*4.3. Laboratory Procedure*

Briefly, the water samples were concentrated 100-fold by filtration through a 0.2-μm polycarbonate filter (Millipore, Billerica, MA, USA). The filter membrane was aseptically placed in one of the bottom corners inside the stomacher bag and rubbed with the finger and thumb of one hand for 1 min with 10 mL Page solution (pH 6.8) to detach the bacteria. A 0.2-mL volume of the concentrated sample was spread on duplicate plates of MWY or BCYE α agar. The plates were incubated at 36 ◦C in a humid 2.5% CO2 chamber and examined after 3, 6, and 10 days of incubation. Suspected colonies were subcultured on blood and BCYE α agar.

#### *4.4. Identification of Legionella spp.*

The presence of background flora was measured through semiquantitative counting. According to the visual density of the colonies spread onto the plate, four categories were determined, where zero represented no background flora and 3+ massive contamination (see supplementary materials). Colonies grown on MWY or BCYE α agar were subsequently identified by agglutination test (*Legionella* latex test; Oxoid). This test allows the separate identification of *L. pneumophila* Serogroup 1 and Serogroups 2 to 14 and detection of seven other species of *Legionella* (polyvalent). Colonies identified as *L. pneumophila* Serogroup 2 to 14 were further tested with *Legionella* agglutination latex reagents (Pro-Lab Diagnostics, Richmond Hill, Canada), which are intended for the identification of a single *L. pneumophila* sero group. Colonies not identified by the agglutination test were tested by polymerase chain reaction (in-house PCR) for the detection of the genus *Legionella*. This PCR assay utilizes specific primers to amplify the 16S rRNA gene of *Legionella* spp. [21]. The plate showing the highest number of confirmed colonies was used to estimate the number of *Legionella* spp. in the original sample (report). Concentrations of *Legionella* spp. in water samples are expressed as colony forming units per liter (CFU/l). According to this method, the lower limit of detection (LOD) is 50 CFU/L.
