Microbial Contamination of Dental Unit Waterlines among Dental Clinics of India- An In vitro Study.

BACKGROUND: Dental Unit Water Line (DUWL) deliver water to different handpieces in a dental unit. The water in DUWL circulates in a closed system, where it is taken from a container. The quality of dental water is of considerable importance since patients and dental staff are regularly exposed to water and aerosols generated from dental equipment. Output water from DUWLs may be a potential source of infection for both dental health care personnel and patients. AIM: To assess the microbial contamination in the DUWL among dental clinics in Chennai. MATERIALS AND METHODS: An in vitro study was conducted on 60 water samples from 20 dental clinics in Chennai in December 2019. Water samples were collected from three different sources of the Dental unit according to ADA guidelines. The collected samples were assessed for the presence of Aspergillus, Acinetobacter, Pseudomonas aeruginosa, and Legionella by agar plate method. The data were analysed using SPSS software version 20. RESULTS: Legionella was the most prevalent microorganism with 70% prevalence in a three-way syringe and 50% in scaler and airotor, followed by Pseudomonas aeruginosa and Acinetobacter with 10% prevalence in scaler and airotor and Aspergillus with a prevalence of 10% in the three-way syringe. CONCLUSION: Most of the dental units were contaminated with Aspergillus, Legionella, Pseudomonas aeruginosa and Acinetobacter which pose a serious threat to the patients as well as the dentists.

Two-year evaluation of Legionella in an aging residential building: Assessment of multiple potable water remediation approaches.

Legionella is an opportunistic waterborne pathogen that is difficult to eradicate in colonized drinking water pipes. Legionella control is further challenged by aging water infrastructure and lack of evidence-based guidance for building treatment. This study assessed multiple premise water remediation approaches designed to reduce Legionella pneumophila within a residential building located in an aging, urban drinking water system over a two-year period. Samples (n = 745) were collected from hot and cold-water lines and quantified via most probable number culture. Building-level treatment approaches included three single heat shocks, three single chemical shocks, and continuous low-level chemical disinfection in the potable water system. The building was highly colonized with L. pneumophila with 71 % L. pneumophila positivity. Single heat shocks had a statistically significant L. pneumophila reduction one day post treatment but no significant L. pneumophila reduction at one week, two weeks, and four weeks post treatment. The first two chemical shocks resulted in statistically significant L. pneumophila reduction at two days and four weeks post treatment, but there was a significant L. pneumophila increase at four weeks following the third chemical shock. Continuous low-level chemical disinfection resulted in statistically significant L. pneumophila reduction at ten weeks post treatment implementation. This demonstrates that in a building highly colonized with L. pneumophila, sustained remediation is best achieved using continuous low-level chemical treatment.

Outbreak of Legionnaires' disease in Rzeszów in 2023. / Ognisko choroby legionistów w Rzeszowie w 2023 roku.

BACKGROUND: Legionnaires' disease is a type of severe pneumonia caused by Legionella bacteria. The case fatality rate in this disease is 5-10%. People with various comorbidities, smokers and the elderly are at greater risk of developing the disease. OBJECTIVE: The aim of the work is to present the results of an epidemiological investigation into the outbreak of Legionnaires' disease that occurred in the city of Rzeszów and the surrounding area in August and September 2023 and to present the threat related to the presence of Legionella bacteria in water supply installations and networks. MATERIAL AND METHODS: The material for this publication was data from an epidemiological investigation conducted in the outbreak of Legionnaires disease in Rzeszów in 2023. RESULTS: Epidemiological investigation revealed 165 cases of Legionnaires' disease in the outbreak, including 152 confirmed cases and 13 probable cases. The case fatality rate in a legionellosis outbreak was 15%. Environmental tests were carried out in residential and public buildings and industrial installations during the investigation. As part of environmental tests, 187 water samples were collected, including 87 warm water samples. CONCLUSIONS: The outbreak of Legionnaires' disease in the city of Rzeszów draws attention to the potential threat from the Legionella bacteria to the health and life of especially elderly people suffering from chronic diseases. The environmental tests carried out confirmed the highest number of Legionella bacteria at medium and high levels in water samples taken in the private apartments of sick people. Despite the lack of strict legal regulations clearly specifying the obligations regarding periodic disinfection of internal hot water supply installations, cooperation with their owners should be undertaken to enforce plans and actions in this area.

