This work aimed to assess and characterize the presence of active efflux systems in clinical isolates of S. aureus using several Trichostatin A nmr methodologies and to understand their role in the development of resistance to fluoroquinolones by S. aureus in the clinical setting, Selonsertib in vivo since fluoroquinolones are considered substrates of the majority of the pumps encoded by the S. aureus chromosome [7]. Results Detection of active efflux systems by the Ethidium

Bromide (EtBr)-agar Cartwheel (EtBrCW) Method For this study, we selected all the S. aureus isolates presenting resistance towards ciprofloxacin received by the Bacteriology Laboratory of one of the largest hospitals in Portugal during a four months period. These corresponded

to a collection of 52 S. aureus isolates. Efflux activity amongst these 52 ciprofloxacin resistant isolates was assessed by means of a fast and practical test, the Ethidum Bromide-agar Cartwheel (EtBrCW) Method that provides information RAAS inhibitor on the capacity of each isolate to extrude EtBr from the cells by efflux, on the basis of the fluorescence emitted by cultures swabbed in EtBr-containing agar plates. Those cultures showing fluorescence at lower EtBr concentrations have potentially less active efflux systems than those for which fluorescence is only detected at higher concentrations of EtBr [11, 12]. The application of this method allowed

the selection of 12 S. aureus isolates showing increased EtBr efflux activity when compared to the non-effluxing control strain ATCC25923 and to the efflux-positive control strain next ATCC25923EtBr [13]. These 12 isolates were designated EtBrCW-positive isolates, whereas the remaining 40 isolates were considered to have no or intermediate efflux activity and therefore designated as EtBrCW-negative isolates (Table 1). Table 1 Genotypic and phenotypic characterization of S. aureus clinical isolates.     QRDR mutationsb MIC (mg/L)c         EtBr CIP NOR NAL Isolate a PFGE pattern GrlA GyrA No + + No + + No + + No + +         EI TZ CPZ EI TZ CPZ EI TZ CPZ EI TZ CPZ ATCC25923 – WT WT 6.25 0.75 0.75 0.25 0.125 0.125 0.5 0.125 0.125 64 n.d. n.d. ATCC25923EtBr – WT WT 200 25 12.5 1 0.25 0.25 2 0.25 0.25 64 n.d. n.d.