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Host actin cytoskeleton is often targeted by pathogenic bacteria through the secretion of effectors.Legionella pneumophilavirulence relies on the injection of the largest known arsenal of bacterial proteins, over 300 Dot/Icm Type 4 Secretion System effectors, into the host cytosol. Here we define the functional interactions between VipA and LegK2, two effectors with antagonistic activities towards actin polymerization that have been proposed to interfere with the endosomal pathway. We confirmed the prominent role of LegK2 effector inLegionellainfection, as the deletion oflegK2results in defects in the inhibition of actin polymerization at theLegionellaContaining Vacuole, as well as in endosomal escape of bacteria and subsequent intracellular replication. More importantly, we observed the restoration of theΔlegK2mutant defects, upon deletion ofvipAgene, making LegK2/VipA the first example of effector-effector suppression pair that targets the actin cytoskeleton and whose functional interaction impactsL. pneumophilavirulence. We demonstrated that LegK2 and VipA do not modulate each other’s activity in a ‘metaeffector’ relationship. Instead, the antagonistic activities of the LegK2/VipA effector pair would target different substrates, Arp2/3 for LegK2 and G-actin for VipA, to temporally control actin polymerization at the LCV and interfere with phagosome maturation and endosome recycling, thus contributing to the intracellular life cycle of the bacterium. Strikingly, the functional interaction between LegK2 and VipA is consolidated by an evolutionary history that has refined the best effector repertoire for the benefit ofL. pneumophilavirulence.

Detection of Legionella pneumophila serogroup 1 urinary antigens is the most widely used technique for the diagnosis of Legionnaires' disease (LD). The aim of this study was to evaluate the performance of the Legionella K-set® immunochromatographic test, in comparison with the BinaxNOW® Legionella urinary antigen card (UAC) on concentrated urine samples (US). A total of 250 concentrated US including 200 prospective US sent to the laboratory for urinary antigens' testing and 50 frozen US from patients with confirmed LD were tested. Positive US were retested after boiling (5 min, 100 °C). Each US leading to discordant results between the two tests was further tested using Binax™ Legionella EIA. Then, 10 additional positive non-concentrated US were tested using both tests. On concentrated US, Legionella K-set® test showed concordant results with that of BinaxNOW® Legionella. All negative US with BinaxNOW® were negative with Legionella K-set® test. For the 50 frozen US, all were positive with BinaxNOW® and 49 were positive with Legionella K-set®, all confirmed after boiling except 3 US which led to uninterpretable results with Legionella K-set®, due to a migration defect. Three of the 10 additional positive non-concentrated US were found negative with Legionella K-set® and only 1 US remained negative after concentration. All these positive non-concentrated US were positive with BinaxNOW® Legionella. The performance of the Legionella K-set® test is comparable to that of BinaxNOW® Legionella UAC, if performed on concentrated US.

The zebrafish has become a powerful model organism to study host-pathogen interactions. Here, we developed a zebrafish model ofLegionella pneumophilainfection to dissect innate immune responses. We show thatL. pneumophilacause zebrafish larvae death in a dose dependent manner, and that macrophages are the first line of defence, with neutrophils cooperating to clear the infection. When either macrophages or neutrophils are depleted, these “immunocompromised” larvae become lethally sensitive toL. pneumophilasimilar to what is known for humans that develop pneumonia. Also as observed in human infections, the adaptor signalling molecule Myd88 is not required to control disease in the larvae. Furthermore, proinflammatory cytokine genesil1ßandtnfαwere upregulated during infection, recapitulating key immune responses seen in human infection. Strikingly, we uncovered a previously undescribed infection phenotype in zebrafish larvae, whereby bloodborne, wild typeL. pneumophilainvade and grow in the larval yolk region, a phenotype not observed with a type IV secretion system deficient mutant that cannot translocate effectors into its host cell. Thus, zebrafish larva represents an innovativeL. pneumophilainfection model that that on one hand mimics important aspects of the human immune response toL. pneumophilainfection and that on the other hand will allow to elucidate the mechanisms by which type IV secretion effectors allowL. pneumophilato cross membranes and to obtain nutrients from nutrient rich environments.

Legionella pneumophila, the causative agent of Legionnaires’ disease, is mostly found in man-made water systems and is one of the most closely monitored waterborne pathogens. With the aim of finding natural ways to control waterborne pathogens and thus further reduce the impact of disinfection by-products on human health, some studies have demonstrated the ability of bacteria to kill Legionella through the production of secondary metabolites or antimicrobial compounds. Here, we describe an unexpected growth inhibition of L. pneumophila when exposed to a physically separated strain of Pseudomonas fluorescens, designated as MFE01. Most of the members of the Legionellaceae family are sensitive to the volatile substances emitted by MFE01, unlike other bacteria tested. Using headspace solid-phase microextraction GC-MS strategy, a volatilome comparison revealed that emission of 1-undecene, 2-undecanone, and 2-tridecanone were mainly reduced in a Tn5-transposon mutant unable to inhibit at distance the growth of L. pneumophila strain Lens. We showed that 1-undecene was mainly responsible for the inhibition at distance in vitro, and led to cell lysis in small amounts, as determined by gas chromatography-mass spectrometry (GC-MS). Collectively, our results provide new insights into the mode of action of bacterial volatiles and highlight them as potent anti-Legionella agents to focus research on novel strategies to fight legionellosis. IMPORTANCE Microbial volatile compounds are molecules whose activities are increasingly attracting the attention of researchers. Indeed, they can act as key compounds in long-distance intrakingdom and interkingdom communication, but also as antimicrobials in competition and predation. In fact, most studies to date have focused on their antifungal activities and only a few have reported on their antibacterial properties. Here, we describe that 1-undecene, naturally produced by P. fluorescens, is a volatile with potent activity against bacteria of the genus Legionella. In small amounts, it is capable of inducing cell lysis even when the producing strain is physically separated from the target. This is the first time that such activity is described. This molecule could therefore constitute an efficient compound to counter bacterial pathogens whose treatment may fail, particularly in pulmonary diseases. Indeed, inhalation of these volatiles should be considered as a possible route of therapy in addition to antibiotic treatment.

