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Legionella, an important cause of pneumonia, is acquired from environmental sources such as potable water. In this article, evidence of person-to-person spread of Legionnaires' disease is reported. To the Editor: Legionnaires’ disease is an often severe form of pneumonia that is typically acquired by susceptible persons (e.g., elderly persons and smokers) through inhalation of aerosols that contain legionella species. 1 – 4 A cluster of cases of this disease occurred in Vila Franca de Xira, Portugal, in 2014. 5 One of the first cases of disease in this cluster occurred in a 48-year-old man (Patient 1), a smoker, who had been employed since October 6, 2014, as a maintenance worker at an industrial cooling tower complex in Vila Franca de Xira that was subsequently found to be contaminated with Legionella . . .

Legionnaires' disease is an important cause of community-acquired and hospital-acquired pneumonia. Although uncommon, Legionnaires' disease continues to cause disease outbreaks of public health significance. The disease is caused by any species of the Gram-negative aerobic bacteria belonging to the genus Legionella; Legionella pneumophila serogroup 1 is the causative agent of most cases in Europe. In this Review we outline the global epidemiology of Legionnaires' disease, summarise its diagnosis and management, and identify research gaps and priorities. Early clinical diagnosis and prompt initiation of appropriate antibiotics for Legionella spp in all patients with community-acquired or hospital-acquired pneumonias is a crucial measure for management of the disease. Progress in typing and sequencing technologies might additionally contribute to understanding the distribution and natural history of Legionnaires' disease, and inform outbreak investigations. Control of Legionnaires' disease outbreaks relies on rapid ascertainment of descriptive epidemiological data, combined with microbiological information to identify the source and implement control measures. Further research is required to define the actual burden of disease, factors that influence susceptibility, key sources of infection, and differences in virulence between strains of Legionella species. Other requirements are improved, specific, sensitive, and rapid diagnostic tests to accurately inform management of Legionnaires' disease, and controlled clinical trials to ascertain the optimum antibiotics for treatment.

BackgroudAlthough not fully investigated, studies show that Legionella pneumophila can develop antibiotic resistance. As there is limited data available for Portugal, we determined the antibiotic susceptibility profile of Portuguese L. pneumophila serogroup 1 (LpnSg1) isolates against antibiotics used in the clinical practice in Portugal.MethodsMinimum inhibitory concentrations (MICs) were determined for LpnSg1 clinical (n = 100) and related environmental (n = 7) isolates, collected between 2006–2022 in the context of the National Legionnaire´s Disease Surveillance Programme, against azithromycin, clarithromycin, erythromycin, levofloxacin, ciprofloxacin, moxifloxacin, rifampicin, doxycycline, tigecycline, and amoxicillin/clavulanic acid, using three different assays. Isolates were also PCR-screened for the presence of the lpeAB gene.ResultsTwelve isolates had azithromycin MICs above the EUCAST tentative highest WT MIC, 9 of which were lpeAB negative; for erythromycin and clarithromycin, all isolates tested within the susceptible range. The number of isolates with MICs above the tentative highest WT MIC for the remaining antibiotics was: ciprofloxacin: 7; levofloxacin: 17; moxifloxacin: 8; rifampicin: 11; doxycycline: 82; tigecycline: 4. EUCAST breakpoints are not available for amoxicillin/clavulanic acid. We estimated the ECOFFs and one isolate had a MIC eightfold higher than the E-test ECOFF. Additionally, a clinical isolate generated three colonies growing on the E-test inhibition zone that resulted in MICs fourfold higher than for the parental isolate.ConclusionsWe report, for the first time, elevated MICs against first-line and other antibiotics (including azithromycin, fluoroquinolones and amoxicillin/clavulanic acid commonly used to treat pneumonia patients in Portugal) in Portuguese L. pneumophila strains. Results point towards decreased susceptibility in circulating strains, justifying further investigation.

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

The combination of copper–metal organic framework (Cu–MOF) with graphene oxide (GO) has received growing interest in electrocatalysis, energy storage and sensing applications. However, its potential as an electrochemical biosensing platform remains largely unexplored. In this study, we introduce the synthesis of GO/Cu–MOF nanocomposite and its application in the simultaneous detection of two biomarkers associated with lower respiratory infections, marking the first instance of its use in this capacity. The physicochemical properties and structural elucidation of this composite were studied with the support of XRD, FTIR, SEM and electrochemical techniques. The immunosensor was fabricated by drop casting the nanocomposite on dual screen-printed electrodes followed by functionalization with pyrene linker. The covalent immobilization of the monoclonal antibodies of the bacterial antigens of Mycoplasma pneumoniae ( M. pneumoniae ; M. p. ) and Legionella pneumophila ( L. pneumophila ; L. p. ) was achieved using EDC-NHS chemistry. The differential pulse voltammetry (DPV) signals of the developed immunosensor platform demonstrated a robust correlation across a broad concentration range from 1 pg/mL to 100 ng/mL. The immunosensor platform has shown high degree of selectivity against antigens for various respiratory pathogens. Moreover, the dual immunosensor was successfully applied for the detection of M. pneumoniae and L. pneumophila antigens in spiked water samples showing excellent recovery percentages. We attribute the high sensitivity of the immunosensor to the enhanced electrocatalytic characteristics, stability and conductivity of the GO–MOF composite as well as the synergistic interactions between the GO and MOF. This immunosensor offers a swift analytical response, simplicity in fabrication and instrumentation, rendering it an appealing platform for the on-field monitoring of pathogens in environmental samples.

