9-, 2.1-, and 3-fold higher, respectively, than those in ATCC 17978, while the deletion of baeR in the wild-type strain decreased the expression levels of these three pump genes by 68.3%, 67.3%, and 73.5%, respectively. The decreased expression of the pump genes can be partially restored by baeR reconstitution. (B) The expression levels of adeB, adeA1, and adeA2 in ABtcm were 51.5%, 42.7%, and 43.7% lower, respectively, than those in ABtc. 16S rRNA gene was used as a control. The results are displayed as the means ± SD from three independent experiments. *, P < 0.05; ***, P < 0.001. #, P < 0.05 between ABtc and ABtcm. Expression analysis of adeAB in induced tigecycline-resistant A.

baumannii and its baeR mutant To further confirm the role of baeR in the tigecycline resistance of A. baumannii via the AdeAB efflux pump, a baeR deletion mutant selleck chemicals of ABtc (ABtcm) was constructed and adeAB expression was analyzed by qRT-PCR. The expression levels of adeB, adeA1, and adeA2 in ABtcm were 51.5, 42.7%, and 43.7% lower, respectively, than those in ABtc (Figure  4B). These data confirmed the contribution of BaeR to the regulation of AdeAB, which is essential to tigecycline resistance in A. baumannii. Time-kill assay To further compare the effects of BaeR on tigecycline susceptibility, time-kill assays were performed using ATCC 17978, AB1026, AB1027, and AB1028. There were CX-5461 no differences in the surviving

colony forming units (CFUs) among these four strains when tigecycline was not added to the LB agar. In the presence of 0.25 μg/mL tigecycline, all tested strains had similar surviving CFU curves; the lowest why value was observed at 4 h, which was followed by regrowth (Figure  5A). Additionally, AB1026 showed a greater reduction in CFUs than the wild-type strain (e.g., 2.9-log10 versus 1.8-log10 reduction, respectively, at 4 h) throughout the assay period, which could be restored by baeR reconstitution. Increasing the tigecycline concentration to 0.5 μg/mL

produced an even more marked 4.7-log10 reduction in the CFUs of AB1026 at 8 h, which was followed by regrowth. In contrast, a smaller reduction (2.1-log10 reduction at 8 h) was observed for the wild-type strain (Figure  5B). However, baeR reconstitution did not fully restore the ability of AB1026 to resist 0.5 μg/mL tigecycline. AB1028 showed a slightly smaller reduction in CFUs than the wild-type strain in the presence of 0.25 and 0.5 μg/mL tigecycline. Therefore, the time-kill assay indicates that the BaeSR TCS plays a role in the tigecycline susceptibility of A. baumannii. Figure 5 Time-kill assays for ATCC 17978, AB1026, AB1027, and AB1028 with 0.25 μ g/mL (A) and 0.5 μ g/mL (B) tigecycline. In the presence of 0.25 μg/mL tigecycline, all tested strains showed similar surviving colony forming unit (CFU) curves, in which the lowest value occurred at 4 h and was followed by regrowth.