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Legionnaires’ Disease (LD) is a severe pneumonia mainly caused in Europe by Legionella pneumophila serogroup 1 (Lp1). Sequence-based typing methods reveal that some sequence types (ST) are overrepresented in clinical samples such as ST1 and ST47, suggesting that some strains are more fit for infection than others. In the present study, a collection of 108 Lp1 clinical isolates were used to evaluate the strain-dependent immune responses from human macrophages. Clinical Lp1 isolates induced differential TNFα secretion from macrophages. ST1 isolates induced a significantly higher TNF-α secretion than non-ST1, whereas ST47 isolates induced a significantly lower TNF-α secretion than non-ST47 isolates. ST1 isolates induced a significantly higher cell death than ST47 isolates evaluated by lactate dehydrogenase activity (cytotoxicity) and caspase-3 activity (apoptosis). Treatment of macrophages with anti-TNF-α antibodies significantly reduced the cell death in macrophages infected with ST1 or ST47 strains. The TNF-α secretion was neither explained by a differential bacterial replication nor by the number or type (bystander or infected) of TNF-α producing cells following infection but by a differential response from macrophages. The Paris ST1 reference strain elicited a significantly higher TNF-α gene transcription and a higher induction of NF-κB signaling pathway than the Lorraine ST47 reference strain. Clinical Lp1 isolates induce a diverse immune response and cell death, which could be related to the genotype. The two predominant sequence-types ST1 and ST47 trigger opposite inflammatory response that could be related to the host susceptibility.

Mycobacterium tuberculosis (Mtb) genetic micro-diversity in clinical isolates may underline mycobacterial adaptation to tuberculosis (TB) infection and provide insights to anti-TB treatment response and emergence of resistance. Herein we followed within-host evolution of Mtb clinical isolates in two cohorts of TB patients, either with delayed Mtb culture conversion (> 2 months), or with fast culture conversion (< 2 months). We captured the genetic diversity of Mtb isolates obtained in each patient, by focusing on minor variants detected as unfixed single nucleotide polymorphisms (SNPs). To unmask antibiotic tolerant sub-populations, we exposed these isolates to rifampicin (RIF) prior to whole genome sequencing (WGS) analysis. Thanks to WGS, we detected at least 1 unfixed SNP within the Mtb isolates for 9/15 patients with delayed culture conversion, and non-synonymous (ns) SNPs for 8/15 patients. Furthermore, RIF exposure revealed 9 additional unfixed nsSNP from 6/15 isolates unlinked to drug resistance. By contrast, in the fast culture conversion cohort, RIF exposure only revealed 2 unfixed nsSNP from 2/20 patients. To better understand the dynamics of Mtb micro-diversity, we investigated the variant composition of a persistent Mtb clinical isolate before and after controlled stress experiments mimicking the course of TB disease. A minor variant, featuring a particular mycocerosates profile, became enriched during both RIF exposure and macrophage infection. The variant was associated with drug tolerance and intracellular persistence, consistent with the pharmacological modeling predicting increased risk of treatment failure. A thorough study of such variants not necessarily linked to canonical drug-resistance, but which are prone to promote anti-TB drug tolerance, may be crucial to prevent the subsequent emergence of resistance. Taken together, the present findings support the further exploration of Mtb micro-diversity as a promising tool to detect patients at risk of poorly responding to anti-TB treatment, ultimately allowing improved and personalized TB management., Author summary Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), bacteria that are able to persist inside the patient for many months or years, thus requiring long antibiotic treatments. Here we focused on TB patients with delayed response to treatment and we performed genetic characterization of Mtb isolates to search for sub-populations that may tolerate anti-TB drugs. We found that Mtb cultured from 9/15 patients contained different sub-populations, and in vitro drug exposure revealed Mtb sub-populations in 6/15 isolates, none related to known drug-resistance mechanisms. By contrast, drug exposure revealed Mtb sup-populations in 2/20 isolates in the control cohort of patients with fast culture conversion. Furthermore, we characterized a Mtb variant isolated from a sub-population growing in the presence of rifampicin (RIF), a major anti-TB drug. We found that this variant featured a modified lipidic envelope, and that it was able to develop in the presence of RIF and inside human macrophage cells. We performed pharmacological modelling and found that this kind of variant may be related to a poor response to treatment. In conclusion, searching for particular Mtb sub-populations may help to detect patients at risk of treatment failure and provide additional guidance for TB management.

