Indian Journal of Marine Sciences Vol. 36(1), March 2007, pp. 43-50

Molecular typing of bacteria Vibrio harveyi and V. alginolyticus from shrimp farming systems

Satendra Kumar, M. Rosalind George*, K. Riji John & M. J. Prince Jeyaseelan

Department of Aquaculture, Fisheries College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Thoothukudi - 628 008, Tamil Nadu, India

*[Email: rosalindmg@yahoo.com]

Received 27 July 2005, revised 16 May 2006

Vibriosis is a commonly encountered disease in cultured shrimps. Vibrio spp. are the natural inhabitants of marine and brackish water ecosystems, which act as opportunistic pathogens in shrimp farms and shrimp hatcheries. Since the pathogenicity of Vibrio harveyi and V. alginolyticus is highly variable, a study was conducted to collect isolates of these two species from shrimp farming systems to analyse their strain variability. The above two bacteria were isolated from shrimp-farm water, sediment, shrimp larvae and hatchery water samples and subjected to polymerase chain reaction (PCR) fingerprinting using insertion sequence (IS) targeted primers. The IS primers were able to differentiate the intraspecies variability existing in both V. alginolyticus and V. harveyi strains efficiently. Twenty strains of V. alginolyticus tested differentiated into nine fingerprint patterns and 14 strains of V. harveyi also differentiated into 8 fingerprint groups. Dendrogram analysis of the two species tested was not able to associate the virulence factors studied, viz. production of amylase, gelatinase and lipase genetically. However, it has been found that all the amylase lacking V. alginolyticus strains except one belonged to a single genogroup. Dendrogram analysis confirmed the source independent genetic variability present within the same species of Vibrio tested.

[Key words: Bacteria, Vibrio harveyi, Vibrio alginolyticus, virulence, molecular typing, insertion-sequence, dendrogram analysis, shrimp]

Introduction Microbial diseases in shrimp farming system have caused serious financial losses to the shrimp farming communities globally. Incidence of diseases by infectious agents is one of the major limiting factors for the development of shrimp culture in India. Apart from the specific viral diseases, shrimp farming also experience the onset of bacterial infections by many opportunistic pathogens at various stages of farming including the hatchery phase. Commonly reported shrimp pathogenic bacteria are members of the genus Vibrio Pacini (family Vibrionaceae) and two taxa V. alginolyticus Sakazaki and V. harveyi (Johnson and Shunk) are potentially serious species causing diseases in the farm and hatchery respectively1,2. Variations in the pathogenicity of these species have been widely reported3 and characterisation of these strains differing in their virulence and other phenotypic characteristics was seldom accomplished

by biochemical tests4. Emergence of strain O139 of V. cholerae as an epidemic strain5 highlighted the limited potential of phenotypic and serotypic characterisation of non-O1 V. cholerae in identifying the pathogenic strains while bringing forth the relevance of genotypic approaches.

Several of the genomic approaches that are currently being used for identification and typing of Vibrio viz., DNA – DNA hybridization, randomly amplified polymorphic DNA polymerase chain reaction (RAPD PCR), amplified fragment length polymorphism (AFLP), ribotyping and arbitrarily primed PCR (AP PCR) fingerprinting have high discriminating power in recognising the strain variation existing among the Vibrio spp.6-10. Among various tests used for strain typing in vibrios, PCR fingerprinting using insertion sequence (IS) based targets are very efficient in differentiating genotypes compared to other methods11. The IS fingerprinting technique produces consistent multiple bands when a single set of primers are used, it is suggested to be a reliable technique for finding out the strain variability.

