The antibiotic stock solutions were prepared by dissolving them in sterile distilled water at concentrations of 256 μg mL−1 (ampicillin, aztreonam, cefotaxime, cefoxitin, ceftazidime, cephalothin, oxacillin, and piperacillin) and serial

dilution (1 : 2) with TSB (pH 7.3). The strains of S. aureus KACC13236, S. aureus CCARM 3080, S. Typhimurium KCCM 40253, and S. Typhimurium CCARM 8009 were anaerobically cultured in TSB at pH 5.5 and 7.3 to obtain planktonic and biofilm cells. In accordance with the CLSI procedure, the planktonic and biofilm cells grown in Palbociclib order TSB at pH 5.5 and 7.3 were incubated in the diluted antibiotic solutions for 18 h at 37 °C to evaluate the susceptibility of cells to antibiotics. Minimum inhibitory concentrations (MICs) were determined at concentrations at which there was no visible growth. The susceptible (S), intermediate (I), and resistant

(R) strains were defined based on MIC values of < 4 μg mL−1, between 4 and 8 μg mL−1, and more than 16 μg mL−1, respectively (Hamilton-Miller & Shah, 1996). The numbers of planktonic and biofilm cells were CT99021 price estimated using the plate count method. For planktonic cell counts, the cell suspensions were collected and the remaining non-adherent cells were rinsed by flooding the plate surface with 10 mL of 0.1% sterile BPW. For biofilm cell counts, the attached cells were collected with a cell scraper (Thermo Scientific Nunc, Rochester, NY) and suspended by sonication at 20 kHz for 10 min in 20 mL of 0.1% sterile BPW. The collected cells were serially diluted (1 : 10) with 0.1% sterile BPW and the proper dilutions were plated on trypticase soy agar (TSA). The agar plates were incubated at 37 °C for 48 h Ribonucleotide reductase for enumeration of planktonic and biofilm cells. Each planktonic or biofilm culture (0.5 mL) was mixed with 1 mL of RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) and centrifuged at 5000 g for 10 min. The collected cells were used for RNA extraction according to the RNeasy® Mini Handbook (Qiagen). The collected cells were disrupted in a buffer containing guanidine isothiocyanate

and lysozyme, mixed with ethanol to adjust proper binding conditions, and then loaded into an RNeasy mini column for RNA isolation. The cDNA was synthesized as described previously (Xu et al., 2010), according to the QuantiTect Reverse Transcription protocol (Qiagen). In brief, the RNA sample was mixed with a master mixture containing Quantiscript Reverse Transcriptase, Quantiscript RT Buffer, RT Primer Mix and RNase-free water, incubated at 42 °C for 15 min, and then immediately incubated at 95 °C for 3 min to inactivate the Quantiscript Reverse Transcriptase. The custom-synthesized oligonucleotide primers using IDT (Integrated DNA Technologies Inc., Coralville, IA) were used in this study (Tables 1 and 2). The PCR mixture (20 μL) containing 2× QuantiTect SYBR Green PCR Master (10 μL), 60 pmol primer (0.6 μL), cDNA (2 μL), and RNase-free water (6.