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%), blaTEM-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.

Molecular comparison of toxigenic clinical & non-toxigenic environmental strains of Vibrio cholerae O1 Ogawa isolated during an outbreak of cholera in south India.

BACKGROUND & OBJECTIVES: While investigating a cholera outbreak in south India, toxigenic and nontoxigenic strains of Vibrio cholerae O1 were isolated from patients and from the environment, respectively. This study was performed to compare the genetic relatedness of the patient and environmental strains to determine clonal relationships among these strains and thereby determine the source of the cholera outbreak. METHODS: The 16 strains of V. cholerae isolated from hospitalized patients and 8 environmental V. cholerae strains isolated from the environment were phenotypically and genotypically characterized using a variety of standard techniques. RESULTS: Sixteen toxigenic clinical strains and 2 nontoxigenic environmental strains belonged to O1 serogroup, Ogawa serotype and El Tor biotype. The remaining 6 nontoxigenic environmental strains were classified as non-O1, non-O139 V. cholerae. The drug resistance pattern of the clinical and environmental strains of V. cholerae showed marked differences with the patient strains being resistant to more number of drugs as compared to the environmental strains. DNA fingerprinting of the strains showed considerable diversity between toxigenic clinical and nontoxigenic environmental O1 Ogawa isolates and between the O1 and non-O1, non-O139 isolates. INTERPRETATION & CONCLUSION: In this outbreak of cholera, the O1 strains of V. cholerae from clinical and environmental sources belonged to two different clones and the environmental strains could perhaps be the future cholera outbreak causing clones.

Cluster-analysis & patterns of dissemination of multidrug resistance among clinical strains of Vibrio cholerae in Calcutta, India.

BACKGROUND & OBJECTIVES: Antimicrobial resistance among Vibrio cholerae has been monitored for several years in Calcutta. To investigate the changing trends in multidrug resistance (MDR) among different serogroups of V. cholerae and to perform software assisted cluster analysis the current study was undertaken. METHODS: Strains isolated from patients with cholera and "cholera-like" diarrhoea admitted in the Infectious Diseases Hospital, Calcutta were analysed. Eight hundred and forty V. cholerae strains isolated from 1992 through 1997 were tested for susceptibility to 11 antibiotics. Cluster analysis was done using SPSS software. RESULTS: Most of the strains exhibited MDR with fluctuating trends as the resistance profile diverged each year. A total of 119 different resistance profiles exhibited by V. cholerae O1, O139 and non-O1, non-O139 serogroups were analysed by cluster combination method. During 1993 and 1994, 53 per cent of V. cholerae O139 and 82 per cent of V. cholerae O1 serogroups, respectively, exhibited maximal number of new resistance patterns. The frequency of new resistance patterns among V. cholerae non-O1, non-O139 was constantly high (33-47%) during 1995 to 1997. INTERPRETATION & CONCLUSIONS: With a few exceptions, preponderance of the resistance profiles was generally not confined to any serogroup. The cluster analysis depicted dissemination of some of the resistance patterns commonly found among V. cholerae non-O1, non-O139 belonging to different serogroups to the O139 serogroup in the succeeding years. In this study we have shown that the V. cholerae strains are resistant to several antibiotics with constant change in the MDR profiles. It is imperative to define the susceptibility pattern of the strains to determine the effective drug of choice for the treatment of cholera.

Shiga-toxin producing Escherichia coli from healthy cattle in a semi-urban community in Calcutta, India.

From October to December 1998, single faecal samples from 67 healthy cattle in a semi-urban community near Calcutta were examined for Shiga toxin producing Esch. coli (STEC) using a multiplex PCR primary screen followed by plating on sorbitol MacConkey agar. STEC was isolated from the faeces of 7 (10.5%) animals. The eight strains isolated belonged to eight serotypes viz, O146:H1, O149:HNT, ONT:H34, ONT:H19, O88:HN, ONT:H2, O82:H8 and O28ac:H21. Bead enzyme linked immunosorbent assay showed that three strains produced Shiga toxin 1, one produced Shiga toxin 2 and four produced both.

Clonal analysis of non-toxigenic Vibrio cholerae O1 associated with an outbreak of cholera.

We examined the clonal relationships among eight clinical isolates of non-toxigenic (NT) V. cholerae O1 associated with a cluster of cases of cholera in Warangal, Andhra Pradesh in south India and compared their relatedness to toxigenic O1 strains of classical and E1Tor biotypes and with O139 Bengal strains of V. cholerae by pulsed-field gel electrophoresis (PFGE). Phylogentic analysis of the NotI restriction fragment length polymorphism showed that all the NT. V. cholerae O1 strains formed a tight cluster with more than 80 per cent similarity. Interestingly, the NT V. cholerae O1 cluster was more closely related to V. cholerae O139 than to classical and E1Tor biotypes of V. cholerae O1 indicating closer genetic relationships between NT V. cholerae 01 and O139 Bengal strains that were isolated during the same time-frame.

Comparison between the multiplex PCR, sensitivity to biotype specific phages & polymyxin B for biotyping of Vibrio cholerae O1.

A total of 196 Vibrio cholerae O1 strains isolated between 1970 and 1996 were biotyped by multiplex PCR, susceptibility to polymyxin B and sensitivity to biotype specific phages. We modified the multiplex PCR by increasing the primer concentration of tcpA to improve the results. Comparison of the results of modified multiplex PCR and sensitivity to biotype specific phages and to polymyxin B showed that multiplex PCR was as efficient as phage typing for biotyping of V. cholerae O1. All the strains of V. cholerae O1 could be accurately distinguished based on polymyxin B sensitivity. Thus our results show that susceptibility of strains of V. cholerae O1 to polymyxin B is the easiest method to biotype V. cholerae O1 and is feasible in most laboratories when compared with multiplex PCR and sensitivity to biotype specific phages.