Differential growth of Legionella pneumophila strains within a range of amoebae at various temperatures associated with in-premise plumbing

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1 Letters in Applied Microbiology ISSN - ORIGINAL ARTICLE Differential growth of Legionella pneumophila strains within a range of amoebae at various temperatures associated with in-premise plumbing National Exposure Research Laboratory, Office of Research and Development, US Environmental Protection Agency, Cincinnati, OH, USA Keywords drinking water, Legionella, protozoa, virulence and water quality. Correspondence Helen Y. Buse, National Exposure Research Laboratory, Office of Research and Development, US Environmental Protection Agency, W Martin Luther King Dr, MS, Cincinnati, OH, USA. buse.helen@epa.gov : received April, revised and accepted May doi:./j.-x...x Abstract Aims: The potential effect of in-premise plumbing temperatures (,, and C) on the growth of five different Legionella pneumophila strains within free-living amoebae (Acanthamoeba polyphaga, Hartmannella vermiformis and Naegleria fowleri) was examined. Methods and Results: Compared with controls that actively fed on Escherichia coli prey, when Leg. pneumophila was used as prey, strains Lp and Bloomington- increased in growth at, and C while strains Philadelphia- and Chicago did not grow at any temperature within A. polyphaga. Strains Lp, Bloomington- and Dallas E did not proliferate in the presence of H. vermiformis nor did strain Philadelphia- in the presence of N. fowleri. Yet, strain Bloomington- grew at all temperatures examined within N. fowleri, while strain Lp proliferated at all temperatures except C. More intriguing, strain Chicago only grew at C within H. vermiformis and N. fowleri suggesting a limited temperature growth range for this strain. Conclusions: Identifying the presence of pathogenic legionellae may require the use of multiple host amoebae and incubation temperatures. Significance and Impact of the Study: Temperature conditions and species of amoeba host supported in drinking water appear to be important for the selection of human-pathogenic legionellae and point to future research required to better understand Legionella ecology. Introduction During, Legionella was identified as the third most common etiologic agent among all US waterborne disease outbreaks, with more than % of legionellosis cases associated with Legionella pneumophila (Craun et al. ). Warm stagnant water, low disinfectant residuals and aerosolization were all features associated with the source of the legionellae outbreaks (Yoder et al. ). Furthermore, outbreaks appear to be primarily attributed to Legionella amplification and dissemination from biofilms within the plumbing of buildings (in-premise plumbing) (Steinert et al. ). While nosocomal cases of legionellosis are more readily identified, sporadic household-acquired infections may go undetected (von Baum et al. ). Nonetheless, in single households, Leg. pneumophila has been isolated from out of homes in the United States (Stout et al. ), homes in Germany (Mathys et al. ) and homes in Canada (Alary and Joly ): with the majority of the isolates recovered from the three studies belonging to serogroups and. In all households studied, a low hot water tank temperature was significantly associated with legionellae occurrence. Hence, as for nosocomial cases, legionellae growth appears to be favoured by warm temperatures, noting that maximum Leg. pneumophila growth has been reported between and C (Mathys et al. ). A second key factor associated with pathogenic legionellae occurrence in drinking water systems is the presence of free-living amoebae (FLA). While the diversity of drinking water FLA remains poorly characterized (Thomas and Ashbolt ), many different genera of FLA have been reported to promote the growth and or survival of legionellae as well as other intracellular Letters in Applied Microbiology, ª The Society for Applied Microbiology

