**1. Introduction**

Several studies have demonstrated that water output from Dental Unit Waterlines (DUWLs) is often contaminated with high densities of microorganisms [1,2], ranging from 10<sup>2</sup> to 10<sup>6</sup> colony-forming units per milliliter (CFU/mL) [3–10]. Bacterial biofilm may be present on the inner surfaces of DUWLs due to contaminations coming from the proximal or distal portion of the circuit. In particular, the presence of small narrow-bore hydrophobic polymeric plastic tubing that facilitate the microorganisms' adhesion (2 mm diameter), electrical components that can heat the water (20–25 ◦C) and the discontinuous and low water flow are all factors that contribute to microbial growth and biofilm formation. The biofilm remains fixed to the tubing wall, but microbes keep spreading from the biofilm into the water as it flows through. For this reason, high microbial levels have been found in output water from handpieces and air/water syringes [11,12]. DUWLs are equipped with a dual water supply system that permits the system to be supplied with only municipal water or sterile water or with both types. Water supply is usually provided by public utilities and its quality must comply with the parameters required by law [13].

The suck-back of biological fluids from the oral cavities of patients (back-contamination) was also reported as important cause of DUWLs contamination [14,15].

*Pseudomonas* spp. are the prevalent bacteria found in DUWLs but a high incidence of *Legionella* was also reported, widely varying from 0% to 68% [16] (including *Legionella pneumophila* serogroup 1) [17]. DUWLs may also be important replication sites for free-living amoebae and protozoa that enable the maintenance of pathogenic intracellular bacteria, increasing their resistance to disinfectants [18]. There is evidence that amoebae huddle around microbial biofilm, and that their concentration is up to 300 times higher in DUWLs' output water than in tap water from the same source [15–17]. The microbial adhesion and biofilm on DUWLs surfaces remain very di fficult to eradicate.

The Centers for Disease Control and Prevention (CDC) recommends that the maximum level of non-coliform bacteria emitted from dental handpieces and air/water syringes should be equal or less than 500 CFU/mL [19]. The Italian legislation on drinking water does not establish a limit for heterotrophic bacteria count; it requires this value not to undergo variations. The water in the operating theatre of healthcare facilities must comply with the target value established by the Italian Workers Compensation Authority guideline, 100 CFU/mL at 22 ◦C and 10 CFU/mL at 37 ◦C, respectively. In 2007, the French "*Ministère de la Santé et des Solidarités*" proposed the same target for drinking water. For this reason, we took it as a reference in our study [20,21].

Because of this contamination, dental units are recognized as a potential source of infection for human health, especially threatening dental sta ff and patients, who are regularly exposed to water and water-aerosol emitted by dental unit handpieces. A study reported the case of a patient who died after being infected with *L. pneumophila* serogroup 1 during a dental practice in Italy [22]. By di fferent typing methods, it was demonstrated that the source of the *Legionella* infection was the DUWL. *L. pneumophila* sg 1 subgroup Benidorm ST593 was isolated in each sample collected from the cold tap water, tap of the DUWL, high-speedturbine and patient's bronchialaspirate. A study of Schönning et al. reported the case of a man diagnosed with leukaemia, who underwent a dental check-up and a high-dose chemotherapy and developed Legionnaires' disease in the following days. The analysis of the environmental samples, from the cup filler resulted to be positive for the same *L. pneumophila* sg 1 specimen detected in the patient's sputum collected through bronchoscopy [23].

The evidence linking exposure to *Pseudomonas aeruginosa* contaminated DUWL during dental treatment and subsequent infection is limited [24] and is based on the results from a single observational study reported in two cancer patients by Martin M.V. et al. [25].

Free-living amoebae were frequently isolated from DUWLs and *Vermamoeba* species were isolated from the throats of humans as long ago as 1967 [26] but it is unknown whether they are associated with a risk in the dental setting, through contaminated aerosols or droplets [27].

In literature there is a single documented fatal case of Legionnaires' disease regarding an American dentist. The infection was attributed to exposure to DUWL aerosols [28]. *L. pneumophilia* and *L. longbeachae* were detected in the dentist's lung tissue and in the DUWLs; however, the dentist's domestic water supplies also had very low levels of *Legionella* spp. Here, the evidence was not conclusive.

A meta-analysis conducted by Petti S et al. demonstrated that there was limited evidence of occupational risk for *Legionella* infection to dental healthcare workers and Legionnaires' disease outbreaks in dental healthcare settings were never reported despite billions of treatments provided each year [29].

To date, there are no known cases of Pontiac fever in patients, resulting from visits to, or treatment in, a dental clinic. This would sugges<sup>t</sup> that the risk to patients posed by *Legionella* spp. from DUWLs is low; however, the risk is not absent [30].

The aim of this study was to report the experience of di fferent dental healthcare settings where a risk managemen<sup>t</sup> plan was implemented and an integrated filtration-disinfection strategy was applied to reduce the DUWLs microbial contamination.
