Dental unit waterlines (DUWLs) represent a complex environment able to promote microbial contamination, due to functional, mechanical and practical risk factors. According to a water safety plan approach, the main goal is to preserve the health of dentists, dental staff and patients. The aim of this study is to develop a DUWLs water safety plan that is able to support correct and effective maintenance and disinfection procedures. Three different water systems serve 60 dental chairs: (i) water that comes directly from municipal water (Type A), (ii) water supplied by municipal water and water bottles (Type B) and (iii) water supplied only via water bottles (Type C). For each type, Legionella and Pseudomonas aeruginosa contamination was studied, by applying a new sampling scheme, based on separate sampling from water bottles, cup filler and handpieces. Type B DUWL is the only type of DUWL contaminated by L. pneumophila (ST 59) and L. anisa (mean contamination: 608.33 ± 253.33 cfu/L) detected in cup filler and handpieces, as well as the high presence of P. aeruginosa (44.42 ± 13.25 cfu/100 mL). Two subsequent shock treatments and resampling procedures were performed by increasing disinfectant dosage and contact time and removing some DUWL components linked to biofilm growth in DUWLs. A significant reduction of contamination was obtained for both microorganisms (Legionella spp.: -100%, p < 0.001 and P. aeruginosa: -99.86%, p = 0.006). The sampling strategy proposed allows us to identify the source of contamination and better focus on the maintenance and disinfection procedures. DUWLs represent an environment that requires a multidisciplinary approach, combining the knowledge of all DUWL components to correct procedures that are able to preserve the health of personnel and patients, as well as guaranteeing DUWLs' safe functionality.

The rapid and accurate detection of SARS-CoV-2 in environmental settings is crucial for effective public health management during the COVID-19 pandemic. This study compares the performance of the Reverse Transcription quantitative polymerase chain reaction (RT-qPCR) and the Reverse Transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection from 100 surface samples collected in healthcare environments. The reference method, RT-qPCR, identified a percentage of 25% of positive samples, while RT-LAMP detected a percentage of 27% of positive surfaces. Our findings reveal a sensitivity of 32% and specificity of 75% for RT-LAMP, with a positive predictive value of 30% and a negative predictive value of 77%. The overall accuracy and concordance with RT-qPCR was 64% for both methods. Despite its lower sensitivity compared to RT-qPCR, RT-LAMP had an advantage due to its rapid screening and environmental surveillance, which is particularly useful for confirming negative results. These results underscore the potential of RT-LAMP not only as a valuable method in the environmental monitoring of SARS-CoV-2 but also as a system to control the sanitation process in ordinary and emergency conditions, providing further optimization and validation for its reliability in routine surveillance and outbreak response efforts.

Two Legionella-like isolates, 8cVS16^T and 9fVS26, were isolated from a water distribution system (WDS) in a healthcare facility. Cells were Gram- and Ziehl Neelsen-stain-negative, rod-shaped, motile, and exhibited a blue-white fluorescence under Wood’s lamp at 365 nm. The strains grew in a range of 32–37 °C on BCYE with L-cysteine (Cys+), GVPC, and MWY agar medium, with a positive reaction for oxidase, catalase, and gelatinase. The dominant fatty acids were summed features 3 (C_16:1ω7c/C_16:1ω6c) (27.7%), C_16:0 iso (17.5%), and C_16:0 (16.3%), and Q13 as the major ubiquinone. The mip and rpoB gene sequences showed a similarity of 96.7% and 92.4%, with L. anisa (ATCC 35292^T). The whole genomes sequencing (WGS) performed displayed a GC content of 38.21 mol% for both. The digital DNA-DNA hybridization (dDDH) analysis demonstrated the separation of the two strains from the phylogenetically most related L. anisa (ATCC 35292^T), with ≤43% DNA-DNA relatedness. The Average Nucleotide Identity (ANI) between the two strains and L. anisa (ATCC 35292^T) was 90.74%, confirming that the two isolates represent a novel species of the genus Legionella. The name proposed for this species is Legionella resiliens sp. nov., with 8cVS16^T (=DSM 114356^T = CCUG 76627^T) as the type strain.

