vibrio cholerae is a gram negative bacterium which lives freely in aquatic environment and causes cholera . cholera is endemic in many parts of the world , especially the countries which lack proper sanitation managements .   in malaysia , cholera outbreaks due to the v. cholerae o1 serotype which occurs periodically .   the ratio of distribution of v. cholerae o139 to o1 serogroups isolated from seafood from 1998 to 1999 was 14  :  1 . non - o1/non - o139 v. cholerae is also frequently isolated from seafood and water sources but has not been implicated in any major outbreaks [ 3 , 4 ] . although non - o1/non - o139 v. cholerae is not associated with any major outbreak , it has been reported to be responsible for sporadic cases of diarrhea [ 57 ] . however , the mechanism of colonization for the non - o1/non - o139 strains remains relatively unknown as they do not harbor ctxa and tcpa . olivier et al .   had reported that accessory toxins such as hemolysin and multifunctional autoprocessing rtx toxin in el tor v. cholerae are involved in prolonged colonization without cholera toxin ( ct ) or toxin - coregulated pili ( tcp ) . as these accessory virulence genes are commonly found in all serogroups of v. cholerae , it is of interest to investigate the involvement of these accessory virulence genes for prolonged colonization in other serogroups of v. cholerae . molecular subtyping of pathogen is important for tracing a new or previously found virulent or multidrug - resistant clone . genomic variation and epidemiological study for different serogroups of v. cholerae have been carried out using many dna - fingerprinting tools . pfge is the most common subtyping tool to define strains from outbreaks and from sporadic cases of cholera as it has the highest discriminatory ability . however , pfge is time consuming and requires strict adherence to standardize protocols for interlaboratory comparison . multiple - locus variable - number of tandem repeat analysis ( mlva ) , a method based on the tandem repeats in multiple loci , was developed and has been popularly adopted for differentiation of bacterial pathogens since 2006 . recently , olsen et al .   evaluated the usefulness of mlva on v. cholerae isolated worldwide based on polymorphism in 6 vntr loci and demonstrated a high level of polymorphism among the strains tested . the objective of the study was to develop a novel mlva assay for genotypic study and to determine the presence of virulence - associated genes among malaysian v. cholerae strains of different serogroups . isolation of v. cholerae strains was carried out from june 2008 to april 2009 . the identification and confirmation of the cultures 43 v. cholerae strains were isolated from human stools ( n = 15 , from various public hospitals in kuala lumpur , labuan , kota kinabalu , kota bharu , tumpat , and alor setar ) , sea water ( n = 13 , from different locations in klang , banting , morib , and bachok ) , household wastewater   ( n = 6 , from different locations in petaling jaya , klang , banting , serdang , and kajang ) , algae ( n = 2 , from costal water in klang and morib ) , and various raw seafood ( n = 7 ) sold in wet markets in petaling jaya and used in this study . crude dna for each strain was prepared by suspending a loopful of bacterial colonies in 50  l of deionized water and boiled at 99c for 5 minutes . after a quick centrifugation , 5  l of the supernatant ( ~50  ng of dna template ) was used for pcr .   a search for potential vntr candidates in the genome of v. cholerae o1 biotype el tor , n16961 , primers were designed for each vntr based on a ~500  bp sequence covering the flanking and tandem repeats region retrieved from the database . the specificity of the primers and vntr polymorphisms were tested with in silico pcr ( http://insilico.ehu.es/ ) and monoplex pcr using dna templates from 5 vibrio strains of different serogroups . primers labeled with fluorescent dyes in the 5-end for the vntrs were synthesized , that is , vctr1-fam , vctr2-vic , vctr3-pet , vctr4-ned , vctr5-fam , vctr6-vic , and vctr7-ned . two multiplex pcrs were performed : vctr1vctr4 ( multiplex 1 ) and vctr5vctr7 ( multiplex 2 ) . each multiplex pcr mixture   in a total volume of 25  l contained 1x pcr buffer , 2  mm  mgcl2 , 160  m dntps , 0.4  m of each primer , 2  l of taq dna polymerase ( promega , usa ) , and 50  ng of dna template . the multiplex pcr was run at   95c for 5 minutes ( 1 cycle ) , 95c for 30  s , 58c for 30  s , 72c for 50  s ( 30 cyclers ) , and 72c for 7 minutes ( 1 cycle ) . the pcr product was then diluted in 1  :  100 ratio with ddh2o . one microlitre of the dilution was mixed with 10  l hidi formamide and 0.2  l genescan 500 liz size standard ( applied biosystem ) . the mixture was boiled at 95c for 5 minutes and