3. Studies of Water Dispenser
Considering the contamination found in the previous study 
, in relation to HPCs at 36 °C and 22 °C, P. aeruginosa
and other pathogenic bacteria, Table 1
displays the status of MWDs’ contamination at the beginning of the study (2017) and the mean contamination found during the period of the study.
Table 1. MWDs’ mean contamination ± standard deviation (cfu/mL ± SD) found at the beginning of the WSP implemented in 2017 until today (2021), with respect to mean contamination found in the previous study (Girolamini et al., 2019), from 2015 to 2017.
|MWDs’ Mean Contamination ± Standard Deviation (cfu/mL ± SD)
||Girolamini et al., 2019 
|Present Study (2017–2021)
|HPCs 36 °C
||1852 ± 0.67
||1014.99 ± 2637.63
||321.67 ± 802.31
||39.05 ± 86.37
||124.83 ± 766.07
||30.02 ± 77.62
|HPCs 22 °C
||2437 ± 0.56
||686.25 ± 1952.55
||453.58 ± 1442.75
||69.78 ± 374.89
||161.50 ± 776.76
||91.75 ± 469.23
||37.4 ± 0.81
||9.48 ± 36.86
||20.81 ± 59.50
||2.18 ± 20.63
||5.04 ± 27.58
||0.67 ± 5.03
||3.3 ± 0.20
In detail, a total of 477 water samples were collected from 57 MWDs and analyzed for HPCs at 36 °C and 22 °C and for pathogenic bacteria, Enterococcus
spp., P. aeruginosa
, Escherichia Coli
, Staphilococcus aureus
and Clostridium perfringens
The overall results showed that the samples were more contaminated by HPCs at 36 °C and 22 °C; instead, P. aeruginosa was detected in 44/477 (9.2%) samples. Total and fecal coliforms, E. coli, S. aureus, and C. perfringens were not detected in any of the investigated samples. The MWDs’ mean contamination found, shown in Table 1, displayed a continuous decrease during the years of the study. The HPCs at 36 °C and 22 °C data were studied to understand the trend of bacteriological contamination. The analysis described below was not performed for other pathogenic parameters found, due to the sample size of data being too small for statistical reporting and for other microbiological parameters never detected in MWDs during the period.
The positive samples were analyzed with respect to the reference values prescribed by the Italian regulations.
Regarding HPCs at 36 °C and 22 °C, the following microbiological profiles were recorded: a total of 187/477 (39.2%) samples, with a range of contamination of 21–14,940 cfu/mL, and 94/477 (19.7%) samples, with a range of contamination of 104–10,960 cfu/mL, exceeded the regulation limit values of 20 cfu/mL and 100 cfu/mL, respectively, for HPCs at 36 °C and 22 °C.
The contamination found was studied also in relation to the temperature measured, considering the temperature as one of the factors that could influence the bacterial growth. According to the Guidelines for Drinking-Water Quality, the reference water temperature was between the range of 12–20 °C, with a limit not to exceed set at 25 °C 
. In particular, in our study, 292/477 (61.2%) samples showed temperature values ≤ 25° C and 185/477 (38.8%) samples showed values > 25° C. The samples showed a minimum value measured of 7.2 °C and a maximum of 32.4 °C, with a median value of 24.1 °C.
shows how most samples were centered around the median value of 24.1 °C, a temperature value close to the reference limit threshold of 25 °C 
Figure 2. Temperature-based distribution, with respect to the interval between 7.2 °C and 32.4 °C and the median value of 24.1 °C (bold row).
The HPCs’ contamination found (for both 36 °C and 22 °C) were separated into two groups based on the temperature values above and below the 25 °C temperature threshold, and compared with each other. In both cases there was no significant difference between the groups with values above and below 25 °C, with a p-value (p) = 0.56 and p = 0.18 for HPCs at 36 °C and 22 °C, respectively.
Moreover, data were evaluated to find a possible correlation between HPCs at 36 °C and 22 °C and the temperature of samples measured, as shown in Figure 3a,b. There was no significant correlation with a p = 0.96 and p = 0.58 for HPCs at 36 °C and 22 °C, respectively.
Figure 3. (a) Correlation between heterotrophic plate counts (HPCs) at 36 °C and the temperatures detected during the drinking water monitoring program. There was no significant correlation with a p-value = 0.96. (b) Correlation between heterotrophic plate counts (HPCs) at 22 °C and the temperatures detected during the drinking water monitoring program. There was no significant correlation with a p-value = 0.58.
