The LysR-type transcriptional regulator LelA co-regulates various effectors in different Legionella species.

The intracellular pathogen Legionella pneumophila translocates more than 300 effector proteins into its host cells. The expression levels of the genes encoding these effectors are orchestrated by an intricate regulatory network. Here, we introduce LelA, the first L. pneumophila LysR-type transcriptional regulator of effectors. Through bioinformatic and experimental analyses, we identified the LelA target regulatory element and demonstrated that it directly activates the expression of three L. pneumophila effectors (legL7, legL6, and legU1). We further found that the gene encoding LelA is positively regulated by the RpoS sigma factor, thus linking it to the known effector regulatory network. Examination of other species throughout the Legionella genus revealed that this regulatory element is found upstream of 34 genes encoding validated effectors, putative effectors, and hypothetical proteins. Moreover, ten of these genes were examined and found to be activated by the L. pneumophila LelA as well as by their orthologs in the corresponding species. LelA represents a novel type of Legionella effector regulator, which coordinates the expression of both adjacently and distantly located effector-encoding genes, thus forming small groups of co-regulated effectors.

Distribution characteristics of the Legionella CRISPR-Cas system and its regulatory mechanism underpinning phenotypic function.

Legionella is a common intracellular parasitic bacterium that infects humans via the respiratory tract, causing Legionnaires' disease, with fever and pneumonia as the main symptoms. The emergence of highly virulent and azithromycin-resistant Legionella pneumophila is a major challenge in clinical anti-infective therapy. The CRISPR-Cas acquired immune system provides immune defense against foreign nucleic acids and regulates strain biological functions. However, the distribution of the CRISPR-Cas system in Legionella and how it regulates gene expression in L. pneumophila remain unclear. Herein, we assessed 915 Legionella whole-genome sequences to determine the distribution characteristics of the CRISPR-Cas system and constructed gene deletion mutants to explore the regulation of the system based on growth ability in vitro, antibiotic sensitivity, and intracellular proliferation of L. pneumophila. The CRISPR-Cas system in Legionella was predominantly Type II-B and was mainly concentrated in the genome of L. pneumophila ST1 strains. The Type II-B CRISPR-Cas system showed no effect on the strain's growth ability in vitro but significantly reduced resistance to azithromycin and decreased proliferation ability due to regulation of the lpeAB efflux pump and the Dot/Icm type IV secretion system. Thus, the Type II-B CRISPR-Cas system plays a crucial role in regulating the virulence of L. pneumophila. This expands our understanding of drug resistance and pathogenicity in Legionella, provides a scientific basis for the prevention of Legionnaires' disease outbreaks and the rational use of clinical drugs, and facilitates effective treatment of Legionnaires' disease.

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.

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.

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.

Evaluating Fourier-transform infrared spectroscopy with IR Biotyper as a faster and simpler method for investigating the sources of an outbreak of legionellosis.

Fourier-transform infrared (FTIR) spectroscopy using the IR Biotyper and core genome single nucleotide polymorphism (cgSNP) analysis were performed on 12 Legionella isolates associated with an outbreak at a spa house in Kanagawa Prefecture, Japan, and 3 non-outbreak isolates. The discriminative power of FTIR spectroscopy for 48-h incubation conditions of L. pneumophila in this outbreak was lower than cgSNP-based typing but higher than serogroup typing. FTIR spectroscopy could screen outbreak isolates from a group of genetically related isolates and may be useful as an initial typing method in Legionella outbreak investigations.

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%.

Estimating the burden of illness caused by domestic waterborne Legionnaires' disease in Canada: 2015-2019.

Legionellosis is a disease caused by the bacterium Legionella that most commonly presents as Legionnaires' disease (LD), a severe form of pneumonia. From 2015 to 2019, an average of 438 LD cases per year were reported in Canada. However, it is believed that the actual number of cases is much higher, since LD may be underdiagnosed and underreported. The purpose of this study was to develop an estimate of the true incidence of illnesses, hospitalizations, and deaths associated with LD in Canada. Values were derived using a stochastic model, based on Canadian surveillance data from 2015 to 2019, which were scaled up to account for underdiagnosis and underreporting. Overall, there were an estimated 1,113 (90% CrI: 737-1,730) illnesses, 1,008 (90% CrI: 271-2,244) hospitalizations, and 34 (90% CrI: 4-86) deaths due to domestically acquired waterborne LD annually in Canada from 2015 to 2019. It was further estimated that only 36% of illnesses and 39% of hospitalizations and deaths were captured in surveillance, and that 22% of illnesses were caused by Legionella serogroups and species other than Legionella pneumophila serogroup 1 (non-Lp1). This study highlights the true burden and areas for improvement in Canada's surveillance and detection of LD.

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.

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.

Improved biosensing of Legionella by integrating filtration and immunomagnetic separation of the bacteria retained in filters.

