Abstract
Mutants of five Salmonella enterica serovars were investigated for structural changes in regulatory regions known to be involved in the up-regulation of efflux pumps. Five Salmonella field isolates and mutants, in which efflux pump inhibitor tests previously pointed towards an up-regulation of efflux, plus one negative control were included in the study. MIC values were determined of antibiotics that were indicative of AcrAB overexpression. The regulatory regions acrRA, soxRS, marORAB, acrSE and ramRA of original strains and mutants were sequenced and compared. The gene expression of acrA, tolC, ramA and soxS was assessed by quantitative real-time PCR. Conjugation experiments and tet gene PCRs were performed to explain unexpected variations in MIC values of tetracycline. In four mutant strains, changes in the ramRA regulatory region, causing up-regulation of ramA, were detected. These changes comprised point mutations and deletions of 10 or 15 bp within the ramR gene and a single bp exchange located in the binding site of the RamR protein in Salmonella Infantis, Paratyphi and Livingstone mutants. An insertion of 49 bp within the soxR gene was involved in soxS up-regulation and enhanced efflux activity in the fifth mutant from Salmonella Virchow. The loss of tetracycline resistance in one Salmonella Paratyphi mutant could be explained by the loss of a plasmid carrying a tet(A) gene. Changes in the ramR-ramA region as well as in the soxR gene occur in mutants of Salmonella serovars other than Typhimurium and seem to be involved in the up-regulation of efflux activity.
Highlights
Resistance to fluoroquinolones is believed to arise from an interplay of different resistance mechanisms, including mutations in topoisomerase genes, increased efflux via transporters, target site protection via Qnr proteins as well as enzymatic inactivation by Aac(60)-Ib-cr.[1,2,3] initial studies focused on the target gene mutations as the major mechanism of resistance, it became obvious during the past decade that active efflux plays a highly relevant role inquinolone resistance
Mutants of five Salmonella enterica serovars were investigated for structural changes in regulatory regions known to be involved in the up-regulation of efflux pumps
Initial studies focused on the target gene mutations as the major mechanism of resistance, it became obvious during the past decade that active efflux plays a highly relevant role inquinolone resistance
Summary
Resistance to fluoroquinolones is believed to arise from an interplay of different resistance mechanisms, including mutations in topoisomerase genes, increased efflux via transporters, target site protection via Qnr proteins as well as enzymatic inactivation by Aac(60)-Ib-cr.[1,2,3] initial studies focused on the target gene mutations as the major mechanism of resistance, it became obvious during the past decade that active efflux plays a highly relevant role in (fluoro)quinolone resistance. Point mutations in ramR, which resulted in either amino acid exchanges or frame shifts, a deletion in the ramA promoter and the inactivation of ramR by the insertion of an IS1 element have been proved to be involved in the up-regulation of efflux-mediated multidrug resistance in Salmonella Typhimurium.[13,14] mutations in the global regulators have recently been investigated in Salmonella Enteritidis,[15] very little is known about the role of ramR mutations or mutations in other regulatory genes, such as acrR, soxR, marR and acrS, that may influence AcrAB expression in Salmonella serovars other than Typhimurium
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