locus was amplified by using polymerase chain reaction (PCR) with primers (Table 1) selected from a conserved region of the locus, as determined by aligning sequences from GenBank. Our primers selectively amplified the orig- inal O139 rstR gene found in all isolates and not the addi- tional one found in some recently inserted CTX elements (19). The presence of amplified products was confirmed on agarose gels. Purification of the products was performed by using Millipore filters. The purified PCR products were sequenced in both directions by using the same primers used for amplification and Big Dye cycle sequencing kit (ABI) in accordance with manufacturer’s instructions. The fluorescently labeled products were separated and detected by using either an ABI 377 or 3700 Automatic Sequencer (ABI). The trace files were read by using Phred (20,21) and Phrap (22). Low-quality sequence at the ends was trimmed, and the contigs from each individual isolate were aligned by using Clustal X (23). Variable nucleotides were identi- fied manually. Isolates with identical alleles were identified from a distance matrix obtained from PAUP (24). The alle- les have been assigned GenBank accession numbers AY297845 to AY297921. The expected number of alleles that were a result of point mutations was calculated. All point mutations were assumed to occur independently; thus, the expected num- ber of alleles with >2 nucleotides (nt) can be calculated, and the excess number of observed alleles was attributed to conspecific LGT of homologous genes. If one assumes that p is the probability of seeing a single mutation in an allele, the chance of seeing two mutations on the same allele is p2; the probability of seeing three or more muta- tions is p3. Probability can be calculated from the data by dividing the number of alleles with a single nucleotide dif- ference, 34, by 785, the number of alleles in which observ- ing a point mutation is possible (the 6-bp deletion; the recombinant gmd, recA alleles; and all duplicate novel alleles were excluded). Thus, p equals 0.043, p2 equals 0.0018, and p3 equals 8 x 10-5. When these probabilities are multiplied by the total number of alleles, 785, the expect- ed number of alleles containing two independent point mutations is 1.45, and the expected number containing three or more is 0.06 (Figure 1). Table 1. Primers used for multilocus sequence typing Locus Primer 1 dnaE CGRATMACCGCTTTCGCCG lap GAAGAGGTCGGTTTGCGAGG rstR CGTGTTAGAGCACAC gmd CCTTATGCKGTGGCRAA recA GAAACCATTTCGACCGGTTC pgm CCKTCSCAYAACCCGCC gyrB GAAGGBGGTATTCAAGC cat ATGGCTTATGAATCGATGGG chi CAYGAYCCRTGGGCWGC RESEARCH Figure 1. Bar graph of the number of novel alleles (y-axis) with a specific number of nucleotide differences from the ancestral allele. Two alleles with 24-bp and 113-bp differences are excluded from the graph. Results Each of the loci examined had a variable number of observed alleles: 9 for dnaE, 20 for lap, 11 for rstR, 11 for gmd, 2 for recA, 8 for pgm, 4 for gyrB, 7 for cat, and 5 for chi. The most variable, lap with 20 alleles, was expected because it is a highly variable locus when analyzed with multilocus enzyme electrophoresis (25). The most com- mon allele was present in 91% of isolates (n=87) for dnaE, 77% (n=86) for lap, 79% (n=90) for rstR, 82% (n=87) for gmd, 99% (n=96) for recA, 90% (n=94) for pgm, 97% (n=92) for gyrB, 93% (n=88) for cat, and 94% (n=89) for chi. Thus, the pattern for each locus consists of a common or ancestral allele and a series of rare alleles, as expected for the expansion of a clone. Of the 64 less frequent alleles, some result from LGT and others from mutation. The three alleles with the largest changes are unlikely to be due to point mutations. First, a gmd allele that differed by 113 of the 360 bp sequenced, when compared with sequences in GenBank using BLAST (available from: URL: www.ncbi.nlm.nih.gov/BLAST/) showed greater similarity to gmd from E. coli (AF061251) than gmd from V. cholerae, consistent with LGT of a homologous gene into the V. cholerae genome. Second, an alternative recA allele that differs by 24 nt is likely to be the result of LGT of a homologous gene. Although sub- stantial, the number of nucleotide differences is not large enough for the allele to be clustered with sequences from Primer 2 GAKATGTGTGAGCTGTTTGC GTTTGAATGGTGAGCGGTTTGCT GAGTGAATCGTCGTG CTWGGATCACCTAACA CCGTTATAGCTGTACCAAGCGCCC TCRACRAACCATTTGAADCC GAGTCACCCTCCACWATGTA TCCCATTGCCATGCACC ACRTCTTCAATCTTGTC Emerging Infectious Diseases • Vol. 9, No. 7, July 2003 811