67) and norC (83.98 ± 19.98) and de novo overexpression of norA (8.36 ± 4.63) and mepA (45.86 ± 13.86). Likewise, exposure of the EtBrCW-negative SM2 to higher ciprofloxacin
concentrations resulted in increased levels of norB expression (4.48 ± 2.48) that was accompanied by de novo overexpression of norC (5.33 ± 0.73) and mepA (10.58 ± 0.73). Discussion The few studies on efflux among S. aureus clinical isolates use the decrease of antibiotic MICs in the presence of EIs, particularly reserpine, as indicative of efflux activity [10]. This approach is laborious and dependent on the susceptibility of the efflux system(s) to reserpine, which varies considerably [19]. More recently, Patel and colleagues have proposed the use of EtBr MICs to identify S.
aureus effluxing strains [20]. This approach has the advantage of assessing efflux activity using a broad range efflux pump substrate, EtBr, which CFTRinh-172 solubility dmso is pumped out by most efflux systems described for S. aureus, and thus, is an useful marker for the detection of efflux pump activity [7, 12, 14, 20]. Nevertheless, it is still an indirect assessment of efflux activity. Selleckchem BEZ235 In the present study, we have applied two methods for the direct assessment of efflux activity among a collection of 52 ciprofloxacin resistant S. aureus clinical isolates, both also based on EtBr efflux. We first applied the EtBr-agar Cartwheel Method to check details select isolates with increased efflux activity. The presence of increased efflux in the 12 isolates selected was supported by the data collected from the semi-automated fluorometric method, which demonstrated that EtBrCW-positive isolates had a higher efflux activity than the EtBrCW-negative isolates. Thus, both methods proved to be adequate to assay efflux activity in S. aureus cells. In particular, the EtBrCW method proved to be a valuable tool for the rapid screening of efflux pump activity, allowing its application to screen large collections of clinical isolates. Thiamet G Furthermore, the use of a broad range efflux pump substrate such as EtBr warrants its wider application as compared to the analysis of EIs effect
on MIC values, which can be severely impaired by the susceptibility of each efflux system to the EI being used and for which the mechanism of action at the cellular level remains, in most cases, to be clarified. In addition, the semi-automated fluorometric method also allowed the characterization of this efflux activity, in terms of maximal concentration of EtBr that the cells were able to extrude without observable accumulation over a 60 min period and susceptibility toward several EIs. The results obtained clearly showed a distinct capacity of the two groups of isolates to extrude EtBr from their cells, with the EtBrCW-positive isolates being able to handle higher EtBr concentrations with no detectable accumulation. It was also observed that for both groups of isolates, EtBr extrusion/accumulation was most affected by the EI verapamil.