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Legionella -like isolates, strains 27fs60, 30fs61 and 30cs62T, were isolated from a hotel water distribution system in the Emilia-Romagna region, Italy. Isolates were Gram- and Ziehl Neelsen-stain-negative, rod-shaped, with transitory flagella presence and able to grow at 32–37 °C (with an optimum at 32 °C) on buffered charcoal–yeast extract agar with l-cysteine, glycine–vancomycin–polymyxin B–cycloheximide agar and Wadowsky–Yee medium agar. The strains showed positive reactions for oxidase, hippurate and gelatinase and a weakly positive reaction for catalase. Based on the EUCAST cut-off, strain 30cs62T was resistant to ciprofloxacin (5 mg l−1). The mip and rpoB gene sequences of the three strains showed close matches to those of Legionella quateirensis ATCC 49507T with similarity values of 98.2 and 94.5 %, respectively. Whole genome sequencing of the three strains was performed, resulting in G+C contents of 39.0, 39.1 and 39.0 mol%, respectively. The identity percentage measured by average nucleotide identity between the three strains and their respective closest strains were: 91.32 % L . quateirensis NCTC 12376T, 91.45 % L . quateirensis ATCC 49507T and 91.45 % L . quateirensis ATCC 49507T, respectively. The digital DNA–DNA hybridization analysis demonstrated how the isolates were separated from the most related phylogenetic Legionella species ( L. quateirensis ATCC 49507T, ≤40.10 % DNA–DNA relatedness). The concatenated phylogenetic tree based on 16S rRNA, mip, rpoB and rnpB genes, shows a close relationship with L. quateirensis ATCC 49507T. The results obtained confirm the status of an independent species. The name proposed for this species is Legionella bononiensis sp. nov. with 30cs62T (=ATCC TSD-262T=DSM 112526T) as the type strain.

We report the draft genome sequences of an environmental Legionella strain isolated from an industrial water distribution system in Italy. Macrophage infectivity potentiator ( mip) and β-subunit of RNA polymerase ( rpoB ) genes were used to perform the species identification. Whole-genome sequencing (WGS) and average nucleotide identity (ANI) identified the isolate as belonging to a presumptive novel Legionella species, with a genome length of 3,281,851 bp.

The use of Microfiltered Water Dispensers (MWDs) is increasing in offices, companies, or commercial facilities, as a response to plastic pollution. Despite their widespread use, poor data are available about the water quality and pathogens developed. Starting from a high contamination found in MWDs, a Water Safety Plan (WSP) was implemented on 57 MWDs to improve the water quality. To assess the effectiveness of WSP during the period 2017–2021, the environmental monitoring of heterotrophic plate counts (HPCs) at 36 °C and 22 °C, Enterococcus spp., Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Clostridium perfringens, as prescribed by Italian regulation for drinking water, was conducted. A high level of HPCs at both temperatures was observed, over the regulation limit, contrarily, the pathogenic bacteria were absent. The contamination found was studied with respect to the temperature threshold of 25 °C, suggested by directives. No significant differences were found between samples above and below the threshold, while a significant decrease over the years was observed for HPCs at 36 °C (p = 0.0000000001) and 22 °C (p = 0.000006). The WSP implementation resulted in a 43.09% decrease for HPCs at 36 °C and a 24.26% decrease for HPCs at 22 °C. Moreover, during the SARS-CoV-2 pandemic, the WSP contributed to limit the microbial contamination, preserving the MWDs’ functionality and hygienic conditions and the drinking water quality.

We report the draft genome sequences of an environmental

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.

We present the draft genome sequences of three Legionella strains that were isolated from a hotel water distribution system. Legionella species identification was performed by macrophage infectivity potentiator ( mip ) and RNA polymerase β subunit ( rpoB ) gene sequencing. Whole-genome sequencing and average nucleotide identity results supported the hypothesis of new Legionella species isolation.

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.

