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.

Comparison of the method of quantitative determination of Legionella pneumophila acc. to PN-EN ISO 11731:2017 with the Legiolert™/Quanti-Tray® (IDEXX).

INTRODUCTION: Legionella pneumophila is the primary etiological agent of Legionnaires' disease. These are opportunistic pathogens causing lung infections by inhalation of contaminated aerosols. Controlling the presence of these bacteria in domestic distribution water systems (mainly hot water systems) is important for reducing the threat they pose to human health. Legionella pathogens are detected and quantified during routine testing of water samples according to procedures included in PN-EN ISO 11731:2017. However, these procedures are labour-intensive, and the results are obtained after a relatively long time. Implementing the Legiolert™/Quanti-Tray® test as an alternative method may constitute a good solution: it simplifies the testing procedure and significantly reduces the time necessary to obtain the final result. OBJECTIVE: The aim of the study was to compare the relative recovery of Legionella from water samples tested according to PN-EN ISO 11731:2017, and the alternative method of the most probable number (MPN) with the Legiolert™/Quanti-Tray® (IDEXX) test, and to assess the suitability of the alternative method for routine testing. MATERIAL AND METHODS: Parallel testing was conducted of 38 hot water samples to detect and determine Legionella acc. to PN-EN ISO 11731:2017 and the Legiolert™/Quanti-Tray® test. Statistical analysis of the results was performed according to PN-EN ISO 17994:2014 and the McNemar's test. RESULTS: The Legiolert™ test was confirmed to be comparable in performance to the reference standardized method in both qualitative and quantitative detection of L. pneumophila in hot water samples. CONCLUSIONS: The study confirmed that the Legiolert™ test is specific and easy to use, and may constitute an alternative to standardized procedures used in the quantification of L. pneumophila in water.

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.

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.

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.

Legionnaires' disease in transportation, construction and other occupations in 39 US jurisdictions, 2014-2016.

BACKGROUND: Certain workers are at increased risk for acquiring Legionnaires' disease compared with other workers. This study aims to identify occupations at increased risk for acquiring Legionnaires' disease. METHODS: Using data from the US Centers for Disease Control and Prevention's Supplemental Legionnaires' Disease Surveillance System, this study identified Legionnaires' disease confirmed patients ≥16 years of age in 39 states with reported symptom onset during 2014-2016. Age-adjusted and sex-adjusted incidence rate ratios (IRR) stratified by occupation group were calculated by comparing Legionnaires' disease patients in an occupation group (eg, transportation) to those in all other occupation groups (eg, non-transportation). RESULTS: A total of 2553 patients had a known occupation group. The two occupations with the highest burden were transportation (N=287; IRR=2.11) and construction (N=269; IRR=1.82). Truck drivers comprised the majority (69.7%) of the transportation occupation group and construction labourers comprised almost half (49%) of the construction occupation group. The healthcare support occupation had the highest IRR (N=75; IRR=2.16). CONCLUSION: Transportation and construction workers, who are generally not covered by guidance related to building water systems, have increased risk of Legionnaires' disease compared with other workers. One hypothesised risk factor for truck drivers is the use of non-genuine windshield cleaner in their vehicles. A simple intervention is to use genuine windshield cleaner with bactericidal properties (ie, includes isopropanol/methanol) which can reduce the risk of Legionella growth and transmission. To improve surveillance of Legionnaires' disease and identification of similar exposures, the authors encourage the collection of occupation and industry information for all patients with Legionnaires' disease.

Analysis of the effectiveness of combination antimicrobial therapy for Legionnaires' disease: A nationwide inpatient database study.

OBJECTIVES: The effectiveness of monotherapy and combination therapy with quinolones and macrolides for treating Legionnaires' disease remains uncertain; this study aimed to assess the comparative effectiveness of three treatment approaches. METHODS: Using a nationwide inpatient database, we analyzed 3560 eligible patients hospitalized for Legionnaires' disease between April 1, 2014, and March 31, 2021; patients were divided into combination therapy, quinolone monotherapy, and macrolide monotherapy groups according to the antibiotics administered within 2 days of admission. We compared in-hospital mortality, total hospitalization costs, and length of stay across these groups using multiple propensity score analysis with inverse probability of treatment weighting. RESULTS: Of the 3560 patients, there were 564 (15.8%), 2221 (62.4%), and 775 (21.8%) patients in the combination therapy, quinolone monotherapy, and macrolide monotherapy groups, respectively. No significant differences were observed in in-hospital mortality between combination therapy and quinolone monotherapy groups, and between combination therapy and macrolide monotherapy groups. There were no significant differences in total hospitalization costs or length of stay among the three groups. CONCLUSION: The study suggests that there may not be a significant advantage in using a combination of quinolones and macrolides over monotherapy for the treatment of Legionnaires' disease. Given the potential for increased side effects, careful consideration is advised when choosing this combination therapy.

