Genetic and transcriptomic analyses of ciprofloxacin-tolerant Staphylococcus aureus isolated by the Replica Plating Tolerance Isolation System (REPTIS) [Mechanisms of Resistance]

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 (R2, R3, R5 and R6) carrying a total of 12 mutations in 12 different genes from a methicillin-sensitive Staphylococcus aureus (MSSA) FDA209P strain. 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 R strains showed a common up-regulation of relA. Overall, transcriptome analysis showed down-regulation of genes related to translation; carbohydrate, fat, and energy metabolism; and nucleotide synthesis, and up-regulation of amino acid biosynthesis and transportation genes in R2, R3, and R6, similar to that observed in 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 mupirocin treatment.

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