**2. Results**

All results are presented, first of all, by considering the general contamination found in SHWOs and, then later, by dividing *Legionella* contamination between hot- and cold-water samples. The data about *Legionella* concentration are expressed in Log10 cfu/L (Log cfu/L).

The same method is used to correlate the microbial contamination found with temperature values measured in SHWOs and their distribution between hot- and cold-water samples.

#### *2.1. Legionella Contamination in SHWOs*

The results of mean *Legionella* concentrations found in 52 SHWOs from 11 hospitals are shown in Figure 1. Seven of the hospitals showed *Legionella* contamination (7/11, 63.6%), where three (3/7, 42.8%) of them showed values over the level of risk indicated by Italian Guidelines, that is, at >100 cfu/L (>2 Log cfu/L) [12]. The contamination was found in hot or cold samples and in both water distribution systems for each hospital.

**Figure 1.** Mean *Legionella* concentrations in 11 hospitals.

The results of microbial contamination from 669 SHWO samples show that *Legionella* was detected in 293/669 (43.8%) of samples.

An analysis of *Legionella* contamination was then performed between hot-water (*n* = 427) and cold-water samples (*n* = 242). The di fferences between the numbers of hot- and cold-water samples were linked to a higher concentration of *Legionella* found in hot-water samples which, according to the suggestions of the Italian Guidelines, requires resampling from the same positive outlets [12].

In particular, the analysis of results between hot- and cold-water distribution systems showed 190/427 (44.5%) of positive hot-water samples and 103/242 (42.6%) positive cold-water samples. The positive samples over the *Legionella* level of risk (>2 Log cfu/L) were 140/190 (73.7%) for hot- and 70/103 (68.0%) for cold-water samples.

In Table 1, the data of mean temperature and disinfectant residue with relative minimum (min) and maximum (max) values, the percentage of *Legionella* positive samples, mean concentrations, and the range of contamination (min–max) found in hot and cold-water samples are listed, respectively. Data about temperature, disinfectant residues, and *Legionella* concentration are expressed as mean ± Standard Deviation (SD).

**Table 1.** Surgical Handwashing Outlet (SHWO) microbiological and physical-chemical parameters measured: hot- vs. cold-water samples.


No significant di fference (*p* = 0.34) is found between hot and cold samples concerning *Legionella* levels.

Regarding the *Legionella* isolates distribution in SHWOs between hot- and cold-positive samples, the results showed samples contaminated only by *Legionella pneumophila (L. pneumophila)*, samples contaminated only by *Legionella* non-*pneumophila* species (other *Legionella* spp.) and others contaminated by both species. Significant di fferences (*p* = 0.001), obtained with the statistical χ2 test, were found concerning the *Legionella* spp. distribution between hot and cold samples as follows: in hot-water samples, the main isolate belonged to *L. pneumophila* 123/190 (64.7%), followed by samples with both species (*L. pneumophila* and other *Legionella* spp.) 41/190 (21.6%) and, finally, by 26/190 (13.7%) showing only the presence of other *Legionella* spp. In cold-water samples, we found the same trend, with 44/103 (42.7%) of samples with *L. pneumophila*, 30/103 (29.1%) contaminated by both species, and finally, 29/103 (28.1%) with only other *Legionella* spp. The isolates of *L. pneumophila* were identified by an agglutination test as belonging to serogroups 1, 3, 4, 6, and 8. The typing of *Legionella* non-*pneumophila* species by *mip* gene sequencing, indicated the presence of *Legionella anisa* (*L. anisa)*, *Legionella rubrilucens (L. rubrilucens), Legionella tauriniensis (L. tauriniensis), Legionella nautarum (L. nautarum),* and *Legionella steelei (L. steelei).*

The study of *Legionella* isolates in terms of mean concentration±standard deviation (Log cfu/L±SD) between hot- and cold-positive samples is presented in Table 2. Multiple comparisons were performed between isolates found in hot- and cold-water samples (horizontal lines), while the comparison between hot- and cold-water samples for each type of *Legionella* isolate is shown in the columns. High *L. pneumophila* concentrations were found in hot-water samples (2.92 ± 1.08 Log cfu/L) with significant di fference compared to samples colonized by only other *Legionella* spp. (*p* = 0.03) and with respect to cold-water samples (*p* = 0.008). In cold-water samples, despite a high other *Legionella* spp. mean concentration (2.47 ± 0.72 Log cfu/L), a significant di fference was found only with respect to samples colonized by both species (*p* = 0.0046).


