Too much ado about data: Continuous remote monitoring of water temperatures, circulation and throughput can assist in the reduction of hospital-associated waterborne infections.

BACKGROUND: National and international guidance provides advice on maintenance and management of water systems in healthcare buildings, however, healthcare-associated waterborne infections (HAWI) are increasing. This narrative review identifies parameters critical to water quality in healthcare buildings and assesses if remote sensor monitoring can deliver safe water systems thus reducing HAWI. METHOD: A narrative review was performed using the following search terms 1) consistent water temperature AND waterborne pathogen control OR nosocomial infection 2) water throughput AND waterborne pathogen control OR nosocomial infection 3) remote monitoring of in-premise water systems AND continuous surveillance for temperature OR throughput OR flow OR use. Databases employed were PubMed, CDSR (Clinical Study Data Request) and DARE (Database of Abstracts of Reviews of Effects) from Jan 2013 - Mar 2024. FINDINGS: Single ensuite-patient rooms, expansion of wash-hand basins, widespread glove use, alcohol gel and wipes have increased water system stagnancy resulting in amplification of waterborne pathogens and transmission risk of Legionella, Pseudomonas and Non-Tuberculous Mycobacteria. Manual monitoring does not represent temperatures across large complex water systems. This review deems that multiple point continuous remote sensor monitoring is effective at identifying redundant and low use outlets, hydraulic imbalance and inconsistent temperature delivery across in-premise water systems. CONCLUSION: As remote monitoring becomes more common there will be greater recognition of failures in temperature control, hydraulics and balancing in water systems and there remains much to learn as we adopt this developing technology within our hospitals.

Comparison of culture media for the detection of Legionella spp. in sanitary water samples: Adaptation to ISO 11731:2017.

INTRODUCTION: Water sample culturing is the reference method to detect Legionella spp. in sanitary facilities. Until 2017, UNE-EN ISO 11731 only included the GVPC medium, which inhibits interfering microbiota but hinders the growth of Legionella spp. To improve its recovery, the new standard incorporates the BCYE medium into the working protocol. METHODS: We inoculated 1306 sanitary water samples onto BCYE and GVPC according to an accredited internal procedure. We compared the number of cfu/L of Legionella spp. detected in both media. RESULTS: The median in BCYE was 2000 cfu/L higher than in GVPC (P = .000). In the presence of high amounts of interfering microbiota, both media were similar; in the absence or low interfering microbiota BCYE was four times more sensitive than GVPC (P = .000). CONCLUSION: Including BCYE in the analysis of sanitary water significantly improves the recovery of Legionella spp. in low contaminated samples.

On-site filtration of large sample volumes improves the detection of opportunistic pathogens in drinking water distribution systems.

In this study, we compared conventional vacuum filtration of small volumes through disc membranes (effective sample volumes for potable water: 0.3-1.0 L) with filtration of high volumes using ultrafiltration (UF) modules (effective sample volumes for potable water: 10.6-84.5 L) for collecting bacterial biomass from raw, finished, and tap water at seven drinking water systems. Total bacteria, Legionella spp., Legionella pneumophila, Mycobacterium spp., and Mycobacterium avium complex in these samples were enumerated using both conventional quantitative PCR (qPCR) and viability qPCR (using propidium monoazide). In addition, PCR-amplified gene fragments were sequenced for microbial community analysis. The frequency of detection (FOD) of Legionella spp. in finished and tap water samples was much greater using UF modules (83% and 77%, respectively) than disc filters (24% and 33%, respectively). The FODs for Mycobacterium spp. in raw, finished, and tap water samples were also consistently greater using UF modules than disc filters. Furthermore, the number of observed operational taxonomic units and diversity index values for finished and tap water samples were often substantially greater when using UF modules as compared to disc filters. Conventional and viability qPCR yielded similar results, suggesting that membrane-compromised cells represented a minor fraction of total bacterial biomass. In conclusion, our research demonstrates that large-volume filtration using UF modules improved the detection of opportunistic pathogens at the low concentrations typically found in public drinking water systems and that the majority of bacteria in these systems appear to be viable in spite of disinfection with free chlorine and/or chloramine.IMPORTANCEOpportunistic pathogens, such as Legionella pneumophila, are a growing public health concern. In this study, we compared sample collection and enumeration methods on raw, finished, and tap water at seven water systems throughout the State of Minnesota, USA. The results showed that on-site filtration of large water volumes (i.e., 500-1,000 L) using ultrafiltration membrane modules improved the frequency of detection of relatively rare organisms, including opportunistic pathogens, compared to the common approach of filtering about 1 L using disc membranes. Furthermore, results from viability quantitative PCR (qPCR) with propidium monoazide were similar to conventional qPCR, suggesting that membrane-compromised cells represent an insignificant fraction of microorganisms. Results from these ultrafiltration membrane modules should lead to a better understanding of the microbial ecology of drinking water distribution systems and their potential to inoculate premise plumbing systems with opportunistic pathogens where conditions are more favorable for their growth.

