A multicenter study of viable PCR using propidium monoazide to detect Legionella in water samples

• DOI: 10.1016/j.diagmicrobio.2016.04.009
• Publication Date: 2016-05-01T06:00:28Z
• Authors: Maria Scaturro; Stefano Fontana; Italo Dell’eva; Fabrizia Helfer; Michele Marchio; Maria Vittoria St...; Mario Cavallaro; Marilena Miglietta; Maria Teresa Mont...; Osvalda De Giglio; Teresa Cuna; Leonarda Chetti; Maria Antonietta ...; Michela Carlotti; Mariagabriella Vi...; Alberta Stenico; Elisa Romanin; Emma Bonanni; Claudio Ottaviano; Laura Franzin; Claudio Avanzini; Valerio Demarie; Marta Corbella; Patrizia Cambieri; Piero Marone; Maria Cristina Rota; Antonino Bella; Maria Luisa Ricci
• Keywords: 0301 basic medicine; Azides; Bacteriological Techniques; Microbial Viability; Culture; Temperature; Legionella; Real-Time Polymerase Chain Reaction; Viable PCR; 6. Clean water; Propidium monoazide; Quantitative PCR; 03 medical and health sciences; Humans; Enzyme Inhibitors; Water Microbiology; Propidium

Legionella quantification in environmental samples is overestimated by qPCR. Combination with a viable dye, such as Propidium monoazide (PMA), could make qPCR (named then vPCR) very reliable. In this multicentre study 717 artificial water samples, spiked with fixed concentrations of Legionella and interfering bacterial flora, were analysed by qPCR, vPCR and culture and data were compared by statistical analysis. A heat-treatment at 55 °C for 10 minutes was also performed to obtain viable and not-viable bacteria. When data of vPCR were compared with those of culture and qPCR, statistical analysis showed significant differences (P < 0.001). However, although the heat-treatment caused an abatement of CFU/mL ≤1 to 1 log10 unit, the comparison between untreated and heat-treated samples analysed by vPCR highlighted non-significant differences (P > 0.05). Overall this study provided a good experimental reproducibility of vPCR but also highlighted limits of PMA in the discriminating capability of dead and live bacteria, making vPCR not completely reliable.