Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide.
OBJECTIVE
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.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Unidade de Micobacterias, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (UNL), Portugal.
Isabel
I
Couto
Sofia Santos
SS
Costa
Miguel
M
Viveiros
Marta
M
Martins
Leonard
L
Amaral
D000222:Adaptation, Physiological; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics*; D002746:Chlorpromazine / Q000494:pharmacology; D027081:DNA Gyrase / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004996:Ethidium / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D020869:Gene Expression Profiling; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012110:Reserpine / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D013881:Thioridazine / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D002746:Chlorpromazine; D027081:DNA Gyrase; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D004996:Ethidium; D005456:Fluorescent Dyes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012110:Reserpine; D013881:Thioridazine; C075040:NorA protein, Staphylococcus