from february 2004 through april 2005 , we systematically collected clinical and environmental v. cholerae from bakerganj and mathbaria , 2 small communities 50 miles apart in the southern part of coastal bangladesh . clinical isolates were collected from 20% of all patients who had symptoms of cholera when seen at the local clinics . environmental isolates were cultured from water , sediment , and plankton samples taken at 6 sites ( ponds or river sites ) in each of the 2 communities . the same sites were used throughout the 15-month study , and the same method was applied at all sites and across all time points . isolation was performed by standard culture methods , and v. cholerae was identified by a combination of biochemical ( 5 ) , molecular , and serologic techniques ( 6 ) . all samples were collected according to protocols approved by institutional review boards at johns hopkins university , university of maryland , and the international centre for diarrheal disease research , bangladesh . for multilocus sequence typing ( mlst ) and variable number of tandem repeat ( vntr ) determinations , each locus was pcr amplified by using standard conditions and appropriate primers from the literature ( 7 ) ( technical appendix ) . the resulting fragments were sequenced by using big dye kit ( applied biosystems , foster city , ca , usa ) . trace files were generated by using an abi 3730xl automatic sequencer and read using either 1 ) the phred ( 8,9 ) , phrap ( www.washington.edu ) , or consed ( 10 ) package or 2 ) sequencher ( agct , gene codes corporation , ann arbor , mi , usa ) . a total of 391 environmental and clinical isolates of v. cholerae were collected and identified from february 2004 through april 2005 . of these , 267 environmental isolates were identified as belonging to non - o1 and non - o139 serogroups and did not carry the gene for cholera toxin ( ctx ) . analysis of these 267 by mlst ( using the 7 loci identified previously ) yielded a genetic background that was distinct from that of the clinical / epidemic strains . the other 68 ( 20% ) of 335 environmental v. cholerae isolates shared a genetic background identical or nearly identical to clinical / epidemic v. cholerae . these 68 and all 56 clinical isolates collected ( all of which were related by mlst ) were further analyzed by examining 5 vntr loci . the loci were identified by those genes in which they occur : vc0147vntr , vc04367vntr ( intergenic ) , vc1650vntr , vc0171vntr , and vca0283vntr . alleles were distinguished by the number of tandem repeats as determined by tandem repeat finder ( 11 ) ( technical appendix ) . sequences from 1 locus with identical numbers of repeats were assigned to the identical allele . the alleles at the 5 loci were ordered to generate a sequence type ( st ) , for example , 3,5,2,2,8 . each locus was polymorphic with 7 , 6 , 6 , 20 , and 16 alleles , respectively . the various sts were defined as related if they were identical at 4 of the 5 loci . when we defined a vntr genetic group as differing by a single locus variant from another member of the group , 3 large vntr genetic groups were identified and 5 vntr genetic groups composed of only 2 isolates and 7 unrelated strains . there was statistically significant agreement between serogroup and vntr genetic group . for v. cholerae o139 , thus , the isolates were considered to be related because x , x = 1,1 ; 2,1 ; or 2,8 , i.e. , a change in a single locus serially connected all isolates . summing the number of isolates of a sequence type there were 10 exceptions , i.e. , 3 non - o1 , non - o139 ctx isolates were in groups ; 3 ctx o139 , 2 ctx o1 inaba , 1 ctx , and 1 ctx o1 ogawa were not . we found that bakerganj and mathbaria yielded distinct v. cholerae populations ; only 2 ( st 3,5,2,2,7 and 1,1,3,9,8 ) of 36 sts identified were found at both locations ( table 1 ; technical appendix ) . there was substantial divergence in sts among strains isolated from patients , compared with strains from the environment in mathbaria ; only 1 ( st 3,5,2,2,7 ) of 16 sts were found in both patient and environmental isolates . similarly , in bakerganj , only 2 ( st 3,5,2,2,6 and 3,5,2,1,5 ) of 24 sts were found in both clinical and environmental isolates . clinical or environmental isolates from a given period were more likely to have a common st ( technical appendix ) . for example , at mathbaria , 49 of the 53 isolates with an st identical to that of another isolate were found in the same or neighboring month . similarly , at bakerganj , 33 of 36 isolates with identical sts were found in the same or neighboring month . variation in the vntr loci appeared to be greater among clinical isolates than among environmental isolates . a total of 29 sts occurred in clinical isolates , whereas only 12 occurred in environmental isolates ( table 1 ) . when we controlled for location and month of collection ( table 2 ) , the total number of sts among environmental isolates ( 7 st/35 isolates ) was less than that among clinical isolates ( 16 st/32 isolates ) (  = 4.4 , df 1 , p = 0.036 ) . common sts were found among environmental isolates , despite the isolates coming from samples from different ponds and distinct subsamples ( e.g. , water , phytoplankton , zooplankton ) . our data do not support the concept of seasonal cholera epidemics occurring by movement of a single clonal wave across the countryside . they are consistent , instead , with the natural occurrence of v. cholerae year - round in the aquatic environment of each site , with each site having its own , distinct grouping of strains ( 12,13 ) . the limited overlap between sts in environmental and clinical isolates is an enigma that remains to be resolved . however , the extensive variation in vntr sts in this short time frame and small geographic area suggests that vntr sts can be useful in assessing genetic relatedness of isolates during outbreaks / epidemics . the strong temporal clustering of the variation arising in the vntr sts of clinical isolates is consistent with the hypothesis that clinical cases reflect the occurrence of multiple small outbreaks . our data are drawn from rural bangladesh ; however , cholera is a global disease . its epidemiology may well differ in sub - saharan africa , the americas , or other parts of asia , or in the mega - cities that are increasingly the hallmark of the developing world .
clinical and environmental vibrio cholerae organisms collected from february 2004 through april 2005 were systematically isolated from 2 rural bangladeshi locales . 
 their genetic relatedness was evaluated at 5 loci that contained a variable number of tandem repeats ( vntr ) . 
 the observed minimal overlap in vntr patterns between the 2 communities was consistent with sequential , small outbreaks from local sources .