ORIGINAL ARTICLE Effect of thermal treatment on free-living amoeba inactivation S. Cervero-Arag  o 1 , S. Rodr  ıguez-Mart  ınez 1 , O. Canals 2 , H. Salvad  o 2 and R. M. Araujo 1 1 Departament de Microbiologia, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain 2 Departament de Biologia Animal, Universitat de Barcelona, Barcelona, Spain Keywords Acanthamoeba, amoeba, dialysis, disinfection, Hartmannella, thermal. Correspondence Rosa Araujo, Departament de Microbiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain. E-mail: raraujo@ub.edu 2013/1617: received 8 August 2013, revised 11 October 2013 and accepted 23 October 2013 doi:10.1111/jam.12379 Abstract Aims: To evaluate the effect of temperature on two amoeba strains of the genera Acanthamoeba and two amoeba strains of the genera Hartmannella separately treated depending on their life stage, trophozoite or cyst, when cells are directly exposed under controlled conditions. Methods and Results: For thermal treatments, three temperatures were selected 50, 60 and 70°C, and a microcosm was designed using dialysis bags. The inactivation of each strain was determined using a method based on the most probable number quantification on agar plates. The results showed that for all amoeba strains, thermal treatment was more effective against trophozoites compared with cyst stages. The inactivation patterns showed statistical differences between the two genera analysed at temperatures above 50°C. The effectiveness of the thermal treatments at 60 and 70°C was higher for both life stages of Hartmannella vermiformis strains compared with Acanthamoeba strains, being the most resistant Acanthamoeba cysts. Conclusions: Free-living amoebae have been isolated in a wide range of environments worldwide due to their capacity to survive under harsh conditions. This capacity is mainly based on the formation of resistant forms, such as double-walled cysts, which confers a high level of resistance as shown here for thermal treatments. Significance and Impact of Study: Free-living amoebae survival can promote a rapid recolonization of drinking water systems and is a likely source of emerging opportunistic pathogens such as Legionella. Because of that a better understanding of the factors that affect micro-organism inactivation in water systems would allow more efficient application of disinfection treatments. Introduction Free-living amoebae (FLA) are widespread protozoa and normal inhabitants of soil, and in particular, fresh-water sources (Rodriguez-Zaragoza 1994). These aquatic sources include environmental water systems such as lakes, ponds (Barbeau and Buhler 2001) and man-made water systems, including water supplies, hospital water networks, cooling towers, swimming pools and hydrotherapy baths (Schmitz-Esser et al. 2008; Thomas et al. 2010). Amoebae are a group of unicellular micro-organisms that have a two-stage life cycle: a replicating and feeding trophozoite stage and a latent cyst stage. In the trophozoite stage, FLA are important predators of prokaryotic and eukaryotic micro-organisms with much influence on the composition of the microbial community feeding on bio- films and detritus (Rodriguez-Zaragoza 1994; Ramaley et al. 2001; Schmitz-Esser et al. 2008). Specifically, they establish symbiotic relationships with other microbes including bacteria of the genera Legionella, Mycobacte- rium, Campylobacter and Listeria, which, apart from being a nutritional source, are able to survive amoeba digestion and multiply within them (Greub and Raoult 2004; Schmitz-Esser et al. 2008). Moreover, some FLA species can infect humans and other mammals, causing serious illnesses like ocular and central nervous system Journal of Applied Microbiology © 2013 The Society for Applied Microbiology 1 Journal of Applied Microbiology ISSN 1364-5072