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

Purpose 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. Methods 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. Results 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. Conclusions 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. Study significance and impact In the presence of one or more suspected cases of nosocomial legionellosis, the application of a rapid molecular method able to identify only the viable and VBNC Lp would be useful in order to quickly identify the source of infection and to intervene with sanitation treatments. However, seeing that in our experience EMA pretreatment on the filter membrane did not come up with the expected results, it would be necessary to proceed with other experiments and/or different dyes. Graphical Abstract

Assessment of Viability of Legionella Pneumophila in Environmental Samples: On Filter Application of Ethidium Monoazide Bromide

Purpose: Culture method, Real-Time PCR (qPCR) and Ethidium Monoazide Bromide (EMA) qPCR have been compared in order to detect Legionella pneumophila (Lp) in water samples, to identify a method able to speed up the procedures, detect the “viable but not cultivable” bacteria (VBNC) and exclude dead bacteria using a commercial kit for extraction and amplification and modifying the protocol.Methods: Using these three methods, 34 environmental water samples and a series of samples artificially spiked with alive, dead and VBNC Lp ATCC 33152 were analysed. ISO 11731-2-2004 culture method was applied, whereas a commercial kit was selected for both qPCR and EMA qPCR pretreatment.Results: 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 dead 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.Conclusions: Discrepancies between culture method and EMA-qPCR were observed and could be due to different causes as membrane-dye interactions, presence of interfering compounds and the relatively low sensitivity of the kit used.Significance and Impact of the Study: In presence of one or more suspected cases of nosocomial legionellosis, the application of a rapid molecular method able to identify only the viable and VBNC Lp would be useful in order to quickly identify the source of infection and to intervene with sanitation treatments. However, because in our experience EMA pretreatment on filter membrane has not given the expected results, it would be necessary to proceed with other experiments and different dyes.

Spiking a Silty-Sand Reference Soil with Bacterial DNA: Limits and Pitfalls in the Discrimination of Live and Dead Cells When Applying Ethidium Monoazide (EMA) Treatment

In the present study, EMA (ethidium monoazide) treatment was applied to a silty-sand reference soil prior to DNA extraction to enable a differentiation between dead and living cells. For this purpose, a reference soil was spiked with Listeria monocytogenes cells or cell equivalents, respectively. With the purpose of evaluating optimum treatment conditions, different EMA concentrations have been tested. However, the results remained largely inconclusive. Furthermore, varied dark incubation periods allowing EMA to penetrate dead cells did not allow the selective removal of DNA from membrane-compromised cells in downstream analyses. In contrast to undiluted soil, an effect of EMA treatment during DNA extraction could be observed when using a 1:10 dilution of the reference soil; however, the effect has not been sufficiently selective to act on heat-treated cells only. Although the application of EMA to soil requires further evaluation, the procedure harbors future potential for improving DNA-based approaches in microbial ecology studies.

EMA-amplicon-based taxonomic characterisation of the viable bacterial community present in untreated and SODIS treated roof-harvested rainwater

Illumina next generation sequencing coupled with ethidium monoazide bromide (EMA) was used to characterise and compare the viable bacterial community present in roof-harvested rainwater pre- and post-treatment.