*4.4. Study Design*

This study was conducted in two experimental phases designated WTP1 and WTP2 in relation to the timing of the introduction of WTP 828, the renovation or enlargement works conducted in the buildings and the acquisition of a new risk assessment plan. The data collected during the WTP1 and WTP2 phases were then compared to evaluate differences in the efficacy of the WTP 828 treatment in the HWNs of the three buildings.

These data were then compared with the data obtained during disinfection with the ClO2 mixture (i.e., ClO2 mixture versus WTP1 phase and ClO2 mixture versus WTP2 phase) to assess the effects of WTP 828 on *Legionella* contamination.

The details of the study period for each building are described below:


During the WTP1 phase, disinfection with WTP 828 started in Building 2 in October 2013 and in some locations within Buildings 1 and 3, which were under construction or undergoing expansion in this period.

Sampling of hot water systems was performed according to the risk assessment plan, which was approved by the MCH Health Director and the Local Authority. There were 29 sampling points spread throughout the three buildings among consulting and diagnostic rooms, wards, common areas, and in-patient rooms, which were monitored every four months on a rotational basis. During this phase, a total of 53 samples were subjected to microbiological analysis for detection of *Legionella, P. aeruginosa* and heterotrophic plate count (HPC) bacteria at 36 ◦C, and data were collected.

During the WTP2 phase, renovation works of Buildings 1 and 3 were completed (January 2015 and April 2015, respectively). WTP 828 treatment was extended to all parts of these buildings and, based on preliminary results regarding WTP 828 efficacy (WTP1 phase), a new risk assessment and monitoring plan were adopted.

In accordance with Italian Guidelines [7], sampling points were chosen at the following three locations: in the vicinity of, mid-way to, and away from the technical room. The location of the sampling points took into account the size of the building, the number of in-patient rooms, the health services provided, the risk of patient, and worker exposure to bacteria and epidemiological data.

Every month, samples were collected from the technical room: one from the aqueduct, two from the cold water reserves, one downstream of the general softener treatment, one from a tap water output, and three from the hot water return lines (1a for Building 1, 1b for Building 2, and 1c for Building 3), and from another 55 sampling points in offices, consulting and diagnostic rooms, wards, common areas, and in-patient rooms (63 points in total). Despite the large number of in-patient rooms, the alternating sampling method enabled sampling of almost all in-patient rooms in the three buildings.

The increased time of monitoring (from once every four months to monthly), extension of the disinfection treatment, and development of a final MCH structure permitted the study of the modulation of microbiological and physical-chemical parameters in a total of 296 hot water and 65 cold water samples.

#### *4.5. Sample Collection and Microbiological Analysis*

Hot water and cold water (2 L) were collected in post-flushing modality (running water for 1 min) in sterile polytetrafluoroethylene (PTFE) bottles containing a sodium thiosulfate solution (10%, v/v). Microbiological analyses were performed in accordance with ISO11731:2017 [69] to detect and enumerate *Legionella*. During *Legionella* surveillance, according to Italian Guidelines [7], the level of risk took into account the concentration of bacteria and percentage of positive samples.

Samples were concentrated using 0.22 μm polycarbonate pre-sterilized filter membranes (Sartorius Stedim Biotech, Göttingen, Germany).

The concentrated samples (filtered, F) were then heated (for 30 min at 50 ◦C) to inhibit interfering microbiota (heated, H). Then, 0.1 mL of the untreated sample (UN) and 0.1 mL of each F and H sample were spread in duplicate onto GVPC agar plates (*Legionella* GVPC selective medium, Thermo Fisher Scientific, Oxoid Ltd., Basingstoke, UK), and incubated at 35.5 ◦C in a humid (2.5% CO2) environment.

The plates were examined after four, eight, and 14 days, and colonies with a typical *Legionella* morphology (presumptive) were enumerated and confirmed by sub-culture on BCYE agar with and without cysteine. The isolates that grew on BCYE but failed to grow on the cysteine-free medium were verified serologically by an agglutination test (*Legionella* latex test kit; Thermo Fisher Scientific, Oxoid Ltd.). The data are expressed as the mean concentration ± standard deviation (SD) of the log10 colony forming units (CFU) per liter of water (log10 CFU/L) including all samples analyzed (positive + negative). The detection limit of the culture technique was 50 CFU/L. The samples with a value of <50 CFU/L were considered negative according to ISO 11731:2017 [69].

Other microorganisms can a ffect the growth of cultivable *Legionella*, and the samples were simultaneously analyzed for the presence of *P. aeruginosa*, a known competitor of *Legionella* that inhibits its growth on medium [70]. The analyses were performed according to UNI EN ISO 16266:2006 [71] using a selective Pseudomonas agar (Biolife, Milan, Italy). The detection limit of the culture technique was 1 CFU/100 mL.