In man-made water distribution systems, Legionella community interactions remain unknown, due to their ability to change from sessile to planktonic states or live in viable but non-culturable forms, in response to anthropic and environmental stress. During 7 years of hospital Legionella surveillance, in 191 hot water positive samples, the interactions among the Legionella species, temperature, and disinfection treatment were evaluated. Legionella was isolated following ISO 11731:2017, and identification was performed by mip gene sequencing and sequence-based typing (SBT) for L. anisa or L. rubrilucens and L. pneumophila, respectively. The species with the higher frequency of isolation was L. pneumophila serogroup 1 (78.53%; 4865.36 ± 25,479.11 cfu/L), followed by L. anisa (54.45%; 558.79 ± 2637.41 cfu/L) and L. rubrilucens (21.99%; 307.73 ± 1574.95 cfu/L), which were sometimes present together. Spearman’s rho correlation test was conducted among the species with respect to temperature and disinfectant (H2O2/Ag+). The results showed a generally positive interaction among these species sharing the same environment, except for competition between L. anisa and L. rubrilucens. High temperature (48.83 ± 2.59 °C) and disinfection treatment (11.58 ± 4.99 mg/L) affected the presence of these species. An exception was observed with L. anisa, which showed disinfection treatment resistance. For the purposes of environmental surveillance, it is fundamental to better understand the interactions and dynamic of the Legionella community in man-made water systems in order to choose the proper physical or chemical treatments. The simultaneous presence of different Legionella species could result in an increased resistance to high temperature and disinfectant treatment, leading to changes in contamination level and species diversity.

In this study, we aimed to associate the molecular typing of Legionella isolates with a culture technique during routine Legionella hospital environmental surveillance in hot water distribution systems (HWDSs) to develop a risk map able to be used to prevent nosocomial infections and formulate appropriate preventive measures. Hot water samples were cultured according to ISO 11731:2017. The isolates were serotyped using an agglutination test and genotyped by sequence-based typing (SBT) for Legionella pneumophila or macrophage infectivity potentiator (mip) gene sequencing for non-pneumophila Legionella species. The isolates&rsquo, relationship was phylogenetically analyzed. The Legionella distribution and level of contamination were studied in relation to temperature and disinfectant residues. The culture technique detected 62.21% of Legionella positive samples, characterized by L. pneumophila serogroup 1, Legionella non-pneumophila, or both simultaneously. The SBT assigned two sequence types (STs): ST1, the most prevalent in Italy, and ST104, which had never been isolated before. The mip gene sequencing detected L. anisa and L. rubrilucens. The phylogenetic analysis showed distinct clusters for each species. The distribution of Legionella isolates showed significant differences between buildings, with a negative correlation between the measured level of contamination, disinfectant, and temperature. The Legionella molecular approach introduced in HWDSs environmental surveillance permits (i) a risk map to be outlined that can help formulate appropriate disinfection strategies and (ii) rapid epidemiological investigations to quickly identify the source of Legionella infections.

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.

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 &deg, 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.

In this study, we aimed to associate the molecular typing of Legionella isolates with a culture technique during routine Legionella hospital environmental surveillance in hot water distribution systems (HWDSs) to develop a risk map able to be used to prevent nosocomial infections and formulate appropriate preventive measures. Hot water samples were cultured according to ISO 11731:2017. The isolates were serotyped using an agglutination test and genotyped by sequence-based typing (SBT) for Legionella pneumophila or macrophage infectivity potentiator (mip) gene sequencing for non-pneumophila Legionella species. The isolates’ relationship was phylogenetically analyzed. The Legionella distribution and level of contamination were studied in relation to temperature and disinfectant residues. The culture technique detected 62.21% of Legionella positive samples, characterized by L. pneumophila serogroup 1, Legionella non-pneumophila, or both simultaneously. The SBT assigned two sequence types (STs): ST1, the most prevalent in Italy, and ST104, which had never been isolated before. The mip gene sequencing detected L. anisa and L. rubrilucens. The phylogenetic analysis showed distinct clusters for each species. The distribution of Legionella isolates showed significant differences between buildings, with a negative correlation between the measured level of contamination, disinfectant, and temperature. The Legionella molecular approach introduced in HWDSs environmental surveillance permits (i) a risk map to be outlined that can help formulate appropriate disinfection strategies and (ii) rapid epidemiological investigations to quickly identify the source of Legionella infections.

Sit Bath Systems (SBSs) are the most common hygiene method for patients who are not self-sufficient. Therefore, the water quality of SBSs in the nosocomial environment plays a fundamental role in controlling infections for both patients and health-care workers. A long-term study on Legionella and Pseudomonas aeruginosa (P. aeruginosa) contamination was performed in SBSs (n = 20) of six Health Care Facilities (HCFs). A total of 254 water samples were analyzed following ISO procedures. The samples were positive for P. aeruginosa (46.85%) and Legionella (53.54%), respectively, both over the directive limits. Legionella isolates were identified as: Legionella pneumophila (L. pneumophila) serogroups 1, 3, and 6 and Legionella non-pneumophila species (L. anisa, L. londiniensis, L. rubrilucens, and L. nagelii). Moreover, the contamination found was studied with respect to median temperature measured (42 °C), from which two groups (A and B) could be distinguished. P. aeruginosa was found in both groups (100% of SBSs), while a higher percentage of Legionella positive samples was found in group A (75% of SBSs), compared to group B (50% of SBSs), showing how Legionella control could be carried out by using temperatures above 42 °C. An analysis of SBS water pipelines, maintenance, and disinfection treatments indicates SBSs as a new source of infection risk for both patients and health-care workers.

