Snapgene version 4

The genetic alterations in the efflux pump gene adeABC and its transcriptional regulatory gene, adeRS, as well as rpsJ, coding for 30S ribosomal protein S10, and tet(X), were assessed by nucleotide sequencing. Genomic DNA was extracted from the wild-type isolates (FA#), tigecycline-resistant subpopulations (FA#-R), and mutants obtained after serial passaging in antibiotic-free medium (FA#-R-p30) using the G-spin genomic DNA extraction kit for bacteria (iNtRON Biotechnology, Seongnam, Korea). PCR and DNA sequencing using the Sanger method were performed using the primers listed in Table S1. The genetic alterations were analyzed using SnapGene version 4.1.9 (GSL Biotech LLC, Chicago, IL, USA).

Genetic alterations within the coding genes of the PmrAB two-component regulatory system, pmrAB and their effector protein-coding gene, pmrC, within the colistin-resistant subpopulations were identified by PCR and sequencing (Additional file 1: Table S1). Genomic DNA was extracted from each isolate using a G-spin™ Genomic DNA Extraction kit for bacteria (iNtRON Biotechnology, Seongnam, Korea); any and all mutations in these colistin-resistant subpopulations were analyzed based on their respective parental strains using SnapGene version 4.1.9 (GSL Biotech LLC, Greater Chicago Area, Great Lakes, Midwestern US) and described as amino acid alterations.