msrA encodes an efflux protein in Staphylococcus spp.that confers inducible resistance to macrolides,such as erythromycin(ERY),but not telithromycin(TEL).Thus,msrA+S.aureus remain susceptible to TEL.Production of msrAis regulated by a 320-bp upstream region,presumably by translational attenuation,similar to regulation of erm.We investigated whether ERY could induce TEL resistance in msrA+S.aureus.MICs of ERY and TEL were determined by broth microdilution(BMD).Inducible TEL resistance was detected by a BMD checkerboard panel containing combinations of ERY and TEL and by a D-test where an ERY disk was placed 15mm from a TEL disk on a blood agar plate.Blunting of the TEL zones of inhibition proximal to the ERY disk indicated inducible resistance and no blunting indicated no inducible resistance.Approximately 400bp of the upstream regulatory region and 200bp of msrA were amplified by PCR and sequenced.The TEL MICs for 10 msrA+isolates ranged from 0.06-2g/mL(MIC90=0.25g/mL);ERY MICs were all≥32g/mL.All isolates showed an increase in TEL MICs of>2doubling dilutions(MIC90>4g/mL)in the presence of ERY;All were also positive by D-test.For 9isolates(initial TEL MIC<1g/mL),constitutive TEL resistance was selected by plating cells on Mueller-Hinton agar with 2g/mL TEL.All 9isolates grew on the selective media and were resistant to TEL(MIC ranged 2-8g/mL).Mutation to constitutive resistance occurred at a rate of 8×10-7 to 2.4×10-6.Eight of the 9resistant isolates had a single nucleotide substitution in the upstream regulatory region relative to the parent strain.Specifically,six isolates had a G to T mutation at position-222;one had a C to A mutation at-228;And one had a C to A mutation at-247.Transformation of the msrAgene with above point mutation in regulatory region into S.aureus RN4220conferred the strain resistant(MIC=8g/mL)to the telithromycin while transformants with the wild-type msrAgene remained susceptible(MIC=1g/mL)to telithromycin.
David ShaferXiao ShaliXia HanWang JueJason LoftZhang WeidongFu Weiling
