Journal of Consumer Protection and Food Safety

, Volume 14, Issue 4, pp 355–364 | Cite as

Pre-enrichment step, incubation temperature and type of selective media affect the pathogenic Vibrio parahaemolyticus detection efficiency in frozen prawns

  • Salima Sadeghi
  • Kwai Lin Thong
  • Lay Ching ChaiEmail author
Research Article


This study evaluates the performance of different detection protocols of Vibrio parahaemolyticus in frozen prawns. The effect of enrichment and incubation temperature on the selectivity of thiosulfate citrate bile sucrose (TCBS) and CHROMagar™ Vibrio (CAV) media were determined. Enumeration of V. parahaemolyticus in frozen prawns by direct-plating on CAV at 37 °C yielded the highest count compared to the other tested direct-plating protocols (p < 0.05). The enrichment step was found to increase the selectivity of TCBS for V. parahaemolyticus but did not affect the selectivity of CAV. Furthermore, trh + V. parahaemolyticus isolates were only successfully recovered from CAV, suggesting that CAV is the better media for the detection of virulent V. parahaemolyticus compared to TCBS. Repetitive Extragenic Palindromic PCR typing (REP-PCR) indicated that CAV and TCBS, as well as direct-plating or enrichment-plating are selective for different subsets of V. parahaemolyticus present in a sample. In conclusion, the current work suggests that CAV is a better selective media for detection and isolation of V. parahaemolyticus in frozen prawns. This study also suggests that direct-plating on CAV with an incubation temperature of 35–37 °C could be an alternative to Most Probable Number (MPN)-PCR for enumeration of V. parahaemolyticus in frozen prawn samples.


CHROMagar™ Vibrio Vibrio parahaemolyticus Direct-plating REP-PCR Frozen prawns 



This research was financially supported by University of Malaya High Impact Research Grant UM (UM.C/625/1/HIR/MOE/SC/20) and UMRG (RP003C-13BIO). We would also like to acknowledge the laboratory facilities provided by faculty of science, University of Malaya.


This study was funded by University of Malaya High Impact Research Grant UM (UM.C/625/1/HIR/MOE/SC/20) and UMRG (RP003C-13BIO) to Lay Ching Chai and Kwai Lin Thong.

