Abstract. – Vibrio parahaemolyticus is amarine bacterium which is also responsible foracute diarrhoeal illness in human beings. Eatingraw seafish or contaminated seafood is respon-sible for acute gastroenteritis. The aim of thisstudy was to investigate the isolation, identifica-tion and molecular characterization of Vibrioparahaemolyticus from the fish samples inKolkata, India.

Materials and Methods: In this study 90 fishsamples were collected from 8 different marketplaces in Kolkata, India. Fish samples collectedwere shrimp, prawn, bhetki, pamfret and hilsa.VP-toxR PCR was performed to confirm thepresence of species specific toxR. tdh and trhgenes PCR for detection of virulence geneswere performed separately. GS-PCR was per-formed in tdh, trh gene positive strains to deter-mine whether they belong to pandemic geno-type. Serotyping was also done on the tdh, trhpositive strains.

Results: Out of the 90 fish samples collectedfrom different local fish markets 60 were positivefor Vibrio parahaemolyticus. 21 (35%) out of 60Vibrio parahaemolyticus isolates from fish sam-ples harboured the tdh gene. 1 (1.7%) out of 60Vibrio parahaemolyticus isolates from fish sam-ples carried trh gene. Out of 22 isolates only 2were positive for GS-PCR. O10:KUT was theserovar maximum isolated.

Conclusion: Considerable percentage ofVibrio parahaemolyticus carrying the viru-lence genes and pandemic genotype amongfish in Kolkata indicates that there is potentialreservoir in Kolkata and consumption of seafish or contaminated fish might cause Vibrioparahaemolyticus mediated diarrhea in this re-gion.

Key Words:

Vibrio parahaemolyticus, Fish, Diarrhoea, Acutegastroenteritis.

European Review for Medical and Pharmacological Sciences

Isolation, identification and molecularcharacterization of Vibrio parahaemolyticusfrom fish samples in Kolkata

D. PAL, N. DAS

Department of Microbiology, All India Institute of Hygiene and Public Health, Kolkata (India)

Corresponding Author: Debaprasad Pal, MD; e-mail: debaprasadpal@hotmail.com 545

Introduction

Vibrio parahaemolyticus is a marine bacteriumwhich is also responsible for acute diarrhoeal ill-ness in human beings. Eating raw seafish or cont-aminated seafood is responsible for acute gas-troenteritis. Vibrio parahaemolyticus was firstimplicated in an outbreak of food poisoning inJapan in 19501. Etiological studies of acute diar-rhoeal illness in Kolkata have shown that gas-troenteritis caused by Vibrio parahaemolyticusranks next to Vibrio cholerae in terms of inci-dence2,3. About 10% of the cases of gastroenteri-tis in patients admitted to Infectious DiseasesHospital in Kolkata are due to Vibrio para-haemolyticus2. Vibrio parahaemolyticus accountsfor about 70% of gastroenteritis cases associatedwith seafood in Japan4. Presence of Vibrio para-haemolyticus infection in seafish, fish productsor in fresh water fishes is of public health impor-tance. Though Vibrio parahaemolyticus infectionis self limited infections, socioeconomic loss andrarely death are some of the problems. Immuno-compromised patients may die due to consump-tion of contaminated raw seafish or under cookedfish products5. Sometimes international travelersare also responsible for spreading infection fromone country to another country6. Fish markets,fish harvesting areas, vectors like flies, sea waterand sometimes fresh water are the main sourcesby which this bacterium may spread to human.Multiplication of Vibrio parahaemolyticus is re-lated to water temperature and season also7,8.

The aim of this study was:

1. To isolate Vibrio parahaemolyticus from fishespurchased from markets in and aroundKolkata.

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2. To identify the serotypes of Vibrio para-haemolyticus isolated in the study.

3. To determine the virulence genes tdh and trhby PCR from Vibrio parahaemolyticus strains.

Materials and Methods

In this study 90 fish samples were collectedfrom 8 different market places in Kolkata, India.Fish samples collected were shrimp, prawn, bhet-ki, pamfret and hilsa. Each fish sample wasplaced in aseptic small plastic bag, labeled andsealed separately to avoid contamination. Thesamples were placed in sealed containers with icecubes and transported to the laboratory. Process-ing of samples were initiated within 2-3 hours ofsample collection. In all specimens swabs of gillsand intestines were taken for analysis9. The swabswere incubated in alkaline peptone water (APW),which contained 3% NaCl at pH 8.0 and incubat-ed for 18 hours at 37°C. This APW was used toenrich the Vibrio parahaemolyticus. Isolation andidentification of Vibrio parahaemolyticus wascarried out as described in Bacteriological Ana-lytical Manual of Food and Drug Administration(FDA 1992). A loopful of broth was placed onThiosulfate Citrate Bile Salts Sucrose (TCBS) at37°C. Next day the colonies were examined fortypical bluish green colour. The typical non-su-crose fermenting green colonies were subjected tostandard biochemical tests in Kaper’s agar slantfor alkali/acidic (K/A) reaction10. The strainswhich produced K/A reaction were stored intonutrient agar stabs containing 3% NaCl for fur-ther confirmation and to carry out further molecu-lar characterization. VP-toxR PCR was performedto confirm the presence of species specific toxR11.