Resume Les infections a Legionella sont causees par une bacterie intracellulaire habitant principalement les environnements aquatiques. La contamination se fait par inhalation d’aerosols. Les facteurs de risques principaux sont le tabac, l’immunodepression et l’âge avance. Les signes extra-respiratoires sont peu specifiques. L’argument clinique le plus solide en faveur d’une legionellose est la resistance aux beta lactamines en cas de pneumopathie. Le diagnostic repose avant tout sur l’antigenurie en cas d’infection a Legionella pneumophila de type 1. L’antigenurie est prise en defaut chez les patients infectes a Legionella non pneumophilia comme les patients immunodeprimes. La culture et la PCR sont alors indispensables au diagnostic. La maladie des legionnaires, forme pulmonaire de l’infection a Legionella, est grave, en particulier chez les sujets âges et/ou les patients immunodeprimes. Le taux de mortalite varie de 10 % dans la population generale, a 50 % en cas d’hospitalisation en reanimation. Le traitement repose sur les macrolides ou les fluoroquinolones. La resistance aux antibiotiques est exceptionnelle.

Significance Natural transformation is a major mechanism of horizontal gene transfer. Although the genes required for natural transformation are nearly ubiquitous in bacteria, it is commonly reported that some isolates of transformable species fail to transform. In Legionella pneumophila, we show that the inability of multiple clinical isolates to transform is caused by a conjugative element that shuts down expression of genes required for transformation. Diverse conjugative elements in the Legionella genus have adopted the same inhibition strategy. We propose that inhibition of natural transformation by episomal and integrated conjugative elements can explain the lack of transformability of isolates and also the apparent lack of natural transformation in some species., Natural transformation (i.e., the uptake of DNA and its stable integration in the chromosome) is a major mechanism of horizontal gene transfer in bacteria. Although the vast majority of bacterial genomes carry the genes involved in natural transformation, close relatives of naturally transformable species often appear not competent for natural transformation. In addition, unexplained extensive variations in the natural transformation phenotype have been reported in several species. Here, we addressed this phenomenon by conducting a genome-wide association study (GWAS) on a panel of isolates of the opportunistic pathogen Legionella pneumophila. GWAS revealed that the absence of the transformation phenotype is associated with the conjugative plasmid pLPL. The plasmid inhibits transformation by simultaneously silencing the genes required for DNA uptake and recombination. We identified a small RNA (sRNA), RocRp, as the sole plasmid-encoded factor responsible for the silencing of natural transformation. RocRp is homologous to the highly conserved and chromosome-encoded sRNA RocR which controls the transient expression of the DNA uptake system. Assisted by the ProQ/FinO-domain RNA chaperone RocC, RocRp acts as a substitute of RocR, ensuring that the bacterial host of the conjugative plasmid does not become naturally transformable. Distinct homologs of this plasmid-encoded sRNA are found in diverse conjugative elements in other Legionella species. Their low to high prevalence may result in the lack of transformability of some isolates up to the apparent absence of natural transformation in the species. Generally, our work suggests that conjugative elements obscure the widespread occurrence of natural transformability in bacteria.

Background Rarely, Legionnaires’ disease (LD) can progress into a slowly or nonresolving form. Methods A nationwide retrospective study was conducted by the French National Reference Center for Legionella (2013–2017) including cases of slowly or nonresolving LD defined as persistent clinical symptoms, computed tomography (CT) scan abnormalities, and Legionella detection in lower respiratory tract specimens by culture and/or real-time (RT) polymerase chain reaction (PCR) >30 days after symptom onset. Results Twelve cases of community-acquired slowly or nonresolving LD were identified among 1686 cases of culture-positive LD. Median (interquartile range [IQR]) age was 63 (29–82) years. Ten (83.3%) patients had ≥1 immunosuppressive factor. Clinically, 9 patients transiently recovered before further deterioration (median [IQR] symptom-free interval, 30 [18–55] days), 3 patients had uniformly persistent symptoms (median [IQR] time, 48 [41.5–54] days). Two patients had >2 recurrences. CT scan imagery found lung abscess in 5 (41.6%) cases. Slowly or nonresolving LD was diagnosed on positive Legionella cultures (n = 10, 83.3%) at 49.5 (IQR, 33.7–79) days. Two cases were documented through positive Legionella RT PCR at 52 and 53 days (cycle threshold detection of 21.5 and 33.7, respectively). No genomic microevolution and no Legionella resistance to antibiotics were detected. The median (IQR) duration of treatment was 46.5 (21–92.5) days. Two empyema cases required thoracic surgery. At a median (IQR) follow-up of 26 (14–41.5) months, LD-attributable mortality was 16.6% (n = 2). Conclusions Slowly or nonresolving LD may occur in immunocompromised patients, possibly leading to lung abscess and empyema.

