Strong alkaline electrolyzed water efficiently inactivates SARS-CoV-2, other viruses, and Gram-negative bacteria.

Air spaces and material surfaces in a pathogen-contaminated environment can often be a source of infection to humans, and disinfection has become a common intervention focused on reducing the contamination levels. In this study, we examined the efficacy of SAIW, a unique electrolyzed water with chlorine-free, high pH, high concentration of dissolved hydrogen, and low oxygen reduction potential, for the inactivation of several viruses and bacteria. Infectivity assays revealed that initial viral titers of enveloped and non-enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, herpes simplex virus type 1, human coronavirus, feline calicivirus, and canine parvovirus, were reduced by 2.9- to 5.5-log10 within 30 s of SAIW exposure. Similarly, the culturability of three Gram-negative bacteria (Escherichia coli, Salmonella, and Legionella) dropped down by 1.9- to 4.9-log10 within 30 s of SAIW treatment. Mechanistically, treatment with SAIW was found to significantly decrease the binding and subsequent entry efficiencies of SARS-CoV-2 on Vero cells. Finally, we showed that this chlorine-free electrolytic ion water had no acute inhalation toxicity in mice, demonstrating that SAIW holds promise for a safer antiviral and antibacterial disinfectant.

Comparison of BCYEα+AB agar and MWY agar for detection and enumeration of Legionella spp. in hospital water samples.

BACKGROUND: This study illustrates for the first time the performance (sensitivity and selectivity) of the selective medium BCYEα +AB suggested by the new edition of ISO 11731 for legionella isolation and enumeration. We compared the efficacy of the selective BCYEα +AB medium with that of the highly selective MWY medium. RESULTS: Legionella spp. was detected in 48.2 and 47.1% of the samples by BCYEα +AB and MWY agar, respectively. For optimal detection of Legionella spp., most protocols recommend using selective media to reduce the number of non-Legionella bacteria. Agreement between the two media was 86.7%. CONCLUSIONS: According to the results, both media have a very similar performance and they both have advantages and disadvantages over each other. In AB medium there is the risk of being less selective so more interfering microbiota may grow but in MWY medium there is the risk of being too selective. The low selectivity of the AB medium could be resolved if other treatments are applied after filtration, e.g. acid and/or heat treatment, but it must be taken into account that these treatments still reduce the number of viable Legionella. In conclusion, we recommend using MWY as a selective medium for the detection of Legionella spp. as it is easier discern suspected colonies and facilitate the final Legionella spp.

The impact of pipeline changes and temperature increase in a hospital historically colonised with Legionella.

Healthcare-related Legionnaires' disease has a devastating impact on high risk patients, with a case fatality rate of 30-50%. Legionella prevention and control in hospitals is therefore crucial. To control Legionella water colonisation in a hospital setting we evaluated the effect of pipeline improvements and temperature increase, analysing 237 samples over a 2-year period (first year: 129, second year: 108). In the first year, 25.58% of samples were positive for Legionella and 16.67% for amoeba. Assessing the distance of the points analysed from the hot water tank, the most distal points presented higher proportion of Legionella colonisation and lower temperatures (nearest points: 6.4% colonised, and temperature 61.4 °C; most distal points: 50% and temperature 59.1 °C). After the first year, the hot water system was repaired and the temperature stabilised. This led to a dramatic reduction in Legionella colonisation, which was negative in all the samples analysed; however, amoeba colonisation remained stable. This study shows the importance of keeping the temperature stable throughout the circuit, at around 60 °C. Special attention should be paid to the most distal points of the circuit; a fall in temperature at these weak points would favour the colonisation and spread of Legionella, because amoeba (the main Legionella reservoir) are not affected by temperature.

Isolation and identification of Legionella spp. from hot spring water in Algeria by culture and molecular methods.