Legionella spp. Monitoring in the Water Supply Systems of Accommodation Facilities in Sardinia, Italy: A Two-Year Retrospective Analysis.

Travel-associated Legionnaires' disease is a significant public health concern worldwide. A high number of cases are reported every year among travellers who stay at guest houses, hotels, and spas. Indeed, hot water systems, showers, and air-conditioning systems can be contaminated by Legionella, which grows at 25-42 °C. Studies have shown that in Sardinia, especially during the summer months, the water circulation in the hotels' pipes is exposed to extremely high temperatures. As a result, this study was conducted to assess the colonization of hotel water systems by Legionella in Sardinia, concerning a recent EU directive 2020/2184 for drinking water with a limit of 1000 CFU /L. Methods. A total of 112 accommodation facilities were analyzed, of which 61.3% were found to be colonized with Legionella, and out of a total of 807 samples, 32.5% were positive for Legionella presence. The results showed a higher number of positive samples in the summer season. This was also associated with the higher concentration presence of >1000 CFU/L in the samples. Consequently, this study confirms that local hotel operators should improve their water safety and prevention plans, especially in spring and summer.

Assessing the impact of the COVID­19 pandemic health protocols on the hygiene status of swimming pools of hotel units.

With the onset of the coronavirus disease 2019 (COVID-19) pandemic, numerous countries imposed strict lockdown measures and travel bans, resulting in the closure of hotels. Over time, the opening of hotel units was gradually allowed, and new strict regulations and protocols were issued to ensure the hygiene and safety of swimming pools in the era of COVID-19. The present study aimed to evaluate the implementation of strict health COVID-19-related protocols in hotel units during the 2020 summer tourist season concerning microbiological hygiene and physicochemical parameters of water, and to compare the data with those from the 2019 tourist season. For this reason, 591 water samples from 62 swimming pools were analyzed, of which 381 samples were for the 2019 tourist season and 210 samples were for the 2020 tourist season. To examine the presence of Legionella spp, 132 additional samples were taken from 14 pools, of which 49 in 2019 and 83 in 2020. In 2019, 2.89% (11/381) of the samples were out of legislative limits (0/250 mg/l) regarding the presence of Escherichia coli (E. coli), 9.45% (36/381) were out of acceptable limits (0/250 mg/l) regarding the presence of Pseudomonas aeruginosa (P. aeruginosa) and 8.92% (34/381) had residual chlorine levels <0.4 mg/l. In 2020, 1.43% (3/210) of the samples were out of the legislative limits as regards the presence of E. coli, 7.14% (15/210) were out of acceptable limits regarding the presence of P. aeruginosa and 3.33% (7/210) of the samples measured residual chlorine levels <0.4 mg/l. The risk ratio (RR) in relation to the presence of E. coli due to incorrect compliance with the requirements for residual chlorine was calculated for 2019 at 8.50, while in 2020 it was calculated at 14.50 (P=0.008). The RR of the presence of P. aeruginosa due to inappropriate residual chlorine requirements was calculated in 2019 at 2.04 (P=0.0814), while in 2020 it was calculated at 2.07 (P=0.44). According to the microbiological hygiene and physicochemical parameters of the water samples studied, there was a significant improvement due to the strict protocols for the swimming pools in the summer season of 2020 compared to the tourist season of 2019, namely 72.72% (E. coli), 58.33% (P. aeruginosa), 79.41% (of residual chlorine <0.4 mg/l) in the three main parameters studied. Finally, an increased colonization by Legionella spp. detected in the internal networks of the hotels due to the non-operation of the hotels during the lockdown, the improper disinfection and stagnant water in the internal water supply networks. Specifically, in 2019, 95.92% (47/49) of the samples tested negative and 4.08% (2/49) tested positive (≥50 CFU/l) for Legionella spp., compared to 2020 where 91.57% (76/83) of the samples tested negative and 8.43% (7/83) tested positive.

