Vibrio cholerae strain typing and phylogeny study based on simple sequence repeats

Abstract

Vibrio cholerae is the etiological agent of cholera. Its natural reservoir is the aquatic environment. To date, practical typing of V. cholerae is mainly serological and requires about 200 antisera. Simple sequence repeats (SSR), also termed VNTR (for variable number of tandem repeats), provide a source of high genomic polymorphism used in bacterial typing. Here we describe an SSR-based typing method that combines the variation in highly mutable SSR loci, with that of shorter, relatively more stable mononucleotide repeat (MNR) loci, for accurate and rapid typing of V. cholerae. In silico screening of the V. cholerae genome revealed thousands of perfect SSR tracts with an average frequency of one SSR every 152 bp. A panel of 32 V. cholerae strains, representing both clinical and environmental isolates, was tested for polymorphism in SSR loci. Two strategies were applied to identify SSR variation: polymorphism of SSR tracts longer than 12 bp (L-SSR) assessed by capillary fragment-size analysis and MNR polymorphism assessed by sequencing. The nine L-SSR loci tested were all polymorphic, displaying 2 to 13 alleles per locus. Sequence analysis of eight MNR-containing loci (MNR-multilocus sequence typing [MLST]) provided information on both variations in the MNR tract itself, and single nucleotide polymorphism (SNP) in their flanking sequences. Phylogenetic analysis of the combined SSR data showed a clear discrimination between the clinical strains belonging to O1 and O139 serogroups, and the environmental isolates. Furthermore, discrimination between 27 strains of the 32 strains was achieved. SSR-based typing methods combining L-SSR and MNR-MLST were found to be efficient for V. cholerae typing.

FIG. 1.

Sequence alignment of representatives isolates of V. cholerae at the VC0929-(G)₈ MNR locus presenting the 10 ST. No product was amplified in O140-20, O140-21, and O49-128 strains. MNR alleles are labeled with different underlines. SNPs at the flanking sequences of the MNR are represented in boldface. -, missing nucleotide; *, nonconserved basepair.

FIG. 2.

Phylogenetic relationships and variation among V. cholerae isolates constructed by UPGMA cluster analysis based on three methods. (a and b) Sequence data of 29 isolates at eight MNR-MLST loci (a) and combined data of eight L-SSR loci and eight MNR-MLST loci (analyzed as sequence types) for 32 isolates (b). The combined analysis was based on 106 polymorphic points (16 loci times the number of alleles in each locus). (c) Dendrogram based on PFGE patterns of 29 isolates digested with NotI.

FIG. 2.

Phylogenetic relationships and variation among V. cholerae isolates constructed by UPGMA cluster analysis based on three methods. (a and b) Sequence data of 29 isolates at eight MNR-MLST loci (a) and combined data of eight L-SSR loci and eight MNR-MLST loci (analyzed as sequence types) for 32 isolates (b). The combined analysis was based on 106 polymorphic points (16 loci times the number of alleles in each locus). (c) Dendrogram based on PFGE patterns of 29 isolates digested with NotI.