Isolation, identification and molecular characterization of Vibrio parahaemolyticus from fish samples in Kolkata

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1 European Review for Medical and Pharmacological Sciences Isolation, identification and molecular characterization of Vibrio parahaemolyticus from fish samples in Kolkata D. PAL, N. DAS 2010; 14: Department of Microbiology, All India Institute of Hygiene and Public Health, Kolkata (India) Abstract. Vibrio parahaemolyticus is a marine bacterium which is also responsible for acute diarrhoeal illness in human beings. Eating raw seafish or contaminated seafood is responsible for acute gastroenteritis. The aim of this study was to investigate the isolation, identification and molecular characterization of Vibrio parahaemolyticus from the fish samples in Kolkata, India. Materials and Methods: In this study 90 fish samples were collected from 8 different market places in Kolkata, India. Fish samples collected were shrimp, prawn, bhetki, pamfret and hilsa. VP-toxR PCR was performed to confirm the presence of species specific toxr. tdh and trh genes PCR for detection of virulence genes were performed separately. GS-PCR was performed in tdh, trh gene positive strains to determine whether they belong to pandemic genotype. Serotyping was also done on the tdh, trh positive strains. Results: Out of the 90 fish samples collected from different local fish markets 60 were positive for Vibrio parahaemolyticus. 21 (35%) out of 60 Vibrio parahaemolyticus isolates from fish samples harboured the tdh gene. 1 (1.7%) out of 60 Vibrio parahaemolyticus isolates from fish samples carried trh gene. Out of 22 isolates only 2 were positive for GS-PCR. O10:KUT was the serovar maximum isolated. Conclusion: Considerable percentage of Vibrio parahaemolyticus carrying the virulence genes and pandemic genotype among fish in Kolkata indicates that there is potential reservoir in Kolkata and consumption of sea fish or contaminated fish might cause Vibrio parahaemolyticus mediated diarrhea in this region. Key Words: Vibrio parahaemolyticus, Fish, Diarrhoea, Acute gastroenteritis. Introduction Vibrio parahaemolyticus is a marine bacterium which is also responsible for acute diarrhoeal illness in human beings. Eating raw seafish or contaminated seafood is responsible for acute gastroenteritis. Vibrio parahaemolyticus was first implicated in an outbreak of food poisoning in Japan in Etiological studies of acute diarrhoeal illness in Kolkata have shown that gastroenteritis caused by Vibrio parahaemolyticus ranks next to Vibrio cholerae in terms of incidence 2,3. About 10% of the cases of gastroenteritis in patients admitted to Infectious Diseases Hospital in Kolkata are due to Vibrio parahaemolyticus 2. Vibrio parahaemolyticus accounts for about 70% of gastroenteritis cases associated with seafood in Japan 4. Presence of Vibrio parahaemolyticus infection in seafish, fish products or in fresh water fishes is of public health importance. Though Vibrio parahaemolyticus infection is self limited infections, socioeconomic loss and rarely death are some of the problems. Immunocompromised patients may die due to consumption of contaminated raw seafish or under cooked fish products 5. Sometimes international travelers are also responsible for spreading infection from one country to another country 6. Fish markets, fish harvesting areas, vectors like flies, sea water and sometimes fresh water are the main sources by which this bacterium may spread to human. Multiplication of Vibrio parahaemolyticus is related to water temperature and season also 7,8. The aim of this study was: 1. To isolate Vibrio parahaemolyticus from fishes purchased from markets in and around Kolkata. Corresponding Author: Debaprasad Pal, MD; debaprasadpal@hotmail.com 545

