Panel B: Assessment of EtBr accumulation in the presence of efflux inhibitors. The EIs were tested at a sub-inhibitory concentration, namely TZ: thioridazine (12.5 mg/L); CPZ: chlorpromazine (25 mg/L); VER: verapamil (200 mg/L) and RES: reserpine (20 mg/L). The arrow indicates the EtBr accumulation in the presence of the most effective EI for each isolate. Panel C: Assessment of EtBr efflux. The assays were done in the presence/absence of 0.4% glucose, with or without the EI verapamil (VER) at a sub-inhibitory concentration of 200 mg/L. The data presented was normalized against the data obtained in conditions of no efflux

(absence of glucose and presence of 200 Belnacasan mg/L of VER). The conditions established by the accumulation assays were then used to load cells with EtBr and perform efflux assays. The assessment of EtBr efflux on a real-time basis (during a 10 min frame) detected a considerable difference between EtBrCW-positive isolates, which showed Luminespib a pronounced efflux pump activity, with a prompt and significant decrease in fluorescence and the EtBrCW-negative isolates, that showed only basal efflux pump activity, similar to the one presented by the reference strain (Figure 1-C). These results confirm the presence of increased efflux activity in the EtBrCW-positive

isolates relatively to the EtBrCW-negative isolates. Effect of efflux inhibitors on MICs of fluoroquinolones and EtBr As expected, Carteolol HCl since all clinical isolates were selected on the basis of resistance to ciprofloxacin, they all presented high MIC values for fluoroquinolones. Nevertheless, the majority of the EtBrCW-positive isolates displayed higher MIC values for the fluoroquinolones tested and EtBr, whilst the EtBrCW-negative isolates presented significantly lower values, although some overlap exists between the two sets of MIC values (Table 1). The EIs reduced the MIC values for fluoroquinolones and EtBr of the EtBrCW-positive isolates to the values presented by the EtBrCW-negative

isolates, confirming the presence of an active efflux component in those isolates (Table 1). The EIs thioridazine (TZ) and chlorpromazine (CPZ) were the most effective in reducing the MIC values. Verapamil (VER) and reserpine (RES) showed a smaller or absent inhibitory effect, while carbonyl cyanide m-chlorophenylhydrazone (CCCP) showed no effect on the MIC values for the compounds tested (data not shown). However, no full reversion of the fluoroquinolone resistance phenotype was obtained with any of the EIs tested, suggesting the contribution of other mechanisms to this resistance, namely, mutations in the target genes. Screening for mutations conferring fluoroquinolone resistance The 25 isolates representing both EtBrCW-positive and negative isolates were screened for the presence of chromosomal mutations most commonly associated with fluoroquinolone resistance in S. aureus, namely the ones occurring in the QRDRs of both grlA and gyrA genes [3, 5, 15, 16].