Multilocus Sequence Typing Has Better Discriminatory Ability for Typing Vibrio cholerae than Does Pulsed-Field Gel Electrophoresis and Provides a Measure of Phylogenetic Relatedness

Abstract

Twenty-two Vibrio cholerae isolates, including some from "epidemic" (O1 and O139) and "nonepidemic" serogroups, were characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) by using three housekeeping genes, gyrB, pgm, and recA; sequence data were also obtained for the virulence-associated genes tcpA, ctxA, and ctxB. Even with the small number of loci used, MLST had better discriminatory ability than did PFGE. On MLST analysis, there was clear clustering of epidemic serogroups; much greater diversity was seen among tcpA-and ctxAB-positive V. cholerae strains from other, nonepidemic serogroups, with a number of tcpA and ctxAB alleles identified.Vibrio cholerae is an environmental species that has been linked with seven pandemics of cholera since 1817 (22). Approximately 200 serogroups of V. cholerae have been identified to date, with classification based on epitopic variations in the heat-stable somatic O antigens of the strains (29). In the modern microbiology era, epidemic cholera has been associated with a limited number of closely related strains in O groups 1 and 139. All such epidemic strains carry genes for cholera toxin (encoded by the ctxAB genes) and the toxin-coregulated pilus (encoded by the tcpA gene) (10,20). Recent studies have indicated that the ctxAB and tcpA genes may also be present in "nonepidemic" (i.e., other than O1 and O139) V. cholerae serogroups (7,8,9,18,23,24,26). While the genetic relatedness of O1 and O139 isolates has been well documented (1, 2, 3, 12), we know less about the genetic relatedness and phylogeny of ctxAB-and tcpA-positive isolates in other serogroups, and such data are critical for understanding why and how pandemic-causing V. cholerae strains emerge.Several molecular typing approaches, including ribotyping (28), insertion sequence-based fingerprinting (3), amplified fragment length polymorphism (17), and pulsed-field gel electrophoresis (PFGE) (6), have been used to characterize the molecular epidemiology of V. cholerae, with PFGE reported (6, 8) to have the most discriminatory power among these methods. These techniques have less utility in defining underlying phylogenetic relationships (11). Multilocus enzyme electrophoresis (MLEE) is of value in this regard (12), although problems with band resolution and the fact that phenotypic expression of the enzyme under study can easily be altered in response to environmental conditions can adversely affect the reproducibility of MLEE results and complicate data analysis and interpretation.More recently, attention has turned to sequence-based approaches. Sequencing of a single gene (19, 31) is not optimal: because evolution occurs by a net-like process, gene trees based on a single gene may not permit accurate determination of the genetic relatedness among various isolates (15). Multilocus sequence typing (MLST), first described in 1998 (25), provides a balance between sequence-based resolution and informativeness and technical feasibility and has been used to characterize several patho...

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

“…We compared the variation found using MLST analyses of housekeeping or virulence genes to that of MNR loci. Both methods revealed the same number of alleles; however, the same degree of nucleotide variation was found on shorter segments (115 to 270 bp) of the MNR loci compared to a longer segment (560 to 1,100 bp) for the housekeeping, 16S-23S rRNA intergenic spacer regions or virulence genes (10,13,29,44). The original MLST method is based on sequence variation at housekeeping genes that are conserved and therefore usually present in all strains but that have limited variation (21,59,69).…”

“…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).…”

“…MLST utilizing differences mainly in housekeeping genes is a rising DNA sequence-based method for bacterial strain typing (14,44,51). DNA sequencing uncovers far more variation per locus than any other method currently used for bacterial strain typing, and it provides a uniform platform for strain comparison analyzed in different laboratories and for database storage.…”

“…Recent advances in biotechnology have resulted in the development of numerous methods for microorganism typing that differ in their sensitivity, rapidity, complexity, discriminatory power, reproducibility, labor-intensity, and cost (7,60,82,85). Several DNA-based methods were used for typing of V. cholerae, such as tests for virulence genes, ribotyping, pulsedfield gel electrophoresis (PFGE), amplified fragment length polymorphism, enterobacterial repetitive intergenic consensus sequence-PCR, random amplification of polymorphic DNA, and multilocus sequence typing (MLST) (2,17,34,44,46,67,73,87).…”

Use of chromosomal integron arrays as a phylogenetic typing system for Vibrio cholerae pandemic strains

“…A dendrogram based on the presence/absence of MGC size classes was able to resolve phylogenetic relationships between pandemic V. cholerae strains that previous singleor multi-locus phylogenetic studies have been unable to do. PFGE and amplified fragment length polymorphism (AFLP) have provided some differentiation of pandemic strains, although observed differences are not statistically supported and cannot be traced to a specific area of the genome (Arakawa et al, 2000;Kotetishvili et al, 2003;Lan & Reeves, 2002). All differences observed using the fingerprinting method are traceable to the integron array and potentially to specific MGCs.…”