AIMS: Due to infectious risk associated with the presence of Legionella in warm water, we determined the prevalence of living Legionella spp. in hot spring water in Algeria. METHODS AND RESULTS: Detection of Legionella by culture was done by using two methods, direct culture on agar plates and co-culture with amoeba. Fifty samples were taken from different hot springs in northern Algeria, including swimming pools, showers and thermal sources. Legionella pneumophila serotypes were predominant, accounting for 60% of positive samples. Direct method allowed the isolation of 13 L. pneumophila only of 50 samples (26%), whereas co-culture using a panel of three free living amoeba allowed the isolation of 119 Legionella species from the same samples (80%) CONCLUSIONS: Amoeba co-culture allowed the isolation of several Legionella sp., while direct culture allowed the isolation of L. pneumophila only. Remarkably, Legionella longbeachae, usually isolated from soil and compost, was isolated for the first time in thermal water in three samples using Vermamoeba vermiformis co-culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Legionella in the water of hot springs in Algeria, which are mainly frequented by individuals at risk of Legionellosis, requires urgent control measures.

Quorum sensing modulates the formation of virulent Legionella persisters within infected cells.

The facultative intracellular bacterium Legionella pneumophila replicates in environmental amoebae and in lung macrophages, and causes Legionnaires' disease. Here we show that L. pneumophila reversibly forms replicating and nonreplicating subpopulations of similar size within amoebae. The nonreplicating bacteria are viable and metabolically active, display increased antibiotic tolerance and a distinct proteome, and show high virulence as well as the capacity to form a degradation-resistant compartment. Upon infection of naïve or interferon-γ-activated macrophages, the nonreplicating subpopulation comprises ca. 10% or 50%, respectively, of the total intracellular bacteria; hence, the nonreplicating subpopulation is of similar size in amoebae and activated macrophages. The numbers of nonreplicating bacteria within amoebae are reduced in the absence of the autoinducer synthase LqsA or other components of the Lqs quorum-sensing system. Our results indicate that virulent, antibiotic-tolerant subpopulations of L. pneumophila are formed during infection of evolutionarily distant phagocytes, in a process controlled by the Lqs system.

Co-Occurrence of Free-Living Amoeba and Legionella in Drinking Water Supply Systems.

Background and Objectives: Legionella is one of the most important water-related pathogens. Inside the water supply systems and the biofilms, Legionella interact with other bacteria and free-living amoeba (FLA). Several amoebas may serve as hosts for bacteria in aquatic systems. This study aimed to investigate the co-occurrence of Legionella spp. and FLA in drinking water supply systems. Materials and Methods: A total of 268 water samples were collected from apartment buildings, hotels, and public buildings. Detection of Legionella spp. was performed in accordance with ISO 11731:2017 standard. Three different polymerase chain reaction (PCR) protocols were used to identify FLA. Results: Occurrence of Legionella varied from an average of 12.5% in cold water samples with the most frequent occurrence observed in hot water, in areas receiving untreated groundwater, where 54.0% of the samples were Legionella positive. The occurrence of FLA was significantly higher. On average, 77.2% of samples contained at least one genus of FLA and, depending on the type of sample, the occurrence of FLA could reach 95%. In the samples collected during the study, Legionella was always isolated along with FLA, no samples containing Legionella in the absence of FLA were observed. Conclusions: The data obtained in our study can help to focus on the extensive distribution, close interaction, and long-term persistence of Legionella and FLA. Lack of Legionella risk management plans and control procedures may promote further spread of Legionella in water supply systems. In addition, the high incidence of Legionella-related FLA suggests that traditional monitoring methods may not be sufficient for Legionella control.

Legionella feeleii: pneumonia or Pontiac fever? Bacterial virulence traits and host immune response.

Gram-negative bacterium Legionella is able to proliferate intracellularly in mammalian host cells and amoeba, which became known in 1976 since they caused a large outbreak of pneumonia. It had been reported that different strains of Legionella pneumophila, Legionella micdadei, Legionella longbeachae, and Legionella feeleii caused human respiratory diseases, which were known as Pontiac fever or Legionnaires' disease. However, the differences of the virulence traits among the strains of the single species and the pathogenesis of the two diseases that were due to the bacterial virulence factors had not been well elucidated. L. feeleii is an important pathogenic organism in Legionellae, which attracted attention due to cause an outbreak of Pontiac fever in 1981 in Canada. In published researches, it has been found that L. feeleii serogroup 2 (ATCC 35849, LfLD) possess mono-polar flagellum, and L. feeleii serogroup 1 (ATCC 35072, WRLf) could secrete some exopolysaccharide (EPS) materials to the surrounding. Although the virulence of the L. feeleii strain was evidenced that could be promoted, the EPS might be dispensable for the bacteria that caused Pontiac fever. Based on the current knowledge, we focused on bacterial infection in human and murine host cells, intracellular growth, cytopathogenicity, stimulatory capacity of cytokines secretion, and pathogenic effects of the EPS of L. feeleii in this review.

