Different Trends in Microbial Contamination between Two Types of Microfiltered Water Dispensers: From Risk Analysis to Consumer Health Preservation

The use of microfiltered water dispensers (MWDs) for treatment of municipal water is increasing rapidly, however, the water quality produced by MWDs has not been widely investigated. In this work a large-scale microbiological investigation was conducted on 46 MWDs. In accordance with Italian regulations for drinking water, we investigated the heterotrophic plate counts at 36 and 22 C for indicator bacteria and pathogenic bacteria, such as Enterococci, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus. Two different MWDs were compared: Type A with Ag+ coated carbon filter and two ultraviolet (UV) lamps, and Type B with a carbon filter and one UV lamp. For each type, the contamination of the input and output points was analyzed. Our findings showed that MWDs are a source of bacteria growth, with output being more contaminated than the input point. Type B was widely contaminated for all parameters tested in both sampling points, suggesting that water treatment by Type A is more effective in controlling bacterial contamination. MWDs are critical devices for water treatment in term of technologies, intended use, and sanitization procedures. The adoption of an appropriate drinking water safety plan associated with clear maintenance procedures and periodic environmental monitoring can ensure the safe and healthy operation of these devices

Occurrence of Legionella spp. in Man-Made Water Sources: Isolates Distribution and Phylogenetic Characterization in the Emilia-Romagna Region

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

First water safety plan approach applied to a Dental Clinic complex: identification of new risk factors associated with Legionella and P. aeruginosa contamination, using a novel sampling, maintenance and management program

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

Comparison between two types of dental unit waterlines: how evaluation of microbiological contamination can support risk containment

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 \ub0C 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 antiretraction valves, and the disinfection procedures performed are the main critical points of Type A DUWLs, confirming 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

The Role of Sensor-Activated Faucets in Surgical Handwashing Environment as a Reservoir of Legionella

Surgical handwashing is a mandatory practice to protect both surgeons and patients in order to control Healthcare-Associated Infections (HAIs). The study is focused on Legionella and Pseudomonas aeruginosa contamination in Surgical Handwashing Outlets (SHWOs) provided by sensor-activated faucets with Thermostatic Mixer Valves (TMVs), as correlated to temperature, technologies, and disinfection used. Samples were analyzed by standard culture techniques, comparing hot- and cold-water samples. Legionella isolates were typed by an agglutination test and by mip sequencing. Legionella contamination showed the same distribution between hot and cold samples concerning positive samples and mean concentration: 44.5% and 1.94 Log10 cfu/L vs. 42.6% and 1.81 Log10 cfu/L, respectively. Regarding the distribution of isolates (Legionella pneumophila vs. Legionella non-pneumophila species), significant di_erences were found between hot- and cold-positive samples. The contamination found in relation to ranges of temperature showed the main positive samples (47.1%) between 45.1–49.6 _C, corresponding to high Legionella concentrations (2.17 Log10 cfu/L). In contrast, an increase of temperature (>49.6 _C) led to a decrease in positive samples (23.2%) and mean concentration (1.64 Log10 cfu/L). A low level of Pseudomonas aeruginosa was found. For SHWOs located in critical areas, lack of consideration of technologies used and uncorrected disinfection protocols may lead to the development of a high-risk environment for both patients and surgeons

Draft Genome Sequence of Legionella Species Isolated from Drinking Water in an Italian Industry

We report the draft genome sequences of an environmentalLegionellastrainisolated from an industrial water distribution system in Italy. Macrophage infectivity poten-tiator (mip)andb-subunit of RNA polymerase (rpoB) genes were used to perform the spe-cies identification. Whole-genome sequencing (WGS) and average nucleotide identity (ANI)identified the isolate as belonging to a presumptive novelLegionellaspecies, with a ge-nome length of 3,281,851 bp

Cooling towers influence in an urban environment: A predictive model to control and prevent Legionella risk and Legionellosis events

Cooling towers (CTs) are used to dissipate excess heat from water by evaporation, common in large facilities as hospital, companies, and hotels. The main risk attributed to CTs is represented by Legionella, a Gram-negative bacterium associated with a severe form of pneumonia known as Legionnaires' disease (LD). The infection route is by inhalation of aerosols reaching the lower respiratory tract. Despite several events associated with CTs, the knowledge in this field is still limited. The aim of this study was to develop a predictive model of bioaerosol dispersion using PM10 particles as a proxy, to generate risk maps of Legionella spread in the surrounding area in several weather and microbiological conditions. The Legionella contamination in the CT basin was 40938 ± 24523 cfu/L, with four peaks independent of the season, associated with an increase in air minimum temperature values (+1–2 °C) and a high relative humidity (66–100%) preceded by rainfall (0.2–30.6 mm/day). The model revealed that the most extensive bioaerosol spread is predicted in winter and summer, with an increase in Legionella risk at a distance of up to 1.5 km from the CT. This method represents a novel integrated approach for the prevention and management of LD risk in CTs