analyzed with abi prism 3130xl genetic analyzer ( applied biosystem ) . the amplicons were identified based on the peak height and colour while the size was determined with the genescan size standard using genemapper software ( applied biosystem ) . the peak tables obtained from gene mapper were imported into bionumerics 6.0 ( applied maths , belgium ) . a minimum spanning tree ( mst ) was also constructed using the categorical coefficient and ward algorithm . the discrimination power of each vntr was calculated individually and combined using simpson 's index of diversity . briefly , equal volumes of standardized cell suspension ( od610 = 0.8 ) of 1% ( w / v ) seakem gold agarose ( cambrex bio science rockland , inc , usa ) were mixed to form plugs . the plugs were lysed in cell lysis buffer ( 50  mm tris ; 50  mm edta [ ph 8.0 ] , 1% sarcosyl , and 1  mg / ml proteinase k ) and incubated at 54c for 3 hours . the plugs were then washed thoroughly with sterile deionised water ( twice ) and te buffer ( 6 times ) . a slice of dna plug was digested overnight with 10  u restriction enzyme not i ( 5-gcggccgc-3 ) ( promega , madison , wis . , the digested chromosomal dna was subjected to pfge on a 1.0 % ( w / v ) agarose gel ( sigma type 1 , st . louis , mo ) with the chef driii ( bio - rad , hercules , ca ) using the following conditions : 200  v for 13 hours with pulse times of 210  s and 6 hours with pulse time 2025  s. gel image was captured by using gel doc xr after staining with ethidium bromide ( 0.5  g / ml ) . pfge banding patterns were analyzed with bionumerics 6.0 ( applied maths , kortrijk , belgium ) . the quantitative differences among the banding patterns were defined by the dice coefficient ; f. cluster analysis was carried out based on the unweighted pair group method with arithmetic averages ( upgma ) using the position tolerance of 0.15 . detections of ctxa , zot , rtxa , rstr , toxr , tcpa , tcpi , and hlya genes were carried out via pcr using published primers [ 6 , 1517 ] . the pcr amplifications were carried out in a master cycler ( eppendorf , usa ) . the pcr mixture in a total volume of 25  l contained 50  ng of dna template , 1x pcr buffer , 2  mm mgcl2 , 200  m of each dntp , 0.3  m of each primer , and 2.5  u of taq dna polymerase ( promega , usa ) . the cycling conditions were set at 94c for 3 minutes ( 1 cycle ) , 94c for 30  s , 60c for 30  s , 72c for 1 minute ( 30 cycles ) , and 72c for 10 minutes ( 1 cycle ) . the products were then analyzed on 1.5% ( w / v ) agarose gel and run at 90  v for 1 hour . gel images were captured and analyzed using gel doc xr ( bio rad , usa ) . representative amplicons of each gene were purified and sent to a commercial facility for sequencing to confirm the identity of amplicons . seven vntr regions were chosen , and primers were designed based on the sequences retrieved from genbank . initially , the primers specificity was determined individually with monoplex pcr using 2 o1 , 1 o139 , and 2 non - o1/non - o139 strains . all 7 vntr regions showed polymorphisms for those tested strains . by using capillary electrophoresis , overall , the size distributions of the 43 v. cholerae strains for each vntr were 212230  bp ( vctr1 ) , 216252  bp ( vctr2 ) , 218276  bp ( vctr3 ) , 278300  bp ( vctr4 ) , 228251  bp ( vctr5 ) , 211269  bp ( vctr6 ) , and 328369  bp ( vctr7 ) . thirty - eight mlva profiles ( f = 0.63 ) were obtained based on the character types ( allelic profiles ) ( figure 3 ) . the 43 strains were grouped into 3 major clusters in the mst ( figure 1 ) . the first cluster ( i ) comprised of 14 o1 strains ( 8 clinical , 6 water ) ( 90.6% similarity ) .   in this cluster , 5 of the o1 strains shared the same profile ( mlva2 : 09 - 08 - 09 - 12 - 05 - 02 - 23 ) . these strains ( vc4 , vc52 , vc63 , vc65 , and vc66 ) were isolated during a cholera outbreak in kota bahru , west malaysia ( n = 2 ) and labuan , east malaysia ( n = 3 ) in 2008 . one environmental o1 strain , vc70 , was highly similar ( 95 % similarity ) with the o1 outbreak strains . cluster ii consisted of 9 o1 strains , 1 non - o1/non - o139 strain , and 1 o139 strain . in this cluster , 4 strains ( vc21 , vc37 , vc38 , and vc40 ) isolated in 2004 from human stools were highly similar with only 1 - 2 alleles difference . vc40 ( o1 clinical ) has 2 and 3 alleles difference from vc11 ( environmental o1 ) and vc4370 ( environmental o139 ) , respectively . the vcpsw ( environmental non - o1/non - o139 ) also has 2 alleles difference from vc11 ( environmental o1 ) . cluster iii comprised of non - o1/non - o139 strains with 83.3% similarity or 15 alleles difference . simpson 's index of diversity for each individual vctr ranged from 0.59 to 0.92 ( table 1 ) while the index for the combined vctr loci was 0.99 . pfge