Genetic and Transcriptomic Analyses of Ciprofloxacin-Tolerant Staphylococcus aureus Isolated by the Replica Plating Tolerance Isolation System (REPTIS).

We developed a simple, efficient, and cost-effective method, named the replica plating tolerance isolation system (REPTIS), to detect the antibiotic tolerance potential of a bacterial strain. This method can also be used to quantify the antibiotic-tolerant subpopulation in a susceptible population. Using REPTIS, we isolated ciprofloxacin (CPFX)-tolerant mutants (mutants R2, R3, R5, and R6) carrying a total of 12 mutations in 12 different genes from methicillin-sensitive Staphylococcus aureus (MSSA) strain FDA209P. Each mutant carried multiple mutations, while few strains shared the same mutation. The R2 strain carried a nonsense mutation in the stress-mediating gene, relA. Additionally, two strains carried the same point mutation in the leuS gene, encoding leucyl-tRNA synthetase. Furthermore, RNA sequencing of the R strains showed a common upregulation of relA. Overall, transcriptome analysis showed downregulation of genes related to translation; carbohydrate, fat, and energy metabolism; nucleotide synthesis; and upregulation of amino acid biosynthesis and transportation genes in R2, R3, and R6, similar to the findings observed for the FDA209P strain treated with mupirocin (MUP0.03). However, R5 showed a unique transcription pattern that differed from that of MUP0.03. REPTIS is a unique and convenient method for quantifying the level of tolerance of a clinical isolate. Genomic and transcriptomic analyses of R strains demonstrated that CPFX tolerance in these S. aureus mutants occurs via at least two distinct mechanisms, one of which is similar to that which occurs with mupirocin treatment.