Evolutionary Genetic Analysis of the Emergence of Epidemic Vibrio cholerae Isolates on the Basis of Comparative Nucleotide Sequence Analysis and Multilocus Virulence Gene Profiles

Abstract

Vibrio cholerae, the causative agent of cholera, is a natural inhabitant of the aquatic ecosystem. We examined a unique collection of V. cholerae clinical and environmental isolates of widespread geographic distribution recovered over a 60-year period to determine their evolutionary genetic relationships based on analysis of two housekeeping genes, malate dehydrogenase (mdh) and a chaperonin (groEL). In addition, the phylogenetic distribution of 12 regions associated with virulence was determined. Comparative sequence analysis of mdh revealed that all V. cholerae O1 and O139 serogroup isolates belonged to the same clonal lineage. Single-strand conformational polymorphism (SSCP) analysis of these O1 and O139 strains at groEL confirmed the presence of an epidemic clonal complex. Of the 12 virulence regions examined, only three regions, Vibrio seventh pandemic island 1 (VSP-I), VSP-II, and RS1, were absent from all classical V. cholerae isolates. Most V. cholerae El Tor biotype and O139 serogroup isolates examined encoded all 12 virulence regions assayed. Outside of V. cholerae O1/O139 serogroup isolates, only one strain, VO7, contained VSP-I. Two V. cholerae El Tor isolates, GP155 and 2164-78, lacked both VSP-I and VSP-II, and one El Tor isolate, GP43, lacked VSP-II. Five non-O1/non-O139 serogroup isolates had an mdh sequence identical to that of the epidemic O1 and O139 strains. These isolates, similar to classical strains, lack both VSP-I and VSP-II. Four of the 12 virulence regions examined were found to be present in all isolates: hlyA, pilE, MSHA and RTX. Among non-O1/non-O139 isolates, however, the occurrence of the additional eight regions was considerably lower. The evolutionary relationships and multilocus virulence gene profiles of V. cholerae natural isolates indicate that consecutive pandemic strains arose from a common O1 serogroup progenitor through the successive acquisition of new virulence regions.

Genomic characterization of non-O1, non-O139 Vibrio cholerae reveals genes for a type III secretion system

“…Indeed, our sequence of the mdh gene of AM-19226 shows five informative polymorphisms (C325T, G606A, T669C, T723C, and C750T) relative to N16961 (data not shown). This pattern most closely matches that of other non-O1, non-O139 strains analyzed by O'Shea et al (52), such as the O141 strain V46. Interestingly, both AM-19226 and V46 carry the TTSS gene cluster described here (see below).…”

“…All four strains are also missing a portion of the nanH region (25), recently described as the VPI-2 pathogenicity island (43). Interestingly, this island is found more frequently in toxigenic O1 strains (43,52) and encodes neuraminidase, an enzyme that can generate additional GM1 receptors for cholera toxin. Each of the four strains appears to lack Ϸ6% (Ͼ250) of the genes that are present in the O1 seventh pandemic strain N16961 (Fig.…”

“…Our earlier microarray analysis found that seventh pandemic O1 El Tor and O139 strains, pre-seventh pandemic O1 El Tor strains, classical biotype strains, and a environmental Gulf Coast O1 strain (2740-80) have strikingly similar gene content (25). By using comparative nucleotide sequence analysis of the mdh gene, O'Shea et al (52) reached similar conclusions about O1 strains but also noted that most non-O1, non-O139 strains appear to have a different phylogenic origin than O1 strains (52). Indeed, our sequence of the mdh gene of AM-19226 shows five informative polymorphisms (C325T, G606A, T669C, T723C, and C750T) relative to N16961 (data not shown).…”

“…In contrast, environmental non-O1, non-O139 strains have typically been reported to be quite diverse. These conclusions were reached by using a variety of techniques, including ribotyping (22), amplified fragment length polymorphism (3), pulsed-field gel electrophoresis (3), and comparative nucleotide sequence analysis (6,52). These analytical methods are useful in assessing relatedness of strains but are limited in their ability to define gene content differences that might have biological and phylogenetic significance.…”

Vibrio cholerae Strain Typing and Phylogeny Study Based on Simple Sequence Repeats

“…By their basic nature, however, housekeeping genes diversify slowly and exhibit only limited sequence variation. Hence, it has been suggested to apply MLST analysis to additional highly polymorphic genomic regions, such as virulence genes, stress response genes, and intergenic regions (11,16,44,61). Recently, MLST of loci harboring MNR at noncoding regions were shown in E. coli to contain high sequence variation, including single-nucleotide polymorphisms (SNPs) in the flanking area of the MNR (20).…”