RESEARCH V. mimicus, the closest sibling species to V. cholerae (8), a finding that suggests that recombination occurred within V. cholerae. Third, a lap allele had a 6-bp deletion and a sin- gle nucleotide difference that may be the result of a dou- ble-strand break repair. We calculated that at least 26 putative conspecific LGT events occurred in the 96 isolates studied. Figure 1 shows the number of nucleotide differences between each novel allele and the ancestral allele. If all point mutations are assumed to occur independently, the expected number of alleles with two or more variable nucleotides can be calcu- lated and the excess number of observed alleles attributed to conspecific LGT of homologous genes. The expected number of alleles containing two independent point muta- tions is 1.45, and the expected number containing three or more is 0.06. Since 11 alleles were observed with 2 nt dif- ferences, 9 more than expected, and 16 were observed with >3 differences, 16 more than expected, all of these alleles probably did not occur through mutation; more likely, these alleles are the result of LGT. Thus, we would esti- mate that 26 alleles (9 + 16 + recA allele above) are puta- tively due to conspecific LGT of homologous genes. The putative conspecific LGT alleles, although fewer in number (26 alleles) than the assumed number of mutation- derived alleles (34 alleles), provide most of the nucleotide differences between alleles. The 120 nt changes introduced by conspecific LGT events are approximately three times the 38 (34 single mutations + 4 2x2 double mutations) introduced by mutation. This calculation is conservative: The 26 conspecific LGT events may represent an underes- timate of the number because some of the alleles differing by <1 nt may have resulted from LGT. The analysis of all nine loci from each isolate was based on the sequence type (ST). Each isolate was defined by a 9-digit number composed of the assigned allele num- ber at each of the nine loci in the following order: dnaE, lap, rstR, gmd, recA, pgm, gyrB, cat, and chi. The most common allele was arbitrarily assigned as number 1. Thus, the ST of all the most common alleles is ST 1,1,1,1,1,1,1,1,1. Missing data were assigned the most common allele. This assumption is conservative, mini- mizes the observed amount of variation, and is consistent with the preponderance of common alleles found at each locus. Fifty-one unique STs were found in the 96 isolates test- ed, reflecting relatively extensive genetic diversity. The overall average of 0.53 unique STs per isolate examined is similar to that seen in every year including 1992 (Table 2). Six STs occur more than once. As expected, the ancestral ST:1,1,1,1,1,1,1,1,1, found in 40 isolates, occurred in all years. Among the others, ST:1,1,2,1,1,1,1,1,1 was found three times, once each in 1995, 1996, and 1997. ST:1,2,1,1,1,1,1,1,1 and ST:1,1,7,1,1,1,1,1,1 were found twice in 1992 and 1994, respectively. ST 1,1,1,6,1,1,1,1,1 was found once in 1998 and again in 1999. ST:1,1,1,1,1,4,1,1,1 was found in 1995 and 1998. Since the number of STs is large (51 types), and number of samples in a collection period is small (8–13 samples; Table 2), STs seen in multiple years must not only persist but also repre- sent a substantial portion of the epidemic O139 V. choler- ae population. Five of the novel STs are related to other novel STs by allelic change at another second or third locus. One sequence type evolved into three related types found in subsequent years (Figure 2a). The starred gmd allele is one related to the E. coli sequence, and its presence in two dis- tinct related STs in two different years demonstrates its establishment in the population. That the pattern seen in Figure 2b of ancestral alleles rstR 1 and chi 1 and two vari- ant alleles, rstR 7 and chi 5, was found in all combinations is indicative of an LGT event. Figure 2c-e shows three additional groups of related sequences. In Figure 2a, b, and d, the ST with the larger number of novel alleles occurred in later years. In contrast, in Figure 2c and e, the ST with the larger number of novel alleles occurred in the earlier years. The lack of an overall temporal relationship may result from the small sample size (8–13 isolates) in any year. One isolate, CRC5, is unusual because it has no sequenced alleles of the ancestral type. Nevertheless, the alleles from this isolate are closely related to those of the ancestral type. Each CRC5 allele differs from the ancestral allele by 7 nt for dnaE, 3 nt for lap, 4 nt for rstR, 24 nt for recA, 6 nt for pgm, 10 nt for gyrB, and 4nt for chi. A com- prehensive survey of the genetic distances for these loci could determine the average distance between alleles for each of these loci. The data would provide insight into whether this isolate represents a second derivation of the O139 clinical type from an environmental strain or if it is a genetic outlier within the clonally related, but diversified, O139 epidemic type. Discussion The emergence and pandemic spread of V. cholerae O139 Bengal represented a chance to examine evolution of a bacterial strain in the midst of a clonal expansion. Our results are consistent with clonal expansion and subse- quent divergence as described by Spratt and Maiden (26). Putative recombinant alleles were found at all nine loci among the 96 clonally related O139 isolates. One gmd allele from V. cholerae was most similar to a gmd allele from E. coli. The number of base-pair differences among other alleles was higher than expected on the basis of a simple computation for the accumulation of independent mutations. This finding suggests that many of these events were due to LGT. When we applied our criteria to the 812 Emerging Infectious Diseases • Vol. 9, No. 7, July 2003