Infection risk management in a dental unit waterline (DUWL) involves healthcare personnel and patients and is related to routine exposure to water and aerosols that may contain bacterial species. To improve water safety plans, maintenance, and sanitation procedures, analyses of heterotrophic plate counts (HPCs) at 36 °C, and two other microorganisms frequently associated with biofilms, Pseudomonas aeruginosa and Legionella spp., were performed in order to evaluate differences in microbiological contamination between two types of DUWLs: Type A, provided by a water tank, and Type B, directly connected to municipal water. The data showed that the water supply and water safety plan differentially influenced microbiological contamination: Type A DUWLs were more contaminated than Type B DUWLs for all microbiological parameters tested, with significant changes in the percentage of positive samples and contamination levels that were beyond the limits of standard guidelines. The results obtained show how the storage tank, the absence of anti-retraction valves, and the disinfection procedures performed are the main critical points of Type A DUWLs, which confirms that dental unit management (maintenance/sanitization) is often missed or not correctly applied by stakeholders, with an underestimation of the real risk of infection for patients and operators.

Detection and enumeration of Legionella in water samples is of great importance for risk assessment analysis. The plate culture method is the gold standard, but has received several well-known criticisms, which have induced researchers to develop alternative methods. The purpose of this study was to compare Legionella counts obtained by the analysis of potable water samples through the plate culture method and through the IDEXX liquid culture Legiolert method. Legionella plate culture, according to ISO 11731:1998, was performed using 1 L of water. Legiolert was performed using both the 10 mL and 100 mL Legiolert protocols. Overall, 123 potable water samples were analyzed. Thirty-seven (30%) of them, positive for L. pneumophila, serogroups 1 or 2–14 by plate culture, were used for comparison with the Legiolert results. The Legiolert 10 mL test detected 34 positive samples (27.6%) and the Legiolert 100 mL test detected 37 positive samples, 27.6% and 30% respectively, out of the total samples analyzed. No significant difference was found between either the Legiolert 10 mL and Legiolert 100 mL vs. the plate culture (p = 0.9 and p = 0.3, respectively) or between the Legiolert 10 mL and Legiolert 100 mL tests (p = 0.83). This study confirms the reliability of the IDEXX Legiolert test for Legionella pneumophila detection and enumeration, as already shown in similar studies. Like the plate culture method, the Legiolert assay is also suitable for obtaining isolates for typing purposes, relevant for epidemiological investigations.

Legionella spp. are widespread bacteria in aquatic environments with a growing impact on human health. Between the 61 species, Legionella pneumophila is the most prevalent in human diseases; on the contrary, Legionella non-pneumophila species are less detected in clinical diagnosis or during environmental surveillance due to their slow growth in culture and the absence of specific and rapid diagnostic/analytical tools. Reliable and rapid isolate identification is essential to estimate the source of infection, to undertake containment measures, and to determine clinical treatment. Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI–TOF MS), since its introduction into the routine diagnostics of laboratories, represents a widely accepted method for the identification of different bacteria species, described in a few studies on the Legionella clinical and environmental surveillance. The focus of this study was the improvement of MALDI–TOF MS on Legionella non-pneumophila species collected during Legionella nosocomial and community surveillance. Comparative analysis with cultural and mip-gene sequencing results was performed. Moreover, a phylogenetic analysis was carried out to estimate the correlations amongst isolates. MALDI–TOF MS achieved correct species-level identification for 45.0% of the isolates belonging to the Legionella anisa, Legionella rubrilucens, Legionella feeleii, and Legionella jordanis species, displaying a high concordance with the mip-gene sequencing results. In contrast, less reliable identification was found for the remaining 55.0% of the isolates, corresponding to the samples belonging to species not yet included in the database. The phylogenetic analysis showed relevant differences inside the species, regruped in three main clades; among the Legionella anisa clade, a subclade with a divergence of 3.3% from the main clade was observed. Moreover, one isolate, identified as Legionella quinlivanii, displayed a divergence of 3.8% from the corresponding reference strain. However, these findings require supplementary investigation. The results encourage the implementation of MALDI–TOF MS in routine diagnostics and environmental Legionella surveillance, as it displays a reliable and faster identification at the species level, as well as the potential to identify species that are not yet included in the database. Moreover, phylogenetic analysis is a relevant approach to correlate the isolates and to track their spread, especially in unconventional reservoirs, where Legionella prevention is still underestimated.

Legionella species distribution in the Emilia-Romagna region, involving hospital (H) and community (C) environments, was conducted. Legionella culture, agglutination test, and mip-gene sequencing were applied on 240 isolates. The analysis showed a higher prevalence of non-Legionellapneumophila (n-Lp) species (84.1%) compared with L. pneumophila (Lp) (15.9%), with a higher frequency of n-Lp with respect to Lp species in both environments (77.6% and 96.4%, in H and C, respectively). The Shannon index showed a significant difference in Legionella distribution (p = 0.00017), with a significant abundance of Lp in the H compared with C environment (p = 0.00028). The continuous disinfection treatment in H could contribute to adaptive survival of the Lp species. Phylogenetic analysis revealed a conservative clade distribution between H and C: L. feeleii clade with three subclades in C and the Lp clade with five subclades in H and two in C, respectively. Our findings suggest the importance of Legionella surveillance both in H and C, with a focus on n-Lp species less connected to human disease. The Legionella prevalence and diversity found here indicate that geographical and temporal isolate evolution should be considered during surveillance, particularly in the light of global warming and changes in population risk factors.

Legionella spp. are a widely spread bacteria that cause a fatal form of pneumonia. While traditional laboratory techniques have provided valuable systems for Legionella pneumophila identification, the amplification of the mip gene has been recognized as the only useful tool for Legionella non- pneumophila species identification both in clinical and environmental samples.