Serendipitous Isolation of Non-Vibrio Bacterial Strains Carrying the Cholera Toxin Gene from Environmental Waters in Indonesia

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) [1]. 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 [2]. 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 [1], 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 [3]. 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 [4]. 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