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 this website 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 CP 690550 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 Sinomenine value was observed at 4 h, which was learn more 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.