Bacterial strains, growth conditions, and minimum inhibitory concentration (MIC) determination
For in vitro resistance-selection studies, P. aeruginosa ATCC27853 was obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in Mueller–Hinton broth (MHB) or on Mueller–Hinton agar plates for 18 h to 24 h in a 5% CO2 atmosphere at 37°C. Antibiotic susceptibility to ciprofloxacin and levofloxacin was determined by the agar dilution method according to Clinical & Laboratory Standards Institute (CLSI)-approved standards. MIC determination was performed in triplicate, using the same experimental conditions for all lineages.
Selection of FQ-resistant P. aeruginosa populations by serial passages
Six independent lineages (L1–L6) for ciprofloxacin and levofloxacin were established and propagated by serial passages with increasing concentrations of antibiotics. Selection of 200 μL of bacteria was performed in flasks containing 20 mL MHB supplemented with various concentration gradients of ciprofloxacin and levofloxacin, starting with an initial concentration of 0.5MIC. Bacteria were sub-cultured for 24 h at 37°C in the flasks, and resistant bacteria were serially transferred to new flasks containing fresh medium with 2-fold serial antibiotic concentrations. The procedure was repeated until growth was markedly inhibited. At each selection passage, a sample of the bacterial population from each passage was collected with 15% sterilized glycerol and frozen in cryotubes at −80°C.
Bacterial DNA extraction
Bacterial DNA extracts used as templates in this study were prepared by the heat-boiling method. Bacterial suspensions were centrifuged at 12,000 rpm for 10 min to collect bacteria, which were resuspended in 500 μL of sterilized water and incubated at 100°C for 10 min. The cooling mixture was then centrifuged at 12,000 rpm for 5 min, and supernatants were collected and stored at −20°C until use.
Gene amplification and Sanger sequencing of DNA gyrase and topoisomerase IV
The QRDRs of the gyrA, gyrB, parC, and parE genes of serially passaged P. aeruginosa populations were amplified by polymerase chain reaction (PCR) using gene-specific primers (Supple. Table 1). To amplify each gene, 1 μg of the DNA template was subjected to PCR amplification in a 30 μL reaction mixture containing 12 μM of each forward and reverse primer, 7.5 μM of dNTPs, 3 μL 10× Ex Taq buffer (with MgCl2), and 1.5 U of Ex Taq DNA polymerase (TaKaRa, Shiga, Japan). PCR amplification was performed under the following conditions: for gyrA and gyrB amplification, denaturation was carried at 94°C for 5 min, followed by 36 cycles of amplification each at 94°C for 30 s, 58°C for 30 s and 72°C for 30 s, and then a final extension at 72°C for 7 min; 2) for parC amplification, denaturation was carried at 94°C for 5 min, followed by 40 cycles of amplification each at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and then a final extension at 72°C for 7 min; and 3) for parE amplification, denaturation was carried at 94°C for 5 min, followed by 36 cycles of amplification each at 94°C for 30 s, 70°C for 30 s, and 72°C for 30 s, and then a final extension at 72°C for 7 min. Amplified PCR products of all genes were visualized on 2% agarose gels containing GoodView (Beijing SBS Genetech Co., Beijing, China) with a DNA marker (TaKaRa) under ultraviolet light. Amplified PCR products were sent to the Beijing Genomics Institute for Sanger sequencing using the above-mentioned PCR primers. Nucleotide sequences of genes were compared with corresponding reference sequences of P. aeruginosa ATCC27853.
Evaluation of the expression of efflux pump genes
Expression levels of mexA, mexC, mexE, mexX, and the housekeeping gene rpoD were determined by quantitative real-time reverse transcription (qRT)-PCR. RNA was isolated using RNAiso reagent (TaKaRa), and cDNA was synthesized using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa), according to manufacturer’s instructions. qRT-PCR was performed in duplicate, in 20 μL volume with 100 ng RNA and primer concentration of 0.4 μM, on a QuantStudio DX Flex system (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq (TaKaRa). The primers for qRT-PCR are listed in Supple. Table 2. Gene expression was determined using the ΔΔCt method and a standard curve to measure PCR efficiency. All results were normalized to the expression of the housekeeping gene rpoD and calibrated relative to expression in P. aeruginosa ATCC27853.