*2.2. Cultivation-Dependent Analysis*

A total of 72 water samples were collected in sterile 1L plastic bottles after a brief flow time (2–3 min). One liter each of cold and hot water was collected for Heterotrophic Plate Counts (HPC) and again for *Legionella* counts from the hospitals. To neutralize residual free chlorine, 0.5 mL of 0.1 N sodium thiosulphate was added to the sterile bottles for *Legionella* plate counts [31].

For HPC, yeas<sup>t</sup> agar plates (Ant. Er. CP63.1, Carl Roth, Karlsruhe, Germany) were used according to the manufacturer's instruction for each type of water in two sets of triplicates. First, 0.1 mL of the water sample was spread on each agar plate using a sterile glass spreader. The plates were inverted and incubated; three plates each were incubated at 37 ◦C for 48 h and at 25 ◦C for 72 h.

Concerning *Legionella* plate counts, 100 mL of water sample was filtered onto a membrane filter (membrane solutions, pore size 0.45 μm, diameter 47 mm, Whatman, England) using sterile filtration unit (Nalgene, Germany). A vacuum of 200 mbar was applied. After filtration, 30 mL of acid buffer (3.9 mL of 0.2 mol/L HCl and 25 mL of sterile 0.2 mol/L KCl were mixed, pH 2.2 ± 0.2) was placed on top of the membrane filter and left for 5 min. The filter was rinsed with 20 mL Page's saline (1.20 g NaCl, 0.04 g MgSO4·7H2O, 0.04 g CaCl2·2H2O), and 1.42 g Na2HPO4 and 1.36 g KH2PO4 were dissolved in ten liters of distilled water and autoclaved. The membrane filter was removed from the filtration unit with sterile forceps and placed onto the relevant agar plate. Duplicates of BCYE and GVPC (M809, Himedia, India) agar plates were used according to the manufacturer's instruction. The plates were incubated inverted at 37 ◦C for 10 days. Plates were checked for growth twice (after three and ten days). Final counts of the triplicates were done after ten days with descriptions of the colonies.

Also, a total of 1136 biofilm swabs from the anterior surfaces of faucets, showerheads or shower hoses in all hospital wards, mainly in areas occupied by high-risk patients (intensive care unit, operating theater, oncology and surgery wards), was obtained using transport medium (Copan, Culture swab transport system, Italy). Swabs for *Legionella* identification were processed immediately by culturing on GVPC agar (medium M809, Himedia, India) based on ISO 11731:2004 [17].

#### *2.3. Cultivation-Independent Analysis (16S rDNA PCR)*

A total of 72 samples (five liters) each of cold and hot water was collected from the main water source from each site for DNA extraction. Water samples were filtered onto sandwich membrane filters composed of nucleopore-filter (Nuclepore Track-Etch Membrane, MB 90 mm, 0.2 μm, Whatman, UK) and glass fiber-microfilter (GF/F) (GFF, 90 mm, Whatman, UK). Also, a total of 225 biofilm swabs from the anterior surfaces of faucets, showerheads or shower hoses was obtained for DNA extraction using sterile cotton swabs (Cotton Tipped Applicator, Beijing, China).

For the extraction of DNA from the filter sandwiches and the swabs, a modified DNeasy protocol (Qiagen kit No. 69506, Hilden, Germany) was used. Briefly, sandwich filters were cut into small pieces and incubated with enzymatic lysis bu ffer (20 mM Tris-HCl, 2 mM EDTA, 1.2% Triton X-100 [pH 8.0]) containing 10 mg/mL lysozyme for 60 min in a 37 ◦C water bath. After the addition of AL bu ffer from the kit, the samples were incubated at 78 ◦C in a shaking water bath for 20 min. After filtration through a cell strainer, i.e., 100 μm (DB falcon 352360, Corning, Glendale, AZ, USA), absolute ethanol was added to the filtrate (ratio of filtrate to ethanol is [2:1]) and the mixture was applied to the spin column of the kit. After this step, the protocol was followed according to the manufacturer's instructions.

Three di fferent PCRs were carried out as follows: (i) for the detection of any bacteria, the bacterial common 16S rRNA gene primers (Com), (ii) for *Legionella* genus-specific primers (Lgsp) and (iii) for *L. pneumophila* species-specific primers (Lp1) were applied [32]. Each PCR reaction was carried out using 3 μL (1 ng/μL) of DNA template in a final volume of 25 μL. Amplification was achieved using PCR-ready Master Mix (GoTaq, Green Master Mix, Promega, Madison, WI, USA).

To test the specificity of *L. pneumophila* primers and confirm species identity, six isolates were identified by amplifying and sequencing an internal fragment of the 16S rRNA gene according to Senderovich et al. [33]. The obtained sequences were compared using the NCBI service to certain closest relatives. The sequences were submitted to the GeneBank database (KX778102-KX778107). Sequencing of the 16S rRNA gene of the six isolates confirmed the presence of *L. pneumophila* (≥99.8% 16S rRNA gene similarities).

#### *2.4. Sero-Grouping of Legionella Isolates*

The serogroups of the 180 *L. pneumophila* isolates were identified by an agglutination test using *Legionella* Latex (Oxoid DR0800, Basingstoke, UK). Using this test, the isolates were sero-grouped as Sg1 and Sg 2–14. Moreover, 47 isolates were sent to the National Reference laboratory for *Legionella* infections in Dresden for analysis by monoclonal antibody subgrouping [34].

#### *2.5. Genotyping of L. pneumophila Isolates*

For molecular typing of *L. pneumophila* at the strain level, MLVA-13 (MLVA-12 plus 1 additional locus from MLVA-8) designated as (MLVA-8(12)) analysis was performed for 180 isolates. DNA extraction was done either directly from living biomass using (Qiagen kit No. 69504, Hilden, Germany) according to the manufacturer protocol, or from biomass on FTA cards (Whatman, Sigma-Aldrich, Darmstadt, Germany).

For DNA extraction from the FTA cards, the area of the card containing the biomass was punched into 3 mm circular pieces. The pieces were transferred to 0.5 mL sterile water (Roth, Karlsruhe, Germany), incubated for 3 min at room temperature and vortexed three times (after water addition, after 1 min and after 3 min incubation). The FTA punch was removed and 1× Tris-EDTA buffer (Sigma-Aldrich, Darmstadt, Germany) was added to the water to preserve the DNA from degradation. More details on the FTA technology are given by Rajendram et al. [35]. DNA was finally quantified by Nanodrop spectrophotometer (NanoDrop, Thermo Scientific, Dreieich, Germany). MLVA-8 and MLVA-12 molecular genotyping assays by multiplex PCR and capillary electrophoresis were carried out for all isolates, as detailed by Pourcel et al., Sobral el al., Visca et al. and Pecellin [28,29,36,37].

For comparison, a subset of strains representing all MLVA-genotypes was characterized by sequence-based typing (SBT) [38]. In addition, several *L. pneumophila* (Lpn) reference strains were used to generate MLVA-8(12) profiles for comparison and interpretation of the results; these reference strains were Lpn str. Philadelphila-1 (ATCC 33152T) [39], Lpn str. Paris (CIP 107629) [40], Lpn str. Bloomington-2 (ATCC 3315) and Lpn str. Corby (NC\_009494) [41].
