ramR Mutations Involved in Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium

INRA, UR1282, Infectiologie Animale et Santé Publique, IASP, Nouzilly, F-37380, France

^* To whom correspondence should be addressed. Email: cloeckae{at}tours.inra.fr.

	Abstract

In the sequenced genome of Salmonella enterica serovar Typhimurium (S. Typhimurium) strain LT2 an open reading frame (STM 0580) coding for a putative regulatory protein of the TetR family is found upstream of the ramA gene. Overxepression of ramA results in increased expression of the AcrAB efflux pump and consequently multidrug resistance (MDR) in several bacterial species. 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 in the MICs of unrelated antibiotics such as quinolones/fluoroquinolones, phenicols, and tetracycline. Inactivation of this gene resulted also in a 4-fold increased expression 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 contrast, inactivation of marR, marA, soxR, and soxS did not affect the susceptibilities of the strain. 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. No mutations in the mar or sox regions were found in the strains studied. In conclusion, mutations in ramR appear to play a major role in upregulation of RamA and AcrAB and consequently efflux-mediated MDR phenotype in S. Typhimurium.