Contamination of Hotel Water Distribution Systems by Legionella Species: Environmental Surveillance in Campania Region, South Italy.

The COVID-19 pandemic period was marked by the absence or reduced circulation of some infectious diseases. Legionellosis may have been affected by the prevention measures adopted to counter COVID-19. Legionellosis is a form of pneumonia interstitial that is normally transmitted via aerosol-containing bacteria (genus Legionella), that could be present in contaminated water sources and is often associated with travel and with staying in hotels. In this work, the data of the environmental surveys carried out by ARPA Campania in accommodation facilities since 2019 were analyzed for a better understanding of the dispersion patterns of L. pneumophila associated with the environment and to evaluate the variation of the data during the pandemic period. The aim was to provide a better understanding of Legionella at different geographic scales and to define a predictive epidemiological method. Results: In 2019, the Legionella genus contaminated 37.7% of all tourist facilities evaluated. In 2020, the Legionella genus contaminated 44.4% of all tourist facilities evaluated. In 2021, the Legionella genus contaminated 54.2% of all tourist facilities evaluated. Conclusions: Legionella pneumophila was the most prevalent species in our community, serogroup 1 was the most frequently isolated and the most implicated risk factor of contamination was the temperature of water in circulation.

Heterotrophic Plate Count Can Predict the Presence of Legionella spp. in Cooling Towers.

Legionella pneumophila (Lp) colonizes aquatic environments and is a potential pathogen to humans, causing outbreaks of Legionnaire's disease. It is mainly associated with contaminated cooling towers (CTs). Several regulations, including Spanish legislation (Sl), have introduced the analysis of heterotrophic plate count (HPC) bacteria and Legionella spp. (Lsp) in management plans to prevent and control Legionella outbreaks from CTs. The 2003 Sl for CTs (RD 865/2003) considered that concentrations of HPC bacteria ≤10,000 cfu/mL and of Lsp ≤100 cfu/L are safe; therefore, no action is required, whereas management actions should be implemented above these standards. We have investigated to what extent the proposed standard for HPC bacteria is useful to predict the presence of Lsp in cooling waters. For this, we analyzed Lsp and HPC concentrations, water temperature, and the levels of chlorine in 1376 water samples from 17 CTs. The results showed that in the 1138 water samples negative for Legionella spp. (LN), the HPC geometric mean was significantly lower (83 cfu/mL, p < 0.05) than in the positive Lsp. samples (135 cfu/mL). Of the 238 (17.3%) LP samples, 88.4% (210/238) were associated with values of HPC ≤10,000 cfu/mL and most of them showed HPC concentrations ≤100 (53.7%). In addition, a relatively low percentage of LP (28/238, 11.6%) samples were associated with HPC bacteria concentrations >10,000 cfu/mL, indicating that this standard does not predict the colonization risk for Legionella in the CTs studied. The present study has demonstrated that a threshold concentration ≤100 cfu/mL of HPC bacteria could better predict the higher concentration of Legionella in CTs, which will aid in preventing possible outbreaks.

Legionnaires' disease in Switzerland: rationale and study protocol of a prospective national case-control and molecular source attribution study (SwissLEGIO).

Switzerland has one of the highest annual Legionnaires' disease (LD) notification rates in Europe (7.8 cases/100,000 population in 2021). The main sources of infection and the cause for this high rate remain largely unknown. This hampers the implementation of targeted Legionella spp. control efforts. The SwissLEGIO national case-control and molecular source attribution study investigates risk factors and infection sources for community-acquired LD in Switzerland. Over the duration of one year, the study is recruiting 205 newly diagnosed LD patients through a network of 20 university and cantonal hospitals. Healthy controls matched for age, sex, and residence at district level are recruited from the general population. Risk factors for LD are assessed in questionnaire-based interviews. Clinical and environmental Legionella spp. isolates are compared using whole genome sequencing (WGS). Direct comparison of sero- and sequence types (ST), core genome multilocus sequencing types (cgMLST), and single nucleotide polymorphisms (SNPs) between clinical and environmental isolates are used to investigate the infection sources and the prevalence and virulence of different Legionella spp. strains detected across Switzerland. The SwissLEGIO study innovates in combining case-control and molecular typing approaches for source attribution on a national level outside an outbreak setting. The study provides a unique platform for national Legionellosis and Legionella research and is conducted in an inter- and transdisciplinary, co-production approach involving various national governmental and national research stakeholders.

