WATER QUALITY
temperature, flow dynamics, and stagnation.5–7
Previous research has
shown that permanent stagnation points in engineered water systems, such as dead ends and dead legs, are known to increase biofilm formation and the risk of Legionnaires disease. However, less is known about the effect of stagnation occurring at end of the line through intermitted usage of outlets. The aim of this research was to utilise an Enware SMART FLOW monitoring system to investigate the water stagnation/flow and temperature profiles at thermostatic mixing values (TMVs) and hand basin outlets on VBNC and culturable Legionella. The relationships between these water parameters and Legionella, heterotrophic plate count (HPC) bacteria, and free-living amoeba were also explored.
Methods Shower and hand basin water and biofilm (slime) samples were collected from the patient en suites within an NSW hospital. A total of 120 water and 46 biofilm samples were collected during four sampling phases from March 2021 to June 2022. The samples were analysis for Legionella using the standard culture based detection method (AS 5132:2017); however, they were also analysed using qPCR ( ISO/TS 12869:2019), which detected Legionella DNA (this includes both alive and dead cells) and our novel method (flow cytometry-cell sorting and qPCR [VFC+qPCR] assay)8
which
detected and enumerated the VBNC Legionella that cannot be detected using the standard culture method. The samples were also analysed for HPC and total amoebae using culture. In addition, the common Legionella host amoeba, Vermamoeba vermiformis and Acanthamoeba, were enumerated using qPCR. Parameters related to water
temperature and flow dynamics were monitored in the hospital water system using the monitoring system (Fig 1). Briefly, the system measures water system delivery temperatures using temperature probes located at the hot water inlet, cold water inlet, and outlet of the thermostatic mixing valves (TMV) and the hot water inlet and cold water inlet of hand basin faucets. Water flow was measured using flow switches located at the hot water inlet and cold water inlet of both the TMVs and hand basin faucets. The temperature data of the hot water supply, cold water supply, and outlet was collected for the entire duration of the sampling period. For analysis, these measurements were separated and then averaged over a period of one week and one month prior to a water sampling event. In terms of flow regime, the total duration (hours) and number (counts) of flushing events for a
IFHE DIGEST 2024
standard microbiological culturing assay returned a false negative result for 32 of them (88.9 per cent) (Fig 2).
Relationships between Legionella, amoeba, and HPC All samples positive for Legionella were also positive for free-living amoebae. This supports a previous study conducted in Australia that
showed that Legionella presence in drinking water always co-occurred with amoeba.9
These results demonstrate the
Figure 2. Legionella detected via the standard culture-based method (AS 5132:2017). The opalescent circles are the Legionella colony with each colony representing a colony forming unit (CFU).
period of one week and one month prior to sampling were recorded. The total duration (hours) of flushing events were divided into low and high flow regimes with categorisation as: low flow regime; 0 to <2 hours per month, and high flow regime; ≥2 to 40 hours per month.
Results
Legionella detection Of the total samples, 21.7 per cent (n=36/ 166) were positive for VBNC Legionella and Legionella DNA (includes both alive and dead cells). With the novel method and qPCR returning the same positive results. However, the standard culture- based method (AS 5132:2017) only found 2.4 per cent (4/166) samples to be positive for Legionella. Therefore, of the 36 samples that were positive for VBNC Legionella using our new method, the
important role that amoebae hosts play in the persistence of Legionella within hospital water systems. Future water management protocols aimed at controlling Legionella should consider incorporating treatments strategies that also target the control of amoebae. Samples with a very high heterotrophic plate count (HPC ≥5 x 103
CFU/L) were
also statistically significantly associated with high concentrations of Legionella DNA (alive and dead cells), VBNC Legionella and V. vermiformis. This supports the use of HPC as an indicator of high-risk microbial water quality. However, despite high concentrations of HPC associated with high concentrations of Legionella, no statistically significant associations were found with the lower concentrations. (Fig 3)
Relationships between microbes and water flow and temperature characteristic As shown in Figure 4, temporary water stagnation arising through intermittent usage (<2 hours of usage per month) significantly (p<0.01) increased the amount of Legionella DNA, VBNC Legionella/L. pneumophila, and V.
5µm
5µm
5µm
5µm Figure 3. Amoeba found in drinking water samples are known hosts for Legionella. 41
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