Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide
Abstract number: P2044
Couto I., Costa S.S., Viveiros M., Martins M., Amaral L.
Objectives: Although over 10 efflux pumps (EPs) are described for S. aureus, their specific role in drug-response is scarcely characterised. Adapting S. aureus to increasing concentrations of a known substrate of EPs, ethidium bromide, in order to increase EPs intrinsic activity and characterising phenotypically and genotypically the resulting progeny, we were able to characterize the molecular mechanisms of adaptation of S. aureus to an EP substrate.
Methods:S. aureus ATCC25923 was grown in increasing, 2-fold concentrations of ethidium bromide (EB), originating ATCC25923-EB. The MICs of 8 classes of antibiotics, 8 biocides and 2 dyes against ATCC25923 and ATCC25923-EB were determined by broth microdilution (CLSI), with/without efflux pumps inhibitors (EPIs) chlorpromazine (CPZ), thioridazine (TZ), reserpine (RES), verapamil (VER), ouabain (OUA) and carbonyl cyanide m-chlorophenylhydrazone (CCCP). Efflux activity, in EPIs presence/absence, was evaluated by a semi-automated real-time fluorometric method. The presence and expression of 8 EP genes were assayed by PCR and RT-PCR, respectively. Mutations in both grlA and gyrA quinolone resistance coding regions (QRDR), as well as in the norA promoter region, were assayed by DNA sequencing.
Results: Compared to its parental strain, ATCC25923-EB had a 32-times higher MIC for EB (6.25 to 200 mg/L), as well as decreased susceptibility to biocides and hydrophilic fluoroquinolones; all of which could be reduced by the EPIs TZ, CPZ and RES. Whereas ATCC25923 showed no detectable efflux activity, ATCC25923-EB had pronounced efflux activity which was inhibited by TZ, CPZ and RES in a concentration-dependent manner. Both ATCC25923 and ATCC25923-EB carried norA, norB, norC, mdeA, mepA and sepA, whereas the genes for EPs QacA and Smr were not detected. RT-PCR showed that norA is 35-fold over-expressed in the ATCC25923-EB culture, whereas the remaining EP genes showed no significant increase in their expression. Sequencing of the norA promoter region detected a 70 bp deletion in ATCC25923-EB. No mutations were detected in grlA and gyrA QRDR regions.
Conclusion: Exposure of S. aureus strains to quaternary compounds such as EB results in a decreased susceptibility of the organism towards a wide variety of compounds, including quinolones, biocides and dyes through an efflux-mediated response, which for strain ATCC25923, is mainly NorA-mediated. This altered expression may result from alterations in norA promoter region.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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