Automated cooling tower detection through deep learning for Legionnaires' disease outbreak investigations: a model development and validation study.

BACKGROUND: Cooling towers containing Legionella spp are a high-risk source of Legionnaires' disease outbreaks. Manually locating cooling towers from aerial imagery during outbreak investigations requires expertise, is labour intensive, and can be prone to errors. We aimed to train a deep learning computer vision model to automatically detect cooling towers that are aerially visible. METHODS: Between Jan 1 and 31, 2021, we extracted satellite view images of Philadelphia (PN, USA) and New York state (NY, USA) from Google Maps and annotated cooling towers to create training datasets. We augmented training data with synthetic data and model-assisted labelling of additional cities. Using 2051 images containing 7292 cooling towers, we trained a two-stage model using YOLOv5, a model that detects objects in images, and EfficientNet-b5, a model that classifies images. We assessed the primary outcomes of sensitivity and positive predictive value (PPV) of the model against manual labelling on test datasets of 548 images, including from two cities not seen in training (Boston [MA, USA] and Athens [GA, USA]). We compared the search speed of the model with that of manual searching by four epidemiologists. FINDINGS: The model identified visible cooling towers with 95·1% sensitivity (95% CI 94·0-96·1) and a PPV of 90·1% (95% CI 90·0-90·2) in New York City and Philadelphia. In Boston, sensitivity was 91·6% (89·2-93·7) and PPV was 80·8% (80·5-81·2). In Athens, sensitivity was 86·9% (75·8-94·2) and PPV was 85·5% (84·2-86·7). For an area of New York City encompassing 45 blocks (0·26 square miles), the model searched more than 600 times faster (7·6 s; 351 potential cooling towers identified) than did human investigators (mean 83·75 min [SD 29·5]; mean 310·8 cooling towers [42·2]). INTERPRETATION: The model could be used to accelerate investigation and source control during outbreaks of Legionnaires' disease through the identification of cooling towers from aerial imagery, potentially preventing additional disease spread. The model has already been used by public health teams for outbreak investigations and to initialise cooling tower registries, which are considered best practice for preventing and responding to outbreaks of Legionnaires' disease. FUNDING: None.

Adherence to Legionella control regulations and guidelines in Norwegian nursing homes: a cross-sectional survey.

BACKGROUND: Infection by Legionella bacteria is a risk to elderly individuals in health care facilities and should be managed by preventing bacterial proliferation in internal water systems. Norwegian legislation calls for a mandatory Legionella-specific risk assessment with the subsequent introduction of an adapted water management programme. The present study investigates adherence to legislation and guidelines on Legionella control and prevention in Norwegian nursing homes. METHODS: A cross-sectional survey was distributed to Norwegian municipalities to investigate the status of Legionella specific risk assessments of internal water distribution systems and the introduction of water management programmes in nursing homes. RESULTS: A total of 55.1% (n = 228) of the participating nursing homes had performed Legionella-specific risk assessments, of which 55.3% (n = 126) stated that they had updated the risk assessment within the last year. 96.5% introduced a water management programme following a risk assessment, whereas 59.6% of the ones without a risk assessment did the same. Nursing homes with risk assessments were more likely to monitor Legionella levels than those without (61.2% vs 38.8%), to remove dead legs (44.7% vs 16.5%), and to select biocidal preventive treatment over hot water flushing (35.5% vs 4.6%). CONCLUSIONS: This study presents novel insight into Legionella control in Norway, suggesting that adherence to mandatory risk assessment in nursing homes is moderate-low. Once performed, the risk assessment seems to be advantageous as an introduction to future Legionella prevention in terms of the scope and contents of the water management programme.