Flushing Temporarily Improves Microbiological Water Quality for Buildings Supplied with Chloraminated Surface Water but Has Little Effect for Groundwater Supplies.

Microbial communities in premise plumbing systems were investigated after more than 2 months of long-term stagnation, during a subsequent flushing event, and during post-flush stagnation. Water samples were collected from showers in buildings supplied with chlorinated groundwater, untreated groundwater, and chloraminated surface water. The building supplied with chlorinated groundwater generally had the lowest bacterial concentrations across all sites (ranging from below quantification limit to 5.2 log copies/L). For buildings supplied with untreated groundwater, bacterial concentrations (5.0 to 7.6 log copies/L) and microbial community diversity index (ACE) values were consistent throughout sampling. Nontuberculous mycobacteria (NTM) and Legionella pneumophila were not detected in any groundwater-supplied buildings. Total bacteria, Legionella spp., and NTM were abundant in the surface water-supplied buildings following long-term stagnation (up to 7.6, 6.2, and 7.6 log copies/L, respectively). Flushing decreased these concentrations by ∼1 to >4 log units and reduced microbial community diversity, but the communities largely recovered within a week of post-flush stagnation. The results suggest that buildings supplied with disinfected surface water are more likely than buildings supplied with treated or untreated groundwater to experience deleterious changes in microbiological water quality during stagnation and that the water quality improvements from flushing with chloraminated water, while substantial, are short-lived.

Surveillance of Legionella pneumophila: Detection in Public Swimming Pool Environment.

The bacterium Legionella pneumophila is a ubiquitous microorganism naturally present in water environments. The actual presence of this opportunistic premise plumbing pathogen in recreational swimming pools and hot tubs in the northwestern part of Croatia has not been investigated. This study aimed to analyze the presence of the opportunistic pathogen L. pneumophila in public swimming pool water in Primorje-Gorski Kotar County (N = 4587) over a four-year period (2018-2021). Additionally, the second aim was to investigate the connection between the presence of L. pneumophila and pool water physicochemical parameters using mathematical predictive models. The presence of L. pneumophila was detected in six pool samples. Five positive samples were found in the water of indoor hot tubs filled with fresh water, and one positive sample in an outdoor recreational saltwater pool. A predictive mathematical model showed the simultaneous influence of chemical parameters dominated by the temperature in saltwater and freshwater pools, as well as the significant influence of free residual chlorine and trihalomethanes. Our results pointed out that keeping all physicochemical parameters in perfect harmony is necessary to reach the best disinfection procedure and to avoid the optimum conditions for L. pneumophila occurrence.

Triclosan Promotes Conjugative Transfer of Antibiotic Resistance Genes to Opportunistic Pathogens in Environmental Microbiome.

Although triclosan, as a widely used antiseptic chemical, is known to promote the transmission of antibiotic resistance to diverse hosts in pure culture, it is still unclear whether and how triclosan could affect the transmission of broad-host-range plasmids among complex microbial communities. Here, bacterial culturing, fluorescence-based cell sorting, and high-throughput 16S rRNA gene amplicon sequencing were combined to investigate contributions of triclosan on the transfer rate and range of an IncP-type plasmid from a proteobacterial donor to an activated sludge microbiome. Our results demonstrate that triclosan significantly enhances the conjugative transfer of the RP4 plasmid among activated sludge communities at environmentally relevant concentrations. High-throughput 16S rRNA gene sequencing on sorted transconjugants demonstrates that triclosan not only promoted the intergenera transfer but also the intragenera transfer of the RP4 plasmid among activated sludge communities. Moreover, triclosan mediated the transfer of the RP4 plasmid to opportunistic human pathogens, for example, Legionella spp. The mechanism of triclosan-mediated conjugative transfer is primarily associated with excessive oxidative stress, followed by increased membrane permeability and provoked SOS response. Our findings offer insights into the impacts of triclosan on the dissemination of antibiotic resistance in the aquatic environmental microbiome.

Legionnaires' Disease: Update on Diagnosis and Treatment.