The 2014–2015 Legionnaires’ disease (LD) outbreak in Genesee County, MI, and the outbreak resolution in 2016 coincided with changes in the source of drinking water to Flint’s municipal water system. Following the switch in water supply from Detroit to Flint River water, the odds of a Flint resident presenting with LD increased 6.3-fold (95% CI: 2.5, 14.0). This risk subsided following boil water advisories, likely due to residents avoiding water, and returned to historically normal levels with the switch back in water supply. During the crisis, as the concentration of free chlorine in water delivered to Flint residents decreased, their risk of acquiring LD increased. When the average weekly chlorine level in a census tract was <0.5 mg/L or <0.2 mg/L, the odds of an LD case presenting from a Flint neighborhood increased by a factor of 2.9 (95% CI: 1.4, 6.3) or 3.9 (95% CI: 1.8, 8.7), respectively. During the switch, the risk of a Flint neighborhood having a case of LD increased by 80% per 1 mg/L decrease in free chlorine, as calculated from the extensive variation in chlorine observed. In communities adjacent to Flint, the probability of LD occurring increased with the flow of commuters into Flint. Together, the results support the hypothesis that a system-wide proliferation of legionellae was responsible for the LD outbreak in Genesee County, MI.

Legionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires’ disease. However, the microorganism is typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 902  L. pneumophila isolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We find that the capacity for human disease is representative of the breadth of species diversity although some clones are more commonly associated with clinical infections. We identified a single gene ( lag-1 ) to be most strongly associated with clinical isolates. lag-1 , which encodes an O -acetyltransferase for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades of L. pneumophila by frequent recent recombination events. The gene confers resistance to complement-mediated killing in human serum by inhibiting deposition of classical pathway molecules on the bacterial surface. Furthermore, acquisition of lag-1 inhibits complement-dependent phagocytosis by human neutrophils, and promoted survival in a mouse model of pulmonary legionellosis. Thus, our results reveal L. pneumophila genetic traits linked to disease and provide a molecular basis for resistance to complement-mediated killing. The bacterium Legionella pneumophila can cause severe respiratory infection, but is typically a symbiont of free-living amoeba. Here, the authors analyse the genomes of 902 clinical and environmental isolates, and identify a bacterial gene that is strongly associated with human infection and confers resistance to complement-mediated killing.

Legionellosis is a resvpiratory disease of public health concern. Identification and quantification from environmental sources are crucial for identifying outbreak origins and providing information for risk assessment and disease prevention. Legionella pneumophila is typically detected and quantified using the culture method, which is considered the gold standard, but it has some critical limitations. The Legioler/Quanti-Tray test can be used as an alternative method to simplify the testing process and reduce the time required to obtain the result. In this study, we compare the new liquid culture method Legiolert™ and real-time PCR with traditional plate culture, assessing the performance of PCR and culture methods for detecting L. pneumophila in potable water samples. We analyzed 75 environmental water samples in parallel using the Standard method (ISO 11731:1998), Legiolert, and real-time PCR for the detection of L. pneumophila. The McNemar test was used to assess the difference in accuracy between the Legiolert and real-time PCR methods, showing that the culture test was more accurate than the molecular biology method. The study confirmed that the Legiolert test is specific, easy to use, and may serve as an alternative to standardized procedures for the quantification of L. pneumophila in water. However, due to its high sensitivity and rapid result acquisition, we believe it could be used as a screening tool to quickly ascertain the absence of the microorganism.

Chlorine and thermal treatments are the most commonly used procedures to control and prevent Legionella proliferation in drinking water systems of large buildings. However, cases of legionellosis still occur in facilities with treated water. The purpose of this work was to model the effect of temperature and free chlorine applied in similar exposure conditions as in drinking water systems on five Legionella spp. strains and two amoebal strains of the genera Acanthamoeba. Inactivation models obtained were used to determine the effectiveness of the treatments applied which resulted more effective against Legionella than Acanthamoeba, especially those in cystic stages. Furthermore, to determine the influence of the relationship between L. pneumophila and Acanthamoeba spp. on the treatment effectiveness, inactivation models of the bacteria-associated amoeba were also constructed and compared to the models obtained for the free living bacteria state. The Legionella-amoeba association did not change the inactivation models, but it reduced the effectiveness of the treatments applied. Remarkably, at the lowest free chlorine concentration, 0.5 mg L-1, as well as at the lowest temperatures, 50°C and 55°C, the influence of the Legionella-amoeba associate state was the strongest in reducing the effectiveness of the treatments compared to the free Legionella state. Therefore, the association established between L. pneumophila and amoebae in the water systems indicate an increased health risk in proximal areas of the system (close to the tap) where lower free chlorine concentrations and lower temperatures are commonly observed.

Given the recent global surge in Legionnaires’ disease cases, the monitoring of Legionella pneumophila becomes increasingly crucial. Epidemiological cases often stem from local outbreaks rather than widespread dissemination, emphasizing the need to study the characteristics of this pathogen at a local level. This study focuses on isolates of L . pneumophila in the Italian region of Friuli Venezia Giulia to assess specific genotype and phenotype distribution over time and space. To this end, a total of 127 L . pneumophila strains isolated between 2005 and 2017 within national surveillance programs were analysed. Rep-PCR, RAPD, and Sau-PCR were used for genotypic characterization, while phenotypic characterization was conducted through fatty acids analysis. RAPD and Sau-PCR effectively assessed genetic characteristics, identifying different profiles for the isolates and excluding the presence of clones. Although Sau-PCR is rarely used to analyse this pathogen, it emerged as the most discriminatory technique. Phenotypically, hierarchical cluster analysis categorized strains into three groups based on varying membrane fatty acid percentages. However, both phenotypic and genotypic analyses revealed a ubiquitous profile distribution at a regional level. These results suggest an absence of correlations between strain profiles, geographical location, and isolation time, indicating instead high variability and strain dissemination within this region.