Search Results (422)

Legionnaires’ disease incidence has risen substantially across industrialised nations since the early 2000s, yet standard control protocols—primarily secondary disinfection verified by culture—rely on an evidence base that warrants further scrutiny. This review offers three interpretations of the available evidence, without claiming they are established conclusions. First, culture-based verification has inherent methodological limitations: treatments that reduce culturability can induce viable-but-non-culturable (VBNC) states undetected by culture alone, creating potential discrepancies between verified culture reduction and complete microbial clearance. This reading rests on evidence that monochloramine at ≥1 mg/L abolishes culturability while genomic units remain essentially unchanged and that starved VBNC Legionella retain the capacity to infect human macrophages after more than 220 days, albeit at roughly 1% the efficiency of culturable cells; whether this translates into material risk in operating buildings remains undetermined. Second, much of the comparative-effectiveness literature evaluating disinfectants relies on observational single-site studies, and early validation frameworks often depended on self-reported survey data; randomised comparison in occupied healthcare buildings is frequently impractical, though recent systematic reviews now supply quantitative synthesis earlier work lacked. Third, regulatory thresholds remain expressed in the units of the method whose deficiencies motivated the alternatives, and the alignment of verification standards with advances in molecular detection remains an ongoing challenge. Full article

Legionella pneumophila is a Gram-negative opportunistic pathogen responsible for Legionnaires’ disease, a severe form of atypical pneumonia associated with high morbidity and mortality, particularly in immunocompromised individuals. Despite its growing global burden and the persistence of environmental reservoirs, no licensed vaccine is currently available. In this study, an integrative immunoinformatics and reverse vaccinology approach was employed to design a multi-epitope vaccine candidate targeting L. pneumophila. The complete proteome was systematically analyzed to identify essential, virulent, and surface-accessible proteins. Subsequent antigenicity assessment and non-homology screening against the human proteome led to the selection of key immunogenic targets, including KDO transferase and TolR. B-cell and T-cell epitopes were predicted and rigorously filtered based on antigenicity, non-allergenicity, and non-toxicity. Selected epitopes were assembled into a multi-epitope vaccine construct using appropriate linkers and adjuvants to enhance immunogenicity and structural stability. The designed construct exhibited favorable physicochemical properties, high antigenicity, and good solubility. Structural modeling and refinement confirmed the reliability of the predicted three-dimensional structure. Molecular docking analysis demonstrated strong binding interactions with immune receptors TLR-2 and TLR-9, which were further validated by molecular dynamics simulations indicating stable complex formation under physiological conditions. In addition, immune simulation predicted robust humoral and cellular immune responses, including memory cell generation. Overall, this study presents a promising computationally designed multi-epitope vaccine candidate against L. pneumophila. However, further in vitro and in vivo studies are required to validate its immunogenicity, safety, and protective efficacy. Full article

Legionella pneumophila, the causative agent of Legionnaires’ disease, remains a major public health concern in water systems. Chlorination is the most widely used disinfection strategy but presents important limitations, including reduced stability in hot water systems and the generation of carcinogenic by-products. This study evaluated ozonation as an alternative disinfection method, comparing its efficacy with chlorination against different physiological states of L. pneumophila, including culturable and viable but non-culturable (VBNC) cells, as well as biofilm-associated bacteria and amoebal hosts. The biocidal effect of ozone and chlorination on planktonic L. pneumophila was assessed by culture and viability-qPCR. Biofilm viability was evaluated using fluorescein diacetate staining, whereas amoeba viability was determined by flow cytometry. Ozonation achieved complete inactivation of both culturable and VBNC L. pneumophila within 5 min. In contrast, chlorination achieved complete loss of culturability only at high concentrations and was associated with the persistence of VBNC cells. Neither ozonation nor chlorination significantly reduced established biofilms or amoebae. These findings indicate that ozonation is a promising strategy for rapid inactivation of planktonic L. pneumophila and may overcome important limitations associated with chlorine-based treatments. Full article