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 differences 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&ndash, 49.6 &deg, C, corresponding to high Legionella concentrations (2.17 Log10 cfu/L). In contrast, an increase of temperature (&gt, 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.

This work aims to describe the development and validation of two low-intensity pulsed ultrasound stimulation systems able to control the dose delivered to the biological target. Transducer characterization was performed in terms of pressure field shape and intensity, for a high-frequency range (500kHz to 5MHz) and for a low-frequency value (38kHz). This allowed defining the distance, on the beam axis, at which biological samples should be placed during stimulation and to exactly know the intensity at the target. Carefully designed retaining systems were developed, for hosting biological samples. Sealing tests proved their impermeability to external contaminants. The assembly/de-assembly time of the systems resulted ~3min. Time-domain acoustic simulations allowed to precisely estimate the ultrasound beam within the biological sample chamber, thus enabling the possibility to precisely control the pressure to be transmitted to the biological target, by modulating the transducer's input voltage. Biologicalin vitrotests were also carried out, demonstrating the sterility of the system and the absence of toxic and inflammatory effects on growing cells after multiple immersions in water, over seven days.

Legionella surveillance is an important issue in public health, linked to the severity of disease and the difficulty associated with eradicating this bacterium from the water environment. Different treatments are suggested to reduce Legionella risk, however long-term studies of their efficiency are lacking. This study focused on the activity of a new formulation of hydrogen peroxide and silver salts, WTP828, in the hospital hot water network (HWN) to contain Legionella contamination during two years of treatment. The effectiveness of WTP828 was tested measuring physical-chemical and microbiological parameters such as Legionella, Pseudomonas aeruginosa (P. aeruginosa), and a heterotopic plate count (HPC) at 36 &deg, C. Legionella isolates were identified by serotyping and genotyping. WTP 828 induced a reduction in Legionella&ndash, positive sites (60% to 36%) and contamination levels (2.12 to 1.7 log10 CFU/L), with isolates belonging to L. pneumophila SG1 (ST1 and ST104), L. anisa and L. rubrilucens widely distributed in HWN. No relevant contamination was found for other parameters tested. The long-term effect of WTP828 on Legionella containment suggest the easy and safe application of this disinfectant, that combined with knowledge of building characteristics, an adequate environmental monitoring and risk assessment plan, become the key elements in preventing Legionella contamination and exposure.

One of the possible ways to improve the operation efficiency of constructed wetlands and to prevent their clogging is the application of earthworms. They have already been successfully applied for vermicomposting and for sludge dewatering and treatment. A few studies have already examined the effect of earthworms on the treatment of wastewater by vertical flow constructed wetlands (VFCWs), but none of them have provided a yearlong research result from an open-air system or compared the effect that different seasons in a temperate climate area can have on these invertebrates. The goal of this research was to estimate the effect that earthworms and plants have on VFCW’s operation. Four mesocosms (a filter, a filter with earthworms, a VFCW and a VFCW with earthworms) were built and their influent and effluent water quality was monitored for a period of 1 year. They were fed with wastewater coming from a building of the University of Bologna (Italy). The results have shown that the presence of earthworms in this specific system did not reduce the organic matter content of the substrate, but it has positively influenced plants’ growth. However, since neither earthworms nor plants had a statistically significant effect on the effluent quality, it can be concluded that the integration of these invertebrates cannot improve wastewater treatment of vertical flow filters or constructed wetlands.

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 &deg, 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.

