Asis Khan

Antibiotic Resistance, Virulence Gene, and Molecular Profiles of Shiga Toxin-Producing Escherichia coli Isolates from Diverse Sources in Calcutta, India

ABSTRACT Antibiotic resistance, virulence gene, and molecular profiles of Shiga toxin-producing Escherichia coli (STEC) non-O157 strains isolated from human stool samples, cow stool samples, and beef samples over a period of 2 years in Calcutta, India, were determined. Resistance to one or more antibiotics was observed in 49.2% of the STEC strains, with some of the strains exhibiting multidrug resistance. The dominant combinations of virulence genes present in the strains studied were stx1 and stx2 (44.5% of strains) and stx1, stx2, and hlyA (enterohemorrhagic E. coli hemolysin gene) (19% of strains). Only 6.4% of the STEC strains harbored eae. The diversity of STEC strains from various sources was assessed by random amplification of polymorphic DNA (RAPD). STEC strains that gave identical or nearly similar DNA fingerprints in RAPD-PCR and had similar virulence genotypes were further characterized by pulsed-field gel electrophoresis (PFGE). Identical RAPD and PFGE profiles were observed in four sets of strains, with each set comprising two strains. There was no match in the RAPD and PFGE profiles between strains of STEC isolated from cows and those isolated from humans. It appears that the clones present in bovine sources are not transmitted to humans in the Calcutta setting although these strains showed evolutionary relatedness. Maybe for this reason, STEC has still not become a major problem in India.

Virulence Genes and Neutral DNA Markers of Helicobacter pylori Isolates from Different Ethnic Communities of West Bengal, India

ABSTRACT Virulence-associated genes and neutral DNA markers of Helicobacter pylori strains from the Santhal and Oroan ethnic minorities of West Bengal, India, were studied. These people have traditionally been quite separate from other Indians and differ culturally, genetically, and linguistically from mainstream Bengalis, whose H. pylori strains have been characterized previously. H. pylori was found in each of 49 study participants, although none had peptic ulcer disease, and was cultured from 31 of them. All strains carried the cag pathogenicity island and potentially toxigenic s1 alleles of vacuolating cytotoxin gene (vacA) and were resistant to at least 8 μg of metronidazole per ml. DNA sequence motifs in vacA mid-region m1 alleles, cagA, and an informative insertion or deletion motif next to cagA from these strains were similar to those of strains from ethnic Bengalis. Three mobile elements, IS605, IS607, and ISHp608, were present in 29, 19, and 10%, respectively, of Santhal and Oroan strains, which is similar to their prevalence in Bengali H. pylori. Thus, there is no evidence that the gene pools of H. pylori of these ethnic minorities differ from those of Bengalis from the same region. This relatedness of strains from persons of different ethnicities bears on our understanding of H. pylori transmission between communities and genome evolution.

Association of Cytolethal Distending Toxin Locus cdtB with Enteropathogenic Escherichia coli Isolated from Patients with Acute Diarrhea in Calcutta, India

ABSTRACT Among Escherichia coli strains isolated from stool specimens from patients with acute diarrhea, 1.4% were found to harbor cdtB by use of enrichment cytolethal distending toxin (CDT) PCR. These isolates were identified as being enteropathogenic E. coli (EPEC). In a retrospective study using a probe hybridization assay, 6 of 138 EPEC strains were found to harbor the cdtB locus. cdtB-positive isolates mostly belong to the O86a and O127a serogroups, with the former being associated with higher expression of CDT. Pulsed-field gel electrophoresis profiles showed that the EPEC strains harboring cdtB strains are genetically diverse.

Escalating Association of Vibrio cholerae O139 with Cholera Outbreaks in India

ABSTRACT Between December 1999 and December 2000, teams from the National Institute of Cholera and Enteric Diseases, Calcutta, India, examined eight outbreaks of cholera, which occurred in different parts of the country distant from each other. In two of these outbreaks each, only V. cholerae O1 biotype ElTor or V. cholerae O139 could be isolated, while in the remaining four outbreaks, both O1 and O139 were isolated. The interesting feature is the escalating association of V. cholerae O139 with outbreaks of cholera; two of the most recent outbreaks, one in Calcutta and one in Orissa, were caused exclusively by O139. The O139 strains from the six different outbreaks were genotypically closely related. These trends indicate a shift in the outbreak propensity of V. cholerae O139.