Introduction La Legionellose est une infection grave, et il existe peu de donnees concernant la legionellose des transplantes d’organes solides (TOS). Materiels et methodes Cette etude retrospective multicentrique rassemble tous les cas d’infections a Legionella, diagnostiques entre 2009 et 2019 chez des TOS dans 8 centres de transplantation francais. Les cas de legionellose ont ete identifies a l’aide du codage PMSI. Le diagnostic de legionellose etait retenu en cas de prelevement respiratoire positif, et/ou d’antigenurie positive et/ou de serologie positive. Resultats Vingt-huit patients ont ete identifies : 17 (61 %) etaient transplantes renaux (dont deux rein–pancreas), 7 (25 %) hepatiques, 4 cardiaques (14 %). L’âge median au diagnostic etait de 61,5 ans [27–81]. L’infection survenait en mediane 5 ans apres la transplantation [14 jours–42 ans] et 25 % etaient associees aux soins (7/28). Dix-neuf patients (68 %) etaient febriles, 22 (79 %) presentaient des symptomes pulmonaires, 13 (46 %) des symptomes digestifs, et 5 (18 %) des symptomes neurologiques. Une imagerie thoracique etait disponible pour 27 patients et montrait un syndrome alveolaire chez 25 (93 %) patients, un syndrome interstitiel chez 19 (70 %) patients. Des complications a types de pleuresie, nodules pulmonaires et excavations etaient presentes chez respectivement 9 (33 %), 7(26 %), et 3 (11 %) des patients. La duree mediane entre le debut des symptomes et le diagnostic etait de 6,5 jours [0–30]. L’antigenurie initiale etait negative chez 14 % des patients (4/28) dont 3 cas positifs sur un controle ulterieur avec une mediane de 7 jours [3–16] entre les deux tests. Les LBA etaient positifs en culture dans 60 % des cas (6/10). Legionella pneumophila serogroupe 1 etait responsable de 96 % des cas (27/28). Au diagnostic, 11 % (3/28) des patients recevaient deja une antibiotherapie probabiliste active sur L. pneumophila. Finalement, 8 (29 %) patients etaient traites par spiramycine seule (8/28), 11 (39 %) par quinolone seule, et 9 (32 %) par quinolone et spiramycine. La duree mediane d’antibiotherapie etait de 19 jours [3–114]. Quinze (54 %) patients ont ete hospitalises en soins intensifs, dont 10 (36 %) ont eu recours a la ventilation mecanique, et 8 (29 %) a des catecholamines. A j30 du diagnostic Le taux de mortalite etait de 11 % (3/28). Conclusion La legionellose en TOS est rare (28 cas en 10 ans dans 8 centres), atypique (26 % de formes nodulaires, 14 % d’antigenuries negatives au diagnostic), et severe (54 % hospitalises en SI, 11 % de mortalite a j30).

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BackgroundLung microbiome analyses have shown that the healthy lung is not sterile but it is colonized like other body sites by bacteria, fungi and viruses. However, little is known about the microbial composition of the lung microbiome during infectious diseases such as pneumonia and how it evolves during antibiotic therapy. To better understand the impact of the composition of the pulmonary microbiome on severity and outcome of pneumonia we analysed the composition and evolution of the human lung microbiome during pneumonia caused by the bacteriumLegionella pneumophila.ResultsWe collected 10 bronchoalveolar lavage (BAL) samples from three patients during long-term hospitalisation due to severe pneumonia and performed a longitudinal in-depth study of the composition of their lung microbiome by high-throughput Illumina sequencing of the 16S rRNA gene (bacteria and archaea), ITS region (fungi) and 18S rRNA gene (eukaryotes). We found that the composition of the bacterial lung microbiome during pneumonia is hugely disturbed containing a very high percentage of the pathogen, a very low bacterial diversity, and an increased presence of opportunistic microorganisms such as species belonging to Staphylococcaceae and Streptococcaceae. The microbiome of antibiotic treated patients cured from pneumonia represented a different perturbation state with a higher abundance of resistant bacteria (mainly Firmicutes) and a significantly different bacterial composition as that found in healthy individuals. In contrast, the mycobiome remains more stable during pneumonia and antimicrobial therapy. Interestingly we identified possible cooperation within and between both communities. Furthermore, archaea (Methanobrevibacter) and protozoa (AcanthamoebaandTrichomonas) were detected.ConclusionsBacterial pneumonia leads to a collapse of the healthy microbiome and a strongly disturbed bacterial composition of the pulmonary microbiome that is dominated by the pathogen. Antibiotic treatment allows some bacteria to regrow or recolonize the lungs but the restoration of a healthy lung microbiome composition is only regained a certain time after the antibiotic treatment. Archaea and protozoa should also be considered, as they might be important but yet overseen members of the lung microbiome. Interactions between the micro- and the mycobiome might play a role in the restoration of the microbiome and the clinical evolution of the disease.

Objective Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 1 is globally widespread in the environment and accounts for a significant proportion of Legionella infections, including nosocomial Legionnaires' disease (LD). This study aimed to design a sensitive and specific detection method for Lp ST1 that will underpin epidemiological investigations and risk assessment. Methods A total of 628 Lp genomes (126 ST1s) were analyzed by comparative genomics. Interrogation of more than 900 accessory genes revealed seven candidate targets for specific ST1 detection and specific primers and hydrolysis probes were designed and evaluated. The analytical sensitivity and specificity of the seven primer and probe sets were evaluated on serially diluted DNA extracted from the reference strain CIP107629 and via qPCR applied on 200 characterized isolates. The diagnostic performance of the assay was evaluated on 142 culture-proven clinical samples from LD cases and a real-life investigation of a case cluster. Results Of seven qPCR assays that underwent analytical validation, one PCR target (lpp1868) showed higher sensitivity and specificity for ST1 and ST1-like strains. The diagnostic performance of the assay using respiratory samples corresponded to a sensitivity of 95% (19/20) (95% CI (75.1–99.9)) and specificity of 100% (122/122) (95% CI (97–100)). The ST1 PCR assay could link two out of three culture-negative hospitalized LD cases to ST1 during a real-time investigation. Conclusion Using whole genome sequencing (WGS) data, we developed and validated a sensitive and specific qPCR assay for the detection of Lp1 belonging to the ST1 clonal complex by amplification of the lpp1868 gene. The ST1 qPCR is expected to deliver an added value for Lp control and prevention, in conjunction with other recently developed molecular assays.

