PCR detection of potentially pathogenic aeromonads in raw and cold-smoked freshwater fish

Aims: Development of a PCR assay for detection of aeromonads carrying the hlyA and/or aerA genes in fish. Methods and Results: The protocol involves an overnight selective enrichment step in tryptic soy broth yeast extract containing 10 mug ml(-1) of ampicillin followed by extraction of DNA and PCR amplification of two haemolysin genes that contribute to the virulence of Aer. hydrophila . This procedure can detect initial populations of 1-10 cfu g(-1) within 24 h in artificially contaminated samples. In naturally contaminated fish, both genes were detected in 13 out of 14 fresh fish lots (aeromonads levels between <1 and 5.42 log cfu g(-1)) and in 4 out of 16 lots of vacuum-packed cold-smoked fish (aeromonads levels between <1 and 3.37 log cfu g(-1)). Before enrichment, dominant species were Aer. hydrophila HG1 (aerA(+) hlyA(+)), Aer. bestiarum HG2 (aerA(+) hlyA(+)) and Aer. caviae HG4 (aer A(-) hlyA(-)). After enrichment, Aer. hydrophila HG1 (aerA(+) hlyA(+)) was dominant. Conclusions: Fresh fish and even smoked fish carry hlyA(+) and/or aerA(+) aeromonads that can be detected by PCR within 24 h. Significance and Impact of the Study: The PCR assay described offers considerable potential as a rapid method with specificity, sensitivity and simplicity.