of not i - digested chromosomal dna yielded 1522 fragments ranging from 40 to 400  kbp ( figure 2 ) . thirty - five pulsotypes ( f = 0.711.0 ) were observed . at a similarity of 80% cluster i comprised of all o1 strains , 1 o139 strain , and 7 non - o1/non - o139 strains . clusters ii and iii comprised of 3 non - o1/non - o139 strains while clusters iv and v were each composed of 2 non - o1/non - o139 strains each . based on the different combination of virulence genes analysed , 5 virulotypes ( v1v5 ) were observed for the 43 v. cholerae strains ( figure 3 ) . virulotype 1   was the most common profile   ( v1 : ctxa , zot , rtxa , rstr , toxr , tcpa , tcpi , and hlya ) and was exhibited by   o139 and 22 o1 strains . virulotype 2 ( v2 : rtxa , rstr , toxr , and hlya ) comprised of 16 non - o1/non - o139 and 1 v. cholerae o1 strain . among the non - o1/non - o139 serogroups , vc310 ( v3 : rtxa , rstr , toxr , tcpi , and hlya ) and vc3477 ( v4 : rtxa , rstr , toxr , tcpi , and hlya ) were negative for tcpa . besides vc3477 , classical hlya was also present in vc77 ( v5 : rtxa , rstr , toxr , and hlya ) . analysis of the dna sequences of the amplicons confirmed their identities as there was a 90100% homology with the targeted genes sequence in the database ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . when the strains were grouped according to serogroups and sources ( clinical o1 , environmental o1 , environmental o139 , and environmental non - o1/non - o139 ) , significant difference in the aggregation was observed ( one way - manova f , wilkes '  = 0.0874 , f6 , 76 = 30.1700 , and p <  .0001 ) . further post hoc tukey hsd test identified that there were significant differences between non - o1/non - o139 environmental vibrio cholerae and the other 3 groups ( p  <  .01 ) . this was pertinent to the fact that all non - o1/non-139 strains lacked 4 virulent genes ( ctxa , zot , tcpa , and tcpi ) . there was no significant difference in the virulotypes among the clinical o1 , environmental o1 , and environmental o139 . the mlva assay was developed based on the 7 loci in chromosomes i and ii of v. cholerae . among these loci , vctr4 demonstrated the highest discriminatory ability ( d = 0.92 ) , followed by vctr7 ( d = 0.89 ) . both loci are situated in the small chromosome of v. cholerae genome ( chromosome ii ) and suggest that there is a higher genetic variation in chromosome ii of v. cholerae genome compared with chromosome i. danin - poleg et al .   and olsen et al . reported that the tandem repeat locus that showed the highest discriminating ability is physically located at 187 kb in chromosome ii ( such as vctr7 in this study ) . however in our study , vctr4 which is located at 303  kb in chromosome ii showed even higher genetic variation ( 17 alleles , d = 0.92 ) among the 43 strains tested . both the pfge and mlva data showed that the v. cholerae o139 strain ( vc4370 ) was closely related with an o1 strain with similarity of more than 90% . strains vcgb and vcsw , isolated from river water and algae , respectively , were indistinguishable by both pfge and mlva . both strains harbored the same virulence genes ( rtxa , rstr , toxr , and hlya ) although they were isolated from different locations . vcgb was isolated from river water in banting , selangor ( august 2008 ) while vcsw was isolated from algae collected at pantai morib , selangor in october 2008 . the result suggests that both river water and algae were probably contaminated with the same clone of non - o1/non - o139 v. cholerae .   among the environmental v. cholerae o1 strains isolated in 2008 , 6 had close allelic similarity to the clinical o1 strains isolated in year 2008 ( cluster i ) while another 2 were indistinguishable to clinical strains isolated in year 2004 ( cluster ii ) . this phenomenon showed that microevolution might have taken place , resulting in new variance of strains since 2004 . the v. cholerae o139 strain was also found to be more clonally related to o1 strains isolated in 2004   than to the o1 strains which were isolated in 2008 . this suggests that the o139 strains might have derived from the 2004 o1 strains due to horizontal gene transfer . with the exception of one strain ( vc35 ) , most of the 2004 clinical v. cholerae o1 strains were very similar with only 1 - 2 alleles difference and were different from the 2008 strains .   