Primary Colonizing Betaproteobacteriales Play a Key Role in the Growth of Legionella pneumophila in Biofilms on Surfaces Exposed to Drinking Water Treated by Slow Sand Filtration.

Slow sand filtration with extensive pretreatment reduces the microbial growth potential of drinking water to a minimum level at four surface water supplies in The Netherlands. The potential of these slow sand filtrates (SSFs) to promote microbial growth in warm tap water installations was assessed by measuring biofilm formation and growth of Legionella bacteria on glass and chlorinated polyvinylchloride (CPVC) surfaces exposed to SSFs at 37 ± 2°C in a model system for up to six months. The steady-state biofilm concentration ranged from 230 to 3,980 pg ATP cm-2 on glass and 1.4 (±0.3)-times-higher levels on CPVC. These concentrations correlated significantly with the assimilable organic carbon (AOC) concentrations of the warm water (8 to 24 µg acetate-C equivalents [ac-C eq] liter-1), which were raised about 2 times by mixing cold and heated (70°C) SSFs. All biofilms supported growth of Legionella pneumophila with maximum concentrations ranging from 6 × 102 to 1.5 × 105 CFU cm-2 Biofilms after ≤50 days of exposure were predominated by Betaproteobacteriales, mainly Piscinibacter, Caldimonas, Methyloversatilis, and an uncultured Rhodocyclaceae bacterium. These rapidly growing primary colonizers most likely served as prey for the host amoebae of L. pneumophilaAlphaproteobacteria, mostly Xanthobacteraceae, e.g., Bradyrhizobium, Pseudorhodoplanes, and other amoeba-resistant bacteria, accounted for 37.5% of the clones retrieved. A conceptual model based on a quadratic relationship between the L. pneumophila colony count and the biofilm concentration under steady-state conditions is used to explain the variations in the Legionella CFU pg-1 ATP ratios in the biofilms.IMPORTANCE Proliferation of L. pneumophila in premise plumbing poses a public health threat. Extended water treatment using physicochemical and biofiltration processes, including slow sand filtration, at four surface water supplies in The Netherlands reduces the microbial growth potential of the treated water to a minimum level, and the distributed drinking water complies with high quality standards. However, heating of the water in warm tap water installations increases the concentration of easily assimilable organic compounds, thereby promoting biofilm formation and growth of L. pneumophila Prevention of biofilm formation in plumbing systems by maintenance of a disinfectant residual during distribution and/or further natural organic matter (NOM) removal is not feasible in the supplies studied. Temperature management in combination with optimized hydraulics and material selection are therefore essential to prevent growth of L. pneumophila in premise plumbing systems. Still, reducing the concentration of biodegradable compounds in drinking water by appropriate water treatment is important for limiting the Legionella growth potential.

The presence and prevalence of Legionella spp in collected rainwater and its aerosolisation during common gardening activities.

AIMS: To determine the presence and prevalence of Legionella spp in domestic rainwater storage butts and to quantify its aerosolisation when collected rainwater is used for common gardening activities. METHODS: Volunteers were asked to take a water sample from their garden rainwater storage butt. The presence of Legionella was determined using quantitative polymerase chain reaction (qPCR). Two new rainwater storage butts were installed on-site at PHE Porton and positioned in sunlight or in the shade. Ambient conditions and those within the two 'experimental' water butts were continually monitored. A cyclone air sampler was used to detect the presence of Legionella in the air when collected rainwater was poured from a watering can or delivered via a hosepipe attached to a submersible water butt pump. RESULTS: A total of 63 volunteers provided water samples from 113 different rainwater storage butts. Legionella spp was detected in 107 of these samples at a mean concentration of 4.7 × 104 genomic units l-1. Two of these samples also contained L. pneumophila. The water butt positioned in the shade stored water at a significantly lower temperature than that exposed to sunlight. While the concentration of Legionella was significantly higher in this cooler water, meteorological conditions rather than conditions within the water butt had the greatest effect upon Legionella concentration. No Legionella was detected in the air when rainwater was poured from a watering can. However, using a hose pipe on a 'fine spray' setting increased both the number of organisms detected in the air and their dissemination. CONCLUSION: In this study, Legionella spp were common contaminants of collected rainwater. However, the use of rainwater for common gardening activities should not be discouraged. Aerosolisation of Legionella when using a watering can is minimal and any increased risk associated with hose pipe use can be mitigated by using a coarse spray setting.

