The overexpression and baeR-reconstituted strains were selected on LB agar containing 10 μg/mL tetracycline and were further verified by PCR (Additional file 5: Figure S5D) and RT-PCR (Additional file 2: Figure S2). Southern blot hybridization Southern blot analysis was performed as reported in a previous publication [45]. Genomic DNA was extracted, and approximately 10 μg was digested with BclI overnight at 50°C. The DNA was then separated on a 0.8% agarose gel containing 1:10,000 SYBR Safe gel stain (Invitrogen, Grand Island, NY), transferred onto a positively
charged nylon CBL0137 membrane (Pall Corporation, Port Washington, NY) via the alkaline transfer method [38], and fixed by baking at 80°C for 2 h. The membrane was hybridized with an [α-32P] dCTP-labeled baeS probe (Additional file 3:
Figure S3A) using prehybridization buffer (6× saline sodium citrate [SSC; 1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 5× Denhardt’s reagent, 0.5% SDS, 100 μg/mL salmon sperm DNA, and 50% formamide) at 42°C overnight. The membrane was then washed and visualized by autoradiography. Time-kill assay The time-kill assays were carried out in duplicate as previously described [46] with some modifications. Briefly, cells were grown to log phase and sub-cultured into 10 mL CAMHB broth without (control) or with tigecycline (0.25 or 0.5 μg/mL) to a cell density of approximately 5 × 105 CFU/mL. The cultures were incubated in an ambient atmosphere SIS3 solubility dmso at 37°C. At different time points (0, 4, 8, 12, and 16 h) after inoculation, 0.1 mL of the culture was removed from each tube and 10-fold serially diluted. Then, 25 μL of each diluted cell suspension was
spotted onto LB agar in duplicate. Viable cell counts were determined, the duplicates were averaged, and the data were plotted. Acknowledgements This study was supported by a grant from the National Venetoclax chemical structure Taiwan University Hospital, Chu-Tung Branch. The authors also thank Dr. Kia-Chih Chang (Tzu Chi University, Taiwan) for providing the clinical A. baumannii strains and Dr. Ming-Li Liou (Yuanpei University, Taiwan) for providing the wild-type strain. We also thank Jeng-Yi Chen for his technical assistance. Electronic supplementary material Additional file 1: Figure S1.: Verification of the baeR deletion mutants. (A) Diagram of the baeR gene and deletion mutant verification using appropriate primers. (B) Successful baeR gene fragment deletion was deduced based on a change in the PCR band size from 4539 bp to 4884 bp. (TIFF 2 MB) Additional file 2: Figure S2.: Southern blot analysis. (A) Genomic DNA from the baeR deletion mutant and the parental strain was digested by BclI. The location of the specific DNA probe is shown. (B) The bands corresponding to 6.7-kb and 2.8-kb 4-Hydroxytamoxifen datasheet fragments are indicated. Four independent clones of AB1026 are included.