2 D. Pal, N. Das 2. To identify the serotypes of Vibrio parahaemolyticus isolated in the study. 3. To determine the virulence genes tdh and trh by PCR from Vibrio parahaemolyticus strains. Materials and Methods In this study 90 fish samples were collected from 8 different market places in Kolkata, India. Fish samples collected were shrimp, prawn, bhetki, pamfret and hilsa. Each fish sample was placed in aseptic small plastic bag, labeled and sealed separately to avoid contamination. The samples were placed in sealed containers with ice cubes and transported to the laboratory. Processing of samples were initiated within 2-3 hours of sample collection. In all specimens swabs of gills and intestines were taken for analysis 9.The swabs were incubated in alkaline peptone water (APW), which contained 3% NaCl at ph 8.0 and incubated for 18 hours at 37 C. This APW was used to enrich the Vibrio parahaemolyticus. Isolation and identification of Vibrio parahaemolyticus was carried out as described in Bacteriological Analytical Manual of Food and Drug Administration (FDA 1992). A loopful of broth was placed on Thiosulfate Citrate Bile Salts Sucrose (TCBS) at 37 C. Next day the colonies were examined for typical bluish green colour. The typical non-sucrose fermenting green colonies were subjected to standard biochemical tests in Kaper s agar slant for alkali/acidic (K/A) reaction 10. The strains which produced K/A reaction were stored into nutrient agar stabs containing 3% NaCl for further confirmation and to carry out further molecular characterization. VP-toxR PCR was performed to confirm the presence of species specific toxr 11. Template DNA Preparation A loopful of culture was plated on 3% NaCl Luria Agar (LA) plate and kept for overnight incubation at 37 C. The small amount of overnight culture was taken with the help of toothpick and placed on sterilized microfuge tube containing 500 µl of triple distilled water. Homogenous suspension was made using vortex. These samples were kept in water bath adjusted at 98 C for 15 minutes to lyse the cells. After 15 minutes the culture lysate was snap cooled on ice for denaturation of template DNA. Amplification was carried out in an automated thermal cycler (Master cycler gradient, Eppendorf) using PrimerF 1781 and PrimerR Reference strain VP-KxV138 was included in PCR assay as a positive control. The amplified PCR products were kept at -20 C until loading into 2% agarose gel µl of the PCR product were mixed with gel-loading dye. In the first well a molecular weight marker (100 bp DNA ladder, Takara, Japan) was loaded. The reference strain PCR product was loaded in the second well. The documentation was done using gel documentation system (Gel Doc 2000, Bio- Rad, Ann Arbor, Michigan, USA). The authenticity of 368 bp amplicon of the toxr gene was confirmed by comparing with the position of the PCR product of the control strain (VP-Kx V138). tdh and trh genes PCR for detection of virulence genes were performed separately 12. The Vp-toxR positive strain culture lysates were used as template DNA. GS-PCR was performed in tdh, trh gene positive strains to determine whether they belong to pandemic genotype of Vibrio parahaemolyticus by detecting toxrs sequence according to method of Matsumoto et al 13. Identification of serotypes of Vibrio parahaemolyticus Currently 13 O and 72 K type of commercial antisera are recognized 14. Serotyping was performed on tdh and trh positive isolates of Vibrio parahaemolyticus by slide agglutination technique using commercially available antisera kits (Denka Seiken Ltd., Tokyo, Japan). Results Out of the 90 fish samples collected from different local fish markets 60 were positive for Vibrio parahaemolyticus. The results of V-toxR confirmed 60 isolates as Vibrio parahaemolyticus. All the 60 isolates of Vibrio parahaemolyticus gave a 368 bp amplicon, which matched with the control strain VP-Kx 138. Isolation of Vibrio parahaemolyticus from prawn, pamfret, bhetki and hilsa are 67.35%, 50%, 69.23% and 100% respectively. 21 (35%) out of 60 Vibrio parahaemolyticus isolates from fish samples harboured the tdh gene. All the 21 isolates of Vibrio parahaemolyticus gave a specific 199 bp amplicon in the tdh PCR which are comparable with the amplicon from the reference strain VP-Kx 138. In the present study 1 (1.7%) out of 60 Vibrio parahaemolyticus isolates from fish samples car- 546

