THE RISK TO HUMAN HEALTH FROM FREE-LIVING AMOEBAE INTERACTION WITH LEGIONELLA IN DRINKING AND RECYCLED WATER SYSTEMS Dissertation submitted by JACQUELINE MARIE THOMAS BACHELOR OF SCIENCE (HONOURS) AND BACHELOR OF ARTS, UNSW In partial fulfillment of the requirements for the award of DOCTOR OF PHILOSOPHY in ENVIRONMENTAL ENGINEERING SCHOOL OF CIVIL AND ENVIRONMENTAL ENGINEERING FACULTY OF ENGINEERING MAY 2012 SUPERVISORS Professor Nicholas Ashbolt Office of Research and Development United States Environmental Protection Agency Cincinnati, Ohio USA and School of Civil and Environmental Engineering Faculty of Engineering The University of New South Wales Sydney, Australia Professor Richard Stuetz School of Civil and Environmental Engineering Faculty of Engineering The University of New South Wales Sydney, Australia Doctor Torsten Thomas School of Biotechnology and Biomolecular Sciences Faculty of Science The University of New South Wales Sydney, Australia ORIGINALITY STATEMENT '1 hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. 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Date ;; 1.~.t1.O'cJ(L..O!b. iv ABSTRACT Treated drinking water in developed countries still causes disease despite meeting current regulations and compliance monitoring needs that focus on faecal contamination. Of growing concern are the increasing rates of community acquired pneumonia (CAP) infections which are in part due to inhalation of treated drinking water aerosols containing opportunistic pathogens including Legionella and Mycobacterium. CAP infections due to Legionella presently are estimated to account for one third of all reported drinking water outbreaks in the USA. If the number of Legionella infections are to be reduced then the means of replication in drinking water need to be better understood as it is apparent that current drinking water disinfection and control mechanism are ineffective. Free-living amoebae (FLA) are eukaryotic microorganisms which are known to feed on bacteria but some microorganisms, such as pathogenic Legionella, effectively avoid digestion and infect the FLA. Microorganisms which can infect FLA are collectively known as amoebae resistant microorganisms (ARM). As hosts FLA effectively facilitate the survival, growth and infectivity of their pathogenic ARM. Despite FLA being an accepted host for pathogenic Legionella and other ARM, very little is known about their density and diversity nor their interactions with Legionella in drinking and recycled water systems and end use applications. The aim of this research was to address the present knowledge gap about FLA by analysing FLA and their interactions with Legionella in a drinking and recycled water systems and applications and then use that information in a novel quantitative microbial risk assessment for FLA and Legionella. Through literature analysis of 29 published studies reporting on FLA in drinking water systems and applications it appears that FLA are ubiquitous. FLA were detected in drinking water tap samples at a mean frequency of 46 % (n = 18, = 28). Overall, the density of FLA was poorly reported but there was a large diversity of FLA with 14 different genera of FLA identified. Already, six different genera of FLA have been isolated from drinking water systems and applications infected with 14 different species of pathogenic ARM, thus highlighting the variety of FLA and ARM interaction occurring in drinking water. From water and biofilm samples FLA were isolated using traditional culture methods utilising non-nutrient agar plates with Escherichia coli overlays. More sensitive molecular techniques were also applied with qPCR targeting Acanthamoeba spp., H. vermiformis and Naegleria spp. Detected FLA were identified by cloning and partial 18S rRNA gene sequencing. Legionella spp. were also isolated using standard culture methods and detected in samples and FLA using PCR and qPCR targeting the Legionella genus. Identification of detected Legionella was achieved by cloning and partial 16S rRNA gene sequencing. Water quality characteristics including biofilm quantity, heterotrophic plate counts and free and combined chlorine were also recorded during sampling and the course of the experiments to ascertain possible trends. To partly address the knowledge gap about FLA in recycled water systems a water recycling plant (WRP) and its recycled water distribution system were sampled for FLA and Legionella. By qPCR H. vermiformis was detected breaking through the WRP at densities of 2.7 amoebae.mL-1 and entering the distribution system. The water and biofilm for both the parallel recycled and drinking systems within the dual distribution system was sampled using a modified Robbins Device (MRD). Acanthamoeba spp. and H. vermiformis were detected by culture and qPCR in drinking and recycled water and biofilm samples with the highest mean densities (4.6 amoebae.cm-2 ) detected in drinking water biofilm. Pathogenic Legionella anisa were detected in low densities in one recycled water sample (40 cells.mL-1) and biofilm samples (14 cells.cm-2) but no Legionella was detected in the drinking water samples. Two of the 16 FLA isolated from drinking and recycled water were shown to be infected with Legionella sp. illuminating how common FLA infections with ARM were. Garden hoses were identified as an exposure pathway for Legionella infection that had been neglected in the literature to date. To evaluate FLA and Legionella density and diversity two garden hoses types (standard green and lilac) supplied with drinking water were sampled over 18 months. Over the three sampling periods the lilac hose type water consistently had higher mean density of Acanthamoeba sp. (324 amoebae.mL-1) and H. vermiformis (300 amoebae.mL-1) compared to the green hose water Acanthamoeba sp. (18 amoebae.mL-1) and H. vermiformis (31 amoebae.mL-1). Similarly, Legionella detection was significantly higher (1 way ANOVA, p < 0.0001) for the lilac hose type water (6.5 103 cells.mL-1) which were higher than all the other Legionella densities reported in the literature. Two thirds of the Legionella detected were identified as unclassified Legionella species with unknown pathogenicity that needs to be explored further. To replicate the conditions in heated applications of drinking water, such as showering, annular reactors were incubated at 42 °C for 13.5 months. Despite having comparable biofilm quantities to the ambient reactors there were consistently less FLA detected in the heated reactor biofilm by both culture and qPCR. A greater diversity of FLA were identified in the reactors including Echinamoeba exudans and Vahlkampfia sp. compared to the MRD drinking water which supplied the reactors. The highest mean detection of Legionella was for heated reactor water (80 cells.mL-1) while heated reactor biofilm had the lowest mean detection (13 cells.mL-1). During heated conditions biofilm appears to be less important in the proliferation of FLA and Legionella than under ambient conditions which was incorporated into the risk assessment. Acanthamoeba sp. and H. vermiformis isolates from the MRD, garden hoses and annular reactors were incubated with fluorescently labeled L. pneumophila at 22 and 37 °C and tracked by fluorescent microscopy over seven days to determine if the FLA would be infected. After seven days at 22 °C all of the Acanthamoeba sp. trophozoites were infected with L. pneumophila but only a maximum mean of 25 % of the H. vermiformis were. Accordingly, the numbers of L. pneumophila increased in the presence of Acanthamoeba sp. by 4.4