Efficacy of chlorine-based disinfectants to control Legionella within premise plumbing systems.

Legionella is an opportunistic waterborne pathogen that causes Legionnaires' disease. It poses a significant public health risk, especially to vulnerable populations in health care facilities. It is ubiquitous in manufactured water systems and is transmitted via inhalation or aspiration of aerosols/water droplets generated from water fixtures (e.g., showers and hand basins). As such, the effective management of premise plumbing systems (building water systems) in health care facilities is essential for reducing the risk of Legionnaires' disease. Chemical disinfection is a commonly used control method and chlorine-based disinfectants, including chlorine, chloramine, and chlorine dioxide, have been used for over a century. However, the effectiveness of these disinfectants in premise plumbing systems is affected by various interconnected factors that can make it challenging to maintain effective disinfection. This systematic literature review identifies all studies that have examined the factors impacting the efficacy and decay of chlorine-based disinfectant within premise plumbing systems. A total of 117 field and laboratory-based studies were identified and included in this review. A total of 20 studies directly compared the effectiveness of the different chlorine-based disinfectants. The findings from these studies ranked the typical effectiveness as follows: chloramine > chlorine dioxide > chlorine. A total of 26 factors were identified across 117 studies as influencing the efficacy and decay of disinfectants in premise plumbing systems. These factors were sorted into categories of operational factors that are changed by the operation of water devices and fixtures (such as stagnation, temperature, water velocity), evolving factors which are changed in-directly (such as disinfectant concentration, Legionella disinfectant resistance, Legionella growth, season, biofilm and microbe, protozoa, nitrification, total organic carbon(TOC), pH, dissolved oxygen(DO), hardness, ammonia, and sediment and pipe deposit) and stable factors that are not often changed(such as disinfectant type, pipe material, pipe size, pipe age, water recirculating, softener, corrosion inhibitor, automatic sensor tap, building floor, and construction activity). A factor-effect map of each of these factors and whether they have a positive or negative association with disinfection efficacy against Legionella in premise plumbing systems is presented. It was also found that evaluating the effectiveness of chlorine disinfection as a water risk management strategy is further complicated by varying disinfection resistance of Legionella species and the form of Legionella (culturable/viable but non culturable, free living/biofilm associated, intracellular replication within amoeba hosts). Future research is needed that utilises sensors and other approaches to measure these key factors (such as pH, temperature, stagnation, water age and disinfection residual) in real time throughout premise plumbing systems. This information will support the development of improved models to predict disinfection within premise plumbing systems. The findings from this study will inform the use of chlorine-based disinfection within premise plumbing systems to reduce the risk of Legionnaires disease.

Residual disinfectant effectively suppresses Legionella species in drinking water distribution systems supplied by surface water in Minnesota, USA.

Seven public water systems in Minnesota, USA were analyzed from one to five times over a two-year period to assess temporal changes in the concentrations of total bacteria, Legionella spp., and Legionella pneumophila from source (i.e., raw water) through the water treatment process to the end water user. Bacterial biomass was collected by filtering large volumes of raw water (12 to 425 L, median: 38 L) or finished and tap water (27 to 1205 L, median: 448 L) using ultrafiltration membrane modules. Quantitative PCR (qPCR) was then used to enumerate all bacteria (16S rRNA gene fragments), all Legionella spp. (ssrA), and Legionella pneumophila (mip). Total coliforms, Escherichia coli, and L. pneumophila also were quantified in the water samples via cultivation. Median concentrations of total bacteria and Legionella spp. (ssrA) in raw water (8.5 and 4.3 log copies/L, respectively) decreased by about 2 log units during water treatment. The concentration of Legionella spp. (ssrA) in water collected from distribution systems inversely correlated with the total chlorine concentration for chloraminated systems significantly (p = 0.03). Although only 8 samples were collected from drinking water distribution systems using free chlorine as a residual disinfectant, these samples had significantly lower concentrations of Legionella spp. (ssrA) than samples collected from the chloraminated systems (p = 5 × 10-4). There was considerable incongruity between the results obtained via cultivation-independent (qPCR) and cultivation-dependent assays. Numerous samples were positive for L. pneumophila via cultivation, none of which tested positive for L. pneumophilia (mip) via qPCR. Conversely, a single sample tested positive for L. pneumophilia (mip) via qPCR, but this sample tested negative for L. pneumophilia via cultivation. Overall, the results suggest that conventional treatment is effective at reducing, but not eliminating, Legionella spp. from surface water supplies and that residual disinfection is effective at suppressing these organisms within drinking water distribution systems.

