The composition of planktonic prokaryotic communities in a hospital building water system depends on both incoming water and flow dynamics.

In recent years, the frequency of nosocomial infections has increased. Hospital water systems support the growth of microbes, especially opportunistic premise plumbing pathogens. In this study, planktonic prokaryotic communities present in water samples taken from hospital showers and hand basins, collected over three different sampling phases, were characterized by 16S rRNA gene amplicon sequencing. Significant differences in the abundance of various prokaryotic taxa were found through univariate and multivariate analysis. Overall, the prokaryotic communities of hospital water were taxonomically diverse and dominated by biofilm forming, corrosion causing, and potentially pathogenic bacteria. The phyla Proteobacteria, Actinobacteriota, Bacteroidota, Planctomycetota, Firmicutes, and Cyanobacteria made up 96% of the relative abundance. The α-diversity measurements of prokaryotic communities showed no difference in taxa evenness and richness based on sampling sites (shower or hand basins), sampling phases (months), and presence or absence of Vermamoeba vermiformis. However, ß-diversity measurements showed significant clustering of prokaryotic communities based on sampling phases, with the greatest difference observed between the samples collected in phase 1 vs phase 2/3. Importantly, significant difference was observed in prokaryotic communities based on flow dynamics of the incoming water. The Pielou's evenness diversity index revealed a significant difference (Kruskal Wallis, p < 0.05) and showed higher species richness in low flow regime (< 13 minutes water flushing per week and ≤ 765 flushing events per six months). Similarly, Bray-Curtis dissimilarity index found significant differences (PERMANOVA, p < 0.05) in the prokaryotic communities of low vs medium/high flow regimes. Furthermore, linear discriminant analysis effect size showed that several biofilm forming (e.g., Pseudomonadales), corrosion causing (e.g., Desulfobacterales), extremely environmental stress resistant (e.g., Deinococcales), and potentially pathogenic (e.g., Pseudomonas) bacterial taxa were in higher amounts under low flow regime conditions. This study demonstrated that a hospital building water system consists of a complex microbiome that is shaped by incoming water quality and the building flow dynamics arising through usage.

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 Legionella, resulting in the potentially fatal Legionnaires' disease. One of the biggest challenges for Legionella management within these systems is that under unfavorable conditions Legionella 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 spp. and microbial communities present in a hospital water system. Water samples were also analyzed for amoebae using culture and Vermamoeba vermiformis and Acanthamoeba 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 Legionella spp., 21% for L. pneumophila, 40.9% for V. vermiformis and 4.2% for Acanthamoeba. All samples that were Legionella spp. positive using qPCR (22%) were also positive for VBNC Legionella spp.; however, only 2.5% of samples were positive for culturable Legionella spp. 18.1% of the samples were positive for free-living amoebae (FLA) using culture. All samples positive for Legionella spp. were also positive for FLA. Samples with a high heterotrophic plate count (HPC ≥ 5 × 103 CFU/L) were also significantly associated with high concentrations of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila (p < 0.01) and V. vermiformis (p < 0.05). Temporary water stagnation arising through intermittent usage (< 2 hours of usage per month) significantly (p < 0.01) increased the amount of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila, and V. vermiformis; 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 spp. (DNA and VBNC) was associated with V. vermiformis, heterotrophic bacteria, and stagnation occurring through intermittent usage. This is the first study to monitor VBNC Legionella spp. within a hospital water system. The high percentage of false negative Legionella spp. results provided by the culture method supports the use of either qPCR or VFC+qPCR to monitor Legionella spp. contamination within hospital water systems.

Real-Time Continuous Surveillance of Temperature and Flow Events Presents a Novel Monitoring Approach for Hospital and Healthcare Water Distribution Systems.

Within hospitals and healthcare facilities opportunistic premise plumbing pathogens (OPPPs) are a major and preventable cause of healthcare-acquired infections. This study presents a novel approach for monitoring building water quality using real-time surveillance of parameters measured at thermostatic mixing valves (TMVs) across a hospital water distribution system. Temperature was measured continuously in real-time at the outlet of 220 TMVs located across a hospital over a three-year period and analysis of this temperature data was used to identify flow events. This real-time temperature and flow information was then compared with microbial water quality. Water samples were collected randomly from faucets over the three-year period. These were tested for total heterotrophic bacteria, Legionella spp. and L. pneumophila. A statistically significant association with total heterotrophic bacteria concentrations and the number of flow events seven days prior (rs[865] = -0.188, p < 0.01) and three days prior to sampling (rs[865] = -0.151, p < 0.01) was observed, with decreased heterotrophic bacteria linked to increased flushing events. Only four samples were positive for Legionella and statistical associations could not be determined; however, the environmental conditions for these four samples were associated with higher heterotrophic counts. This study validated a simple and effective remote monitoring approach to identifying changes in water quality and flagging high risk situations in real-time. This provides a complementary surveillance strategy that overcomes the time delay associated with microbial culture results. Future research is needed to explore the use of this monitoring approach as an indicator for different opportunistic pathogens.