The composition and dynamics of the lung microbiome during pneumonia are not known, although the lung microbiome might influence the severity and outcome of this infectious disease, similar to what was shown for the microbiome at other body sites. Here we report the findings of a comprehensive analysis of the lung microbiome composition of three patients with long-term pneumonia due to L. pneumophila and its evolution during antibiotic treatment. This work adds to our understanding of how the microbiome changes during disease and antibiotic treatment and points to microorganisms and their interactions that might be beneficial. In addition to bacteria and fungi, our analyses included archaea and eukaryotes (protozoa), showing that both are present in the pulmonary microbiota and that they might also play a role in the response to the microbiome disturbance., Despite the importance of pneumonia to public health, little is known about the composition of the lung microbiome during infectious diseases, such as pneumonia, and how it evolves during antibiotic therapy. To study the possible relation of the pulmonary microbiome to the severity and outcome of this respiratory disease, we analyzed the dynamics of the pathogen and the human lung microbiome during persistent infections caused by the bacterium Legionella pneumophila and their evolution during antimicrobial treatment. We collected 10 bronchoalveolar lavage fluid samples from three patients during long-term hospitalization due to pneumonia and performed a unique longitudinal study of the interkingdom microbiome, analyzing the samples for presence of bacteria, archaea, fungi, and protozoa by high-throughput Illumina sequencing of marker genes. The lung microbiome of the patients was characterized by a strong predominance of the pathogen, a low diversity of the bacterial fraction, and an increased presence of opportunistic microorganisms. The fungal fraction was more stable than the bacterial fraction. During long-term treatment, no genomic changes or antibiotic resistance-associated mutations that could explain the persistent infection occurred, according to whole-genome sequencing analyses of the pathogen. After antibiotic treatment, the microbiome did not recover rapidly but was mainly constituted of antibiotic-resistant species and enriched in bacteria, archaea, fungi, or protozoa associated with pathogenicity. The lung microbiome seems to contribute to nonresolving Legionella pneumonia, as it is strongly disturbed during infection and enriched in opportunistic and/or antibiotic-resistant bacteria and microorganisms, including fungi, archaea, and protozoa that are often associated with infections.

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; Three urinary antigen tests were compared using purified Legionella pneumophila (Lp) LPS. For Lp serogroup1, Sofia®FIA and Binax®EIA limits of detection (LOD) were similar; that of BinaxNOW® lower. For all tests the LOD was higher with LPS from non-Pontiac compared to Pontiac-strains. The LOD was variable for other Lp serogroups.

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.

Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila . Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings.

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.

International audience; In France, approximately 1200 cases of Legionnaires disease (LD) are reported annually, and isolates are available for approximately 20% of cases identified since 2000. All Legionella pneumophila serogroup 1 (sg1) isolates are characterized by sequence-based typing at the National Reference Centre. LD cases caused by L.~pneumophila sg1 reported from 2008 through 2012 were considered for the study. Our study objective was to describe cases according to their sequence type (ST). We also constructed multivariable modified Poisson regression models to estimate the incidence rate ratio (IRR) and to identify characteristics potentially associated with ST23 clones compared to ST1 and ST47 clones. We studied 1192 patients infected by ST1 (n~= 109), ST23 (n~= 236), ST47 (n~= 123) or other STs (n~= 724). The geographic distribution of the ST23 cases across the country was significantly different compared to other ST groups. This genotype was significantly associated with the absence of corticosteroid therapy compared to ST1 (IRR~= 0.56; p 0.016). Concerning exposure, the ST23 genotype was significantly less associated with hospital-acquired infections compared to ST1 (IRR~= 0.32; p 0.001), but it was more associated with infections acquired in hospitals and elderly settings compared with ST47. Finally, the ST23 genotype was less frequently associated with travel than other STs. Despite the large number of cases of ST23 infection, we did not identify any characteristics specific to this ST. However, we identified independent associations between ST1 and nosocomial transmission and steroid therapy. These findings should encourage further exploration, especially in terms of environmental diffusion, strain virulence and host factors.

