Document Type : Original Research

Authors

1 epartment of Fishery and Environment, Faculty of Natural Resources, University of Tehran, Karaj, Iran

2 Department of Fishery and Environment, Faculty of Natural Resources, University of Tehran, Karaj, Iran

3 Department of Pathology, Tehran University of Medical Sciences, Tehran, Iran

Abstract

 
Objectives: The presence of E.coli in fish intended for human consumption may constitute a potential danger, not only in causing disease, but also because of the possible transfer of antibiotic resistance from aquatic bacteria to those infecting humans. The objective of this study was to develop an improved PCR method based on species – specific 16 S rRNA gene primers (FES, RES) for detection of E. coli from agar plates and fish tissues.                         
Materials and Methods: In this study, For the rapid detection of E .coli from fish a set of primers (FES, RES), targeting 16S rRNA gene sequences of the specific microorganism was  designed, and fifty two rainbow trout were obtained from Karaj fish farm. Then  1mL of bacterial concentration of 106CFU/ml was injected into intraperitoneal cavity. Samples were collected from liver and kidney after 48h injection. The PCR reaction conditions were optimized to permit detection of organism from agar plates and fish tissue in a day.
Results: All tissue samples were positive for microbiological and PCR identification. DNA was successfully extracted by a boiled – extraction method or by phenol – chloroform – isoamyl alcohol. The BLAST analysis from sequencing of 4 amplicons randomly selected showed similar results, with the match being E .coli with a 100% similarity (not shown here).
Conclusion: It is concluded that this method is fast, specific and sensitive to detect E.coli in infected and asymptomatic animals, fish product, and may have a positive impact on public and environmental health.
 