A novel approach is presented that combines filtration and the direct immunomagnetic separation of the retained bacteria Legionella in filters, for further electrochemical immunosensing. This strategy allows for the separation and preconcentration of the water-borne pathogen from high-volume samples, up to 1000 mL. The limit of detection of the electrochemical immunosensor resulted in 100 CFU mL-1 and improved up to 0.1 CFU mL-1 when the preconcentration strategy was applied in 1 L of sample (103-fold improvement). Remarkably, the immunosensor achieves the limit of detection in less than 2.5 h and simplified the analytical procedure. This represents the lowest concentration reported to date for electrochemical immunosensing of Legionella cells without the need for pre-enrichment or DNA amplification. Furthermore, the study successfully demonstrates the extraction of bacteria retained on different filtering materials using immunomagnetic separation, highlighting the high efficiency of the magnetic particles to pull out the bacteria directly from solid materials. This promising feature expands the applicability of the method beyond water systems for detecting bacteria retained in air filters of air conditioning units by directly performing the immunomagnetic separation in the filters.

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.

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.

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.

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.

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.

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.

The impact of DNA extraction on the quantification of Legionella, with implications for ecological studies.

Monitoring the levels of opportunistic pathogens in drinking water is important to plan interventions and understand the ecological niches that allow them to proliferate. Quantitative PCR is an established alternative to culture methods that can provide a faster, higher-throughput, and more precise enumeration of the bacteria in water samples. However, PCR-based methods are still not routinely applied for Legionella monitoring, and techniques, such as DNA extraction, differ notably between laboratories. Here, we quantify the impact that DNA extraction methods had on downstream PCR quantification and community sequencing. Through a community science campaign, we collected 50 water samples and corresponding shower hoses, and compared two commonly used DNA extraction methodologies to the same biofilm and water phase samples. The two methods showed clearly different extraction efficacies, which were reflected in both the quantity of DNA extracted and the concentrations of Legionella enumerated in both the matrices. Notably, one method resulted in higher enumeration in nearly all samples by about one order of magnitude and detected Legionella in 21 samples that remained undetected by the other method. 16S rRNA amplicon sequencing revealed that the relative abundance of individual taxa, including sequence variants of Legionella, significantly varied depending on the extraction method employed. Given the implications of these findings, we advocate for improvement in documentation of the performance of DNA extraction methods used in drinking water to detect and quantify Legionella, and characterize the associated microbial community.IMPORTANCEMonitoring for the presence of the waterborne opportunistic pathogen Legionella is important to assess the risk of infection and plan remediation actions. While monitoring is traditionally carried on through cultivation, there is an ever-increasing demand for rapid and high-throughput molecular-based approaches for Legionella detection. This paper provides valuable insights on how DNA extraction affects downstream molecular analysis such as the quantification of Legionella through droplet digital PCR and the characterization of natural microbial communities through sequencing analysis. We analyze the results from a risk-assessment, legislative, and ecological perspective, showing how initial DNA processing is an important step to take into account when shifting to molecular-based routine monitoring and discuss the central role of consistent and detailed reporting of the methods used.

Correlation between bacterial microbiome and Legionella species in water from public bath facilities by 16S rRNA gene amplicon sequencing.

Public bath facilities are a major source of Legionella infections in Japan. In this study, we performed 16S rRNA gene amplicon sequencing to characterize the bacterial community in bath and shower water from public bath facilities, along with chemical parameters, and investigated the effect of the bacterial microbiome on the presence of Legionella species. Although no significant difference in bacterial community richness was observed between bath and shower water samples, there was a remarkable difference in the bacterial community structure between them. Distance-based redundancy analysis revealed that several factors (free residual chlorine, pH, and conductivity) were correlated with the bacterial community in bath water. The most abundant bacterial genera in the samples were Pseudomonas (13.7%) in bath water and Phreatobacter (13.6%) in shower water, as indicated by the taxonomic composition, and the dominant bacteria differed between these environmental samples. Legionella pneumophila was the most frequently detected Legionella species, with additional 15 other Legionella species detected in water samples. In Legionella-positive water samples, several unassigned and uncultured bacteria were enriched together. In addition, the co-occurrence network showed that Legionella was strongly interconnected with two uncultured bacteria. Corynebacterium and Sphingomonas negatively correlated with Legionella species. The present study reveals the ecology of Legionella species, especially their interactions with other bacteria that are poorly understood to date. IMPORTANCE: Public bath facilities are major sources of sporadic cases and outbreaks of Legionella infections. Recently, 16S rRNA gene amplicon sequencing has been used to analyze bacterial characteristics in various water samples from both artificial and natural environments, with a particular focus on Legionella bacterial species. However, the relationship between the bacterial community and Legionella species in the water from public bath facilities remains unclear. In terms of hygiene management, it is important to reduce the growth of Legionella species by disinfecting the water in public bath facilities. Our findings contribute to the establishment of appropriate hygiene management practices and provide a basis for understanding the potential health effects of using bath and shower water available in public bath facilities.

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.