Legionellosis is the infection caused by bacteria of the genus Legionella, including a non-pneumonic influenza-like syndrome, and Legionnaires' disease is a more serious illness characterized by pneumonia. Legionellosis is becoming increasingly important as a public health problem throughout the world; although it is an underreported disease, studies have consistently documented a high incidence. In addition, health costs associated with the disease are high. Diagnosis of Legionnaires' disease is based mainly on the detection of Legionella pneumophila serogroup 1 antigen in urine. However, there have been advances in detection tests for patients with legionellosis. New methodologies show greater sensitivity and specificity, detect more species and serogroups of Legionella spp., and have the potential for use in epidemiological studies. Testing for Legionella spp. is recommended at hospital admission for severe community-acquired pneumonia, and antibiotics directed against Legionella spp. should be included early as empirical therapy. Inadequate or delayed antibiotic treatment in Legionella pneumonia has been associated with a worse prognosis. Either a fluoroquinolone (levofloxacin or moxifloxacin) or a macrolide (azithromycin preferred) is the recommended first-line therapy for Legionnaires' disease; however, little information is available regarding adverse events or complications, or about the duration of antibiotic therapy and its association with clinical outcomes. Most published studies evaluating antibiotic treatment for Legionnaires' disease are observational and consequently susceptible to bias and confounding. Well-designed studies are needed to assess the usefulness of diagnostic tests regarding clinical outcomes, as well as randomized trials comparing fluoroquinolones and macrolides or combination therapy that evaluate outcomes and adverse events.

Legionella endocarditis: A case report and review.

BACKGROUND: Legionella is a well known but infrequent cause of bacterial endocarditis. METHODS: We report a case of endocarditis caused by Legionella spp. We reviewed previously reported cases in PubMed, Google Scholar and in references included in previous reports, and summarized relevant clinical data. RESULTS: A 63-year-old man with a history of aortic valve replacement developed persistent fever and monoarthritis. Transesophageal echocardiography showed perivalvular abscess. He died during surgery. Blood and valve cultures were negative. Legionella spp. was demonstrated with 16S-rRNA PCR from the resected material. Twenty cases of Legionella endocarditis have been reported. Harboring a prosthetic valve was the main risk factor. Prognosis was favorable, both for patients treated with or without surgical valve replacement. Overall mortality was <10%. CONCLUSIONS: Legionella is an infrequent cause of endocarditis. It frequently requires surgical treatment. Prognosis is good. Molecular techniques are likely to become the gold standard for diagnosis.

Legionella endocarditis: A case report and review / Endocarditis por Legionella: caso clínico y revisión

BackgroundLegionella is a well known but infrequent cause of bacterial endocarditis.MethodsWe report a case of endocarditis caused by Legionella spp. We reviewed previously reported cases in PubMed, Google Scholar and in references included in previous reports, and summarized relevant clinical data.ResultsA 63-year-old man with a history of aortic valve replacement developed persistent fever and monoarthritis. Transesophageal echocardiography showed perivalvular abscess. He died during surgery. Blood and valve cultures were negative. Legionella spp. was demonstrated with 16S-rRNA PCR from the resected material. Twenty cases of Legionella endocarditis have been reported. Harboring a prosthetic valve was the main risk factor. Prognosis was favorable, both for patients treated with or without surgical valve replacement. Overall mortality was <10%.ConclusionsLegionella is an infrequent cause of endocarditis. It frequently requires surgical treatment. Prognosis is good. Molecular techniques are likely to become the gold standard for diagnosis.

IntroducciónLegionella es una causa bien conocida, aunque infrecuente, de endocarditis bacteriana.MétodosA continuación, presentamos un caso de endocarditis por Legionella spp. Hemos revisado los casos publicados hasta la fecha en PubMed, Google Scholar y otras referencias incluidas en las comunicaciones previas, y resumimos aquí los datos clínicos relevantes. Un varón de 63 años con historia de recambio valvular aórtico desarrolló fiebre y monoartritis por lo que se realizó un ecocardiograma transesofágico que demostró un absceso perivalvular. Se decidió tratamiento quirúrgico, y el paciente falleció durante la cirugía. Los cultivos tanto de sangre como valvulares fueron negativos. El análisis mediante 16S rRNA PCR del material valvular resecado durante la cirugía demostró Legionella spp. Se han publicado un total de veinte casos de endocarditis por Legionella en la literatura médica. El principal factor de riesgo observado fue la presencia de una válvula protésica. El pronóstico fue bueno tanto para los pacientes tratados de manera conservadora como para aquellos sometidos a recambio valvular quirúrgico. La mortalidad global fue <10%.ConclusionesLegionella es una causa infrecuente de endocarditis. Generalmente requiere tratamiento quirúrgico. El pronóstico es bueno. Las técnicas moleculares podrían convertirse en el método de referencia para su diagnóstico.