We assessed the subsistence-related variation of the human gut microbiome at a fine resolution for two of the main dimensions of microbiome variation, age and geography. For this, we investigated the fecal microbiome and metabolome in rural Bassa and urbanized individuals from Nigeria, including infants, and compared data with worldwide populations practicing varying subsistence. Our data highlight specific microbiome traits that are progressively lost with urbanization, such as the dominance of pristine fiber degraders and the low inter-individual variation. For the Bassa, this last feature is the result of their subsistence-related practices favoring microbial dispersal, such as their extensive environmental contact and the usage of untreated waters from the Usuma River. The high degree of microbial dispersal observed in the Bassa meta-community nullifies the differences between infant and adult intestinal ecosystems, suggesting that the infant-type microbiome in Western populations could be the result of microbiome-associated neotenic traits favored by urbanization. Ayeni et al. characterize the fecal microbiome and metabolome of rural Bassa and urban individuals from Nigeria, including infants. Their findings stress the loss of ancient signatures along with urbanization and support distinct trajectories of development of the intestinal ecosystem in early life, depending on human subsistence.

The contamination of industrial cooling towers has been identified as one cause of legionellosis, but the real risk has been underestimated. Two different disinfection treatments were tested on Legionella colonization in an industrial Cooling Tower System (CTS). Environmental monitoring of Legionella, P. aeruginosa, and a heterotrophic plate count (HPC) at 36 °C was performed from June to October 2016. The disinfection procedures adopted were based on hydrogen peroxide (H2O2) and silver salts (Ag+), in addition to an anti-algal treatment, then using hyperclorination as a shock, and then continuous treatment by sodium hypochlorite (NaClO). L. pneumophila serogroup 8 was found at a concentration of 5.06 Log cfu/L after the CTS filling; a shock treatment performed by H2O2/Ag+ produced a rapid increase in contamination up to 6.14 Log cfu/L. The CTS activity was stopped and two subsequent shock treatments were performed using NaClO, followed by continuous hyperclorination. These procedures showed a significant decrease (p < 0.05) in Legionella concentration (1.77 Log cfu/L). The same trend was observed for P. aeruginosa (0.55 Log cfu/100 mL) and HPC (1.95 Log cfu/mL) at 36 °C. Environmental monitoring and the adoption of maintenance procedures, including anti-scale treatment, and physical, chemical, and microbiological control, ensure the good performance of a CTS, reducing the Legionella risk for public health.

Contamination of hot water distribution systems by Legionella represents a great challenge due to difficulties associated with inactivating microorganisms, preserving the water characteristics. The aim of this study was to examine over the course of 1 year in 11 fixed sites, the impact of monochloramine disinfection on Legionella, heterotrophic bacteria (36 °C), Pseudomonas aeruginosa contamination, and chemical parameters of a plumbing system in an Italian hospital. Three days after installation (T0), in the presence of monochloramine concentration between 1.5 and 2 mg/L, 10/11 sites (91%) were contaminated by L. pneumophila serogroups 3 and 10. After these results, the disinfectant dosage was increased to between 6 and 10 mg/L, reducing the level of Legionella by three logarithmic unit by 2 months postinstallation (T2) until 6 months later (T3). One year later (T4), there was a significant reduction (p = 0.0002) at 8/11 (73%) sites. Our data showed also a significant reduction of heterotrophic bacteria (36 °C) in 6/11 (55%) sites at T4 (p = 0.0004), by contrast the contamination of P. aeruginosa found at T0 in two sites persisted up until T4. The results of the present study show that monochloramine is a promising disinfectant that can prevent Legionella contamination of hospital water supplies.

Background. Recent preclinical evidence and data from adult subjects suggests that statins could improve the proinflammatory profile of hypercholesterolemic subjects. Objectives. We aim to compare the serum levels of a set of proinflammatory chemokines in elderly statistical twins taking or not taking statins. Material and Methods. Among the historical cohort of the Brisighella Heart Study, we chose 40 healthy elderly subjects continuously treated with statins for at least 1 year and 40 cross-matched subjects not treated with statins (M : F = 1 : 1) characterized by similar age, body mass index (BMI), leisure-time and working activity, smoking habits, history of cardiovascular disease, systolic and diastolic blood pressure, fasting plasma glucose, plasma lipids, uric acid, and creatinine. Results. The proinflammatory chemokine serum level is similar in statin untreated and treated statistical twins. The OR to have a serum level of monocyte chemoattractant protein (MCP-1) lower than the 50 th percentile of the distribution in statin-treated subjects compared to the statin untreated subjects is 0.669 (95% CI 0.193; 2.327), the OR for interleukin-8 (IL-8) = 0.818 (95% CI 0.236; 2.835), the OR for γ-interferon inducible protein-10 (IP-10) = 1.361 (95% CI 0.358; 5.175), and for interleukin-18 (IL-18) = 0.545 (95% CI 0.155; 1.914). Conclusions. In relatively healthy, elderly subjects selected from a randomized general population sample, we did not observe differences in the serum levels of the selected set of proinflammatory chemokines in statin treated and untreated subjects with similar LDL-C level, suggesting that cholesterol reduction per se could be a main determinant of statin anti-inflammatory effects (Adv Clin Exp Med 2014, 23, 5, 723–728).