Despite the recommendation of purified water, dental unit waterlines (DUWLs) readily form biofilm and suffer persistent microbial contamination, threatening both patients and dental staff. This study aimed to evaluate the microbial contamination and practical effectiveness of chemical disinfection in reverse osmosis (RO) water-supplied DUWLs. A single-center, sequential longitudinal study involving 15 dental chairs and 60 terminal outlets was conducted. Two disinfection strategies were sequentially evaluated: shock disinfection with a chlorine-based agent and continuous disinfection using slightly acidic electrolyzed water (SAEW). Heterotrophic plate counts (HPCs) were quantified via R2A culture, and microbial communities were characterized by metagenomic next-generation sequencing (mNGS) on 12 water samples (four terminal outlets and two source-water points, both pre- and post-disinfection). Water quality assessment revealed severe microbial contamination at the terminal outlets, with 73.33% of samples exceeding the threshold (500 CFU/mL) and a median HPC level of 805 CFU/mL. Notably, mNGS identified nucleic acid sequences of Legionella pneumophila, Pseudomonas aeruginosa, and Acanthamoeba in four pre-disinfection terminal-outlet samples. Chlorine-based shock disinfection achieved 100% compliance immediately after treatment; however, by week 5, the percentage above the threshold value had risen to 73.33%, with a median HPC level of 600 CFU/mL. The continuous SAEW disinfection protocol sustained effective microbial control over 55 weeks, with an overall percentage above threshold value of only 3.33%. mNGS analysis further confirmed a reduction in microbial relative abundance following one year of continuous SAEW disinfection. In conclusion, purified RO water alone could not guarantee DUWL microbiological safety, and this study provided practical evidence to support decision-making for DUWL maintenance. Full article

Background: Legionnaires’ disease (LD) is most often described as caused by a single Legionella pneumophila (Lp) serogroup (SG), typically SG1. However, mixed infections involving multiple SGs or even different Legionella species can occur within a single host. Such cases remain underrecognized and pose challenges for diagnosis, clinical management, and outbreak investigation. Case presentation: A 61-year-old woman developed acute fever, malaise, and productive cough after visiting a wellness spa in France, with radiological findings consistent with a lower respiratory tract infection. The urinary antigen test (UAT) for Lp was negative, but respiratory multiplex PCR was weakly positive. Subsequent testing at the Belgian National Reference Centre (NRC) for Legionella revealed an unexpected discordance between an in-house SG1-specific PCR and culture-based findings. Further analysis identified co-infection with two distinct Lp strains, SG1 (sequence type, ST146) and SG10 (ST1267), using a combination of direct sequence-based typing (SBT) and culture with serogrouping of multiple colonies. The patient improved rapidly after treatment with oral levofloxacin. Conclusions: Mixed Lp SG infections are likely underrecognized in routine practice, as neither UAT, commercial molecular assays, nor characterization of a single cultured isolate may adequately capture within-host strain diversity. Comprehensive diagnostic approaches combining culture with analysis of multiple colonies and molecular tests are essential for accurate case detection and source attribution. Referral to NRCs is recommended when advanced typing methods are unavailable. This first reported Belgian case illustrates the potential implications for patient management and epidemiological investigations, underscoring the need for broader diagnostic strategies. Full article

Background: Legionella pneumophila is a leading cause of community- and hospital-acquired pneumonia (HAP/CAP), although the clinical and radiological features associated with infection remain incompletely defined. This study aimed to identify clinical, comorbidity-related, and chest computed tomography (CT) features associated with L. pneumophila pneumonia in hospitalized adults with community-acquired pneumonia (CAP). Methods: A retrospective case–control study was conducted at a tertiary-care university hospital in Italy between January 2019 and August 2025. Eighty patients positive for L. pneumophila were compared with 79 controls negative for L. pneumophila. Demographic, clinical, microbiological, and radiological data were collected, and factors independently associated with infection were evaluated using multivariable logistic regression analysis. Results: Patients with L. pneumophila infection exhibited a greater comorbidity burden, particularly heart failure, and were more frequently smokers. Bilateral lung involvement and hilar/mediastinal lymphadenopathy were significantly more common among Legionella-positive patients, whereas overall pulmonary complications and bilateral pleural effusion were more frequently observed in controls. Multivariable analysis identified comorbidity burden as an independent positive predictor of L. pneumophila infection, while intensive care unit admission, oxygen therapy, and pulmonary complication score were independently associated with a lower likelihood of infection. Conclusions: The integration of comorbidity assessment with readily available chest CT findings may help raise clinical suspicion of L. pneumophila pneumonia and support consideration of appropriate diagnostic testing. Full article

