AUTHOR=Nisar Muhammad Atif , Ros Kirstin E. , Brown Melissa H. , Bentham Richard , Best Giles , Xi James , Hinds Jason , Whiley Harriet 

TITLE=Stagnation arising through intermittent usage is associated with increased viable but non culturable Legionella and amoeba hosts in a hospital water system

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=13

YEAR=2023

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2023.1190631

DOI=10.3389/fcimb.2023.1190631

ISSN=2235-2988

ABSTRACT=<p>Hospital water systems are a significant source of <italic>Legionella</italic>, resulting in the potentially fatal Legionnaires’ disease. One of the biggest challenges for <italic>Legionella</italic> management within these systems is that under unfavorable conditions <italic>Legionella</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 <italic>Legionella</italic> spp. and microbial communities present in a hospital water system. Water samples were also analyzed for amoebae using culture and <italic>Vermamoeba vermiformis</italic> and <italic>Acanthamoeba</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<sup>®</sup> monitoring system. qPCR analysis demonstrated that 21.8% samples were positive for <italic>Legionella</italic> spp., 21% for <italic>L. pneumophila</italic>, 40.9% for <italic>V. vermiformis</italic> and 4.2% for <italic>Acanthamoeba</italic>. All samples that were <italic>Legionella</italic> spp. positive using qPCR (22%) were also positive for VBNC <italic>Legionella</italic> spp.<italic>;</italic> however, only 2.5% of samples were positive for culturable <italic>Legionella</italic> spp. 18.1% of the samples were positive for free-living amoebae (FLA) using culture. All samples positive for <italic>Legionella</italic> spp. were also positive for FLA. Samples with a high heterotrophic plate count (HPC ≥ 5 × 10<sup>3</sup> CFU/L) were also significantly associated with high concentrations of <italic>Legionella</italic> spp. DNA, VBNC <italic>Legionella</italic> spp./<italic>L. pneumophila</italic> (<italic>p</italic> &lt; 0.01) and <italic>V. vermiformis</italic> (<italic>p</italic> &lt; 0.05). Temporary water stagnation arising through intermittent usage (&lt; 2 hours of usage per month) significantly (<italic>p</italic> &lt; 0.01) increased the amount of <italic>Legionella</italic> spp. DNA, VBNC <italic>Legionella</italic> spp./<italic>L. pneumophila</italic>, and <italic>V. vermiformis;</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, <italic>Legionella</italic> spp. (DNA and VBNC) was associated with <italic>V. vermiformis</italic>, heterotrophic bacteria, and stagnation occurring through intermittent usage. This is the first study to monitor VBNC <italic>Legionella</italic> spp. within a hospital water system. The high percentage of false negative <italic>Legionella</italic> spp. results provided by the culture method supports the use of either qPCR or VFC+qPCR to monitor <italic>Legionella</italic> spp. contamination within hospital water systems.</p>