Molecular epidemiology of Vibrio cholerae O139 in China: polymorphism of ribotypes and CTX elements

Abstract

Vibrio cholerae O139, the second etiological serogroup of cholera, triggered the first outbreak of O139 cholera in China in 1993. To analyze the clone polymorphism of O139 isolates in China, 117 strains of V. cholerae O139, isolated from different areas in China between 1993 and 1999, were selected to characterize the phylogenetic relationships by molecular techniques. Analysis of restriction fragment length polymorphism in the conserved 16S rRNA gene revealed seven different ribotypes within the 117 strains. Among these strains, there were eight that lacked the cholera toxin gene (ctxAB), zot, and the repetitive sequence (RS); these eight strains belonged to three individual ribotypes. Our results suggested that V. cholerae O139 strains in China had clone diversity in phylogeny. The results of our hybridization patterns for CTX genetic elements (ctxAB, zot, and RS) showed that CTXPhi genomes in most V. cholerae O139 strains had two or more copies and had extensive restriction patterns even for the strains which belong to the same ribotype. For 22 (20.1%) strains, the copies of ctxAB were different from those of zot, suggesting that a ctxAB-negative CTXPhi genome may exist in O139 strains. This ctxAB-negative CTXPhi genome may coexist with the intact CTXPhi genome in a strain. In addition, the dendrogram for I-CeuI-generated pulsed-field gel electrophoresis patterns showed that V. cholerae serogroup O139 has a closer relationship with one strain of serogroup O22 than with the strains of serogroup O1. The results of this study showed the clonal diversity and the distribution of O139 strains in China, suggesting multiple origins of the O139 cholera epidemic or sporadic events.

FIG. 1.

Southern hybridization analysis of rRNA genes in 117 V. cholerae O139 strains isolated from 1993 to 1999 in China. Genomic DNA was digested with BglI and hybridized with a digoxigenin-labeled 16S rRNA gene probe. The ribotyping patterns are rb1 to rb7. The numbers indicating the molecular sizes of the bands correspond to a λ DNA HindIII marker.

FIG. 2.

Southern hybridization analysis of the elements in the CTXΦ genome (ctxAB [top], zot [middle], and RS [bottom]) in V. cholerae O139 strains isolated from 1993 to 1999. Genomic DNA was digested with PstI and probed with digoxigenin-labeled ctxAB, zot, and RS, respectively. The numbers on the left indicate the molecular sizes of the bands and correspond to a λ DNA HindIII marker.

FIG. 3.

PFGE profiles (left) of the genomic DNAs of V. cholerae strains belonging to different serogroups and biotypes digested with I-CeuI and schematic diagram (right) of dendrogram analysis of the strains. Lane 1, 94001 (O139; cholera toxin negative; China); lane 2, 1837 (O139; ctxAB⁺; Bangladesh); lane 3, MO45 (O139; ctxAB⁺; India); lane 4, 22s (O22; cholera toxin negative; China); lane 5, 86015 (O1; El Tor biotype; cholera toxin negative; China); lane 6, Bin-43 (O1; El Tor biotype; ctxAB⁺; China); lane 7, Wujiang-2 (O1; El Tor biotype; ctxAB⁺; China); lane 8, 1119 (O1; classical biotype; ctxAB⁺; India); lane 9, O395 (O1; classical biotype; ctxAB⁺; Bangladesh); lane 10, 569B (O1; classical biotype; ctxAB⁺; Bangladesh); lane M, λ DNA ladder molecular size standard.