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

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