In accordance with the international and national guidelines, the Emilia-Romagna Region (Italy) has established regional guidelines for the surveillance and prevention of legionellosis based on the concept of risk assessment, with particular attention to environmental monitoring. The aim of this study was to verify how environmental surveillance in the context of risk assessment plans could help to guide decisions about preventive strategies against Legionella infections in Long Term Care Facilities (LTCF). In six LTCFs in the city of Bologna (Emilia-Romagna Region) a self-control plan was implemented that included the environmental monitoring of Legionella spp. and the surveillance of hospital-acquired Legionnaires’ Disease. At baseline, four hot water systems were colonized by Legionella pneumophila (3 LCTFs) and Legionella londiniensis (1 LCTF). In each establishment specific control measures were adopted based on the characteristics of the system, the virulence of the strain and the level of the contamination. The monitoring, carried out for around two years, was also extended to the ways in which the system and the distal water distribution points were used and maintained with respect to the good practices in operation and management. The adopted actions (shock and/or continuous disinfection treatments) and the implementation of the good practice measures reduced the contamination to acceptable and stable levels. No cases of hospital-acquired legionellosis occurred during the period of study. The environmental surveillance was successful in evaluating the risk and identifying the most suitable preventive strategies.

Obbiettivi: Nel poliedrico ambito del rischio professionale a cui gli operatori del settore odontoiatrico sono esposti, quello biologico costituisce senza dubbio uno dei più rilevanti ed al contempo più studiati. La conoscenza delle normative che lo regolamentano, associata ad una approfondita confidenza con le varie procedure sviluppate per il suo controllo, sono requisiti fondamentali per un’efficace e razionale gestione dell’attività clinica. E’ quindi obbiettivo del presente lavoro riassumere le conoscenze ad oggi acquisite e le norme legislative a cui attenersi. Materiali e metodi: Dopo una attenta introduzione alle normative legislative in materia di rischio biologico, vengono descritti i criteri per d’identificazione dei livelli di rischio e la classificazione nonché la corretta gestione dei molteplici prodotti di scarto, dei materiali d’uso e degli ambienti di lavoro. Conclusioni: Una razionale gestione del rischio biologico vede la sua attuazione in una chiara e condivisa conoscenza della tematica, nel riconoscimento di specifiche mansioni e nella rigorosa attinenza a protocolli operativi. Solo in questo modo sarà possibile un approccio sicuro e professionale a tale importante aspetto dell’attività odontoiatrica.

Aims: To compare the standard culture method with a new, rapid test (ScanVIT-Legionella™) using fluorescently labelled gene probes for the detection and enumeration of Legionella spp. The new technique was validated through experiments conducted on both artificially and naturally contaminated water and through an inter-laboratory comparison. Methods and Results: All samples were processed by the ScanVIT test according to the manufacturer’s instructions and by a culture method (ISO 11731). ScanVIT detected significantly more positive samples, although concentrations were similar and a strong positive correlation between the two methods was observed (r = 0·888, P

We evaluated the role of CCL20 (MIP-3α) chemokine in cells directly involved in the remodeling of bone tissue (osteoblasts and osteoclasts) and we confirmed its expression in the subchondral bone tissue of rheumatoid arthritis (RA) patients. The expression of CCL20 and of its receptor CCR6 was evaluated in osteoblasts isolated from bone tissue of post-traumatic (PT) patients. Functional tests were performed to evaluate osteoblast proliferation and matrix protein modulation. Immunohistochemical analysis for CCR6, CCL20, and RANKL was performed on bone samples from RA patients. The role of CCL20 was then analyzed in osteoclast differentiation. We found that in basal conditions CCR6, but not its ligand CCL20, was highly expressed by osteoblasts. Functional analysis on osteoblasts showed that CCL20 significantly increased cellular proliferation but did not affect matrix protein expression. Pro-inflammatory cytokines significantly induced the release of CCL20 and RANKL by human osteoblasts but did not modulate CCR6 expression. Increased expression of CCR6, CCL20, and RANKL was confirmed in RA subchondral bone tissue biopsies. We demonstrated that CCL20 was also an earlier inducer of osteoclast differentiation by increasing the number of pre-osteoclasts, thus favoring cell fusion and MMP-9 release. Our results add new insight to the important role of the CCL20/CCR6, RANKL system in the bone tissue of RA. The contemporary action of CCL20 on osteoblasts and osteoclasts involved in the maintenance of bone tissue homeostasis demonstrates the important role of this compartment in the evolution of RA, by showing a clear uncoupling between new bone formation and bone resorption. J. Cell. Physiol. 210: 798–806, 2007. © 2006 Wiley-Liss, Inc.

