This study describes a multiplex PCR assay for the detection of antibiotic resistance genes and the SXT element in Vibrio cholerae. Conditions were optimized to amplify fragments of sulII (encoding sulfamethoxazole resistance), dfrA1 (O1-specific trimethoprim resistance), dfr18 (O139-specific trimethoprim resistance), strB (streptomycin B resistance) and the SXT element simultaneously in one PCR. This multiplex PCR was evaluated on 142 V. cholerae isolates and the results correlated with the phenotypic antibiotic data obtained using a disc diffusion assay and a colony blot assay. Thus this one-step PCR can be used as a simple, rapid and accurate method for identification of antibiotic resistance profiles and could be used for the surveillance of the spread of antibiotic resistance determinants in epidemiological and environmental studies.

A collection of ten strains of Vibrio cholerae O139, comprising six isolates from Eichhornia crassipes, two from water of the River Ganga, and one each from a well and a hand pump, were characterized. All the strains carried the CTX genetic element (ctxA, zot, and ace) except for the st gene and carried structural and regulatory genes for toxin-coregulated pilus (tcpA, tcpI, and toxR), adherence factor (ompU), and accessory colonization factor (acfB); all produced cholera toxin (CT). These strains were resistant to trimethoprim, sulfamethoxazole, streptomycin, and to the vibriostatic agent pteridine. Results obtained by ribotyping and enterobacterial repetitive intergenic consensus sequence-PCR fingerprint analysis indicate that multiple clones of toxigenicpathogenic V. cholerae O139 were present in the aquatic environment.Until 1992, Vibrio cholerae belonging to serogroup O1 was considered to be the only causative agent of epidemic cholera (2). However, V. cholerae non-O1 serogroups were associated mostly with sporadic cases of diarrhea and extraintestinal infections (4, 24). It was found recently that a highly epidemic form of cholera-like disease on the Indian subcontinent was strongly associated with non-O1 strains of V. cholerae designated V. cholerae O139 Bengal (1, 21, 25), which subsequently spread to the different parts of the world (1, 6, 11). Studies of V. cholerae from environmental surface water indicated that O139 Bengal, like O1 vibrios and non-O1, non-O139 vibrios, may survive better in the aquatic environment in association with aquatic plants (13,14,26) and that environmental water may be a reservoir for infectious V. cholerae O139 (14, 29).In the present study, we used molecular techniques to analyze V. cholerae O139 Bengal isolated between January 1992 and September 1994 from water and the aquatic plant Eichhornia crassipes (water hyacinth) from the river Ganga, Varanasi, India, to study genomic diversity and to examine the presence of virulence and regulatory genes, pathogenicity islands, and antibiotic resistance. Results obtained by ribotyping, enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR, PCR analysis, and antibiotic susceptibility testing indicated that the cholera outbreaks caused by O139 vibrios between 1992 and 1994 were caused by toxigenic-pathogenic and antibiotic-resistant V. cholerae O139 strains identical to that found in the aquatic environment.Bacterial strains. A total of 10 strains of V. cholerae O139 were included in this study. Six isolates (EC1, EC2, EC3, EC6, EC7, and EC8) were from the aquatic plant Eichhornia crassipes and were collected between 28 January and 6 April 1992; two (GW3 and GW4) were from surface water of the River Ganga and were collected between 3 August and 29 August 1993 from different ghats on the bank of the River Ganga in Varanasi; and one each was from a well (WO4, collected on 10 September 1994 from Bhadaini, Varanasi) and a hand pump (HP11, collected on 5 August 1992 from Assi, Varanasi). The samples from the aquatic plants ...

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We characterized a Vibrio cholerae O139 strain isolated from a diarrheal patient admitted to Taluk Hospital, Cherthala, Alleppey, Kerala, India, on 9 June 2000. The V. cholerae O139 strain possesses the core of the CTX genetic element, colonization toxin-coregulated pilus, the adherence outer membrane protein, and the central regulatory protein encoded by toxR and produces cholera toxin (200 pg/ml). We provide molecular evidence showing that toxigenic V. cholerae O139 strain ALO95 belongs to a distinct genotype characterized by a unique ribotype designated B-VII and has a unique enterobacterial repetitive intergenic consensus sequence PCR fingerprint profile designated E-V.Vibrio cholerae belonging to serogroup O1 biotype El Tor was considered the causative agent of diarrhea until the emergence of V. cholerae O139 Bengal in 1992. This organism caused an explosive epidemic in India and Bangladesh and subsequently in neighboring countries (1, 9, 10). Molecular studies using pulsed-field gel electrophoresis (8), ribotyping (4), and restriction fragment length polymorphism of the CTX genetic element of V. cholerae O139 Bengal have demonstrated the emergence of new clones with temporal changes in phenotypic and genetic properties (2, 4). During the height of the V. cholerae O1 biotype El Tor outbreak in Kerala, India, in the year 2000, we isolated V. cholerae O139 Bengal strain ALO95 from a 66-year-old female diarrheal patient admitted to the Taluk Hospital, Cherthala, Alleppey, Kerala, India, on 9 June 2000.In this report, we present molecular evidence that toxigenic V. cholerae O139 strain ALO95 belongs to a distinct genotype characterized by a unique ribotype and has a unique enterobacterial repetitive intergenic consensus sequence (ERIC) PCR fingerprint profile.A V. cholerae strain was isolated from a stool sample from a diarrheal patient and identified by using standard bacteriologic techniques (16). This strain agglutinated with monoclonal O139 antiserum supplied by the World Health Organization Regional Office of Southeast Asia, New Delhi, India, and was confirmed to belong to V. cholerae serogroup O139.A hexaplex PCR was used to determine the presence of virulence and regulatory genes, including ctxA, zot, ace, tcpA, ompU, and toxR, as described earlier (12). Briefly, the amplification program began with initial denaturation at 94°C for 2 min, followed by 20 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 1 min and 10 cycles of denaturation at 94°C for 1 min, annealing at 54°C for 1 min, and extension at 72°C for 1 min. A final extension was done at 72°C for 10 min. V. cholerae O1 serotype Inaba biotype classical strain 569B and V. cholerae O1 serotype Ogawa biotype El Tor strain 20 were used as the PCR positive controls for ctxA, zot, ace, tcpA, ompU, and toxR. Aliquots of PCR products were analyzed by agarose (1.8%, wt/vol) gel electrophoresis in 0.5ϫ Tris-borate-EDTA buffer, stained in ethidium bromide, and visualized with a Fluoro-S MultiImager (Bio-Rad, In...

Until the emergence of Vibrio cholerae O139 in 1992, toxigenic strains of V. cholerae O1 were considered to be the only causative agents of epidemic and pandemic cholera (3). Molecular studies using pulsed-field gel electrophoresis (4), ribotyping (3), enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR (1), and restriction fragment length polymorphism analysis of the CTX genetic element of toxigenic V. cholerae O139 Bengal have demonstrated the emergence of new clones with temporal changes in phenotypic and genetic properties. However, cholera toxin gene-negative O139 strains that failed to hybridize with ctxA, zot, and ace probes either shared the ribotype of toxigenic strains or belonged to a different ribotype distinct from those of toxigenic O139 strains (3).We report the characterization of a cholera toxin genenegative V. cholerae O139 Bengal strain CO788 isolated from a stool sample of a diarrheal patient and provided