Environmental Monitoring and Assessment

, Volume 186, Issue 10, pp 6935–6945 | Cite as

Occurrence of virulence genes among Vibrio cholerae and Vibrio parahaemolyticus strains from treated wastewaters

  • Sadok Khouadja
  • Elisabetta Suffredini
  • Besma Baccouche
  • Luciana Croci
  • Amina Bakhrouf


Pathogenic Vibrio species are an important cause of foodborne illnesses. The aim of this study was to describe the occurrence of potentially pathogenic Vibrio species in the final effluents of a wastewater treatment plant and the risk that they may pose to public health. During the 1-year monitoring, a total of 43 Vibrio strains were isolated: 23 Vibrio alginolyticus, 1 Vibrio cholerae, 4 Vibrio vulnificus, and 15 Vibrio parahaemolyticus. The PCR investigation of V. parahaemolyticus and V. cholerae virulence genes (tlh, trh, tdh, toxR, toxS, toxRS, toxT, zot, ctxAB, tcp, ace, vpi, nanH) revealed the presence of some of these genes in a significant number of strains. Intraspecies variability and genetic relationships among the environmental isolates were analyzed by random amplified polymorphic DNA-PCR (RAPD-PCR). We report the results of the first isolation and characterization of an environmental V. cholerae non-O1 non-O139 and of a toxigenic V. parahaemolyticus strain in Tunisia. We suggest that non-pathogenic Vibrio might represent a marine reservoir of virulence genes that can be transmitted between strains by horizontal transfer.


Vibrio parahaemolyticus Vibrio cholerae Virulence genes PCR 



The authors thank Pr. Mauro M Colombo for assistance with reference strains.


