Legionella pneumophila and macrolides : from susceptibility testing to characterization of molecular mechanisms conferring resistance

Les macrolides sont recommandés seuls ou en association dans le traitement des légionelloses. Cependant, malgré une antibiothérapie adaptée et l'absence de souches résistantes décrites, des échecs thérapeutiques sont régulièrement observés. L'isolement de souches de Legionella à partir de prélèvements respiratoires est indispensable à la réalisation d'antibiogrammes mais il n'est obtenu que dans 24% des cas en France. Au cours de ce travail, nous avons tout d'abord amélioré le rendement d'isolement des souches cliniques, notamment par des techniques de co-culture amibienne, avant de nous intéresser aux mécanismes pouvant expliquer les échecs thérapeutiques (antagonisme entre antibiotiques, sélection de résistance). Nous avons pu montrer, dans un modèle de croissance intracellulaire de Legionella pneumophila, une absence d'antagonisme entre macrolides et fluoroquinolones ou rifampicine invalidant la première hypothèse. Nous avons ensuite généré des lignées de mutants hautement résistants aux macrolides. Nous avons mis en évidence des mutations dans les gènes codant les protéines ribosomales L4 et L22 associées à un faible niveau de résistance. Des mutations dans les gènes codant l'ARN ribosomal 23S étaient associées à un niveau plus élevé de résistance, dépendant de la nature de la mutation et du nombre de copies du gène mutées. La facilité d'obtention de mutants résistants aux macrolides in vitro suggère une potentielle acquisition de résistance in vivo au décours de l'antibiothérapie et justifie une recherche systématique de cette résistance dans les prélèvements cliniquesMacrolides, alone or in combination, are recommended as first-line therapy of Legionnaires’ disease. Legionella pneumophila is characterized by a constant in vitro susceptibility to macrolides. Resistant variants have never been detected, in contrast with several therapeutic failures observed in patients receiving an adequate treatment. Legionellae isolation from respiratory secretions is needed for antibiotic susceptibility testing, but the reported culture sensitivity reaches only 24% in France. In this work, we improved Legionellae isolation rate by culture and amoebal co-culture. In a second time, we were interested in hypothetic mechanisms involved in therapeutic failures like antibiotic antagonisms and acquired resistance. We found no antagonistic effects for macrolide/fluoroquinolones and macrolide/rifampin combinations in an intracellular Legionella model, which made irrelevant our first hypothesis. In a last step, we propagated Legionella strains with increasing macrolides concentrations. This procedure provided us high-level macrolides-resistant mutants. We found that mutations in the genes encoding L4 and L22 proteins were correlated with low-level resistance. Mutations in domain V of the 23S rRNA were associated with intermediate or high-level resistance. We found a correlation between the kind of nucleotide substitution or the number of mutated ribosomal operons and the resistance level. The speed and efficiency of in vitro selection of L. pneumophila macrolide-resistant mutants suggest a potential acquisition of macrolide resistance in patients under therapy and emphasize the need to explore clinical Legionella macrolide susceptibility in order to track down resistanc

Legionella pneumophila et macrolides : de l’antibiogramme à la caractérisation des mécanismes moléculaires impliqués dans la résistance