_________ *Corresponding author Phone : 094422 11792 Fax : 0461 2340574

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The present study was therefore undertaken to find out the extent of the molecular variability existing among different isolates of V. alginolyticus and V. harveyi isolated from shrimp farming systems. Materials and Methods Pond sediment, water and larvae samples were collected from shrimp farms and hatcheries located in coastal areas of Tamilnadu, Andhra Pradesh and Kerala. Samples were collected from different locations in sterile containers and brought to laboratory within 6-24 h in ice. Viable postlarvae were transported from the hatchery in oxygen-filled polythene bags and brought to the laboratory within 24 h. The samples were inoculated into thiosulphate citrate bile salt sucrose (TCBS) agar (Hi media, Mumbai) and incubated at room temperature for 24 h. Colonies were selected at random and identified using standard biochemical tests according to the scheme of Alsina & Blanch12,13. Twenty strains of V. alginolyticus and 14 strains of V. harveyi were used for molecular typing. The selected strains were grown in Luria – Bertani (LB) broth (1.0% tryptone, 0.5% yeast, 1.0% NaCl, pH 7.5) and incubated at 32°C for 16-20 h. The fully-grown cultures were harvested at 48 h by centrifuging at 6000 rpm for 10 minutes in sterile polypropylene tubes and washed twice in phosphate buffered saline. The cell pellet was then mixed well with 100 µl of SDS (0.025%) and 100 µl of NaOH (0.05 N) by aspirating through a micropipette tip and immersed in boiling water bath for 4 minutes followed by cooling immediately on ice. The suspension was centrifuged at 10000 rpm for 10 minutes and the supernatant was used for PCR or stored at –20°C until taken up for analysis. IS-PCR (Insertion sequence targeted polymerase chain reaction) was conducted with forward and reverse primers at 75 picomoles each in 50 µl reaction mix for 35 cycles with initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C for 45 sec, 52°C for 45 sec, and 72°C for 1 min. with a final extension at 72°C for 20 min. in a Master cycler (Eppendorf, Hamburg, Germany). For PCR, the reaction mix contained 5 µl of 10× buffer comprising 160 mM (NH4)2SO4, 670 mM Tris-HCl, 25 mM MgCl2 and 0.1% Tween-20, 2.0 µl of 20 mM of deoxy nucleic acid triphosphates (dNTPs) (Medox Biotech India Pvt. Ltd., Chennai), 33.5 µl of deionised water (Millipore, Cedex, France) and 3 µl of diluted bacterial DNA sample having 20 ng/µl concentration

as template along with 2.5 units of Taq polymerase. The PCR products were resolved by 1.2% agarose gel electrophoresis at 5 Vcm-1 and scanned using a gel documentation system (UVI Tec Ltd., UK). The fingerprint patterns obtained were analysed by unweighted pair group method of mathematic averages (UPGMA) by Dice Coefficient cluster analysis using UVI Bandmap software in the gel documentation system (UVI Tec, UK).

Results Details of the source of isolation of the 20 isolates of V. alginolyticus and 14 isolates of V. harveyi are given in Table 1. All the isolates were gram negative, motile, oxidase positive, oxidative and fermentative, produced ornithine and lysine decarboxylase enzymes while unable to produce arginine dihydrolase. All the V. alginolyticus isolates exhibited swarming on solid media and were able to produce acetoin from glucose. All but one isolate (SK025) of V. harveyi were luminescent and all the isolates were unable to produce acetoin from glucose and swarming on solid media. Select virulence characters viz. production of gelatinase, amylase and lipase were also examined (Table 1). While all the isolates of V. alginolyticus and V. harveyi isolates were gelatinase positive, six isolates of V. alginolyticus and seven isolates of V. harveyi were amylase negative. Lipase negative isolates were, however, comparatively less with five of V. alginolyticus and none of V. harveyi. Only one isolate of V. alginolyticus (SK166) was negative to both amylase and lipase production. Both V. alginolyticus (Fig. 1) and V. harveyi (Fig. 2) isolates produced characteristic fingerprint patterns against the IS primers used for amplification. The banding pattern observed indicated the presence of variation existing among the different strains of both the species tested. A common band of about 0.55 kbp size was present in 18 of the 20 V. alginolyticus strains examined indicating certain degree of similarity of the insertion sequence associated genetic material of V. alginolyticus strains. Fingerprints of V. alginolyticus had as high as 11 bands in some strains (SK245) and as low as 3 bands in certain strains (SK061 and SK155). All but 4 strains had a very thick band at 0.14 kbp. DNA fingerprints generated by IS primers for V. harveyi strains (Fig. 2) were equally characteristic as in the case of V. alginolyticus. However, unlike in V. alginolyticus, there was no amplicon common to all the 14 V. harveyi strains when these IS primers

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Table 1⎯Virulence characteristics of the V. alginolyticus and V. harveyi isolates obtained

