Genomic profile of antibiotic resistant, classical ctxB positive Vibrio cholerae O1 biotype El Tor isolated in 2003 and 2005 from Puri, India: A retrospective study.

Objectives: To examine eight strains of Vibrio cholerae O1 isolated in 2003 and 2005 from Puri, India, for antibiotic susceptibility, presence of virulence and regulatory genes, cholera toxin (CT) production, CTX arrangement and genomic profiles. Materials and Methods: Bacterial strains were tested for antibiotic susceptibility using disc diffusion assay. Polymerase chain reaction determined the presence of antibiotic resistance, virulence and regulatory genes. To determine the type of cholera toxin subunit B (ctxB), nucleotide sequencing was performed. Southern hybridisation determined the number and arrangement of CTXΦ. Ribotyping and pulsed‑field gel electrophoresis (PFGE) were used to determine the genomic profile of isolates. Results: All the eight strains, except one strain, showed resistant to nalidixic acid, sulphamethoxazole, streptomycin and trimethoprim and possessed the sullI, strB, dfrA1 and intSXT genes. All the strains carried the toxin‑co‑regulated pilus pathogenicity island, the CTX genetic element, the repeat in toxin and produced CT. Restriction fragment length polymorphism (RFLP) analysis showed that V. cholerae O1 possess a single copy of the CTX element flanked by tandemly arranged RS element. Nucleotide sequencing of the ctxB gene showed the presence of classical ctxB. RFLP analysis of conserved rRNA gene showed two ribotype patterns. PFGE analysis also showed at least three PFGE patterns, irrespective of year of isolations, indicating the genomic relatedness among them. Conclusion: Overall, these data suggest that classical ctxB‑positive V. cholerae O1 El Tor strains that appeared in 2003 continue to cause infection in 2005 in Puri, India, and belong to identical ribotype(s) and/or pulsotype(s). There is need to continuous monitor the emergence of variant of El Tor because it will improve our understanding of the evolution of new clones of variant of V. cholerae.

Characteristics of Vibrio cholerae O1 isolated from water of the River Ganga, Varanasi, India.

Background: Vibrio cholerae is an autochthonous inhabitant of fresh and brackish water and estuarine system. Investigation of V. cholerae from the River Ganga seems important to find variation in CTX arrangement and genomic diversity. Objectives: To investigate V. cholerae O1 strains for the presence of virulence and regulatory genes, variation in number and organisation of the pre‑CTXФ and/or CTXФ, and for the genomic diversity. Materials and Methods: Polymerase chain reaction (PCR) was used to detect virulence and regulatory genes, type of rstR and location of CTXΦ on the chromosome. Southern hybridisation was conducted to see the number and arrangement of pre‑CTXΦ and CTXΦ. Ribotyping and pulsed‑field gel electrophoresis were used to find genetic relatedness. Results: Seven strains gave positive results by PCR for the gene encoding for ctxA, zot, ace, tcpA (El Tor), ompU, and toxR, except one strain that was negative for the ctxA. Three strains were positive for the tcpA (El Tor), ompU and toxR genes. Determination of CTX organisation showed that among the ctx‑positive strains, four harboured two copies of CTXETФ arranged in tandem and two harboured one copy of CTXETФ, and one ctx‑negative strain harboured only one copy of pre‑CTXETФ. Pulsotype and ribotype analysis showed existence of at least three pulsotype and ribotypes indicating diversity in genomic content among them. Conclusion: This study thus indicates that multiple clones (ribotypes/pulsotypes) of V. cholerae O1 carrying pre‑CTXΦ and/or CTXΦ and ctx‑negative strains were present in the water of the River Ganga, Varanasi, India.