Template DNA PreparationA loopful of culture was plated on 3% NaCl

Luria Agar (LA) plate and kept for overnight in-cubation at 37°C. The small amount of overnightculture was taken with the help of toothpick andplaced on sterilized microfuge tube containing500 µl of triple distilled water. Homogenous sus-pension was made using vortex. These sampleswere kept in water bath adjusted at 98°C for 15minutes to lyse the cells. After 15 minutes theculture lysate was snap cooled on ice for denatu-ration of template DNA. Amplification was car-ried out in an automated thermal cycler (Mastercycler gradient, Eppendorf) using PrimerF 1781

and PrimerR 1782. Reference strain VP-KxV138was included in PCR assay as a positive control.The amplified PCR products were kept at -20°Cuntil loading into 2% agarose gel. 5-10 µl of thePCR product were mixed with gel-loading dye.In the first well a molecular weight marker (100bp DNA ladder, Takara, Japan) was loaded. Thereference strain PCR product was loaded in thesecond well. The documentation was done usinggel documentation system (Gel Doc 2000, Bio-Rad, Ann Arbor, Michigan, USA). The authen-ticity of 368 bp amplicon of the toxR gene wasconfirmed by comparing with the position of thePCR product of the control strain (VP-Kx V138).

tdh and trh genes PCR for detection of viru-lence genes were performed separately12. TheVp-toxR positive strain culture lysates were usedas template DNA.

GS-PCR was performed in tdh, trh gene posi-tive strains to determine whether they belong topandemic genotype of Vibrio parahaemolyticusby detecting toxRS sequence according tomethod of Matsumoto et al13.

Identification of serotypes ofVibrio parahaemolyticus

Currently 13 O and 72 K type of commercialantisera are recognized14. Serotyping was per-formed on tdh and trh positive isolates of Vibrioparahaemolyticus by slide agglutination tech-nique using commercially available antisera kits(Denka Seiken Ltd., Tokyo, Japan).

Results

Out of the 90 fish samples collected from dif-ferent local fish markets 60 were positive for Vib-rio parahaemolyticus. The results of V-toxR con-firmed 60 isolates as Vibrio parahaemolyticus.All the 60 isolates of Vibrio parahaemolyticusgave a 368 bp amplicon, which matched with thecontrol strain VP-Kx138. Isolation of Vibrio para-haemolyticus from prawn, pamfret, bhetki andhilsa are 67.35%, 50%, 69.23% and 100% re-spectively. 21 (35%) out of 60 Vibrio para-haemolyticus isolates from fish samples har-boured the tdh gene. All the 21 isolates of Vibrioparahaemolyticus gave a specific 199 bp ampli-con in the tdh PCR which are comparable withthe amplicon from the reference strain VP-Kx138.In the present study 1 (1.7%) out of 60 Vibrioparahaemolyticus isolates from fish samples car-

D. Pal, N. Das

ried trh gene. The trh positive strain gave a spe-cific 249 bp amplicon from the reference strainVP-V287 (Table I). Distribution of virulence genesamong prawn, pamfret, bhetki, hilsa are 42.42%,40%, 33.33% and 12.5% respectively. The tdh,trh gene positive isolates of Vibrio para-haemolyticus from fish samples were selected forgroup specific PCR. Out of 22 isolates only 2were positive for GS-PCR that produced 651 bpamplicon. This result was comparable to refer-ence strain VP-KxV224. Percentage of Vibrioparahaemolyticus harboring tdh, trh and pan-demic strains among fish isolates are 35%, 1.7%and 3.3% respectively. The tdh and trh gene posi-tive isolates of Vibrio parahaemolyticus fromfish sample were selected for serology. In thisstudy O1:KUT serovar was identified with 2 iso-lates, O10:KUT serovar with 4 isolates andO1:K33 with 2 isolates (Table II).

