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The objective of the study was to evaluate all available systematic reviews on the use of prone positional ventilation in adult patients with acute respiratory distress syndrome (ARDS). An umbrella review on the efficacy of prone positional ventilation in adult patients ventilation in adult patients with acute respiratory distress syndrome was conducted. We performed a systematic search in the database of Medline (Pubmed), Scopus, Cochrane Library, Web of Science, and Epistemonikos. The ROBIS tools and GRADE methodology were used to assess the risk of bias and certainty of evidence. We estimated the necessary number of patients to be treated to have benefit. For the synthesis of the result, we selected the review with the lowest risk of bias. Sixteen systematic reviews including 64 randomized clinical trials and evaluating the effect of prone positional ventilation, with or without other ventilation strategies were included. Aoyama 2019 observed prone positioning, without complementary ventilation strategies, leading to a reduction in the 28-day mortality only when compared to high-frequency oscillatory ventilation (RR 0.61; 95% CI 0.39-0.95) and lung-protective ventilation in the supine position (RR 0.69; 95% CI 0.48-0.98), with an ARR of 9.32% and 14.94%, an NNTB of 5.89 and 8.04, and a low and moderate certainty of evidence, respectively. Most reviews had severe methodological flaws that led to results with very low certainty of evidence. The review with the lowest risk of bias presented results in favor of prone positional ventilation compared with high-frequency oscillatory ventilation and lung-protective ventilation. There is a need to update the available reviews to obtain more accurate results.

Mycobacterium abscessus is an environmental non-tuberculous mycobacteria causing severe lung infections in cystic fibrosis patients. Its intrinsic resistance to antibiotics renders treatments extremely challenging. This pathogen has developed a wide panel of strategies to resist to antibiotics, including efflux pumps and target-modifying enzymes. Standard antibiotherapy combines an aminoglycoside (amikacin (AMK)), -lactams (imipenem (IPM)) and macrolides (clarithromycin (CLR) or azithromycin (AZM)). My thesis was focusing on the regulation of antibiotic resistance mechanisms involving efflux pumps from the MmpL family as well as an enzyme modifying the macrolide target. Selection of resistant mutants against thiacetazone derivatives (TACd) and clofazimine (CFZ) unraveled mutations in the TetR regulators MAB_4384 and MAB_2299c, respectively. The CFZ-mutants, also co-resistant to bedaquiline (BDQ), overexpress two distinct MmpS/MmpL, MAB_135c/1134c and MAB_2300/2301 while the TACd mutants overproduce the MAB_4383c/4382c efflux pump. Biochemical, genetic and structural approaches confirmed their involvement in drug resistance mechanisms. MAB_2299c could therefore represent a potential resistance marker to monitor in strains isolated from patients under CFZ/BDQ therapy. The methyltransferase Erm(41) modifies the adenosine 2058 of the ribosomal 23S rRNA, protecting the ribosome from the macrolides. WhiB7 may represent a major actor in inducible resistance, observed in 40% of clinical cases, often leading to treatment failure. We confirmed the role of WhiB7 in this process and showed that inducible resistance occurred also in vivo in the zebrafish model. Our data suggest that AZM is a stronger and faster resistance inducer than CLR in vitro and that both drugs show antagonism with AMK, thus reducing drugs’ efficacy. This work enriched our knowledge regarding both MmpL-mediated and macrolides inducible resistance mechanism against M. abscessus. It also provides new efficient tools to investigate the function of proteins through a novel unmarked gene deletion approach and to rapidly assay new antibiotics to counteract inducible macrolide resistance.; Mycobacterium abscessus est une bactérie environnementale non-tuberculeuse causant des infections pulmonaires sévères chez les patients atteints de mucoviscidose. Sa résistance intrinsèque aux antibiotiques rend les infections à M. abscessus extrêmement difficiles à traiter. Ses mécanismes de résistance sont variés et incluent des pompes à efflux expulsant les antibiotiques ainsi que des enzymes modifiant leurs cibles. La trithérapie standarde contre M. abscessus implique un aminoglycoside (amikacine (AMK)), une -lactamine (imipénème (IPM)) et un macrolide (clarithromycine (CLR) ou azithromycine (AZM). Ma thèse a porté sur la régulation de nouveaux mécanismes d’antibiorésistance via des pompes à efflux de la famille MmpL ainsi que sur une enzyme modifiant la cible des macrolides. La sélection de mutants résistants aux dérivés du thiacétazone (TACd) et à la clofazimine (CFZ) a permis d’identifier des mutations dans les régulateurs TetR MAB_4384 et MAB_2299c, respectivement. Les mutants CFZ, également co-résistants à la bédaquiline (BDQ), surexpriment deux MmpS/MmpL distincts, MAB_1135c/1134c et MAB_2300/2301 tandis que les mutants TACd surproduisent le système MmpS/MmpL MAB_4383c/4382c. Des approches biochimiques, génétiques et structurales ont confirmé le rôle de ces protéines dans la résistance à ces composés et ont permis de disséquer le mode de régulation. Ainsi, MAB_2299c représenterait un marqueur de résistance à exploiter chez des patients traités par CFZ ou BDQ. La méthyltransférase Erm(41) modifie l’adénosine 2058 de l’ARNr 23S du ribosome, bloquant l’action des macrolides. WhiB7 serait l’activateur de cette résistance inductible observée chez 40% des isolats cliniques, conduisant souvent à l’échec thérapeutique. Nous avons confirmé le rôle de WhiB7 dans ce mécanisme et montré que la résistance inductible opérait in vivo chez le modèle zebrafish. Nos résultats indiquent que l’AZM induit une résistance plus rapide et forte que la CLR in vitro et qu’elles exercent un effet antagoniste sur l’AMK, réduisant l’efficacité du traitement. Ces travaux décrivent l’export d’antibiotique via des MmpL et la résistance adaptative aux macrolides chez M. abscessus. Enfin, ils apportent des nouveaux outils génétiques performants pour étudier la fonction de protéines d’intérêt via une nouvelle technique de délétion génique et pour tester rapidement de nouveaux antibiotiques contournant la résistance induite aux macrolides.

International audience; Mycobacterium abscessus is an emerging human pathogen causing severe pulmonary infections and is refractory to standard antibiotherapy, yet few drug resistance mechanisms have been reported in this organism. Recently, mutations in MAB_4384 leading to up-regulation of the MmpS5/MmpL5 efflux pump were linked to increased resistance to thiacetazone derivatives. Herein, the DNA-binding activity of MAB_4384 was investigated by electrophoretic mobility shift assays using the palindromic sequence IR S5/L5 located upstream of mmpS5/mmpL5. Introduction of point mutations within IR S5/L5 identified the sequence requirements for optimal binding of the regulator. Moreover, formation of the protein/IR S5/L5 complex was severely impaired for MAB_4384 harboring D14N or F57L substitutions. IR S5/L5 /lacZ reporter fusions in M. abscessus demonstrated increased β-galactosidase activity either in strains lacking a functional MAB_4384 or in cultures treated with the TAC analogs. In addition, X-ray crystallography confirmed a typical TetR homodimeric structure of MAB_4384 and unraveled a putative ligand binding site in which the analogs could be docked. Overall, these results support drug recognition of the MAB_4384 TetR regulator, alleviating its binding to IR S5/L5 and steering up-regulation of MmpS5/MmpL5. This study provides new mechanistic and structural details of TetR-dependent regulatory mechanisms of efflux pumps and drug resistance in mycobacteria.