Legionella Serositis: A Rare Presentation.

BACKGROUND: Legionella has a higher prevalence in India than in the world. Legionaries' disease most commonly involves the lungs but because of increased awareness, extrapulmonary manifestations are also being diagnosed more frequently. CASE DESCRIPTION: We present a case of a young female with acute onset of fever and chest pain. On initial investigation, an electrocardiogram (ECG) reported widespread pulse rate (PR) depression suggestive of pericarditis which was confirmed by ECG. High-resolution computed tomography (HRCT) thorax suggested mild bilateral pleural effusion with normal lung parenchyma. elevated erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) added to the diagnosis of serositis. Serological study for atypical organisms was remarkable for positive immunoglobulin M (IgM) for Legionella. She was treated with a high dose of steroids and azithromycin successfully. CONCLUSION: Isolated extrapulmonary presentation of legionaries disease is often overlooked and is common. So it should be always included in the diagnostic armamentarium as treatment is highly efficacious if started early.

Applications of Quantitative Microbial Risk Assessment to Respiratory Pathogens and Implications for Uptake in Policy: A State-of-the-Science Review.

BACKGROUND: Respiratory tract infections are major contributors to the global disease burden. Quantitative microbial risk assessment (QMRA) holds potential as a rapidly deployable framework to understand respiratory pathogen transmission and inform policy on infection control. OBJECTIVES: The goal of this paper was to evaluate, motivate, and inform further development of the use of QMRA as a rapid tool to understand the transmission of respiratory pathogens and improve the evidence base for infection control policies. METHODS: We conducted a literature review to identify peer-reviewed studies of complete QMRA frameworks on aerosol inhalation or contact transmission of respiratory pathogens. From each of the identified studies, we extracted and summarized information on the applied exposure model approaches, dose-response models, and parameter values, including risk characterization. Finally, we reviewed linkages between model outcomes and policy. RESULTS: We identified 93 studies conducted in 16 different countries with complete QMRA frameworks for diverse respiratory pathogens, including SARS-CoV-2, Legionella spp., Staphylococcus aureus, influenza, and Bacillus anthracis. Six distinct exposure models were identified across diverse and complex transmission pathways. In 57 studies, exposure model frameworks were informed by their ability to model the efficacy of potential interventions. Among interventions, masking, ventilation, social distancing, and other environmental source controls were commonly assessed. Pathogen concentration, aerosol concentration, and partitioning coefficient were influential exposure parameters as identified by sensitivity analysis. Most (84%, n=78) studies presented policy-relevant content including a) determining disease burden to call for policy intervention, b) determining risk-based threshold values for regulations, c) informing intervention and control strategies, and d) making recommendations and suggestions for QMRA application in policy. CONCLUSIONS: We identified needs to further the development of QMRA frameworks for respiratory pathogens that prioritize appropriate aerosol exposure modeling approaches, consider trade-offs between model validity and complexity, and incorporate research that strengthens confidence in QMRA results. https://doi.org/10.1289/EHP12695.

Initial waterline contamination by Pseudomonas aeruginosa in newly installed dental chairs.

