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.