A 42-plex clustered regularly interspaced short palindromic repeat (CRISPR)-based typing technique (spoligotyping) was recently developed at the French National Reference Center for Legionella . It allows the subtyping of the Legionella pneumophila sequence type 1/Paris pulsotype. In this report, we present the transfer of the membrane-based spoligotyping technique to a microbead-based multiplexed format. This microbead-based high-throughput assay uses devices such as Luminex 200 or the recently launched Magpix system (Luminex Corp., Austin, TX). We designated this new technique LP-SPOL (for L. p neumophila spol igotyping). We used two sets of samples previously subtyped by the membrane-based spoligotyping method to set up and validate the transfer on the two microbead-based systems. The first set of isolates ( n = 56) represented the whole diversity of the CRISPR patterns known to date. These isolates were used for transfer setup (determination of spacer cutoffs for both devices). The second set of isolates ( n = 245) was used to validate the transfer to the two microbead-based systems. The results obtained by the Luminex 200 system were 100% concordant with those obtained by the Magpix system for the 2 sets of isolates. In total, 10 discrepant results were observed when comparing the membrane-based method to the microbead-based method. These discrepancies were further resolved by repeating either the membrane-based or the microbead-based assay. This new assay is expected to play an emerging role for surveillance of L. pneumophila , starting with one of the most frequent genotypes, the sequence type 1/Paris pulsotype. However, the generalization of this typing method to all L. pneumophila strains is not feasible, since not all L. pneumophila strains contain CRISPRs.

Background In France, Legionnaires disease is mainly caused by Legionella pneumophila. Here, we investigated possible host factors associated with susceptibility to community-acquired Legionnaires disease caused by the endemic Paris and Lorraine strains. Methods We conducted a double-nested exploratory case-control study with use of data from the French national surveillance network of incident Legionnaires disease cases notified from 1998 through 2007. Patients with community-acquired Legionnaires disease and an L. pneumophila serogroup 1 isolate were eligible. Case patients were patients infected by the Paris or Lorraine strain, and control patients were those infected by sporadic strains. Epidemiological and clinical factors associated with infection with the Paris and Lorraine strains were assessed by calculating adjusted odds ratios (aOR) in multivariate logistic regression models. Results We studied 1090 patients infected by sporadic strains (n = 920), the Paris strain (n = 80), or the Lorraine strain (n = 90). Infection with the Paris strain was significantly associated with female sex (aOR, 1.98; 95% confidence interval [CI], 1.19-3.28), steroid therapy (aOR, 3.16; 95% CI, 1.76-5.68), and a history of cancer or hematologic malignancies (aOR, 2.08; 95% CI, 1.15-3.76). In addition, the mortality rate was higher among patients infected with the Paris strain than in the control group (38% vs. 25.5%). The Lorraine strain was associated with smoking (aOR, 1.82; 95% CI, 1.14-2.91) and reduced mortality (9.9%). . Conclusion Several host characteristics were associated with the risk of infection by endemic strains of L. pneumophila serogroup 1. These findings may help to guide preventive measures. Factors predisposing patients to infection by specific strains need to be explored further.

