In order to select IBG-resistant mutants, ∼1010 CFU of S. aureus ATCC 29213 cells were plated onto MH agar containing 2.5×, 5×, and 10×MIC of IBG. After 48 h of incubation at 37°C, resistant colonies were calculated and the MICs of IBG were determined. When this approach proved unsuccessful, development of resistant mutants by serial passage in liquid medium was conducted previously (Ling et al., 2015). The bacteria culture (OD600 = 0.01) was treated with IBG or CIP at different concentrations. Cells were incubated at 37°C and passaged at 24 h intervals in the presence of IBG or CIP. The MIC was determined by broth microdilution. Experiments were performed with three replicates (SP1, SP2, and SP3).

The genomic DNA from the strains with elevated MIC of IBG was extracted using a Hipure bacterial DNA kit (Magen, Shanghai, China). A paired-end sequencing library (2 bp × 250 bp) was created using a VAHTS Universal DNA Library Prep kit for Illumina® (Illumina, San Diego, CA, United States) and sequenced on an Illumina HiSeq system (Illumina Inc.). Processed reads were de novo assembled into draft genomes using CLC Genomics Workbench 10.1 (CLC Bio, Aarhus, Denmark) and annotated by using Prokka pipeline. These genomes were subjected to SNP analysis by utilizing snippy pipeline. S. aureus ATCC 29213 genome of the starting strain was used as a reference genome in SNP analysis.

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