Paper Title
Mutant Prevention Concentration and Resistance Mechanism of Fluoroquinoloneagainstsalmonella Typhimurium

Abstract
The aims of this study were to investigate the current antibacterial activity and mutant prevention concentration (MPC) of marbofloxacinas well as to establish mechanisms of FQ-resistance with reduced susceptibility and different phenotypes against clinical isolates of Salmonella Typhimurium from pigs in Korea.To determinethe resistant phenotypes, MICs inclinical isolatesand representative mutants arisenfrom exposure to marbofloxacin with sub-MPC were evaluatedin the presence or absence ofefflux pump inhibitor (EPI). The mechanism of fluoroquinolone resistance was determined by sequencing analyzing target mutation in QRDRs,quantifying the overexpression of efflux pump and their regulators using qRT-PCR. MICs and MPCs of marbofloxacinagainst all isolates (n=15) showed 0.03-2 g/mL and 0.125-5 g/mL, respectively. Detection of mutation in thegyrA(Ser83Phe orAsp87His)showed in clinical isolates (43.8%) with MIC > 0.06 g/mL and observed in most of laboratory-derived mutants(81.3%) suggesting that gyrA mutation could be a frequent cause to loss of susceptibility in S. Typhimurium. The overexpression of acrAB-tolC could also be contributed to develop the resistant regardlessof gyrAmutation, but no positive correlation to expression of global regulators, namely marA/soxS/ramA.Interestingly, a novel mutation in acrRgene founded in two isolates and 43.8% of single-step mutants withouttarget mutation was responsible partially for efflux pump activation. Taken together with the present results, the current dosing regimen ofmarbofloxacin, however, was founded inadequate to restrict emergence of resistant S. Typhimurium. Therefore the reassessment the current dosing regimen and continuous monitoring of the emergence of resistant S. Typhimurium in farm would be required. Keywords- Mutant prevention concentration, Fluoroquinolones, S. Typhimurium, gyrA, acrAB-tolC, acrR