Legionella-related disease is caused by an intracellular bacteria mainly living in water. Contamination results from inhalation of Legionella sp containing aerosolized water. Main risk factors are tobacco, immunodeficiency, and advanced age. Antigenuria is the cornerstone of the diagnosis. Immunocompromised patients, more commonly infected with non pneumophilaLegionella, present negative antigenuria, and culture and PCR are essential for the diagnosis. Legionnaires' disease may be severe, especially in elderly and/or immunocompromised patients. Mortality rate varies from 10 % in the general population to 50 % in intensive care. Treatment is based on macrolides or fluoroquinolones. Antibiotic resistance is very rare.

Summary Whole-genome sequencing can be used to support or refute suspected links between hospital water systems and Legionnaires’ disease cases. However, caveats regarding the interpretation of genomic data from Legionella pneumophila are described that should be considered in future investigations., Background. Legionnaires’ disease is an important cause of hospital-acquired pneumonia and is caused by infection with the bacterium Legionella. Because current typing methods often fail to resolve the infection source in possible nosocomial cases, we aimed to determine whether whole-genome sequencing (WGS) could be used to support or refute suspected links between cases and hospitals. We focused on cases involving a major nosocomial-associated strain, L. pneumophila sequence type (ST) 1. Methods. WGS data from 229 L. pneumophila ST1 isolates were analyzed, including 99 isolates from the water systems of 17 hospitals and 42 clinical isolates from patients with confirmed or suspected hospital-acquired infections, as well as isolates obtained from or associated with community-acquired sources of Legionnaires’ disease. Results. Phylogenetic analysis demonstrated that all hospitals from which multiple isolates were obtained have been colonized by 1 or more distinct ST1 populations. However, deep sampling of 1 hospital also revealed the existence of substantial diversity and ward-specific microevolution within the population. Across all hospitals, suspected links with cases were supported with WGS, although the degree of support was dependent on the depth of environmental sampling and available contextual information. Finally, phylogeographic analysis revealed that hospitals have been seeded with L. pneumophila via both local and international spread of ST1. Conclusions. WGS can be used to support or refute suspected links between hospitals and Legionnaires’ disease cases. However, deep hospital sampling is frequently required due to the potential coexistence of multiple populations, existence of substantial diversity, and similarity of hospital isolates to local populations.

The genus Legionella comprises 65 species, among which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes and Legionella . Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens.

International audience; Transplant recipients are at risk of developing Legionnaires' disease (LD) because of impaired cellular immunity. Here, we describe a renal transplant recipient who developed LD at least 10 days after hospital admission and transplantation. The hospital water network was initially suspected, but further testing determined that the probable source was the patient's domestic water supply. Our report also suggests that the patient's immunosuppressed state may have switched potential colonization to pneumonia.

International audience; Legionnaires' disease is a severe pneumonia mainly caused by Legionella pneumophila that is treated by antibiotics. The purpose of this study was to describe the susceptibility of clinical strains of L. pneumophila to eight antibiotics used for treatment of legionellosis. The minimum inhibitory concentrations (MICs) of 109 well-characterised clinical strains of L. pneumophila serogroup 1 were determined by the broth microdilution method without charcoal and were compared with antibiotic-resistant strains selected in vitro. All strains were inhibited by low concentrations of fluoroquinolones, macrolides and rifampicin. The epidemiological cut-off values (ECOFFs) were 0.064 mg/L for ciprofloxacin, 0.064 mg/L for moxifloxacin, 0.032 mg/L for levofloxacin, 1 mg/L for erythromycin, 2 mg/L for azithromycin, 0.064 mg/L for clarithromycin, 2 mg/L for doxycycline and 0.001 mg/L for rifampicin. However, MIC distributions revealed a subpopulation of strains displaying reduced susceptibility to some macrolides (especially azithromycin), which correlated with the presence of the lpeAB genes encoding a macrolide efflux pump found specifically in sequence type (ST) ST1, ST701 and closely related STs. Thus, all isolates could be considered susceptible to the tested antibiotics, although macrolides were less active against some strains harbouring a specific efflux system.

International audience; OBJECTIVES: Legionella pneumophila serogroup 1 (Lp1) sequence type 47 is the leading cause of legionellosis in north-western Europe, but, surprisingly, it is rarely isolated from environmental samples. Comparative genomics was applied to develop a PCR assay and to better understand the evolution of this strain. METHODS: Comparative analysis of 36 genomes representative of the Lp species was used to identify specific PCR targets, which were then evaluated in silico on 545 sequenced genomes and in~vitro on 436 Legionella strains, 106 respiratory samples, and three environmental samples from proven ST47 sources. Phylogenetic analyses were performed to understand the evolution of ST47. RESULTS: The gene LPO\₁073 was characterized as being 100% conserved in all 129 ST47 genomes analysed. A real-time PCR designed to detect LPO\₁073 was positive for all 110 ST47 strains tested and agreed with culture and typing results previously obtained for 106 respiratory samples. The three environmental samples were also positive. Surprisingly, 26 of the 44 ST109 strains tested among 342 non-ST47 strains scored positive for LPO\₁073. SNP-based phylogenetic analysis was undertaken to understand this result: the PCR-positive ST109 genomes were almost identical to ST47 genomes, with the exception of a recombined region probably acquired by ST47 from a ST62(-like) strain. CONCLUSION: The genomic analysis allowed the design of a highly specific PCR assay for rapid detection of ST47 strains. Furthermore, it allowed us to uncover the evolution of ST47 strains from ST109 by homologous recombination with ST62. We hypothesize that this recombination generated the leading cause of legionellosis in north-western Europe.

The contamination of aerosols by washbasin water colonized by Legionella in a hospital was evaluated. Aerosol samples were collected by two impingement technologies. Legionella was never detected by culture in all the (aerosol) samples. However, 45% (18/40) of aerosol samples were positive for Legionella spp. by polymerase chain reaction, with measurable concentrations in 10% of samples (4/40). Moreover, immunoassay detected Legionella pneumophila serogroup 1 and L. anisa, and potentially viable bacteria were seen on viability testing. These data suggest that colonized hospital washbasins could represent risks of exposure to Legionella aerosol inhalation, especially by immunocompromised patients.

The development of a method for the quantification of Legionella pneumophila genomic deoxyribonucleic acid is considered. The method is based on the quantification by inductively coupled plasma mass spectrometry (ICP-MS) of the mass fraction of phosphorus, stoichiometrically presented in the DNA molecules. Through the DNA sequencing data, it was possible to convert the ICP-MS analysis results into DNA genome units. L. pneumophila DNA samples were analyzed using ICP-sector field MS and ICP-quadrupole MS with a collision/reaction cell. Spectrophotometric measurements of the absorbance at 260nm and real-time PCR techniques were used to independently confirm the ICP-MS results. The comparison of the methods showed that the ICP-MS method provides better accuracy with respect to currently applied analytical techniques such as UV spectrophotometry, fluorescent dye methods, and real-time PCR. Moreover, with the use of calibration standards whose values are traceable to the International System of Units and the possibility of evaluating the contribution to the overall uncertainty of each step of the measurement procedure, the method enables long-term comparability of the measurement results. These advantages make the ICP-MS method suitable for nucleic acid investigation, from nucleotides to genomic DNA, as well as for the certification of the reference materials containing nucleic acids.

International audience; Objectives: A previous study on 12 in vitro -selected azithromycin-resistant Legionella~pneumophila lineages showed that ribosomal mutations were major macrolide resistance determinants. In addition to these mechanisms that have been well described in many species, mutations upstream of lpeAB operon, homologous to acrAB in Escherichia~coli , were identified in two lineages. In this study, we investigated the role of LpeAB and of these mutations in macrolide resistance of L.~pneumophila . Methods: The role of LpeAB was studied by testing the antibiotic susceptibility of WT, deleted and complemented L.~pneumophila Paris strains. Translational fusion experiments using GFP as a reporter were conducted to investigate the consequences of the mutations observed in the upstream sequence of lpeAB operon. Results: We demonstrated the involvement of LpeAB in an efflux pump responsible for a macrolide-specific reduced susceptibility of L.~pneumophila Paris strain. Mutations in the upstream sequence of lpeAB operon were associated with an increased protein expression. Increased expression was also observed under sub-inhibitory macrolide concentrations in strains with both mutated and WT promoting regions. Conclusions: LpeAB are components of an efflux pump, which is a macrolide resistance determinant in L.~pneumophila Paris strain. Mutations observed in the upstream sequence of lpeAB operon in resistant lineages led to an overexpression of this efflux pump. Sub-inhibitory concentrations of macrolides themselves participated in upregulating this efflux and could constitute a first step in the acquisition of a high macrolide resistance level.

International audience; Legionella are opportunistic pathogens that develop in aquatic environments where they multiply in protozoa. When infected aerosols reach the human respiratory tract they may accidentally infect the alveolar macrophages leading to a severe pneumonia called Legionnaires' disease (LD). The ability of Legionella to survive within host-cells is strictly dependent on the Dot/Icm Type 4 Secretion System that translocates a large repertoire of effectors into the host cell cytosol. Although Legionella is a large genus comprising nearly 60 species that are worldwide distributed, only about half of them have been involved in LD cases. Strikingly, the species Legionella pneumophila alone is responsible for 90% of all LD cases. The present review summarizes the molecular approaches that are used for L. pneumophila genotyping with a major focus on the contribution of whole genome sequencing (WGS) to the investigation of local L. pneumophila outbreaks and global epidemiology studies. We report the newest knowledge regarding the phylogeny and the evolution of Legionella and then focus on virulence evolution of those Legionella species that are known to have the capacity to infect humans. Finally, we discuss the evolutionary forces and adaptation mechanisms acting on the Dot/Icm system itself as well as the role of mobile genetic elements (MGE) encoding T4ASSs and of gene duplications in the evolution of Legionella and its adaptation to different hosts and lifestyles.

Culture media performance is a critical factor in the isolation of Legionellae from respiratory samples. We showed that BMPA and MWY media yielded significantly higher isolation rates than GVPC and BCYE media in regard to performance with samples that harbored low Legionella inocula and high contamination levels.

Resume Les legionelloses se presentent essentiellement comme des infections pulmonaires aigues causees par Legionella pneumophila et plus rarement par d’autres especes. Elles peuvent etre nosocomiales ou communautaires souvent liees a un sejour hotelier. Generalement l’inhalation d’aerosols generes par des sources d’eau contaminees : douches, systeme de distribution, tours aerorefrigerantes…sont a l’origine de ces infections. Notre etude porte sur le typage moleculaire de 35 souches de L. pneumophila dont quatre souches isolees de prelevements cliniques et 31 souches isolees a partir du reseau hydrique de 14 etablissements hoteliers dans le but de comparer les profils genetiques. Le serogroupe 1 est retrouve pour tous les isolats etudies. Une electrophorese en champ pulse est effectuee sur ces souches. Parmi les quatre souches cliniques, deux presentent le meme profil, cependant elles sont toutes differentes des souches environnementales etudiees. Pour les 31 souches environnementales, dix profils sont obtenus, parmi lesquels un meme pulsotype est retrouve chez quatre souches provenant de quatre etablissements differents. Par ailleurs, deux profils differents sont retrouves pour des souches isolees dans un meme etablissement. L’electrophorese en champ pulse a demontre l’existence de differents profils moleculaires. Bien que des cas de legionellose aient ete declares dans ces hotels, il n’existe pas de liens epidemiologiques entre les souches cliniques et environnementales pouvant determiner l’origine de la contamination humaine concernant ces cas.

Resume La legionellose est une pathologie dont la description est recente pour laquelle la surveillance a ete renforcee ces dernieres annees. En 2005, 1527 cas ont ete notifies correspondant a un taux d'incidence de 2,5 pour 100 000 habitants. L'âge median etait de 61 ans [5–100] et le sex-ratio homme/femme de 3,0. La letalite etait de 11 %. Un ou plusieurs facteurs favorisants ont ete retrouves chez 1084 cas (71 %). La majorite des cas (91 %) avaient ete diagnostiques par un test de detection de l'antigene urinaire et une souche avait ete isolee pour 276 cas (18 %). L'espece Legionella pneumophila serogroupe 1 representait 95 % des cas. Une exposition a risque lors de la periode d'incubation a ete reportee pour 39 % des cas. Une notion de voyage etait notee pour 17 % des cas et 7 % avaient effectue un sejour en partie ou en totalite dans un hopital ou une clinique. De nombreuses investigations de cas groupes ont ete effectuees. La plus importante est survenue debut mai dans le nord de l'agglomeration lyonnaise avec 34 cas. Depuis 1997, l'incidence des cas declares de legionellose est en constante augmentation. Cela reflete vraisemblablement une meilleure sensibilite et une amelioration globale du systeme de surveillance. Depuis ces dernieres annees, de nombreuses actions visant a ameliorer le controle et la prevention de cette maladie ont ete mises en place. Ces efforts doivent desormais se poursuivre dans le domaine de la recherche pour ameliorer les connaissances sur la maladie afin d'en limiter a terme, l'impact sur les populations exposees.

The aim of this review was to describe the current knowledge of Legionnaires' disease (LD) illustrated by the epidemiological situation in France in 2013. LD is a severe pneumonia commonly caused by Legionella pneumophila serogroup 1. The diagnosis is usually based on the urinary antigen test. This rapid method reduces the delay between clinical suspicion and initiation of an appropriate treatment. However, the availability of a clinical strain is important to improve knowledge of circulating bacteria, to document case clusters, and to identify the sources of contamination. The source of contamination is unknown in most cases. The main contamination sources generating aerosols are water network systems and cooling towers. Thanks to the strengthening of clinical and environmental monitoring and to several guidelines, no epidemic has been reported in France since 2006. Despite these efforts, the number of LD cases has not decreased in recent years. It is essential that applied research continue to better understand the spatial and temporal dynamics of the disease and its characteristics (impact of environmental factors, sources of exposure, strains, host, etc.). Fundamental knowledge has been greatly improved (pathogenesis, immune mechanisms, etc.). The results of this research should help define new strategies for the diagnosis, prevention, and control to decrease the number of LD cases diagnosed every year.

Resume Chez Staphylococcus aureus , la synthese de la majorite des exoproteines, incluant les facteurs de virulence et les enzymes necessaire a l'acquisition de nutriments, est sous le controle du systeme agr (accessory gene regulator) qui met en jeu un mecanisme de ≪ quorum sensing ≫. Ce systeme code un peptide auto-inducteur (PAI) dont l'accumulation dans l'environnement extracellulaire conduit a l'activation d'un systeme de transduction du signal a deux composants, AgrC (senseur) et AgrA (regulateur de reponse). Trois alleles du systeme agr ont ete mis en evidence chez S. aureus . Dans chaque allele, le peptide auto-inducteur induit l'activation specifique d'AgrC du meme allele et inhibe les recepteurs AgrC des autres alleles. Les sequences d'alleles agr ont egalement ete decrites chez d'autres especes que S. aureus . La signification biologique et clinique de ce polymorphisme n'est pas encore connue. II existerait une relation entre les alleles agr et les capacites a induire specifiquement certains syndromes infectieux. Par ailleurs, la variabilite du systeme agr pourrait etre associee a des interferences de colonisation et d'infection entre des souches appartenant a differents alleles. Enfin, une modelisation des interactions entre le peptide auto-inducteur et AgrC contribuerait a la comprehension des relations structure-activite au sein de ce couple, et pourrait permettre la synthese de peptides inhibiteurs du systeme agr . Ces peptides synthetiques presenteraient l'avantage d'attenuer la virulence de souches de staphylocoques pathogenes, en ne perturbant pas ou peu l'equilibre ecologique.

We report a case of severe Legionnaires' disease (LD) complicated by a lung abscess in an immunocompetent patient who required ECMO therapy and thoracic surgery. The results of repeatedLegionellaquantitative real-time PCR performed on both sera and respiratory samples correlated with the LD severity and the poor clinical outcome. Moreover, the PCR allowed for the detection ofLegionellaDNA in the lung abscess specimen, which was negative when cultured forLegionella. This case report provides a logical basis for further investigations to examine whether theLegionellaquantitative PCR could improve the assessment of LD severity and constitute a prognostic marker.

Aims The characterization and certification of a Legionella DNA quantitative reference material as a primary measurement standard for Legionella qPCR. Methods and Results Twelve laboratories participated in a collaborative certification campaign. A candidate reference DNA material was analysed through PCR-based limiting dilution assays (LDAs). The validated data were used to statistically assign both a reference value and an associated uncertainty to the reference material. Conclusions This LDA method allowed for the direct quantification of the amount of Legionella DNA per tube in genomic units (GU) and the determination of the associated uncertainties. This method could be used for the certification of all types of microbiological standards for qPCR. Significance and Impact of the Study The use of this primary standard will improve the accuracy of Legionella qPCR measurements and the overall consistency of these measurements among different laboratories. The extensive use of this certified reference material (CRM) has been integrated in the French standard NF T90-471 (April 2010) and in the ISO Technical Specification 12 869 (Anon 2012 International Standardisation Organisation) for validating qPCR methods and ensuring the reliability of these methods.

SUMMARYThe notification rate of Legionnaires' disease (LD) in France was 2·4/100 000 population in 2010, varying across regions with an increasing rate from west to east. Two sources [mandatory notifications (MN) and a survey of hospital laboratories] were used in a capture–recapture study to estimate the number of LD cases and the sensitivity of the MN system at national and regional levels in 2010. The number of missed cases was estimated using Chapman's method. The estimated sensitivity of MN was 88·5% (95% CI 88·0–89·0) and ranged from 70% to 100% by region. The estimated incidence was 2·7/100 000 population. Sensitivity of the MN system improved since the previous capture–recapture estimates (10% in 1995, 33% in 1998). This study confirmed that the observed west–east gradient is not related to regional notification disparities. Ecological studies should be conducted to better understand the observed spatial variations in LD incidence.

We evaluated the contribution of amoebic coculture to the recovery of Legionella spp. from 379 respiratory samples. The sensitivity of axenic culture was 42.1%. The combination of axenic culture with amoebic coculture increased the Legionella isolation rate to 47.1%. Amoebic coculture was particularly efficient in isolating Legionella spp. from respiratory samples contaminated with oropharyngeal flora.

The staphylococcal virulon is activated by the density-sensing agr system, which is autoinduced by a short peptide (autoinducing peptide [AIP]) processed from a propeptide encoded by agrD . A central segment of the agr locus, consisting of the C-terminal two-thirds of AgrB (the putative processing enzyme), AgrD, and the N-terminal half of AgrC (the receptor), shows striking interstrain variation. This finding has led to the division of Staphylococcus aureus isolates into three different agr specificity groups and to the division of non- aureus staphylococci into a number of others. The AIPs cross-inhibit the agr responses between groups. We have previously shown that most menstrual toxic shock strains belong to agr specificity group III but that no strong clinical identity has been associated with strains of the other two groups. In the present report, we demonstrate a fourth agr specificity group among S. aureus strains and show that most exfoliatin-producing strains belong to this group. A striking common feature of group IV strains is activation of the agr response early in exponential phase, at least 2 h earlier than in strains of the other groups. This finding raises the question of the biological significance of the agr autoinduction threshold.

In May 2010, a cluster of three cases of Legionnaires’ disease was identified in France. The results of the epidemiological, environmental and microbiological investigations allowed the rapid identification of a public whirlpool spa as the most probable source of contamination and the implementation of appropriate control measures. This investigation has stressed the need for good cooperation between partners and the importance of the molecular analysis of Legionella strains.

The relationship between the presence/absence of the virulence-associated MAb3/1 epitope of sixteen Legionella pneumophila serogroup 1 strains and their respective surface physicochemical properties is evidenced from electrokinetic measurements (microelectrophoresis) performed as a function of KNO(3) electrolyte concentration (range 1-100mM, pH∼6.5). Among the bacteria selected, nine original strains constitute the Dresden reference panel and differ according to the presence/absence of the virulence-associated monoclonal antibody MAb3/1 of the O-specific chain of the lipopolysaccharides (LPS). Five isogenic Lens strains, also investigated in the current study, present the epitope MAb3/1 of their LPS and were involved to some extent in the outbreak that stroke the Nord Pas-de-Calais region (France) in 2004. All bacteria exhibit the typical electrokinetic features of soft (permeable) particles. On the basis of Ohshima's model, analysis of the electrophoretic mobility data allows evaluating the intraparticular flow penetration length 1/λ(0) and the (negative) volume charge density ρ(0) that both reflect the structure and chemical composition of the soft bacterial component. Our results show that the virulent MAb3/1 positive strains are characterized on average by 1/λ(0) and ǀρ(0)ǀ values that are about 1.5 times larger and 5 times lower, respectively, than those derived for lesser virulent (MAb3/1 negative) strains. In other words, on average the soft surface layer of MAb3/1 positive strains is significantly less charged and more permeable than those of MAb3/1 negative strains. The intimate correlation between virulence-associated MAb3/1 epitope and charge density carried by the bacterial envelop was further confirmed by lower 1/λ(0) and greater ǀρ(0)ǀ values for lag-1 mutant CS332 strain, lacking the MAb3/1 epitope, compared to the parental strain AM511. A closer inspection of the dispersion in 1/λ(0) and ǀρ(0)ǀ data over the ensemble of analysed bacteria together with the reported number of Legionnaires' disease cases they are responsible for, points out the charge density ǀρ(0)ǀ as the parameter that is most suitable for discriminating highly virulent (MAb3/1 positive) from less virulent (MAb3/1 negative) strains. Although short-range interaction determines infection process, our results suggest that the infection potential of Legionella pneumophila serogroup 1 may be also controlled significantly by non-specific long-range electrostatic repulsion the bacteria undergo when approaching negatively charged host cells to be infected.

Legionella pneumophila is a common cause of hospital and community-acquired pneumonia, being transmitted by inhalation of aqueous aerosols. Most outbreaks are linked to contaminated hot water systems and cooling towers. Our study was about the molecular typing of 35 strains of L. pneumophila including four clinical isolates and 31 environmental strains isolated from the distribution systems of 14 hotels. Among the clinical strains, two have the same pattern, however, all were different from the studied environmental strains. For the 31 environmental strains, ten patterns were obtained. Among which, a same pulsotype was found for four strains isolated from four different establishments. In addition, two different pulsotypes were found for strains isolated from the same establishment. The pulsed-field gel electrophoresis showed the existence of various patterns. Although cases of legionellosis were declared in these hotels, there are no epidemiological links between the clinical and environmental strains.

Legionnaire's disease is a recently described infection and surveillance in France was implemented in 1987. In 2005, 1,527 cases were notified corresponding to a population incidence rate of 2.5 per 100,000. The median age of cases was 61 years [5-100] and the male to female sex ratio was 3.0. The case fatality rate was 11%. One or more risk factors were identified for 1,084 (71%) cases. The majority of cases (91%) was diagnosed by urinary antigen detection and a strain was identified in 276 cases (18%). Legionella pneumophila serogroup 1 infection was confirmed in 95% of cases. A specific exposure during the incubation period was reported for 39% of cases. Travel exposure was reported for 17% and hospital exposure for 7%. Multiple clusters and outbreaks were investigated. The largest one reported was identified in the north Lyon with 34 cases. Since 1997 the incidence of LD has steadily increased. This probably suggests a better detection of cases and an improvement in the surveillance system. In recent years, several new measures concerning prevention and control were implemented. However these efforts must continue particularly in the research domain to improve knowledge of the disease, in order to limit its impact on exposed populations.

We evaluated improvements made to the mandatory notification surveillance system for Legionnaires' disease in France by estimating its sensitivity in 1995 and 1998 using a repeat capture–recapture method. A case of Legionnaires' disease was defined as a person treated for pneumonia in whom legionella had been detected. Patient details were collected from (1) mandatory notifications; (2) the National Reference Centre for Legionella; (3) a postal survey of all hospital laboratories. The three sources were cross-matched and 715 individual cases were identified. A log-linear model, which included an interaction term between mandatory notifications and both the National Reference Centre and Laboratory sources, provided an estimated total of 1124 cases (95% CI 973–1275) in 1998, a twofold increase compared with 1995. The sensitivity of the surveillance system improved from 10% in 1995 to 33% (95% CI 29–38%) in 1998. Capture-recapture methods are important tools in the evaluation of surveillance systems.

The mission of the National Reference Centre for Staphylococcal Toxemia (CNRTS) is to participate in the epidemiological surveillance of staphylococcal toxemia in France. As these syndromes do not have to be declared, the data collected remain incomplete. Comparison of the clinical data with the results of molecular analysis of the causal strains has nonetheless enabled advances in our understanding of the present-day epidemiology of these syndromes, a clearer knowledge of their pathophysiology and isolation of a hitherto unknown entity, staphylococcal necrotizing pneumonia.

Two proteins of Klebsiella pneumoniae, termed Yor5 and Yco6, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, protein Yco6 was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive adenosine triphosphate, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, protein Yor5 was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Yor5 was able to dephosphorylate protein Yco6 previously autophosphorylated. Together, these data demonstrate that similarly to other bacterial species including Acinetobacter johnsonii and Escherichia coli, the cells of K. pneumoniae contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. Since Yco6 and Yor5 are both involved in the synthesis of capsular polysaccharide and since capsules are essential to the virulence of K. pneumoniae, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.Two proteins of Klebsiella pneumoniae, termed Yor5 and Yco6, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, protein Yco6 was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive adenosine triphosphate, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, protein Yor5 was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Yor5 was able to dephosphorylate protein Yco6 previously autophosphorylated. Together, these data demonstrate that similarly to other bacterial species including Acinetobacter johnsonii and Escherichia coli, the cells of K. pneumoniae contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. Since Yco6 and Yor5 are both involved in the synthesis of capsular polysaccharide and since capsules are essential to the virulence of K. pneumoniae, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.

Objectives To determine the incidence of Staphylococcus aureus isolates with reduced susceptibility to glycopeptides among all clinical isolates collected consecutively in two French hospitals between November 1998 and April 1999. Methods Methicillin-resistant and -susceptible S. aureus isolates were screened on vancomycin- or teicoplanin-supplemented agar plates. Glycopeptide MICs were determined by the E test procedure with a high inoculum and by an agar dilution technique. Glycopeptide-intermediate S. aureus isolates were identified as homogeneously or heterogeneously resistant to vancomycin by performing population analysis. Results Of the 640 isolates recovered from 518 patients, three from the same patient and two from two different patients showed homogeneous or heterogeneous intermediate resistance to vancomycin. Conclusion The incidence of glycopeptide-intermediate S. aureus (homogeneously or heterogeneously resistant) in a non-selected patient population, i.e. regardless of predisposing factors and glycopeptide therapeutics, remains low in the two French hospitals involved in the study, representing 0.6% of isolates.