Antimicrobial susceptibilities of Vibrio cholerae strains isolated from cholera patients admitted to the Infectious Diseases Hospital, Calcutta, India for 6 years were analysed to determine the changing trends; 840 V. cholerae strains isolated in 1992-1997 were included in this study. Among V. cholerae serogoup O1 and O139, ampicillin resistance increased from 1992 (35 and 70%, respectively) to 1997 (both serogroups 100%). Resistance to furazolidone and streptomycin was constantly high among V. cholerae O1 strains with gradual increase in resistance to other drugs such as ciprofloxacin, co-trimoxazole, neomycin and nalidixic acid. V. cholerae O139 strains exhibited susceptibilities to furazolidone and streptomycin comparable with those of O1 strains. However, after initial increase in resistance to chloramphenicol and co-trimoxazole, all the V. cholerae O139 strains became susceptible to these two drugs from 1995 onwards. Both V. cholerae O1 and O139 remained largely susceptible to gentamicin and tetracycline. V. cholerae non-O1, non-O139 strains, in contrast, exhibited high levels of resistance to virtually every class of antimicrobial agents tested in this study especially from 1995. Kruskal-Wallis one-way analysis showed that V. cholerae O1 Ogawa serogroup exhibited significant yearly increase in resistance to nine antibiotics followed by non-O1 non-O139 and O139 strains to six antibiotics and two antibiotics respectively. Interesting observation encountered in this study was the dissipation of some of the resistant patterns commonly found among V. cholerae non-O1 non-O139 or O1 serogroups to the O139 serogroup and vice versa during the succeeding years.

The ctxAB genes encoding cholera toxin, reside in the genome of a filamentous bacteriophage CTXphi. The presence of CTX prophage in non-epidemic environmental Vibrio cholerae strains is rare. The CTX prophage, the lysogenic form of CTXphi in V. cholerae, is comprised of the 'RS2' and the 'Core'. Analysis of the rstR gene present in the RS2 region of the CTX prophage revealed the presence of new alleles of the prophages in four environmental non-O1, non-O139 strains VCE22 (O36), VCE228 (O27), VCE232 (O4) and VCE233 (O27), and the CTX prophages are located in the small chromosomes. Phylogenetic analysis based on the nucleotide sequences of the rstR and orfU (present in the core) genes of these prophages placed them in a single unique cluster, which is distally located compared with that of epidemic V. cholerae O1 strains. Further analysis indicated that the genome of the prophage present in the strain VCE22 is devoid of the ctxAB genes, called pre-CTX prophage and the strain also possess the toxin-coregulated pilus protein coding gene tcpA of classical type, another important pathogenicity determining locus of the epidemic V. cholerae strains. Comparative analysis of the nucleotide sequences of the rstR and orfU genes indicated that the pre-CTX prophage of VCE22 might be the progenitor of new alleles of the CTX prophages present in these environmental strains.

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Toxigenic Vibrio cholerae O1 and O139 serogroups have the capacity of causing epidemic and pandemic cholera but are infrequently found in the environment. The other serogroups are abundant in aquatic environments but do not possess the virulence genes necessary for causing the disease. Of the 559 environmental strains of V. cholerae, collected during different periods from environmental samples in Calcutta, 9 (1.6%) harboured the heat-stable enterotoxin gene (stn). Six of the 9 strains belonged to the O14 serogroup. Thus, V. cholerae strains carrying the stn gene revealed preferential association with the O14 serogroup. Three of the six strains harboured the tcpA gene of the E1 Tor type, which is an unusual feature among environmental V. cholerae strains. A strain that possessed the E1 Tor type tcpA also had the CTX prophage. Pulsed field gel electrophoresis (PFGE) revealed that the stn gene positive O14 strains of V. cholerae were not clonal.