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Legionella pneumophila utilizes the Dot/Icm type IVB secretion system to deliver hundreds of effector proteins inside eukaryotic cells to ensure intracellular replication. Our understanding of the molecular functions of the largest pathogenic arsenal known to the bacterial world remains incomplete. By leveraging advancements in 3D protein structure prediction, we provide a comprehensive structural analysis of 368 L. pneumophila effectors, representing a global atlas of predicted functional domains summarized in a database ( https://pathogens3d.org/legionella-pneumophila ). Our analysis identified 157 types of diverse functional domains in 287 effectors, including 159 effectors with no prior functional annotations. Furthermore, we identified 35 cryptic domains in 30 effector models that have no similarity with experimentally structurally characterized proteins, thus, hinting at novel functionalities. Using this analysis, we demonstrate the activity of thirteen functional domains, including three cryptic domains, predicted in L. pneumophila effectors to cause growth defects in the Saccharomyces cerevisiae model system. This illustrates an emerging strategy of exploring synergies between predictions and targeted experimental approaches in elucidating novel effector activities involved in infection.

The bacterium Legionella pneumophila secretes numerous effector proteins that manipulate endoplasmic reticulum (ER)-derived vesicles to form the Legionella-containing vacuole (LCV). Despite extensive studies, whether the LCV membrane is separate from or connected to the host ER network remains unclear. Here, we show that the smooth ER (sER) is closely associated with the LCV early in infection. Remarkably, Legionella forms a distinct rough ER (rER) niche at later stages, disconnected from the host ER network. We discover that host small GTPases Rab10 and Rab4 and an ER protein, BAP31, play crucial roles in transitioning the LCV from an sER to an rER. Additionally, we have identified a Legionella effector, Lpg1152, that binds to BAP31. Interestingly, the optimal growth of Legionella is dependent on both BAP31 and Lpg1152. These findings detail the complex interplay between host and pathogen in transforming the LCV membrane from a host-associated sER to a distinct rER.

The intracellular bacterial pathogen Legionella pneumophila (L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.'s ∼330 secreted effector proteins are ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p. hijacks host cell ubiquitin signaling, we generated a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection increases ubiquitination of host regulators of subcellular trafficking and membrane dynamics, most notably ∼40% of mammalian Ras superfamily small GTPases. We determine that these small GTPases undergo nondegradative ubiquitination at the Legionella-containing vacuole (LCV) membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central role in cross-family small GTPase ubiquitination, and that these effectors function upstream of SidE family ligases in the polyubiquitination and retention of GTPases in the LCV membrane. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. Our findings position L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.

The KEAP1-NFE2L2 axis is essential for the cellular response against metabolic and oxidative stress. KEAP1 is an adaptor protein of CUL3 (cullin 3) ubiquitin ligase that controls the cellular levels of NFE2L2, a critical transcription factor of several cytoprotective genes. Oxidative stress, defective autophagy and pathogenic infections activate NFE2L2 signaling through phosphorylation of the autophagy receptor protein SQSTM1, which competes with NFE2L2 for binding to KEAP1. Here we show that phosphoribosyl-linked serine ubiquitination of SQSTM1 catalyzed by SidE effectors of Legionella pneumophila controls NFE2L2 signaling and cell metabolism upon Legionella infection. Serine ubiquitination of SQSTM1 sterically blocks its binding to KEAP1, resulting in NFE2L2 ubiquitination and degradation. This reduces NFE2L2-dependent antioxidant synthesis in the early phase of infection. Levels of serine ubiquitinated SQSTM1 diminish in the later stage of infection allowing the expression of NFE2L2-target genes; causing a differential regulation of the host metabolome and proteome in a NFE2L2-dependent manner. Abbreviation: ARE: antioxidant response element; Dup: deubiquitinase specific for phosphoribosyl-linked serine ubiquitination; ER: endoplasmic reticulum; h.p.i: hours post infection; HIF1A/HIF-1α: hypoxia inducible factor 1 subunit alpha; KEAP1: kelch like ECH associated protein 1; KIR: KEAP1-interacting region; LIR: LC3-interacting region; NES: nuclear export signal; NFKB/NF-κB: nuclear factor kappa B; NLS: nuclear localization signal; NFE2L2/Nrf2: NFE2 like bZIP transcription factor 2; PB1 domain: Phox1 and Bem1 domain; PR-Ub: phosphoribosyl-linked serine ubiquitination; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; tBHQ: tertiary butylhydroquinone; TUBE2: tandem ubiquitiin binding entity 2; UBA domain: ubiquitin-associated domain. [ABSTRACT FROM AUTHOR]

Quantitative polymerase chain reaction (qPCR) offers a rapid, automated, and potentially on-site method for quantifying L. pneumophila in building potable water systems, complementing and potentially replacing traditional culture-based techniques. However, its application in assessing human health risks is complicated by a tendency to overestimate risks due to the detection of genomic copies unassociated with viable, infectious bacteria. This study examines the relationship between L. pneumophila measurements via qPCR and culture-based methods, aiming to establish qPCR-to-culture concentration ratios needed to inform associated health risks. Eligible studies collected quantitative data on L. pneumophila concentrations using molecular and culture-based methods within paired water samples. We developed a Poisson lognormal ratio model and a random-effects meta-analysis model to analyze variations in qPCR-to-culture ratios within and across sites. Of the 17 studies in the systematic review, seven, including 23 site-specific data sets, were used for meta-analysis. Our findings indicate these ratios typically vary from 1:1 to 100:1, with ratios close to 1:1 predicted at all sites. Consequently, adopting a default 1:1 conversion factor appears necessary as a cautious approach to convert qPCR concentrations to culturable concentrations for use in health risk models, such as quantitative microbial risk assessment (QMRA). Where this approach may be too conservative, viability-qPCR could improve the accuracy of qPCR-based QMRA. Standardizing qPCR and culture-based methods and reporting site-specific environmental factors affecting L. pneumophila culturability would improve understanding of the relationship between the two methods. The ratio model introduced here advances beyond simple correlation analyses, facilitating investigations of temporal and spatial heterogeneities in the relationship. This analysis is a step forward in the integration of QMRA and molecular biology, and the framework demonstrated for L. pneumophila is applicable to other pathogens monitored in the environment. [ABSTRACT FROM AUTHOR]

