 isolates. Three V. cholerae O139 isolated in 2001 had a new ribotype BVIII. Pulsed-field gel electrophoresis analysis revealed clonal variation in 2001 isolates compared to the 2004–2006 isolates. Molecular changes in V. cholerae O139 have to be closely monitored as this information may help in understanding the changing genetic features of this pathogen in relation to the epidemiology of cholera

Dipstick Test for Rapid Diagnosis of Shigella dysenteriae 1 in Bacterial Cultures and Its Potential Use on Stool Samples

International audienceBACKGROUND: We describe a test for rapid detection of S. dysenteriae 1 in bacterial cultures and in stools, at the bedside of patients. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S. dysenteriae 1 lipopolysaccharide (LPS) using serotype 1-specific monoclonal antibodies coupled to gold particles and displayed on a one-step immunochromatographic dipstick. A concentration as low as 15 ng/ml of LPS was detected in distilled water and in reconstituted stools in 10 minutes. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 1.6×10⁶ CFU/ml and 4.9×10⁶ CFU/ml of S. dysenteriae 1, respectively. Optimal conditions to read the test have been determined to limit the risk of ambiguous results due to appearance of a faint yellow test band in some negative samples. The specificity was 100% when tested with a battery of Shigella and unrelated strains in culture. When tested on 328 clinical samples in India, Vietnam, Senegal and France by laboratory technicians and in Democratic Republic of Congo by a field technician, the specificity (312/316) was 98.7% (95% CI:96.6-99.6%) and the sensitivity (11/12) was 91.7% (95% CI:59.8-99.6%). Stool cultures and the immunochromatographic test showed concordant results in 98.4 % of cases (323/328) in comparative studies. Positive and negative predictive values were 73.3% (95% CI:44.8-91.1%) and 99.7% (95% CI:98-100%). CONCLUSION: The initial findings presented here for a simple dipstick-based test to diagnose S. dysenteriae 1 demonstrates its promising potential to become a powerful tool for case management and epidemiological surveys

Genetic traits of Vibrio cholerae O1 Haitian isolates that are absent in contemporary strains from Kolkata, India.

The world's worst cholera epidemic in Haiti (2010) coerced to trace the origin and dissemination of the causative agent Vibrio cholerae O1 for proper management of cholera. Sequence analysis of the Haitian strain showed several variations in the genes encoding cholera toxin B subunit (ctxB); toxin-co-regulated pilus (tcpA), repeat in toxins (rtxA), quinolone resistance-determining region (QRDR) of gyrase A (gyrA), rstB of RS element along with the change in the number of repeat sequences at the promoter region of ctxAB. Our earlier studies showed that variant tcpA (tcpA CIRS) and ctxB (ctxB7) first appeared in Kolkata during 2003 and 2006, respectively. The present study revealed that a variant rtxA was first isolated in Kolkata during 2004 and probably formed the genetic background for the emergence of the ctxB7 allele as we were unable to detect a single strain with the combination of El Tor rtxA and ctxB7. The variant gyrA was first time detected in Kolkata during 1994. The Kolkata strains contained four heptad repeats (TTTTGAT) in their CT promoter regions whereas Haitian strains carried 5 heptad repeats. Haitian strains had 3 nucleotide deletions at the rstB gene, which is a unique feature of the classical biotype strains. But the Kolkata strains did not have such deletion mutations in the rstB. Our study demonstrated the existence of some Haitian genetic traits in Kolkata isolates along with the dissimilarities in genomic content with respect to rstB and ctxAB promoter region. Finally, we conclude that Haitian variant strain may be evolved due to sequential event in the Indian subcontinent strain with some cryptic modification in the genome

Species diversity and antimicrobial resistance of Shigella spp. isolated between 2001 and 2004 from hospitalized children with diarrhoea in Kolkata (Calcutta), India.

The incidence, phenotypic characteristics and antimicrobial resistance patterns of 193 Shigella strains isolated from 2489 hospitalized children with acute diarrhoea were studied during January 2001 to August 2004. S. flexneri (60%) was the most prevalent serogroup, followed by S. sonnei (23.8%), S. dysenteriae (9.8%) and S. boydii (5.7%). Since 2002, S. flexneri 2a was the most dominant serotype. Almost all S. flexneri strains exhibited resistance to ampicillin, co-trimoxazole, tetracycline, nalidixic acid and fluoroquinolones. After a lapse of almost 14 years, S. dysenteriae type 1 strains reemerged for the first time during 2002 and these strains were resistant to more than two antibiotics (multidrug resistance), including fluoroquinolones. An upsurge of similar resistance patterns was also noted among S. flexneri type 2a since December 2003. Resistance to fluoroquinolone increased year on year among S. dysenteriae type 1 and S. flexneri, but not in S. boydii or S. sonnei. Monitoring of antimicrobial susceptibility through a surveillance programme is recommended to select appropriate antibiotics for the effective treatment of shigellosis in this region