Isolate No. Source Virulence characteristics Farm Sample Gelatinase Amylase Lipase

A) V. alginolyticus

SK 011 Farm 1, TN Sediment + - + SK 061 Hatchery, TN Water + + - SK 087 Farm, AP Water + + + SK 155 Farm 2, TN Pond 4 Water + + + SK 156 Farm 2, TN Pond 4 Shrimp gut + + + SK 157 Farm 2, TN Pond 4 Shrimp gut + + + SK 160 Farm 2, TN Pond 1 Water + + + SK 161 Farm 2, TN Pond 1 Water + + + SK 162 Farm 2, TN Pond 1 Water + + + SK 164 Farm 2, TN Pond 1 Water + + + SK 165 Farm 2, TN Pond 1 Water + + + SK 166 Farm 2, TN Pond 1 Water + - - SK 182 Farm 2, TN Pond 1 Sediment + - + SK 184 Farm 2, TN Pond 1 Sediment + - + SK 187 Farm 2, TN Pond 2 Sediment + - + SK 192 Farm 2, TN Pond 2 Sediment + - + SK 201 Farm 2, TN Pond 4 Sediment + + - SK 202 Farm 2, TN Pond 4 Sediment + + +

SK 209 Y Farm 2, TN Pond 4 Sediment + + - SK 245* + + -

B) V. harveyi

SK 025 Farm, Cochin Sediment + + + SK 094 Farm 2, TN Pond 1 Water + + + SK 096 Farm 2, TN Pond 1 Water + + + SK 101 Farm 2, TN Pond 1 Water + - + SK 102 Farm 2, TN Pond 2 Water + - + SK 104 Farm 2, TN Pond 2 Water + + + SK 105 Farm 2, TN Pond 2 Water + - + SK 106 Farm 2, TN Pond 2 Water + + + SK 107 Farm 2, TN Pond 2 Water + - + SK 108 Farm 2, TN Pond 2 Water + - + SK 111 Farm 2, TN Pond 3 Water + + + SK 112 Farm 2, TN Pond 3 Water + + + SK 119 Farm 2, TN Pond 2 Water + - + SK 120 Farm 2, TN Pond 7 Water + - +

*from the farm sample isolates of bacteriology laboratory, Department of Aquaculture

were used. Unlike V. alginolyticus, V. harveyi had seemingly less binding sites for the IS primers as the highest number of bands noticed in any strain was only five, that too only in three of the 14 strains (SK094, SK101, SK096) examined. However, the discriminating potential of the IS primers was sufficiently high for V. harveyi as the fingerprints were unambiguously different for 64% of the strains (8 out of 14) examined. UPGMA cluster analysis at 80% homogeneity showed the presence of three major groups in V. alginolyticus comprising of nine strains (45%) in

Group 1 (SK160, SK187, SK161, SK166, SK182Y, SK162, SK184, SK192 and SK165), three strains (15%) in Group 2 (SK156, SK164 and SK202) and two strains (10%) in Group 3 (SK155 and SK61) at the threshold of 80% (Fig. 3). Remaining six strains (SK11, SK209Y, SK245, SK157, SK201 and SK87) had unique fingerprinting pattern forming six different genotypes. While the Group 1 cluster had seven strains (SK160, SK161, SK162, SK165, SK166, SK182Y, and SK 184) coming from the same pond water and sediment, one of the strains (SK164) from the same sample was in a different cluster (Group 2).

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Fig. 1⎯DNA fingerprint patterns generated by PCR amplification of Vibrio alginolyticus genomic DNA using primers targeted at insertion sequences in 1.2% agarose gel. a) Lane 1-SK162, lane 2-SK165, lane 3-SK192, lane 4-SK201, lane 5-100 bp ladder, lane 6-SK11, lane 7-SK209, lane 8-SK245, lane 9-SK160, lane 10-SK161, lane 11-SK182, lane 12-SK166, lane 13-molecular weight marker (λ DNA Eco RI/Hind III digest). b) Lane 1-SK245, lane 2-SK187, lane 3-SK165, lane 4-SK184, lane 5-SK156, lane 6-SK164, lane 7-SK202, lane 8-100 bp ladder and λ DNA Eco RI/Hind III digest, lane 9-SK157, lane 10-SK87, lane 11-SK61, lane 12-SK155, lane 13-SK201, lane 14-SK182, lane 15-molecular weight marker (λ DNA Eco RI/Hind III digest)

