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. Vibrio cholerae, a gram-negative bacterium, is the causal agent of the severe diarrheal disease cholera. Its natural reservoir is the aquatic environment (49, 84). Cholera pandemics are caused by specific serogroups of V. cholerae that are pathogenic only to humans (35,84). Since 1817, V. cholerae has caused a number of pandemics. The first seven were presumed to be caused by O1 serogroup of V. cholerae (68). In October 1992, a new serogroup, defined O139, caused a severe outbreak of cholera in southeast India. Within 10 months, the O139 serogroup was disseminated all over the Indian subcontinent and soon thereafter spread to 11 neighboring countries, temporarily displacing the O1 serogroups (64). Since then both serogroups coexist and are responsible for large outbreaks. Genomic studies indicated that O139 epidemic strain arose by horizontal acquisition of unique DNA (15, 47).Traditionally, V. cholerae classification is serological and requires about 200 antisera based on the somatic O antigen (72). Isolates of V. cholerae are divided into three major subgroups: O1, O139, and non-O1/non-O139, of which only the O1 and O139 serogroups are associated with cholera pandemics and epidemics. Non-O1, non-O139 serogroups are recognized as causative agents of sporadic and localized outbreaks (73). Pathogenic V. cholerae isolates carry virulence genes, such as the toxins genes ctxAB (25,65,68). The environmental V. cholerae strains from non-O1, non-O139 serogroups are a possible natural reservoir of potentially new emerging epidemic strains (67,73). This assumption is supported by the finding that some of these environmental strains harbor virulence genes (23) and thus are likely to evolve into novel pathogenic strains by horizontal gene transfer (24,25). The emergence of new pathogenic V. cholerae strains requires not only an efficient, rapid, and accurate identification tool but also a means for determining genetic relationships among environmental and clinical isolates.Genome-based bacterial identification and typing is essential for several disciplines, including taxonomy, epidemiology, determining phylogenetic relationships, and the study of evolutionary mechanisms. It allows distinguishing among strains within a species to monitor epidemics and routes of contamination. Recent advances in biotechnology have resulted in the development of numerous methods for microorganism typing that differ in their sensitivity, rapidity, complexity, discrimin...