Chondrogenesis is a complex process characterized by a sequence of different steps that start with the condensation of the cells, followed by the expression of specific components, such as collagens and proteoglycans. We evaluated in vitro chondrogenic differentiation of C3H10T1/2 murine mesenchymal cells and compared them with human mesenchymal stromal cells (h-MSCs) in a hyaluronic acid scaffold. We analyzed (from day 0 to day 28) cellular morphology, proliferation, and chondrogenic/osteogenic gene expression at different time points. Our data demonstrate that, during chondrogenic differentiation, murine cells proliferate both in the absence and presence of TGF beta, while h-MSCs require the presence of this activating factor. Murine cells, even if viable, differentiate on hyaluronan scaffold, maintain a fibroblastic morphology, and form a capsule outside the scaffold. At the mRNA level, murine cells showed a decrease in collagen type 1 combined with a significant increase in collagen type H (from day 0), and aggrecan (on day 28), as found for h-MSCs. Immunohistochemical data confirmed that chondrogenic differentiation of murine cells, induced by TGF beta, occurred only in some restricted areas inside the scaffold that were positive to collagen type II, but did not show a cartilage-like tissue structure, as we had found using h-MSCs. These data demonstrate that C3H10T1/2 murine cells, widely used as a chondrogenic model, show a different sequence of chondrogenic events in hyaluronic acid scaffold, compared with primary h-MSCs.

To evaluate the role of CXC chemokines CXCL8 (IL8), CXCL10 (IP-10), CXCL12 (SDF-1), and CXCL13 (BCA-1) in bone remodeling, we analyzed their effects on osteoblasts (OBs) obtained from subchondral trabecular bone tissue of osteoarthritis (OA) and post-traumatic (PT) patients. The expression of CXC receptors/ligands (CXCR1/CXCL8, CXCR2/CXCL8, CXCR3/CXCL10, CXCR4/CXCL12, and CXCR5/CXCL13) was analyzed in cultured OBs by flow cytometry and immunocytochemistry. Functional assays on CXC chemokine-treated-OBs in the presence or absence of their specific inhibitors were performed to analyze cellular proliferation and the enzymatic response to chemokine activation. The expression of chemokine ligands/receptors was also confirmed in bone tissue samples by immunohistochemical analysis. Collagen type I and alkaline phosphatase mRNA expression were analyzed on CXCL12- and CXCL13-treated OBs by real-time PCR. OBs from both OA and PT patients expressed high levels of CXCR3 and CXCR5 and lower amounts of CXCR1 and CXCR4. CXCL12 and CXCL13, only in OBs from OA patients, induced a significant proliferation that was also confirmed by specific blocking experiments. Moreover, OBs from OA patients released a higher amount of CXCL13 than those of PT patients while no differences were found for CXCL12. In the remodeling area of bone tissue samples, immunohistochemical analysis confirmed that OBs expressed CXCL12/CXCR4 and CXCL13/CXCR5 both in OA and PT samples. CXCL12 and CXCL13 upregulated collagen type I mRNA expression in OBs from OA patients. These data suggest that CXCL12 and CXCL13 may directly modulate cellular proliferation and collagen type I in OA patients, so contributing to the remodeling process that occurs in the evolution of this disease. © 2005 Wiley-Liss, Inc.

