We initially attempted to isolate a Vibrio cholerae O1 El Tor biotype that carries a novel variant of the cholera toxin gene (ctxAB) from environmental waters of Indonesia, where the seventh cholera pandemic by V. cholerae O1 El Tor biotype began. Nested PCR targeting the gene revealed that a total of eight strains were found to carry ctxAB. However, sequencing of the 16S rRNA genes of these isolates showed they were not V. cholerae but were either Klebsiella, Enterobacter, Pantoea, or Aeromonas. Subsequent nested PCR assays targeting all genes known to be encoded on the CTX phage (i.e., zot, ace, orfU, cep, rstB, rstA, and rstR) showed that one isolate belonged to the Enterobacter genus carried all the genes tested, while the other isolates lacked either 2, 3, or 5 of the genes. This evidence suggests that phages with ctxAB are genetically diverse and can infect not only V. cholerae and V. mimicus but also other species and genera in the form of a pseudolysogen. 1. Introduction Vibrio cholerae is a gastrointestinal pathogen that causes cholera, a notorious enteric disease with serious morbidity and mortality worldwide. The clinical strains belonging to serogroups O1 and O139 are responsible for all the major cholera epidemics and pandemics on record. The main virulence factor causing the disease, cholera toxin (CTX), is encoded by ctxA and ctxB (ctxA and ctxB; collectively referred to as ctxAB) . The ctxAB genes are present on a filamentous phage, called CTX phage, which has been shown to lysogenize only V. cholerae, V. mimicus, and other Vibrio species . To date, the world has experienced seven major pandemics of cholera since the early 19th century. The 5th and 6th pandemics were caused by toxigenic strains belonging to the classical biotype of serogroup O1 that possesses the classical type of ctxB, whereas the ongoing 7th pandemic, which began in 1961 on the island of Sulawesi in Indonesia , is caused by the El Tor biotype that carries the El Tor type of ctxB that has an amino acid sequence slightly different from that of the classical type . Over the past two decades, several V. cholerae strains of different serogroups carrying ctxB with amino acid sequences slightly different from each other have emerged. As of 2009, a total of nine genomic variants of ctxB including the classical type have been reported . We therefore attempted to isolate the V. cholerae O1 El Tor biotype that carried the novel ctxB variant gene from environmental waters of Indonesia. During the course of this attempt, we isolated bacterial strains that also
M. Das, A. Jaiswal, S. Pal, et al., “Dynamics of classical-El Tor switch of Vibrio cholerae strains isolated from 1961–2010,” International Journal of Antimicrobial Agents, vol. 40, no. 6, pp. 570–571, 2012.
E. F. Boyd, A. J. Heilpern, and M. K. Waldor, “Molecular analyses of a putative CTXφ precursor and evidence for independent acquisition of distinct CTXφs by toxigenic Vibrio cholerae,” Journal of Bacteriology, vol. 182, no. 19, pp. 5530–5538, 2000.
J. Chun, A. Huq, and R. R. Colwell, “Analysis of 16S-23S rRNA intergenic spacer regions of Vibrio cholerae and Vibrio mimicus,” Applied and Environmental Microbiology, vol. 65, no. 5, pp. 2202–2208, 1999.
C. L. Tarr, J. S. Patel, N. D. Puhr, E. G. Sowers, C. A. Bopp, and N. A. Strockbine, “Identification of Vibrio isolates by a multiplex PCR assay and rpoB sequence determination,” Journal of Clinical Microbiology, vol. 45, no. 1, pp. 134–140, 2007.
K. Khemayan, T. Pasharawipas, O. Puiprom, S. Sriurairatana, O. Suthienkul, and T. W. Flegel, “Unstable lysogeny and pseudolysogeny in Vibrio harveyi siphovirus-like phage 1,” Applied and Environmental Microbiology, vol. 72, no. 2, pp. 1355–1363, 2006.
E. F. Boyd and M. K. Waldor, “Alternative mechanism of cholera toxin acquisition by Vibrio cholerae: generalized transduction of CTXΦ by bacteriophage CP-T1,” Infection and Immunity, vol. 67, no. 11, pp. 5898–5905, 1999.
P. Kumar, A. Thulaseedharan, G. Chowdhury, T. Ramamurthy, and S. Thomas, “Characterization of novel alleles of toxin co-regulated pilus a gene (tcpA) from environmental isolates of Vibrio cholerae,” Current Microbiology, vol. 62, no. 3, pp. 758–763, 2011.
A. Sarkar, R. K. Nandy, G. B. Nair, and A. C. Ghose, “Vibrio pathogenicity island and cholera toxin genetic element-associated virulence genes and their expression in non-O1 non-O139 strains of Vibrio cholerae,” Infection and Immunity, vol. 70, no. 8, pp. 4735–4742, 2002.
E. F. Boyd, K. E. Moyer, L. Shi, and M. K. Waldor, “Infectious CTXΦ and the Vibrio pathogenicity island prophage in Vibrio mimicus: evidence for recent horizontal transfer between V. mimicus and V. cholerae,” Infection and Immunity, vol. 68, no. 3, pp. 1507–1513, 2000.
L. A. Sechi, I. Duprè, A. Deriu, G. Fadda, and S. Zanetti, “Distribution of Vibrio cholerae virulence genes among different Vibrio species isolated in Sardinia, Italy,” Journal of Applied Microbiology, vol. 88, no. 3, pp. 475–481, 2000.
M. Snoussi, E. Noumi, D. Usai, L. A. Sechi, S. Zanetti, and A. Bakhrouf, “Distribution of some virulence related-properties of Vibrio alginolyticus strains isolated from Mediterranean seawater (Bay of Khenis, Tunisia): investigation of eight Vibrio cholerae virulence genes,” World Journal of Microbiology and Biotechnology, vol. 24, no. 10, pp. 2133–2141, 2008.