Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR

Abstract

One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50 μM PMA followed by 5 min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells.

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

“…Interestingly, PCR in clinical applications may have a higher PPV (than for environmental samples) despite the lower sensitivity with nonrespiratory specimens (379). Two potential remedies for the low PPV with samples from environmental sources include reverse transcription-PCR, amplifying labile RNA targets present in metabolically active bacteria, and the use of a cell-impermeant chemical, such as ethidium monoazide (EMA) or propidium monoazide (PMA), to inhibit PCR amplification from nonviable cells or extracellular nucleic acids (385)(386)(387)(388)(389)(390). Notably, neither alternative protocol alone will discriminate VBNC legionellae; however, this cell population still poses a potential human health risk (391)(392)(393)(394).…”

Molecular viability testing of viable but non‐culturable bacteria induced by antibiotic exposure

“…Alternatively, rapid and versatile nucleic acid‐based techniques that detect specific DNA and RNA genes have been used to identify and quantify bacteria in environmental samples (Malorny et al ., 2003; Yanez et al ., 2011). Despite the advantages of culture‐independent methods, the limitation of molecular assessment (especially for DNA‐based methods) is the possible overestimation of viable cell densities because DNA can persist for an extended period after cell death in environments (Rudi et al ., 2005).…”

First study in Qatar to reveal high Legionella counts in cooling towers

“…The detection of Legionella in water samples is conventionally performed using a selective culture method (15), however, interference of background microorganisms may lead to false low counts (16), not to mention the necessary expertise in interpreting growth (14). Additionally, culture methods do not detect viable but nonculturable bacteria, which may also represent a health hazard (14).…”

“…However, qRT-PCR has the disadvantage that it cannot differentiate between live and dead bacteria, therefore it may yield false high counts (15). In accordance with the universal standards for reporting Legionella counts (CFU/mL), qRT-PCR results were converted to CFU/ mL (14).…”