Contribution of Resistance-Nodulation-Cell Division Efflux Systems to Antibiotic Resistance and Biofilm Formation in Acinetobacter baumannii

International audienceABSTRACT : Acinetobacter baumannii is a nosocomial pathogen of increasing importance due to its multiple resistance to antibiotics and ability to survive in the hospital environment linked to its capacity to form biofilms. To fully characterize the contribution of AdeABC, AdeFGH, and AdeIJK resistance-nodulation-cell division (RND)-type efflux systems to acquired and intrinsic resistance, we constructed, from an entirely sequenced susceptible A. baumannii strain, a set of isogenic mutants overexpressing each system following introduction of a point mutation in their cognate regulator or a deletion for the pump by allelic replacement. Pairwise comparison of every derivative with the parental strain indicated that AdeABC and AdeFGH are tightly regulated and contribute to acquisition of antibiotic resistance when overproduced. AdeABC had a broad substrate range, including β-lactams, fluoroquinolones, tetracyclines-tigecycline, macrolides-lincosamides, and chloramphenicol, and conferred clinical resistance to aminoglycosides. Importantly, when combined with enzymatic resistance to carbapenems and aminoglycosides, this pump contributed in a synergistic fashion to the level of resistance of the host. In contrast, AdeIJK was expressed constitutively and was responsible for intrinsic resistance to the same major drug classes as AdeABC as well as antifolates and fusidic acid. Surprisingly, overproduction of AdeABC and AdeIJK altered bacterial membrane composition, resulting in decreased biofilm formation but not motility. Natural transformation and plasmid transfer were diminished in recipients overproducing AdeABC. It thus appears that alteration in the expression of efflux systems leads to multiple changes in the relationship between the host and its environment, in addition to antibiotic resistance.IMPORTANCE: Increased expression of chromosomal genes for RND-type efflux systems plays a major role in bacterial multidrug resistance. Acinetobacter baumannii has recently emerged as an important human pathogen responsible for epidemics of hospital-acquired infections. Besides its remarkable ability to horizontally acquire resistance determinants, it has a broad intrinsic resistance due to low membrane permeability, endogenous resistance genes, and antibiotic efflux. The study of isogenic mutants from a susceptible A. baumannii clinical isolate overproducing or deleted for each of the three major RND-type pumps demonstrated their major contribution to intrinsic resistance and to the synergism between overproduction of an efflux system and acquisition of a resistance gene. We have also shown that modulation of expression of the structural genes for the efflux systems results in numerous alterations in membrane-associated cellular functions, in particular, in a decrease in biofilm formation and resistance gene acquisition

Fitness Cost of VanA-Type Vancomycin Resistance in Methicillin-Resistant Staphylococcus aureus▿

We have quantified the biological cost of VanA-type glycopeptide resistance due to the acquisition of the resistance operon by methicillin-resistant Staphylococcus aureus (MRSA) from Enterococcus sp. Exponential growths of recipient strain HIP11713, its transconjugant VRSA-1, VRSA-5, and VRSA-6 were compared in the absence or, except for HIP11713, in the presence of vancomycin. Induction of resistance was performed by adding vancomycin in both the preculture and the culture or the culture at only 1/50 the MIC. In the absence of vancomycin, the growth rates of the vancomycin-resistant S. aureus (VRSA) strains were similar to that of susceptible MRSA strain HIP11713. When resistance was induced, and under both conditions, there was a significant reduction of the growth rate of the VRSA strains relative to that of HIP11713 and to those of their noninduced counterparts, corresponding to a ca. 20% to 38% reduction in fitness. Competition experiments between isogenic VRSA-1 and HIP11713 mixed at a 1:1, 1:100, or 100:1 ratio revealed a competitive disadvantage of 0.4% to 3% per 10 generations of the transconjugant versus the recipient. This slight fitness burden can be attributed to the basal level of expression of the van genes in the absence of induction combined with a gene dosage effect due to the presence of the van operon on multicopy plasmids. These data indicate that VanA-type resistance, when induced, is highly costly for the MRSA host, whereas in the absence of induction, its biological cost is minimal. Thus, the potential for the dissemination of VRSA clinical isolates should not be underestimated

