Rapid quantification of viable legionellae in water and biofilm using ethidium monoazide coupled with real‐time quantitative PCR

Abstract

Aims: To optimize ethidium monoazide (EMA) coupled with real‐time quantitative PCR (qPCR) and to evaluate its environmental applicability on quantifying viable legionellae in water and biofilm of cooling towers and hot water systems. Methods and Results: EMA (0·9–45·5 μg ml−1) and propidium monoazide (PMA, 0·9 and 2·3 μg ml−1) combined with qPCR (i.e. EMA‐qPCR and PMA‐qPCR, respectively) were applied to unheated and heated (70°C for 30 min) Legionella pneumophila to quantify viable cells, which was also simultaneously determined by BacLight Bacterial Viability kit with epifluorogenic microscopic enumeration (BacLight‐EM). The effects of nontarget microflora and sample matrix on the performance of EMA‐qPCR were also evaluated. In comparison with BacLight‐EM results, qPCR with EMA at 2·3 μg ml−1 was determined as the optimal EMA‐qPCR assay, which performed equally well as PMA‐qPCR for unheated Leg. pneumophila but better than PMA‐qPCR for heated Leg. pneumophila (P < 0·05). Moreover, qPCR with EMA at 2·3 μg ml−1 accurately quantified viable Leg. pneumophila, Legionella anisa and Legionella‐like amoebal pathogens 6 (LLAP 6) without interferences by heated legionellae, unheated nonlegionellae cells and cooling tower water matrix (P > 0·05). As for water and biofilm samples collected from cooling towers and hot water systems, the viable legionellae counts determined by EMA‐qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR. Conclusions: The qPCR with EMA at 2·3 μg ml−1 may accurately quantify viable legionellae (including fastidious LLAP 6) and Leg. pneumophila pretreated with superheating and is applicable for water and biofilm samples obtained from cooling towers and hot water systems. Significance and Impact of the Study: The EMA‐qPCR assay may be useful in environmental surveillance for viable legionellae and in evaluation of superheating efficacy against legionellae.

Specific Real-Time PCR for Simultaneous Detection and Identification of Legionella pneumophila Serogroup 1 in Water and Clinical Samples

“…This hidden risk would not have been identified by culture until favorable conditions of growth were restored (such as a dip in the temperature or the failure of the chlorination system) and L. pneumophila could proliferate to high numbers. The combination of qPCR with extraction methods that allow the exclusion of dead bacteria could improve the significance of a positive signal (10)(11)(12)17).…”

Detection of <i>Legionella</i> Species in Environmental Water by the Quantitative PCR Method in Combination with Ethidium Monoazide Treatment

“…EMA-treated DNA is not a target for amplification by PCR, and therefore viable Legionella are selectively detected by PCR when combined with EMA treatment. The effect of EMA treatment on Legionella detection by PCR has been published Chang et al, 2009;Chen et al, 2010;Delgado-Viscogliosi et al, 2009;Qin et al, 2012 . In this paper, we report a comparison of the detection of Legionella spp. by the plate culture method, quantitative PCR qPCR method and qPCR method in combination with EMA treatment EMA-qPCR .…”

Optimization of Propidium Monoazide Quantitative PCR for Evaluating Performances of Bioaerosol Samplers for Sampling AirborneStaphylococcus aureus

“…These compounds may also induce membrane damage. Another EMA dye combined with qPCR and heating has also been optimized for the detection of L. pneumophila, and this assay was subsequently used to monitor L. pneumophila in air and water (Chen and Chang 2010;Chang and Chou 2011b). However, PMA-qPCR was considered inadequate for identifying inactivated bacteria after a short exposure to ultraviolet radiation (Nocker et al 2007).…”