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Effect of ramR mutations on efflux genes expression and on fluoroquinolone susceptibility in Salmonella enterica serotype Kentucky ST198
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Edité par CCSD -
Poster - Session 4 : Blanche. National audience. Background. Efflux is a mechanism that has been previously reported to increase fluoroquinolone (FQ) resistance levels when it is upregulated in clinical isolates of Salmonella enterica mainly of serotype Typhimurium (1). In this study efflux related genes were investigated in the emerging FQ-resistant epidemic S. enterica serotype Kentucky ST198 clone (2) for the presence of mutations in their regulatory genes, and for their expression levels. Methods. Among a representative panel of thirty serotype Kentucky strains from Egypt or east Africa with decreased FQ susceptibility, three strains overproducing the AcrAB-TolC efflux system were detected and studied (3). Two FQ-resistant strains with substitutions in QRDR and with basal expression level of AcrABTolC and the susceptible reference strain 98K were used as control. Genetic relatedness was determined by XbaI-pulsed field gel electrophoresis and multilocus sequence typing. Presence of mutations in the ramRA, soxRS, marOR loci and acrR, acrS genes were assessed by sequencing. The six strains and their wild-type ramR gene complemented derivatives were analysed by (i) qRT-PCR for gene expression of regulatory and efflux genes and by (ii) MIC determinations of quinolones, FQ and florfenicol as other substrate of AcrAB-TolC. Results. All serotype Kentucky strains studied were of sequence type X1-ST198, excepted the 98K strain which was X4-ST198. Among the thirty S. Kentucky strains, three overproduce AcrAB-TolC (3). All three strains presented different mutations in the ramR gene in comparison to the reference strain 98K. The three detected mutations (deletion of 91 bp, insertion of 1 bp or 4 bp) resulted in frame shift of the ramR gene. All other efflux regulatory genes were not mutated. As confirmed by complementation with a wild-type ramR gene, all three mutations were responsible for increased expression of ramA and acrAB. Increased expression of tolC and acrEF genes was observed in 2 out of the 3 strains. All three mutations were shown to increase two-fold the MICs of FQ and florfenicol in comparison to the ramR complemented derivatives and the reference strain. The two strains with a basal expression level of AcrAB-TolC presented any mutation in sequenced genes. The only strain presenting 3 substitutions in QRDR associated with 1 mutation in ramR upregulating AcrAB-TolC leaded to the higher level FQ resistance Conclusion. Various novel ramR mutations, responsible for increased efflux, were detected in the emerging epidemic serotype Kentucky ST198 clone. As previously reported in other FQ-resistant strains of serotypes Typhimurium or Schwarzengrund, ramR mutations seem to be sporadic (10 % in this study) and contribute only to a little extent to the decreased FQ susceptibility
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