2 Host and temperature effects on Leg. pneumophila replication pathogens (Lau and Ashbolt ; Thomas et al. ). Furthermore, various FLA have been shown to enhance the virulence, survival and or amplification of Leg. pneumophila (Barker et al., ; Brieland et al. ; Steinert et al. ; Cirillo et al. ). Acanthamoeba, Naegleria, Vahlkampfia, Hartmannella spp. and Leg. pneumophila have all been isolated from the same domestic water tap in a community drinking water system, although seasonally influenced (via water temperature) (Marciano-Cabral et al. ) and from recreational waters such as mud springs, hot spring facilities and various sources of spring water (Hsu et al. ; Huang and Hsu ). Overall, Acanthamoeba and Hartmannella appear to have a lower growth temperature tolerance while Naegleria fowleri may dominate in hot water samples (> C) (Griffin ). Collectively, these studies suggest that temperatures amenable to suitable amoebae hosts play an important role in Legionella pathogenesis (Lau and Ashbolt ). In this study, five strains of Leg. pneumophila representing several serogroups were tested for their ability to infect and replicate within three different FLA at various temperatures known to occur within in-premise plumbing. The goal of this work was to illustrate the importance of using multiple host amoebae and incubation temperatures to recover pathogenic Leg. pneumophila from water distribution systems. Materials and methods Legionella and Amoebae strain preparation The Leg. pneumophila stains used in this study (Table ) were grown overnight with continuous shaking as pure cultures at C in buffered yeast extract (BYE) broth ( g ACES, g yeast extract, Æ g l-cysteine and Æ g ferric nitrate per l). Bacterial density was estimated by absorbance at nm calibrated to a predetermined standard curve of colony forming units (CFU). Bacteria were then diluted to the desired concentration in Page s amoebae saline (PAS: Æ mmol l ) NaCl, mmol l ) KH PO, Æ mmol l ) Na HPO, lmol l ) CaCl ÆH O and lmol l ) MgSO ÆH O). To determine Legionella densities (as measured by CFU), a small aliquot of the bacterial suspension was serially diluted and plated on buffered charcoal yeast extract (BCYE) agar plates (BD Diagnostics, Franklin Lakes, NJ, USA). Plates were incubated for h at C, and Legionella colonies were counted. The amoebae strains used in this study (Table ) were grown as monolayers at C in either ATCC medium for Acanthamoeba polyphaga or ATCC medium for Hartmannella vermiformis and N. fowleri. Amoebae growth with Escherichia coli prey Amoebae cells were harvested on the day of the experiment, washed twice with ml of PAS, counted with the aid of a haemacytometer and then seeded into -well plates at a density of approx. cells per well. An overnight culture of E. coli (ATCC ) grown in Luria broth (BD Diagnostics, Franklin Lakes, NJ) at C was washed twice with ml of PAS and then added to the appropriate wells at an E. coli amoeba ratio of : in replicates of five. Plates were incubated at either room temperature (±Æ), (±Æ), (±Æ) or (±Æ) C. At various time points ( h), the total number of amoebae cells (both trophozoites and cysts) in each well were quantified using an inverted microscope. Legionella survival and proliferation within amoebae Amoebae cells were harvested as described elsewhere and then seeded into -well plates at a density of approx. cells per well, and PAS-diluted Legionella cells were added to duplicate wells to provide bacteria to one amoeba and incubated at the same five temperatures as the E. coli controls but sampled at,, and h and processed as described previously. Results are presented as a ratio of bacterial CFU in the presence of amoebae (Tn) divided by the legionellae CFU in bacteria only wells (Tc) at each time point. Bacterial CFU from only the control wells were used to assess Legionella growth at various temperatures after days in PAS. The presence of Legionella-infected ameobae was confirmed microscopically at each time point under an inverted Nikon Eclipse TF microscope. Statistical analysis Statistical significance was determined using the unpaired, two-tailed Student s t test, anova for multiple group comparisons using the Student Newman Keuls post-test and Fisher s exact test. Where appropriate, the unpaired, nonparametric Mann Whitney test was utilized. Calculations and graphs were generated using InStat and Prism (GraphPad Software, San Diego, CA, USA). Results Growth of amoebae hosts at various temperatures in the presence of Escherichia coli prey At C, the total number of A. polyphaga trophozoites reached a maximum of ± by day with % of those cells encysting by day (Fig. a). Similarly, Letters in Applied Microbiology, ª The Society for Applied Microbiology

