Detection of &lt;i&gt;Legionella&lt;/i&gt; Species in Environmental Water by the Quantitative PCR Method in Combination with Ethidium Monoazide Treatment

Inoue, Hiroaki; Takama, Tomoko; Yoshizaki, Miwa; Agata, Kunio

doi:10.4265/bio.20.71

Cited by 14 publications

(16 citation statements)

References 10 publications

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“…In general, EMA/PMA-qPCR reduces the detection rates compared to conventional qPCR, but demonstrates equal or higher detection rates and cell counts relative to culturing due to inclusion of VBNC cells (Delgado-Viscogliosi et al, 2009; Inoue et al, 2015; Mansi et al, 2014). The effectiveness of EMA/PMA-qPCR is associated with a variety of factors, such as dye selection and dosage (Chen and Chang, 2010; Yanez et al, 2011), incubation time (Yanez et al, 2011), sample types (Inoue et al, 2015), target-cell loads and background flora (Gensberger et al, 2013; Slimani et al, 2012), as well as characteristics of target microorganisms (e.g., bacterial or eukaryotic cells (Fittipaldi et al, 2011)). Therefore, optimization processes and extra adaptation steps for each sample and/or microorganism type may be needed prior to application of EMA/PMA-qPCR.…”

Section: Detection Methodsmentioning

confidence: 97%

“…These intercalating dyes are intended to enter damaged cell membranes and covalently bind to DNA after photo activation, preventing downstream PCR amplification of DNA from membrane-compromised cells. Several studies have indicated promise of an EMA/PMA-based qPCR method for Legionella (Adela Yanez et al, 2011; Chen and Chang, 2010; Delgado-Viscogliosi et al, 2009; Inoue et al, 2015; Mansi et al, 2014; Slimani et al, 2012), mycobacteria (Lee et al, 2015; Nocker et al, 2007), P. aeruginosa (Gensberger et al, 2013, 2014), and Acanthamoeba (Chang et al, 2013) cells by testing against heat-treated, chlorine-treated cells and/or environmental water samples (e.g. drinking water, spa water, swimming pool water) as controls.…”

Section: Detection Methodsmentioning

confidence: 99%

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Methodological approaches for monitoring opportunistic pathogens in premise plumbing: A review

et al. 2017

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Opportunistic pathogens inhabiting premise (i.e., building) plumbing (OPPPs, e.g., L. pneumophila, M. avium complex, P. aeruginosa, Acanthamoeba, and N. fowleri) are a significant and growing source of disease. Because OPPPs establish and grow as part of the native drinking water microbiota, they do not correspond to fecal indicators, presenting a significant challenge to common and effective monitoring strategies. Further, different OPPPs present distinct requirements for sampling, preservation, and analysis, creating a significant impediment to their parallel detection. The aim of this critical review is to synthesize the state of the science of monitoring OPPPs and to identify a path forward for their simultaneous detection and quantification in a manner commensurate with the need for reliable data to inform risk assessment and mitigation. Water and biofilm sampling procedures, as well as factors influencing sample representativeness and detection sensitivity, are critically evaluated with respect to the five representative bacterial and amoebal OPPPs noted above. Available culturing and molecular approaches are discussed in terms of their advantages, limitations, and applicability. Knowledge gaps and research needs are identified.

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Section: Detection Methodsmentioning

confidence: 97%

Section: Detection Methodsmentioning

confidence: 99%

Methodological approaches for monitoring opportunistic pathogens in premise plumbing: A review

et al. 2017

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“…Another promising strategy to assess cell viability relies on the use of nucleic acid intercalating dyes such as ethidium monoazide (EMA) or propidium monoazide (PMA) in combination with specific PCR amplification (Elizaquível et al 2014;Inoue et al 2015;Lee et al 2015;Cattani et al 2016). This method is based on the membrane integrity of cells, as intact cell membranes (i.e., viable cells) are not permeable to these dyes contrarily to compromised cell membranes (i.e., dead cells).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of propidium monoazide–based qPCR to detect viable oocysts of Toxoplasma gondii