The lvh region of the Legionella pneumophila genome, which encodes a type IV secretion system, is located on a plasmid-like element in strains Paris (pP36) and Philadelphia (pLP45). The pP36 element has been described either integrated in the chromosome or excised as a multi-copy plasmid, in a similar manner to pLP45. In this paper, the chromosomal integration of pP36 in the Paris strain genome was described, occurring through site-specific recombination at the 3′ end of a transfer-messenger RNA gene by recombination between attachment sites, in a similar manner to pathogenicity islands. This integration was growth-phase dependent, occurring during the exponential phase. Several pP36-borne genes were expressed during the lag phase of bacterial growth, coinciding with the peak amount of the episomal form of pP36. Expression of the same genes decreased during the exponential and stationary phases, owing to the integration phenomenon and a loss of episomal copies of pP36. A similar plasmid-like element was described in the Lens strain genome, suggesting that the mobility of the lvh region is a phenomenon widespread among Legionella sp.

Legionella pneumophila (Lp) is an accidental human pathogen which can infect alveolar macrophages and pneumocytes. During infection, Legionella have to deal with to various types of antibacterial agents, such as antimicrobial peptides (AMPs) produced by the host, and antibiotics with intracellular activity administered to patients. The mechanism of action of human AMPs against Legionella, and the resistance level to antibiotics of the bacterium are still poorly described. Our work aimed to contribute to a better understanding of the anti-Legionella activity of these molecules. The first part of this study consisted in the evaluation of the susceptibility of clinical Lp sg1 isolates to 8 antibiotics, to determine the epidemiological cut-off values of these different molecules. We demonstrated that all clinical isolates are susceptible to the tested antibiotics. The results revealed the presence of a subpopulation displaying a reduced susceptibility to macrolides. The analysis of the genomes allowed us to correlate this reduced susceptibility to le presence of the LpeAB macrolides efflux pump, found specifically in the sequence types ST1, ST701 and ST1335.The second part of this study was dedicated to the characterization of the antibacterial activity of the human AMPs LL-37 and HBD-3, and to the identification of their mechanism(s) of action against Legionella. All of the experiments show that LL-37 and HBD-3 induce a loss of cultivability by different mode of action. The results suggest that LL-37 is able to permeabilize the membrane of the L. pneumophila cells. Our findings also show that both peptides inhibit the intracellular replication of L. pneumophila, in part through collaboration with the host cell; Legionella pneumophila (Lp) est un pathogène accidentel de l'homme capable d'infecter les macrophages alvéolaires et les pneumocytes. Au cours de l'infection, Legionella se confronte à différents types d'agents antibactériens, dont les peptides antimicrobiens (PAMs) produit par l'hôte et les antibiotiques à activité intracellulaire administrés aux patients. Le mécanisme d'action des PAMs humains à l'encontre de Legionella, ainsi que le niveau de résistance aux antibiotiques de la bactérie sont à ce jour encore peu documentés. Mes travaux ont pour but de contribuer à une meilleure connaissance de l'activité anti-Legionella de ces molécules. La première partie de cette étude a consisté à évaluer la sensibilité d'isolats cliniques de Lp sg 1 à 8 antibiotiques, afin de déterminer le seuil épidémiologique de sensibilité de la bactérie à ces différentes molécules. Nous avons démontré que l'ensemble des isolats cliniques sont sensibles aux antibiotiques testés. Les résultats ont révélé l'existence d'une sous-population présentant une sensibilité réduite aux macrolides. L'analyse des génomes a permis de corréler cette sensibilité diminuée à la présence de la pompe à efflux LpeAB spécifique des macrolides. Cette pompe est présente uniquement dans trois complexes clonaux centrés sur le ST1, le ST701 et le ST1335.La seconde partie de cette étude a été consacrée à la caractérisation de l'activité antibactérienne des PAMs humains LL-37 et HBD-3, ainsi qu'à l'identification de leur(s) mécanisme(s) d'action contre Legionella. L'ensemble des tests réalisés montre que LL-37 et HBD-3 induisent une perte de cultivabilité des légionelles par des modes d'action différents. Les résultats suggèrent que LL-37 agit par perméabilisation des membranes de L. pneumophila. Nos résultats ont également montré que les deux peptides exercent une activité inhibitrice sur la réplication intracellulaire des légionelles, au moins en partie grâce à une collaboration avec la cellule hôte

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 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 cliniques