{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,19]],"date-time":"2026-10-19T11:56:01Z","timestamp":1792410961458,"version":"4.4.0"},"reference-count":57,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,1,30]],"date-time":"2023-01-30T00:00:00Z","timestamp":1675036800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p><jats:italic>Legionella pneumophila<\/jats:italic> is a waterborne pathogen and, as the causative agent of Legionnaires\u2019 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) <jats:italic>Legionella<\/jats:italic>. The management of engineered water systems to prevent Legionnaires\u2019 disease is hindered by the presence of VBNC <jats:italic>Legionella<\/jats:italic> 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 <jats:italic>Legionella<\/jats:italic> from environmental water samples using a \u201cviability based flow cytometry-cell sorting and qPCR\u201d (VFC\u2009+\u2009qPCR) assay. This protocol was then validated by quantifying the VBNC <jats:italic>Legionella<\/jats:italic> 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 <jats:italic>Legionella<\/jats:italic> 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 <jats:italic>Legionella<\/jats:italic> 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 <jats:italic>Legionella<\/jats:italic> from environmental sources. This will significantly improve future research evaluating <jats:italic>Legionella<\/jats:italic> risk management approaches for the control of Legionnaires\u2019 disease.<\/jats:p>","DOI":"10.3389\/fmicb.2023.1094877","type":"journal-article","created":{"date-parts":[[2023,1,30]],"date-time":"2023-01-30T04:51:31Z","timestamp":1675054291000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":36,"title":["Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR"],"prefix":"10.3389","volume":"14","author":[{"given":"Muhammad Atif","family":"Nisar","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kirstin E.","family":"Ross","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melissa H.","family":"Brown","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard","family":"Bentham","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giles","family":"Best","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Harriet","family":"Whiley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2023,1,30]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"7813","DOI":"10.1128\/AEM.01364-08","article-title":"Use of flow cytometry to monitor Legionella viability","volume":"74","author":"Allegra","year":"2008","journal-title":"Appl. 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