  1. Ahmed, A. M., Nakagawa, T., Arakawa, E., Ramamurthy, T., Shinoda, S., & Shimamoto, T. (2004). New aminoglycoside acetyltransferase gene, aac(3)-Id, in a class 1 integron from a multiresistant strain of Vibrio fluvialis isolated from an infant aged 6 months. Journal of Antimicrobial Chemotherapy, 53(6), 947–951.CrossRefGoogle Scholar
  2. Alsina, M., & Blanch, A. R. (1994). A set of keys for biochemical identification of environmental Vibrio species. Journal of Applied Bacteriology, 76, 79–85.CrossRefGoogle Scholar
  3. Bej, A. K., Patterson, D. P., Brasher, C. W., Vickery, M. C., Jones, D. D., & Kaysner, C. A. (1999). Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh. Journal of Microbiological Methods, 36(3), 215–225.CrossRefGoogle Scholar
  4. Brauns, L. A., Hudson, M. C., & Oliver, J. D. (1991). Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells. Applied and Environmental Microbiology, 57(9), 2651–2655.Google Scholar
  5. Ceccarelli, D., Salvia, A. M., Sami, J., Cappuccinelli, P., & Colombo, M. M. (2006). New cluster of plasmid-located class 1 integrons in Vibrio cholerae O1 and a dfrA15 cassette-containing integron in Vibrio parahaemolyticus isolated in Angola. Antimicrobial Agents and Chemotherapy, 50(7), 2493–2499. doi: 10.1128/AAC.01310-05.CrossRefGoogle Scholar
  6. Chen, Y., Johnson, J. A., Pusch, G. D., Morris, J. G., Jr., & Stine, O. C. (2007). The genome of non-O1 Vibrio cholerae NRT36S demonstrates the presence of pathogenic mechanisms that are distinct from those of O1 Vibrio cholerae. Infection and Immunity, 75(5), 2645–2647.CrossRefGoogle Scholar
  7. Colombo, M. M., Mastrandrea, S., Santona, A., de Andrade, A. P., Uzzau, S., Rubino, S., & Cappuccinelli, P. (1994). Distribution of the ace, zot, and ctxA Foxin genes in clinical and environmental Vibrio cholerae. Journal of Infectious Diseases, 170(3), 750–751.CrossRefGoogle Scholar
  8. Dawyndt, P., Vancanneyt, M., Meyer, H. D., & Swings, J. (2005). Knowledge accumulation and resolution of data inconsistencies during the integration of microbial information sources. IEEE Transactions on Knowledge and Data Engineering, 17, 1111–1126.CrossRefGoogle Scholar
  9. Di Pinto, A., Ciccarese, G., Tantillo, G., Catalano, D., & Forte, V. T. (2005). A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus. Journal of Food Protection, 68(1), 150–153.Google Scholar
  10. Farina, C., Gnecchi, F., Luzzi, I., & Vailati, F. (2000). Vibrio cholerae O2 as a cause of a skin lesion in a tourist returning from Tunisia. Journal of Travel Medicine, 7(2), 92–94.CrossRefGoogle Scholar
  11. Folgosa, E., Mastrandrea, S., Cappuccinelli, P., Uzzau, S., Rappelli, P., Brian, M. J., & Colombo, M. M. (2001). Molecular identification of pathogenicity genes and ERIC types in Vibrio cholerae O1 epidemic strains from Mozambique. Epidemiology and Infection, 127(1), 17–25.CrossRefGoogle Scholar
  12. Gil, A. I., Louis, V. R., Rivera, I. N., Lipp, E., Huq, A., Lanata, C. F., Taylor, D. N., Russek-Cohen, E., Choopun, N., Sack, R. B., & Colwell, R. R. (2004). Occurrence and distribution of Vibrio cholerae in the coastal environment of Peru. Environmental Microbiology, 6(7), 699–706.CrossRefGoogle Scholar
  13. Iyer, L., Vadivelu, J., & Puthucheary, S. D. (2000). Detection of virulence associated genes, haemolysin and protease amongst Vibrio cholerae isolated in Malaysia. Epidemiology and Infection, 125(1), 27–34.CrossRefGoogle Scholar
  14. Jermyn, W. S., & Boyd, E. F. (2002). Characterization of a novel Vibrio pathogenicity island (VPI-2) encoding neuraminidase (nanH) among toxigenic Vibrio cholerae isolates. Microbiology, 148(Pt 11), 3681–3693.Google Scholar
  15. Jermyn, W. S., & Boyd, E. F. (2005). Molecular evolution of Vibrio pathogenicity island-2 (VPI-2): mosaic structure among Vibrio cholerae and Vibrio mimicus natural isolates. Microbiology, 151(Pt 1), 311–322.CrossRefGoogle Scholar
  16. Khouadja, S., Snoussi, M., Saidi, N., & Bakhrouf, A. (2012). Phenotypic characterization and RAPD fingerprinting of Vibrio parahaemolyticus and Vibrio alginolyticus isolated during Tunisian fish farm outbreaks. Folia microbiologica, 58(3). doi: 10.1007/s12223-012-0174-x.