Background: Legionella pneumophila is an uncommon pathogen causing community-acquired atypical pneumonia. Acinetobacter baumannii is a major pathogen responsible for hospital-acquired pneumonia, but it rarely causes serious infections in a community setting. Without prompt and appropriate treatments, infection from either of these two pathogens can cause a high mortality rate. Concurrent infection from both L. pneumophila and A. baumannii can cause serious outcomes, but it has rarely been reported previously. Case presentation: A 45-year-old male presented to our hospital with a productive cough and fever after staying in a local hotel. His chest computed tomography (CT) scan showed bilateral lower-lobe infiltration and left pleural effusion. Empirical antibiotics, including piperacillin-tazobactam, levofloxacin, meropenem, and doxycycline, were administered to him to treat community-acquired pneumonia. However, his condition deteriorated very rapidly, and he required endotracheal intubation and mechanical ventilation for respiratory support. Finally, metagenomic next-generation sequencing (mNGS) of his bronchoalveolar lavage fluid identified L. pneumophila and A. baumannii. The sputum culture demonstrated multidrug-resistant A. baumannii. He was diagnosed with pneumonia by concurrent infections from both L. pneumophila and A. baumannii. After careful consideration of the antibiotic susceptibility results and the antibacterial mechanism of each antibiotic, we switched the antibiotics to omadacycline and cefoperazone/sulbactam. His clinical symptoms gradually subsided. The repeat chest CT image showed no infiltration or pleural effusion. Conclusions: Community-acquired pneumonia can be caused by concurrent infections of both L. pneumophila and A. baumannii. Close clinical monitoring, early pathogen detection and antibiotic susceptability tests, and appropriate antibiotic regimen adjustments should be applied to these patients who failed initial antibiotic treatments. [ABSTRACT FROM AUTHOR]

Production and use of growing media involve a trade-off between sustainability and public health. We claim that a paradigm shift is needed, focusing on properties supporting environmental sustainability and human health, with a holistic approach involving One Health expertise. We use Legionella in growing media, soil, and gardening as an example to highlight key knowledge gaps. [ABSTRACT FROM AUTHOR]

Investigating human pathogens in wastewater is crucial for identifying and predicting potential occupational health risks faced by wastewater treatment plant (WWTP) workers. This study aimed to determine the occurrence and levels of Legionella pneumophila, Mycobacterium spp., Arcobacter butzleri, and Aeromonas hydrophila in untreated municipal wastewater. Grab influent, activated sludge, and secondary settling tank (SST) effluent samples were collected bi-weekly over 6 months from 5 WWTPs in Tshwane, South Africa. Mycobacterium spp., A. butzleri, and A. hydrophila were detected using quantitative PCR (qPCR), while Legionella was detected using both a culture method and qPCR. The four pathogens were identified in most samples at varying levels. Legionella pneumophila had a positivity rate of 92%, ranging from 2 to 5.4 log10 MPN/100 mL. Detection rates of Legionella spp., L. pneumophila, and L. pneumophila serogroup 1 were 97%, 75%, and 69%, respectively, with up to 5.3 log10 gene copies (GC)/mL. Importantly, this study demonstrates molecular typing of L. pneumophila serogroup 1 in wastewater, a topic that has been rarely documented. Mycobacterium spp. were detected in all samples at varying levels (log10 GC/mL) in influent (2.8–7.6), activated sludge (4.8–8.9), and SST effluent (3.8–8.9) samples. Arcobacter butzleri and A. hydrophila were detected in 96% and 82% of the samples, respectively, with GC levels in influent, activated sludge, and SST effluent ranging from 0.8 to 6.6, 1.5 to 6.5, and 0.7 to 6.6 log10 GC/mL for A. butzleri, and similar levels for A. hydrophila. These findings underscore the presence of respiratory and enteric pathogens at various treatment points, suggesting potential occupational exposure for WWTP workers. This emphasises the need for microbiological risk assessments (RAs) or reviewing existing RAs and implementing necessary control measures to protect WWTP workers. [ABSTRACT FROM AUTHOR]

Pathogenic bacteria trigger complex molecular interactions in hosts that are characterized mainly by an increase in reactive oxygen species (ROS) as well as an inflammation-associated response. To counteract oxidative damage, cells respond through protective mechanisms to promote resistance and avoid tissue damage and infection; among these cellular mechanisms the activation or inhibition of the nuclear factor E2-related factor 2 (Nrf2) is frequently observed. The transcription factor Nrf2 is considered the master regulator of several hundred cytoprotective and antioxidant genes. Under normal conditions, the Keap1/Nrf2 signaling protects the cellular environment by sensing deleterious oxygen radicals and inducing the expression of genes coding for proteins intended to neutralize the harmful effects of ROS. However, bacteria have developed strategies to harness Nrf2 activity to their own benefit, complicating the host response. This review is aimed to present the most recent information and probable mechanisms employed by a variety of bacteria to modulate the Keap1/Nrf2 activity in order to survive in the infected tissue. Particularly, those utilized by the Gram-positive bacteria Staphylococcus aureus , Streptococcus pneumoniae , Listeria monocytogenes , and Mycobacterium tuberculosis as well as by the Gram-negative bacteria Escherichia coli , Helicobacter pylori , Legionella pneumophila , Pseudomonas aeruginosa and Salmonella typhimurium. We also discuss and highlight the beneficial impact of the Keap1/Nrf2 antioxidant and anti-inflammatory role in bacterial clearance. [ABSTRACT FROM AUTHOR]

