Successful construction of the AB1027 and AB1028 strains was verified by RT-PCR. The expression of baeR was comparable in the MLN2238 in vitro Wild-type and the baeR-reconstituted AB1027 strains, whereas baeR was overexpressed in AB1028 relative to the wild-type strain (data not shown). Table 2 Bacterial strains PLX4032 concentration and plasmids used in this study Strain or plasmid Relevant feature(s) Source or reference A. baumannii strains ATCC 17978 Wild-type strain ATCC AB1026 (ΔbaeR::kan r ) Derived from ATCC 17978. baeR mutant obtained by kan r gene replacement This study AB1027 AB1026 baeR::pWH1266 This study AB1028 ATCC 17978 baeR::pWH1266 This study AB1029 ATCC 17978 kan:: pWH1266 This study
ABtc Induced tigecycline resistant ATCC 17978 This study ABtcm (ΔbaeR::kan r ) Derived from ABtc. baeR mutant obtained by kan r gene replacement This study
ABhl1 Tigecycline resistant clinical isolate This study E. coli strains XL1 blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F’ proAB lacI q ZΔM15 Tn10 (Tetr)] Stratagene S17-1 (ATCC 47055) thi pro hsdR hsdM recA[RP42-Tc::Mu- Km::Tn7 (TprSmr)Tra+] ATCC Plasmids pEX18Tc Suicide vector containing sacB, Tcr 40 pSFS2A Containing kan r , an FRT site, FLP1, and CaSAT1 as a SAT1 flipper 41 pEX18Tc-Δbae::kan r pEX18Tc containing baeR upstream and downstream fragments joined by a kan r cassette This study pWH1266 (ATCC 77092) E. coli-A. baumannii shuttle cloning vector, containing Ampr, Tetr 43 pC2HP Provided kan r for pWH1266 42 pWH1266-kan r pWH1266 check details containing kan r This study pWH1266-kan r -baeR pWH1266-kan r containing baeR This study Minimal inhibitory concentration (MIC) determination To correlate BaeR with tigecycline susceptibility, the MIC of tigecycline was determined. For A. baumannii ATCC 17978, the MIC of tigecycline was 0.5 μg/mL. However, the MIC of tigecycline for the baeR deletion mutant was 0.25 μg/mL; baeR reconstitution Thymidylate synthase restored the MIC to the wild-type level (MIC 0.5 μg/mL).
Moreover, the overexpression of baeR in AB1028 raised the MIC of tigecycline to 1 μg/mL. The introduction of pWH1266 alone did not affect the MIC of tigecycline, whereas the MICs obtained with the induced tigecycline-resistant strain ABtc and the clinical tigecycline-resistant strain ABhl1 were 256 and 16 μg/mL, respectively. These results indicate that BaeR is closely related to the tigecycline susceptibility of A. baumannii. Expression of the adeAB and baeSR genes in strains with different levels of tigecycline resistance To further decipher the role of the BaeSR TCS and AdeAB in tigecycline resistance, we analyzed gene expression in the wild-type A. baumannii strain ATCC 17978 as well as the ABtc and ABhl1 strains. The quantitative real-time PCR (qRT-PCR) results showed that the expression levels of adeB were 216- and 53-fold higher in ABtc and ABhl1, respectively, than in the wild-type strain.