Macrolides, alone or in combination, are recommended as first-line therapy of Legionnaires’ disease. Legionella pneumophila is characterized by a constant in vitro susceptibility to macrolides. Resistant variants have never been detected, in contrast with several therapeutic failures observed in patients receiving an adequate treatment. Legionellae isolation from respiratory secretions is needed for antibiotic susceptibility testing, but the reported culture sensitivity reaches only 24% in France. In this work, we improved Legionellae isolation rate by culture and amoebal co-culture. In a second time, we were interested in hypothetic mechanisms involved in therapeutic failures like antibiotic antagonisms and acquired resistance. We found no antagonistic effects for macrolide/fluoroquinolones and macrolide/rifampin combinations in an intracellular Legionella model, which made irrelevant our first hypothesis. In a last step, we propagated Legionella strains with increasing macrolides concentrations. This procedure provided us high-level macrolides-resistant mutants. We found that mutations in the genes encoding L4 and L22 proteins were correlated with low-level resistance. Mutations in domain V of the 23S rRNA were associated with intermediate or high-level resistance. We found a correlation between the kind of nucleotide substitution or the number of mutated ribosomal operons and the resistance level. The speed and efficiency of in vitro selection of L. pneumophila macrolide-resistant mutants suggest a potential acquisition of macrolide resistance in patients under therapy and emphasize the need to explore clinical Legionella macrolide susceptibility in order to track down resistanceLes macrolides sont recommandés seuls ou en association dans le traitement des légionelloses. Cependant, malgré une antibiothérapie adaptée et l'absence de souches résistantes décrites, des échecs thérapeutiques sont régulièrement observés. L'isolement de souches de Legionella à partir de prélèvements respiratoires est indispensable à la réalisation d'antibiogrammes mais il n'est obtenu que dans 24% des cas en France. Au cours de ce travail, nous avons tout d'abord amélioré le rendement d'isolement des souches cliniques, notamment par des techniques de co-culture amibienne, avant de nous intéresser aux mécanismes pouvant expliquer les échecs thérapeutiques (antagonisme entre antibiotiques, sélection de résistance). Nous avons pu montrer, dans un modèle de croissance intracellulaire de Legionella pneumophila, une absence d'antagonisme entre macrolides et fluoroquinolones ou rifampicine invalidant la première hypothèse. Nous avons ensuite généré des lignées de mutants hautement résistants aux macrolides. Nous avons mis en évidence des mutations dans les gènes codant les protéines ribosomales L4 et L22 associées à un faible niveau de résistance. Des mutations dans les gènes codant l'ARN ribosomal 23S étaient associées à un niveau plus élevé de résistance, dépendant de la nature de la mutation et du nombre de copies du gène mutées. La facilité d'obtention de mutants résistants aux macrolides in vitro suggère une potentielle acquisition de résistance in vivo au décours de l'antibiothérapie et justifie une recherche systématique de cette résistance dans les prélèvements clinique

Détermination de l'activité intracellulaire d'antibiotiques seuls ou en association sur legionella pneumophila sérogroupe 1

LYON1-BU Santé (693882101) / SudocSudocFranceF

Identification of legionella in clinical samples.

International audienceCurrently, several methods are used for the detection of Legionella in clinical samples, and these methods constitute part of the criteria for defining legionellosis cases. Urinary antigen detection is the first-line diagnostic test, although this test is limited to L. pneumophila serogroup 1 (Lp1) (Helbig et al., J Clin Microbiol 41:838-840, 2003). The use of molecular techniques can improve Legionaire's disease (LD) diagnosis by detecting other serogroups and species (Diederen et al., J Clin Microbiol 46:671-677, 2008). The isolation of Legionella strains from pulmonary samples by axenic culture is still required to perform further epidemiological investigations (Blyth et al., N S W Public Health Bull 20:157-161, 2009; Fields et al., Clin Microbiol Rev 15:506-526, 2002) but demonstrates various sensitivities. Amoebic coculture has been described as a method to recover Legionella from clinical culture-negative specimens (La Scola et al., J Clin Microbiol 39:365-366, 2001; Rowbotham, J Clin Pathol 36:978-986, 1983) and can be proposed for optimizing Legionella strain isolation from samples contaminated by oropharyngeal flora. Identification of Legionella isolates is based on serological characterization, genotypic methods (with sequencing of the mip gene as the standard method) and, more recently, the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method.This chapter is limited to the identification of Legionella in clinical samples; antibody detection in human serum will not be discussed

Macrolide resistance in Legionella pneumophila: the role of LpeAB efflux pump

International audienceObjectives: 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

KKL-35 Exhibits Potent Antibiotic Activity against Legionella Species Independently of trans-Translation Inhibition

International audiencetrans-Translation is a ribosome-rescue system that is ubiquitous in bacteria. Small molecules defining a new family of oxadiazole compounds that inhibit trans-translation have been found to have broad-spectrum antibiotic activity. We sought to determine the activity of KKL-35, a potent member of the oxadiazole family, against the human pathogen Legionella pneumophila and other related species that can also cause Legionnaires' disease (LD). Consistent with the essential nature of trans-translation in L. pneumophila, KKL-35 inhibited the growth of all tested strains at submicromolar concentrations. KKL-35 was also active against other LD-causing Legionella species. KKL-35 remained equally active against L. pneumophila mutants that have evolved resistance to macrolides. KKL-35 inhibited the multiplication of L. pneumophila in human macrophages at several stages of infection. No resistant mutants could be obtained, even during extended and chronic exposure. Surprisingly, KKL-35 was not synergistic with other ribosome-targeting antibiotics and did not induce the filamentation phenotype observed in cells defective for trans-translation. Importantly, KKL-35 remained active against L. pneumophila mutants expressing an alternate ribosome-rescue system and lacking transfer-messenger RNA, the essential component of trans-translation. These results indicate that the antibiotic activity of KKL-35 is not related to the specific inhibition of trans-translation and its mode of action remains to be identified. In conclusion, KKL-35 is an effective antibacterial agent against the intracellular pathogen L. pneumophila with no detectable resistance development. However, further studies are needed to better understand its mechanism of action and to assess further the potential of oxadiazoles in treatment

