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we evaluated nine loci  dnae , lap , reca , pgm , gyrb , cat , chi , rstr , and gmd  from 96 v. cholerae o139 isolated from patients seen at the infectious diseases hospital , calcutta , from 1992 to 2000 ( see appendix , online only ) .
each 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 original o139 rstr gene found in all isolates and not the additional one found in some recently inserted ctx elements ( 19 ) .
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 identified manually . isolates with identical alleles were identified from a distance matrix obtained from paup ( 24 ) .
all point mutations were assumed to occur independently ; thus , the expected number 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 p ; the probability of seeing three or more mutations is p. probability can be calculated from the data by dividing the number of alleles with a single nucleotide difference , 34 , by 785 , the number of alleles in which observing a point mutation is possible ( the 6-bp deletion ; the recombinant gmd , reca alleles ; and all duplicate novel alleles were excluded ) .
when these probabilities are multiplied by the total number of alleles , 785 , the expected number of alleles containing two independent point mutations is 1.45 , and the expected number containing three or more is 0.06 ( figure 1 ) .
bar graph of the number of novel alleles ( y - axis ) with a specific number of nucleotide differences from the ancestral allele .
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 common 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 substantial , the number of nucleotide differences is not large enough for the allele to be clustered with sequences from 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 single nucleotide difference that may be the result of a double - 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 calculated and the excess number of observed alleles attributed to conspecific lgt of homologous genes .
if all point mutations are assumed to occur independently , the expected number of alleles with two or more variable nucleotides can be calculated and the excess number of observed alleles attributed to conspecific recombination of homologous genes .
the expected number of alleles containing two independent point mutations is 1.45 , and the expected number containing three or more is 0.06 . since
11 alleles were observed with 2 nt differences , 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 estimate that 26 alleles ( 9 + 16 + reca allele above ) are putatively 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 underestimate 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 number at each of the nine loci in the following order : dnae , lap , rstr , gmd , reca , pgm , gyrb , cat , and chi .
this assumption is conservative , minimizes 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 tested , 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 ) .
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 .
since the number of sts is large ( 51 types ) , and number of samples in a collection period is small ( 813 samples ; table 2 ) , sts seen in multiple years must not only persist but also represent a substantial portion of the epidemic o139 v. cholerae 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 distinct 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 variant 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 ( 813 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 comprehensive survey of the genetic distances for
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 .
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 subsequent 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 .
when we applied our criteria to the novel alleles identified in a previous study ( 18 ) , 11 of the 13 would be considered to have resulted from lgt , since the number of nucleotide differences to the ancestral allele varied from 4 to 19 .
thus , for v. cholerae , like neisseria , streptococcus , and other bacterial species ( 1114 ) , conspecific recombination of homologous genes appears to be common and responsible for most of the alleles with multiple nucleotide differences and the majority of the nucleotide differences .
the genetic variability at the nine loci alters our understanding of evolution in bacteria , showing that recombination in v. cholerae occurs frequently and most nucleotide changes occur by means of a recombination that can alter any gene .
the proportion of recombinants from conspecific recombination , 3.5% ( 28/785 ) is greater than that from transgeneric recombination ( 0.01% from the acquisition of e. coli gmd by one isolate ) .
one potential implication of a greater rate of conspecific recombination may be that , over time , it will maintain the species identity of each individual bacterium , despite the constant bombardment of homologous genes from other genera .
although at first glance the frequency of the novel sequence types appears to conflict between our study and an earlier study ( 18 ) , the observations may be reconciled on the basis of both the observed frequencies and the timing of the observations . both studies identified a common ancestral allele in from 77% to 99% of isolates in our study and a series of rare alleles with 119 variant alleles for each locus .
these studies reported 10% novel sequence types in 29 isolates that were collected from  the first epidemic period ,  from 1992 to 1993 ( 18 ) .
these data are not statistically different ( chi - square test=2.4 , p=0.12 ) . however , the researchers estimate of frequency ( 18 ) is more likely to be correct because of the larger sample size .
the dates of collection may also be important because our collection of isolates from 1993 began in march , when the number of o139 cases at the infectious diseases hospital rose from < 10 to > 80 per month , corresponding to a rapid population expansion or flush .
the genetic diversity was greater in the v. cholerae o139 isolates than in other clinically associated clones . in v. parahaemolyticus o3:k6 , a pandemic strain ,
coli o157 , all 77 isolates were identical at seven loci in spite of variation between isolates on pfge ( 17 ) .
although v. parahaemolyticus and e. coli are widespread pathogens , they differ from v. cholerae o139 because their population size has expanded much more slowly . among o139 isolates ,
the substantial genetic diversity found in the first year of the epidemic may reflect a 
i.e. , a flush , any novel genotype with similar or even slightly deleterious fitness compared to the founder genotype will produce sufficient offspring to become established in the population ( 27 ) .
a founder flush appears to have occurred in the establishment of helicobacter pylori in a single person ( 28 ) .
although other previous descriptions of this phenomenon have been limited to insects , specifically butterflies ( 29 ) and drosophilids ( 30 ) , we believe that the founder flush phenomenon may become the paradigm for epidemic bacterial expansion in individual patients and populations .
this founder flush phenomenon , in turn , has implications for our interpretation of  clonality  among epidemic isolates and for our understanding of factors that contribute to the emergence of new pathogenic strains .
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. cholerae 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 .