Considering the period of analysis, from 2017 to 2021, to assess the compliance of monitoring and maintenance measures undertaken, researchers studied the microbiological contamination trend of HPCs at 36 °C and 22 °C (Figure 4a,b).
Figure 4. (a) Boxplot graph showing a decreasing trend for heterotrophic plate counts (HPCs) at 36 °C observed during the period of analysis, from 2017 to 2021. Outliers are not displayed. (b) Boxplot graph showing a decreasing trend for heterotrophic plate counts (HPCs) at 22 °C observed during the period of analysis, from 2017 to 2021. Outliers are not displayed.
Figure 4a,b show how the continuous water quality monitoring and the MWDs’ maintenance, according to International Regulations and Guidelines, led to a decrease in bacterial contamination. The same contamination trend was observed in terms of percentage of positive samples that exceeded the regulation limits. We found 68/109 (62.4%) for HPCs at 36 °C and 36/109 (33.0%) for HPCs at 22 °C positive samples that exceeded the regulation limits in 2017, compared to 11/57 (19.3%) and 5/57 (8.8%) in 2021.
The value of mean contamination ± SD found were 1014.99 ± 2637.63 cfu/mL and 686.25 ± 1952.55 cfu/mL for HPCs at 36 °C and 22 °C, respectively, in 2017, compared to 30.02 ± 77.62 cfu/mL and 91.75 ± 469.23 cfu/mL for HPCs at 36 °C and 22 °C, respectively, in 2021.
These data are supported by the statistical analysis applied on HPCs at 36 °C and 22 °C detected in 2017 compared to 2021, when it was possible to assess a significant decrease of HPCs at 36 °C (p = 0.0000000001) and 22 °C (p = 0.000006) in 2021 compared to 2017.
The WSP implementation resulted in a 43.09% decrease for HPCs at 36 °C and 24.26% decrease for HPCs at 22 °C.
Finally, in order to verify the impact of the lockdown period, due to the global SARS-CoV-2 pandemic that occurred during 2020, which also affected the industrial company involved in this study, causing the closure of MWDs’ devices, we focused on the analysis of the data, both for HPCs at 36 °C and 22 °C, comparing the contamination found in the pre-COVID (year 2019) and post-COVID (year 2021) periods.
The extended period of inactivity did not affect the MDWs’ contamination, as evidenced by a significant decreasing trend of HPCs’ parameters, with a p-value of 0.000005 for 36 °C and 0.001 for 22 °C, respectively. Moreover, a decreasing number of positive samples over the directive limits was observed: 31/94 (33.0%) for HPCs at 36 °C and 9/94 (9.6%) for HPCs at 22 °C in 2019 compared to 11/57 (19.3%) and 5/57 (8.8%) in 2021.
Regarding the contamination found, it was possible to evaluate again a decrease, such as 39.05 ± 86.37 cfu/mL and 69.78 ± 347.89 cfu/mL for HPCs at 36 °C and 22 °C, respectively, in 2019 compared to 30.02 ± 77.62 cfu/mL and 91.75 ± 469.23 cfu/mL for HPCs at 36 °C and 22 °C, respectively, in 2021.
The facts prove the long-term effectiveness of the WSP followed by the company and the consumers, in addition to the role of a multidisciplinary approach established to assure the drinking water quality, according to the International and National directives. In addition, researchers suggests a need to implement more rigorous microbiological monitoring to apply to MWDs’ environment, underestimated to date, despite the large diffusion of these devices in the communities.
Lack of awareness and knowledge on these issues, in addition to missing systematical control by users as well as by public health authorities, can lead to a high risk to human health, especially in hospitals and, in general, in all health-care settings, where the use of MWDs is already widespread and the presence of immunocompromised people could increase the risk of infections.
Moreover, these findings assume a relevant importance even across new scenarios: the higher request for plastic reduction, the introduction, starting from January 2021, of the new European Directive 2184/2020 on the quality of water intended for human consumption focused to implement a WSP approach in all facilities, and the introduction of new microbiological parameters in order to assure the water quality, such as, for example, Legionella
. Considering the habitat, the pathogenic role, and the impact on public health demonstrated for these bacteria, the sanitation and maintenance procedures of MWDs will play a relevant role to preserve the water quality.