Higher Prevalence of PldA, a Pseudomonas aeruginosa Trans-Kingdom H2-Type VI Secretion System Effector, in Clinical Isolates Responsible for Acute Infections and in Multidrug Resistant Strains

Pseudomonas aeruginosa can manipulate eukaryotic host cells using secreted effectors delivered by the type III or the type VI Secretion Systems (T3SS and T6SS). The T3SS allows the injection of bacterial effectors (Exo toxins) into eukaryotic cell. P. aeruginosa, encodes three T6SSs, H1-, H2- and H3-T6SS. The H1-T6SS is mainly involved in delivering toxins to kill bacterial competitors. Recently, two T6SS-secreted phospholipases D, PldA (H2-T6SS) and PldB (H3-T6SS), were identified as trans-kingdom virulence effectors, triggering both killing of bacterial competitors and internalization into non-phagocytic cells. We deciphered the prevalence of T3SS and T6SS effectors encoding genes in 185 clinical isolates responsible for infections (septicaemia, pulmonary infections, urinary tract infections, and chronic infections in CF patients), 47 environmental strains, and on 33 carbapenemase-producers. We included 107 complete genomes of P. aeruginosa available in public databases. The prevalence of pldA is increased in clinical isolates responsible for severe acute infection and particularly in multi-drug resistant strains. In contrast, the pldB prevalence was high (96.8%) in all isolates. Regarding T3SS effectors, exoT and exoY are present in nearly all isolates while exoS and exoU were found to be exclusive with a higher prevalence of exoU+ strains in severe acute infections. The hypervirulent exoU+ isolates are more prone to be pldA+, suggesting a role of PldA in virulence. Finally, we observed that extremely drug resistant isolates producing an IMP-type carbapenemase were all pldA+. Our results suggest that PldA might have a role during pulmonary infections and have been co-selected in multidrug resistant strains particularly IMP-producers

Drug susceptibility testing by dilution methods.

International audienceSerial twofold dilution methods are widely used to assess the bacteriostatic activities of antibiotics. This can be achieved by dilution of considered drugs in agar medium or in culture broth, and inoculation by calibrated inoculums. Although seemingly simple, these methods are greatly influenced by the experimental conditions used and may lead to discrepant results, in particular with untrained investigators. The present step-by-step protocol has been validated for Pseudomonas species, including P. aeruginosa. Introduction of appropriate control strains is crucial to ascertain minimal inhibitory concentration values and compare the results of independent experiments

Polymorphisme de la protéine PcrV chez Pseudomonas Aeruginosa (une cible pour le développement de nouvelles stratégies thérapeutiques ?)

BESANCON-BU Médecine pharmacie (250562102) / SudocSudocFranceF

Intérêt du milieu McKay pour la détection des streptocoques du groupe milleri chez les patients atteints de mucoviscidose

BESANCON-BU Médecine pharmacie (250562102) / SudocSudocFranceF

CONSOMMATION D'ANTIBIOTIQUES ET RESISTANCE BACTERIENNE EN VILLE

BESANCON-BU Médecine pharmacie (250562102) / SudocSudocFranceF

Mutations in gene fusA1 as a novel mechanism of aminoglycoside resistance in clinical strains of Pseudomonas aeruginosa.

International audienceResistance of clinical strains of Pseudomonas aeruginosa to aminoglycosides can result from production of transferable aminoglycoside-modifying enzymes, of 16S rRNA methylases, and/or mutational derepression of intrinsic multidrug efflux pump MexXY(OprM). The present study reports on characterization of a new type of mutants 4- to 8-fold more resistant to 2-deoxystreptamine derivatives (e.g., gentamicin, amikacin, tobramycin), as compared with wild-type strain PAO1. The genetic alterations of three in vitro mutants were mapped on fusA1, and found to result in single amino acid substitutions in domains II, III and V of elongation factor G (EF-G1A), a key component of translational machinery. Transfer of the mutated fusA1 alleles into PAO1 reproduced the resistance phenotype. Interestingly, fusA1 mutants with other amino acid changes in domains G, IV and V of EF-G1A were identified among clinical strains with decreased susceptibility to aminoglycosides. Allelic exchange experiments confirmed the relevance of these latter mutations and of three other previously reported alterations located in domains G and IV. Pump MexXY(OprM) was found to partly contribute to the resistance conferred by the mutated EF-G1A variants, and to have additive effects on aminoglycosides MICs when mutationally up-regulated. Altogether, our data demonstrate that cystic fibrosis (CF) and non-CF strains of P. aeruginosa can acquire a therapeutically significant resistance to important aminoglycosides, via a new mechanism involving mutations in elongation factor EF-G1A

Adaptation des mécanismes de résistance par efflux actif chez les souches de pseudomonas aeruginosa dans la mucoviscidose

BESANCON-BU Médecine pharmacie (250562102) / SudocSudocFranceF

Résistance non enzymatique aux aminosides chez Pseudomonas aeruginosa

Bactérie opportuniste bien connue, Pseudomonas aeruginosa est responsable d'infection bronchopulmonaire chronique chez les patients atteints de mucoviscidose. Afin de mieux appréhender les mécanismes mis en jeu par la bactérie pour résister aux nombreuses cures d'antibiothérapie par les aminosides (AGs), nous avons construit une banque d'insertion chez la souche sauvage de référence PAO1. Différents mécanismes de résistance ont ainsi été caractérisés, correspondant à : (I) l'augmentation de l'efflux actif des AGs; (II) la diminution de la perméabilité de la membrane externe aux AGs ; (III) la baisse du transport actif des AGs à travers la membrane cytoplasmique et (IV) l'altération de protéines ribosomales. Chacun des mécanismes identifiés confère à la bactérie une résistance de bas niveau aux AGs. Les hauts niveaux de résistance observés chez certaines souches cliniques pourraient résulter de l'addition de plusieurs mécanismes, ce qui a été démontré chez des doubles, triples et quadruples mutants construits in vitroPseudomonas aeruginosa is an opportunistic pathogen responsible for chronic lung infection in patients with cystic fibrosis. ln order to identify aminoglycoside resistance mechanisms, an insertional library was built in reference strain PAO 1 of P. aeruginosa with a transposon. Screening of 12,000 clones on agar medium containing gentamicin led to the isolation of four groups of aminoglycoside resistant mutants showing either an alteration of the LPS, a defective electron transport chain, a lack in ribosomal protein L25, or overproduction of the efflux system MexXY. Each of these mechanisms promoted a 2-fold increase in the MICs of various aminoglycosides compared to PAO1. When combined, these alterations had addictive or multiplicative effects on resistance to aminoglycosides, resulting in MICs up to 16 fold higher than in PAO1 for clinical relevant drugs such as tobramycin. Altogether, these results show that high level resistance to aminoglycosides in P. aeruginosa may result from the interplays of several low-level resistance mechanismsBESANCON-BU Médecine pharmacie (250562102) / SudocSudocFranceF

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

International audienceThe global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps