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Fluoroquinolone efflux by the plasmid-mediated multidrug efflux pump QacB variant QacBIII in Staphylococcus aureus.

ABSTRACT: Plasmids that carry the multidrug efflux genes qacA and qacB are widely distributed in methicillin-resistant Staphylococcus aureus (MRSA). Although the QacA and QacB proteins are similar to each other, their respective substrate specificities may differ. We investigated the variability and structure-function relationships of QacA and QacB in MRSA isolates. The amino acid sequences of 7 QacA and 25 QacB proteins showed that QacB was present in three variants, designated QacBII, QacBIII, and QacBIV, that were different from the prototypic QacB variant encoded by plasmid pSK23, which was named QacBI, while QacA was present in two variants. When cloned and expressed in S. aureus, the strain carrying qacBIII exhibited higher susceptibility to dyes and decreased susceptibility to norfloxacin and ciprofloxacin compared to strains carrying the other QacB variants. Site-directed mutagenesis experiments revealed that the residue at position 320 in QacB plays an important role in the resistance phenotypes to dyes and fluoroquinolones. Furthermore, the accumulation of norfloxacin and ciprofloxacin in the strain carrying qacBIII was significantly decreased. Our data demonstrate that the plasmid-mediated multidrug efflux pump QacB variant QacBIII confers the capability for fluoroquinolone efflux on S. aureus.

SUBMITTER: Nakaminami H

PROVIDER: S-EPMC2944592 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Fluoroquinolone efflux by the plasmid-mediated multidrug efflux pump QacB variant QacBIII in Staphylococcus aureus.

Nakaminami Hidemasa H Noguchi Norihisa N Sasatsu Masanori M

Antimicrobial agents and chemotherapy 20100726 10

Plasmids that carry the multidrug efflux genes qacA and qacB are widely distributed in methicillin-resistant Staphylococcus aureus (MRSA). Although the QacA and QacB proteins are similar to each other, their respective substrate specificities may differ. We investigated the variability and structure-function relationships of QacA and QacB in MRSA isolates. The amino acid sequences of 7 QacA and 25 QacB proteins showed that QacB was present in three variants, designated QacBII, QacBIII, and QacBI ...[more]

PMID: 20660673

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Project description:Staphylococcus aureus is a versatile human commensal bacteria and pathogen that causes various community and hospital-acquired infections. The S. aureus efflux pump NorA which belongs to the major facilitator superfamily, confers resistance to a range of substrates. Many efflux pump inhibitors (EPIs) have been discovered, but none is clinically approved due to their undesirable toxicities. In this study, we have screened clinically approved drugs for possible NorA EPI-like activity. We identified six drugs that showed the best efflux pump inhibition in vitro, with a fractional inhibitory concentration index of ≤0.5, indicating synergism with hydrophilic fluoroquinolones. The mechanistic validation of efflux inhibitory potential was demonstrated in ethidium bromide-based accumulation and efflux inhibition assays. We further confirmed the functionality of EPIs by norfloxacin accumulation assay depicting more realistic proof of the conjecture. None of the EPIs disturbed membrane function or depleted the ATP synthesis levels in bacteria. Both raloxifene and pyrvinium displayed an increase in bactericidal activity of ciprofloxacin in time-kill kinetics, prolonged its post-antibiotic effect, and reduced the frequency of spontaneous resistant mutant development. The combination of EPIs with ciprofloxacin caused significant eradication of preformed biofilms. Moreover, in the murine thigh infection model, a single dose of pyrvinium combined with ciprofloxacin reduced the bacterial burden significantly compared to untreated control and ciprofloxacin alone, indicating the efficacy of the combination. Conclusively, this study represents approved drugs that can be repurposed and combined with antibiotics as NorA EPIs, having anti-biofilm properties to treat severe S. aureus infections at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus is a frequent pathogen bacterium and the predominant cause of worsened nosocomial infections. Efflux pumps contribute to drug efflux and are reportedly associated with biofilm formation, thereby promoting difficult-to-treat biofilm-associated S. aureus infections. One strategy to combat these bacteria is to reduce active efflux and increase pathogen sensitivity to existing antibiotics. Repurposing approved drugs may solve the classical toxicity issues with previous efflux pump inhibitors and help reach sufficient plasma concentrations. We describe the in silico-based screening of FDA-approved drugs that identified six different molecules able to inhibit NorA pump (Major Facilitator Superfamily). Our study highlights that these compounds bind to and block the activity of the NorA pump and increase the sensitivity of S. aureus and methicillin-resistant S. aureus to fluoroquinolones. These drugs combined with fluoroquinolones significantly reduced the preformed biofilms and displayed significant efficacy in the murine thigh infection model when compared to untreated control and ciprofloxacin alone.

Dataset Information

Fluoroquinolone efflux by the plasmid-mediated multidrug efflux pump QacB variant QacBIII in Staphylococcus aureus.

Publications

Fluoroquinolone efflux by the plasmid-mediated multidrug efflux pump QacB variant QacBIII in Staphylococcus aureus.

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