Analyzing genomic alterations involved in fluoroquinolone-resistant development in Staphylococcus aureus

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 proteins played an important role in the emergence of MDR in S. aureus . Author summary Antimicrobial resistance is a major public health problem worldwide. Multiple studies have been performed to understand how bacteria develops resistance during the antibiotic therapy in vitro and in vivo . Here we revealed how Staphylococcus aureus , a stubborn human pathogen, changed its genome and expression of important genes in responding with sub-MIC exposure to flouroquinolone antibiotics. Mutations were found in the target of flouroquinolones such as GrlA (R570H) and interestingly in some hypothetical regions which may be important for gene expression regulation. We have observed an marked 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 in all exposed strains. Importantly, the expression change still remained when the bacteria were no longer exposed to the antibiotics. This study is important to understand response of S. aureus to flouroquinolone and how it obtains the resistance phenotype under antibiotic exposure. ### Competing Interest Statement The authors have declared no competing interest.