Background: Legionella pneumophila is a rare but severe cause of community-acquired pneumonia and can lead to acute respiratory distress syndrome (ARDS). Awake prone positioning (APP) has been recognized as an effective adjunct for non-intubated patients with hypoxemic respiratory failure, primarily studied in coronavirus disease 2019 (COVID-19) and other ARDS etiologies. Its application in Legionella-associated ARDS remains poorly documented. Therefore, in this work, we present a case of Legionella-associated ARDS successfully managed with APP, high-flow nasal cannula (HFNC), non-invasive ventilation (NIV), levofloxacin, and corticosteroids. Case presentation: A 62-year-old male with multiple comorbidities, including type 2 diabetes mellitus (T2DM) and a history of coronary artery bypass surgery, presented with a 3-day history of productive cough, exertional dyspnea, and general malaise. Oxygen saturation on admission was 90%, with fever and tachycardia. Inflammatory markers were markedly elevated. Chest computed tomography (CT) revealed extensive bilateral pulmonary infiltrates. Despite a negative urinary Legionella antigen test, sputum polymerase chain reaction (PCR) confirmed Legionella pneumophila on day 2. A Horowitz index of 147 mmHg on day 2 established moderate ARDS. The patient was treated with HFNC oxygen therapy, NIV, and APP for up to 12 h daily. Antibiotic therapy was initiated with amoxicillin/clavulanic acid and clarithromycin, subsequently streamlined to levofloxacin upon microbiological confirmation. Methylprednisolone 40 mg/day was administered for 8 days as adjunctive ARDS therapy. The patient demonstrated gradual clinical and respiratory improvement without requiring endotracheal intubation. Follow-up chest CT on day 7 showed regression of bilateral consolidations and ground-glass opacities (GGOs). The patient was transferred to pulmonary rehabilitation on day 11 and completed antibiotic therapy as an outpatient. Conclusions: This case illustrates the successful use of APP combined with HFNC and NIV alongside standard medical therapies, including appropriate antibiotics and corticosteroids, to avoid intubation in moderate ARDS secondary to Legionella pneumonia. Early initiation of APP may be a valuable strategy in Legionella-associated ARDS in carefully selected patients. Full article

Legionella pneumophila (L. pneumophila) persists in engineered water systems and is the causative agent of Legionnaires’ disease. Despite its clinical importance, little is known about whether environmental populations are undergoing gradual changes in their antimicrobial susceptibility. Here, we compared the antimicrobial susceptibility of 95 environmental L. pneumophila isolates collected in 2013–2014 with those obtained in 2024–2025 from the same sites in a drinking water distribution system (DWDS) using Epsilometer (E-test). The analyses showed both temporal and spatial variation: Ciprofloxacin activity remained relatively stable, whereas levofloxacin exhibited a broader minimum inhibitory concentration (MIC) range among contemporary isolates, with the minimum inhibitory concentration required to inhibit the growth of 90% of isolates (MIC₉₀) doubling from 0.5 to 1.0 mg/L, suggesting increased variability and the emergence of less susceptible subpopulations. Tigecycline showed a marked site-specific increase in MIC values, with the mean MIC at site D increasing from 0.26 mg/L in the 2013–2014 isolates to 1.86 mg/L in the 2024–2025 isolates, indicating reduced susceptibility at this site. Among macrolides, erythromycin exhibited the lowest MIC values across both isolate collections, whereas azithromycin showed higher MIC values. Moreover, isolates recovered from adjacent sites within the same DWDS exhibited significantly different susceptibility profiles, possibly reflecting the influence of local environmental conditions. These findings emphasize that L. pneumophila susceptibility is not static but is likely shaped by ecological and temporal factors. The study underscores the importance of long-term environmental surveillance to track the evolution of this pathogen and provides new insights into how micro-scale dynamics within water systems can influence antimicrobial susceptibility patterns. Full article

