DNA manipulation Plasmid DNA was prepared with the FavorPrep™ Plasmid DNA Extraction Mini Kit (Favorgen, Ping-Tung, Taiwan). A. baumannii genomic DNA was extracted as described previously [38]. PCR amplification of the DNA was performed in

a Thermo Hybaid PXE 0.2 HBPX02 Thermal Cycler (Thermo Scientific, Redwood, CA), using ProTaq™ DNA Polymerase (Protech, Taipei, Taiwan) or the KAPA HiFi™ PCR Kit (Kapa Biosystems, Boston, MA). DNA fragments were extracted from agarose gels and purified using the GeneKlean Gel Recovery & PCR CleanUp Kit (MDBio, Inc., Taipei, Taiwan). Nucleotide sequences of the PCR products were verified using an ABI 3730XL DNA Analyzer (Applied Biosystems, South San Francisco, CA). RNA isolation, RT-PCR, and qRT-PCR For total RNA isolation, A. baumannii ATCC 17978 was

grown overnight in LB broth (37°C, 220 rpm, 16 h) to reach an OD600 of approximately 6.5. The overnight cultures were sub-cultured at a 1:100 dilution www.selleckchem.com/products/epz-6438.html in 25 mL fresh LB medium. The cells were grown to mid-log phase and harvested by centrifugation at 4°C. The cell pellets were resuspended VX-770 supplier in 200 μL ice-cold RNA extraction buffer (0.1 M Tris-Cl [pH 7.5], 0.1 M LiCl, 0.01 M ethylenediaminetetraacetic acid [pH 8.0], 5% sodium dodecyl sulfate [SDS], 2% β-mercaptoethanol), and 200 μL ice-cold phenol-chloroform-isoamyl alcohol (PCIA [25:24:1], pH 4.5) was added and vortexed for 2 min. The supernatants were then collected PD184352 (CI-1040) by centrifugation, added to 200 μL ice-cold PCIA, and mixed well. This step was repeated three times. Then, RNA was precipitated with ethanol at -80°C overnight and collected by centrifugation at maximum speed for 5 min. The RNA pellets were dissolved in 25–100 μL diethylpyrocarbonate-treated water. DNA was removed using Ambion® TURBO™ DNase (Life Technologies, Grand Island, NY), and cDNA was synthesized by reverse transcription using High-Capacity cDNA Reverse Transcriptase

Kits (Applied Biosystems). The cDNAs were used in PCR reactions with different primers (Table  1). qRT-PCR was carried out with a StepOne™ Real-Time PCR System (Life Technologies). The primers used for qRT-PCR are listed in Table  1. Briefly, each 20-μL reaction mixture contained 25 ng cDNA, 10 μL Power SYBR green PCR master mix (Life Technologies), and 300 nM each forward and reverse primer. The reactions were performed with 1 cycle at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The 16S rRNA transcript was used as an endogenous control for the qRT-PCR. The data were analyzed using StepOne v2.1 software (Life Technologies). Induction of tigecycline resistance To induce tigecycline resistance, serial passaging was performed as previously described [39] with some modifications. Briefly, on day 1, 3 mL of LB broth Fedratinib solubility dmso containing tigecycline at the MIC was inoculated with A. baumannii (passage 1), and the cultures were incubated at 37°C with shaking (220 rpm).