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Regulation of efflux-mediated multidrug resistance by the ramR-ramA region in Salmonella enterica serovar Typhimurium

Abstract number: P2029

Baucheron S., Abouzeed Y.M., Cloeckaert A.

Objectives: To study the regulation of efflux system expression in multidrug resistant (MDR) Salmonella enterica serovar Typhimurium (S. Typhimurium) DT104 and DT204 strains.

To determine the involvement of two transcriptional regulators in multidrug resistance and in overexpression of the AcrAB efflux pump.

Methods: Identification in the sequenced genome of S. Typhimurium strain LT2 of an open reading frame coding for a putative regulatory protein of the TetR family upstream of the ramA gene whose gene product, homologous to MarA, when overxepressed confers a multidrug resistance (MDR) phenotype in several bacterial species.

Characterisation of the role of this putative regulatory protein gene was done by its inactivation and by complementing experiments. The measure of the expression level of ramA was done by RT-PCR and that of the AcrAB efflux pump by Western blotting. Sequencing of this region was also performed in strains overproducing AcrAB to identify possible mutations involved in deregulation of ramA expression.

Results: Inactivation of the putative regulatory protein gene upstream of ramA in a susceptible S. Typhimurium strain resulted in a MDR phenotype with a 4-fold increase of resistance levels to unrelated antibiotics such as quinolones, fluoroquinolones, phenicols, and tetracycline. Inactivation of this gene resulted also in a 4-fold increased transcription of ramA and a 4-fold increased expression of the AcrAB efflux pump. These results indicated that the gene encodes a local repressor of ramA and was thus named ramR.

In quinolone- or fluoroquinolone-resistant strains of S. Typhimurium overexpressing AcrAB several mutations were identified in ramR which consisted of point mutations resulting in amino acid changes or an in frame shift and also of interruption of ramR by an IS1 element in high-level fluoroquinolone resistant S. Typhimurium DT204 strains. One S. Typhimurium DT104 isolate had a two nucleotide deletion in the putative RamR binding site found upstream of ramA. These mutations were confirmed to play a role in the MDR phenotype by complementing with an intact ramR or inactivation of their respective ramA gene.

Conclusions: The ramR gene coding a transcriptional repressor was identified in S. Typhimurium to control the transcription of ramA. Mutations in ramR appear to play a major role in upregulation of RamA and AcrAB and consecutive efflux-mediated MDR phenotype in S. Typhimurium.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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