Propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay for rapid detection of viable and viable but non-culturable (VBNC) Pseudomonas aeruginosa in swimming pools.

Abstract

Lack of culturability in the viable but non-culturable (VBNC) bacteria and the ability to regain infectivity in favourable conditions is one of the new challenges of public health providers for Pseudomonas aeruginosa monitoring in environmental samples. Propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) is one of the promising methods for timely detection of VBNC pathogens in environmental samples. We developed and used a method for the first time to detection of VBNC P. aeruginosa in swimming pool water samples using a membrane filter (MF). Moreover, the dominant model of the distribution of colonies on the MF and the effect of the culture medium and MF type on colony recovery by MF were evaluated. Swimming pool samples were subjected to conventional culture-based, qPCR and PMA-qPCR methods and the results were compared for the presence of VBNC P. aeruginosa in the samples. The positivity rate was 21% and 75% for P. aeruginosa in water samples as confirmed by standard culture-based and qPCR methods, respectively. Furthermore, of 24 samples, 9 (37.5%) were positive for VBNC P. aeruginosa. The developed qPCR/PMA-qPCR assay can detect the VBNC bacteria directly from aquatic samples and may result in better monitoring of recreational waters.