Differential development of Legionella sub-populations during short- and long-term starvation.

Legionellae are among the most important waterborne pathogens in industrialized countries. Monitoring and surveillance of Legionella in engineered water systems is usually performed with culture-based methods. Since the advent of culture-independent techniques, it has become clear that Legionella concentrations are often several orders of magnitude higher than those measured by culture-based techniques and that a variable proportion of these non-culturable cells are viable. In engineered water systems, the formation of these viable but non-culturable (VBNC) cells can be caused by different kinds of stress, such as, and most importantly, nutrient starvation, oxidative stress and heat. In this study, the formation of VBNC cells of six Legionella strains under conditions of starvation was monitored in mono-species microcosms for up to one year using a combination of different viability indicators. Depending on the strain, complete loss of culturability was observed from 11 days to 8 weeks. During the starvation process, three distinct phases and different sub-populations of VBNC cells were identified. Until complete loss of culturability, the number of membrane-intact cells decreased rapidly to 5.5-69% of the initial cell concentration. The concentration of the sub-population with low esterase activity dropped to 0.03-55%, and the concentration of the highly esterase-active sub-population dropped to 0.01-1.2% of the initial concentration; these sub-populations remained stable for several weeks to months. Only after approximately 200 days of starvation, the number of VBNC cells started to decrease below detection limits. The most abundant VBNC sub-populations were characterized by partially damaged membranes and low esterase-activity. With this study, we showed that upon starvation, a stable VBNC Legionella community may be present over several months in a strain-dependent manner even under harsh conditions. Even after one year of starvation, a small proportion of L. pneumophila cells with high esterase-activity was detected. We speculate that this highly active VBNC subpopulation is able to infect amoebae and human macrophages.

Adaptation of Amoeba Plate Test To Recover Legionella Strains from Clinical Samples.

The isolation of Legionella from respiratory samples is the gold standard for diagnosis of Legionnaires' disease (LD) and enables epidemiological studies and outbreak investigations. The purpose of this work was to adapt and to evaluate the performance of an amoebic coculture procedure (the amoeba plate test [APT]) for the recovery of Legionella strains from respiratory samples, in comparison with axenic culture and liquid-based amoebic coculture (LAC). Axenic culture, LAC, and APT were prospectively performed with 133 respiratory samples from patients with LD. The sensitivities and times to results for the three techniques were compared. Using the three techniques, Legionella strains were isolated in 46.6% (n = 62) of the 133 respiratory samples. The sensitivity of axenic culture was 42.9% (n = 57), that of LAC was 30.1% (n = 40), and that of APT was 36.1% (n = 48). Seven samples were positive by axenic culture only; for those samples, there were <10 colonies in total. Five samples, all sputum samples, were positive by an amoebic procedure only (5/5 samples by APT and 2/5 samples by LAC); all had overgrowth by oropharyngeal flora with axenic culture. The combination of axenic culture with APT yielded a maximal isolation rate (i.e., 46.6%). Overall, the APT significantly reduced the median time for Legionella identification to 4 days, compared with 7 days for LAC (P < 0.0001). The results of this study support the substitution of LAC by APT, which could be implemented as a second-line technique for culture-negative samples and samples with microbial overgrowth, especially sputum samples. The findings provide a logical basis for further studies in both clinical and environmental settings.

Plasmas ozone inactivation of Legionella in deionized water and wastewater.

