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

Abstract

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 μg 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 (aerA-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.