An acetyltransferase effector conserved across Legionella species targets the eukaryotic eIF3 complex to modulate protein translation.

The survival of Legionella spp. as intracellular pathogens relies on the combined action of protein effectors delivered inside their eukaryotic hosts by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IVb secretion system. The specific repertoire of effector arsenals varies dramatically across over 60 known species of this genera with Legionella pneumophila responsible for most cases of Legionnaires' disease in humans encoding over 360 Dot/Icm effectors. However, a small subset of "core" effectors appears to be conserved across all Legionella species raising an intriguing question of their role in these bacteria's pathogenic strategy, which for most of these effectors remains unknown. L. pneumophila Lpg0103 effector, also known as VipF, represents one of the core effector families that features a tandem of Gcn5-related N-acetyltransferase (GNAT) domains. Here, we present the crystal structure of the Lha0223, the VipF representative from Legionella hackeliae in complex with acetyl-coenzyme A determined to 1.75 Å resolution. Our structural analysis suggested that this effector family shares a common fold with the two GNAT domains forming a deep groove occupied by residues conserved across VipF homologs. Further analysis suggested that only the C-terminal GNAT domain of VipF effectors retains the active site composition compatible with catalysis, whereas the N-terminal GNAT domain binds the ligand in a non-catalytical mode. We confirmed this by in vitro enzymatic assays which revealed VipF activity not only against generic small molecule substrates, such as chloramphenicol, but also against poly-L-lysine and histone-derived peptides. We identified the human eukaryotic translation initiation factor 3 (eIF3) complex co-precipitating with Lpg0103 and demonstrated the direct interaction between the several representatives of the VipF family, including Lpg0103 and Lha0223 with the K subunit of eIF3. According to our data, these interactions involve primarily the C-terminal tail of eIF3-K containing two lysine residues that are acetylated by VipF. VipF catalytic activity results in the suppression of eukaryotic protein translation in vitro, revealing the potential function of VipF "core" effectors in Legionella's pathogenic strategy.IMPORTANCEBy translocating effectors inside the eukaryotic host cell, bacteria can modulate host cellular processes in their favor. Legionella species, which includes the pneumonia-causing Legionella pneumophila, encode a widely diverse set of effectors with only a small subset that is conserved across this genus. Here, we demonstrate that one of these conserved effector families, represented by L. pneumophila VipF (Lpg0103), is a tandem Gcn5-related N-acetyltransferase interacting with the K subunit of human eukaryotic initiation factor 3 complex. VipF catalyzes the acetylation of lysine residues on the C-terminal tail of the K subunit, resulting in the suppression of eukaryotic translation initiation factor 3-mediated protein translation in vitro. These new data provide the first insight into the molecular function of this pathogenic factor family common across Legionellae.

An Epidemiological Study on Legionnaires' Disease in Gyeonggi Province, Korea: Occurrence, Infection Routes, and Risk Factors for Mortality (2016-2022).

Background and Objectives: Legionnaires' disease (LD) is an acute respiratory disease with increasing annual numbers of reported domestic and global cases. This study aimed to establish foundational data for the prevention and control of LD by investigating the occurrence and infection routes of reported and suspected cases of LD in Gyeonggi Province, Korea, from January 2016 to December 2022, and by and analyzing the risk factors for death. Materials and Methods: A sex-and-age standardization was performed on LD patients and suspected cases reported in Gyeonggi Province. The monthly average number of confirmed cases was visualized using graphs, and a survival analysis was performed using Kaplan-Meier survival curves. The mortality risk ratio was estimated using the Cox proportional hazards model. Results: The incidence of LD in Gyeonggi Province mirrored the national trend, peaking in July with the highest number of confirmed and suspected cases. While there was no significant difference in survival rates by age, the survival rate was higher for suspected cases when analyzed separately. Comparing the death ratio by infection route, nosocomial infections showed the highest death ratio, and intensive care unit (ICU) admission and the presence of coinfections were significantly correlated with mortality. Factors such as nosocomial infection, admission within 1 to 3 days following diagnosis, and the development of complications were factors contributing to a higher risk of death. Conclusions: The general characteristics of patients with LD were similar to those suggested by previous studies. The proportion of community-acquired infections was lower than in previous studies, but the length of hospital stay was similar for survivors and the deceased, and the mortality rate within 30 days after diagnosis was higher for nosocomial infections. In conclusion, nosocomial infection, a period of up to 3 days from admission to diagnosis, and complications were significantly related to the mortality rate of LD.