The occurrence of Legionella bacteria in drinking water distribution systems poses an important public health concern, particularly in the context of opportunistic premise plumbing pathogens (OPPPs) and the implementation of EU Directive 2020/2184. This study evaluated the prevalence of Legionella using a “source-to-tap” approach and compared culture-based and molecular detection methods. A total of 120 water samples were collected between February and November 2025 from water intakes, treatment plants, distribution networks, and premise plumbing systems in Poland. Analyses were performed using the ISO 11731 culture method and quantitative PCR (qPCR) targeting the ssrA, mip, and wzm genetic markers. The highest prevalence of Legionella was observed in premise plumbing systems, with positivity rates of 64.0% by qPCR and 30.0% by culture. Overall, qPCR detected Legionella in 37.5% of samples compared with 14.2% using the culture method, demonstrating significantly higher sensitivity (p < 0.001). Seasonal variation was observed, with the highest detection frequencies during summer months. Positive samples were associated with higher water hardness as well as elevated iron and manganese concentrations. Significant differences in Legionella occurrence were found among sampling points and seasons (p < 0.05). The results indicate that premise plumbing systems constitute the primary reservoir of Legionella within drinking water distribution systems. Furthermore, qPCR proved to be a rapid and sensitive tool supporting microbiological risk assessment and monitoring within the “source-to-tap” framework recommended by EU Directive 2020/2184. Full article

Accurate species-level identification of Legionella during routine environmental surveillance remains challenging, as standard proteomic screening methods like MALDI-TOF MS often lack the resolution to distinguish closely related taxa. This study introduces a novel, cost-effective, and stepwise taxonomic framework that bridges routine environmental monitoring with multi-layered, genome-resolved analysis. We expanded upon our previous screening of four novel candidate lineages by focusing on the three remaining, uncharacterized environmental isolates, while using the recently validly described Legionella sheltonii (strain PATHC038) as a reference framework control. By applying nucleotide- and protein-based overall genomic relatedness indices (ANI, dDDH, AAI, and POCP) alongside core-genome phylogenomics, our framework independently confirmed the genomic distinctiveness of the three isolates, which exhibited AAI values of 94.3–96.6% and POCP values of 86.5–91.0%, with POCP values lying close to the proposed species delineation threshold. By defining clear criteria for escalating isolates from culture to targeted sequencing, this workflow prevents diagnostic misidentification during epidemiological outbreak investigations and provides public health agencies with a scalable tool to monitor hidden Legionella diversity, ultimately enhancing proactive water safety and risk assessment. Full article

Legionella pneumophila is widely recognized as the principal organism responsible for Legionnaires’ disease. Modifications of surface-exposed lipopolysaccharide in L. pneumophila are key determinants of bacterial adaptation to host cells. Using a mutant strain deficient in N-methylation of legionaminic acid and fluorescence-based biophysical analyses, it was demonstrated that N-methyl groups linked to the acetimidoylamino group of legionaminic acid modulate the physicochemical properties of the bacterial cell surface and influence the interaction of L. pneumophila with macrophages. Loss of N-methyl groups reduced the efficiency of bacterial interaction with THP-1-derived macrophages and impaired intracellular proliferation. In addition, the presence of N-methyl groups in legionaminic acid contributes to increased TNF-α production in THP-1 macrophages stimulated with L. pneumophila sg1 LPS, without affecting IL-6 induction, suggesting that N-methylation of legionaminic acid may skew the early pro-inflammatory response toward TNF-α-dominated signaling during L. pneumophila infection. Full article