Compliance with ethical standards

Conflict of interest

Salima Sadeghi, Kwai Lin Thong, Lay Ching Chai declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Anupama KP, Deeksha K, Deeksha A, Karunasagar I, Karunasagar I, Maiti B (2019) Comparative performance of TCBS and TSA for the enumeration of trh + Vibrio parahaemolyticus by direct colony hybridization. J Microbiol Methods 157:37–42CrossRefGoogle Scholar
  2. Banerjee SK, Farber JM (2017) Detection, enumeration, and isolation of Vibrio parahaemolyticus and V. vulnificus from seafood: development of a multidisciplinary protocol. J AOAC Int 100(2):445–453CrossRefGoogle Scholar
  3. Bej AK, Patterson DP, Brasher CW, Vickery MC, Jones DD, Kaysner CA (1999) Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh. J Microbiol Methods 36:215–225CrossRefGoogle Scholar
  4. Beuchat L (1977) Suitability of some enrichment broths and diluents for enumerating cold-and heat-stressed Vibrio parahaemolyticus. Can J Microbiol 23:630–633CrossRefGoogle Scholar
  5. Blanco-Abad V, Ansede-Bermejo J, Rodriguez-Castro A, Martinez-Urtaza J (2009) Evaluation of different procedures for the optimized detection of Vibrio parahaemolyticus in mussels and environmental samples. Int J Food Microbiol 129:229–236CrossRefGoogle Scholar
  6. Canizalez-Roman A, Flores-Villaseñor H, Zazueta-Beltran J, Muro-Amador S, León-Sicairos N (2011) Comparative evaluation of a chromogenic agar medium–PCR protocol with a conventional method for isolation of Vibrio parahaemolyticus strains from environmental and clinical samples. Can J Microbiol 57:136–142CrossRefGoogle Scholar
  7. Centers for Disease Control and Prevention (2017) Vibrio species causing Vibriosis. Accessed 02 Mar 2017
  8. DePaola A, Hopkins L, Peeler J, Wentz B, McPhearson R (1990) Incidence of Vibrio parahaemolyticus in US coastal waters and oysters. Appl Environ Microbiol 56:2299–2302PubMedPubMedCentralGoogle Scholar
  9. Di Pinto A, Terio V, Novello L, Tantillo G (2011) Comparison between thiosulphate-citrate-bile salt sucrose (TCBS) agar and CHROMagar Vibrio for isolating Vibrio parahaemolyticus. Food Control 22:124–127CrossRefGoogle Scholar
  10. García K, Torres R, Uribe P, Hernández C, Rioseco ML, Romero J, Espejo RT (2009) Dynamics of clinical and environmental Vibrio parahaemolyticus strains during seafood-related summer diarrhea outbreaks in southern Chile. Appl Environ Microbiol 75:7482–7487CrossRefGoogle Scholar
  11. Hara-Kudo Y, Nishina T, Nakagawa H, Konuma H, Hasegawa J, Kumagai S (2001) Improved method for detection of Vibrio parahaemolyticus in seafood. Appl Environ Microbiol 67:5819–5823CrossRefGoogle Scholar
  12. Jiang X, Chai TJ (1996) Survival of Vibrio parahaemolyticus at low temperatures under starvation conditions and subsequent resuscitation of viable, nonculturable cells. Appl Environ Microbiol 62:1300–1305PubMedPubMedCentralGoogle Scholar
  13. Kaufman G, Blackstone G, Vickery M, Bej A, Bowers J, Bowen MD, Meyer RF, DePaola A (2004) Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix. J Food Prot 67:2424–2429CrossRefGoogle Scholar
  14. Kaysner CA, Tamplin ML, Wekell MM, Stott RF, Colburn KG (1989) Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and C. virginica) and effects of isolation medium on recovery. Appl Environ Microbiol 55:3072–3079PubMedPubMedCentralGoogle Scholar
  15. Kim DG, Ahn SH, Kim LH, Park KJ, Hong YK, Kong IS (2008) Application of the rpoS gene for species-specific detection of Vibrio vulnificus by real-time PCR. J Microbiol Biotechnol 18:1841–1847PubMedGoogle Scholar
  16. Kourany M (1983) Medium for isolation and differentiation of Vibrio parahaemolyticus and Vibrio alginolyticus. Appl Environ Microbiol 45:310–312PubMedPubMedCentralGoogle Scholar
  17. Lucht L, Blank G, Bousa J (1998) Recovery of foodborne microorganisms from potentially lethal radiation damage. J Food Prot 61:586–590CrossRefGoogle Scholar
  18. Mackey BM (1984) Lethal and sublethal effects of refrigeration, freezing and freeze-drying on micro-organisms. In: Andrew MHE, Russell AD (eds) The revival of injured microbes. Academic Press, London, pp 45–75Google Scholar
