Analyzing genomic alterations involved in fluoroquinolone-resistant development inStaphylococcus aureus

Abstract

Aim: Recently, the rise in Staphylococcal infection incidence accompanied by a rise of antibiotic-resistant strains is a major threat to public health. In this study, mechanisms leading to the occurrence of high-level multidrug-resistant (MDR) Staphylococcus aureus (S. aureus) strains after fluoroquinolone (FQ) exposure were investigated. Methodology: Serially exposing S. aureus ATCC 29213 to ciprofloxacin (CIP), ofloxacin (OFL), or levofloxacin (LEV) at sub-minimum inhibitory concentrations (sub-MICs) for 12 days was performed to obtain S. aureus -1 strains and culturing for another 10 days without antibiotics to obtain S. aureus-2 strains. The genomic alterations in FQ-exposed strains were reached using whole genome sequencing and target sequencing. The expressions of efflux-related genes, alternative sigma factors, and genes involved in FQ resistance were evaluated using RT-qPCR. Results: After serial FQ exposure, we observed a strong and irreversible increase of MICs to all applied FQs, i.e 32 to 128 times in all S. aureus-1 and remained 16 to 32 times in all S. aureus-2. WGS indicated 10 significant mutations including 2 deletions, 1 insertion, and 7 missense mutations that occur in all S. aureus-1 and -2 but not in initial strain. The FQ target, GrlA, was also mutated (R570H) in all S. aureus-1 and -2 which can partly explain the development of FQ resistance over the FQ exposure. Besides, FQ exposure also resulted in overexpression of genes encoding for (1) efflux pumps and their regulator (norA, norB, norC, and mgrA); (2) alternative sigma factors (sigB and sigS); (3) acetyltransferase (rimI); (4) methicillin resistance (fmtB); and (5) hypothetical protein BJI72_0645. Conclusion: The mutations occurred in the FQ-target sequence were associated with high-level FQ resistance while the activation of efflux pump systems and post-translational modification proteins played an important role in the emergence of MDR in S. aureus.