Abstract
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. Deletion mutation and RT-PCR techniques were employed to study the role of efflux pumps in fluoroquinolone resistance and their regulation cascades. 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. 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.
Highlights
Non-typhoidal salmonellae are an important cause of food-borne gastroenteritis worldwide
The role of efflux pumps in fluoroquinolone resistance of Salmonella was evaluated by comparing the expression levels of acrB and acrF in fluoroquinolone-resistant strains LTL and LTH with ciprofloxacin MICs of 4 and 64 mg/L, respectively, with those of LT2
As ramA and soxS were highly up-regulated in fluoroquinoloneresistant LTL and LTH, their role in drug resistance in Salmonella was determined by comparing antimicrobial susceptibility of Salmonella Typhimurium strains with or without deletion of these genes (Table 1)
Summary
Non-typhoidal salmonellae are an important cause of food-borne gastroenteritis worldwide. High-level fluoroquinolone resistance in Salmonella remains rare, outbreaks of fluoroquinolone-resistant Salmonella infections have been reported in the USA,[5,6] Taiwan[7] and Japan.[2]. Bacterial resistance to antimicrobials can be conferred by horizontal transfer of mobile elements carrying resistance genes, such as plasmids, transposons and bacteriophage, by target gene mutations, and by increased expression of multidrug efflux pumps resulting in reduced intracellular concentrations of various antibiotics, including b-lactams, macrolides, tetracycline, chloramphenicol and quinolones.[8,9,10,11,12,13] Studies in Escherichia coli have shown that transcriptional activators, such as MarA, SoxS and
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