Ubiquitination represents a critical post-translational modification of proteins, capable of inducing alterations in the stability, cellular localisation and activity of substrate proteins. Consequently, it plays a pivotal role in a multitude of essential cellular processes. The intracellular parasite Legionella pneumophila releases in excess of 300 effector proteins into its host cell via its distinctive type IVB secretion system. These effector proteins regulate the physiological activity of host cells, thereby facilitating the growth and reproduction of Legionella and ultimately resulting in Legionella infection in humans. In the context of host infection, a number of effector proteins have been identified as regulators of the host cell ubiquitination system. Together with SidJ, SdjA, DupA/DupB, LnaB, and MavL, SidEs precisely and dynamically modulate the ubiquitination pathway of host cells, thereby providing a suitable environment for L. pneumophila to survive. The clarification of the biological functions of LnaB and MavL has led to the elucidation of this complex non-canonical ubiquitination regulatory cycle in L. pneumophila. This review presents a summary of the structural and enzymatic basis of non-classical ubiquitination mediated by SidEs, along with an examination of its biological significance in regulating endoplasmic reticulum rearrangement and promoting Legionella-containing vacuole formation in host cells; the mechanism by which SidJ/SdjA regulates the phosphoribosylation activity of SidEs; and DupA/DupB, Lnaß and MavL reverse the ubiquitination of host substrate proteins by SidEs through a multi-step catalytic reaction. In conclusion, this study will provide a reference for further understanding the detailed mechanism and biological significance of this type of non-classical ubiquitination modification, as well as offering insights into the pathogenic mechanism of L. pneumophila. [ABSTRACT FROM AUTHOR]

HtpB, the chaperonin of the bacterial pathogen L. pneumophila, is found in extracellular locations, even the cytoplasm of host cells. Although chaperonins have an essential cytoplasmic function in protein folding, HtpB exits the cytoplasm to perform extracellular virulence-related functions that support L. pneumophila's lifestyle. The mechanism by which HtpB reaches extracellular locations is not currently understood. To address this experimental gap, immunoelectron microscopy, trypsin-accessibility assays, and cell fractionation were used to localize HtpB in various L. pneumophila secretion mutants. Dot/Icm type IV secretion mutants displayed less surface-exposed HtpB and more periplasmic HtpB than parent strains. The analysis of periplasmic extracts and outer membrane vesicles of these mutants, where HtpB co-localized with bona fide periplasmic proteins, confirmed the elevated levels of periplasmic HtpB. Genetic complementation of the mutants recovered parent strain levels of surface-exposed and periplasmic HtpB. The export of GSK-tagged HtpB into the cytoplasm of infected cells was also Dot/Icm-dependent. The translocating role of the Dot/Icm system was not specific for HtpB because GroEL, the chaperonin of Escherichia coli, was found at the cell surface and accumulated in the periplasm of Dot mutants when expressed in L. pneumophila. These findings establish that a functional Dot/Icm system is required for HtpB to reach extracellular locations, but the mechanism by which cytoplasmic HtpB reaches the periplasm remains partially unidentified. [ABSTRACT FROM AUTHOR]

Evolving technology and the development of new devices that can aerosolize water present a risk for new sources of Legionella bacteria growth and spread within industrial settings. We investigated a cluster of legionellosis among employees of a manufacturing facility in South Carolina, USA, and found 2 unique equipment sources of Legionella bacteria. The cluster of cases took place during August-November 2022; a total of 34 cases of legionellosis, including 15 hospitalizations and 2 deaths, were reported. Legionella pneumophila was isolated from 3 devices: 2 water jet cutters and 1 floor scrubber. L. pneumophila sequence type 36 was identified in environmental isolates and 1 patient specimen, indicating that those devices were the likely source of infection. Remediation was ultimately achieved through the development and implementation of a device-specific water management program. Manufacturing facilities that use aerosol-generating devices should consider maintaining updated Legionella water management programs to prevent Legionella bacterial infections. [ABSTRACT FROM AUTHOR]

In June 2022, a 73-year-old man with a history of laryngeal and esophageal carcinoma was admitted to the emergency unit with sudden fever, confusion, and general condition deterioration. Initial assessments showed a fever of 38.5 °C, elevated C-reactive protein (CRP) at 209 mg/L, and a neutrophil count of 10.4 G/L, with negative results for urine analysis, blood cultures, and multiple infectious pathogens, including Legionella pneumophila, pneumococcal antigen, and SARS-CoV-2. Computed tomography (CT) scans revealed no significant infectious focus. Empirical treatment with Ceftriaxone and Ciprofloxacin was initiated. Despite treatment, the patient's condition remained unchanged, and a lumbar puncture revealed turbid cerebrospinal fluid (CSF) with 14,300 white blood cells (WBC)/mm³, predominantly neutrophils, elevated proteins, and decreased glucose. Gram staining suggested Neisseria meningitidis, but further testing was necessary. Antibiotic therapy was switched to Cefotaxime and Dexamethasone, and the patient was transferred to the Tropical and Infectious Disease Unit. Multiplex PCR assays and additional CSF tests were negative for common pathogens. Sequencing of 16S ribosomal RNA identified Gemella sp. The patient's condition improved with continued Cefotaxime treatment, and he recovered without neurological sequelae. Subsequent dental CT revealed poor dental hygiene but no signs of osteo-meningeal breach or bone lysis. A literature review identified 22 reported cases of central nervous system (CNS) infections caused by various Gemella species from 1980 to 2022. Of these, 59% presented with meningitis, and 41% had additional encephalitis or brain abscesses. Complete recovery occurred in 77% of cases, with 9% resulting in neurological damage and another 9% in fatal outcomes. Relapses occurred in 14% of the cases. The review highlighted that CNS infections by Gemella spp. primarily affect immunocompromised adults with ENT (ear nose throat) or neurological breaches, although some cases involved healthy individuals. This case underscores the diagnostic challenges posed by uncommon pathogens like Gemella and highlights the utility of molecular microbiology in identifying causative agents, thus guiding appropriate treatment. The patient's history of ENT and esophageal cancers, along with recent radiotherapy and chemotherapy, likely contributed to the infection's development. The case emphasizes the importance of thorough investigation in febrile confusion cases and the potential role of Gemella spp. in CNS infections. [ABSTRACT FROM AUTHOR]