The clinical presentation of Legionella arthritis reveals the mode of infection and the bacterial species: case report and literature review

International audienceBACKGROUND:While Legionella is a common cause of pneumonia, extrapulmonary infections like arthritis are scarce. Here, we describe a case of monoarthritis due to Legionella bozemanii, with no history of pneumonia. We provide a literature review of the 9 previously published Legionella arthritis and highlight a dichotomous epidemiology suggesting different physiopathological pathways leading to joint infection.CASE PRESENTATION:A 56-year old woman under immunosuppressive treatment by oral and intra-articular corticosteroids, methotrexate, and tocilizumab for an anti-synthetase syndrome was hospitalized for worsening pain and swelling of the left wrist for 3 days. Clinical examination showed left wrist synovitis and no fever. The arthritis occurred a few days after an accidental fall on wet asphalt responsible for a cutaneous wound followed by a corticosteroid intra-articular injection. Due to both the negativity of conventional culture of articular fluid and suspicion of infection, 16S rRNA and specific PCRs were performed leading to the identification of L. bozemanii. Legionella-specific culture of the articular fluid was performed retrospectively and isolated L. bozemanii. The empiric antibiotic therapy was switched for oral levofloxacin and rifampin and the patient recovered after a 12-week treatment.CONCLUSION:We report a case of L. bozemanii monoarthritis in an immunosuppressed woman, following a fall on wet asphalt and intra-articular corticosteroid injection. The review of the literature found that the clinical presentation reveals the mode of infection and the bacterial species. Monoarthritis more likely occurred after inoculation in patients under immunosuppressive therapy and were associated with non-Legionella pneumophila serogroup 1 (Lp1) strains that predominate in the environment. Polyarthritis were more likely secondary legionellosis localizations after blood spread of Lp1, the most frequently found in pneumonia. In both settings, 16S rRNA and Legionella-specific PCR were key factors for the diagnosis

Legionella pneumophila sequence type 1/Paris pulsotype subtyping by spoligotyping.

International audienceEndemic strains of Legionella pneumophila sequence type 1 (ST1), in particular the ST1/Paris pulsotype, are dispersed worldwide and represent about 10% of culture-proven clinical cases of Legionnaires' disease in France. The high rate of isolation of this strain from both clinical and environmental samples makes identification of the source of infection difficult during epidemiological investigations. The full-length genome sequence of this strain was recently determined, and it revealed the presence of a CRISPR/cas complex. The aim of this study was to develop and evaluate a spoligotyping tool based on the diversity of this CRISPR locus that would allow the accurate subtyping of the L. pneumophila serogroup 1 ST1/Paris pulsotype. The CRISPR loci of 28 L. pneumophila ST1/Paris pulsotype isolates were sequenced, and 42 different spacers regions were characterized. A membrane-based spoligotyping method was developed and used to determine the subtypes of 406 L. pneumophila isolates, including 233 with the ST1/Paris pulsotype profile that were collected in France from 2000 to 2011. A total of 46 different spoligotypes were detected, and 41 of these were specifically identified in the ST1/Paris pulsotype isolates. In 27 of 33 epidemiological investigations, the environmental source of contamination was confirmed by comparing spoligotypes of clinical isolates with those of environmental isolates. With an index of discrimination of 79.72% (95% confidence interval, 75.82 to 83.63), spoligotyping of the L. pneumophila ST1/Paris pulsotype has the potential to be a useful complementary genotyping tool for discriminating isolates with undistinguishable pulsed-field gel electrophoresis (PFGE) and ST genotypes, which could help to identify environmental sources of infection

Macrolide-Resistant Bordetella pertussis Infection in Newborn Girl, France

A macrolide antimicrobial drug was administered to a newborn with cough. On day 23 of hospitalization, macrolide-resistant Bordetella pertussis was isolated from nasopharyngeal aspirates. DNA sequencing and PCR–restriction fragment length polymorphism showed a 2047 A-to-G mutation in the 3 copies of the 23S rRNA gene. Monitoring for macrolide resistance is essential in infants <6 months of age