Water contamination in dental unit waterlines (DUWLs) is a potential source of healthcare-associated infection during dental care. The aim of this study was to evaluate the microbiological quality of DUWLs water from newly installed dental chairs in a French University Hospital. The microbiological quality of water from 24 new DUWLs initially disinfected by ICX Renew-prior to use of the dental units for patient treatment-was assessed for total culturable aerobic bacteria at 22°C and 36°C, Legionella sp., Pseudomonas aeruginosa, and total coliforms. Among the 24 samples analyzed, 21 were compliant with the water quality levels: 19 had no bacteria, and 2 contained only 4 and 1 CFU/mL for total culturable aerobic bacteria at 22°C and 36°C, respectively. Three samples were non-compliant due to contamination by P. aeruginosa (4, 2, and 2 CFU/100 mL). Controlling and preventing the microbiological contamination of DUWLs, especially by pathogenic bacteria, at the time of the installation of the new dental chairs are crucial to prevent healthcare-associated infection in dentistry. IMPORTANCE: Dental unit waterlines (DUWLs) of new dental chairs may be contaminated before their first clinical use, so an initial shock disinfection is crucial at the time of their installation. The microbiological analyses are crucial to control the water quality of DUWLs before their first clinical use because their disinfection does not guarantee the elimination of all bacteria.

Virulence Genes and Biofilm Formation Among Legionella pneumophila Isolates Collected from Hospital Water Sources.

Legionella pneumophila can be transmitted to people, especially immunocompromised patients, via hospital water pipe systems and cause severe pneumonia. The aim of our study was to investigate the presence of major virulence factor genes, ability of biofilms formation, and correlation between presence of Legionella isolates and temperature, pH, and residual chlorine of water. Hundred water samples were collected from nine hospitals in Tehran, Iran. Temperature, pH, and residual chlorine were determined during sampling. Different virulence genes and the ability to form biofilms were subsequently analyzed among the L. pneumophila isolates. Results showed that 12 (12%) samples were positive in culture method and all of the isolates were positive as L. pneumophila species (mip). A correlation was found between Legionella culture positivity and temperature and pH of water, but there was no significant correlation between residual chlorine of water samples and the presence of Legionella. The isolation of Legionella rate in summer and spring was higher than winter and autumn. Twelve (100%) isolates were positive for mip genes, 9 (75%) for dot genes, 8 (66.66%) for hsp, 6 (50%) for lvh, and 4 (33.33%) for rtx. All of the isolates displayed strong ability for biofilm production every three days. Two of these isolates (16.6%) displayed weak ability to form biofilm on the first day of incubation. This study revealed that water sources in hospitals were colonized by virulent Legionella and should be continuously monitored to avoid elevated concentrations of Legionella with visible biofilm formation.

Quantitative Microbial Risk Assessment Framework Incorporating Water Ages with Legionella pneumophila Growth Rates.

Water age in drinking water systems is often used as a proxy for water quality but is rarely used as a direct input in assessing microbial risk. This study directly linked water ages in a premise plumbing system to concentrations of Legionella pneumophila via a growth model. In turn, the L. pneumophila concentrations were used for a quantitative microbial risk assessment to calculate the associated probabilities of infection (Pinf) and clinically severe illness (Pcsi) due to showering. Risk reductions achieved by purging devices, which reduce water age, were also quantified. The median annual Pinf exceeded the commonly used 1 in 10,000 (10-4) risk benchmark in all scenarios, but the median annual Pcsi was always 1-3 orders of magnitude below 10-4. The median annual Pcsi was lower in homes with two occupants (4.7 × 10-7) than with one occupant (7.5 × 10-7) due to more frequent use of water fixtures, which reduced water ages. The median annual Pcsi for homes with one occupant was reduced by 39-43% with scheduled purging 1-2 times per day. Smart purging devices, which purge only after a certain period of nonuse, maintained these lower annual Pcsi values while reducing additional water consumption by 45-62%.

Upstream land use with microbial downstream consequences: Iron and humic substances link to Legionella spp.