from the analysis of mst , those strains which belonged to the same pfge profile ( n11 , n13 , n14 , and n16 ) were distinguishable by mlva mutations at the restriction sites may result in variation in pfge profiles while gene mutations may affect the number and frequency of tandem repeats . hence , a combination of pfge and mlva analysis may yield more information about the clonality of bacterial pathogens . for example , 3 environmental strains ( vc87 , vc88 , and vc90 ) and 1 clinical strain ( vc66 ) had identical pulsotype but were distinguishable by mlva . five strains ( vc4 , vc52 , vc63 , vc65 , and vc66 ) were indistinguishable by mlva as they shared identical allelic profile . however , pfge was able to separate 2 of kota bahru outbreak strains ( vc4 and vc65 ) ( west malaysia ) from the labuan outbreak strains in east malaysia . pfge is the most commonly used subtyping method to determine the epidemiological relatedness of the strains . in this study , the 23 o1 strains were subtyped into 18 pulsotypes . based on visual comparison of the published pulsotypes , our pulsotypes n22 and n11 appeared to be similar to those pulsotypes of hong kong strains isolated during an outbreak in 2001 . pulsotype n13 which comprised of 3 environmental strains ( vc87 , vc88 , and vc90 ) and 1 clinical strain ( vc66 ) was similar to the pulsotype of a philippine strain obtained during an outbreak in 2001 . the malaysian strains isolated from sea water had similar pulsotype with those indonesian outbreak strains in 1995 . overall , comparison of the pulsotype distributions among v. cholerae in different neighboring countries would provide important information and further facilitate the epidemiological study of cholera in southeast asia . however , as different pfge conditions were used by different researchers in the region , direct comparison was difficult . adoption of a standardized pfge protocol such as the pulsenet pfge protocol proposed by cdc pulsenet , usa would greatly enhance interlaboratory comparison and improve tracking of v. cholerae strains among the endemic countries in the region .   in this study , 8 environmental strains harbored all virulence - associated genes , indicating the toxigenic potential and risk of infection from environmental sources . the presence of accessory toxigenic genes in non - o1/non - o139 strains indicates the relative importance of these strains as they have the potential to cause infections .   two non - o1/non - o139 strains , vc3477 and vc310 isolated from seawater and seafood , respectively , harbored tcpi but not tcpa . tcpi gene is the first gene in tcp cluster and is associated with the synthesis of tcpa . it may function as a regulator to determine the virulence of vpi [ 6 , 21 ] . therefore , the presence of this gene in the strains which do not harbor tcpa is not surprising . in addition , vc3477 harbored the classical hlya gene while vc310 harbored the el tor hlya gene . however , these 2 strains could not be distinguished from other non - o1/non - o139 strains using mlva , but pfge distinguished vc3477 from other strains . vc 35 was isolated from a patient during a cholera outbreak in kota kinabalu , sabah . this strain did not harbor the major virulence genes ( ctxa , zot , tcpa , and tcpi ) which are essential for colonization . hence , the finding was in agreement with the finding of olivier et al .   that secreted accessory toxins such as hemolysin and rtx toxin in el tor v. cholerae would modify the host environment and prolong colonization without cholera toxin ( ct ) or toxin - coregulated pili ( tcp ) . overall , the mlva assay developed in this study was a promising method to study the relatedness of malaysian v. cholera , and the discriminatory ability was comparable with pfge . in addition , further combination of mlva and pfge analysis may provide more information of the relatedness of strains and distinguish strains from different sources and geographical regions .
molecular analysis of malaysian vibrio cholerae was carried out using a multiple - locus variable - number tandem repeat analysis ( mlva ) assay based on 7 loci of v. cholerae . 
 the discriminatory ability of the assay was compared with pulsed - field gel electrophoresis ( pfge ) using 43 malaysian v. cholerae isolated from various sources . 
 in addition , the virulotypes of the strains were determined . 
 based on mlva , 38 allelic profiles were obtained ( f = 0.63 ) while pfge generated 35 pulsotypes ( f = 0.71 ) . 
 simpson 's index of diversity for different vntr loci ranged from 0.59 to 0.92 . 
 the combined loci increased the discriminatory index to 0.99 which was comparable with pfge ( d = 0.99 ) . 
 most of the environmental non - o1/non - o139 strains harbored rtxa , rstr , toxr , and hlya only , and the virulotype of this serogroup was significantly different ( p < .01 ) from clinical / environmental o1 and environmental o139 strains . in conclusion , 
 the mlva assay developed in this study was a useful genotyping tool with comparable discriminatory power with pfge . 
 in addition , the combination of the two approaches can further distinguish the strains from different sources and geographical regions of isolation .