3 Host and temperature effects on Leg. pneumophila replication Table Amoebae and Legionella pneumophila strains used in this study Name Strain Description Serogroup Source Bacteria Leg. pneumophila Philadelphia- Clinical isolate, human lung ATCC Lp Clinical isolate, derivative of Philadelphia- strain Dr Michele Swanson Bloomington- Environmental isolate, creek water ATCC Dallas E Environmental isolate, cooling tower ATCC Chicago Clinical isolate, human lung ATCC Amoebae Acanthamoeba polyphaga Puschkarew Clinical isolate, human corneal scrapings ATCC Hartmannella vermiformis CDC- Environmental isolate, hospital cooling tower drain ATCC Naegleria fowleri Lee Clinical isolate, cerebrospinal fluid ATCC A. polyphaga growth reached a maximum by day at C with total cell numbers reaching ± and decreasing to ± by day with % of amoebae in the cyst form. Growth of A. polyphaga was minimal at C with lysis occurring within several hours of incubation at C, consistent with expectations. For example, strains of A. polyphaga, Acanthamoeba castellanii and Acanthamoeba astronyxis have been reported to grow up to and C for strains Acanthamoeba culbertsoni and Acanthamoeba rhysodes (Griffin ). In contrast, there was no significant growth of H. vermiformis at C and lysis occured within several hours at C (Fig. b). Although H. vermiformis has been described to only growth at temperatures C (Gianinazzi et al. ), others have suggested that it can tolerate temperatures of over C (Kuchta et al. ; Rohr et al. ). Hence, temperature acclimation of H. vermiformis cells (rather than our isothermal culture at C) may provide different results to what we reported. Naegleria fowleri was able to grow at all temperatures investigated and also did not encyst in the presence of E. coli prey for the duration of the experiments, with most growth occurring in the first few days at, and C (Fig. c). Hence, conditions did not trigger encystment as seen in the other two amoebal strains, rather lysis occurred. In the absence of E. coli prey, all amoebal strains rapidly lysed from day to the last time point for each temperature, being %, % and % for A. polyphaga, H. vermiformis and N. fowleri, respectively. Figure a and Table show that, in the presence of A. polyphaga, both Leg. pneumophila Lp and Bloomington- grew significantly at, and C, but no growth was observed at C ( h Tn Tc = Æ and Æ, respectively). Interestingly, strains Lp and Bloomington- not only proliferated at, and C but also at C ( h Tn Tc = Æ and Æ, respectively) in the presence of N. fowleri (Fig. c). The ability of both strains to grow at C with N. fowleri as the host but not with A. polyphaga illustrates that host, bacterial strain and temperature play key roles in Leg. pneumophila proliferation. Further highlighting this is the observation that neither strain Lp nor Bloomington- grew in the presence of H. vermiformis, suggesting an incompatibility of this host as a vehicle for their proliferation (Fig. b, Tn Tc at h). Strain Chicago exhibited a Tn Tc ratio of at h for all amoebae hosts at all temperatures except at C in the presence of H. vermiformis (Tn Tc = Æ, P <Æ) and N. fowleri (Tn Tc = Æ, P <Æ), indicating a limited temperature growth range for this strain. The variability of Leg. pneumophila growth is further highlighted with strain Dallas E where Tn Tc ratio at all time points and temperatures for A. polyphaga indicated no growth of this strain (Fig. a, Tn Tc <). There were also differences in growth rates observed for strain Philadelphia- within each amoeba host; with Tn Tc ratio at h <Æ for each FLA (Fig. ). Significant growth of strain Philadelphia- was observed at h in the presence of H. vermiformis (Table, P < Æ). Furthermore, strain Philadelphia- displayed a higher growth rate at C at h (Fig. b, Tn Tc = Æ) compared with A. polyphaga (Fig. a, Tn Tc = Æ) and N. fowleri (Tn Tc = Æ). Proliferation of Legionella pneumophila strains within amoebae hosts at five temperatures Discussion To date, very few studies have addressed the growth potential of FLA in the presence of prey bacteria. Pickup et al. (a) compared growth of A. castellanii and H. vermiformis at C when fed either suspended or attached E. coli K, and both FLA had a preference for attached E. coli. Subsequent work also performed at C Letters in Applied Microbiology, ª The Society for Applied Microbiology

