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.