Distribution and characterization of integrons in various serogroups of Vibrio cholerae strains isolated from diarrhoeal patients between 1992 and 2000 in Kolkata, India.

A total of 133 clinical strains of Vibrio cholerae comprising 44 strains of O1, 45 strains of O139 and 44 strains of non-O1, non-O139 serogroups isolated from hospitalized patients in Kolkata, India, from 1992 to 2000 was examined for the presence of class 1, 2 and 4 integrons. By PCR and DNA sequencing, seven strains of O1, one strain of O139 and six strains of non-O1, non-O139 serogroups were found to contain class 1 integron-harbouring genes aadA1, aadA2 (encoding resistance to streptomycin and spectinomycin), blaP1 (resistance to beta-lactams), aar-3 (resistance to rifampicin), aacA4 (resistance to kanamycin and gentamicin), and dfrA1 and dfrA15 (resistance to trimethoprim). Most strains produced one or two bands using primers specific for the amplification of the variable region where the antibiotic-resistance genes are located, and their sizes ranged from 700 to 1250 bp. However, one strain of V. cholerae O1 isolated in 1994 gave a 2483 bp fragment, the largest fragment so far found in a class 1 integron of V. cholerae O1. No strain was positive for the intI2 gene. All V. cholerae strains, regardless of serogroup, were positive for the intI4 gene by PCR and using a colony hybridization test. Amplification of the intI4 gene by PCR yielded a 2200 bp fragment (1260 bp larger than the expected size) from three V. cholerae O139 strains isolated in 1999. Sequence analysis of this amplicon revealed an insertion of IS1359 in the middle of the intI4 gene. These data indicate that a class 1 integron is present in some clinical strains of V. cholerae isolated in Kolkata, India, and that a class 4 integron is ubiquitously distributed among V. cholerae strains regardless of serogroup.

Genomic sequence and receptor for the Vibrio cholerae phage KSF-1phi: evolutionary divergence among filamentous vibriophages mediating lateral gene transfer.

KSF-1phi, a novel filamentous phage of Vibrio cholerae, supports morphogenesis of the RS1 satellite phage by heterologous DNA packaging and facilitates horizontal gene transfer. We analyzed the genomic sequence, morphology, and receptor for KSF-1phi infection, as well as its phylogenetic relationships with other filamentous vibriophages. While strains carrying the mshA gene encoding mannose-sensitive hemagglutinin (MSHA) type IV pilus were susceptible to KSF-1phi infection, naturally occurring MSHA-negative strains and an mshA deletion mutant were resistant. Furthermore, d-mannose as well as a monoclonal antibody against MSHA inhibited infection of MSHA-positive strains by the phage, suggesting that MSHA is the receptor for KSF-1phi. The phage genome comprises 7,107 nucleotides, containing 14 open reading frames, 4 of which have predicted protein products homologous to those of other filamentous phages. Although the overall genetic organization of filamentous phages appears to be preserved in KSF-1phi, the genomic sequence of the phage does not have a high level of identity with that of other filamentous phages and reveals a highly mosaic structure. Separate phylogenetic analysis of genomic sequences encoding putative replication proteins, receptor-binding proteins, and Zot-like proteins of 10 different filamentous vibriophages showed different results, suggesting that the evolution of these phages involved extensive horizontal exchange of genetic material. Filamentous phages which use type IV pili as receptors were found to belong to different branches. While one of these branches is represented by CTXphi, which uses the toxin-coregulated pilus as its receptor, at least four evolutionarily diverged phages share a common receptor MSHA, and most of these phages mediate horizontal gene transfer. Since MSHA is present in a wide variety of V. cholerae strains and is presumed to express in the environment, diverse filamentous phages using this receptor are likely to contribute significantly to V. cholerae evolution.