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First description of an RND-type multidrug efflux pump in Achromobacter xylosoxidans, AxyABM.

ABSTRACT: Achromobacter xylosoxidans is an emerging pathogen in cystic fibrosis patients. The multidrug resistance of these bacteria remains poorly understood. We have characterized in a clinical strain the first resistance-nodulation-cell division (RND)-type multidrug efflux pump in this species: AxyABM. The inactivation of the transporter component axyB gene led to decreased MICs of cephalosporins (except cefepime), aztreonam, nalidixic acid, fluoroquinolones, and chloramphenicol.

SUBMITTER: Bador J

PROVIDER: S-EPMC3186983 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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First description of an RND-type multidrug efflux pump in Achromobacter xylosoxidans, AxyABM.

Bador Julien J Amoureux Lucie L Duez Jean-Marie JM Drabowicz Anthony A Siebor Eliane E Llanes Catherine C Neuwirth Catherine C

Antimicrobial agents and chemotherapy 20110801 10

Achromobacter xylosoxidans is an emerging pathogen in cystic fibrosis patients. The multidrug resistance of these bacteria remains poorly understood. We have characterized in a clinical strain the first resistance-nodulation-cell division (RND)-type multidrug efflux pump in this species: AxyABM. The inactivation of the transporter component axyB gene led to decreased MICs of cephalosporins (except cefepime), aztreonam, nalidixic acid, fluoroquinolones, and chloramphenicol. ...[more]

PMID: 21807978

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Project description:RND (Resistance-Nodulation-Division) family transporters are widespread especially among Gram-negative bacteria, and catalyze the active efflux of many antibiotics and chemotherapeutic agents. They have very large periplasmic domains, and form tripartite complexes with outer membrane channels and periplasmic adaptor proteins. AcrAB-TolC complex of Escherichia coli, which pumps out a very wide range of drugs, has been studied most intensively. Early studies showed that the transporter captures even those substrates that cannot permeate across the cytoplasmic membrane, such as dianionic beta-lactams, suggesting that the capture can occur from the periplasm. It was also suggested that the capture occurs from the cytoplasmic membrane/periplasm interface, because most substrates contain a sizable hydrophobic domain; however, this may simply be a reflection of the nature of the binding site within AcrB. Genetic studies of chimeric transporters showed that much of the substrate specificity is determined by their periplasmic domains. Biochemical studies with intact cells recently led to the determination of the kinetic constants of AcrB for some beta-lactams, and the result confirms the old prediction that AcrB is a rather slow pump. Reconstitution of purified AcrB and its relatives showed that the pump is a drug/proton antiporter, that AcrA strongly stimulates the activity of the pump, and that AcrB seems to have a highest affinity for conjugated bile salts. Structural study with mutants of the network of charged residues in the transmembrane domain showed that protonation here produced a far-reaching conformational change, which was found to be present in one of the protomers in the asymmetric crystal structure of the wild-type AcrB. The functional rotatory hypothesis then predicts that the drug bound in the periplasmic domain is extruded through this conformational change initiated by the protonation of one of the residues in the aforementioned network, an idea that was recently supported by disulfide cross-linking as well as by the behavior of linked AcrB protomers.

Project description:The emergence of multidrug-resistant Klebsiella pneumoniae is a worldwide problem. K. pneumoniae possesses numerous resistant genes in its genome. We isolated mutants resistant to various antimicrobials in vitro and investigated the importance of intrinsic genes in acquired resistance. The isolation frequency of the mutants was 10-7-10-9. Of the multidrug-resistant mutants, hyper-multidrug-resistant mutants (EB256-1, EB256-2, Nov1-8, Nov2-2, and OX128) were identified, and accelerated efflux activity of ethidium from the inside to the outside of the cells was observed in these mutants. Therefore, we hypothesized that the multidrug efflux pump, especially RND-type efflux pump, would be related to changes of the phenotype. We cloned all RND-type multidrug efflux pumps from the K. pneumoniae genome and characterized them. KexEF and KexC were powerful multidrug efflux pumps, in addition to AcrAB, KexD, OqxAB, and EefABC, which were reported previously. It was revealed that the expression of eefA was increased in EB256-1 and EB256-2: the expression of oqxA was increased in OX128; the expression of kexF was increased in Nov2-2. It was found that a region of 1,485 bp upstream of kexF, was deleted in the genome of Nov2-2. K. pneumoniae possesses more potent RND-multidrug efflux systems than E. coli. However, we revealed that most of them did not contribute to the drug resistance of our strain at basic levels of expression. On the other hand, it was also noted that the overexpression of these pumps could lead to multidrug resistance based on exposure to antimicrobial chemicals. We conclude that these pumps may have a role to maintain the intrinsic resistance of K. pneumoniae when they are overexpressed. The antimicrobial chemicals selected many resistant mutants at the same minimum inhibitory concentration (MIC) or a concentration slightly higher than the MIC. These results support the importance of using antibiotics at appropriate concentrations at clinical sites.

Dataset Information

First description of an RND-type multidrug efflux pump in Achromobacter xylosoxidans, AxyABM.

Publications

First description of an RND-type multidrug efflux pump in Achromobacter xylosoxidans, AxyABM.

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