  17. Kim, Y. B., Okuda, J., Matsumoto, C., Takahashi, N., Hashimoto, S., & Nishibuchi, M. (1999). Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxR gene. Journal of Clinical Microbiology, 37(4), 1173–1177.Google Scholar
  18. Koch, W. H., Payne, W. L., Wentz, B. A., & Cebula, T. A. (1993). Rapid polymerase chain reaction method for detection of Vibrio cholerae in foods. Applied and Environmental Microbiology, 59(2), 556–560.Google Scholar
  19. Nandi, B., Nandy, R. K., Mukhopadhyay, S., Nair, G. B., Shimada, T., & Ghose, A. C. (2000). Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW. Journal of Clinical Microbiology, 38(11), 4145–4151.Google Scholar
  20. Nishibuchi, M., Janda, J. M., & Ezaki, T. (1996). The thermostable direct hemolysin gene (tdh) of Vibrio hollisae is dissimilar in prevalence to and phylogenetically distant from the tdh genes of other vibrios: implications in the horizontal transfer of the tdh gene. Microbiology and Immunology, 40(1), 59–65.CrossRefGoogle Scholar
  21. Okoh, A. I., & Igbinosa, E. O. (2010). Antibiotic susceptibility profiles of some Vibrio strains isolated from wastewater final effluents in a rural community of the Eastern Cape Province of South Africa. BMC Microbiology, 10, 143.CrossRefGoogle Scholar
  22. Ottaviani, D., Santarelli, S., Bacchiocchi, S., Masini, L., Ghittino, C., & Bacchiocchi, I. (2005). Presence of pathogenic Vibrio parahaemolyticus strains in mussels from the Adriatic Sea, Italy. Food Microbiology, 22, 585–590.CrossRefGoogle Scholar
  23. Quindos, G., Salesa, R., Carrillo-Munoz, A. J., Lipperheide, V., Jaudenes, L., San Millan, R., Torres-Rodriguez, J. M., & Ponton, J. (1994). Multicenter evaluation of ATB fungus: a standardized micromethod for yeast susceptibility testing. Chemotherapy, 40(4), 245–251.CrossRefGoogle Scholar
  24. Reidl, J., & Klose, K. E. (2002). Vibrio cholerae and cholera: out of the water and into the host. FEMS Microbiology Review, 26(2), 125–139.CrossRefGoogle Scholar
  25. Ripabelli, G., Sammarco, M. L., McLauchlin, J., & Fanelli, I. (2003). Molecular characterisation and antimicrobial resistance of Vibrio vulnificus and Vibrio alginolyticus isolated from mussels (Mytilus galloprovincialis). Systematic and Applied Microbiology, 26(1), 119–126.CrossRefGoogle Scholar
  26. Sechi, L. A., Dupre, I., Deriu, A., Fadda, G., & Zanetti, S. (2000). Distribution of Vibrio cholerae virulence genes among different Vibrio species isolated in Sardinia, Italy. Journal of Applied Microbiology, 88(3), 475–481.CrossRefGoogle Scholar
  27. Thompson, C. C., Vicente, A. C., Souza, R. C., Vasconcelos, A. T., Vesth, T., Alves, N., Jr., Ussery, D. W., Iida, T., & Thompson, F. L. (2009). Genomic taxonomy of Vibrios. BMC Evolutionary Biology, 9, 258.CrossRefGoogle Scholar
  28. Thompson, F. L., Iida, T., & Swings, J. (2004). Biodiversity of vibrios. Microbiology and Molecular Biology Reviews, 68(3), 403–431. table of contents.CrossRefGoogle Scholar
  29. WHO. (2002). Cholera, 2001. Weekly Epidemiological Record, 77, 257–268.Google Scholar
  30. Zanetti, S., Deriu, A., Volterra, L., Falchi, M. P., Molicotti, P., Fadda, G., & Sechi, L. (2000). Virulence factors in Vibrio alginolyticus strains isolated from aquatic environments. Annali d’Igiene, 12(6), 487–491.Google Scholar
  31. Zhang, X. H., & Austin, B. (2005). Haemolysins in Vibrio species. Journal of Applied Microbiology, 98(5), 1011–1019.CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2014

Authors and Affiliations

  • Sadok Khouadja
    • 1
  • Elisabetta Suffredini
    • 2
  • Besma Baccouche
    • 1
  • Luciana Croci
    • 2
  • Amina Bakhrouf
    • 1
  1. 1.Laboratoire d’Analyse, Traitement et Valorisation des Polluants de l’Environnement et des Produits, Département de Microbiologie, Faculté de PharmacieMonastirTunisia
  2. 2.Reparto Adempimenti Comunitari e Sanità Pubblica, Dipartimento di Sanità Pubblica Veterinaria e Sicurezza AlimentareIstituto Superiore di SanitàRomeItaly

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