Ligaments are complex structures that maintain the mechanical stability of the joint. Healing of injured ligaments involves the interactions of different cell types, local cellular environment, and the use of devices. To gain new information on the complex interactions between mesenchymal stem cells (MSCs) and a specific hyaluronan-based prototype scaffold (HYAFF, useful for ligament tissue engineering, short time-course experiments were performed to analyze the proliferation, vitality, and phenotype of MSCs grown on the scaffold. MSC proliferation was analyzed using the MTT test, during the early time points (2, 4, 6, days). Viability was assessed using calcein/acetyloxymethylester immunofluorescence dye and confocal microscopy analysis. Hyaluronic acid receptor (CD44), typical matrix ligament proteins (collagen type I, type III, laminin, fibronectin, actin), and chondrogenic/osteogenic markers (collagen type II and bone sialoprotein) were evaluated by immunohistochemistry. Our data demonstrated that MSC growth and viability were cell density-dependent. MSCs completely wrapped the fibers of the scaffold, expressed CD44, collagen type I, type III, laminin, fibronectin, and actin, and were negative to collagen type II and bone sialoprotein. These data demonstrate that MSCs survive well in the hyaluronan-based prototype ligament scaffold, as assessed after 2 days from seeding, and express CD44, a receptor important for scaffold interaction, and proteins responsible for the functional characteristics of the ligaments.

Chemokines are involved in a number of inflammatory pathologies and some of them show a pivotal role in the modulation of osteoclast development. Therefore, we evaluated the role of CXCL12 chemokine on osteoclast differentiation and function and we analyzed its expression on synovial and bone tissue biopsies from rheumatoid arthritis (RA) patients. Osteoclasts were obtained by 7 days in vitro differentiation with RANKL and M-CSF of CD11b positive cells in the presence or absence of CXCL12. The total number of osteoclast was analyzed by Tartrate-resistant acid phosphatase (TRAP)-staining and bone-resorbing activity was assessed by pit assay. MMP-9 and TIMP-1 release was evaluated by ELISA assay. CXCL12 expression on biopsies from RA patients was analyzed by immunohistochemistry. Osteoclasts obtained in the presence of CXCL12 at 10 nM concentration displayed a highly significant increase in bone-resorbing activity as measured by pit resorption assay, while the total number of mature osteoclasts was not affected. The increased resorption is associated with overexpression of MMP-9. Immunostaining for CXCL12 on synovial and bone tissue biopsies from both rheumatoid arthritis (RA) and osteoarthritis (OA) samples revealed a strong increase in the expression levels under inflammatory conditions. CXCL12 chemokine showed a clear activating role on mature osteoclast by inducing bone-resorbing activity and specific MMP-9 enzymatic release. Moreover, since bone and synovial biopsies from RA patients showed an elevated CXCL12 expression, these findings may provide useful tools for achieving a full elucidation of the complex network that regulates osteoclast function in course of inflammatory diseases.

The expression and functional activity of CXC chemokine receptors were evaluated in human osteoblasts (OB) obtained post-trauma from old donors compared to very young donors. It was found that CXCR1 and CXCR4 were only expressed by old but not young donors' cells. In contrast, CXCR3 and CXCR5 were expressed by both young and old donors. We functionally evaluated CXCR3/CXCL10 and CXCR5/CXCL13 receptor/ligand pairs by analysing cell proliferation and the release of N-acetyl-β-D-glucosaminidase (NAG), an enzyme that degrades glycosaminoglycans and hyaluronic acid. CXCL10 and CXCL13 induced a dose-dependent increase of cell proliferation in OB from young donors while cell proliferation of OB in old donors was not affected. By contrast, CXCL10 and CXCL13 induced a significantly higher NAG release in OB from old donors compared to young ones. These data demonstrate a significant age-dependent difference in the response of OB to CXCL10 and CXCL13 stimulation. These chemokines induce an inverse response of OB from old and young donors, which suggests a role of ageing in the modulation of cellular response of bone cells.

In the present study we analyse chemokine expression in the remodelling of subchondral bone in arthritis patients. Trabecular bone biopsies were tested by immunohistochemistry to identify interleukin (IL)-8, GRO-alpha, MCP-1, RANTES, MIP-1alpha and MIP-1beta expression. Subsequently, we evaluated by immunoassay the effect of interferon (IFN)-gamma and IL-6 on chemokine production by osteoarthritis (OA), rheumatoid arthritis (RA) and post-traumatic (PT) patients' isolated osteoblasts (OB). OB constitutively produced in situ IL-8, GRO-alpha, MCP-1, RANTES and MIP-1alpha. MIP-1beta was positive only in mononuclear cells. In RA many of these chemokines were also produced by mononuclear cells. IFN-gamma significantly down-regulated IL-8 and up-regulated MCP-1 produced by OB from all patients tested, whereas it did not affect the other chemokines analysed. Moreover, IFN-gamma reduced IL-1beta-stimulated IL-8 production but significantly increased both MCP-1 and RANTES. Interestingly, IL-6 significantly downregulated IFN-gamma-induced MCP-1 production, that was significantly lower in OA compared to RA patients. OB expressed chemokines both in vivo and in vitro suggesting that these cells are primary effectors in the bone capable of regulating autocrine/paracrine circuits that affect bone remodelling in these diseases.

