Virulence Factors and Antimicrobial Susceptibility of Vibrio parahaemolyticus Isolated from the Oyster Crassostrea gigas

양식 굴(Crassostrea gigas)에서 분리된 장염비브리오균의 독소 유전자 보유 및 항균제 감수성

Kim, Sukyung;An, Sera;Park, Bomi;Oh, Eun-Gyoung;Song, Ki Cheol;Kim, Jung-Wan;Yu, Hongsik

  • Received : 2016.02.29
  • Accepted : 2016.03.30
  • Published : 2016.04.30


This study investigated the prevalence of Vibrio parahaemolyticus in the oyster Crassostrea gigas, which is commonly consumed raw. The presence of virulence factors and the antimicrobial susceptibility of isolates were also investigated. The overall prevalence rate of V. parahaemolyticus in oysters was 37.5% (36/96) and the range of concentrations was 30-11,000 MPN/100 g. PCR-based assays indicated that 9.6% (11/115) of the isolates were positive for the thermostable direct hemolysin-related hemolysin gene (trh), while none of the isolates were positive for the thermostable direct hemolysin gene (tdh). The Multiple Antibiotics Resistance (MAR) index was measured for 16 common antimicrobial agents and 46.1% (53/115) of the isolates had a MAR index > 0.2. The MAR index ranged from 0.07 to 0.73. The highest MAR index was observed with strain s150608, isolated in June 2015, which exhibited resistance to 11 antimicrobial agents. Our results demonstrate that oysters are high-risk sources of V. parahaemolyticus, although no antimicrobial agent was being used to promote growth or to treat bacterial infections in the sampled oyster-growing areas.


Vibrio parahaemolyticus;Oyster;Virulence factors;Antimicrobial susceptibility;MAR index