Discussion

Vibrio parahaemolyticus is a marine bacteriumthat occurs naturally in shellfish, oyster, prawn,crab, raw fish, blue mussels, salted herring,roe,fresh water fish, sea fish, etc. Fish is a goodprotein source and Kolkata is one of the places inIndia where consumption is very high. In Kolkatasome of the fish variety is delivered from otherstates of India. Moreover this bacterium is com-monly found in other South East Asian countries.In India the first outbreak of Vibrio para-haemolyticus mediated diarrhea was reported inVellore, Tamilnadu15. In 1996 an abrupt increasein diarrhoeal cases were reported in InfectiousDisease Hospital, Kolkata due to pandemic strainO3:K616. PCR used to identify VPtoxR gene isspecies specific for Vibrio parahaemolyticus11.As both pathogenic and nonpathogenic strains ofVibrio parahaemolyticus exist in seafood, PCRspecific for virulenc genes (tdh, trh) will help in

detection of the pathogenic strains12. In the pre-sent study, 66.7% isolates were positive for theVibrio parahaemolyticus, VP-toxR confirmed94% species specific tox-R gene and 22 (36.7%)out of 60 isolates were positive for virulencegenes such as tdh or trh. Early works of Sakzakiet al17 reported that 1-2% of environmental sam-ples contain virulent isolates. Recently Deepan-jali et al8 reported that the environmental samples(oysters) contain 93.87% Vibrio parahaemolyti-cus and most of them harbor trh (59.3%) and afew with tdh (6.1%) in South West coast of India.Presence of 35% tdh, 1.66% trh and two pan-demic genotype isolates in this region, stronglysuggest that fish (seafish, fresh water fish) pro-duce a conducive environment for this pathogen,which not only support its survival but also to re-tain virulence genes. Normally estuarine or seawater fish carries the Vibrio parahaemolyticus. Inthis study fresh water Hilsa collected fromKolkata were contaminated with pathogenic Vib-rio parahaemolyticus. GS-PCR performed with22 fish samples showed that 2 (3.33%) were pos-itive for the 651 bp amplicon. GS-PCR has beenutilized for detection of pandemic strains of Vib-rio parahaemolyticus, utilizing nucleotide varia-tion for ToxRS sequence. GS-PCR distinguishesnew pandemic clones from old clones13. GS-PCRpositive strains belonged to O1:KUT is theserotype, which is responsible for increasingnumbers of diarrhea among patients in as manyas 12 countries18,19,20. In contrast to Vibrio choler-ae, Vibrio parahaemolyticus infection is not as-sociated with exclusive serovar in the past. TheO1:KUT and O8:KUT serovars isolated in thisstudy belonged to pandemic genotype. The pan-demic O3:K6 clone was isolated from environ-mental samples in Thailand21 and India8. In vitroadherence and cytotoxicity studies with humanepithelial cells showed that O3:K6 strains exhib-ited higher level of adherence and cytotoxicity tohost cells than non O3:K6 strains contributing tounique pathogenic potential. Thermostable Di-

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Isolation, identification and molecular characterization of Vibrio parahaemolyticus

No. of samples screened 90No. of positive isolates 60 66.6%(V. parahaemolyticus)

No. of pathogenic strains 22 36.6%(21 tdh +1 trh)

No. of pandemic strains 2 9%(GS-PCR)

Table I.

Table II. Different serotypes of Vibrio parahaemolyticusobtained.

Serotype Number

O1: KUT 2

O10: KUT 4

O1: K33 2

548

rect Haemolysin (TDH) and TDH relatedhaemolysin (TRH), capsular polysaccharide(CPS), lipopolysaccharide (LPS) and serine pro-tease are the four virulence factors playing animportant role in pathogenicity of Vibrio para-haemolyticus infections22,23. TDH is capable ofproducing β haemolysis on Wagatsumas agarwhich is called Kanagawas phenomena24. Over90% of TDH produced by Kanagawa positivestrains are attributed to tdh-2 and tdh-1 gene di-rect the synthesis of less than 10% of total TDH.TRH is another virulence factor has been discov-ered in clinical strains lacking tdh gene25. TRH isa 23kDa protein, immunologically related toTDH and environmental strains producing thisgene also produces urease. Considerable per-centage of Vibrio parahaemolyticus carryingthe virulence genes and pandemic genotypeamong fish in Kolkata indicates that there ispotential reservoir in Kolkata and consumptionof sea fish or contaminated fish might causeVibrio parahaemolyticus mediated diarrhea inhumans.

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––––––––––––––––––––Acknowledgements

The Authors wish to express their sincere thanks to Dr.M. Saravanan, Student MVPH for performing the lab-oratory techniques and to Dr. T. Ramamurthy, DeputyDirector, Division of Molecular Microbiology, Nation-al Institute of Cholera and Enteric Diseases, Kolkatafor helping to do the PCR in his Laboratory.

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