In this study, we demonstrated that analyzed strains of Vibrio mimicus and Vibrio cholerae could be separated in two groups by using multilocus enzyme electrophoresis (MEE) data from 14 loci. We also showed that the combination of four enzymatic loci enables us to differentiate these two species. Our results showed that the ribosomal intergenic spacer regions PCR-mediated identification system failed, in some cases, to differentiate between V. mimicus and V. cholerae. On the other hand, MEE proved to be a powerful molecular tool for the discrimination of these two species even when atypical strains were analyzed.Vibrio mimicus is a species closely related to Vibrio cholerae. Phenotypically, most of the features of this organism are identical or similar to those found in V. cholerae, and sucrose fermentation is the main trait differentiating them biochemically (10, 11). They share somatic antigens and virulence-related genes and are associated with sporadic and epidemic cholera diarrhea (4, 10, 15). Both species are natural inhabitants of aquatic environments, such as seawater, freshwater, and brackish water. They may constitute the microbiota of zooplankton, crustaceans, and filter-feeding molluscs but are recognized mainly as human pathogens (6, 9).In 1991 a large cholera outbreak started in Latin America, and the etiological agent was V. cholerae O1 biotype El Tor. Interestingly, during this epidemic, cases of severe diarrhea associated with the presence of V. mimicus were reported in Costa Rica (5). At the same time, in French Guyana and in the northern part of the Brazilian Amazon region, a sucrose-negative variant of V. cholerae was identified in most of the cholera cases reported (8). Therefore, the emergence of V. mimicus as a pathogen and its coexistence with non-sucrose-fermenting V. cholerae isolates highlight the necessity for precise discrimination between these two species.After the characterization of V. mimicus as a new pathogenic species, only a few attempts to identify it on a molecular basis have been reported. One of these previous studies applied multilocus enzyme electrophoresis (MEE) to characterize V. cholerae strains, and the results suggested the possibility of using this approach to differentiate V. cholerae from V. mimicus (13). Chun et al. (7) recently developed a PCR-mediated identification system based on the analysis of nucleotide sequences of 16S-23S ribosomal intergenic spacer regions (ISR) that would be useful in distinguishing between these two species. However, it is important to observe that in both studies only a limited number of V. mimicus strains were considered, since V. cholerae was the main interest.Reported here are the results of an analysis by MEE of V. mimicus isolates from distinct sources and geographic regions. Using these data, we determined the genetic variation within this species and the relationship between V. mimicus and V. cholerae. In addition, we evaluated the efficiency of the ISR-PCR approach in the separation of these two closely related species.

Zymovar analysis of 260 strains of Vibrio cholerae plus 3 reference strains of V. mimicus, using 13 structural loci, led to the grouping of strains in 73 zymovars (strain or group of strains sharing the same alleles). Effective separation of strains, distinction of V. cholerae strains from closely related V. mimicus and the detection of 2 vibrio strains, including one with two O1 serovars, in supposedly pure collection cultures, illustrate the potential of zymovar analysis in the identification of V. cholerae isolates. Two El Tor strains from USA, one CT+ and the other CT-, shared the same zymovar 71, while 127 typical El Tor strains belonged to zymovar 14.

Thirty-two strains of Bacillus spp. were examined in a multilocus enzyme study by agarose gel electrophoresis. The organisms were Bacillus thuringiensis (21 strains, B. cereus (8), including two of var. mycoides, and B. megaterium (3). Strains having similar enzyme variants were grouped into zymovars. A total of 10 of 11 enzyme loci studied were polymorphic and 27 zymovars were distinguished among the 32 strains. The results were subjected to numerical analysis, phenetic affinities and genetic distances between the strains were calculated. The numerical analysis was unable to differentiate between B. thuringiensis and B. cereus. Our results indicated that based on this multilocus enzyme study these zymovars should be considered as belonging to the same species. A mycoides variant of B. cereus was the most distinctive strain studied and clearly belonged to a separate species, B. mycoides. The technique also allowed for identification of contamination and mislabelling of strains.

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Zymovar analysis was used to study 50 strains of Vibrio cholerae O1 and 40 strains of V. cholerae non-O1 isolated in Australia. The strains were assigned to 42 zymovars; the O1 strains to 9 types and the non-O1 strains to 33 types, with no overlapping between serovars. All the human O1 isolates, regardless of their ability to produce cholera toxin (CT), and all the CT-producing O1 environmental isolates, were type Z14. The remaining O1 strains and the non-O1 strains belonged to a variety of zymovars, and more than one zymovar was present in some rivers.