  1. Alam MJ, Tomochika KI, Miyoshi SI and Shinoda S. 2002. Environ- mental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea,Japan. FEMS Microbiol Lett 208, 83-87.
  2. Acar JF and Goldstein FW. 1991. Disk susceptibility test. In: Antibiotics in laboratory medicine. Lorian V, ed. Williams & Wilkins, Baltimore, U.S.A., 17-52.
  3. Broberg CA, Calder TJ and Orth K. 2011. Vibrio parahaemolyticus cell biology and pathogenicity determinants. Microbes Infect 13, 992-1001.
  4. Cabello FC, Godfrey HP, Tomova A, Ivanova L, Dölz H, Millanao A and Buschmann AH. 2013. Antimicrobial use in aquaculture re-examined: its relevance to antimicrobial resistance and to animal and human health. Appl Eviron Microbiol 15, 1917-1942.
  5. Canizalez RA, Flores VH, Zazueta BJ, Muro AS and Leon SN. 2011.Comparative evaluation of achromogenic agar medium-PCR protocol with a conventional method for isolation of Vibrio parahaemolyticus strains from environmental and clinical samples. Can J Microbiol 57, 136-142.
  6. Ceccarelli D, Hasan NA, Hug A and Colwell RR. 2013. Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors. Front Cell Infect Microbio 3, 1-9.
  7. Daniels NA, MacKinnon L, Bishop R, Altekruse S, Ray B, Hammond RM, Thompson S, Wilson S, Bean NH, Griffin PM and Slutsker L. 2000. Vibrio parahaemolyticus infection in the United States, 1973-1998. J Infect Dis 181, 1661-1666.
  8. Chakraborty RD, Surendran PK and Joseph TC. 2008. Isolation and characterization of Vibrio parahaemolyticus from seafoods along the southwest coast of India. World J Microbiol Biotechnol 24, 2045-2054.
  9. Chao G, Jiao X, Zhou X, Yang Z, Huan J, Pan Z, Zhou L and Qian X. 2009. Serodiversity, pandemic O3:K6 clone, molecular typing, and antibiotic susceptibility of foodborne and clinical Vibrio parahaemolyticus isolates in Jiangsu, China. Foodborne Pathog Dis 6, 1021-1028.
  10. CLSI (Clinical and Laboratory Standard Institute). 2015. Susceptibility testing process. In: Performance standards for antimicrobial disk susceptibility tests. Approved standard 12th edition. CLSI document M2-A10. Jean BP, ed. Clinical and Laboratory Standard Institute, Wayne Pa, U.S.A., 15-39.
  11. Deepanjali A, Kumar HS, Karunasagar I and Karunasagar I. 2005. Seasonal variation in abundance of total and pathogenic Vibrio parahaemolyticus bacteria in oysters along the south west coast of India. Appl Environ Microbiol 71, 3575-3580.
  12. DePaola A, Kaysner CA, Bowers J and Cook DW. 2000. Environmental investigations of Vibrio parahaemolyticus in oysters after outbreaks in Washington, Texas, and New York (1997 and 1998). Appl Environ Microbiol 66, 4649-4654.
  13. Elliot EL, Kaysner CA, Jackson L and Tamplin ML. 1995. Vibrio cholerae, V. parahaemolyticus, V. vulnificus and other Vibrio spp. In: Bacteriological Analytical Manual. Association of Official Analytical Chemists ed. FDA, Arlington, U.S.A., 9.01-9.27.
  14. Haley BJ, Kokashvili T, Tskshvediani A, Janelidze N, Mitaishvili N, Grim CJ, Constantin de MG, Chen AJ, Taviani E, Eliashvili T, Tediashvili M, Whitehouse CA, Colwell RR, Huq A. 2014. Molecular diversity and predictability of Vibrio parahaemolyticus along the Georgian coastal zone of the BlackSea. Front Microbiol 5, 1-9.
  15. Furtula V, Charlene RJ, Erin GF, John BB, Lari MH and Patricia AC. 2013. Antimicrobial Resistance in Enterococcus spp. Isolated from Environmental Samples in an Area of Intensive Poultry Production Int J Environ Res Public Health 10, 1020-1036. ijerph10031020.
  16. Gode-Potratz CJ, Kustusch RJ, Breheny PJ, Weiss DS and McCarter LL. 2011. Surface sensing in Vibrio parahaemolyticus triggers a programme of gene expression that promotes colonization and virulence. Mol Microbiol 79, 240-263.
  17. Gutierrez WCK, Klein SL and Lovell CR. 2013. High frequency of virulence factor genes tdh, trh, and tlh in Vibrio parahaemolyticus strains isolated from a pristine estuary. Appl Environ Microbiol 79, 2247-2252.
  18. Han F, Walker RD, Janes ME, Prinyawinwatkul W and Ge B. 2007. Antimicrobial susceptibilities of Vibrio parahaemolyticus and Vibrio vulnificus isolates from Louisiana Gulf and retail raw oysters. Appl Environ Microbiol 73, 7096-7098.
  19. Hervio-Heath D, Colwell RR, Derrien JM, Robert-Pillot A, Fournier JM and Pommepuy M. 2002. Occurrence of pathogenic vibrios in coastal areas of France. J Appl Microbiol 92, 1123-1135.
  20. Johnson CN, Flowers AR, Young VC, Gonzalez-Escalona N, DePaola A, Norie NFIII and Grimes DJ. 2008. Genetic relatedness among tdh+ and trh+ Vibrio parahamolyticus cultured from Gulf of Mexico oysters (Crassostrea virginica) and surrounding water and sediment. Microb Eco 57, 437-443. 008-9418-3.
  21. Letchumanan V, Yin WF, Lee LH and Chan KG. 2015. Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shrimps in Malaysia. Front Microbiol 6, 1-11.
  22. KFDA. 2016. Food poisoning statistics by pathogen and month. Retrieved from on February 22.
  23. Kirs M, DePaola A, Fyfe R, Jones JL, Krantz J, Van Laanen A, Cotton D and Castle M. 2011. A survey of oysters (Crassostrea gigas) in New Zealand for Vibrio parahaemolyticus and Vibrio vulnificus. Int J Food Microbiol 147, 149-153.
  24. Krumperman PH. 1983. Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Appl Environ Microbiol 46, 165-170.
  25. Makino K, Oshima K, Kurokawa K, Yokoyama K, Uda T, Tagomori K, Iijima Y, Najima M, Nakano M, Yamashita A, Kubota Y, Kimura S, Yasunaga T, Honda T, Shinagawa H, Hattori M and Iida T. 2003. Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V. cholerae. Lancet 361, 743-749.
