Abstract
By adapting an antibiotic-susceptible Staphylococcus aureus strain to increasing concentrations of ethidium bromide, a known substrate of efflux pumps (EPs), and by phenotypically and genotypically analysing the resulting progeny, we characterized the molecular mechanisms of S. aureus adaptation to ethidium bromide. S. aureus ATCC 25923 was grown in increasing concentrations of ethidium bromide. The MICs of representatives of eight classes of antibiotics, eight biocides and two dyes against ATCC 25923 and its ethidium bromide-resistant progeny ATCC 25923(EtBr) were determined with or without six efflux pump inhibitors (EPIs). Efflux activity in the presence/absence of EPIs was evaluated by real-time fluorometry. The presence and expression of eight EP genes were assayed by PCR and quantitative RT-PCR (qRT-PCR), respectively. Mutations in grlA, gyrA and norA promoter regions were screened by DNA sequencing. Compared with its parental strain, ATCC 25923(EtBr) was 32-fold more resistant to ethidium bromide and also more resistant to biocides and hydrophilic fluoroquinolones. Resistance to these could be reduced by the EPIs chlorpromazine, thioridazine and reserpine. Increased efflux of ethidium bromide by ATCC 25923(EtBr) could be inhibited by the same EPIs. qRT-PCR showed that norA was 35-fold over-expressed in ATCC 25923(EtBr), whereas the remaining EP genes showed no significant increase in their expression. Sequencing of the norA promoter region revealed a 70 bp deletion in ATCC 25923(EtBr). Exposure of S. aureus to quaternary compounds such as ethidium bromide results in decreased susceptibility of the organism to a wide variety of compounds, including quinolones and biocides through an efflux-mediated response, which for strain ATCC 25923 is mainly NorA-mediated. This altered expression may result from alterations in the norA promoter region.
Highlights
Staphylococcus aureus is one of the most common human pathogens, being responsible for a wide variety of infections, many of which can be life-threatening
The study was conducted with S. aureus strain ATCC 25923, a clinical isolate collected at Seattle in 1945, fully antibiotic-susceptible, b-lactamase-negative, with an ethidium bromide MIC of 6.25 mg/L
After 82 days, the resistance of this strain to ethidium bromide was raised from an MIC of 6.25 to 200 mg/L and this ethidium bromide-adapted culture was designated as ATCC 25923EtBr
Summary
Staphylococcus aureus is one of the most common human pathogens, being responsible for a wide variety of infections, many of which can be life-threatening. This bacterium shows many different mechanisms of resistance towards antibiotics. Several of these mechanisms are well known, and have been characterized: resistance to b-lactam antibiotics mediated by PBP2a, encoded by the mecA gene, or resistance to fluoroquinolones resulting from mutations in either topoisomerase IV or gyrase genes.[1] On the other hand, antibiotic resistance based on efflux systems capable of extruding the drug or other noxious agents from the cell is less well characterized for these bacteria. Most of these pumps belong to the major facilitator superfamily, namely the chromosomally encoded NorA, NorB, NorC, MdeA and SdrM as well as the plasmid-encoded QacA/B pumps.[3,4,5,6,7,8] Other types of pumps have been described for S. aureus such as MepA, a member of Ethidium bromide-induced efflux in S. aureus the multidrug and toxic compound extrusion family, as well as Smr, which belongs to the small multidrug resistance (SMR) family, and SepA.[9,10,11] these pumps show different substrate specificity, most of them are capable of extruding compounds of different chemical classes, providing the cell with the means to develop a multidrug resistance (MDR) phenotype or to survive in a hostile environment
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