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

Abstract 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...

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Autores principales:	Michela Consonni, Anna Grassi, Stefania Scuri, Maria Gori, Elisabetta Tanzi, Marina Tesauro
Formato:	article
Lenguaje:	EN
Publicado:	BMC 2021
Materias:	Legionella Detection Rapid methods Water Environmental health Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/7904b27086424d71b95876d278714d3f
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spelling	oai:doaj.org-article:7904b27086424d71b95876d278714d3f2021-11-14T12:07:14ZAssessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide10.1186/s13213-021-01653-51590-42611869-2044https://doaj.org/article/7904b27086424d71b95876d278714d3f2021-11-01T00:00:00Zhttps://doi.org/10.1186/s13213-021-01653-5https://doaj.org/toc/1590-4261https://doaj.org/toc/1869-2044Abstract 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 AbstractMichela ConsonniAnna GrassiStefania ScuriMaria GoriElisabetta TanziMarina TesauroBMCarticleLegionellaDetectionRapid methodsWaterEnvironmental healthMicrobiologyQR1-502ENAnnals of Microbiology, Vol 71, Iss 1, Pp 1-7 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Legionella Detection Rapid methods Water Environmental health Microbiology QR1-502
spellingShingle	Legionella Detection Rapid methods Water Environmental health Microbiology QR1-502 Michela Consonni Anna Grassi Stefania Scuri Maria Gori Elisabetta Tanzi Marina Tesauro Assessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide
description	Abstract 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
format	article
author	Michela Consonni Anna Grassi Stefania Scuri Maria Gori Elisabetta Tanzi Marina Tesauro
author_facet	Michela Consonni Anna Grassi Stefania Scuri Maria Gori Elisabetta Tanzi Marina Tesauro
author_sort	Michela Consonni
title	Assessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide
title_short	Assessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide
title_full	Assessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide
title_fullStr	Assessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide
title_full_unstemmed	Assessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide
title_sort	assessing the viability of legionella pneumophila in environmental samples: regarding the filter application of ethidium monoazide bromide
publisher	BMC
publishDate	2021
url	https://doaj.org/article/7904b27086424d71b95876d278714d3f
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Assessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide

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