  26. Malcolm TTH, Cheah YK, Radzi CWJWM, Kasim FA, Kantilal HK, John TYH, Martinez-Urtazaf J, Nakaguchig Y, Nishibuchig M and Sona R. 2015. Detection and quantification of pathogenic Vibrio parahaemolyticus in shellfish by using multiplex PCR and loop-mediated isothermal amplification assay. Food Control 47, 664-671.
  27. Martinez-Urtaza J, Huapaya B, Gavilan RG, Blanc-Abad V, Ansede-Bermejo J, Cadarso-Suarez C, Figueiras A, Trinanes J. 2008. Emergence of Asiatic vibrio disease in south America in phase with ElNino. Epidemiol 19, 829-837.
  28. Okamoto AS, AndreattiFilho RL, Rocha TS, Menconi A and Marietto-Goncalves GA. 2009. Detection and transfer of antimicrobial resistance gene integron in Salmonella enteritidis derived from avian material. Rev Brasil Ciên Avíc 11, 195-201.
  29. Neela FA, Nonaka L and Suzuki S. 2007. The diversity of multidrug resistance profiles in tetracycline-resistant Vibrio species isolated from coastal sediments and seawater. J Microbiol 45, 64-68.
  30. Oh EG, Son KT, Ha KS, Yoo HD, Yu HS, Shin SB, Lee HJ and Kim JH. 2009. Antimicrobial resistance of Vibrio strains from brackish water on the coast of gyeongsangnamdo. J Kor Fish Soc 42, 335-343.
  31. Oh EG, Son KT, Yu HS, Lee TS, Lee HJ, Shin SB, Kwon JY, Park KBW and KIM JH. 2011. Antimicrobial resistance of Vibrio parahaemolyticus and Vibrio alginolyticus strains isolated from Farmed Fish in Korea during 2005-2007. J Food Protec 74, 380-386.
  32. Panicker G, Myers ML and Bej AK. 2004. Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR. Appl Environ Microbiol 70, 498-507. 10.1128/AEM.70.1.498-507.2004.
  33. Parveen S, Hettiarachchi KA, Bowers JC, Jones JL, Tamplin ML, McKay R, Beatty W, Brohawn K, Dasilva LV, Depaola A. 2008. Seasonal distribution of total and pathogenic Vibrio parahaemolyticus in Chesapeake Bay oysters and waters. Int J Food Microbiol 128, 354-361.
  34. Rico A, Satapornvanit K, Haque MM, Min J, Nguyen PT, Telfer T and van den Brink PJ. 2012. Use of chemicals and biological products in Asian aquaculture and their potential environmental risks: acritical review. Rev Aquac 4, 75-93.
  35. Su CY and Liu C. 2007. Vibrio parahaemolyticus: a concern of seafood safety. Food Microbiol 24, 549-558.
  36. Sahilah AM, Laila RA, Sallehuddin HM, Osman H, Aminah A and Ahmah AA. 2014. Antibiotic resistance and molecular typing among cockle (Anadaragranosa) strains of Vibrio parahaemolyticus by polymerase chain reaction (PCR)-based analysis. World J Microbiol Biotech 30, 649-659.
  37. Sani NA, Ariyawansa S, Babji AS and Hashim JK. 2013. The risk assessment of Vibrio parahaemolyticus in cooked black tiger shrimps (Penaeusmonodon) in Malaysia. Food Control 31, 546-552.
  38. Shaw KS, RosenbergGoldstein RE, He X, Jacobs JM, Crump BC and Sapkota AR. 2014. Antimicrobial susceptibility of Vibrio vulnificus and Vibrio parahaemolyticus recovered from recreational and commercial areas of Cheaspeake Bay and Maryland coastal bay. PLoS One 9:e89616.
  39. Sudha S, Mridula C, Silvester R and Hatha AAM. 2014. Prevalence and antibiotic resistance of pathogenic vibrios in shellfishes from Cochin market. Indian J Mar Sci 43, 815-824.
  40. Tang HJ, Chang MC, Ko WC, Huang KY, Lee CL and Chuang YC. 2002. In vitro and in vivo activities of newer fluoroquinolones against Vibrio vulnificus. Antimicrob Agents Chemother 46, 3580-3584.
  41. Vuddhakul V, Chowdhury A, Laohaprertthisan V, Pungrasamee P, Patararun-grong N and Thianmontri P. 2000. Isolation of a pandemic O3:K6 clone of a Vibrio parahaemolyticus strain from environmental and clinical sources in Thailand. Appl Environ Microbiol 66, 2685-2689. 2689.2000.
  42. Yano Y, Hamano K, Satomi M, Tsutsui I, Ban M and Aue-umneoy D. 2014. Prevalence and antimicrobial susceptibility of Vibrio species related to food safety isolated from shrimp cultured at inland ponds in Thailand. Food Control 38, 30-45.
  43. WHO/FAO, 2002. Hazard identification, exposure assessment and hazard characterization of Vibrio spp. in seafood In: Joint FAO/WHO Expert Consultation on Risk Assessment of Microbiological Hazards in Foods. Geneva, Switzerland, 3-27.
  44. Xu X, Wu Q, Zhang J, Cheng J, Zhang S and Wu K. 2014. Prevalence, pathogenicity, and serotypes of Vibrio parahaemolyticus in shrimp from Chinese retail markets. Food Control 46, 81-85.
  45. Yamamoto A, Iwahori J, Vuddhakul V, Charernjiratrag W, Vose D, Osaka K, Shigematsu M, Toyofuku H, Yamamoto S, Nishibuchi M and Kasuga F. 2008. Quantitative modeling for risk assessment of Vibrio parahaemolyticus in bloody clams in southern Thailand. Int J Food Microbiol 124, 70-78.
  46. Yu HS, Oh EG, Shin SB, Park YS, Lee HJ, Kim JH and Song KC. 2014. Distribution and Antimicrobial Resistance of Vibrio parahaemolyticus Isolated from Korean Shellfish. Korean J Fish Aquat Sci 47, 508-515.
  47. Zarei M, Borujeni MP, Jamnejad A and Khezrzadeh M. 2012. Seasonal prevalence of Vibrio species in retail shrimps with an emphasis on Vibrio parahaemolyticus. Food Control 25, 107-109.
  48. Zhang L and Orth K. 2013. Virulence determinants for Vibrio parahaemolyticus infection. Curr Opin Microbiol 16, 70-77.

Cited by

  1. Food-borne outbreaks, distributions, virulence, and antibiotic resistance profiles of Vibrio parahaemolyticus in Korea from 2003 to 2016: a review vol.21, pp.1, 2018,