3 Isolation, identification and molecular characterization of Vibrio parahaemolyticus ried trh gene. The trh positive strain gave a specific 249 bp amplicon from the reference strain VP-V 287 (Table I). Distribution of virulence genes among prawn, pamfret, bhetki, hilsa are 42.42%, 40%, 33.33% and 12.5% respectively. The tdh, trh gene positive isolates of Vibrio parahaemolyticus from fish samples were selected for group specific PCR. Out of 22 isolates only 2 were positive for GS-PCR that produced 651 bp amplicon. This result was comparable to reference strain VP-KxV 224. Percentage of Vibrio parahaemolyticus harboring tdh, trh and pandemic strains among fish isolates are 35%, 1.7% and 3.3% respectively. The tdh and trh gene positive isolates of Vibrio parahaemolyticus from fish sample were selected for serology. In this study O1:KUT serovar was identified with 2 isolates, O10:KUT serovar with 4 isolates and O1:K33 with 2 isolates (Table II). Discussion Vibrio parahaemolyticus is a marine bacterium that occurs naturally in shellfish, oyster, prawn, crab, raw fish, blue mussels, salted herring, roe,fresh water fish, sea fish, etc. Fish is a good protein source and Kolkata is one of the places in India where consumption is very high. In Kolkata some of the fish variety is delivered from other states of India. Moreover this bacterium is commonly found in other South East Asian countries. In India the first outbreak of Vibrio parahaemolyticus mediated diarrhea was reported in Vellore, Tamilnadu 15. In 1996 an abrupt increase in diarrhoeal cases were reported in Infectious Disease Hospital, Kolkata due to pandemic strain O3:K6 16. PCR used to identify VPtoxR gene is species specific for Vibrio parahaemolyticus 11. As both pathogenic and nonpathogenic strains of Vibrio parahaemolyticus exist in seafood, PCR specific for virulenc genes (tdh, trh) will help in Table I. No. of samples screened 90 No. of positive isolates % (V. parahaemolyticus) No. of pathogenic strains % (21 tdh + 1 trh) No. of pandemic strains 2 9% (GS-PCR) Table II. Different serotypes of Vibrio parahaemolyticus obtained. Serotype Number O1: KUT 2 O10: KUT 4 O1: K33 2 detection of the pathogenic strains 12. In the present study, 66.7% isolates were positive for the Vibrio parahaemolyticus, VP-toxR confirmed 94% species specific tox-r gene and 22 (36.7%) out of 60 isolates were positive for virulence genes such as tdh or trh. Early works of Sakzaki et al 17 reported that 1-2% of environmental samples contain virulent isolates. Recently Deepanjali et al 8 reported that the environmental samples (oysters) contain 93.87% Vibrio parahaemolyticus and most of them harbor trh (59.3%) and a few with tdh (6.1%) in South West coast of India. Presence of 35% tdh, 1.66% trh and two pandemic genotype isolates in this region, strongly suggest that fish (seafish, fresh water fish) produce a conducive environment for this pathogen, which not only support its survival but also to retain virulence genes. Normally estuarine or sea water fish carries the Vibrio parahaemolyticus. In this study fresh water Hilsa collected from Kolkata were contaminated with pathogenic Vibrio parahaemolyticus. GS-PCR performed with 22 fish samples showed that 2 (3.33%) were positive for the 651 bp amplicon. GS-PCR has been utilized for detection of pandemic strains of Vibrio parahaemolyticus, utilizing nucleotide variation for ToxRS sequence. GS-PCR distinguishes new pandemic clones from old clones 13. GS-PCR positive strains belonged to O1:KUT is the serotype, which is responsible for increasing numbers of diarrhea among patients in as many as 12 countries 18,19,20. In contrast to Vibrio cholerae, Vibrio parahaemolyticus infection is not associated with exclusive serovar in the past. The O1:KUT and O8:KUT serovars isolated in this study belonged to pandemic genotype. The pandemic O3:K6 clone was isolated from environmental samples in Thailand 21 and India 8. In vitro adherence and cytotoxicity studies with human epithelial cells showed that O3:K6 strains exhibited higher level of adherence and cytotoxicity to host cells than non O3:K6 strains contributing to unique pathogenic potential. Thermostable Di- 547