Coût biologique de la résistance à la vancomycine chez les cocci à Gram positif

Le coût biologique est un des facteurs qui influence la stabilité et la dissémination de la résistance des bactéries aux antibiotiques. La résistance acquise aux glycopeptides de type VanA et VanB est due à un mécanisme sophistiqué impliquant 5 gènes de résistance (vanHAXYZ ou vanYBWHB,BXB) qui combinent la synthèse de précurseurs du peptidoglycane modifiés avec l élimination de la voie de synthèse chromosomique des précurseurs sensibles, et 2 gènes de regulation (vanRS ou vanRBSB) codant pour un système à deux composantes responsables de l expression inductible des gènes de résistance. Le coût biologique résultant de ce mécanisme de résistance est présumé élevé. Pourtant, les entérocoques résistants à la vancomycine (VRE) ont disséminé à travers le monde. En revanche, les souches de Staphylococcus aureus résistants à la méthicilline (MRSA) ayant acquis récemment les gènes van des entérocoques (VRSA) ont étonnamment faiblement disséminé. La détermination de la vitesse de croissance de la souche réceptrice de MRSA et son transconjugant VRSA-1 a révélé une réduction minimale de la vitesse de croissance de VRSA-1 par rapport à la souche sensible de MRSA en absence d induction tandis qu en absence d induction, une importante réduction est observée pour VRSA-1 et deux autres souches, VRSA-5 et VRSA-6, par rapport à la souche non-induite correspondante. En revanche, en compétition avec la souche réceptrice de MRSA, VRSA-1 présente un désavantage de compétitivité qui a été attribué au niveau basal d expression des gènes vanA combiné avec un effet de dosage de gènes dû à la présence de l opéron van sur un plasmide multicopie. Ce désavantage compétitif de la souche résistante versus la souche sensible pourrait participer à la faible dissémination des VRSA. Le coût biologique de la résistance de type VanB due à l acquisition du transposon conjugatif Tn1549 a été évalué in vitro et in vivo. L impact de la présence du transposon étant variable suivant le site d intégration dans le chromosome, plusieurs dérivés isogéniques de E. faecalis JH2-2 ont été construits afin de déterminer le coût de l expression inductible et constitutive de l opéron vanB ou le coût dû à l acquisition de Tn1549. Le gène lucR de la luciférase issue de la luciole Photinus pyralis, utilisé comme gène rapporteur après integration dans le chromosome des souches isogéniques, a permis de les différencier en co-culture in vitro et d évaluer leur fitness dans un modèle de souris gnotobiotique. La présence de Tn1549 non-induit n est pas coûteuse pour l hôte tandis que l induction par la vancomycine ou l expression constitutive de la résistance de type VanB entraîne une réduction significative de la fitness de E. faecalis à la fois in vitro et in vivo, ce qui est en accord avec le faible nombre de souches constitutives rapportées à ce jour en clinique. Même si le coût biologique, à lui seul, n est pas suffisant pour élucider la capacité de dissémination de la résistance, ces résultats montrent que la régulation inductible des mécanismes de résistance de type VanA et VanB s avère très efficace pour réduire le coût énergétique de son expression et permet ainsi le maintien de la résistance en absence de pression de sélection.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Introduction

Le présent volume est le produit d’une initiative de recherche lancée par le Groupe de recherche sur les pouvoirs et sociétés dans l’Occident médiéval et moderne (Grepsomm) en 2006-2008, sur le thème général de « L’écrit et la ville ». Fondé en 2004, le Grepsomm s’intéresse, dans une perspective pluridisciplinaire, aux fondements du lien social et politique durant la longue période de réorganisation de la société occidentale, depuis la fragmentation féodale des pouvoirs au xie siècle jusqu’à ..

Contribution of the Ade Resistance-Nodulation-Cell Division-Type Efflux Pumps to Fitness and Pathogenesis of Acinetobacter baumannii

Overexpression of chromosomal resistance-nodulation-cell division (RND)-type efflux systems with broad substrate specificity contributes to multidrug resistance (MDR) in Acinetobacter baumannii. We have shown that modulation of expression of the structural genes for the efflux systems AdeABC and AdeIJK confers MDR and results in numerous alterations of membrane-associated cellular functions, in particular biofilm formation. However, the contribution of these RND pumps to cell fitness and virulence has not yet been studied. The biological cost of an antibiotic resistance mechanism is a key parameter in determining its stability and dissemination. From an entirely sequenced susceptible clinical isolate, we have generated a set of isogenic derivatives having single point mutations resulting in overexpression of each efflux system or with every pump deleted by allelic replacement. We found that overproduction of the pumps results in a significant decrease in fitness of the bacterial host when measured by competition experiments in vitro. Fitness and virulence were also evaluated in vivo both in systemic and pulmonary infection models in immunocompetent mice. A diminished competitiveness of the AdeABC-overexpressing mutant was observed only after intraperitoneal inoculation, but not after intranasal inoculation, the latter mimicking the most frequent type of human A. baumannii infection. However, in mice infected intranasally, this mutant was more virulent and stimulated an enhanced neutrophil activation in the lungs. Altogether, these data account for the observation that adeABC overexpression is common in MDR A. baumannii frequently found in ventilator-associated pneumonia

Aminoglycoside resistance 16S rRNA methyltransferases block endogenous methylation, affect translation efficiency and fitness of the host.

International audienceIn Gram-negative bacteria, acquired 16S rRNA methyltransferases ArmA and NpmA confer high-level resistance to all clinically useful aminoglycosides by modifying, respectively, G1405 and A1408 in the A-site. These enzymes must coexist with several endogenous methyltransferases that are essential for fine-tuning of the decoding center, such as RsmH and RsmI in Escherichia coli, which methylate C1402 and RsmF C1407. The resistance methyltransferases have a contrasting distribution-ArmA has spread worldwide, whereas a single clinical isolate producing NpmA has been reported. The rate of dissemination of resistance depends on the fitness cost associated with its expression. We have compared ArmA and NpmA in isogenic Escherichia coli harboring the corresponding structural genes and their inactive point mutants cloned under the control of their native constitutive promoter in the stable plasmid pGB2. Growth rate determination and competition experiments showed that ArmA had a fitness cost due to methylation of G1405, whereas NpmA conferred only a slight disadvantage to the host due to production of the enzyme. MALDI MS indicated that ArmA impeded one of the methylations at C1402 by RsmI, and not at C1407 as previously proposed, whereas NpmA blocked the activity of RsmF at C1407. A dual luciferase assay showed that methylation at G1405 and A1408 and lack of methylation at C1407 affect translation accuracy. These results indicate that resistance methyltransferases impair endogenous methylation with different consequences on cell fitness

Bacterial and Host Factors Implicated in Nasal Carriage of Methicillin-Resistant Staphylococcus aureus in Mice

Nasal carriage is a major risk factor for Staphylococcus aureus infection, especially for methicillin-resistant strains (MRSA). Using a mouse model of nasal carriage, we have compared several S. aureus strains and demonstrated increased colonization levels by MRSA in cystic fibrosis transmembrane conductance regulator-deficient mice and Toll-like receptor 2 (TLR2)-deficient mice but not TLR4-deficient mice