Background. Pulmonary superinfections increase the mortality risk among COVID-19 patients, highlighting the need for enhanced understanding to enable early and accurate diagnosis. Methods. We present the case of a patient, a 76-year-old man, hospitalized for a severe form of COVID-19, with a ground-glass pneumonia, involving 40–45% of lung surfaces. Results. In evolution, the clinical condition worsened, presenting leukocytosis with neutrophilia, imaging towards resorption, and computer tomography images showing the appearance of pulmonary condensations in the right lower lobe, the posterior portion of the left lower lobe and pleural collections. Carbapenemase-producing Klebsiella pneumoniae was isolated from the tracheal aspirate, and the real-time polymerase chain reaction test was positive for Klebsiella pneumoniae and Legionella pneumophila. The investigations that were carried out allowed us to establish the coinfections as a probable case of Legionnaire's disease and a ventilator-associated pneumonia with Klebsiella pneumoniae. Conclusions. The case analysis revealed that rare pneumonias may remain undiagnosed, and coinfections may be conditioned by pathophysiological factors or components of COVID-19 critical form treatment. Enhanced understanding of these aspects in clinical practice may contribute to reducing mortality risk in COVID-19 patients. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance is an emerging problem in hospitals and long-term healthcare facilities. Early detection of susceptibility pattern changes in pathogenic bacteria can prevent treatment failures. Therefore, this study chose to investigate the antibiotic susceptibility situation of Legionella pneumophila isolates from hospitals and long-term healthcare facilities in Southern Germany. Serogroups and minimal inhibitory concentrations (MICs) of nine antibiotics were determined from 41 L. pneumophila strains. In total, 28% of the collected strains belonged to the more pathogenic serogroup 1, whereas 72% belonged to serogroups 2–14. Among the tested antibiotics, rifampicin had the lowest MIC90 value. The MIC90 values can be summarized in the following order: rifampicin < levofloxacin < moxifloxacin < ciprofloxacin < clarithromycin < azithromycin < erythromycin < doxycycline < tigecycline. [ABSTRACT FROM AUTHOR]

Legionella pneumophila is different from traditional drinking water contaminants because it presents a latent public health risk for public and private drinking water systems and for the building water systems they supply. This paper reviews information on the likelihood of occurrence of L. pneumophila in public water systems to lay a foundation for public water systems, as a stakeholder in public health risk management, to better manage L. pneumophila. Important to this approach is a literature review to identify conditions that could potentially promote L. pneumophila being present in drinking water systems at either an elevated abundance or at an increased frequency of occurrence, and/or water quality and supply conditions that would contribute to its amplification. The literature review allows the development of an inventory of hazardous conditions that a public water system could experience and, therefore, can be used by water systems to develop control and monitoring strategies. However, effective L. pneumophila risk management programs are hampered by significant data and knowledge gaps. Priority research to advance public water system’s risk assessments and management of L. pneumophila is proposed. [ABSTRACT FROM AUTHOR]

Hot water systems are the most frequent environment associated with the prevalence and growth of opportunistic premise plumbing pathogens (OPPPs). Previous studies identified water heaters as a source of waterborne diseases and concluded that design variables may contribute to their prevalence. A multifaceted approach was used to investigate the vertical stratification of the microbiome and selected OPPPs in an electric water heater tank connected to a home plumbing system simulator. Results show that the microbiome is highly diverse with evidence of temperature stratification and temporal structuring influenced by the partial drainage of the tank. Representatives of the Mycobacterium spp. were the most prevalent taxa, followed by Legionella spp., and a relatively low prevalence of free-living amoeba Vermamoeba vermiformis. Higher concentrations of Legionella pneumophila at the bottom of the tank indicated the potential growth and protection of this opportunistic pathogen at this location. Overall, partial drainage of the water tank (60% of the tank capacity) did not significantly mitigate the microbiome and selected OPPPs. The outcome of this study sheds light on the role of vertical stratification on water quality and demonstrates the resilience of the microbial community residing in an electric water heater tank and the implications for public health. [ABSTRACT FROM AUTHOR]

Ubiquitination and ADP-ribosylation are two types of post-translational modification (PTM) involved in regulating various cellular activities. In a striking example of direct inter- play between ubiquitination and ADP-ribosylation, the bacterial pathogen Legionella pneumophila uses its SidE family of secreted effectors to catalyze an NAD+ -dependent phosphoribosyl ubiquitination of host substrates in a process involving the intermediary formation of ADP-ribosylated ubiquitin (ADPR-Ub). This noncanonical ubiquitination pathway is finely regulated by multiple Legionella effectors to ensure a balanced host subjugation. Among the various regulatory effectors, the macrodomain effector MavL has been recently shown to reverse the Ub ADP-ribosylation and regenerate intact Ub. Here, we briefly outline emerging knowledge on ubiquitination and ADP-ribosylation and tap into cases of direct cross-talk between these two PTMs. The chemistry of ADP-ribose in the context of the PTM and the reversal mechanisms of ADP-ribosylation are then highlighted. Lastly, focusing on recent structural studies on the MavL-mediated reversal of Ub ADP-ribosylation, we strive to deduce distinct mechanisms regarding the catalysis and product release of this reaction. [ABSTRACT FROM AUTHOR]

The Covid 19 pandemic has significantly affected the health, economy, and social impact of humanity. The continuous mutations of the virus variants have accelerated the demand for scientific research on disinfection techniques for a safer indoor environment. Among all the available surface disinfection techniques, ultraviolet germicidal irradiance at 254 nm wavelength has been proven for its disinfection efficacy; however, its usage is limited to unoccupied conditions due to the risk of ultraviolet exposure. This study investigated the efficacy of far-UVC-222 nm experimentally in both controlled environment and field setting. Staphylococcus epidermidis and Mycobacterium smegmatis were employed for surface disinfection in both the laboratory and a meeting room. Total plate count was used to determine the disinfection efficacy by a 20 W unfiltered far-UVC lamp. At 1.1 µW/cm2 far-UVC irradiation, a 1-log10 reduction of Staphylococcus epidermidis and Mycobacterium smegmatis contamination on tabletop can be achieved by 31.3 min and 101.8 min of far-UVC irradiation, respectively. Other pathogens of interest such as Staphylococcus aureus, Mycobacterium tuberculosis, Legionella pneumophila, SARS-CoV-2, and the Measles virus were also referred and compared in this study. This study carefully examined how far-UVC irradiation performs effectively for surface disinfection in a real meeting room setting. The results offer useful recommendations for alternatives to upper-room ultraviolet germicidal irradiance for continuous disinfection within the ultraviolet threshold limit value, with the goal of preventing the spread of any diseases in the future. [ABSTRACT FROM AUTHOR]

Multiple human and plant pathogens are dispersed and transmitted as bioaerosols (e.g., Mycobacterium tuberculosis, SARS-CoV-2, Legionella pneumophila, Aspergillus fumigatus, Phytophthora spp., and Fusarium graminearum). Rapid, on-site methods to detect airborne pathogens would greatly enhance our ability to monitor exposure and trigger early mitigation measures across different settings. Analysis of air samples for microorganisms in a regulatory context is often based on culture-based methods, which are slow, lack specificity, and are not suitable for detecting viruses. Molecular methods (based on nucleic acids) could overcome these challenges. For example, loop-mediated isothermal amplification (LAMP) is rapid, sensitive, specific, and may detect microbial pathogens from air samples in under 60 min. However, the low biomass in air samples makes recovering sufficient nucleic acids for detection challenging. To overcome this, we present a simple method for concentrating bioaerosols collected through liquid impingement (one of the most common methods for bioaerosol collection). This method paired with LAMP (or other molecular approaches) offers simple, rapid, and sensitive detection of pathogens. We validated this method using three airborne pathogens (Mycobacterium tuberculosis, Legionella pneumophila, and Aspergillus fumigatus), and we were able to detect fewer than five cells in a 15 mL liquid impinger air sample in under 60 min. This simple method offers rapid pathogen detection without the use of specialist equipment, and it can be used across healthcare, education, environmental monitoring, and military settings. [ABSTRACT FROM AUTHOR]

Background: Legionella species are the causative agent of Legionnaires' disease and, as ubiquitous waterborne bacteria, are prone to antimicrobial resistance gene (ARG) acquisition and dissemination due to the antimicrobial contamination of natural environments. Given the potential health risks associated with ARGs, it is crucial to assess their presence in the Legionella population. Methods: The ARGs lpeAB and tet56 were detected in 348 samples, isolates, and DNA extracts using conventional PCR. In a subset of lpeAB-positive isolates, azithromycin (AZT) MIC values were obtained using the EUCAST protocol and LpeAB activity was evaluated through an efflux pump inhibition assay. Results: The lpeAB gene was found in 19% (66/348) of samples, with higher detection rates in the L. pneumophila and L. pneumophila sg1 subgroups, at 30% and 41%, respectively. A positive association between lpeAB and L. pneumophila sg1 was found. The MIC values of the lpeAB-positive isolates ranged from 0.064 to 2 mg/L. LpeAB inhibition resulted in 2- and 4-fold MIC reductions in 10 of the 13 isolates analyzed. One sample each of L. longbeacheae and L. bozemanae was found to possess the tet56 gene. Conclusions: The lpeAB gene is predominant in L. pneumophila sg1. A few isolates with the lpeAB gene exhibited MIC values below the EUCAST tentative highest MIC values for wild-type isolates. Expanding ARG monitoring in Legionella is essential to assess the public health risk of Legionnaires' disease. [ABSTRACT FROM AUTHOR]

Abstract Background Legionella pneumophila is an uncommon pathogen causing community-acquired atypical pneumonia. Acinetobacter baumannii is a major pathogen responsible for hospital-acquired pneumonia, but it rarely causes serious infections in a community setting. Without prompt and appropriate treatments, infection from either of these two pathogens can cause a high mortality rate. Concurrent infection from both L. pneumophila and A. baumannii can cause serious outcomes, but it has rarely been reported previously. Case presentation A 45-year-old male presented to our hospital with a productive cough and fever after staying in a local hotel. His chest computed tomography (CT) scan showed bilateral lower-lobe infiltration and left pleural effusion. Empirical antibiotics, including piperacillin-tazobactam, levofloxacin, meropenem, and doxycycline, were administered to him to treat community-acquired pneumonia. However, his condition deteriorated very rapidly, and he required endotracheal intubation and mechanical ventilation for respiratory support. Finally, metagenomic next-generation sequencing (mNGS) of his bronchoalveolar lavage fluid identified L. pneumophila and A. baumannii. The sputum culture demonstrated multidrug-resistant A. baumannii. He was diagnosed with pneumonia by concurrent infections from both L. pneumophila and A. baumannii. After careful consideration of the antibiotic susceptibility results and the antibacterial mechanism of each antibiotic, we switched the antibiotics to omadacycline and cefoperazone/sulbactam. His clinical symptoms gradually subsided. The repeat chest CT image showed no infiltration or pleural effusion. Conclusions Community-acquired pneumonia can be caused by concurrent infections of both L. pneumophila and A. baumannii. Close clinical monitoring, early pathogen detection and antibiotic susceptability tests, and appropriate antibiotic regimen adjustments should be applied to these patients who failed initial antibiotic treatments.