The results show that ozone concentration determination using ultraviolet spectrophotometry (UV-2450) at 258 nm is easier than using indigo method at 600 nm. A strong linear relationship was found between purge time and O3 concentration in deionized water. Ozone concentration can be predicted in deionized water. A higher O3 flow rate or lower temperature led to a higher O3 concentration. Ozone concentration was stable in 60 min, so that ozone self-decomposition could be ignored at ozone concentrations below 0.4 mg L-1. A higher temperature led to a higher inactivation efficiency and rate, and that a lower temperature led to a lower ozone decay rate and inactivation efficiency even if ozone solubility increased when temperature decreased. The fastest inactivation rate occurred before c0t = 165 µg L-1 s, but the inactivation rate decreased after c0t = 165 µg L-1 s with tail phenomena. The tail phenomena were clearly observed and may be caused by oxidization of lipopolysaccharides (LPS), cell membrane, etc. The activation energy Ea = 55,404 ± 0.3 J mol-1 were obtained for Legionella inactivation with ozone in deionized water. Ozone maximum decay rate was positively proportional to COD concentration. COD impacted on ozone concentration seriously. Higher COD concentration resulted in higher ozone decay rate. COD could result in ozone concentration decrement rapidly to a steady value in 5 s. Higher initial ozone concentration resulted in higher germ inactivation rate. Higher initial COD concentration resulted in lower Legionella inactivation efficiency. COD was easier to react with ozone than Legionella. The relationship among the initial COD concentrations COD0, the initial O3 concentration c0, and the O3 contact time t necessary for a 99.999% reduction of Legionella in wastewater can be expressed in some equations. O3 disinfection time t necessary for a 99.999% reduction of Legionella can be predicted by Eqs. (10) and (11). Graphical abstract ᅟ.

Temperature-driven growth of Legionella in lab-scale activated sludge systems and interaction with protozoa.

The occurrence of Legionella pneumophila in activated sludge systems has been reported in the literature. However, the factors triggering its growth are not yet well understood. This knowledge is needed to develop strategies to minimize the risk of the spread of Legionnaires' disease that originates in these systems. In the present study, L. pneumophila multiplied exponentially at 35 °C in activated sludge, but lower temperatures (24 °C and 15 °C) did not favour the growth of the pathogen despite the presence of its protozoan hosts (free-living amoeba and ciliates). L. non-pneumophila species, which are adapted to low temperature, prevailed at 15 °C. Legionella was found dispersed in the activated sludge, forming clusters of different sizes and also inside protozoa. This study shows that temperature is a key parameter triggering the growth of L. pneumophila in activated sludge. The detection of infected protozoa suggests that these are important vehicles for the multiplication of Legionella. However, in this study it was not possible to exclude the growth of Legionella directly on the substrate or on the activated sludge matrix.

Deposition pattern of aerosolized Legionella using an ex vivo human-porcine respiratory model.

Legionella are bacteria responsible for severe lung pathologies. However how they enter and are deposited within the respiratory tract remains poorly documented. Data using animal testing led to the establishment of mathematical models allowing the estimation of aerosol dispersion risks. But direct extrapolation to humans is questionable and experimental models more physiologically representative of the inhalation route are welcome. The aim of this study was to develop a model as close as possible to the human anatomy and physiology allowing determining the deposition pattern of aerosolized Legionella while limiting in vivo experiments. To that purpose, we adapted the chimeric respiratory tract model we previously developed. This original model consisted of a replica of the human upper respiratory airways made by additive manufacturing connected to ex vivo porcine lungs ventilated by passive expansion, as for humans in physiological conditions. These experiments didn't imply specific animal sacrifices as pigs were bred for human consumption and lungs were considered as wastes by the slaughterhouse. Fluorescent Legionella were aerosolized and visualized using Cellvizio® Lab (probe-based confocal fluorescence microscope). Legionella were found in the whole respiratory tract. Broncho-alveolar lavages were also performed and the amount of Legionella reaching the thoracic region was quantified by culture and qPCR. Legionella were found preferentially in the left upper lobe compared to the right lower lobe. To our knowledge, it is the first time that experiments mimicking so closely human exposure by inhalation are performed while limiting animal experiments and providing a model for further Legionella infectious risk assessment.