4 Host and temperature effects on Leg. pneumophila replication (a) Total cell numbers (b) Total cell numbers (c) Total cell numbers C C C C Time (days) Time (days) Time (days) Time (days) Figure Amoeba growth rates at various temperatures in the presence of prey bacteria. Acanthamoeba polyphaga (a), Hartmannella vermiformis (b) and Naegleria fowleri (c) were incubated with (grey bars) and without (white bars) Escherichia coli grown at,, and C. At designated time points, the total number of amoeba cells in trophozite (nonhatched) and cyst (hatched) forms in each well were quantified. Data were generated from two independent experiments. demonstrated that the same FLA grew at least % faster when fed live prey compared with heat-killed prey (Pickup et al. b). Because of the likely importance of FLA in shaping biofilm communities in drinking water systems (Huws et al. ), the interactions of FLA with both bacterial prey and potential human pathogens and their growth responses at different temperatures were investigated. In our study, it is unclear why Lp, a derivative of the Philadelphia- strain, would proliferate to high titres in the presence of A. polyphaga and N. fowleri while strain Philadelphia- displayed no significant growth in either hosts. Besides the fact that strain Lp is a rpsl (S ribosomal subunit protein S) thya (thymidylate synthetase) hsdr (endonuclease R type I restriction enzyme) derivative of Philadelphia-, there are no other genetic differences to explain the behaviours observed (Berger and Isberg ). Ratios of Philadelphia- Leg. pneumophila to A. polyphaga were increased to, and :, but no change in growth potential was observed (data not shown), indicating that lack of growth was not because of insufficient numbers of bacteria. In fact, Ohno et al. () reported decreased intracellular growth of Leg. pneumophila strains Lp and Suzuki within A. castellanii at cell ratios of and compared with to at C, further suggesting that lack of strain Philadelphia- proliferation, compared with its Lp derivative, is not attributable to a low bacterial inocula but rather a difference in genetic composition. Intriguingly, strain Philadelphia- was reported to grow significantly within Acanthamoeba lenticulata with a Æ% bacterial increase per amoeba observed (Molmeret et al. ) emphasizing the importance of testing various Letters in Applied Microbiology, ª The Society for Applied Microbiology

5 Host and temperature effects on Leg. pneumophila replication (a) (b) (c) Lp Lp Lp Ratio (Tn/Tc) Ratio (Tn/Tc) Ratio (Tn/Tc) Ratio (Tn/Tc) Ratio (Tn/Tc) Philadelphia- Philadelphia- Philadelphia- Bloomington- Bloomington- Bloomington- Chicago Dallas E Chicago Dallas E Time (h) Time (h) Time (h) Chicago Dallas E Figure Legionella pneumophila proliferation at various temperatures in the presence of amoebae. Acanthamoeba polyphaga (a), Hartmannella vermiformis (b) and Naegleria fowleri (c) were infected with different Leg. pneumophila strains at C (d), C (j), C (m), C ( ) and C ()). Legionella proliferation was determined at,, and h. Data are presented as a ratio of bacterial colony forming unit (CFU) at each time point (Tn) divided by the bacterial CFU levels in control wells (Tc). Data were generated from two independent experiments. *P < Æ; **P <Æ; P <Æ. Letters in Applied Microbiology, ª The Society for Applied Microbiology

6 Host and temperature effects on Leg. pneumophila replication Table Summary of Legionella pneumophila proliferation data Lp Philadelphia- Bloomington- Chicago Dallas E C C C C C C C C C C C C C C C C C C C C C C C C C ) * * n.d. ) ) ) ) n.d. ) à * n.d. ) ) ) ) n.d. ) ) ) ) n.d. Acanthamoeba polyphaga ) ) ) ) n.d. ) ) * ) n.d. ) ) ) ) n.d. ) n.s. à ) n.d. ) n.s. n.s. ) n.d. Hartmannella vermiformis Naegleria fowleri n.s. * ) ) ) ) ) ) à * * * * ) ) * ) ) ) n.s. ) ) ) n.d., No data; n.s., not significant; ), no growth observed (Tn Tc ). *P <Æ; P <Æ; àp <Æ. hosts suitable for legionellae amplification. In the same study, four Leg. pneumophila serogroup environmental isolates displayed large increases in intracellular growth within A. lenticulata ranging from to % with three serogroup clinical and environmental isolates exhibiting increases from ) to % (Molmeret et al. ). Dey et al. () showed that while A. castellanii and H. vermiformis were permissive to growth of Leg. pneumophila strains Paris, Lens and Philadelphia- at C, Willaertia magna was resistant to strain Paris growth but not the other two strains. Hence, data from the current and previous studies indicate that legionellae density in drinking water environments may vary in response to both the density and composition of amoebae present factors that may have significant downstream effects on legionellosis outbreaks. A further consideration is the transition of cells to an active or viable but nonculturable (VBNC) state, as described for numerous