Journal of Microbiology

The Microbiological Society of Korea (한국미생물학회)
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Rapid Detection of Virulence Factors of Aeromonas Isolated from a Trout Farm by Hexaplex-PCR

  • Nam, In-Young (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Joh, Ki-Seong (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
  • Published : 2007.08.30
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Abstract

The detection of virulence factors of Aeromonas is a key component in determining potential pathogenicity because these factors act multifunctionally and multifactorially. In this study water samples were collected from a trout farm on a seasonal basis, and diseased fish and Aeromonas species were isolated and identified. For rapid detection of six virulence factors of isolated Aeromonas, a hexaplex-polymerase chain reaction (hexaplex-PCR) assay was used. The detected virulence factors include aerolysin (aer), GCAT (gcat), serine protease (ser), nuclease (nuc) lipase (lip) and lateral flagella (laf). The dominant strain found in our isolates was Aeromonas sobria, and the dominant virulence factors were aer and nuc for all seasons. We confirmed that A. sobria and two of the virulence genes (aer and nuc) are related. We proposed a method by which one can identify the major strains of Aeromonas: A. hydrophila, A. sobria, A. caviae, and A. veronii, using hexaplex-PCR.

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References

  1. Abrami, L., M. Firaz, E. Decroly, N. Seidah, J. Jean, and G. Thomas.1998. The pore forming toxin aerolysin is activated by furin. J. Biol. Chem. 271, 32656-32661
  2. Abrami, L., M. Fivaz, and F.G. van der Goot. 2000. Adventures of a pore-forming toxin at the target cell surface. Trends Microbiol. 8, 168-172 https://doi.org/10.1016/S0966-842X(00)01722-4
  3. Anguita, J., L.B. Rodrıguez-Aparicio, and G. Naharro. 1993. Purification, gene cloning, amino acid sequence analysis, and expression of an extracellular lipase from an Aeromonas hydrophila human isolate. Appl. Environ. Microbiol. 59, 2411-2417
  4. Borrell, N., M.J. Figueras, and J. Guarro. 1998. Phenotypic identification of Aeromonas genomospecies from clinical and environmental sources. Can. J. Microbiol. 44, 7-12
  5. Borrell, N., S.G. Acinas, M.J. Figueras, and A.J. Martinez-Murcia. 1997. Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes. J. Clin. Microbiol. 35, 1671-1674
  6. Buckley, J.T. and S.P. Howard. 1999. The cytotoxic enterotoxin Aeromonas hydrophila is aerolysin. Infect. Immun. 67, 466-467
  7. Cascon, A., J. Fregenda, M. Allen, J. Yugueros, A. Temprano, and C. Hernanz. 2000. Cloning, characterization and insertional inactivation of a major extracellular serine protease gene with elastolytic activity for Aeromonas hydrophila. J. Fish. Dis. 23, 49-59 https://doi.org/10.1046/j.1365-2761.2000.00206.x
  8. Chacon, M.R., M.J. Figuras, G. Castro-Escarpulli, L. Soler, and J. Guarro. 2003. Distribution of virulence genes in clinical and environmental isolates of Aeromonas spp. Antonie van Leeuwenhoek 84, 269-278 https://doi.org/10.1023/A:1026042125243
  9. Chang, M.C., S.Y. Chang, S.L. Chen, and S.M. Chuang. 1992. Cloning and expression in Escherichia coli of the gene encoding an extracellular deoxyribonuclease (DNase) from Aeromonas hydrophila. Gene 94, 175-180
  10. Chuang, Y.C., S.F. Chiou, J.H. Su, M.L. Wu, and M.C. Chang. 1997. Molecular analysis and expression of the extracellular lipase of Aeromonas hydrophila MCC-2. Microbiology 143, 803-812 https://doi.org/10.1099/00221287-143-3-803
  11. Dodd, H. and J.M. Pemberton. 1996. Cloning, sequencing and characterization of the nucH gene encoding an extracellular nuclease from Aeromonas hydrophila JMP636. J. Bacteriol. 178, 3926-3933 https://doi.org/10.1128/jb.178.13.3926-3933.1996
  12. Figueras, M.J., L. Soler, M.R. Chacon, J. Guarro, and A.J. Martinez-Murcia. 2000. Extended method for discrimination of Aeromonas spp. by 16S rDNA RFLP analysis. Int. J. Syst. Evol. Microbiol. 50, 2069-2073 https://doi.org/10.1099/00207713-50-6-2069
  13. Fivaz, M., L. Abrami, Y. Tsitrin, and F.G. van der Goot. 2001. Not as simple as just punching a hole. Trends Microbiol. Toxicon. 39, 1637-1645
  14. Gavin, R., S. Merino, M. Altarriba, R. Canals, J.G. Shaw, and J.M. Tomas. 2003. Lateral flagella are required for increased cell adherence, invasion and biofilm formation by Aeromonas spp. FEMS Microbiol. Lett. 224, 77-83 https://doi.org/10.1016/S0378-1097(03)00418-X
  15. Heuzenroeder, M.W., C.Y. Wong, and R.L. Flower. 1999. Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.: correlation with virulence in a suckling mouse model. FEMS Microbiol. Lett. 174, 131-136 https://doi.org/10.1111/j.1574-6968.1999.tb13559.x
