When these clinical isolates and ATCC25923 were exposed to an efflux pump substrate, either ciprofloxacin or EtBr, at ½ their

MICs, and gene expression levels determined against the respective unexposed condition, overexpression of efflux pump genes was detected in six clinical isolates, three EtBrCW-negative and three EtBrCW-positive as well as in the reference strain itself (Table 2). Table 2 EP gene expression analysis by RT-qPCR of representative S. aureus exposed to CIP or EtBr.   Overexpression levels* and no. of isolates** showing gene overexpression   ½ CIP MIC ½ EtBr MIC     EtBrCW- RG-7388 EtBrCW+   EtBrCW- EtBrCW+ Gene ATCC25923 isolates isolates ATCC25923 isolates isolates     (n = 4) (n = 6)   (n = 4) (n = 6) norA – - – 4.51 ± 0.77 – -     0 0   0 0 norB 13.80 ± 6.50 5.43 ± 2.39 5.47 ± 0.19 7.07 ± 2.78 5.33 ± 0.73 –     2 a, b 1 e   1 a 0 norC – - 4.92 ± 0.00 5.89 ± 0.71 4.99 ± 1.51 –     0 1 e   1 a 0 mepA – - 8.59 ± 0.59 3.90 ± 0.13 5.94 ± 1.02 –     0 1 f   1 a 0 mdeA – 4.97 ± 0.68 – 3.96 ± 2.10 – 4.15 ± 1.12     1 c 0   0 1 d smr n.a. n.a. – n.a. n.a. 7.66 ± 3.66       0     1 f * Gene expression was measured in the presence of ciprofloxacin and EtBr relatively to the drug-free condition. The results are expressed in terms of the mean ± standard deviation of at least three independent assays performed GSK1120212 datasheet with independently extracted RNAs and correspond

to the range of values obtained for isolates showing overexpression of that gene. **The numbers Carnitine palmitoyltransferase II in bold correspond to the number of isolates overexpressing that gene: a isolate SM2; b SM3; c SM5; dSM25; e SM50; f SM52. Overexpression was considered for values ≥4 [10]. (-): no overexpression was detected; n.a.: not applicable. The majority of the isolates showed overexpression of a single efflux pump gene, most frequently, norB or mdeA. One isolate showed overexpression of two efflux pump genes (norB/norC) and another one overexpressed three EP genes (norB/norC/mepA).

Overall, isolates showed to be more responsive to ciprofloxacin. The smr gene was found to be overexpressed only in the presence of EtBr, in accordance to the substrate specificity described in the literature for this pump [18]. These same agents had a distinct effect on ATCC25923, which showed significant overexpression of all efflux pump genes tested in the presence of EtBr, and a higher overexpression of norB when exposed to ciprofloxacin (Table 2). The effect of drug exposure on the expression level of the efflux pump genes was further explored by increasing the ciprofloxacin concentration to ¾ the MIC. Isolates that showed EP gene overexpression with ½ the MIC of ciprofloxacin showed either an increase in that expression level or the overexpression of additional genes. For instance, EtBrCW-positive isolate SM50 overexpressing norB/norC with ½ MIC of ciprofloxacin, now showed even higher expression of norB (37.