bacterial pathogens (Oliver ). For example, reactivation of VBNC Leg. pneumophila cells has been demonstrated within A. polyphaga and A. castellanii after starvation in tap water for more than days in the presence of chlorine disinfectant (Steinert et al. ; Garcia et al. ). In the current study, it is unclear whether any Leg. pneumophila strain entered the VBNC state because of the exposure to either low nutrient culture conditions for days or high or low incubation temperature. Nonetheless, culturability did not seem to affect a strain s ability to proliferate within amoebae. Amplification of Leg. pneumophila in drinking water systems is multifactorial, being dependent not only on the availability of suitable amoeba, ciliate and possibly nematode hosts but also environmental conditions conducive to the survival and growth of both host and bacteria. Although it is commonly assumed that all Legionella isolates from waters are potentially human pathogens, the current study highlights the difficulties in describing a virulent Legionella strain; a case made stronger by the fact that it is still unclear as to how many legionellae bacteria and in what delivered form will result in a disease outcome (Armstrong and Haas ; Lau and Ashbolt ). Infectivity is also known to change based on the incubation temperature of macrophage cells (Edelstein et al. ; Mauchline et al. ). The results presented highlight the growth dependence of legionellae to both host and temperature and support the case for the selection of virulent Leg. pneumophila strains associated with conditions expected with in-premise plumbing. As observed with just three different genera of FLA, the suitability and susceptibility of a host can have significant impacts on the ability of Leg. pneumophila to amplify. Furthermore, the bacterial strain-to-strain variability can also dictate if bacterial amplification will occur under specific conditions. Letters in Applied Microbiology, ª The Society for Applied Microbiology

7 Host and temperature effects on Leg. pneumophila replication Future studies will aim to determine the diversity and density of FLA that support Legionella spp. growth within drinking water systems and to better understand the ecological interactions between legionellae and their in situ hosts. All of which should shed light on how opportunistic pathogens are selected for and released into drinking waters. Acknowledgements The authors thank Drs Gene Rice and Mary Schoen for their critical review on this manuscript. The United States Environmental Protection Agency through its Office of Research and Development reviewed and approved this work for publication. References Alary, M. and Joly, J.R. () Risk factors for contamination of domestic hot water systems by legionellae. Appl Environ Microbiol,. Armstrong, T.W. and Haas, C.N. () Legionnaires disease: evaluation of a quantitative microbial risk assessment model. J Water Health,. Barker, J., Brown, M.R., Collier, P.J., Farrell, I. and Gilbert, P. () Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation. Appl Environ Microbiol,. Barker, J., Scaife, H. and Brown, M.R. () Intraphagocytic growth induces an antibiotic-resistant phenotype of Legionella pneumophila. Antimicrob Agents Chemother,. von Baum, H., Bommer, M., Forke, A., Holz, J., Frenz, P. and Wellinghausen, N. () Is domestic tap water a risk for infections in neutropenic patients? Infection,. Berger, K.H. and Isberg, R.R. () Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol,. Brieland, J.K., Fantone, J.C., Remick, D.G., LeGendre, M., McClain, M. and Engleberg, N.C. () The role of Legionella pneumophila-infected Hartmannella vermiformis as an infectious particle in a murine model of Legionnaire s disease. Infect Immun,. Cirillo, J.D., Cirillo, S.L., Yan, L., Bermudez, L.E., Falkow, S. and Tompkins, L.S. () Intracellular growth in Acanthamoeba castellanii affects monocyte entry mechanisms and enhances virulence of Legionella pneumophila. Infect Immun,. Craun, G.F., Brunkard, J.M., Yoder, J.S., Roberts, V.A., Carpenter, J., Wade, T., Calderon, R.L., Roberts, J.M. et al. () Causes of outbreaks associated with drinking water in the United States from to. Clin Microbiol Rev,. Dey, R., Bodennec, J., Mameri, M.O. and Pernin, P. () Free-living freshwater amoebae differ in their susceptibility to the pathogenic bacterium Legionella pneumophila. FEMS Microbiol Lett,. Edelstein, P.H., Beer, K.B. and DeBoynton, E.D. () Influence of growth temperature on virulence