  16. Hirono, I. and T. Aoki. 1993. Cloning and characterization of three hemolysin genes from Aeromonas salmonicida. Microb. Pathog. 15, 269-282 https://doi.org/10.1006/mpat.1993.1077
  17. Hirono, I., T. Aoki, T. Asao, and S. Kozaki. 1992. Nucleotide sequence and characterisation of hemolysin gene from Aeromonas hydrophila and Aeromonas sobria. Microb. Pathog. 13, 433-446 https://doi.org/10.1016/0882-4010(92)90011-C
  18. Husslein, V., T. Chakraborty, A. Carnahan, and S.W. Joseph. 1992. Nucleotide sequence and transcriptional analysis of the aerC, aerA region of Aeromonas sobria encoding aerolysin and its regulator region. Clin. Infect. Dis. 14, 1061-1068 https://doi.org/10.1093/clinids/14.5.1061
  19. Kingombe, C.I.B., G. Huys, M. Tonolla, M.J. Alvert, J. Swings, R. Peduzzi, and T. Jemmi. 1999. PCR detection, characterization, and distribution of virulence genes in Aeromonas spp. Appl. Environ. Microbiol. 65, 5293-5302
  20. Kirov, S.M., M. Castrisios, and J.G. Sha. 2004. Aeromonas flagella (polar and lateral) are enterocyte adhesions that contribute to biofilm formation on surfaces. Infect. Immun. 72, 1939-1945 https://doi.org/10.1128/IAI.72.4.1939-1945.2004
  21. Krovacec, K., V. Pasquale, S.B. Baloda, V. Soprano, M. Conte, and S. Dumontet. 1994. Comparison of putative virulence factors in Aeromonas hydrophila strains isolated from the marine environment and human diarrheal cases in Southern Italy. Appl. Environ. Microbiol. 60, 1379-1382
  22. Lafont, F., L. Abrami, and F.G. van der Goot. 2004. Bacterial subversion of lipid rafts. Cur. Oprin. Microbiol. 7, 4-10 https://doi.org/10.1016/j.mib.2003.12.007
  23. Lee, C., J.C. Cho, S.H. Lee, D.G. Lee, and S.J. Kim. 2002. Distribution of Aeromonas spp. as identified by 16S rDNA restriction fragment length polymorphism analysis in a trout farm. J. Appl. Microbiol. 93, 976-985 https://doi.org/10.1046/j.1365-2672.2002.01775.x
  24. Martins, L.M., R.F. Marquez, T. Yano, 2002. Incidence of toxic Aeromonas isolated from food and human infection. FEMS Immunol. Med. Microbiol. 32, 237-242 https://doi.org/10.1111/j.1574-695X.2002.tb00559.x
  25. Nam, I.-Y., H. Myung, and K. Joh. 2004. Molecular cloning, purification, and characterization of an extracellular nuclease from Aeromonas hydrophila ATCC 14715. J. Microbiol. Biotechnol. 14, 178-181
  26. Nerland, A.H. 1996. The nucleotide sequence of the gene encoding GCAT from Aeromonas salmonicida ssp. salmonicida. J. Fish Dis. 19, 145-150 https://doi.org/10.1111/j.1365-2761.1996.tb00693.x
  27. Pemberton, J.M., S.P. Kidd, and R. Schmidt. 1997. Secreted enzymes of Aeromonas. FEMS Microbiol. Lett. 152, 1-10 https://doi.org/10.1111/j.1574-6968.1997.tb10401.x
  28. Rahman, M., P. Colque-Navaro, I. Kuhn, G. Huys, J. Swings, and R. Mollby. 2002. Identification and characterization of pathogenic Aeromonas veronii biovar sobria associated with epizootic ulcerative syndrome in fish in Bangladesh. Appl. Environ. Microbiol. 68, 650-655 https://doi.org/10.1128/AEM.68.2.650-655.2002
  29. Sen, M. and M. Rodgers. 2004. Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification. J. Appl. Microbiol. 97, 1077-1086 https://doi.org/10.1111/j.1365-2672.2004.02398.x
  30. Sugita, H., K. Tanaka, M. Yoshinami, and Y. Deguchi. 1995. Distribution of Aeromonas species in the intestinal tracts of river fish. Appl. Environ. Microbiol. 61, 4128-4130
  31. Thornton, J., S.P. Howard, and J.T. Buckley. 1988. Molecular cloning of a glycerophospholipid cholesterol acyltransferase from Aeromonas: Sequence homologies with lecitin cholesterol acyltransferase and other lipases. Biochim. Biophys. Acta. 959, 153-159 https://doi.org/10.1016/0005-2760(88)90026-4
  32. Wahli, T., S.E. Burr, D. Pugovkin, O. Mueller, and J. Frey. 2005. Aeromonas sobria, a causative agent of disease in farmed perch, Perca fluviatilis L. Fish Dis. 28, 141-150 https://doi.org/10.1111/j.1365-2761.2005.00608.x
  33. Wang, G., C.G. Clark, C. Liu, D. Pucknell, C.K. Munro, T.M.A.C. Kruk, R. Caldeira, D.L. Woodward, and R.G. Rodgers. 2003. Detection and characterization of the aerolysin genes in Aeromonas hydrophila and Aeromonas sobria by multiplex PCR. J. Clin. Microbiol. 41, 1048-1054 https://doi.org/10.1128/JCM.41.3.1048-1054.2003
  34. Whitby, P.W., M. Landon, and G. Coleman. 1992. The cloning and nucleotide sequence of the serine protease gene (aspA) of Aeromonas salmonicida ssp. salmonicida. FEMS Microbiol. Lett. 99, 65-72 https://doi.org/10.1111/j.1574-6968.1992.tb05543.x
  35. Yu, H.B., Y.L. Zang, Y.L. Lau, F. Yao, S. Vilches, S. Merino, J.M. Tomas, S.P. Howard, and K.Y. Leung. 2005. Identification and characterization of putative virulence genes and gene clusters in Aeromonas hydrophila PPD134/91. Appl. Environ. Microbiol. 71, 4469-4477 https://doi.org/10.1128/AEM.71.8.4469-4477.2005