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Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR

Legionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires’ disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC)...

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Autores principales: Nisar, Muhammad Atif, Ross, Kirstin E., Brown, Melissa H., Bentham, Richard, Best, Giles, Whiley, Harriet
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9922708/
https://www.ncbi.nlm.nih.gov/pubmed/36793878
http://dx.doi.org/10.3389/fmicb.2023.1094877
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author Nisar, Muhammad Atif
Ross, Kirstin E.
Brown, Melissa H.
Bentham, Richard
Best, Giles
Whiley, Harriet
author_facet Nisar, Muhammad Atif
Ross, Kirstin E.
Brown, Melissa H.
Bentham, Richard
Best, Giles
Whiley, Harriet
author_sort Nisar, Muhammad Atif
collection PubMed
description Legionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires’ disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC) Legionella. The management of engineered water systems to prevent Legionnaires’ disease is hindered by the presence of VBNC Legionella that cannot be detected using the standard culture (ISO11731:2017-05) and quantitative polymerase reaction (ISO/TS12869:2019) methods. This study describes a novel method to quantify VBNC Legionella from environmental water samples using a “viability based flow cytometry-cell sorting and qPCR” (VFC + qPCR) assay. This protocol was then validated by quantifying the VBNC Legionella genomic load from hospital water samples. The VBNC cells were unable to be cultured on Buffered Charcoal Yeast Extract (BCYE) agar; however, their viability was confirmed through their ATP activity and ability to infect amoeba hosts. Subsequently, an assessment of the ISO11731:2017-05 pre-treatment procedure demonstrated that acid or heat treatment cause underestimation of alive Legionella population. Our results showed that these pre-treatment procedures induce culturable cells to enter a VBNC state. This may explain the observed insensitivity and lack of reproducibility often observed with the Legionella culture method. This study represents the first time that flow cytometry-cell sorting in conjunction with a qPCR assay has been used as a rapid and direct method to quantify VBNC Legionella from environmental sources. This will significantly improve future research evaluating Legionella risk management approaches for the control of Legionnaires’ disease.
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spelling pubmed-99227082023-02-14 Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR Nisar, Muhammad Atif Ross, Kirstin E. Brown, Melissa H. Bentham, Richard Best, Giles Whiley, Harriet Front Microbiol Microbiology Legionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires’ disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC) Legionella. The management of engineered water systems to prevent Legionnaires’ disease is hindered by the presence of VBNC Legionella that cannot be detected using the standard culture (ISO11731:2017-05) and quantitative polymerase reaction (ISO/TS12869:2019) methods. This study describes a novel method to quantify VBNC Legionella from environmental water samples using a “viability based flow cytometry-cell sorting and qPCR” (VFC + qPCR) assay. This protocol was then validated by quantifying the VBNC Legionella genomic load from hospital water samples. The VBNC cells were unable to be cultured on Buffered Charcoal Yeast Extract (BCYE) agar; however, their viability was confirmed through their ATP activity and ability to infect amoeba hosts. Subsequently, an assessment of the ISO11731:2017-05 pre-treatment procedure demonstrated that acid or heat treatment cause underestimation of alive Legionella population. Our results showed that these pre-treatment procedures induce culturable cells to enter a VBNC state. This may explain the observed insensitivity and lack of reproducibility often observed with the Legionella culture method. This study represents the first time that flow cytometry-cell sorting in conjunction with a qPCR assay has been used as a rapid and direct method to quantify VBNC Legionella from environmental sources. This will significantly improve future research evaluating Legionella risk management approaches for the control of Legionnaires’ disease. Frontiers Media S.A. 2023-01-30 /pmc/articles/PMC9922708/ /pubmed/36793878 http://dx.doi.org/10.3389/fmicb.2023.1094877 Text en Copyright © 2023 Nisar, Ross, Brown, Bentham, Best and Whiley. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Nisar, Muhammad Atif
Ross, Kirstin E.
Brown, Melissa H.
Bentham, Richard
Best, Giles
Whiley, Harriet
Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR
title Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR
title_full Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR
title_fullStr Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR
title_full_unstemmed Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR
title_short Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR
title_sort detection and quantification of viable but non-culturable legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative pcr
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9922708/
https://www.ncbi.nlm.nih.gov/pubmed/36793878
http://dx.doi.org/10.3389/fmicb.2023.1094877
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