Intensified land use can disturb water quality, potentially increasing the abundance of bacterial pathogens, threatening public access to clean water. This threat involves both direct contamination of faecal bacteria as well as indirect factors, such as disturbed water chemistry and microbiota, which can lead to contamination. While direct contamination has been well described, the impact of indirect factors is less explored, despite the potential of severe downstream consequences on water supply. To assess direct and indirect downstream effects of buildings, farms, pastures and fields on potential water sources, we studied five Swedish lakes and their inflows. We analysed a total of 160 samples in a gradient of anthropogenic activity spanning four time points, including faecal and water-quality indicators. Through species distribution modelling, Random Forest and network analysis using 16S rRNA amplicon sequencing data, our findings highlight that land use indirectly impacts lakes via inflows. Land use impacted approximately one third of inflow microbiota taxa, in turn impacting ∼20-50 % of lake taxa. Indirect effects via inflows were also suggested by causal links between e.g. water colour and lake bacterial taxa, where this influenced the abundance of several freshwater bacteria, such as Polynucleobacter and Limnohabitans. However, it was not possible to identify direct effects on the lakes based on analysis of physiochemical- or microbial parameters. To avoid potential downstream consequences on water supply, it is thus important to consider possible indirect effects from upstream land use and inflows, even when no direct effects can be observed on lakes. Legionella (a genus containing bacterial pathogens) illustrated potential consequences, since the genus was particularly abundant in inflows and was shown to increase by the presence of pastures, fields, and farms. The approach presented here could be used to assess the suitability of lakes as alternative raw water sources or help to mitigate contaminations in important water catchments. Continued broad investigations of stressors on the microbial network can identify indirect effects, avoid enrichment of pathogens, and help secure water accessibility.

Susceptibility of Legionella gormanii Membrane-Derived Phospholipids to the Peptide Action of Antimicrobial LL-37-Langmuir Monolayer Studies.

LL-37 is the only member of the cathelicidin-type host defense peptide family in humans. It exhibits broad-spectrum bactericidal activity, which represents a distinctive advantage for future therapeutic targets. The presence of choline in the growth medium for bacteria changes the composition and physicochemical properties of their membranes, which affects LL-37's activity as an antimicrobial agent. In this study, the effect of the LL-37 peptide on the phospholipid monolayers at the liquid-air interface imitating the membranes of Legionella gormanii bacteria was determined. The Langmuir monolayer technique was employed to prepare model membranes composed of individual classes of phospholipids-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL)-isolated from L. gormanii bacteria supplemented or non-supplemented with exogenous choline. Compression isotherms were obtained for the monolayers with or without the addition of the peptide to the subphase. Then, penetration tests were carried out for the phospholipid monolayers compressed to a surface pressure of 30 mN/m, followed by the insertion of the peptide into the subphase. Changes in the mean molecular area were observed over time. Our findings demonstrate the diversified effect of LL-37 on the phospholipid monolayers, depending on the bacteria growth conditions. The substantial changes in membrane properties due to its interactions with LL-37 enable us to propose a feasible mechanism of peptide action at a molecular level. This can be associated with the stable incorporation of the peptide inside the monolayer or with the disruption of the membrane leading to the removal (desorption) of molecules into the subphase. Understanding the role of antimicrobial peptides is crucial for the design and development of new strategies and routes for combating resistance to conventional antibiotics.

A novel hybridization capture method for direct whole genome sequencing of clinical specimens to inform Legionnaires' disease investigations.

The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires' disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations.IMPORTANCELegionnaires' disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella-contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.

Controlling Legionella pneumophila growth in hot water systems by reducing dissolved oxygen levels.

