Author: Muhammad Atif Nisar
Nisar, Muhammad Atif, 2023 Legionella pneumophila and protozoan hosts from the engineered water systems, Flinders University, College of Science and Engineering
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Legionella is an opportunistic premise plumbing pathogen and the causative agent of Legionnaires’ disease and Pontiac fever. Globally, the incidence of Legionella infections has been increasing. The persistence of Legionella in engineered water systems is associated with protozoan hosts, biofilms, failure of disinfection treatments, and water stagnation.
Globally, routine water testing for Legionella is recommended to manage hospital water systems to prevent outbreaks of Legionnaires’ disease. The International Organization for Standardization (ISO) recommends two protocols for the detection and quantification of Legionella in engineered water systems. ISO11731:2017-05 detects only culturable Legionella, whereas ISO/TS12869:2019 is a quantitative PCR (qPCR) based method which detect its genomic DNA. However, both methods are unable to quantify and characterize VBNC Legionella. In this study, a culture-independent “viability-based flow cytometry-cell sorting and qPCR (VFC+qPCR)” assay was designed to detect and quantify VBNC Legionella from environmental samples. This was the first time that flow cytometry-cell sorting in conjunction with a qPCR assay has been used as a direct and rapid method to quantify VBNC Legionella from engineered water systems.
Protozoan hosts, specifically free-living amoebae, are natural hosts and reservoirs of Legionella. In this study, a systematic literature review identified that free-living amoebae, most commonly Acanthamoeba and Vermamoeba vermiformis, are the major hosts of Legionella in building water distribution systems. Based on the findings from the systematic literature review, water and biofilm samples from Australian hospital and domestic water systems were screened for the presence of free-living amoebae and Legionella. Both culture-dependant and culture-independent approaches were used for screening and characterization of samples. Direct qPCR assays demonstrated that 41% of samples were positive for Legionella, 33% for L. pneumophila, 11% for Acanthamoeba, and 55% for V. vermiformis. Only 7% of samples were positive for culturable Legionella. In contrast, 41% of samples were positive for culturable free-living amoebae, which were identified as V. vermiformis, Acanthamoeba, Stenamoeba, and Allovahlkampfia. These culturable free-living amoebae were highly thermotolerant and osmotolerant and harboured strong broad spectrum bacteriogenic activity. Importantly, all Legionella/L. pneumophila positive samples were also positive for free-living amoeba, and this co-occurrence was statistically significant (p<0.05). Furthermore, using qPCR and fluorescence in situ hybridization it was identified that V. vermiformis and Allovahlkampfia harboured intracellular L. pneumophila. Importantly, this is the first time Allovahlkampfia and Stenamoeba have been demonstrated to be hosts of L. pneumophila in engineered potable water systems. In conclusion, the high frequency of free-living amoebae in Australian engineered water systems is a significant public health concern.
Stagnation and flow dynamics are important parameters which affect the water quality in engineered water systems. In this study, a systematic literature review demonstrated that both permanent and temporary stagnation promotes the growth of Legionella in engineered water systems. Based on the findings from the systematic literature review, a laboratory scale biofilm model study, and a real world (hospital water system) investigation into the effect of intermittent stagnation was conducted. In both of these studies the new method described above was used to quantify the VBNC Legionella present. In the laboratory scale study, a model plumbing system consisting of a water tank and two biofilm reactors was used to investigate the effect of stagnation, and intermitted usage, on Legionella and free-living amoebae. Initially, both biofilm reactors were left stagnant for 147 days to allow the formation of biofilm. This was followed by the operational phase during which one biofilm reactor was flushed once a day with 70 L of potable water, and the other biofilm reactor was flushed once a week. It was identified that once-a-day flushing for 28 days significantly (p<0.001) reduced the amount of biofilm-associated alive (potentially culturable based on VFC+qPCR) and culturable Legionella and increased the amount of VBNC Legionella compared with the once-a-week flushing. Furthermore, it was observed that the concentration of culturable Legionella (p<0.001) was positively correlated with heterotrophic plate count (HPC) and the concentration of VBNC Legionella (p<0.001) was positively correlated with the concentration of V. vermiformis. This laboratory scale study demonstrated that a reduction of water stagnation, and an increase in usage/flushing, significantly decreased the population of total, alive and culturable Legionella. The effect of stagnation and flow dynamics was also investigated on water (n=120) and biofilm (n=46) samples collected from an Australian hospital water distribution system. The shower and hand basin water/biofilm samples were collected over 16 months from one hospital and water flushing data was obtained from Enware™ using their Smart Flow® monitoring system. The molecular analysis showed that 22% samples were positive for Legionella and 41% for V. vermiformis using qPCR. This investigation also suggested that temporary stagnation (<2 hours water flushing/month) significantly (p<0.01) increased the quantity of VBNC and total (genomic unit) Legionella. Moreover, it was also identified that high HPC load was significantly (p<0.01) associated with increased concentrations of Legionella and V. vermiformis in engineered water system. These three studies demonstrate that stagnation arising through intermitted usage is an important factor influencing the risk of Legionella in engineered water systems.
Engineered water systems are a complex environment with a range of variables that can influence the growth and persistence of microbes, especially opportunistic premise plumbing pathogens. In this study, 16S rRNA sequence analysis was used to examine the prokaryotic communities present throughout a hospital distribution system. A total of 46 water samples from showers and hand basins collected during three different sampling periods were examined. The influence of temperature and water flow dynamics (number and total duration of flow events) for one week and six months prior to sample collection was examined. It was found that the hospital water primarily contained six bacterial phyla i.e., Proteobacteria, Actinobacteriota, Bacteroidota, Planctomycetota, Firmicutes, and Cyanobacteria. The diversity of prokaryotic communities present was significantly (p<0.05) affected by sampling phase (month) and flow dynamics. Importantly, it was also observed that several biofilm forming, corrosion responsible, extremely resistant, and potentially pathogenic bacterial taxa were enriched in low flow regimes. This study showed that hospital water system consists of complex prokaryotic communities that is shaped by incoming water quality and the building flow dynamics.
Keywords: Legionella pneumophila, Legionnaires' disease, Free-living amoebae, Biofilms, Engineered water systems, Water stagnation, Water microbiome
Subject: Microbiology & Infectious Diseases thesis
Thesis type: Doctor of Philosophy
Completed: 2023
School: College of Science and Engineering
Supervisor: Associate Professor Harriet Whiley