Keywords

  1. Adams  A., Thompson  KD. Bio technology offers revolution to fish health management. TRENDS in biotechnology 2006; 24: 201-205
  2. Almedia A, Cunha  A, Gomes N, Alves E, Costa L, Faustino M. Phage Therapy and photodynamic Therapy: Low Environmental impact Approaches to Inactivate Microorganisms in Fish Farming plants. Mar Drugs2009;7(3):263-313.
  3. Altinok I, Capkin E, Kayis S. Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens. Vet Microbiol 2008 Oct 15;131(3-4):332-8.
  4. Aoki T., Hirono I. 1995.Detection of the fish pathogenic bacteria edwardsiella trada by polymeras chain reaction in proceedings of the international symposium on biotechnology applications in aquaculture, pp, 135-146, Asian fisheries society special publication No.10, Quezon city Philippines
  5. Argenton D, De Mas S, Malocco C, Dalla valle L, Ogiorgetti G, Columbo L. Use of random DNA amplification to generate specific molecular probes for hybridization test and PCR, based diagnosis of yersinia ruckeri. Disease Aquat Org 1996;24:121-7.
  6. Arias CR, Garay E, Aznar R. Nested PCR method for rapid and sensitive detection of Vibrio vulnificus in fish, sediments, and water. Appl Environ Microbiol 1995;61(9):3476-8
  7. Bader JA, Shoemaker CA, Klesius PH. Rapid detection of columnaris disease in channel catfish (Ictalurus punctatus) with a new species-specific 16-S rRNA gene-based PCR primer for Flavobacterium columnare. J Microbiol Methods 2003;52(2):209-20
  8. Blackwood  CM. Microbiological quality of fishery products, role and environment Canada of fisheries inspection Branch Canadian Institute of Science and Technology Journal 1978.;1: A42-A49.
  9. Blanco M, Gibello A, Fernandez-Garayzabal J.F. Influence of fish health management: Bases, Procedures and economic implications. Cahiers Options Méditerranéennes 2000;51:45-9.
  10. Brown L., Iwama G.K., Eve;yn T.P.T, Nelson, W.S, Levine R.P. Use of the polymerase chain reaction(PCR) to detect DNA from Reni-bacterium salmoninarum within individual salmonid eggs, Diseases of aquatic organisms1994; 18: 165-171.
  11. Buras N, Duek L, Niv S. Reactions of fish to microorganisms in wastewater. Appl Environ Microbiol 1985;50(4):989-95.
  12. Cascon A, Anguita J, Hernanz C, Sanchez M, Fernandez M, Naharro G. Identification of Aeromonas hydrophila hybridization group 1 by PCR assays. Appl Environ Microbiol 1996;62(4):1167-70.
  13. Darwish AM, Ismaiel AA, Newton JC, Tang J. Identification of Flavobacterium columnare by a species-specific polymerase chain reaction and renaming of ATCC43622 strain to Flavobacterium johnsoniae. Mol Cell Probes 2004;18(6):421-7
  14. Edward J. Mechanisms of resistance development in aquatic microorganism in chemotherapy in aquaculture. 2 ed. New York: Blackwell; 2010
  15. Fapohunda A, Macmillian KW, Marshall DL, waites WM. Growth of sleeted cross contaminating bacterial pathogens on beef and fish at 15oC and 35oC. J food protec1994;57:337-
  16. Fattahi F. Rapid detection of Aeromonas hydrophila  Haemorrahgic  septicemia in Rainbow trout (Oncorhynchus  mykiss) using duplex-PCR Technique. Iran: University of Tehran; 2011.
  17. Fattal  B, Dotan A, Tchorch Y. Rates of experimental microbiological contamination of fish exposed to polluted water. water res. 1992; 7: 1621-1627.
  18. Forbes B, Sahm D, Weisfeld A. Induction of tumor-specific immunity by multi-epitope rat HER2/neu-derived peptides encapsulated in LPD Nanoparticles. 10 ed. New York: Mosby; 1998.
  19. French GL, Ling J, Chow KL, Mark KK. Occurrence of multiple antibiotic resistance and R-plasmids in gram-negative bacteria isolated from faecally contaminated fresh-water streams in Hong Kong. Epidemiol Infect 1987;98(3):285-99
  20. Geldreich EE, Clarke NA. Bacterial pollution indicators in the intestinal tract of freshwater fish. Appl Microbiol 1966;14(3):429-37.
  21. Gustafson CE, Thomas CJ, Trust TJ. Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene. Appl Environ Microbiol 1992;58(12):3816-25.
  22. Guzman MC, Bistoni ML, Tamagnini LM, Gonzalez RD. Recovery of Escherichia coli in fresh water fish, Jenynsia multidentata and Bryconamericus iheringi. Water Res 2004 May;38(9):2367-73.
  23. Hiney M, Dawson MT, Heery DM, Smith PR, Gannon F, Powell R. DNA probe for Aeromonas salmonicida. Appl Environ Microbiol 1992;58(3):1039-42.
  24. Janssen WA. Fish as potential vectors of human bacterial diseases of fishes and shellfishes. Am Fish Soc Spec Publ 1970;5: 90-284.
  25. Martinez-Picado J, Alsina M, Blanch AR, Cerda M, Jofre J. Species-Specific Detection of Vibrio anguillarum in Marine Aquaculture Environments by Selective Culture and DNA Hybridization. Appl Environ Microbiol 1996;62(2):443-9.
  26. Miranda CD, Zemelman R. Antibiotic resistant bacteria in fish from the Concepcion Bay, Chile. Mar Pollut Bull 2001;42(11):1096-102.
  27. Mukherjee S, Chakraborty R. Conjugation potential and class 1 integron carriage of resident plasmids in river water copiotrophs. Acta microbiological immunologica hungarica 2007; 54: 379- 397.
  28. Olayemi A.B, Dedayo O, Ojo A. Microbial flora of six fresh water fish species from Asa river, Nigeria. Revista de biologia tropical 1991; 39: 165- 167.
  29. Ouattara NK, Passerat J, Servais P. Faecal contamination of water and sediment in the rivers of the Scheldt drainage network. Environ Monit Assess 2011 Dec;183(1-4):243-57.
  30. Pal D, Dasgupta Ck. Interaction of some city sewage bacteria with Indian major carp cirrhinus migala. aquat anim health 1991; 3: 124-129.
  31. Sabat G, Rose P, Hickey W, Harkin J.M. Selective and Sensitive Method for PCR Amplification of Escherichia coli 16Sr RNA Genes in soil. Applied  and Environmental Microbiology 2000; 66: 844-849.
  32. Schmidt C.Q. 2002. Antibiotic resistance in livestock: more at stake than steak, environmental health perspectives. 11, A39-A402.
  33. Sqrum H, Labee-Lund T.M. Antibiotic resistance in food related bacteria a result of interfering with global web of bacterial genetics. International journal of food microbiology 2000; 78: 43- 56.
  34. Strauss M, Blumenthal UJ, 1990, Human waste use in agriculture and aquaculture e-utilization practice and health perspectives. International reference center for waste disposal (IRCWD), report 9/1990 Duebendorf, Switzerland, 6-34.
  35. Tang Y.W, Stratton C. Advanced techniques in diagnostic microbiology. USA, Springer; 2006. 1-551.
  36. Toranzo A.E, combarro P, Lemos M.L, Barja J.L. Plasmid coding for transferable drug resistance in bacteria isolated from cultured rainbow trout, Applied and environmental microbiology 1984; 48: 872- 877
  37. Wegener H.C. The consequences for food safety of the use of fluoroquinolons in food animals, New England Journal of Medicine 1999; 340: 1581- 1582.