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Fig. 2⎯DNA fingerprint patterns generated by PCR amplification of Vibrio harveyi genomic DNA using primers targeted at insertion sequences in 1.2% agarose gel. Lane 1-SK94, lane 2-SK101, lane 3-SK96, lane 4-SK25, lane 5-SK107, lane 6-SK112, lane 7-100 bp ladder & λ DNA Eco RI/Hind III digest molecular marker, lane 8-SK106, lane 9-SK104, lane 10-SK105, lane 11-SK111, lane 12-SK102, lane 13-SK108, lane 14-SK119 lane 15-SK120

Fig. 3⎯A) Dendrogram derived by UPGMA cluster analysis using UVI bandmap software for the PCR fingerprint profiles of 20 strains of V. alginolyticus. Percentage homogeneity scale is indicated. Strain numbers are given following the gel numbers. B) Clustering of 3 are groups identified above 80% threshold limit from fig. 3a. Group 1-SK160, SK187, SK161, SK166, SK182Y, SK162, SK184, SK192, SK165; Group 2-SK156, SK164, SK202; Group 3-SK155, SK61.

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Comparative analysis of the fingerprints of V. alginolyticus strains showed that no specific correlation could be associated with the generated fingerprint pattern and their virulence characteristics. However, it has been found that all amylase lacking V. alginolyticus strains (SK166, SK182, SK184, SK187 and SK192) except one (SK11) belonged to a single genogroup (Group 1). But no such similarity was observable in case of lipase lacking V. alginolyticus strains (SK201, SK209Y, SK245, SK61 and SK166), which were spread among different genogroups. Similar to the above, two strains (SK187 and SK245), which were unable to grow at 11%, had no specific fingerprint pattern. While one strain (SK187) had perfect homogeneity with another strain (SK160) growing at 11% salt, the second strain (SK245) differed from the other 19 V. alginolyticus strains analysed by a homology coefficient of 62%. Vibrio harveyi formed two main groups and six unique patterns in the dendrogram analysis forming eight different fingerprint clusters above 80%

homogeneity (Fig. 4). While one of the groups had four strains of 100% homogeneity, the other had four strains above 80% homogeneity. Of the fourteen strains tested, one (SK25) isolate came from a geographically different source and it formed an independent genogroup from the rest. However, other isolates originated from a single geographical source also had independent genogroups, some being 100% different from the rest (SK119). Among the virulence tests carried out for the 14 isolates analysed for fingerprinting, seven (SK101, SK102, SK105, SK107, SK108, SK119 and SK120) were not able to produce amylase. These seven isolates were differently distributed among all the fingerprint clusters without any specific correlation. Discussion Analysis of select characters in two species of vibrios revealed that there is a variation in the production of enzymes in certain isolates. Ramaiah et al.14 observed that hydrolytic or catalytic enzymes

Fig. 4⎯Dendrogram derived by UPGMA cluster analysis using UVI bandmap software for the PCR fingerprint profiles of 14 strains of V. harveyi. Percentage homogeneity scale is indicated.