The heterotrophic plate count (HPC) at 36 ◦C was used as an indicator of the actual level of bacterial contamination at the sampling points. The HPC is a useful indicator of increased microbial growth, increased biofilm activity, extended retention times, water stagnation, or breakdown of the integrity of the system [33,72]. The analyses were performed using a standard plate method based on tryptic glucose yeas<sup>t</sup> agar (Biolife) in accordance with UNI EN ISO 6222:2001 [73]. The data are expressed as the mean concentration ± SD of the log10 CFU per milliliter of water (log10 CFU/mL) including all samples (positive + negative).

The detection limit of the culture technique was 1 CFU/mL.

## *4.6. Legionella Typing*

Colonies identified by the agglutination test as belonging to the genus *Legionella* were subsequently analyzed by DNA sequencing. In particular, all strains identified as *L. pneumophila* were analyzed by sequence-based typing (SBT) to determine the sequence type (ST); strains identified as *Legionella* species were analyzed by *mip* sequencing. Genomic DNA was extracted from cultures using the InstaGene Purification Matrix (Bio-Rad, Hercules, CA, USA). SBT was performed according to an ELDSNet protocol (http://bioinforatics.phe.org.uk/legionella/legionella\_sbt/php/sbt\_homepage.php). The protocol was based on the sequencing of seven genes (flaA, pilE, asd, mip, mompS, proA, and neuA) and on the assignment of a ST allelic profile by the ELDSNet database (http://www.hpabioinformatics. org.uk/cgibin/legionella/sbt/seq\_assemble\_legionella1.cgi).

The strains that were serotyped by agglutination as *L. species* were then genotyped by *mip* gene amplification via the polymerase chain reaction (PCR) using degenerate primers, as described in 1998 by Ratcli ff et al. [74] and modified by M13 tailing to avoid noise in the DNA sequence [75]. Gene amplification was carried out in a 50 μL reaction volume containing DreamTaq Green PCR Master Mix 2x (Thermo Fisher Scientific, OxoidLtd., Basingstoke, UK) and 40 pmol of each primer; 100 ng of DNA extracted from the presumptive colonies of *Legionella* was added as template. The same amounts

of DNA from *Legionella pneumophila* (*L. pneumophila*) type strain EUL00137 provided by the European Working Group for *Legionella* Infections [76] and fetal bovine serum were used as positive and negative controls, respectively.

Following purification, DNA was sequenced using BigDye Chemistry and analyzed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Specifically, *mip* amplicons (661–715 bp) were sequenced using M13 forward and reverse primers (M13 FW, 5-TGTAAAACGACGGCCAGT-3; M13 RW, 3-CAGGAAACAGCTATGACC-5) to obtain complete coverage of the sequenced region of interest. Raw sequencing data were assembled using CLC Main Workbench 7.6.4 software (https://www.qiagenbioinformatics.com/). The sequences were compared with sequences deposited in the *Legionella mip* gene sequence database using a similarity analysis tool (http://bioinformatics.phe.org.uk/cgi-bin/legionella/mip/mip\_id.cgi). The identification at the species level was conducted based on 98% similarity to a sequence in the database [77].

#### *4.7. Physical and Chemical Parameters of Water*

The physical and chemical parameters of water were analyzed only during the WTP2 phase, before this phase the hospital did not have any data on water quality as prescribed by WHO [49].

Cold water samples (1 L) were collected from each of the following locations: the aqueduct, water reserves, softener, and tap water output. Hot water samples (1 L) were collected from each of the three hot water return lines and distal outlets. The pH, hardness (◦f), conductivity (μS/cm), turbidity (nephelometric turbidity units), total iron content (mg/L), total phosphorus content (mg/L of P2O5), and Ag+ content (μg/L) were monitored monthly during the session sampling.

The analysis of total iron and phosphorus content (orthophosphate, condensed phosphate, and organic phosphate) allowed us to monitor the maintenance of anti-scale and corrosion treatment.

Temperature (◦C) and residual WTP 828 levels [the peroxide component (mg/L)] were measured and recorded at distal outlets weekly in each building. WTP 828 (peroxide component) was measured using an MQuant™ Peroxide Test (Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions.

Other parameters were measured using different techniques according to standardized APAT CNR IRSA methods [78].

In our study the disinfection treatment was performed by a disinfectant based on H2O2/Ag<sup>+</sup>, therefore the dosage of DBPs release in water is not necessary. The chemical water compounds measured are listed in Table 4. The results are expressed as the mean value ± SD.
