Characterization of Vibrio cholerae O1 El Tor Biotype Variant Clinical Isolates from Bangladesh and Haiti, Including a Molecular Genetic Analysis of Virulence Genes

Abstract

Vibrio cholerae serogroup O1, the causative agent of the diarrheal disease cholera, is divided into two biotypes: classical and El Tor. Both biotypes produce the major virulence factors toxin-coregulated pilus (TCP) and cholera toxin (CT). Although possessing genotypic and phenotypic differences, El Tor biotype strains displaying classical biotype traits have been reported and subsequently were dubbed El Tor variants. Of particular interest are reports of El Tor variants that produce various levels of CT, including levels typical of classical biotype strains. Here, we report the characterization of 10 clinical isolates from the International Centre for Diarrhoeal Disease Research, Bangladesh, and a representative strain from the 2010 Haiti cholera outbreak. We observed that all 11 strains produced increased CT (2-to 10-fold) compared to that of wild-type El Tor strains under in vitro inducing conditions, but they possessed various TcpA and ToxT expression profiles. Particularly, El Tor variant MQ1795, which produced the highest level of CT and very high levels of TcpA and ToxT, demonstrated hypervirulence compared to the virulence of El Tor wild-type strains in the infant mouse cholera model. Additional genotypic and phenotypic tests were conducted to characterize the variants, including an assessment of biotype-distinguishing characteristics. Notably, the sequencing of ctxB in some El Tor variants revealed two copies of classical ctxB, one per chromosome, contrary to previous reports that located ctxAB only on the large chromosome of El Tor biotype strains.Vibrio cholerae is a Gram-negative, curved-rod-shaped bacterium that is the causative agent of the watery diarrheal disease cholera. The structure of the cell surface lipopolysaccharide O antigen is used to classify V. cholerae into more than 200 serogroups, of which only two, O1 and O139, possess the potential to cause epidemic or pandemic cholera. The O1 serogroup is further divided into two biotypes, classical and El Tor, which evolved from independent lineages (20, 22), and they display genotypic and phenotypic differences.V. cholerae O1 is distinguished by two of its major virulence factors, cholera toxin (CT) and the toxin-coregulated pilus (TCP). The cholera toxin is encoded by ctxA and ctxB, which are found on the CTX prophage (49), and is responsible for the manifestation of diarrheal disease with severe water and electrolyte loss. The TCP, encoded by the tcp operon in the Vibrio pathogenicity island (VPI), is required for V.

Allele Variants of Enterotoxigenic Escherichia coli Heat-Labile Toxin Are Globally Transmitted and Associated with Colonization Factors

“…We were initially interested in determining whether LT evolves into morevirulent variants over time and whether we could follow this in a set of strains isolated over 3 decades. The recent discovery of several natural variants in the LT sequence among ETEC strains isolated in Brazil (15), as well as the emergence of novel variants of the related cholera toxin (27), prompted us to evaluate the diversity of LT (LT-I) in human ETEC strains on a global scale. We identified novel LT variants and could show that both known and novel LT variants are associated with specific ETEC CF profiles, but no association with time was found.…”

Phenotypic Analysis Reveals that the 2010 Haiti Cholera Epidemic Is Linked to a Hypervirulent Strain

“…Clinical reports have revealed that the AET strains induce a more dehydrating diarrhea (22), and isolation of AET strains has been specifically associated with patients with severe diarrheal symptoms (17). Human studies have revealed a proinflammatory phase of cholera disease (23), a deviation from the traditional definition of ET cholera as "noninflammatory," although no comparative studies have been conducted.…”

“…The tcpA gene for the major colonization factor toxin-coregulated pilus (TCP) gained the polymorphism N89S, although the functional consequence of this change is unknown (7,17). The rtxA gene for the multifunctional, autoprocessing repeats-in-toxin (MARTX) toxin acquired a stop codon (rtxA°) and the toxin became nonfunctional (18).…”

Molecular Characterization of High-Level-Cholera-Toxin-Producing El Tor Variant Vibrio cholerae Strains in the Zanzibar Archipelago of Tanzania

“…The amount of CT produced was measured as described previously (32,33) during the growth of the representative strains from Zanzibar in AKI medium and compared with prototype El Tor and classical strains. It was found that all the El Tor variant strains from Zanzibar produced significantly larger amounts of CT in vitro than most strains of prototype El Tor (P Ͻ 0.001, MannWhitney U test) (Fig.…”