Filtration procedure and isolation of bacteria
A portion of 100 ml of ATSW was filtered through seven 0.20 µm pore cellulose nitrate membranes (Nalgene 0.2 Analytical Filter Unit; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Each membrane was plated on differential and selective media (Tryptic Soy Agar Blood BD, Chocolate agar BD, McConkey agar Oxoid, Mannitol Salt agar Oxoid, Sabouraud dextrose agar Oxoid, Colistin Nalidixic acid agar Biolife, Schaedler agar BD) incubated in aerobic condition at 37°C for 2-3 days and in anaerobic condition at 37°C for 3 days. For autumn sampling, the bacterial plates were also incubated at 55 °C in order to mimic the temperature of thermal water. Microbial growth was then recorded and each pure culture was subsequently prepared and stored at -80° C. At the same times, 300 ml of water sample was filtered through 0.20 µm pore cellulose nitrate membrane and the filter was soaked in 5ml of 1X phosphate-buffered saline (PBS, containing 140mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 18mM KH2PO4, pH 7.4) in a centrifugal tube (Di Natale et al., 2018). Vortexing was performed for 2–3 min with a vortex touch mixer (HeidolphREAX 2000) to evaluate the presence ofLegionella pneumophila antigen (ALERE BINAXNOW® Legionella antigen card) following the instructions of the manufacturer.