et al. 2019

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Information on the viability of Toxoplasma gondii oocysts is crucial to establish the public health significance of this environmental transmission stage that can contaminate water and foods. Interest for molecular-based methods to assess viability is growing and the aim of our study was to assess, for the first time, a propidium monoazide (PMA)-qPCR approach to determine the viability of T. gondii oocysts. Untreated and heat-killed (99°C, 5 min) oocysts were incubated with PMA, a photoreactive DNA binding dye, and analyzed by confocal microscopy and flow cytometry to characterize oocysts' dye permeability. Different PMA concentrations (50 to 150 μM), incubation temperatures (22, 37, and 45°C), amplicon length, selected targeted gene, and dyes (PMA, PMAxx™) were evaluated to define optimal conditions to discriminate specifically viable oocysts by PMA-qPCR. In theory, PMA binding to DNA would inhibit PCR amplification in dead but not in viable oocysts. Incubation at 22°C with 100 μM PMA coupled to qPCR targeting a 123-bp sequence of the 529-bp repeat element allowed the distinction between viable and heated oocysts. However, the reduction of viability following heating of oocysts at high temperature was slight and, contrarily to reverse transcriptase-qPCR, the qPCR signal was not totally suppressed in heated suspensions. Therefore, PMA-qPCR is able to assess the impact of heating on T. gondii oocysts' viability but underestimates the efficacy of this treatment. The relevance of this technique to evaluate the efficacy of other inactivation processes and assess exposure of humans to this pathogen requires further investigations.

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“…After treatment with EMA using the Viable Legionella Selection Kit for PCR Ver. 2.0 (Takara Bio) and LED CrossLinker 12 (Takara Bio), the DNA was extracted with Lysis Buffer for Legionella (Takara Bio) [38]. All protocols were carried out according to the manufacturer's instructions.…”

Section: Ema-qpcrmentioning

confidence: 99%

Detection of Legionella Species and the Influence of Precipitation on the Amount of Legionella DNA Present in Aerosols from Outdoor Sites Near Asphalt Roads in Toyama Prefecture, Japan, with Microbiome Analysis

Kanatani

Watahiki

Kimata

et al. 2020

Preprint

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Background: Legionellosis can be caused by the inhalation of aerosolized water contaminated with Legionella. In this study, we investigated the prevalence of Legionella species in aerosols collected from outdoor sites near asphalt roads, bathrooms in public bath facilities, and other indoor sites such as buildings and private homes using culture methods, quantitative PCR with ethidium monoazide treatment (EMA-qPCR), and 16S rRNA gene amplicon sequencing. Results: Legionella species were not detected in culture. However, Legionella DNA was detected in 114/151 (75.5%) air samples collected near roads (geometric mean ± standard deviation was 1.80 ± 0.52 log10 copies/m3); these numbers were comparable to those obtained from bathrooms [15/21 (71.4%), 1.82 ± 0.50] and higher than those obtained from other indoor sites [11/30 (36.7%), 0.88 ± 0.56] (P < 0.05). By EMA-qPCR, Legionella DNA was detected in 20/30 (66.7%) samples collected near roads, indicating the presence of membrane-intact Legionella cells in the air. The amount of Legionella DNA correlated with the monthly total precipitation (r = 0.25, P < 0.01). It was also directly and inversely correlated with the daily total precipitation for seven days (r = 0.21, P = 0.01) and one day (r = −0.29, P < 0.01) before the sampling day, respectively. In addition, 16S rRNA gene amplicon sequencing revealed that the three most abundant bacterial genera in the samples collected near roads were Sphingomonas (21.1%), Streptococcus (14.6%), and Methylobacterium (1.6%); Legionella species were detected in 9/30 samples (30%) collected near roads (mean proportion of reads, 0.11%). At the species level, L. pneumophila was detected in 2/30 samples collected near roads (mean proportion of reads, 0.11% and 0.09% in the detected samples).Conclusions: DNA from Legionella species, including Legionella pneumophila, were widely detected in aerosols collected from outdoor sites near asphalt roads, especially during the rainy season. Our findings suggest that there may be a risk of exposure to Legionella species in the areas surrounding asphalt roads.

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

Cited by 14 publications

References 10 publications

Methodological approaches for monitoring opportunistic pathogens in premise plumbing: A review

Methodological approaches for monitoring opportunistic pathogens in premise plumbing: A review

Evaluation of propidium monoazide–based qPCR to detect viable oocysts of Toxoplasma gondii

Detection of Legionella Species and the Influence of Precipitation on the Amount of Legionella DNA Present in Aerosols from Outdoor Sites Near Asphalt Roads in Toyama Prefecture, Japan, with Microbiome Analysis

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