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were important in microbes in anthropogenically related ecosystems which experience physiological stress affecting biodegradation and resulting in accumulation of materials in the marine environment. Vibrios generally show high gelatinase activity, moderate amylase activity and very low lipase activity15. A slightly different observation was noticed in the present study where 100% isolates showed gelatinase activity, 61.8% showed amylase activity while 85.3% showed lipase activity. Of the 14 V. harveyi strains selected in the present study, 13 were found to produce bioluminescence, which is one of the virulence characteristics of V. harveyi16. In the present study, molecular typing of twenty V. alginolyticus strains and fourteen V. harveyi strains were carried out. The fingerprints generated by IS primers when analysed by UPGMA analysis showed that the primers were capable of identifying the strain specific variability in both the species investigated with high discriminating potential. While each fingerprint pattern generated by IS PCR could be considered as a strain by itself11, a cut off threshold of 80% similarity has been used to discriminate the different strains in ERIC PCR17 and IS PCR18. In the present study, we have also considered a threshold cut off of 80% to differentiate between the strains. Existence of strain variability among V. alginolyticus isolated from different sources was reported by various workers10,11,18. George et al.18 however pointed out that the V. alginolyticus strains isolated from even a single source could have genomic variations that could be exhibited by IS based fingerprinting. Present study also supports the above finding that the strain variation in V. alginolyticus could be present even among the strains isolated from a single source (genogroups of SK157, Group3 which include SK155 and Group2 which include SK156). This probably could be an indicator of the nature of variability happening in the bacterial strains when present in the aquatic environment where the bacteria are subjected to various types of stressors and also due to the ability of the bacterial strains in mutation and recombination to promote genetic divergence for adaptation to stressors and functional repair system19. The above primers were also able to discriminate the V. harveyi strains into eight genogroups with two of the groups having four strains each and six unique fingerprint patterns. As noticed in V. alginolyticus, V. harveyi also showed source independent genetic variation among the different strains. The strain

SK119 was a unique genogroup with 0% homogeneity with the rest of the strains from the same source (SK102, SK104, SK105, SK106, SK107 and SK108). Existence of strain variation among V. harveyi isolates was observed earlier and FAFLP-PCR (Fluorescent amplified fragment length polymorphism - PCR) was able to identify the intraspecific strain variation among this species3,20. Investigations similar to the present study leading to the molecular profiling of Vibrio strains attains significance due to the less reliability associated with biochemical analysis especially in case of V. harveyi4,21. Although earlier reports have indicated the usefulness of IS based fingerprinting in V. alginolyticus, this is the first report of the effectiveness of this technique in V. harveyi. Molecular markers based on the phenotypic virulence characters have been reported for different Vibrio spp.22,23. In the present study, no conclusive evidence could be obtained to link the genetic fingerprint patterns and the tested biochemical characteristics of the strains. However, in V. alginolyticus, all amylase-lacking strains except one isolate clustered into a single genogroup. The lack of association of the genetic markers and the phenotypic characteristics noticed in the present study could be due to the less number of parameters studied and also probably due to the limited representation of the generated amplicons in the genetic expression of various phenotypic traits. Since the IS PCR could generate different fingerprint patterns for V. alginolyticus and V. harveyi, this technique could be used to classify the strains into different genogroups using UPGMA cluster analysis and identify strains with specific phenotypic characteristics including virulence factors. Further studies would, therefore, could generate specific molecular markers/ fingerprints associated with these two Vibrio species that could be used for identifying virulent strains from among the omnipotent Vibrio species of the marine environment. Acknowledgement The paper has formed part of the MFSc thesis of the first author submitted to the Tamil Nadu Veterinary and Animal Sciences University. The authors thank Dr. R. Santhanam, Dean, FCRI and Dr. G. Sanjeeviraj, Head, Department of Aquaculture for their keen interest and support in the study.

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References 1 Jiravanichpaisal P & Miyazaki T, Histopathology,

biochemistry and pathogenicity of Vibrio harveyi infecting black tiger shrimp Penaeus monodon, J Aquat Anim Health, 6 (1994) 27-35.

2 Karunasagar I, Pai R, Malathi G R & Karunasagar I, Mass mortality of Penaeus monodon larvae due to antibiotic–resistant Vibrio harveyi infection. Aquaculture, 128 (1994) 203-206.

3 Vandenberghe J, Verdonck L, Arozarena R R, Rivera G, Boll A, Balladares M, Gomez Gil B, Calderon J, Sorgeloos P & Swings J, Vibrios associated with Litopenaeus vannamei larvae, postlarvae, broodstock and hatchery probionts, Appl Environ Microbiol, 65 (1999) 2592-2597.

4 Vandenberghe J, Thompson F L, Gomez-Gil B & Swings J, Phenotypic diversity amongst Vibrio isolates from, marine aquaculture systems, Aquaculture, 219 (2003) 9-20.

