Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW

Abstract

The distribution of genes for an outer membrane protein (OmpW) and a regulatory protein (ToxR) in Vibrio cholerae and other organisms was studied using respective primers and probes. PCR amplification results showed that all (100%) of the 254 V. cholerae strains tested were positive for ompW and 229 ( approximately 98%) of 233 were positive for toxR. None of the 40 strains belonging to other Vibrio species produced amplicons with either ompW- or toxR-specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay. These studies were extended with representative number of strains using ompW- and toxR-specific probes in DNA dot blot assay. While the V. cholerae strains reacted with ompW probe, only one (V. mimicus) out of 60 other bacterial strains tested showed weak recognition. In contrast, several strains belonging to other Vibrio species (e.g., V. mimicus, V. splendidus, V. alginolyticus, V. fluvialis, V. proteolyticus, V. aestuarianus, V. salmonicida, V. furnissii, and V. parahaemolyticus) showed weak to strong reactivity to the toxR probe. Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that the ompW sequence is highly conserved among V. cholerae strains belonging to different biotypes and/or serogroups. All of these results suggest that the ompW gene can be targeted for the species-specific identification of V. cholerae strains. The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplification of ompW and ctxA genes which should be of considerable value in the screening of both toxigenic and nontoxigenic V. cholerae strains of clinical as well as environmental origin.

FIG. 1

PCR amplification results obtained with bacterial strains using ompW-specific primer pairs 1 and 2 (A), 1 and 4 (B), and 2 and 3 (C). The bacterial strains used were V. cholerae O1 classical (lane 2), O1 El Tor (lane 3), O139 (lane 4), rough (lane 5), and non-O1/non-O139 (lanes 6 and 7). Other bacteria used were V. parahaemolyticus (lane 8), V. mimicus (lane 9), V. anguillarum (lane 10), V. alginolyticus (lane 11), V. furnissii (lane 12), Aeromonas spp. (lane 13), and enteroaggregative E. coli (lane 14). Lane 1 represents marker DNA of known molecular weights. The amplicon sizes are indicated by arrows.

FIG. 2

DNA dot blot hybridization test carried out with V. cholerae strains using ompW (A) and toxR (B) gene probes. The test strains used were V. cholerae O1 classical (O395 [blot 1], 569B [blot 2], and ATCC 14035 [blot 3]), O1 El Tor (PG27 [blot 4] and ATCC 39315 [blot 5]), O139 (Arg3 [blot 6] and SG25 [blot 7]), rough ALO46 (blot 8), non-O1/non-O139 (ATCC 25872 [blot 9], ATCC 25874 [blot 10], V5 [blot 11], and S7 [blot 12]), V. mimicus ATCC 33653 (blot 13), V. tyrogens (blot 14), V. alginolyticus ATCC 17749 (blot 15), V. anguillarum ATCC 19264 (blot 16), V. furnissii ATCC 35016 (blot 17), V. proteolyticus ATCC 15338 (blot 18), V. mimicus (blot 19), V. vulnificus (ATCC 33816 [blot 20] and ATCC 27562 [blot 21]), V. salmonicida ATCC 43839 (blot 22), V. parahaemolyticus (121 [blot 23] and RIMD 2210001 [blot 24]), V. splendidus ATCC 33125 (blot 25), V. carchariae ATCC 35084 (blot 26), V. aestuarianus ATCC 35048 (blot 27), V. nereis ATCC 25917 (blot 28), V. natriegens ATCC 14048 (blot 29), V. tubiashii ATCC 19109 (blot 30), V. fluvialis ATCC 33809 (blot 31), Aeromonas sp. (blot 32), E. coli ATCC 25922 (blot 33), Shigella sp. (blot 34), Salmonella sp. (blot 35), and P. aeruginosa ATCC 27853 (blot 36).

FIG. 3

RFLP analysis of ompW amplicons of different V. cholerae strains using the restriction enzymes HindIII (A), NdeI (B), and HpaI (C). The bacterial strains used were V. cholerae O1 classical (lane 1), O1 El Tor (lane 2), O139 (lane 3), and non-O1/non-O139 (lanes 4 to 9). Lane 10 represents the uncut ompW amplicon shown for a comparison. The fragment sizes are indicated by arrows.

FIG. 4

Multiplex-PCR analysis of V. cholerae strains using primers 1 and 2 for ompW and primers 7 and 8 for ctxA. Bacterial strains used were V. cholerae O1 (lane 3), O139 (lane 4), non-O1/non-O139 (lane 5), a nontoxigenic non-O1/non-O139 (lane 6), and a V. mimicus strain (lane 7). The positions of the ompW and ctxA amplicons generated by the use of individual primer pairs are shown in lanes 1 and 2, respectively.