BackgroundThe detection rates of Legionella pneumophila in metro cooling tower water in various cities are relatively high, while studies investigating the trends of Legionella pneumophila pollution and analyzing its influencing factors are still limited. ObjectiveTo investigate the status of Legionella pneumophila contamination in the water of metro cooling towers in a city, to analyze its influencing factors, and to provide a basis for preventing and controlling Legionella pneumophila contamination in metro cooling tower water. MethodsAfter categorizing the metro lines into three groups (>10 years, >5-10 years, and ≤5 years) according to the operating years of each metro line in a city, we randomly selected one line from each group and the selection resulted a total of 58 cooling towers. We collected water samples from the cooling towers during the early, middle, and late stages of cooling tower operation to detect Legionella pneumophila and water quality related indicators. Information on the environment and hygiene management measures of the cooling towers was obtained through on-site investigation and questionnaire survey. Generalized mixed linear models were used to analyze the influencing factors of Legionella pneumophila contamination in cooling tower water. ResultsThe detection rates of Legionella pneumophila in the early, middle, and late stages of cooling tower operation were 25.9%, 77.6%, and 81.0%, respectively. The detection rates in the middle and late stages of operation were significantly higher than that in the early stage (P

HEPN–MNT, a type VII TA module, comprises the HEPN toxin and the MNT antitoxin, which acts as a nucleotidyltransferase that transfers the NMP moiety to the corresponding HEPN toxin, thereby interfering with its toxicity. Here, we report crystal structures of the Legionella pneumophila HEPN–MNT module, including HEPN, AMPylated HEPN, MNT, and the HEPN–MNT complex. Our structural analysis and biochemical assays, suggest that HEPN is a metal-dependent RNase and identify its active site residues. We also elucidate the oligomeric state of HEPN in solution. Interestingly, L. pneumophila MNT, which lacks a long C-terminal α4 helix, controls the toxicity of HEPN toxin via a distinct binding mode with HEPN. Finally, we propose a comprehensive regulatory mechanism of the L. pneumophila HEPN–MNT module based on structural and functional studies. These results provide insight into the type VII HEPN–MNT TA system. HEPN–MNT is a bacterial type VII toxin-antitoxin (TA) system, comprising the HEPN toxin and the MNT antitoxin. Crystal structures and functional assays of the HEPN–MNT module suggest that HEPN is a metal-dependent RNase and identify its active site residues and regulatory mechanism. [ABSTRACT FROM AUTHOR]

We report a fatal case of Legionella feeleii endocarditis in a post–lung transplant patient. The diagnosis was delayed, as routine microbiological testing of nonrespiratory specimens does not account for extrapulmonary Legionella , and urine antigen testing only reliably detects Legionella pneumophila serogroup 1. This case also illustrates the utility of molecular sequencing for blood culture–negative endocarditis. [ABSTRACT FROM AUTHOR]

Legionella is an important waterborne pathogen that causes Legionnaires’ disease (LD). Several outbreaks associated with wastewater treatment plants (WWTPs) have been reported in recent years; however, the prevalence of Legionella in WWTPs in Japan has rarely been investigated. In this study, we investigated the distribution of Legionella in influent wastewater collected from two WWTPs in Kobe, Japan between April 2023 and March 2024. The concentrations for Legionella in all seasons varied between 104 and 106 copies/100 mL in all seasons. Among the 10 Legionella species detected in the influent wastewater, Legionella pneumophila was the most commonly isolated. Genotyping revealed that pathogenic L. pneumophila strains were widely distributed in the influent wastewater in Japan with genetic diversity. LD is one of the most important infectious diseases during natural disasters. This study highlights the importance of influent wastewater as a potential source of LD in Japan, where natural disasters occur frequently. [ABSTRACT FROM AUTHOR]

Diagnosing the cause of death can be challenging, particularly for patients with no prior history of visits to the treating hospital. We encountered a case involving a 76‐year‐old male who was discovered in a state of cardiopulmonary arrest at his home and subsequently declared deceased in our hospital due to severe pneumonia. He had exhibited symptoms of fever over 37°C and severe coughing for several days. Despite consulting a primary care physician one day prior, his symptoms worsened. Autopsy findings revealed an increase in lung weight and diffuse changes in parenchyma. Histological analysis showed numerous inflammatory cells and exudate within the alveoli. Gram and Periodic acid‐Schiff staining were negative, but slight staining was observed in the cytoplasm of macrophages by Warthin‐starry and Gimenez stains. Tests using a pan bacterial/viral detection kit and qualitative polymerase chain reaction (PCR) for Legionella pneumophila were negative. However, using deoxyribonucleic acid extracted from formalin‐fixed paraffin‐embedded lung tissue, PCR amplification of the ssrA gene of congeneric Legionella species yielded positive results. The results suggest that the cause of death was likely due to bacterial pneumonia caused by Legionella species. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae and Legionella pneumophila are common Gram-negative bacteria that can cause lung infections. The multidrug resistance of K. pneumoniae presents a significant challenge for treatment. This study focuses on isocitrate dehydrogenase (IDH), a key enzyme in the oxidative metabolic pathway of these two bacteria. KpIDH and LpIDH were successfully overexpressed and purified, and their biochemical characteristics were thoroughly investigated. The study revealed that KpIDH and LpIDH are homodimeric enzymes with molecular weights of approximately 70 kDa. They are completely dependent on the coenzyme NADP+ and are inactive towards NAD+. KpIDH exhibits the highest catalytic activity at pH 8.0 in the presence of Mn2+ and at pH 7.8 in the presence of Mg2+. Its optimal catalytic performance is achieved with both ions at 55 °C. LpIDH exhibited its highest activity at pH 7.8 in the presence of Mn2+ and Mg2+, respectively, and exhibits optimal catalytic performance at 45 °C. Heat inactivation studies showed that KpIDH and LpIDH retained over 80% of their activity after being exposed to 45 °C for 20 min. Furthermore, we successfully altered the coenzyme specificity of KpIDH and LpIDH from NADP+ to NAD+ by replacing four key amino acid residues. This study provides a comprehensive biochemical characterization of two multidrug-resistant bacterial IDHs commonly found in hospital environments. It enhances our understanding of the characteristics of pathogenic bacteria and serves as a reference for developing new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Two sporadic cases of legionellosis occurring in consecutive years were confirmed by positive antigenuria to Legionella pneumophila serogroup 1 in individuals with limited mobility who were confined to their homes. Both cases had a history of using ultrasonic humidifiers and of low exposure to other possible sources of infection. This study was conducted through an expanded epidemiological survey and home inspection. Samples were collected from domestic hot water and humidifiers. Environmental isolates were characterised by immunoagglutination and immunofluorescence. The Dresden panel is employed for the determination of groups and subgroups of serogroup 1. The amoebae were isolated by water filtration and subsequent cultivation of the filters. Identification of the isolates was conducted through the sequencing of PCR products. In both cases, epidemiological studies identified the ultrasonic humidifiers as the probable source of infection. The presence of Legionella pneumophila was confirmed in the sanitary water installation and in the humidifiers, where high values of Legionella pneumophila serogroup 1 counts were also obtained. The isolates were characterised as Olda Oxford and Olda OLDA. Furthermore, in addition to the Legionella isolates, the presence of Vermamoeba vermiformis was also identified in samples obtained from domestic hot water and humidifiers in one of the residential addresses under investigation. It has been demonstrated that humidifiers can act as an amplification mechanism for pathogenic microorganisms when they are not cleaned and maintained correctly. This can pose a health danger, especially to people with previous respiratory pathologies, the immunosuppressed, and the elderly. Therefore, it is of the utmost importance that professionals who recommend the therapeutic use of this equipment should issue warnings regarding the importance of its correct use, cleaning, and disinfection. Finally, humidifiers should be considered in epidemiological surveys and in the study of sporadic cases. [ABSTRACT FROM AUTHOR]