KKL-35 Exhibits Potent Antibiotic Activity against Legionella Species Independently of trans-Translation Inhibition.

trans-Translation is a ribosome-rescue system that is ubiquitous in bacteria. Small molecules defining a new family of oxadiazole compounds that inhibit trans-translation have been found to have broad-spectrum antibiotic activity. We sought to determine the activity of KKL-35, a potent member of the oxadiazole family, against the human pathogen Legionella pneumophila and other related species that can also cause Legionnaires' disease (LD). Consistent with the essential nature of trans-translation in L. pneumophila, KKL-35 inhibited the growth of all tested strains at submicromolar concentrations. KKL-35 was also active against other LD-causing Legionella species. KKL-35 remained equally active against L. pneumophila mutants that have evolved resistance to macrolides. KKL-35 inhibited the multiplication of L. pneumophila in human macrophages at several stages of infection. No resistant mutants could be obtained, even during extended and chronic exposure. Surprisingly, KKL-35 was not synergistic with other ribosome-targeting antibiotics and did not induce the filamentation phenotype observed in cells defective for trans-translation. Importantly, KKL-35 remained active against L. pneumophila mutants expressing an alternate ribosome-rescue system and lacking transfer-messenger RNA, the essential component of trans-translation. These results indicate that the antibiotic activity of KKL-35 is not related to the specific inhibition of trans-translation and its mode of action remains to be identified. In conclusion, KKL-35 is an effective antibacterial agent against the intracellular pathogen L. pneumophila with no detectable resistance development. However, further studies are needed to better understand its mechanism of action and to assess further the potential of oxadiazoles in treatment.

Growth of Legionella anisa in a model drinking water system to evaluate different shower outlets and the impact of cast iron rust.

Legionella continues to be a problem in water systems. This study investigated the influence of different shower mixer faucets, and the influence of the presence of cast iron rust from a drinking water system on the growth of Legionella. The research is conducted using a model of a household containing four drinking water systems. All four systems, which contained standard plumbing components including copper pipes and a water heater, were filled with unchlorinated drinking water. Furthermore, all systems had three different shower faucets: (A) a stainless-steel faucet, (B) a brass-ceramic faucet, and (C) a brass thermostatic faucet. System 1 was solely filled with drinking water. System 2 was filled with drinking water, and cast iron rust. System 3 was contaminated with Legionella, and system 4 was contaminated with a Legionella, and cast iron rust. During a period of 34 months, 450 cold water samples were taken from 15 sample points of the four drinking water systems, and tested for Legionella according to the Dutch Standard (NEN 6265). In system 4, with added cast iron rust, the stainless-steel mixer faucet (A) had the highest concentration of Legionella at >4.3log10CFU/l (>20,000CFU/l) and was positive in 46.4% of samples. In contrast, the stainless-steel mixer faucet (A) of system 3 without cast iron rust showed 14.3% positive samples with a maximum concentration of 3.9log10CFU/l (7600CFU/l) Legionella. Additionally, both contaminated systems (3 and 4), with the brass thermostatic faucet (C), tested positive for Legionella. System 3 in 85.7% of the samples, with a maximum concentration of 4.38log10CFU/l (24,200CFU/l), and system 4 in 64.3% of the samples with a maximum concentration of 4.13log10CFU/l (13.400CFU/l). These results suggest that both the type of faucet used in a drinking water system and the presence or absence of cast iron rust influence the growth of Legionella.

Kinetic analysis of Legionella inactivation using ozone in wastewater.

Legionella inactivation using ozone was studied in wastewater using kinetic analysis and modeling. The experimental results indicate that the relationship between the ozone concentration, germ concentration, and chemical oxygen demand (COD) can be used to predict variations in germ and COD concentrations. The ozone reaction with COD and inactivation of Legionella occurred simultaneously, but the reaction with COD likely occurred at a higher rate than the inactivation, as COD is more easily oxidized by ozone than Legionella. Higher initial COD concentrations resulted in a lower inactivation rate and higher lnN/N0. Higher temperature led to a higher inactivation efficiency. The relationship of the initial O3 concentration and Legionella inactivation rate was not linear, and thus, the Ct value required for a 99.99% reduction was not constant. The initial O3 concentration was more important than the contact time, and a reduction of the initial O3 concentration could not be compensated by increasing the contact time. The Ct values were compared over a narrow range of initial concentrations; the Ct values could only be contrasted when the initial O3 concentrations were very similar. A higher initial O3 concentration led to a higher inflection point value for the lnN/N0 vs C0t curve. Energy consumption using a plasma corona was lower than when using boron-doped diamond electrodes.

