This study introduces an innovative electrochemical aptasensor designed for the highly sensitive and rapid detection of Legionella pneumophila serogroup 1 (L. pneumophila SG1), a particularly virulent strain associated with Legionellosis. Employing a rigorous selection process utilizing cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX), we identified new high-affinity aptamers specifically tailored for L. pneumophila SG1. The selection process encompassed ten rounds of cell-SELEX cycles with live L. pneumophila, including multiple counter-selection steps against the closely related Legionella sub-species. The dissociation constant (Kd) of the highest affinity sequence to L. pneumophila SG1 was measured at 14.2 nM, representing a ten-fold increase in affinity in comparison with the previously reported aptamers. For the development of electrochemical aptasensor, a gold electrode was modified with the selected aptamer through the formation of self-assembled monolayers (SAMs). The newly developed aptasensor exhibited exceptional sensitivity, and specificity in detecting and differentiating various Legionella sp., with a detection limit of 5 colony forming units (CFU)/mL and an insignificant/negligible cross-reactivity with closely related sub-species. Furthermore, the aptasensor effectively detected L. pneumophila SG1 in spiked water samples, demonstrating an appreciable recovery percentage. This study shows the potential of our aptamer-based electrochemical biosensor as a promising approach for detecting L. pneumophila SG1 in diverse environments.

BACKGROUND: Infection by Legionella bacteria is a risk to elderly individuals in health care facilities and should be managed by preventing bacterial proliferation in internal water systems. Norwegian legislation calls for a mandatory Legionella-specific risk assessment with the subsequent introduction of an adapted water management programme. The present study investigates adherence to legislation and guidelines on Legionella control and prevention in Norwegian nursing homes.
METHODS: A cross-sectional survey was distributed to Norwegian municipalities to investigate the status of Legionella specific risk assessments of internal water distribution systems and the introduction of water management programmes in nursing homes.
RESULTS: A total of 55.1% (n = 228) of the participating nursing homes had performed Legionella-specific risk assessments, of which 55.3% (n = 126) stated that they had updated the risk assessment within the last year. 96.5% introduced a water management programme following a risk assessment, whereas 59.6% of the ones without a risk assessment did the same. Nursing homes with risk assessments were more likely to monitor Legionella levels than those without (61.2% vs 38.8%), to remove dead legs (44.7% vs 16.5%), and to select biocidal preventive treatment over hot water flushing (35.5% vs 4.6%).
CONCLUSIONS: This study presents novel insight into Legionella control in Norway, suggesting that adherence to mandatory risk assessment in nursing homes is moderate-low. Once performed, the risk assessment seems to be advantageous as an introduction to future Legionella prevention in terms of the scope and contents of the water management programme.

BACKGROUND: Legionnaires\' disease (LD) is a common but under-diagnosed cause of community-acquired pneumonia (CAP), although rapid detection of urine antigen testing (UAT) and advances in molecular testing have improved the diagnosis. LD entails intensive care unit (ICU) admission in almost one-third of cases, and the mortality rate ranges from 4% to 40%. This review aims to discuss recent advances in the study of this condition and to provide an update on the diagnosis, pathogenesis and management of severe LD.
RESULTS: The overall incidence of LD has increased worldwide in recent years due to the higher number of patients with risk factors, especially immunosuppression, and to improvements in diagnostic methods. Although LD is responsible for only around 5% of all-cause CAP, it is one of the three most common causes of CAP requiring ICU admission. Mortality in ICU patients, immunocompromised patients or patients with a nosocomial source of LD can reach 40% despite appropriate antimicrobial therapy. Regarding pathogenesis, no Legionella-specific virulence factors have been associated with severity; however, recent reports have found high pulmonary Legionella DNA loads, and impairments in immune response and lung microbiome in the most severe cases. The clinical picture includes severe lung injury requiring respiratory and/or hemodynamic support, extrapulmonary symptoms and non-specific laboratory findings. LD diagnostic methods have improved due to the broad use of UAT and the development of molecular methods allowing the detection of all Lp serogroups. Therapy is currently based on macrolides, quinolones, or a combination of the two, with prolonged treatment in severe cases.
CONCLUSIONS: Numerous factors influence the mortality rate of LD, such as ICU admission, the underlying immune status, and the nosocomial source of the infection. The host immune response (hyperinflammation and/or immunoparalysis) may also be associated with increased severity. Given that the incidence of LD is rising, studies on specific biomarkers of severity may be of great interest. Further assessments comparing different regimens and/or evaluating host-directed therapies are nowadays needed.

BACKGROUND: In case of pneumonia, some biological findings are suggestive for Legionnaire\'s disease (LD) including C-reactive protein (CRP). A low level of CRP is predictive for negative Legionella Urinary-Antigen-Test (L-UAT).
METHODS: Observational retrospective study in Nord-Franche-Comté Hospital with external validation in Besançon University Hospital, France which included all adults with L-UAT performed during January 2018 to December 2022. The objective was to determine CRP optimal threshold to predict a L-UAT negative result.
RESULTS: URINELLA included 5051 patients (83 with positive L-UAT). CRP optimal threshold was 131.9 mg/L, with a negative predictive value (NPV) at 100%, sensitivity at 100% and specificity at 58.0%. The AUC of the ROC-Curve was at 88.7% (95% CI, 86.3-91.1). External validation in Besançon Hospital patients showed an AUC at 89.8% (95% CI, 85.5-94.1) and NPV, sensitivity and specificity was respectively 99.9%, 97.6% and 59.1% for a CRP threshold at 131.9 mg/L; after exclusion of immunosuppressed patients, index sensitivity and NPV reached also 100%.
CONCLUSIONS: In case of pneumonia suspicion with a CRP level under 130 mg/L (independently of the severity) L-UAT is useless in immunocompetent patients with a NPV at 100%. We must remain cautious in patients with symptoms onset less than 48 h before CRP dosage.