Biofilms in cooling towers represent a common habitat for the human pathogen Legionella pneumophila. Within the biofilm consortium, frequent interactions with protozoa, i.e. amoebae and ciliates, were reported, while nematodes have only recently been considered as potential environmental reservoir for the pathogenic bacteria. This study is the first approach to investigate the biotic interactions between L. pneumophila and bacterial-feeding nematodes in a semi-natural biofilm model. The species were Diploscapter coronatus, Diploscapter pachys, Plectus similis and Plectus sp., which all co-occur with L. pneumophila in the environment. Biofilms derived from cooling towers were either inoculated with mCherry-labeled L. pneumophila solely or in combination with GFP-labeled Escherichia coli. All experiments were conducted in single-species set-ups and multi-species (D. coronatus and P. similis) set-ups, to account for interspecific competition. Bacterial ingestion was assessed after 24 and 96 h as fluorescence patterns in the digestive tract of the nematodes using confocal laser scanning microscopy. L. pneumophila cells were ingested by all nematode species, with D. coronatus having the highest pathogen load. The fluorescence intensity (i.e. bacterial load) varied between compartments within the digestive tract and was independent of incubation time. Bacterial cells accumulated mostly around the cardia and in the intestine, while less cells were found within stoma and pharynx. Interspecific competition changed the pattern, i.e. with incubation of D. coronatus and P. similis in the same biofilm a significantly higher pathogen load occurred in the intestine of D. coronatus than P. similis after 24 h and 96 h. Remarkably, when given a choice between L. pneumophila and E. coli, P. similis was the only nematode species containing both bacteria after incubation for 24 h. None of the other nematode species contained E. coli after 24 h and 96 h incubation, while L. pneumophila was present. This study thus provides the first evidence, that under environmental conditions L. pneumophila is a frequent diet of bacterial-feeding nematodes, highlighting their potential as pathogen vectors or even host in cooling tower habitats. [ABSTRACT FROM AUTHOR]

Introduction: Legionnaires' disease is a severe pneumonia predominantly caused by Legionella pneumophila (Lp), whose major reservoirs are artificial water systems. As most human infections are caused by L. pneumophila serogroup 1 (Lp1), a reliable method for Lp distinction can be crucial for bacterial spread prevention. As the ability to withstand in environments and to cause the waterborne disease is strongly related to specific genes, the identification of virulent strains can be of great relevance to implement water environmental monitoring and to contain harmful outbreaks to public health. We aimed to test an assay for Lp identification among different Legionella species, and to determine the serogroups. Additionally, we investigated the carriage of virulence and antimicrobial resistance genes. Methods: A total of 90 Legionella spp. isolates identified by phenotypic tests were subjected to the designed quantitative PCR assay targeting specific mip for Lp, wzm for Lp1, pvcA and ahpD for biofilm production. Eleven serogroups were investigated in all our isolates tested positive for mip gene, subsequently analyzed for 12 virulence and 8 antimicrobial resistance genes. Results: Only the 70 Lp isolates were positive for mip. Out of 27 Lp isolates belonging to serogroup 1 based on agglutination test, 23 (85.2%) carried wzm. The presence of ahpD and pvcA was found in 94.3 and 98.6% of Lp isolates, respectively. By multiplex PCR, all 23 wzm-positive strains were confirmed as serogroup 1 that was the most predominant (33%). At least one virulence gene was detected in all Lp isolates. The most frequent gene was ispE (100%), followed by issD (96%), icmK and enhC (93%), cpxA (91%), rtxA2 (74%), lvhB8-B9 (61%), and prpA (54%). The other genes were less diffused in Lp strains (rtxA1, 44%; lvhB3-B4, 47%; pvcB, 27%; lvrE, 24%). Of the macrolide resistance genes, the ereA was found in 84% of Lp strains, while only 14 (20%) harbored the lpeAB among the efflux pump genes. Conclusion: The assays validated in this study enable the simultaneous Lp and Lp1 detection. The differentiation of Lp strains according to their virulence properties could be useful to predict the bacterial ability to survive and to cause the disease. [ABSTRACT FROM AUTHOR]

