Molecular screening and characterization of Legionella pneumophila associated free-living amoebae in domestic and hospital water systems

Abstract

Free-living amoebae are ubiquitous in the environment and cause both opportunistic and non-opportunistic infections in humans. Some genera of amoebae are natural reservoirs of opportunistic plumbing pathogens, such as Legionella pneumophila. In this study, the presence of free-living amoebae and Legionella was investigated in 140 water and biofilm samples collected from Australian domestic (n = 68) and hospital water systems (n = 72). Each sample was screened in parallel using molecular and culture-based methods. Direct quantitative polymerase chain reaction (qPCR) assays showed that 41% samples were positive for Legionella, 33% for L. pneumophila, 11% for Acanthamoeba, and 55% for Vermamoeba vermiformis gene markers. Only 7% of samples contained culturable L. pneumophila serogroup (sg)1, L. pneumophila sg2-14, and non-pneumophila Legionella. In total, 69% of samples were positive for free-living amoebae using any method. Standard culturing found that 41% of the samples were positive for amoeba (either Acanthamoeba, Allovahlkampfia, Stenamoeba, or V. vermiformis). V. vermiformis showed the highest overall frequency of occurrence. Acanthamoeba and V. vermiformis isolates demonstrated high thermotolerance and osmotolerance and strong broad spectrum bacteriogenic activity against Gram-negative and Gram-positive bacteria. Importantly, all Legionella positive samples were also positive for amoeba, and this co-occurrence was statistically significant (p < 0.05). According to qPCR and fluorescence in situ hybridization, V. vermiformis and Allovahlkampfia harboured intracellular L. pneumophila. To our knowledge, this is the first time Allovahlkampfia and Stenamoeba have been demonstrated as hosts of L. pneumophila in potable water. These results demonstrate the importance of amoebae in engineered water systems, both as a pathogen and as a reservoir of Legionella. The high frequency of gymnamoebae detected in this study from Australian engineered water systems identifies an issue of significant public health concern. Future water management protocols should incorporate treatments strategies to control amoebae to reduce the risk to end users.