of Legionella pneumophila. Infect Immun,. Garcia, M.T., Jones, S., Pelaz, C., Millar, R.D. and Abu Kwaik, Y. () Acanthamoeba polyphaga resuscitates viable nonculturable Legionella pneumophila after disinfection. Environ Microbiol,. Gianinazzi, C., Schild, M., Zumkehr, B., Wuthrich, F., Nuesch, I., Ryter, R., Schurch, N., Gottstein, B. et al. () Screening of Swiss hot spring resorts for potentially pathogenic free-living amoebae. Exp Parasitol,. Griffin, J.L. () Temperature tolerance of pathogenic and nonpathogenic free-living amoebas. Science,. Hsu, B.M., Lin, C.L. and Shih, F.C. () Survey of pathogenic free-living amoebae and Legionella spp. in mud spring recreation area. Water Res,. Huang, S.W. and Hsu, B.M. () Survey of Naegleria and its resisting bacteria-legionella in hot spring water of Taiwan using molecular method. Parasitol Res,. Huws, S.A., McBain, A.J. and Gilbert, P. () Protozoan grazing and its impact upon population dynamics in biofilm communities. J Appl Microbiol,. Kuchta, J.M., Navratil, J.S., Shepherd, M.E., Wadowsky, R.M., Dowling, J.N., States, S.J. and Yee, R.B. () Impact of chlorine and heat on the survival of Hartmannella vermiformis and subsequent growth of Legionella pneumophila. Appl Environ Microbiol,. Lau, H.Y. and Ashbolt, N.J. () The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water. J Appl Microbiol,. Marciano-Cabral, F., Jamerson, M. and Kaneshiro, E.S. () Free-living amoebae, Legionella and Mycobacterium in tap water supplied by a municipal drinking water utility in the USA. J Water Health,. Mathys, W., Stanke, J., Harmuth, M. and Junge-Mathys, E. () Occurrence of Legionella in hot water systems of single-family residences in suburbs of two German cities with special reference to solar and district heating. Int J Hyg Environ Health,. Mauchline, W.S., James, B.W., Fitzgeorge, R.B., Dennis, P.J. and Keevil, C.W. () Growth temperature reversibly modulates the virulence of Legionella pneumophila. Infect Immun,. Molmeret, M., Jarraud, S., Mori, J.P., Pernin, P., Forey, F., Reyrolle, M., Vandenesch, F., Etienne, J. et al. () Different growth rates in amoeba of genotypically related environmental and clinical Legionella pneumophila strains isolated from a thermal spa. Epidemiol Infect,. Ohno, A., Kato, N., Sakamoto, R., Kimura, S. and Yamaguchi, K. () Temperature-dependent parasitic relationship between Legionella pneumophila and a free-living amoeba Letters in Applied Microbiology, ª The Society for Applied Microbiology

8 Host and temperature effects on Leg. pneumophila replication (Acanthamoeba castellanii). Appl Environ Microbiol,. Oliver, J.D. () Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol Rev,. Pickup, Z.L., Pickup, R. and Parry, J.D. (a) A comparison of the growth and starvation responses of Acanthamoeba castellanii and Hartmannella vermiformis in the presence of suspended and attached Escherichia coli K. FEMS Microbiol Ecol,. Pickup, Z.L., Pickup, R. and Parry, J.D. (b) Growth of Acanthamoeba castellanii and Hartmannella vermiformis on live, heat-killed and DTAF-stained bacterial prey. FEMS Microbiol Ecol,. Rohr, U., Weber, S., Michel, R., Selenka, F. and Wilhelm, M. () Comparison of free-living amoebae in hot water systems of hospitals with isolates from moist sanitary areas by identifying genera and determining temperature tolerance. Appl Environ Microbiol,. Steinert, M., Emody, L., Amann, R. and Hacker, J. () Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR by Acanthamoeba castellanii. Appl Environ Microbiol,. Steinert, M., Ockert, G., Luck, C. and Hacker, J. () Regrowth of Legionella pneumophila in a heat-disinfected plumbing system. Zentralbl Bakteriol,. Stout, J.E., Yu, V.L., Yee, Y.C., Vaccarello, S., Diven, W. and Lee, T.C. () Legionella pneumophila in residential water supplies: environmental surveillance with clinical assessment for Legionnaires disease. Epidemiol Infect,. Thomas, J.M. and Ashbolt, N.J. () Do free-living amoebae in treated drinking water systems present an emerging health risk? Environ Sci Technol,. Thomas, V., McDonnell, G., Denyer, S.P. and Maillard, J.Y. () Free-living amoebae and their intracellular pathogenic microorganisms: risks for water quality. FEMS Microbiol Rev,. Yoder, J., Roberts, V., Craun, G.F., Hill, V., Hicks, L.A., Alexander, N.T., Radke, V., Calderon, R.L. et al. () Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking United States,. MMWR Surveill Summ,. Letters in Applied Microbiology, ª The Society for Applied Microbiology