Natural transformation is the only mechanism of genetic exchange controlled by the recipient bacteria. We quantified its rates in 786 clinical strains of the human pathogens Legionella pneumophila (Lp) and 496 clinical and environmental strains of Acinetobacter baumannii (Ab). The analysis of transformation rates in the light of phylogeny revealed they evolve by a mixture of frequent small changes and a few large quick jumps across 6 orders of magnitude. In standard conditions close to half of the strains of Lp and a more than a third in Ab are below the detection limit and thus presumably non-transformable. Ab environmental strains tend to have higher transformation rates than the clinical ones. Transitions to non-transformability were frequent and usually recent, suggesting that they are deleterious and subsequently purged by natural selection. Accordingly, we find that transformation decreases genetic linkage in both species, which might accelerate adaptation. Intragenomic conflicts with chromosomal mobile genetic elements (MGEs) and plasmids could explain these transitions and a GWAS confirmed systematic negative associations between transformation and MGEs: plasmids and other conjugative elements in Lp, prophages in Ab, and transposable elements in both. In accordance with the hypothesis of modulation of transformation rates by genetic conflicts, transformable strains have fewer MGEs in both species and some MGEs inactivate genes implicated in the transformation with heterologous DNA (in Ab). Innate defense systems against MGEs are associated with lower transformation rates, especially restriction-modification systems. In contrast, CRISPR-Cas systems are associated with higher transformation rates suggesting that adaptive defense systems may facilitate cell protection from MGEs while preserving genetic exchanges by natural transformation. Ab and Lp have different lifestyles, gene repertoires, and population structure. Nevertheless, they exhibit similar trends in terms of variation of transformation rates and its determinants, suggesting that genetic conflicts could drive the evolution of natural transformation in many bacteria. Bacterial species exhibit large variations in their transformation rates. This study of Legionella pneumophila and Acinetobacter baumannii shows that their transformation rates evolve by sudden quick changes, likely caused by intragenomic conflicts between mobile genetic elements and the host bacterium. [ABSTRACT FROM AUTHOR]

The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is a large, multisubunit complex that exports a vast array of substrates into eukaryotic host cells. DotO, a distant homolog of the T4ASS ATPase VirB4, associates with the bacterial inner membrane despite lacking hydrophobic transmembrane domains. Employing a genetic approach, we found DotO's membrane association is mediated by three inner-membrane Dot/Icm components, IcmT, and a combined DotJ-DotI complex (referred to as DotJI). Although deletion of icmT or dotJI individually does not affect DotO's membrane association, the simultaneous inactivation of all three genes results in increased amounts of soluble DotO. Nevertheless, deleting each receptor separately profoundly affects positioning of DotO, disrupting its link with the Dot/Icm complex at the bacterial poles, rendering the receptors nonredundant. Furthermore, a collection of dotO point mutants that we isolated established that DotO's N-terminal domain interacts with the membrane receptors and is involved in dimerization, whereas DotO's C-terminal ATPase domain primarily contributes to the protein's formation of oligomers. Modeling data revealed the complex interaction between DotO and its receptors is responsible for formation of DotO's unique "hexamer of dimers" configuration, which is a defining characteristic of VirB4 family members. [ABSTRACT FROM AUTHOR]

Legionnaires' disease (LD) is a severe and potentially fatal form of bacterial pneumonia caused by Legionella spp. We evaluated the use of UV-light for detecting Legionella non- pneumophila in water samples according to the NEN-EN-ISO 11731:2017 methodology (reference method) in a collaborative effort involving 10 laboratories. First, a test panel was constructed of 298 strains: 157 Legionella strains and 141 non- Legionella strains were cultured on buffered charcoal yeast extract (BCYE)-medium and confirmed according to ISO 11731: 2017 (cultured on BCYE agar plates with and without l -cysteine), and by matrix-assisted laser desorption-ionization time of flight or next generation sequencing. All strains were additionally exposed to an UV-light to assess if they showed a bright blue fluorescence effect (UV-positive) or not (UV-negative). Second, in an interlaboratory study, 10 laboratories analyzed a blinded set of 16 Legionella strains and 8 non- Legionella strains using both methods. The test panel analyses showed 100% accordance between the UV-light and reference method. In addition, the interlaboratory study results showed full agreement between both methods. Our results support the implementation of UV-light detection to confirm Legionella presumptive colonies during analyses of water samples according to the NEN-EN-ISO 11731:2017 methodology. Implementation of UV-light confirmation could reduce workload, time-to-result and costs for the analyses of water samples for the presence of Legionella. [ABSTRACT FROM AUTHOR]

Background: The incidence of Legionnaires' disease (LD) is increasing steadily in Europe. Its early diagnosis by general practitioners (GPs) is crucial for better patient outcomes. Study objectives: This study assessed Belgian GPs' knowledge about LD and the accessibility of diagnostic tests in their practices. Methods: A specifically designed questionnaire was distributed to actively practicing GPs, including primary care trainees, between 31 January 2022 and 13 March 2022. This survey targeted approximately 4200 GPs with an estimated population catchment of 30% of the actively working Belgian GPs. Results: The response rate was estimated at 3%. Over 70% of the GPs correctly identified the LD occurrence peak, major risk factors, and clinical manifestations. While 62% of participants preferred the Legionella pneumophila urinary antigen test (UAT) as a primary diagnostic method, 75% were unsure about its availability within their laboratories and 82% had not prescribed it in the last year. Finally, 76% expressed a desire for additional information on this topic. Conclusions: Belgian GPs should evaluate the possibility of conducting UAT testing in their laboratories to enhance LD case management and improve their preparedness. Furthermore, initiatives should be implemented to improve communication between specialists and GPs and develop educational programs directed at Belgian GPs. [ABSTRACT FROM AUTHOR]