On day 5, 30 μL of the culture was transferred into LB broth containing tigecycline at 16× the MIC (passage 3), and the cultures

were again incubated at 37°C with shaking (220 rpm). This passaging was repeated on day 7 (passage 4). On day 9, aliquots (3 mL) of the cultures were mixed with 10% glycerol and stored at -80°C until use. Daily passaging in tigecycline-free LB was conducted for 30 days for both ATCC 17978 and the clinical strain. Construction of baeR deletion mutants and baeR reconstituted strains To assess the contribution of BaeR to the regulation of tigecycline resistance, baeR deletion mutants of P5091 datasheet A. baumannii ATCC 17978 were constructed as previously described [23] with some modifications. The suicide vector pEX18Tc [40] was first cloned with a 953-bp DNA fragment carrying a kanamycin resistance cassette, which was PCR-amplified from the pSFS2A plasmid [41], to generate pEX18Tc-kan r . DNA fragments carrying the upstream and downstream regions of the baeR gene, referred to as baeR-up and baeR-dw, were independently amplified by PCR using the primer pairs baeR-up-SalI-F and baeR-up-BamHI-R or baeR-dw-KpnI-F and baeR-dw-SacI-R (Table  1). The baeR-up fragment (1,119 bp) was digested with SalI and BamHI enzymes, whereas SB-715992 the baeR-dw fragment (1,120 bp) was digested with KpnI and SacI enzymes (Additional file 4: Figure S4A). Both enzyme-digested DNA fragments

were then independently cloned into the corresponding restriction sites of pEX18Tc-kan r , generating pEX18Tc-kan r -baeR-flanking. The resultant plasmid was then transformed into the E. coli S17-1 strain using the standard CaCl2/heat shock method [38]. Then, trans-conjugation was performed between E. coli S17-1 donor cells and A. baumannii ATCC

17978 recipient cells Tobramycin to transfer and integrate pEX18Tc-kan r -baeR-flanking into the chromosome of ATCC 17978 (Additional file 4: Figure S4B). By growing the ATCC 17978 conjugate cells on LB agar containing 10% sucrose, the cells were able to resolve the suicide plasmid pEX18Tc (Additional file 4: Figure S4C). Sucrose-resistant colonies were examined to verify that they had the kanamycin-resistant phenotype as a Natural Product Library clinical trial result of plasmid eviction. The absence of the baeR gene sequence in the genome was verified by PCR and RT-PCR and further confirmed by Southern blot hybridization. To reconstitute the baeR gene in the baeR-deleted mutants, a DNA fragment carrying the entire baeR gene sequence was generated by PCR using the genomic DNA of A. baumannii ATCC 17978 as the template. Briefly, a kanamycin resistance cassette was first amplified from the pC2HP vector [42] and cloned into the E. coli/Acinetobacter shuttle vector pWH1266 [43, 44] (Additional file 5: Figure S5A and S5B). Subsequently, the baeR DNA fragment was cloned into the XbaI/XhoI restriction sites (Additional file 5: Figure S5C).