  19. Martinez-Urtaza J, Lozano-Leon A, Varela-Pet J, Trinanes J, Pazos Y, Garcia-Martin O (2008) Environmental determinants of the occurrence and distribution of Vibrio parahaemolyticus in the rias of Galicia, Spain. Appl Environ Microbiol 74:265–274CrossRefGoogle Scholar
  20. Miwa N, Nishio T, Arita Y, Kawamori F, Masuda T, Akiyama M (2003) Evaluation of MPN method combined with PCR procedure for detection and enumeration of Vibrio parahaemolyticus in seafood. Shokuhin Eiesigaku Zasshi 44:289–293CrossRefGoogle Scholar
  21. Navia MM, Capitano L, Ruiz J, Vargas M, Urassa H, Schellemberg D, Gascon J, Vila J (1999) Typing and characterization of mechanisms of resistance of Shigella spp. isolated from feces of children under 5 years of age from Ifakara, Tanzania. J Clin Microbiol 37:3113–3117PubMedPubMedCentralGoogle Scholar
  22. Nemoto J, Ikedo M, Kojima T, Momoda T, Konuma H, Hara-Kudo Y (2011) Development and evaluation of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Vibrio parahaemolyticus. J Food Prot 74:1462–1467CrossRefGoogle Scholar
  23. Newton A, Kendall M, Vugia DJ, Henao OL, Mahon BE (2012) Increasing rates of vibriosis in the United States, 1996–2010: review of surveillance data from 2 systems. Clin Infect Dis 54:S391–S395CrossRefGoogle Scholar
  24. Nigro OD, Steward GF (2015) Differential specificity of selective culture media for enumeration of pathogenic vibrios: advantages and limitations of multi-plating methods. J Microbiol Methods 111:24–30CrossRefGoogle Scholar
  25. Nordin N, Yusof NA, Abdullah J, Radu S, Hushiarian R (2017) A simple, portable, electrochemical biosensor to screen shellfish for Vibrio parahaemolyticus. Amb Express 7:41CrossRefGoogle Scholar
  26. Nordstrom JL, DePaola A (2003) Improved recovery of pathogenic Vibrio parahaemolyticus from oysters using colony hybridization following enrichment. J Microbiol Methods 52:273–277CrossRefGoogle Scholar
  27. Paydar M, Teh CSJ, Thong KL (2013) Prevalence and characterisation of potentially virulent Vibrio parahaemolyticus in seafood in Malaysia using conventional methods, PCR and REP-PCR. Food Control 32:13–18CrossRefGoogle Scholar
  28. Raghunath P, Acharya S, Bhanumathi A, Karunasagar I, Karunasagar I (2008) Detection and molecular characterization of Vibrio parahaemolyticus isolated from seafood harvested along the southwest coast of India. Food Microbiol 25:824–830CrossRefGoogle Scholar
  29. Ray B, Hawkins SM, Hackney CR (1978) Method for the detection of injured Vibrio parahaemolyticus in seafoods. Appl Environ Microbiol 35:1121–1127PubMedPubMedCentralGoogle Scholar
  30. Su Y-C, Liu C (2007) Vibrio parahaemolyticus: a concern of seafood safety. Food Microbiol 24 (6):549-558CrossRefGoogle Scholar
  31. Su YC, Duan J, Wu WH (2005) Selectivity and specificity of a chromogenic medium for detecting Vibrio parahaemolyticus. J Food Prot 68:1454–1456CrossRefGoogle Scholar
  32. Tada J, Ohashi T, Nishimura N, Shirasaki Y, Ozaki H, Fukushima S, Takano J, Nishibuchi M, Takeda Y (1992) Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction. Mol Cell Probes 6:477–487CrossRefGoogle Scholar
  33. Ward LN, Bej AK (2006) Detection of Vibrio parahaemolyticus in shellfish by use of multiplexed real-time PCR with TaqMan fluorescent probes. Appl Environ Microbiol 72:2031–2042CrossRefGoogle Scholar
  34. Wesche AM, Gurtler JB, Marks BP, Ryser ET (2009) Stress, sublethal injury, resuscitation, and virulence of bacterial foodborne pathogens. J Food Prot 72:1121–1138CrossRefGoogle Scholar
  35. Yamazaki W, Kumeda Y, Uemura R, Misawa N (2011) Evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Vibrio parahaemolyticus in naturally contaminated seafood samples. Food Microbiol 28:1238–1241CrossRefGoogle Scholar
  36. Yu WT, Jong KJ, Lin YR, Tsai SE, Tey YH, Wong HC (2013) Prevalence of Vibrio parahaemolyticus in oyster and clam culturing environments in Taiwan. Int J Food Microbiol 160:185–192CrossRefGoogle Scholar

Copyright information

© Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (BVL) 2019

Authors and Affiliations

  • Salima Sadeghi
    • 1
  • Kwai Lin Thong
    • 1
  • Lay Ching Chai
    • 1
    Email author
  1. 1.Institute of Biological Science, Faculty of ScienceUniversity of MalayaKuala LumpurMalaysia

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