4 D. Pal, N. Das rect Haemolysin (TDH) and TDH related haemolysin (TRH), capsular polysaccharide (CPS), lipopolysaccharide (LPS) and serine protease are the four virulence factors playing an important role in pathogenicity of Vibrio parahaemolyticus infections 22,23. TDH is capable of producing β haemolysis on Wagatsumas agar which is called Kanagawas phenomena 24. Over 90% of TDH produced by Kanagawa positive strains are attributed to tdh-2 and tdh-1 gene direct the synthesis of less than 10% of total TDH. TRH is another virulence factor has been discovered in clinical strains lacking tdh gene 25.TRH is a 23kDa protein, immunologically related to TDH and environmental strains producing this gene also produces urease. Considerable percentage of Vibrio parahaemolyticus carrying the virulence genes and pandemic genotype among fish in Kolkata indicates that there is potential reservoir in Kolkata and consumption of sea fish or contaminated fish might cause Vibrio parahaemolyticus mediated diarrhea in humans. References 1) FUJINO T, OKUNO Y, NAKADA D, AOYAMA A, FUKAI K, MUKAI T, UKBO T. On the bacteriological examination of Shirasen food poisoning. Med J Osaka Univ : ) CHATTERJEE BD, NEOGY KN, GORBACH SL. Study of Vibrio parahaemolyticus from cases of diarrhea in Calcutta. Indian J Med Res 1970; 58: ) SAKAZAKI RK, TAMURA L, PRESCOTT M, BENC Z, SANYAL SC, SINHA R. Bacteriological examination of diarrhoeal stools in Calcutta. Indian J Med Res 1971; 54: ) KANEKO T, COLWELL R. Ecology of Vibrio parahaemolyticus in Chesapeake Bay. J Bacteriol 1973; 113: ) NG TC, CHIANG PC, WU TL, LEU HS. Vibrio parahaemolyticus bacteraemia: case report. Changgeng Vi Xue, Za Zhi 1999; 22: ) LAOHAPRERTTHISAN V,C HOWDHURY A, KONGMUANG U, KALNAUWAKUL S, ISHIBASHI M, MATSUMATO C, NISHS- BUCHI M. Prevalence of serodiversitybof the pandemic clone among the clinical strains of Vibrio parahaemolyticus isolated from Thailand. Epidemiol Infect 2003; 130: ) SARKAR B, CHOWDHURY NR, NAIR GB, NISHIBUCHI M, YAMASAKI S, TAKEDA Y, GUPTA SK, BHATTACHARYA SK, RA- MAMURTHY T. Molecular characterization of Vibrio parahaemolyticus of similar serovars isolated from sewage and clinical cases of diarrhea in Calcutta, India. World J Microbiol Biotechnol 2003; 19: ) DEEPANJALI A, SANATH KUMAR H, KARUNASAGAR I. Seasonal variation in abundance of total and pathogenic Vibrio parahaemolyticus bacteria in oysters along the Southwest coast of India. Appl Environ Microbiol 2005; 71: ) NAIR GB, ABRAHAM M, NATARAJAN R. Distribution of Vibrio parahaemolyticus in finfish harvested from Porto Novio (S. India) environs: a seasonal study. Canadian J Microbiol 1980; 26: ) KAPER JB, LOCKENAN H, COLWELL R, JOSEPH SW. Ecology, serology and enterotoxin production of Vibrio cholera in Chesapeak Bay. Appl Environ Microbiol 1979; 37: ) KIM YB, OKUDA J, MATSUMOTO C, TAKAHASI N, HASHIMOTO S, NISHIBUICHI M. Identification of Vibrio parahaemolyticus strains at species level by PCRtargetted to toxr gene. J Clin Microbiol 1999; 37: ) DILEEP V, KUMAR HS, NISHIBUCHI M, KARUNASAGAR I. Application of polymerase chain reaction for detection of Vibrio parahaemolyticus associated with tropical seafoods and coastal environment. Lett Appl Microbiol 2003; 36: ) MATSUMOTO C, OKUDA J, ISHIBASHI M, IWANAGA M, GARG P, R AMAMURTHY T, WONG H, DEPAOLA A, KIM YB, ALBERT MJM, NISHIBUSHI M. Pandemic spread of an O3:K6 clone of Vibrio parahaemolyticus and emergence of related strains evidenced by arbitrarily primed PCR and toxrs sequence analysis. J Clin Microbiol 2000; 38: ) WONG HC, HO CY, KUO LP, WONG TK, LEE CL, SHIH DY. Ribotyping of Vibrio parahaemolyticus obtained from food poisoning outbreaks in Taiwan. Microbiol Immunol 1999; 43: ) LALITHA MK, WALTER NM, JESUDASAN M, MATHAN VI. An outbreak of gastroenteritis due to Vibrio parahaemolyticus in Vellore. Indian J Med Res 1983; 78: ) OKUDA J, ISHIBASHI M, HAYAKAWA E, NISHINO T, TAKEDA Y, MUKHOPADHYAY AK, GARG S, BHATTACHARYA SK, NAIR GB, NISHIBUCHI M. Emergence of a unique O3:K6 clone of Vibrio parahaemolyticus in Calcutta, India and isolation of strains from the same clonal group from Southeast Asian travelers arriving in Japan. J Clin Microbiol 1997; 35: ) SAKAZAKI R, TAMURA K, KATO T, OBARA Y, YAMAI S, HOBO K. Studies of enteropathogenic facultative halophilic bacteria Vibrio parahaemolyticus III. Enteropathogenicity. Jpn J Med Sci Biol 1968; 21: ) WORLD HEALTH ORGANISATION. Vibrio parahaemolyticus Japan, Wkly Epidemiol Rec 1999; 74: ) CHOWDHURY NR, CHAKRABORTY S, EAMPOKALAP B, CHAICUMPA WM, CHONGSANGUAN M, MOOLASART P, MI- 548

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