Identification of a vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage.

We identify and characterize a gene cluster in El Tor Vibrio cholerae that encodes a cytotoxic activity for HEp-2 cells in vitro. This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the V. cholerae genome. We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity. The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif. Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD. In V. cholerae strains of the classical biotype, a deletion within the gene cluster removes rtxC and eliminates cytotoxic activity. Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity. Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence. Furthermore, the RTX toxin of V. cholerae may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines.

Use of representational difference analysis to identify genomic differences between pathogenic strains of Vibrio cholerae.

Representational difference analysis (RDA) is a recently developed technique used for amplifying genetic differences between two closely related genomes. We compared RDA and a modified version of RDA to examine genomic differences between the two Vibrio cholerae serogroups that cause epidemic cholera, O1 and O139, and between the two biotypes of the O1 serogroup. With both techniques, we recovered several sequences known to be found only in V. cholerae O139 but absent in its presumed progenitor, V. cholerae O1 El Tor. A greater number of unique fragments were generated in comparing the two V. cholerae O1 biotypes, consistent with the probable greater genetic differences between the two biotypes.

Primary septicemia caused by Vibrio cholerae non-O1 acquired on Cape Cod, Massachusetts.

We describe a patient with non-O1, non-O139 Vibrio cholerae septicemia associated with hemorrhagic bullous skin lesions of the lower extremities. The patient had underlying liver disease, and he probably acquired the organism through ingestion of raw clams. Although his condition rapidly improved during appropriate therapy, the patient's cellulitis and skin lesions persisted and he developed a fluid collection of the lower extremity that required drainage. Molecular methods were used to examine the non-O1 V. cholerae isolate for several known virulence factors of V. cholerae O1. The isolate failed to express cholera toxin and toxin-coregulated pilus (Tcp) and was negative in Southern hybridizations for ctxB, tcpA, toxR, and toxT. The vast majority of vibrio infections in the United States are clustered in the Gulf Coast area. This patient acquired the infection on Cape Cod. To our knowledge, this is the first case of non-O1 V. cholerae septicemia reported to have occurred in Massachusetts. Given the high fatality rate of this infection, it is important for physicians to consider this diagnosis in patients who have underlying risk factors and appropriate epidemiologic exposures, even when they reside as far north as the New England states.

Comparison of Vibrio cholerae O139 with V. cholerae O1 classical and El Tor biotypes.

Vibrio cholerae O139 is a recently identified non-O1 V. cholerae strain responsible for outbreaks of epidemic cholera in India, Bangladesh, and Thailand in the past 2 years. Other workers have demonstrated the presence of the cholera toxin genetic element in V. cholerae O139, unlike the situation for other non-O1 V. cholerae strains. We sought to compare further this strain with strains of V. cholerae O1, classical and El Tor biotypes, by classic microbiologic methods, Southern blot analysis for restriction fragment length polymorphisms with probes for iron-regulated genes of V. cholerae O1, and comparisons of outer membrane protein profiles. Our results were similar for V. cholerae O139 and the El Tor biotype of V. cholerae O1, with the exception of the constitutive expression in V. cholerae O139 of OmpS, an outer membrane protein that was maltose inducible in comparison strains of V. cholerae O1.