Objectives: Multidrug resistance (MDR) including fluoroquinolone resistance in Salmonella Typhimurium can result from overexpression of efflux pumps. We examined the mechanisms of fluoroquinolone resistance among in vitro-induced ciprofloxacin-resistant Salmonella Typhimurium mutants, LTL and LTH, derived from laboratory strain LT2.
Methods: Deletion mutation and RT-PCR techniques were employed to study the role of efflux pumps in fluoroquinolone resistance and their regulation cascades.
Results: In addition to point mutations in DNA gyrase (gyrA, gyrB) and topoisomerase IV (parC, parE) genes, increased expression of efflux pump genes, such as acrAB and acrEF, was observed in fluoroquinolone-resistant Salmonella strains. Constitutive expression of ramA containing a 9 bp deletion in the promoter region was directly associated with the overexpression of acrAB and acrEF and conferred an MDR phenotype in LTL. Inactivation of ramA increased the antimicrobial susceptibility of LTL, whereas complementation with the mutant allele induced an MDR phenotype in drug-susceptible Salmonella Typhimurium LT2, as demonstrated by 2- to 64-fold increases in resistance to fluoroquinolones, tetracycline and chloramphenicol. On the other hand, inactivation of mutant soxRS resulted in a slight increase in the susceptibility of LTH to several fluoroquinolone drugs, and the introduction of the mutant allele had no effect on antimicrobial susceptibility of LT2, indicating that constitutive expression of soxRS played a minimum role in fluoroquinolone resistance.
Conclusions: Mutations in the promoter region of ramA appear to play a role in the up-regulation of RamA and AcrAB, and RamA is an activator of the MDR regulation cascade in Salmonella Typhimurium.