Incidence, virulence factors, and clonality among clinical strains of non-O1, non-O139 Vibrio cholerae isolates from hospitalized diarrheal patients in Kolkata, India

Abstract

The incidence of Vibrio cholerae non-O1, non-O139 strains from hospitalized patients with acute diarrhea constituted 27.4% (n = 54) of the total 197 V. cholerae strains isolated from patients in Kolkata, India, in 2003. Of 197 strains, 135 were identified as O1 serotype Ogawa and 2 were identified as O139. In the same time period, six O1 background rough strains that possessed all known virulence factors were identified. Serotype analysis of the non-O1, non-O139 strains placed 42 strains into 19 serogroups, while 12 remained O nontypeable (ONT); the existing serotyping scheme involved antisera to 206 serogroups. Detection of a good number of ONT strains suggested that additional serogroups have arisen that need to be added to the current serotyping scheme. The non-O1, non-O139 strains were nontoxigenic except for an O36 strain (SC124), which regulated expression of cholera toxin as O1 classical strains did. Additionally, strain SC124 carried alleles of tcpA and toxT that were different from those of the O1 counterpart, and these were also found in five clonally related strains belonging to different serogroups. Strains carrying tcpA exhibited higher colonization in an animal model compared to those lacking tcpA. PCR-based analyses revealed remarkable variations in the distribution of other virulence factors, including hlyA, rtxA, Vibrio seventh pandemic island I (VSP-I), VSP-II, and type III secretion system (TTSS). Most strains contained hlyA (87%) and rtxA (81.5%) and secreted cytotoxic factors when grown in vitro. Approximately one-third of the strains (31.5%) contained the TTSS gene cluster, and most of these strains were more motile and hemolytic against rabbit erythrocytes. Partial nucleotide sequence analysis of the TTSS-containing strains revealed silent nucleotide mutations within vcsN2 (type III secretion cytoplasmic ATPase), indicating functional conservation of the TTSS apparatus.

FIG. 1.

Monthly isolation profile of non-O1, non-O139 V. cholerae strains isolated from diarrheal patients admitted to Infectious Diseases and Beliaghata General Hospital and BC Roy Children Hospital in Kolkata, India, in 2003.

FIG. 2.

Lipopolysaccharide profiles of representative V. cholerae strains. The O1 background rough (A) and smooth non-O1, non-O139 (B) strains were isolated in 2003. (A) V. cholerae rough strains (lanes 3 to 8) included SC6, SC117, SC122, SC135, SC145, and SC156, respectively. (B) Smooth V. cholerae non-O1, non-O139 strains (lanes 3 to 8) included SC191 (O48), SC132 (O48), SC124 (O36), SC107a (O128), SC103a (O48), and SC72 (ONT), respectively (B). In panels A and B, lanes 1 and 2 show LPS profiles obtained with E. coli ATCC 25922 and V. cholerae O1 classical strain O395, respectively.

FIG. 3.

The BglI ribotype patterns of V. cholerae O1 background rough (A) and smooth non-O1, non-O139 (B) strains isolated in 2003. (A) V. cholerae rough strains (lanes 1 to 6) included SC6, SC117, SC122, SC135, SC145, and SC156, respectively. The smooth variant of O1 strains with RIII ribotype pattern (9) (lane 7, CO840) was included as reference in panel A. (B) Smooth V. cholerae non-O1, non-O139 strains (lanes 1 to 9) included SC17 (O37), SC110 (O34), SC182 (O15), SC72 (ONT), SC103a (O48), SC107a (O128), SC132 (O48), SC191 (O48), and SC124 (O36), respectively. The positions of the HindIII-digested λ molecular size markers (in kilobases) are indicated at the sides of the gels in the figure.

FIG. 4.

RFLP patterns of the CTX genetic elements of toxigenic non-O1, non-O139 V. cholerae strain SC124 (O36). Genomic DNA was digested with different restriction enzymes as indicated in the figure. DNA probes specific for ctxA (A) and cep (B) were used. The positions of the HindIII-digested λ molecular size markers (in kilobases) are indicated at the sides of the gels in the figure.

FIG. 5.

DNA fingerprinting patterns generated with IS1004 (A) and vcsN2 (B) probes against BglI-digested genomic DNA obtained from non-O1, non-O139 V. cholerae strains. V. cholerae strains include SC17 (O37), SC110 (O34), SC182 (O15), SC72 (ONT), SC103a (O48), SC107a (O128), SC132 (O48), SC191 (O48), and SC124 (O36) in lanes 1 to 9, respectively. The positions of the HindIII-digested λ molecular size markers (in kilobases) are indicated at the sides of the gels in the figure.