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.

High performance Legionella pneumophila source attribution using genomics-based machine learning classification.

Fundamental to effective Legionnaires' disease outbreak control is the ability to rapidly identify the environmental source(s) of the causative agent, Legionella pneumophila. Genomics has revolutionized pathogen surveillance, but L. pneumophila has a complex ecology and population structure that can limit source inference based on standard core genome phylogenetics. Here, we present a powerful machine learning approach that assigns the geographical source of Legionnaires' disease outbreaks more accurately than current core genome comparisons. Models were developed upon 534 L. pneumophila genome sequences, including 149 genomes linked to 20 previously reported Legionnaires' disease outbreaks through detailed case investigations. Our classification models were developed in a cross-validation framework using only environmental L. pneumophila genomes. Assignments of clinical isolate geographic origins demonstrated high predictive sensitivity and specificity of the models, with no false positives or false negatives for 13 out of 20 outbreak groups, despite the presence of within-outbreak polyclonal population structure. Analysis of the same 534-genome panel with a conventional phylogenomic tree and a core genome multi-locus sequence type allelic distance-based classification approach revealed that our machine learning method had the highest overall classification performance-agreement with epidemiological information. Our multivariate statistical learning approach maximizes the use of genomic variation data and is thus well-suited for supporting Legionnaires' disease outbreak investigations.IMPORTANCEIdentifying the sources of Legionnaires' disease outbreaks is crucial for effective control. Current genomic methods, while useful, often fall short due to the complex ecology and population structure of Legionella pneumophila, the causative agent. Our study introduces a high-performing machine learning approach for more accurate geographical source attribution of Legionnaires' disease outbreaks. Developed using cross-validation on environmental L. pneumophila genomes, our models demonstrate excellent predictive sensitivity and specificity. Importantly, this new approach outperforms traditional methods like phylogenomic trees and core genome multi-locus sequence typing, proving more efficient at leveraging genomic variation data to infer outbreak sources. Our machine learning algorithms, harnessing both core and accessory genomic variation, offer significant promise in public health settings. By enabling rapid and precise source identification in Legionnaires' disease outbreaks, such approaches have the potential to expedite intervention efforts and curtail disease transmission.

Legionnaires' Disease Outbreak Associated With a Hot Tub Display at the North Carolina Mountain State Fair, September 2019.

OBJECTIVES: On September 23, 2019, the North Carolina Division of Public Health identified a legionellosis increase in western North Carolina; most patients had recently attended the North Carolina Mountain State Fair. We conducted a source investigation. METHODS: Cases were fair attendees with laboratory-confirmed legionellosis and symptom onset within 2 to 14 days (Legionnaires' disease) or ≤3 days (Pontiac fever). We conducted a case-control study matching cases to non-ill fair attendees as control participants and an environmental investigation, and we performed laboratory testing (Legionella bacteria culture and polymerase chain reaction) of 27 environmental samples from fairgrounds and hot tubs and 14 specimens from case patients. We used multivariable unconditional logistic regression models to calculate adjusted odds ratios for potential Legionella exposure sources and risk factors. RESULTS: Of 136 people identified with fair-associated legionellosis, 98 (72%) were hospitalized and 4 (3%) died. Case patients were more likely than control participants to report walking by hot tub displays (adjusted odds ratio = 10.0; 95% CI, 4.2-24.1). Complete hot tub water treatment records were not kept, precluding evaluation of water maintenance conducted on display hot tubs. Legionella pneumophila sequence types (STs) were consistent among 10 typed clinical specimens (ST224) but distinct from the only positive environmental sample from the fair (ST7 and ST8). CONCLUSIONS: Hot tub displays were identified as the most likely outbreak source, making this the largest hot tub-associated Legionnaires' disease outbreak worldwide. Following the investigation, the North Carolina Division of Public Health and the Centers for Disease Control and Prevention released guidance on mitigating risk of Legionella exposure from hot tub displays. Results highlight the importance of properly maintaining equipment that aerosolizes water, including hot tubs intended for display purposes only.

Legionella pneumonia in hospitalized adults with respiratory failure: Quinolones or macrolides?

