Clinical isolates of Vibrio cholerae O1 El Tor Ogawa of 2009 from Kolkata, India: preponderance of SXT element and presence of Haitian ctxB variant.

BACKGROUND: Increase in the number of multidrug resistant pathogens and the accompanied rise in case fatality rates has hampered the treatment of many infectious diseases including cholera. Unraveling the mechanisms responsible for multidrug resistance in the clinical isolates of Vibrio cholerae would help in understanding evolution of these pathogenic bacteria and their epidemic potential. This study was carried out to identify genetic factors responsible for multiple drug resistance in clinical isolates of Vibrio cholerae O1, serotype Ogawa, biotype El Tor isolated from the patients admitted to the Infectious Diseases Hospital, Kolkata, India, in 2009. METHODOLOGY/PRINCIPAL FINDINGS: One hundred and nineteen clinical isolates of V. cholerae were analysed for their antibiotic resistance phenotypes. Antibiogram analysis revealed that majority of the isolates showed resistance to co-trimoxazole, nalidixic acid, polymixin B and streptomycin. In PCR, SXT integrase was detected in 117 isolates and its sequence showed 99% identity notably to ICEVchInd5 from Sevagram, India, ICEVchBan5 from Bangladesh and VC1786ICE sequence from Haiti outbreak among others. Antibiotic resistance traits corresponding to SXT element were transferred from the parent Vibrio isolate to the recipient E. coli XL-1 Blue cells during conjugation. Double-mismatch-amplification mutation assay (DMAMA) revealed the presence of Haitian type ctxB allele of genotype 7 in 55 isolates and the classical ctxB allele of genotype 1 in 59 isolates. Analysis of topoisomerase sequences revealed the presence of mutation Ser83 → Ile in gyrA and Ser85→ Leu in parC. This clearly showed the circulation of SXT-containing V. cholerae as causative agent for cholera in Kolkata. CONCLUSIONS: There was predominance of SXT element in these clinical isolates from Kolkata region which also accounted for their antibiotic resistance phenotype typical of this element. DMAMA PCR showed them to be a mixture of isolates with different ctxB alleles like classical, El Tor and Haitian variants

Synergistic effect of various virulence factors leading to high toxicity of environmental V. cholerae non-O1/ non-O139 isolates lacking ctx gene : comparative study with clinical strains.

Vibrio cholerae non-O1/ non-O139 serogroups have been reported to cause sporadic diarrhoea in humans. Cholera toxins have been mostly implicated for hypersecretion of ions and water into the small intestine. Though most of the V. cholerae non-O1/ non-O139 strains lack these cholera toxins, several other innate virulence factors contribute towards their pathogenicity. The environmental isolates may thus act as reservoirs for potential spreading of these virulence genes in the natural environment which may cause the emergence of epidemic-causing organisms.The environmental isolates of vibrios were obtained from water samples, zooplanktons and phytoplanktons, from a village pond in Gandhinagar, Gujarat, India. They were confirmed as Vibrio cholerae non-O1/ non-O139 using standard biochemical and serotyping tests. PCR experiments revealed that the isolates lacked ctxA, ctxB, tcpA, zot and ace genes whereas other pathogenicity genes like toxR, rtxC, hlyA, hapA and prtV were detected in these isolates. Compared with epidemic strain V. cholerae O1 El Tor N16961, culture supernatants from most of these isolates caused higher cytotoxicity to HT29 cells and higher hemolytic, hemagglutinin and protease activities. In rabbit ileal loop assays, the environmental isolates showed only 2-4 folds lesser fluid accumulation in comparison to N16961 and a V. cholerae clinical isolate IDH02365 of 2009. Pulsed Field Gel electrophoresis and Random amplification of Polymorphic DNA indicated that these isolates showed considerable diversity and did not share the same clonal lineage even though they were derived from the same water source. All the isolates showed resistance to one or more antibiotics.Though these environmental isolates lacked the cholera toxins, they seem to have adopted other survival strategies by optimally utilising a diverse array of several other toxins. The current findings indicate the possibility that these isolates could cause some gastroenteric inflammation when ingested and may serve as progenitors for overt disease-causing organisms

Agarose gel (1%) analysis of PCR product of SXT integrase from IDH isolates and their transconjugants.

<p>PCR products obtained using genomic DNA templates from clinical isolates or their transconjugants have been electrophoresed in different lanes as follows: Lane M : 1 kb ladder (Fermentas); Lane 1: Positive control <i>V.cholerae</i> O139 MO10; Lane 2: Recipient <i>E. coli</i> XL-1 Blue; Lanes 3 and 4: Negative controls of no DNA template and SXT-negative IDH02095 isolate respectively; Lanes 5 and 6 : IDH01572 (SXT-positive) isolate and its transconjugant respectively; Lanes 7 and 8 : IDH01738 (SXT-positive) isolate and its transconjugant respectively.</p

Antimicrobial susceptibility of IDH01572, IDH01738 and their transconjugants.

<p>The antibiotic names are as described in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056477#pone-0056477-g001" target="_blank">figure 1</a>.</p><p>Bold face indicates the resistance traits from recipient XL-1Blue cells.</p

Antibiotic susceptibility profile of 119 clinical isolates from Kolkata, India, in 2009.

<p>AMP, Ampicillin; CHL, Chloramphenicol; CIP, Ciprofloxacin; COT, Co-Trimoxazole; GEN, Gentamicin; KAN, Kanamycin; NAL, Nalidixic Acid; NEO, Neomycin; NOR, Norfloxacin; PB, Polymixin B; STR, Streptomycin; SUL, Sulfisoxazole; TET, Tetracycline; TRI, Trimethoprim.</p

Cytotoxic effect of environmental isolates on HT29 cells.

<p>HT29 cells not treated with supernatant from any of the isolates (A); Cell rounding in HT29 cells treated with the supernatant from P1(B); Clumping of HT29 cells treated with the supernatant from Z2 (C); Bar diagram showing cytotoxic effect of culture supernatants from environmental isolates and the controls <i>V. cholerae</i> N16961 and <i>E. coli</i> DH5α in terms of percentage of dead cell (D). The experiment was independently performed three times. The error bars indicate standard deviation.</p

Rabbit ileal loop assay to assess entrotoxigenic activity.

<p>Pictorial view of rabbit ileal loop of different clinical and environmental strains (A). Analysis of fluid accumulation of different <i>V. cholerae</i> strains (B). Rabbit ileal loops were inoculated with 10⁸ CFU of each strain N16961 (O1 El tor, clinical), IDH02365(O1 El tor, clinical), Z2 (non-O1/non-O139, Environmental) and W3(non-O1/non-O139, Environmental) in PBS and incubated for 18 h. Results are expressed as fluid accumulation (FA) (in milliliters) per loop length (in centimeters). Shown are means ± standard deviations; <i>n</i> = 3.</p

Analysis of protease activity of the environmental isolates.

<p>SDS PAGE (10%) analysis of culture supernatants (A). Lane 1: Protein markers (Pageruler, Fermentas) with molecular mass in kDa indicated on the left; Lanes 2 to 8, culture supernatants of Z2, Z3, P2, W1, W3, <i>V. cholerae</i> N16961 and <i>E. coli</i> DH5α, respectively. Protease activity of culture supernatants on 1.5% Skim milk agar plate (B). Sample identity has been indicated near each well.</p