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Free-Living Protozoa in Drinking Water Supplies: Community Composition and Role As Hosts for Legionella Pneumophila

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Free-Living Protozoa in Drinking Water Supplies: Community Composition and Role As Hosts for Legionella Pneumophila Free-living protozoa in drinking water supplies: community composition and role as hosts for Legionella pneumophila Rinske Marleen Valster Thesis committee Thesis supervisor Prof. dr. ir. D. van der Kooij Professor of Environmental Microbiology Wageningen University Principal Microbiologist KWR Watercycle Institute, Nieuwegein Thesis co-supervisor Prof. dr. H. Smidt Personal chair at the Laboratory of Microbiology Wageningen University Other members Dr. J. F. Loret, CIRSEE-Suez Environnement, Le Pecq, France Prof. dr. T. A. Stenstrom,¨ SIIDC, Stockholm, Sweden Dr. W. Hoogenboezem, The Water Laboratory, Haarlem Prof. dr. ir. M. H. Zwietering, Wageningen University This research was conducted under the auspices of the Graduate School VLAG. Free-living protozoa in drinking water supplies: community composition and role as hosts for Legionella pneumophila Rinske Marleen Valster Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Monday 20 June 2011 at 11 a.m. in the Aula Rinske Marleen Valster Free-living protozoa in drinking water supplies: community composition and role as hosts for Legionella pneumophila, viii+186 pages. Thesis, Wageningen University, Wageningen, NL (2011) With references, with summaries in Dutch and English ISBN 978-90-8585-884-3 Abstract Free-living protozoa, which feed on bacteria, play an important role in the communities of microor- ganisms and invertebrates in drinking water supplies and in (warm) tap water installations. Several bacteria, including opportunistic human pathogens such as Legionella pneumophila, are able to sur- vive and replicate within protozoan hosts, and certain free-living protozoa are opportunistic human pathogens as well. However, not much is known about the occurrence of these organisms in drinking water supplies in relation to water quality and other environmental conditions. This lack of knowl- edge may in part be attributed to the limitations of microscopic techniques and cultivation methods for detection and identification of protozoa. In the investigations described in this thesis, molecular methods were applied to identify communities of free-living protozoa in drinking water supplies and to elucidate the effects of environmental conditions on the growth of these organisms. Molecular methods were also used to detect and identify protozoan hosts, both known and yet-undescribed, for L. pneumophila. The eukaryotic communities were studied using terminal restriction fragment length polymorphism (T-RFLP), clone library analyses of partial 18S rRNA gene fragments, and qPCR assays for Acanthamoeba spp. and Hartmannella vermiformis. In two groundwater supplies in the Netherlands (T < 18◦C) and three supplies in the Caribbean (T ∼ 30◦C) using seawater as source, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. A total of 127 operational taxonomic units (OTUs, each OTU contains sequences with ≥ 99% similarity) related to free-living protozoa were obtained from the sup- plies in the Netherlands and 59 OTUs were identified from the supplies in the Caribbean. Free-living protozoa related to the phyla Amoebozoa, Cercozoa, Choanozoa, Ciliophora and Stramenopiles were observed in both regions, whereas the phyla Euglenozoa and Myzozoa were only found in the supplies in the Netherlands. H. vermiformis, a described host for L. pneumophila was observed in all five sup- plies, and was a predominant protozoan in the treated water and in the distribution system in one of the examined supplies. Acanthamoeba spp. were detected at low concentrations in only a few samples. In one drinking water supply in the Caribbean, the concentration of H. vermiformis correlated with the concentration of Legionella spp., and clones related to Amoebozoa predominated the protozoan com- munity. These observations, in combination with the low turbidity and the varying ATP concentrations, indicated that biofilms in this system promoted growth of amoebae and also Legionella spp. Ciliophora represented 25% of the protozoan OTUs in another Caribbean supply with elevated ATP concentrations v and turbidity, related to corroding cast-iron pipes. Thus, sediments seem to favor growth of ciliates. Cercozoan types predominated in one of the Caribbean supplies with low ATP concentrations (< 1 ng liter−1) and turbidity (< 1 NTU) in most samples of distributed water. Growth of H. vermiformis was also observed in a drinking water supply in the Netherlands with a high concentration of natural organic matter (NOM) combined with an elevated concentration of active biomass (ATP), whereas no growth of this amoeba was observed in the groundwater supply with a low concentration of ATP (< 1 ng liter−1) and NOM (< 0.5 mg C liter−1). A biofilm batch test (BBT) was applied to amplify and subsequently identify protozoan hosts for L. pneumophila. In this test 21 freshwater samples, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated in duplicate at 37◦C. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan H. vermi- formis had been added. Indigenous H. vermiformis was indicated as host in 12 of the 19 BBT flasks with growth of L. pneumophila. In none of the water samples incubated at 37◦C in the BBT system, growth of Acanthamoeba spp. was observed. In several flasks with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. To assess the effects of water temperature on indigenous free-living protozoan communities, four wa- ter types were incubated in a BBT system at 20, 30, 37 and 42◦C. Only two of the 53 OTUs related to free-living protozoa obtained from these freshwater types at day 0 were also detected after incu- bation. Multiplication of indigenous Acanthamoeba spp. was observed at 20 and 30◦C, with only limited growth in one flask at 37◦C. Indigenous H. vermiformis multiplied at 20, 30 and 37◦C, but at 42◦C proliferation was only observed in water collected from the river Rhine. Growth of organisms related to Amoebozoa and Stramenopiles was observed at all four temperatures, whereas Cercozoan and Euglenozoan types predominated in the BBT system incubated at 37 and 30◦C, respectively. The Amoebozoan Arachnula sp. was identified as candidate host for L. pneumophila at 42◦C, but in vitro studies with this organism are needed for confirmation. These observations indicate that H. vermiformis, the predominating protozoan host for Legionella spp., is ubiquitous in water supplies and other engineered water installations. This organism can grow at temperatures ranging from less than 20 to 42◦C. Proliferation is limited at active biomass concen- trations < 1 ng ATP liter−1 in combination with NOM concentrations < 0.5 mg C liter−1. However, production of drinking water with such low concentrations of NOM and ATP is not achievable in most cases. Therefore, a combination of measures in water treatment (reducing the NOM concentration) and distribution (prevention of sediment accumulation, disinfection) is needed to reduce the potential for growth of free-living protozoa in water supplies and in water installations. vi Contents 1 General introduction 1 2 Free-living protozoa in two unchlorinated drinking water supplies, identified by phylogenic analysis of 18S rRNA gene sequences 15 3 Detection of protozoan hosts for Legionella pneumophila in engineered water systems by using a biofilm batch test 33 4 Relationships between free-living protozoa, cultivable Legionella spp. and water quality parameters in three drinking water supplies in the Caribbean 51 5 Effect of temperature on the communities of free-living protozoa in four freshwater types in a biofilm batch system 69 6 General Discussion 85 A Supplemental material to Chapter 2 105 B Supplemental material to Chapter 3 113 C Supplemental material to Chapter 4 123 D Supplemental material to Chapter 5 131 Bibliography 149 Summary 167 Samenvatting 172 Dankbetuiging/Acknowledgements 177 vii Contents Curriculum Vitae 179 VLAG PhD Education form 180 List of publications 182 Addendum 184 viii Chapter 1 General introduction 2 1. General introduction 1.1 Background of research Free-living protozoa are ubiquitous in natural freshwater environments, but also multiply in engineered water systems, including wastewater treatment plants, drinking water treatment systems, drinking wa- ter distribution systems and tap water installations inside buildings [175, 217, 256, 274]. Free-living protozoa are unicellular heterotrophic eukaryotes, which feed mainly on bacteria and other small biomass particles that are suspended in the water, or are present in biofilms and sediments in natural and engineered freshwater environments [196]. Consequently, free-living protozoa have a consid- erable impact on the microbial communities in aquatic environments. Many protozoa are selective consumers that can recognize various traits of their potential prey [55, 113]. They play an impor- tant role as decomposers and nutrient regenerators, and their metabolization of organic molecules leads to the release of phosphate, ammonia, nitrate and amino acids into the environment [130, 201]. Furthermore, free-living protozoa are an
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