Legionella pneumophila, the leading cause of Legionnaires' disease in the United States, is found in lakes, ponds, and streams but poses a health risk when it grows in building water systems. The growth of L. pneumophila in hot water systems of healthcare facilities poses a significant risk to patients, staff, and visitors. Hospitals and long-term care facilities account for 76% of reported Legionnaires' disease cases with mortality rates of 25%. Controlling L. pneumophila growth in hot water systems serving healthcare and hospitality buildings is currently achieved primarily by adding oxidizing chemical disinfectants. Chemical oxidants generate disinfection byproducts and can accelerate corrosion of premise plumbing materials and equipment. Alternative control methods that do not generate hazardous disinfection byproducts or accelerate corrosion are needed. L. pneumophila is an obligate aerobe that cannot sustain cellular respiration, amplify, or remain culturable when dissolved oxygen (DO) concentrations are too low (< 0.3 mg/L). An alternative method of controlling L. pneumophila growth by reducing DO levels in a hot water model system using a gas transfer membrane contactor was evaluated. A hot water model system was constructed and inoculated with L. pneumophila at DO concentrations above 0.5 mg/L. Once the model system was colonized, DO levels were incrementally reduced. Water samples were collected each week to evaluate the effect of reducing dissolved oxygen levels when all other conditions favored Legionella amplification. At DO concentrations below 0.3 mg/L, L. pneumophila concentrations were reduced by 1-log over 7 days. Under conditions in the hot water model system, at favorable temperatures and with no residual chlorine disinfectant, L. pneumophila concentrations were reduced by 1-log, indicating growth inhibition by reducing DO levels as the sole control measure. In sections of the model system where DO levels were not lowered L. pneumophila continued to grow. Reducing dissolved oxygen levels in hot water systems of healthcare and other large buildings to control L. pneumophila could also lower the risk of supplemental chemical treatment methods currently in use.

The role of HDAC6 in enhancing macrophage autophagy via the autophagolysosomal pathway to alleviate legionella pneumophila-induced pneumonia.

Legionella pneumophila (L. pneumophila) is a prevalent pathogenic bacterium responsible for significant global health concerns. Nonetheless, the precise pathogenic mechanisms of L. pneumophila have still remained elusive. Autophagy, a direct cellular response to L. pneumophila infection and other pathogens, involves the recognition and degradation of these invaders in lysosomes. Histone deacetylase 6 (HDAC6), a distinctive member of the histone deacetylase family, plays a multifaceted role in autophagy regulation. This study aimed to investigate the role of HDAC6 in macrophage autophagy via the autophagolysosomal pathway, leading to alleviate L. pneumophila-induced pneumonia. The results revealed a substantial upregulation of HDAC6 expression level in murine lung tissues infected by L. pneumophila. Notably, mice lacking HDAC6 exhibited a protective response against L. pneumophila-induced pulmonary tissue inflammation, which was characterized by the reduced bacterial load and diminished release of pro-inflammatory cytokines. Transcriptomic analysis has shed light on the regulatory role of HDAC6 in L. pneumophila infection in mice, particularly through the autophagy pathway of macrophages. Validation using L. pneumophila-induced macrophages from mice with HDAC6 gene knockout demonstrated a decrease in cellular bacterial load, activation of the autophagolysosomal pathway, and enhancement of cellular autophagic flux. In summary, the findings indicated that HDAC6 knockout could lead to the upregulation of p-ULK1 expression level, promoting the autophagy-lysosomal pathway, increasing autophagic flux, and ultimately strengthening the bactericidal capacity of macrophages. This contributes to the alleviation of L. pneumophila-induced pneumonia.

Legionella metaeffector MavL reverses ubiquitin ADP-ribosylation via a conserved arginine-specific macrodomain.

