Effect of transcriptional activators RamA and SoxS on expression of multidrug efflux pumps AcrAB and AcrEF in fluoroquinolone-resistant Salmonella Typhimurium.

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.

Zheng J ，Cui S ，Meng J 《-》

Role of an acrR mutation in multidrug resistance of in vitro-selected fluoroquinolone-resistant mutants of Salmonella enterica serovar Typhimurium.

Olliver A ，Vallé M ，Chaslus-Dancla E ，Cloeckaert A ... -  《FEMS MICROBIOLOGY LETTERS》

Overexpression of the multidrug efflux operon acrEF by insertional activation with IS1 or IS10 elements in Salmonella enterica serovar typhimurium DT204 acrB mutants selected with fluoroquinolones.

High-level fluoroquinolone (FQ) resistance in Salmonella enterica serovar Typhimurium phage type DT204 has been previously shown to be essentially due to both multiple target gene mutations and active efflux by the AcrAB-TolC efflux system. In this study we show that in intermediatly resistant acrB-inactivated serovar Typhimurium DT204 mutants, high-level resistance to FQs can be restored on in vitro selection with FQs. In each FQ- resistant mutant selected from serovar Typhimurium DT204 acrB mutant strains, an insertion sequence (IS1 or IS10) was found integrated upstream of the acrEF operon, coding for AcrEF, an efflux pump highly homologous to AcrAB. In one of the strains, transposition of IS1 caused partial deletion of acrS, the putative local repressor gene of the acrEF operon. Sequence analysis showed that both IS1 and IS10 elements contain putative promoter sequences that might alter the expression of adjacent acrEF genes. Indeed, reverse transcription-PCR experiments showed an 8- to 10-fold increase in expression of acrF in these insertional mutants, relative to their respective parental strain, which correlated well with the resistance levels observed to FQs and other unrelated drugs. It is noteworthy that AcrEF did not contribute to the intrinsic drug resistance of serovar Typhimurium, since acrF deletion in wild-type strains did not result in any increase in drug susceptibility. Moreover, deletion of acrS did not cause any acrF overexpression or any decrease in drug susceptibility, suggesting that acrEF overexpression is mediated solely by the IS1 and IS10 promoter sequences and not by inactivity of AcrS. Southern blot experiments showed that the number of chromosomal IS1 and IS10 elements in the serovar Typhimurium DT204 genome was about 5 and 15 respectively. None were detected in epidemic serovar Typhimurium DT104 strains or in the serovar Typhimurium reference strain LT2. Carrying IS1 and/or IS10 elements in their chromosome may thus be a selective advantage for serovar Typhimurium DT204 strains as opposed to DT104 strains for which no high-level FQ resistance nor insertional mutations were found. Taken together, the results of the present study indicate that the IS1- or IS10- activated AcrEF efflux pump may relay AcrAB in serovar Typhimurium, and underline the importance of transposable elements in the acquisition of FQ and multidrug resistance.

Olliver A ，Vallé M ，Chaslus-Dancla E ，Cloeckaert A ... -  《-》

Decreased fluoroquinolone susceptibility in mutants of Salmonella serovars other than Typhimurium: detection of novel mutations involved in modulated expression of ramA and soxS.

Kehrenberg C ，Cloeckaert A ，Klein G ，Schwarz S ... -  《-》

The role of intra- and extragenic compensatory mutations in the suppression of fluoroquinolone resistance in a Salmonella Typhimurium gyrA mutant (D87G).

Preisler A ，Heisig P 《-》