The risk to human health from free-living amoebae interaction with Legionella in drinking and recycled water systems

Treated drinking water in developed countries still causes disease despite meeting current regulations. Community acquired pneumonia (CAP) infections due to the bacterium Legionella presently account for one third of all reported drinking water outbreaks in the USA. Free-living amoebae (FLA) are eukaryotic microorganisms that are known to intracellularly harbour Legionella and thus facilitate their survival, growth and human infectivity. Analysis of 29 studies revealed that FLA were detected in drinking water taps at a mean frequency of 46 % (n = 18, &#963; = 28) across the world. Despite FLA being an accepted host for pathogenic Legionella very little is known about their interactions in drinking and recycled water systems. This research aimed to address this knowledge gap. Utilising traditional culture methods and qPCR the density and diversity of FLA and Legionella in a water recycling plant (WRP) and a dual drinking and recycled water distribution system was analysed. By qPCR the FLA H. vermiformis was detected breaking through the WRP and entering the distribution system at densities of 2.7 amoebae.mL-1. Pathogenic Legionella anisa were detected in low densities in recycled water (40 cells.mL-1) and biofilm samples (14 cells.cm-2). Garden hoses were identified as an exposure pathway for Legionella infection that had been neglected in the current literature. FLA and Legionella detection was significantly higher (1 way ANOVA, p < 0.0001) for the lilac hose type water and mean Legionella densities (6.5 × 103 cells.mL-1) were higher than all other Legionella densities reported in the literature. To replicate the conditions in heated drinking water applications, such as showers, annular reactors were incubated at 42 °C for 13.5 months. Heated reactor biofilm had the lowest detection mean of FLA and Legionella (13 cells.mL-1) compared to ambient reactors. Acanthamoeba spp. isolated from the drinking water were found to increase the growth of L. pneumophila two fold more than H. vermiformis isolates. FLA density and diversities in water and biofilm were incorporated to form a novel quantitative microbial risk assessment (QMRA) for Legionella infection. The probability of human infection exceeded the recommended benchmark risk of 1 × 10-4 for both garden hose use and showering. Hence, behavioural changes were recommended to reduce exposure for those people who are at risk of Legionella infection, such as the elderly and people with suppressed immune systems