Contamination of environmental surfaces by nosocomial pathogens like Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), and Enterococcus spp. poses significant health risks worldwide. However, gold-standard detection methods are too time-consuming and labor-intensive. This study aimed to optimize loop-mediated isothermal amplification (LAMP) as a rapid, innovative, and cost-effective approach, comparing its effectiveness with the gold-standard cultural method. Sterile surfaces (24 cm²) were contaminated in duplicate with different concentrations of P. aeruginosa, S. aureus, and Enterococcus faecalis (E. faecalis) reference stains. For each pair of contaminated surfaces, one was analyzed using the agar contact plate method (UNI EN 17141:2021), while the other was analyzed using LAMP, following three different pre-incubation times (three, six, and nine hours). The sensitivity and accuracy of LAMP for P. aeruginosa improved with longer incubation times, reaching a value of 1.00 at nine hours, while the specificity and positive predictive value (PPV) remained at 1.00 regardless of the incubation time. For S. aureus, LAMP achieved a sensitivity, specificity, accuracy, PPV, and negative predictive value (NPV) of 1.00 across all incubation times. Finally, for E. faecalis, sensitivity increased from 0.57 at three hours to 1.00 at six and nine hours, with a high specificity, accuracy, PPV, and NPV from six hours onwards. These findings showed that LAMP can be used as a rapid and reliable alternative to gold-standard methods for detecting pathogens on surfaces. The high sensitivity and specificity achieved, especially at six and nine hours of pre-incubation, suggested its use for real-time monitoring in healthcare settings. Further research in real-world environments is needed to confirm these findings.

The World Health Organization (WHO) recommends the introduction of a water safety plan (WSP) approach on drinking water, in all types of settings. This study represents the first WSP developed on the Neptune Fountain, in Bologna (Italy), based on an interdisciplinary approach, integrating hydraulic and microbiological features, in a Building Information Modeling (BIM). The aim was to develop a dynamic and digital platform to update and share the maintenance program, promoting collaboration among microbiologists, engineers, and municipal staff. Water samples were collected along fountain water distribution systems (WDS) from 2016 to 2021 to monitor water quality through the heterotrophic bacteria at 22 °C and 37 °C, as well as to conduct an Enterococci, Coliform bacteria, Escherichia coli, Pseudomonas aeruginosa, Clostridium perfringens, and Staphylococcus aureus assessment. Simultaneously, hydraulic measures were performed, and advanced geomatics techniques were used to detect the WDS structural components, with a focus on the water treatment system (WTS). The WTS consisted of 10 modules corresponding to specific treatments: descaling, carbon–sand filtration, reverse osmosis, and ultraviolet disinfection. Fecal indicators, heterotrophic bacteria, and P. aeruginosa exceeded the reference limits in most of the modules. Several disinfections and washing treatments, other than changing the maintenance procedure scheduling, were performed, improving the WTS and controlling the contamination. The developed microbiological results, hydraulic measurements, and maintenance procedures were integrated in the BIM model to optimize the data storage, updating procedures and the real-time data sharing. This approach improved the fountain management, operation, and material conservation, ultimately preserving the health of daily visitors.

This study presents the characterization of a novel Legionella species isolated in Italy over three different years from one company and two hospitals. Starting from standard techniques such as culture of water samples, agglutination test, MALDI-TOF MS and gene sequencing analysis used to identify the isolates, genomic and metabarcoding approaches were subsequently employees to further characterize the species. Legionella contamination ranged from 400 to 700 CFU/L. The tiny colonies displayed atypical morphology compared to typical Legionella features, although they grew on BCYE medium supplemented with L-cysteine. Microscopic and phenotypic analyses revealed Gram-stain negative, Ziehl-Neelsen-negative, rod-shaped, motile cells capable of growing at 32-37 °C, including on selective media such as GVPC and MWY. The isolates tested positive for oxidase and gelatinase activity. Fatty acid profiling identified the dominant components as Summed Features 3 (C16:1 ω7c/C16:1 ω6c, 28.9%), C16:0 iso (18.4%), and C15:0 anteiso (15.4%). Ubiquinone Q13 was the major quinone. Sequence analysis of the mip and rpoB genes showed 98.2% and 95.1% similarity, respectively, to L. feeleii (WO-44C ATCC 35072^T). Whole genome sequencing (WGS) revealed a GC content of 41.5%, a dDDH value of ≤54.9%, and an ANI of 94.06% with L. feeleii (WO-44C ATCC 35072^T), supporting the classification of a novel species within the genus Legionella. Furthermore, taxonomic resolution of water samples revealed the presence of 168 bacterial genera, including several respiratory, opportunistic, and zoonotic pathogens, as well as seven Legionella species. The name Legionella petroniana sp. nov. is proposed, with strain 31fI33^T (=DSM 114357^T=CCUG 76442^T) designated as type strain.