New Insight regarding Legionella Non- Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene

The identification of Legionella non-pneumophila species (non-Lp) in clinical and environmental samples is based on the mip gene, although several studies suggest its limitations and the need to expand the classification scheme to include other genes. In this study, the development of a new classification scheme targeting the rpoB gene is proposed to obtain a more reliable identification of 135 Legionella environmental isolates. All isolates were sequenced for the mip and rpoB genes, and the results were compared to study the discriminatory power of the proposed rpoB scheme. Complete concordance between the mip and rpoB results based on genomic percent identity was found for 121/135 (89.6%) isolates; in contrast, discordance was found for 14/135 (10.4%) isolates. Additionally, due to the lack of reference values for the rpoB gene, inter- and intraspecies variation intervals were calculated based on a pairwise identity matrix that was built using the entire rpoB gene (∼4,107 bp) and a partial region (329 bp) to better evaluate the genomic identity obtained. The interspecies variation interval found here (4.9% to 26.7%) was then proposed as a useful sequence-based classification scheme for the identification of unknown non-Lp isolates. The results suggest that using both the mip and rpoB genes makes it possible to correctly discriminate between several species, allowing possible new species to be identified, as confirmed by preliminary whole-genome sequencing analyses performed on our isolates. Therefore, starting from a valid and reliable identification approach, the simultaneous use of mip and rpoB associated with other genes, as it occurs with the sequence-based typing (SBT) scheme developed for Legionella pneumophila, could support the development of multilocus sequence typing to improve the knowledge and discovery of Legionella species subtypes

Use of Fourier-Transform Infrared (FTIR) Spectroscopy with IR Biotyper® System for Legionella pneumophila serogroups identification

Legionella spp. are Gram-negative bacteria that inhabit freshwater environments representing a serious risk for human health. Legionella pneumophila (Lp) is the species most frequently responsible for a severe pneumonia, known as Legionnaires’ disease. Lp consists of 15 serogroups (Sgs), usually identified by monoclonal or polyclonal antibodies. Concerning Lp serogrouping, it is well known that phenotyping methods do not have a sufficiently high discriminating power, while genotypic methods although very effective, are expensive and laborious. Recently, mass spectrometry and infrared spectroscopy have proved to be rapid and successful approach for the microbial identification and typing. Different biomolecules (e.g., lipopolysaccharides) adsorb infrared radiation originating a specific microbial fingerprint. The development of a classification system based on the intra-species identification features allows a rapid and reliable typing of strains for diagnostic and epidemiological purposes. The aim of the study was the evaluation of Fourier Transform Infrared Spectroscopy using the IR Biotyper® system (Bruker Daltonik, Germany) for the identification of Lp at serogroup (Sg) level for diagnostic purposes as well as in outbreak events. A large dataset of Lp isolates (n=133) and ATCC reference strains representing the 15 Lp serogroups were included. The discriminatory power of instrument’s classifier, by Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) was tested. All isolates were classified as follow: 12/133 (9.0 %) Lp Sg1 and 115/133 (86.5%) as Lp Sg 2-15 (including both ATCC and environmental Lp serogroup). Moreover, a mis-classification for 2/133 (1.5%) isolates of Lp Sg 2-15 returned as Lp Sg1 was observed and 4/133 (3.0%) isolates were not classified. The accuracy of 95.49% and an error rate of 4.51% were calculated. IR Biotyper® is able provide a quick and cost-effective reliable Lp classification with advantages compared to agglutination tests that show ambiguous and unspecific results. Further studies including a larger number of isolates could be useful to implement the classifier obtaining a robust and reliable tool for the routine Lp serogrouping. IR Biotyper® could be a powerful and easy-to-use tool to identify Lp Sgs especially during cluster/outbreak investigations, to trace the source of the infection and promptly adopt preventive and control strategies

Case Report: First Report of Fatal Legionella pneumophila and Klebsiella pneumoniae Coinfection in a Kidney Transplant Recipient

A very rare case of pulmonary Klebsiella pneumoniae-Legionella pneumophila coinfection in a double kidney transplanted man affected by the chronic renal disease is described. Cases of Legionnaires’ disease with an incubation period of 14 days have rarely been documented. Despite the long period of hospitalization, typing of clinical and environmental L. pneumophila strains demonstrated that the patient’s home water distribution system was the source of infection, highlighting that Legionella house contamination can be a hidden risk, especially for immune-compromised people