5 Albert M J, Vibrio cholerae O139 Bengal, J Clin Microbiol, 32 (1994) 2345-2349.

6 Welsh J & McClelland M Fingerprinting genomes using PCR with arbitrary primers, Nucleic Acids Res, 18 (1990) 7213-7218

7 Arias C R, Verdonek. L, Swings J, Garay E & Aznar R, Intraspecific differentiation of Vibrio vulnificus biotypes by amplified fragment length polymorphism and ribotyping, Appl Environ Microbiol, 63 (1997) 2600-2606.

8 Arias C R, Pujalte M J, Garay E & Aznar R, Genetic relatedness among environmental, clinical and diseased Vibrio vulnificus isolates from different geographic regions by ribotyping and randomly amplified polymorphic DNA PCR, Appl Environ Microbiol, 64 (1998) 3403-3410.

9 Goarant C, Merien F, Berthe F, Mermoud I & Perolat P, Arbitrarily primed PCR to type Vibrio spp. pathogenic for shrimp, Appl Environ Microbiol, 65 (1999) 1145-1151.

10 Sudheesh P S & Xu H S, Pathogenicity of Vibrio parahaemolyticus in tiger prawn Penaeus monodon Fabricius: possible role of extracellular proteases, Aquaculture, 196 (2001) 37-46.

11 Zanetti S, Ilaria A D, Maurizio S, Giovanni F & Sechi L A, Differentiation of Vibrio alginolyticus strains isolated from Sardinian waters by ribotyping and a new rapid PCR fingerprinting method, Appl Environ Microbiol, 65 (1999) 1871-1875.

12 Alsina M & Blanch A R, A set of keys for the biochemical identification of environmental Vibrios, J Appl Bacteriol, 76 (1994) 79-85.

13 Alsina M & Blanch A R, Improvement and update of a set of keys for biochemical identification of environmental Vibrio species, J Appl Bacteriol, 77 (1994) 719-721.

14 Ramaiah N, Raghukumar C, Sheetu G & Chandramohan D, Bacterial abundance communities and heterotrophic activities in the coastal waters off Tamilnadu, Indian J Mar Sci, 25 (1996) 234-239.

15 Sudheesh P S, Jie K & Xu H S, Random amplified polymorphic DNA-PCR typing of Vibrio parahaemolyticus and V. alginolyticus isolated from cultured shrimps, Aquaculture, 207 (2002) 37-46.

16 Manefield M, Harris L, Rice S A, Denys R & Kielleberg S, Inhibition of luminescence and virulence in the black tiger prawn (P. monodon) pathogen Vibrio harveyi by intercellular signal antagonists, Appl Environ Microbiol, 66 (2000) 2079-2084.

17 Maluping R P, Ravelo C, Lavilla-Pitogo C R, Krovacek K & Romalde J L, Molecular typing of Vibrio parahaemolyticus strains isolated from the Philippines by PCR-based methods, J Appl Microbiol, 99 (2005) 383-391.

18 George M R, John K R, Iyappan T & Jeyaseelan M J P, Genetic heterogeneity among Vibrio alginolyticus isolated from shrimp farms by PCR fingerprinting, Letters Appl Microbiol, 40 (2005) 369-372.

19 Vulic M, Lenski R E & Radman M, Mutation, recombination and incipient speciation of bacteria in the laboratory, Proc Natl Acad Sci USA, 96(1999) 7348-7351.

20 Gomez-Gil B, Soto-Rodriguez A S, Garcia Gasca A, Roque A, Vazquez -Juarez F, Thompson F L & Swings J, Molecular identification of Vibrio harveyi related isolates associated with diseased aquatic organisms, Microbiology, 150 (2004) 1769-1777.

21 Gauger E J & Gomez Chiarri M, 16S ribosomal DNA sequencing confirms the synonymy of Vibrio harveyi and V. carchariae, Dis Aquat Org, 52 (2002) 39-46.

22 Aguirre-Guzmán G, Humberto Mejia R & Felipe A, A review of extracellular virulence product of Vibrio species important in diseases of cultivated shrimp, Aquacult Res, 15 (2004) 1395-1404.

23 O’Shea Y A, Reen F J, Quirke A M & Boyd E F, Evolutionary genetic analysis of the emergence of epidemic Vibrio cholerae isolates on the basis of comparative nucleotide sequence analysis and multilocus virulence gene profiles, J Clin Microbiol, 42 (2004) 4657-4671.