The optimal antimicrobial regimen for adults with respiratory failure due to Legionella pneumonia remains controversial. A systematic review was performed to assess the impact on outcomes comparing quinolones versus macrolides. A literature search was conducted in PubMed, Cochrane Library and Web of Science between 2012 and 2022. It yielded 124 potentially articles and ten observational studies met the inclusion criteria. A total of 4271 patients were included, 2879 (67 %) were male. A total of 1797 (42 %) subjects required intensive care unit (ICU) admission and 942 (52 %) mechanical ventilation. Fluoroquinolones and macrolides alone were administered in 1397 (33 %) and 1500 (35 %) subjects, respectively; combined therapy in 204 (4.8 %) patients. Overall mortality was 7.4 % (319 patients), with no difference between antibiotics. When data from the three studies with severe pneumonia were pooled together, mortality with fluoroquinolones alone was statistically superior to macrolides alone (72.8 % vs 30.8 %, p value 0.027). Hospital length of stay and complications were comparable. Our findings suggest that macrolides and quinolones were comparable for hospitalized Legionella pneumonia. However, in severe pneumonia, a randomized clinical trial is an unmet clinical need. PROSPERO registration number: CRD42023389308.

Association between the Timing of Urinary Antigen Testing and Outcomes in Legionella Pneumonia Patients: A Nationwide Database Study.

Objective Recommendations on the timing of Legionella urinary antigen tests for community-acquired pneumonia patients differ among guidelines in Japan, the United States, and European nations. We therefore evaluated the association between the timing of urinary antigen tests and in-hospital mortality in patients with Legionella pneumonia. Methods We conducted a retrospective cohort study using the Diagnosis Procedure Combination database, a nationwide database of acute care inpatients in Japan. Patients who underwent Legionella urinary antigen tests on the day of admission formed the tested group. Patients who were tested on day 2 of admission or later or were unexamined formed the control group. We performed a propensity score matching analysis to compare in-hospital mortality, length of hospital stay and duration of antibiotics use between the two groups. Results Of the 9,254 eligible patients, 6,933 were included in the tested group. One-to-one propensity score matching generated 1,945 pairs. The tested group had a significantly lower 30-day in-hospital mortality than the control group (5.7 vs. 7.7%; odds ratio, 0.72; 95% confidence intervals, 0.55-0.95; p=0.020). The tested group also showed a significantly shorter length of stay and duration of antibiotics use than the control group. Conclusion Urine antigen testing upon admission was associated with better outcomes in patients with Legionella pneumonia. Urine antigen tests upon admission may be recommended for all patients with severe community-acquired pneumonia.

Incidence of Legionnaires' Disease among Travelers Visiting Hotels in Germany, 2015-2019.

We determined whether the incidence rates of travel-associated Legionnaires' disease (TALD) in hotels in Germany increased after a previous occurrence and whether control measures required by the European Legionnaires' Disease Surveillance Network after a cluster (>2 cases within 2 years) restored the rate to baseline. We analyzed TALD surveillance data from Germany during 2015-2019; a total of 307 TALD cases (163 domestic, 144 nondomestic) in hotels were reported. The incidence rate ratio was 5.5 (95% CI 3.6-7.9) for a second case and 25 (95% CI 11-50) for a third case after a cluster had occurred, suggesting that control measures initiated after the occurrence of TALD clusters might be inadequate to restore the incidence rate to baseline. Our findings indicate that substantial LD preventive measures should be explored by hotels or other accommodations after the first TALD case occurs to reduce the risk for future infections.

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.

Large Community Outbreak of Legionnaires Disease Potentially Associated with a Cooling Tower - Napa County, California, 2022.

Legionnaires disease is a serious infection acquired by inhalation of water droplets from human-made building water systems that contain Legionella bacteria. On July 11 and 12, 2022, Napa County Public Health (NCPH) in California received reports of three positive urinary antigen tests for Legionella pneumophila serogroup 1 in the town of Napa. By July 21, six Legionnaires disease cases had been confirmed among Napa County residents, compared with a baseline of one or two cases per year. NCPH requested assistance from the California Department of Public Health (CDPH) and CDC to aid in the investigations. Close temporal and geospatial clustering permitted a focused environmental sampling strategy of high-risk facilities which, coupled with whole genome sequencing results from samples and investigation of water system maintenance, facilitated potential linking of the outbreak with an environmental source. NCPH, with technical support from CDC and CDPH, instructed and monitored remediation practices for all environmental locations that tested positive for Legionella. The investigation response to this community outbreak illustrates the importance of interdisciplinary collaboration by public health agencies, laboratory support, timely communication with the public, and cooperation of managers of potentially implicated water systems. Timely identification of possible sources, sampling, and remediation of any facility testing positive for Legionella is crucial to interrupting further transmission.

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes.

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.