Legionnaires\' disease (LD) is a severe form of pneumonia (∼10-25% fatality rate) caused by inhalation of aerosols containing Legionella, a pathogenic gram-negative bacteria. These bacteria can grow, spread, and aerosolize through building water systems. A recent dramatic increase in LD incidence has been observed globally, with a 9-fold increase in the United States from 2000 to 2018, and with disproportionately higher burden for socioeconomically vulnerable subgroups. Despite the focus of decades of research since the infamous 1976 outbreak, substantial knowledge gaps remain with regard to source of exposure and the reason(s) for the dramatic increase in LD incidence. Here, we rule out factors indicated in literature to contribute to its long-term increases and identify a hitherto unexplored explanatory factor. We also provide an epidemiological demonstration that the occurrence of LD is linked with exposure to cooling towers (CTs). Our results suggest that declining sulfur dioxide air pollution, which has many well-established health benefits, results in reduced acidity of aerosols emitted from CTs, which may prolong the survival duration of Legionella in contaminated CT droplets and contribute to the increase in LD incidence. Mechanistically associating decreasing aerosol acidity with this respiratory disease has implications for better understanding its transmission, predicting future risks, and informed design of preventive and interventional strategies that consider the complex impacts of continued sulfur dioxide changes.

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

暂无摘要。

Legionella is an important waterborne pathogen that causes legionellosis. Public baths are considered the primary cause of legionellosis infection in Japan. We investigated the prevalence and genetic distribution of 338 Legionella spp. isolates from 81 public bath facilities, including 35 hot springs and 46 other facilities, through annual periodic surveillance in Kobe, Japan, from 2016 to 2021. In addition, the genotypes of nine clinical strains of unknown infectious source from the same period were compared to those of bathwater isolates. We elucidated the differences in the distribution of Legionella species, serogroups, and genotypes between hot springs and other public baths. Legionella israelensis, L. londiniensis, and L. micdadei colonized hot springs along with L. pneumophila. The minimum spanning tree analysis based on multiple-locus variable number tandem repeat analysis (MLVA) also identified four major clonal complexes (CCs) in L. pneumophila SG1 and found that CC1 of the four CCs is a specific novel genotype with the lag-1 gene in hot springs. The same MLVA genotypes and sequence types as those of the clinical strains were not present among the strains isolated from bath water. Thus, our surveillance is useful for estimating the sources of legionellosis infection in Japan and developing prevention strategies.

Bacterial persisters are a transient subpopulation of non-growing, antibiotic-tolerant cells. There is increasing evidence that bacterial persisters play an important role in treatment failure leading to recurring infections and promoting the development of antibiotic resistance. Current research reveals that recurring legionellosis is often the result of relapse rather than reinfection and suggests that the mechanism of bacterial persistence may play a role. The development of single-cell techniques such as the Timerbac system allows us to identify potential persister cells and investigate their physiology. Here, we tested the persister forming capacity of 7 pairs of Legionella pneumophila (Lp) clinical isolates, with isolate pairs corresponding to two episodes of legionellosis in the same patient. We distinguished non-growing subpopulations from their replicating counterparts during infection in an amoeba model. Imaging flow cytometry allowed us to identify single non-growing bacteria within amoeba cells 17 h post-infection, thus corresponding to this subpopulation of potential persister cells. Interestingly the magnitude of this subpopulation varies between the 7 pairs of Lp clinical isolates. Biphasic killing kinetics using ofloxacin stress confirmed the persister development capacity of ST1 clinical isolates, highlighting enhanced persister formation during the host cell infection. Thus, persister formation appears to be strain or ST (sequence type) dependent. Genome sequence analysis was carried out between ST1 clinical isolates and ST1 Paris. No genetic microevolution (SNP) linked to possible increase of persistence capacity was revealed among all the clones tested, even in clones issued from two persistence cycle experiments, confirming the transient reversible phenotypic status of persistence. Treatment failure in legionellosis is a serious issue as infections have a 5-10% mortality rate, and investigations into persistence in a clinical context and the mechanisms involved may allow us to combat this issue.

Legionellosis is a respiratory disease of bacterial and environmental origin that usually presents two distinct clinical entities, \"Legionnaires\' disease\" (LD) and \"Pontiac fever\". LD is an important cause of hospital-acquired pneumonia (HAP). The objective of this study is to describe the epidemiology of legionellosis-associated hospitalization (L-AH) in Spain from 2002 to 2021 and the burden of hospitalization due to legionellosis. Discharge reports from the Minimum Basic Data Set (MBDS) were used to retrospectively analyze hospital discharge data with a diagnosis of legionellosis, based on the ICD-9-CM and ICD-10-CM diagnosis codes, from 2002 to 2021. 21,300 L-AH occurred throughout the year during 2002-2021. The incidence of hospitalization associated per 100,000 inhabitants by month showed a similar trend for the 2002-2011, 2012-2021, and 2002-2021 periods. In Spain, during 2002-2021, the hospitalization rate (HR) in the autonomous communities ranged from 4.57 (2002-2011) to 0.24 (2012-2021) cases per 100,000 inhabitants. The HR of legionellosis in Spain has substantially increased across the 2002-2021 period, and the estimate is consistent with available European data. It is considered that in-depth epidemiological surveillance studies of legionellosis and improvements in the prevention and control of the disease are required in Spain.