Isolation and identification of Legionella spp. from non-hospital facilities: a preliminary one-year surveillance study in the urban area of Pesaro-Urbino (Central Italy).

BACKGROUND: Legionella is considered one of the most important causes of potentially preventable morbid-ity and mortality. These microorganisms are ubiquitous, but incomplete information is available on the geographic distribution of Legionella species in our region. STUDY DESIGN: For the mentioned reasons, in this work the distribution of Legionella spp. in non-hospital facilities of the urban area of Pesaro-Urbino (Central Italy), including public fountains, residential build-ings, public and private offices and retirement homes, was investigated. METHODS: A total of 298 water samples were collected from the different facilities and subjected to standard Legionella isolation and identification protocols. RESULTS: As reported, 17.8% of the collected water samples resulted positive for Legionella spp. (28.6% from retirement homes, 21.3% from residential buildings, 15.3% from private and public offices). The highest per-centage of positive samples (14.4%) was found in hot water from retirements homes (58.8%) and residential buildings (31.8%); the most frequent isolated serogroups were L. pneumophila 2-14 (71.7%). CONCLUSIONS: This work is the first describing the distribution of Legionella spp. in non-hospital facilities in the province of Pesaro-Urbino, and highlights a condition of potential risk for susceptible categories. From our data, we can point that a regular and constant control to prevent microbiological risk from legionellosis, particularly in facilities housing the elderly, is recommended.

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. IMPORTANCELegionella 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. Several studies focused on the mip gene classification scheme showed its limitations and the need to improve the classification scheme, including other genes. Our study provides significant advantages on Legionella identification, providing a reproducible new rpoB gene classification scheme that seems to be more accurate than mip gene sequencing, bringing out greater genetic variation on Legionella species. In addition, the combined use of both the mip and rpoB genes allowed us to identify presumed new Legionella species, improving epidemiological investigations and acquiring new understanding on Legionella fields.

Amoebal coculture and enrichment methods as a proposal for water quality control in Brazil.

Free living amoebae (FLA) can be found in different environments, where they feed on diverse microorganisms. Some bacteria preyed by FLA are called amoeba-resistant bacteria (ARB), as they can resist to lysosomal fusion and are capable of multiplying and evading FLA after internalization, propagating in the environment. Despite the health risks due to the existence of pathogenic and opportunistic species that are ARB and the pathogenicity of some FLA species, there are no water quality protocols to analyze the presence of ARB or FLA. In this sense, our study aimed to isolate FLA through amoebal enrichment and to identify ARB using amoebal coculture in water samples from a public park and two hospitals in southern Brazil. As a result, 9 different microorganisms genera have been identified through amoebal coculture, including fastidious Legionella spp. and Bosea vestrisii. From the positive samples for FLA, by amoebal enrichment, Acanthamoeba spp., Vermamoeba vermiformis and Naegleria spp. were identified in 14 amoebic isolates. The methodologies used in this work proved to be effective as simple and low-cost methods to be used in the implementation in water quality control of anthropogenic environments.

Assessment of hygienic conditions of recreational facility restrooms: an integrated approach.

INTRODUCTION: Microbiological quality of recreational environments included restrooms, is generally assessed by water and surface monitoring. In this study, an environmental monitoring, conducted in spring, of swimming pool restrooms of a recreation center located in the Marche region has been carried out. Seven water samples and seven surface swabs were collected. Moreover, six air samples have been included. The aim of this study was to evaluate if air microbiological monitoring, along with molecular detection in real-time PCR, could give additional useful information about the hygienic conditions of the facility. METHODS: Heterotrophic Plate Count (HPC) both at 22°C (psychrophilic) and 37°C (mesophilic) was determined by separate cultures in all samples. The presence of Legionella pneumophila and Pseudomonas aeruginosa was evaluated by both culture and real-time PCR. RESULTS: The analysis of shower water recorded a HPC load of mesophilic bacteria (37°C) more than 10-fold higher in men restroom, respect to women's one (> 100 vs < 10 CFU/ml), while in air samples was between < 100 and > 500. Concerning pathogen presence, both species Legionella pneumophila and Pseudomonas aeruginosa were detected only in men restroom, but in different sample types by using different methods (culture and real-time PCR). CONCLUSIONS: Air sampling may offer the advantage of giving more representative data about microbial presence in restrooms, including bacterial species transmitted through aerosol, like Legionella. Moreover, the concurrent use of molecular and microbiological detection in an integrated approach could offer the advantage of greater sensitivity.

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.