Several pathologies of respiratory tract are referred to be related to bacteria belonging to Legionella genus1. This pathology mainly affects immunocompromized people as nosocomial or community patients. Surveillance programs are required for monitoring bacteria diffusion and optimization of periodic decontamination action. Detection of Legionella pneumophila in water is based on well-standardized cultural and morphological analysis indicated in national guidelines2. Species assignment is typically performed by rapid agglutination tests3, which can be realized in few minutes. They usually provide univocal results in discrimination and serogrouping of L. pneumophila versus L. species including L. longbeachae, L. bozemanii, L. dumofii, L. jordanis, L. micdadei and L. anisa. This test is based on recognition of lipopolysaccharide present on the outer membrane4,5. We report some evidences where phenotypic procedures failed. During surveillance programs6,7 some unclearly identified bacteria from different regions were isolated. They showed morphological and cultivation features of legionellaceae but species classification, performed by agglutination test, gave negative or equivocal results (Figure 1). In particular they provide an absent or ambiguous agglutination of latex particles as can be observed in the representative Figure 1 (panels 1 and 3) where, positive results were obtained from a clear formation of precipitates (Figure 1, panel 2), as suggested by manufacturing practices and described by Dennis8 and related references. In principle this kind of equivocal results have to be considered negative. Bacteria that showed these results were definitely identified by DNA sequencing.

Human mesenchymal stem cells (hMSCs) are an alternative cell source in bioconstruct production for cartilage regeneration, and hyaluronic acid (HA) is a widely-used bioabsorbable scaffold material used for cartilage regeneration. In this work, the aims were to evaluate the mechanical competency of hMSC-seeded HA scaffolds compared with native intact human articular cartilage, and in relation to its cellular properties. Human MSCs were grown under static conditions in HA scaffolds and then tested, in stepwise, stress-relaxation indentation, 7, 14, and 21 days later. Scaffolds at days 14 and 21 showed a significant increase in mechanical measures when compared with day 7 and unseeded scaffold material, but did not achieve the same levels as human cartilage. There was consistent stiffness within the scaffold, with a decreased stiffness around the edge. In vitro culture of hMSC-seeded HA scaffolds over 3 weeks produces a white, solid tissue compared with unseeded constructs. Increased cell proliferation and collagen type II expression were also seen over this period of time. These results demonstrate the competency of the neo-formed cartilage-like tissue in relation to its mechanical and cellular properties, and further, the importance, for future clinical use, of implanting this construct after 14 days of culture.

Chemokines contribute to the maintenance of cartilage homeostasis. To evaluate the role of CXC chemokines CXCL8 (interleukin-8), CXCL10 (interferon-gamma-inducible protein-10), CXCL12 (stroma-derived factor-1) and CXCL13 (B-cell attracting chemokine-1) and their receptors, respectively CXCR1-2, CXCR3, CXCR4, and CXCR5, during chondrogenic differentiation of human mesenchymal stromal cells (h-MSCs), we used a well-defined in vitro model. Chondrogenic differentiation was analyzed on h-MSCs grown on hyaluronic acid-based biomaterial in the presence or absence of transforming growth factor-beta, and the expression and modulation of CXC chemokines and receptors were evaluated at different time points. Real-time polymerase chain reaction was performed to analyze their expression at the messenger ribonucleic acid (mRNA) level, and immunohistochemistry and enzyme-linked immunosorbent assay were used to evaluate their expression at the protein level. Human articular cartilage biopsies were used to evaluate chemokine and receptor expression in normal tissue. We found no expression of CXCR1, CXCR2, CXCR3, or CXCL10 at the mRNA level. CXCL8 mRNA was down-modulated, whereas at the protein level, we found greater release of this chemokine. CXCR4 and its ligand CXCL12 were down-modulated during chondrogenesis. By contrast, CXCR5 was up-regulated, whereas its ligand CXCL13 was lower. These data were also confirmed on human articular cartilage. These findings show that, during in vitro h-MSC chondrogenic differentiation, chemokine and receptor expression was specifically induced or repressed. This was in line with what the authors also found in normal articular cartilage, suggesting a role in differentiation and maturation of a cartilage-like structure in vitro and consequently the regulation of cartilage homeostasis.