Using a real-time quantitative polymerase chain reaction (PCR) method for reliable enumeration of Aeromonas hydrophila in water samples

Aeromonas hydrophila is an opportunistic pathogen of human and animals with a multifactorial pathogenesis. Control of A. hydrophila numbers in water is recommended for water quality estimation. Consequently, we developed a SYBR Green method for rapid quantification of A. hydrophila, targeting three genes: 16SrRNA gene, the cytolytic enterotoxin gene (aerA) and the heat-stable cytotonic enterotoxin gene (ast). The sensitivity and the specificity of the method were tested using 34 positive and negative controls. The improved level of detection was established with serial dilution of genomic DNA of type strain CIP 7614T. Linear relation between Ct-value and bacterial cell concentration were obtained. The limit of detection found in this study corresponds to 10 cells of A. hydrophila. Finally, the method was tested on seven artificially contaminated waters and on 30 unknown water samples. A significant similarity was observed when comparing results of SYBR Green method (concerning specially the 16SrRNA gene) with microbiological enumeration. Only 13% of natural samples were contaminated with A. hydrophila. The qPCR protocol developed in this study allows quantifying of A. hydrophilain water samples with a good level of detectability.   Key words: Aeromonas hydrophila, water, real time PCR, SYBR Green.