Development of a Simple Latex Agglutination Assay for Detection of Shiga Toxin-Producing Escherichia coli (STEC) by Using Polyclonal Antibody against STEC

ABSTRACT Rabbit antiserum raised against the whole-cell antigen of Shiga toxin-producing Escherichia coli (STEC) strain VT3 (stx1+stx2+eae+) was repeatedly adsorbed with heat-killed cells of different non-STEC strains and other enteric bacteria. Thus, the antiserum obtained was designated VT3 antiserum. VT3 antiserum reacted with intimin type γ. We assessed the reactivity of VT3 antiserum to whole-cell lysates of 87 strains of E. coli and other enteric bacteria by immunoblotting. The antiserum recognized the 97-kDa protein in whole-cell lysate from strain VT3, and 36 (83.7%) of the 43 STEC strains were positive for the STEC antigen. None of the non-STEC strains or strains of other species examined tested positive by immunoblotting. Based on this result, we developed a latex agglutination assay for the detection of STEC strains. Thirty-five (81.4%) of the 43 STEC strains tested positive for the STEC antigen by the latex agglutination assay. One (3.3%) of the 30 non-STEC strains and none of the strains of the other enteric bacteria included in this study tested positive by the latex agglutination assay. The corresponding specificity of the latex agglutination assay was approximately 98%. Results of this study showed the production of STEC antiserum and the generation of a simple, cost-effective, sensitive, and specific latex agglutination assay for establishing an etiological diagnosis of STEC.

Environmental isolates of Aeromonas spp. harboring the cagA-like gene of Helicobacter pylori.

ABSTRACT We investigated the presence of cagA-like gene of Helicobacter pylori in environmental isolates of Aeromonas spp. from different water samples of Calcutta, India, by colony hybridization using a cagA-specific DNA probe and by PCR with cagA-specific primers. Nucleotide sequencing of five PCR products revealed 97 to 98% homology to canonical cagA of H. pylori 26695 as well as to four clinical H. pylori strains from Calcutta. The cagA-like gene of the environmental isolates was unstable in laboratory conditions and tended to be lost upon subculturing.

Dairy farm investigation on Shiga toxin-producing Escherichia coli (STEC) in Kolkata, India with emphasis on molecular characterization

An investigation was conducted to determine the distribution, virulence gene profile and phenotypes of Shiga toxin-producing Escherichia coli (STEC) strains within a dairy farm in Kolkata, India by characterizing the STEC strains isolated from healthy dairy cow and calf stool samples, raw milk and farm floor swabs from July 2001 to March 2002. Primary screening by multiplex-PCR detected stx1 and stx2, the common virulence genes of STEC, in 18.9% of cow faeces, 32.4% of calf stool samples, 21.6% of farm floor swabs and 4.5% of raw milk samples and viable STEC were recovered from 4.5, 9.9, 8.1 and 1.8% of the corresponding PCR-positive samples. Strains harbouring stx1 (63.3%) and hlyA (53.3%) were frequently detected compared to eae (13.3%). Most of the strains harboured similar sets of reported virulence genes common among isolates from diarrhoea patients. Most of the strains also exhibited multidrug resistance, sorbitol fermentation and produced enterohaemolysin. The randomly amplified polymorphic DNA-PCR (RAPD-PCR) profile of the STEC strains isolated from the farm milieu revealed diverse banding patterns and clonal analysis demonstrated that the strains from different sources were not identical but showed some genetic relatedness. The study demonstrates the potential of dairy farm for housing virulent STEC.

Environmental isolates of Citrobacter braakii that agglutinate with Escherichia coli O157 antiserum but do not possess the genes responsible for the biosynthesis of O157 somatic antigen.

While searching for Escherichia coli O157 in the aquatic environment of Calcutta using an immunodetection procedure, we fortuitously detected five strains of Citrobacter braakii, which cross-reacted with the commercially available O157 polyvalent antiserum. The five C. braakii isolates gave positive results when a sensitive dot-ELISA was performed with E. coli O157 monoclonal antibody. Further, the O157 monoclonal antibody recognized the bands of proteinase K treated whole cells of lipopolysaccharide of all the C. braakii isolates. Apart from weak reactions with two or three of the DNA probes, all the C. braakii strains did not hybridize with the other probes spanning the minimum region required for O157 O-antigen biosynthesis. These strains did not possess any of the virulence genes that are commonly found in the Shiga toxin-producing E. coli (STEC) specially the serotype O157: H7. Therefore, it appears that the serological cross-reaction between C. braakii and E. coli O157 antiserum is based on structural mimicry between the O-polysaccharide of C. braakii and E. coli O157.