{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T15:28:21Z","timestamp":1791386901091,"version":"4.3.3"},"reference-count":57,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,6,7]],"date-time":"2023-06-07T00:00:00Z","timestamp":1686096000000},"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. Cell. Infect. Microbiol."],"abstract":"Hospital water systems are a significant source of Legionella<\/jats:italic>, resulting in the potentially fatal Legionnaires\u2019 disease. One of the biggest challenges for Legionella<\/jats:italic> management within these systems is that under unfavorable conditions Legionella<\/jats:italic> transforms itself into a viable but non culturable (VBNC) state that cannot be detected using the standard methods. This study used a novel method (flow cytometry-cell sorting and qPCR [VFC+qPCR] assay) concurrently with the standard detection methods to examine the effect of temporary water stagnation, on Legionella<\/jats:italic> spp. and microbial communities present in a hospital water system. Water samples were also analyzed for amoebae using culture and Vermamoeba vermiformis<\/jats:italic> and Acanthamoeba<\/jats:italic> specific qPCR. The water temperature, number and duration of water flow events for the hand basins and showers sampled was measured using the Enware Smart Flow\u00ae<\/jats:sup> monitoring system. qPCR analysis demonstrated that 21.8% samples were positive for Legionella<\/jats:italic> spp., 21% for L. pneumophila<\/jats:italic>, 40.9% for V. vermiformis<\/jats:italic> and 4.2% for Acanthamoeba<\/jats:italic>. All samples that were Legionella<\/jats:italic> spp. positive using qPCR (22%) were also positive for VBNC Legionella<\/jats:italic> spp.;<\/jats:italic> however, only 2.5% of samples were positive for culturable Legionella<\/jats:italic> spp. 18.1% of the samples were positive for free-living amoebae (FLA) using culture. All samples positive for Legionella<\/jats:italic> spp. were also positive for FLA. Samples with a high heterotrophic plate count (HPC \u2265 5 \u00d7 103<\/jats:sup> CFU\/L) were also significantly associated with high concentrations of Legionella<\/jats:italic> spp. DNA, VBNC Legionella<\/jats:italic> spp.\/L. pneumophila<\/jats:italic> (p<\/jats:italic> &lt; 0.01) and V. vermiformis<\/jats:italic> (p<\/jats:italic> &lt; 0.05). Temporary water stagnation arising through intermittent usage (&lt; 2 hours of usage per month) significantly (p<\/jats:italic> &lt; 0.01) increased the amount of Legionella<\/jats:italic> spp. DNA, VBNC Legionella<\/jats:italic> spp.\/L. pneumophila<\/jats:italic>, and V. vermiformis;<\/jats:italic> however, it did not significantly impact the HPC load. In contrast to stagnation, no relationship was observed between the microbes and water temperature. In conclusion, Legionella<\/jats:italic> spp. (DNA and VBNC) was associated with V. vermiformis<\/jats:italic>, heterotrophic bacteria, and stagnation occurring through intermittent usage. This is the first study to monitor VBNC Legionella<\/jats:italic> spp. within a hospital water system. The high percentage of false negative Legionella<\/jats:italic> spp. results provided by the culture method supports the use of either qPCR or VFC+qPCR to monitor Legionella<\/jats:italic> spp. contamination within hospital water systems.<\/jats:p>","DOI":"10.3389\/fcimb.2023.1190631","type":"journal-article","created":{"date-parts":[[2023,6,7]],"date-time":"2023-06-07T06:25:11Z","timestamp":1686119111000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":19,"title":["Stagnation arising through intermittent usage is associated with increased viable but non culturable Legionella and amoeba hosts in a hospital water system"],"prefix":"10.3389","volume":"13","author":[{"given":"Muhammad Atif","family":"Nisar","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kirstin E.","family":"Ros","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":"James","family":"Xi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jason","family":"Hinds","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":[[2023,6,7]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"21660","DOI":"10.3390\/ijms141121660","article-title":"Biofilms: the stronghold of Legionella pneumophila","volume":"14","author":"Abdel-Nour","year":"2013","journal-title":"Int. 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