RESEARCH Molecular Epidemiology of O139 Vibrio cholerae: Mutation, Lateral Gene Transfer, and Founder Flush Pallavi Garg,*† Antonia Aydanian,‡ David Smith,‡ J. Glenn Morris, Jr.,‡ G. Balakrish Nair,*† and O. Colin Stine‡ Vibrio cholerae in O-group 139 was first isolated in 1992 and by 1993 had been found throughout the Indian subcontinent. This epidemic expansion probably resulted from a single source after a lateral gene transfer (LGT) event that changed the serotype of an epidemic V. choler- ae O1 El Tor strain to O139. However, some studies found substantial genetic diversity, perhaps caused by multiple origins. To further explore the relatedness of O139 strains, we analyzed nine sequenced loci from 96 isolates from patients at the Infectious Diseases Hospital, Calcutta, from 1992 to 2000. We found 64 novel alleles distributed among 51 sequence types. LGT events produced three times the number of nucleotide changes compared to mutation. In contrast to the traditional concept of epidemic spread of a homogeneous clone, the establishment of variant alleles generated by LGT during the rapid expansion of a clonal bacterial population may be a paradigm in infections and epidemics. Aand within a year had spread across the Indian sub- n epidemic of cholera began in Madras, India, in 1992 continent, with cases numbering in the millions (1,2). Vibrio cholerae isolates from this epidemic had a previous- ly unidentified serotype, subsequently designated as O139 Bengal (1,2). This new serotype appears to have resulted when a lateral gene transfer (LGT) event occurred that replaced the 22 kb of the wbf region (encoding the O1 anti- gen) of a seventh pandemic V. cholerae O1 El Tor strain with a 37-kb region encoding the O139 polysaccharide (3–5). The epidemic spread rapidly through all age groups, as persons with previous exposure to V. cholerae O1 were not immune to O139 infection. Since 1992, O139 strains have established endemicity in this geographic region and account for a variable percentage of cholera cases every year (6). Genetic variation observed in O139 isolates has been attributed to many causes. Variation in restriction fragment *National Institute of Cholera and Enteric Diseases, Calcutta, India; †International Centre for Diarrheal Diseases Research, Dacca, Bangladesh; and ‡University of Maryland School of Medicine, Baltimore, Maryland, USA length polymorphism (RFLP) analysis of rDNA genes (7) and in recA sequence (8) has been interpreted as evidence for multiple origins. Genetic variability in RFLP of the CTX element (6) has been attributed to phage-mediated recombination. Variation in antimicrobial susceptibility (9) has been attributed to plasmid exchange in response to selective pressure from drug use. The variation in pulsed- field gel electrophoresis (PFGE) analysis of genomic restriction fragments (6,10) has been attributed to point mutations. Multilocus sequence typing (MLST), which has been used in the evaluation of a number of other bacterial species (11–14), provides an alternative method for meas- uring genetic relatedness and has provided data for identi- fying both point mutations and LGT events (14). MLST has improved discriminatory power over PFGE in some cases, e.g., Enterococcus (15) and Salmonella (16); how- ever, in the case of Escherichia coli O157, it does not because of an absence of sequence variation in the clonal- ly derived isolates (17). A small MLST study of O139 iso- lates of V. cholerae did not identify any LGT events (18). To understand the evolutionary dynamics of V. choler- ae O139, we sequenced segments from nine loci, including seven that may be classified as traditional housekeeping genes, one that carries the genes for cholera toxin, and another that is next to the insertion sequence within the O139 wbf region (3–5). Thus, the last two loci might be expected to show LGT, because they are associated with known mobile elements, but the other seven loci would not be expected to show LGT. However, we found putative LGT alleles at all nine loci in the 96 clonally related O139 isolates. Materials and Methods We evaluated nine loci—dnaE, lap, recA, pgm, gyrB, cat, chi, rstR, and gmd—from 96 V. cholerae O139 isolat- ed from patients seen at the Infectious Diseases Hospital, Calcutta, from 1992 to 2000 (see Appendix, online only). DNA was prepared from overnight cultures by using PrepMan Ultra (Applied Biosystems Inc., Foster City, CA) at the University of Maryland School of Medicine. Each 810 Emerging Infectious Diseases • Vol. 9, No. 7, July 2003