**Table 2.** *Legionella* isolate mean concentration comparison in SHWOs: hot- vs. cold-water samples.

> Values are statistically significant at *p* ≤ 0.05.

#### *2.2. Legionella Contamination in Relation to Water Temperature*

\*

Regarding the temperature measured between hot and cold samples, we found a range between 21.9–60.1 ◦C (mean value of 47.7 ◦C) and a range between 9.2–44.7 ◦C (mean value of 19.1 ◦C) for hot and cold samples, respectively.

The *Legionella* contamination found considering all SHWOs samples was distributed in four ranges of temperature, which were linked to relevant considerations about the environment of *Legionella* as follows:


A multiple comparison was performed between each range by an ANOVA test, showing significant differences, as indicated in Table 3 with the (\*) symbol.



\* Values are statistically significant at *p* ≤ 0.05.

The contamination of samples in relation to the temperature measured during the sampling reveals that the main *Legionella* positive samples (47.1%) were in the third range (III), which was also the main contaminated source in terms of *Legionella* mean concentration (2.17 Log cfu/L). By contrast, the lowest percentage of positive samples (23.2%) and mean concentration (1.64 Log cfu/L) were found in the fourth range (IV).

In Figure 2, the distribution of mean *Legionella* concentration in relation to temperature values measured is represented, with hot and cold samples separately considered, in ranges between 21.9–60.1 ◦C (mean value of 47.7 ◦C) and between 9.2–44.7 ◦C (mean value of 19.1 ◦C).

**Figure 2.** Mean *Legionella* concentration distribution in relation to water sample temperatures measured (◦C).

An analysis of the results considering only samples in the range of 21–45 ◦C (e.g., the range for SHWO mixed water) showed 98/427 (23.0%) and 81/242 (33.5%) contaminated hot- and cold-water samples, with mean concentrations of 2.12 ± 1.22 Log cfu/L and 1.87 ± 0.92 Log cfu/L, respectively.

Considering only *Legionella*-positive samples, we found 52/98 (53.0%) in hot water—respectively 39/81 (48.1%) in cold water—with mean *Legionella* concentration higher in hot (2.94 ± 1.17 Log cfu/L) than cold samples (2.60 ± 0.87 Log cfu/L). The nonsignificant difference was found using the Mann–Whitney test (*p* = 0.22).

#### *2.3. Legionella Contamination before and after the SHWO Replacement*

In three hospitals (called 1, 8, and 11), following renovation works, replacement of sensor-activated faucets with TMVs by clinical valves without TMVs was carried out. The reassessment of *Legionella* contamination on the same SHWOs after replacement permitted us to observe changes in the *Legionella* concentration. Analyzing the contamination found in 110 of 669 total samples collected in these hospitals, we compared the contamination before (*n* = 55) and after (*n* = 55) replacement. As shown in Table 4, we observed a significant decrease in terms of *Legionella* contamination (*p* = 0.001) with the same significant trend in each hospital, other than with an increase of hot-water temperature and a consequent decrease of *Legionella* levels.


**Table 4.** Mean *Legionella* concentration in three hospitals before and after the replacement of sensor-activated faucets with Thermostatic Mixer Valves (TMVs).

> \* Values are statistically significant at *p* ≤ 0.05.

#### *2.4. P. aeruginosa Contamination in SHWOs*

The data about *P. aeruginosa* contamination indicated that 27/669 (4.0%) samples were contaminated. Considering the contamination in relation to hot- and cold-water circuits, we found a higher contamination in cold-water samples compared to hot-water samples: 22/242 (9.0%) and 5/427 (1.2%), respectively. However, the low number of positive samples did not permit us to find a statistical correlation between the data analyzed (*p* = 0.65).

#### *2.5. Disinfectant Residue Analysis*

Concerning the disinfectant residue measured, the mean concentration of hydrogen peroxide (H2O2) component was about 2.5 mg/<sup>L</sup> and 10 mg/<sup>L</sup> in cold- and hot-water samples, respectively. Although only the hot water network is treated with hydrogen peroxide/Ag<sup>+</sup> (H2O2/Ag+), we found the presence of disinfectant residue in all cold-water samples, with a range between 0.5–5 mg/L.