Aerobiology of the built environment: Synergy between Legionella and fungi.

BACKGROUND: The modern built environment (BE) design creates unique ecological niches ideal for the survival and mutual interaction of microbial communities. This investigation focused on the synergistic relations between Legionella and the fungal species commonly found in BEs and the impact of these synergistic relationships on the survival and transmission of Legionella. METHODS: A field study was conducted to identify the types and concentrations of fungi in BEs. The fungal isolates purified from BEs were cocultured with Legionella to study their synergistic association. Cocultured Legionella cells were aerosolized in an air-tight chamber to evaluate the efficacy of ultraviolet (UV) to inactivate these cells. RESULTS: Aspergillus, Alternaria, and Cladosporium were the most common fungi detected in samples that tested positive for Legionella. After coculturing, Legionella cells were detected inside fungal hyphae. The microscopic observations of Legionella internalization in fungal hyphae were confirmed by molecular analyses. UV disinfection of the aerosolized Legionella cells that were cocultured with fungi indicated that fungal spores and propagules act as a shield against UV radiation. The shield effect of fungal spores on Legionella cells was quantified at >2.5 log10. CONCLUSIONS: This study provides the first evidence, to our knowledge, of Legionella cell presence inside fungi detected in an indoor environment. This symbiotic relationship with fungi results in longer survival of Legionella under ambient conditions and provides protection against UV rays.

Eliminating Legionella by inhibiting BCL-XL to induce macrophage apoptosis.

Human pathogenic Legionella replicate in alveolar macrophages and cause a potentially lethal form of pneumonia known as Legionnaires' disease(1). Here, we have identified a host-directed therapeutic approach to eliminate intracellular Legionella infections. We demonstrate that the genetic deletion, or pharmacological inhibition, of the host cell pro-survival protein BCL-XL induces intrinsic apoptosis of macrophages infected with virulent Legionella strains, thereby abrogating Legionella replication. BCL-XL is essential for the survival of Legionella-infected macrophages due to bacterial inhibition of host-cell protein synthesis, resulting in reduced levels of the short-lived, related BCL-2 pro-survival family member, MCL-1. Consequently, a single dose of a BCL-XL-targeted BH3-mimetic therapy, or myeloid cell-restricted deletion of BCL-XL, limits Legionella replication and prevents lethal lung infections in mice. These results indicate that repurposing BH3-mimetic compounds, originally developed to induce cancer cell apoptosis, may have efficacy in treating Legionnaires' and other diseases caused by intracellular microbes.

Are there effective interventions to prevent hospital-acquired Legionnaires' disease or to reduce environmental reservoirs of Legionella in hospitals? A systematic review.

BACKGROUND: Legionnaires' disease (LD) is recognized as an important hospital-acquired disease. Despite the several methods available, the optimal method to control hospital-acquired LD is not well established and their overall efficacy requires further evaluation. OBJECTIVE: To systematically review all controlled trials evaluating the efficacy of interventions to prevent hospital-acquired LD in patients at high risk of developing the disease and its effects on environmental colonization. METHODS: A database search was performed through PubMed and the Cochrane Central Register of Controlled Trials (inception-November 2014). Eligible studies included all controlled studies evaluating interventions to prevent hospital-acquired LD in patients at high risk or evaluating the effect on environmental colonization. Both individual and pooled risk estimates were reported using risk ratio (RR) and 95% confidence intervals (95% CIs). RESULTS: There were no studies evaluating the risk reduction in hospital-acquired LD, but 4 studies evaluated the influence of copper-silver ionization and ultraviolet light in the reduction of environmental reservoirs of Legionella. The meta-analysis showed a significant 95% risk reduction of Legionella positivity in environmental samples using copper-silver ionization (RR, 0.05; 95% CI, 0.01-0.17) and 97% risk reduction with ultraviolet light (RR, 0.03; 95% CI, 0.002-0.41). CONCLUSIONS: The best available evidence suggests that copper-silver ionization and ultraviolet light are effective in reducing Legionella positivity in environmental samples. Nevertheless, the low quality of evidence weakens the robustness of conclusions.