ADP-ribosylation is a reversible post-translational modification involved in various cellular activities. Removal of ADP-ribosylation requires (ADP-ribosyl)hydrolases, with macrodomain enzymes being a major family in this category. The pathogen Legionella pneumophila mediates atypical ubiquitination of host targets using the SidE effector family in a process that involves ubiquitin ADP-ribosylation on arginine 42 as an obligatory step. Here, we show that the Legionella macrodomain effector MavL regulates this pathway by reversing the arginine ADP-ribosylation, likely to minimize potential detrimental effects caused by the modified ubiquitin. We determine the crystal structure of ADP-ribose-bound MavL, providing structural insights into recognition of the ADP-ribosyl group and catalytic mechanism of its removal. Further analyses reveal DUF4804 as a class of MavL-like macrodomain enzymes whose representative members show unique selectivity for mono-ADP-ribosylated arginine residue in synthetic substrates. We find such enzymes are also present in eukaryotes, as exemplified by two previously uncharacterized (ADP-ribosyl)hydrolases in Drosophila melanogaster. Crystal structures of several proteins in this class provide insights into arginine specificity and a shared mode of ADP-ribose interaction distinct from previously characterized macrodomains. Collectively, our study reveals a new regulatory layer of SidE-catalyzed ubiquitination and expands the current understanding of macrodomain enzymes.

Ethambutol inhibited the growth of acid-fast bacteria and enhanced the detection of Legionella in environmental water samples.

The growth of acid-fast bacteria often hinders the detection of Legionella in water samples on agar plates by the plate culture method. We studied whether anti-tubercular agents inhibit acid-fast bacteria growth on agar plates. First, the antimicrobial activities of isoniazid, ethionamide, and ethambutol were evaluated against Mycobacterium and Legionella. We found that ethambutol at ≥ 100 µg/mL completely inhibited Mycobacterium growth, but ethambutol at 1,000 µg/mL did not inhibit Legionella growth. Next, the effect of ethambutol dissolved in acid buffer was examined. Cell suspensions of L. pneumophila and Mycobacterium spp. were mixed, and ethambutol-acid buffer was added. After 5 min, mixtures were inoculated on GVPC agar plates and incubated at 36℃ for 6 d. We found that ethambutol inhibited Mycobacterium growth on agar plates, but the Legionella colonies recovered. The effect of ethambutol was also significant in the evaluation using bathwaters. Comparing 1,302 bathwaters, the addition of ethambutol reduced the detection rate of acid-fast bacteria from 30.6% to 0% and increased the detection rate of Legionella from 7.1% to 7.5%. Ethambutol, which selectively inhibited acid-fast bacteria growth, enhanced the detection of Legionella on agar plates and will contribute to improving the accuracy of Legionella testing by the plate culture method.

Identification rate of Legionella species in non-purulent sputum culture is comparable to that in purulent sputum culture in Legionella pneumonia.

Many Legionella pneumonia patients do not produce sputum, and it is unknown whether purulent sputum is required for the identification of Legionella species. This study aimed to evaluate the identification rate of Legionella species based on sputum quality and the factors predictive of Legionella infection. This study included Legionella pneumonia patients at Kurashiki Central Hospital from November 2000 to December 2022. Sputum quality, based on gram staining, was classified as the following: Geckler 1/2, 3/6 and 4/5. Geckler 4/5 was defined as purulent sputum. The sputa of 104 of 124 Legionella pneumonia patients were cultured. Fifty-four patients (51.9%) were identified with Legionella species, most of which were Legionella pneumophila serogroup 1 (81.5%). The identification rates of Legionella species according to sputum quality were 57.1% (16/28) in Geckler 1/2 sputum, 50.0% (34/68) in Geckler 3/6 sputum, and 50.0% (4/8) in Geckler 4/5 sputum, which were not significantly different (P = 0.86). On multivariate analysis, pre-culture treatment with anti-Legionella antimicrobials (odds ratio [OR] 0.26, 95% confidence interval [CI] 0.06-0.91), Pneumonia Severity Index class ≥IV (OR 2.57 [95% CI 1.02-6.71]), and intensive care unit admission (OR 3.08, 95% CI 1.06-10.09) correlated with the ability to identify Legionella species, but sputum quality did not (OR 0.88, 95% CI 0.17-4.41). The identification rate of Legionella species in non-purulent sputum was similar to that in purulent sputum. For the diagnosis of Legionella pneumonia, sputum should be collected before administering anti-Legionella antibiotics and cultured regardless of sputum quality.