Background: The biliary and pancreatic tract is increasingly recognized as a microbial ecosystem rather than a sterile environment. Dysbiosis contributes to inflammation, bile acid alterations, and carcinogenesis, with distinct microbiota profiles linked to progression from benign to malignant conditions. Clinical factors, including gut–liver axis disruption and biliary stenting, may further exacerbate microbial imbalance. Objective: The objective of this study is to synthesize current evidence and identify knowledge gaps on the role of biliary microbiota in pancreaticobiliary carcinogenesis and its implications for diagnosis, prognosis, and therapy. Methods: This scoping review was conducted following PRISMA-ScR guidelines. A systematic search of PubMed, Web of Science, and Scopus was performed for studies published between January 2015 and December 2025, guided by the PICo framework. Results: Included studies primarily characterized changes in microbiota composition to identify microbial biomarkers associated with pancreaticobiliary diseases. Predictive bioinformatics analyses suggest that dysbiosis may promote carcinogenesis through metabolic and inflammatory pathways. Machine learning approaches identified microbiota-based signatures with potential diagnostic value for precancerous lesions, although discrimination remains limited. Biliary dysbiosis was also associated with outcomes related to biliary stenting, chemoprophylaxis, postoperative complications, and responses to chemotherapy or surgery. Conclusions: Integration of microbiota profiling with predictive bioinformatics and machine learning may improve understanding of pancreaticobiliary carcinogenesis. Identifying microbial and functional biomarkers could enable personalized diagnostic and therapeutic strategies, ultimately improving patient outcomes. Full article

Background: Acanthamoeba is a free-living protozoan widely distributed in the environment and causes Acanthamoeba keratitis, skin, and brain disease. Acanthamoeba can exchange genes, potentially increasing antimicrobial resistance and virulence. Therefore, this systematic review aimed to summarize published studies on horizontal gene transfer (HGT) between Acanthamoeba and its intracellular microorganisms and to evaluate the impact of HGTs on the pathogenicity of Acanthamoeba. Methods: This systematic review was conducted following the recommended reporting guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement guideline. The electronic databases PubMed, Embase, and Web of Science were used to search for relevant published research articles. Results: Nineteen studies that fulfilled the inclusion criteria were included in this systematic review. A total of 14 (73.6%) studies reported evidence of HGT involving Acanthamoeba, and five studies of the nineteen (26.3%) analysed the presence of intracellular microorganisms on the pathological effects of the host Acanthamoeba. Horizontally transferred genes were predominantly reported from Pseudomonas species, Legionella pneumophila, and Chlamydia species. Conclusions: HGT can occur among intracellular microorganisms and their host Acanthamoeba. Acanthamoeba harbouring intracellular microbes showed enhanced pathogenic effects on human corneal epithelial cells and in a mouse model. However, heterogeneity among the included studies precluded meta-analysis. Studies using clinical and environmental samples are needed to characterize the horizontal transfer of virulence and antimicrobial resistance genes. Full article

Travel-associated Legionnaires’ disease (TALD) investigations in hotels have generated extensive environmental monitoring data. However, the occupational implications for workers who operate, maintain, clean, or inspect the same systems are rarely assessed. We developed a hybrid framework integrating a semi-quantitative environmental hazard model with deterministic Quantitative Microbial Risk Assessment (QMRA). In the first model, culture concentration bands were combined with physicochemical deviation indicators (temperature, free residual chlorine, and pH) to derive point-level hazard ($H_i$) and zone-level hazard (${\overline{H}}_z$). In the second model, a job-based presence matrix was combined with zone-specific serogroup-based severity using a simplified World Health Organization (WHO)-style 3 × 3 likelihood–severity approach. Legionella pneumophila (≥50 CFU/L) was detected in 29.94% of water samples and was significantly associated with low chlorine (<0.2 mg/L; RR 2.90) and hot water temperature < 55 °C (RR 3.07). To support comparative occupational exposure stratification, QMRA was applied to estimate the daily inhaled dose ($\mathrm{d}$) for 15 worker groups, indicating variability in modeled biological exposure across occupational categories. Within this framework, modeled occupational exposure potential was shaped by the combined influence of pathogen concentration and assumed exposure duration. Under the hazard model, the highest zone-level hazard estimate was observed in kitchens and food and beverage (F&B) areas (${\overline{H}}_z$ = 2.607), followed by machinery rooms (${\overline{H}}_z$ = 2.022) and guest rooms (${\overline{H}}_z$ = 1.874). These findings support the integration of worker protection into water safety management, particularly in areas and groups overlooked in routine investigations. Full article