Assessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide

• Published in: Annals of microbiology 2021-11, Vol.71 (1), p.1-7, Article 43
• Main Authors: Consonni, Michela, Grassi, Anna, Scuri, Stefania, Gori, Maria, Tanzi, Elisabetta, Tesauro, Marina
• Format: Article
• Language: English
• Subjects: Bacteria; Detection; Dyes; Environmental health; Laboratories; Legionella; Legionella pneumophila; Methods; Polymerase chain reaction; Rapid methods; Sanitation; Strains (organisms); Toxicity; Water
• ISSN: 1590-4261; 1869-2044
• DOI: 10.1186/s13213-021-01653-5

Analyses of 34 water samples from 13 healthcare structures revealed how culture method and quantitative PCR (qPCR) often differ in the detection of Legionella pneumophila (Lp). With these considerations in hand, culture method, PCR and Ethidium Monoazide Bromide (EMA) qPCR have all been compared in order to detect Lp in water samples, identify a method able to speed up the procedures, detect the "viable but not cultivable" bacteria (VBNC) and exclude non-viable bacteria using a commercial kit for extraction and amplification as well as modification of the protocol. Pure water samples artificially spiked with viable, non-viable and VBNC Lp ATCC 33152 were analyzed using a commercial kit for both qPCR and EMA-qPCR, while ISO 11731-2-2004 was used for culture method. Only 35% (12/34) of the environmental samples were positive in both culture and qPCR methods. With regard to EMA-qPCR, results showed the absence of dye toxicity on viable and VBNC strains and an incomplete effectiveness on the non-viable ones. In both viable and VBNC strains, a decrease of bacterial DNA amplification was recorded as a function of sample dilution but not of EMA concentration. Discrepancies between culture method and EMA-qPCR were observed and may be due to different causes such as membrane-dye interactions, presence of interfering compounds and the sensitivity of the kit used.