Type M Resistance to Macrolides Is Due to a Two-Gene Efflux Transport System of the ATP-Binding Cassette (ABC) Superfamily

Abstract

The <i>mef</i>(A) gene was originally identified as the resistance determinant responsible for type M resistance to macrolides, a phenotype frequently found in clinical isolates of <i>Streptococcus pneumoniae</i> and <i>Streptococcus pyogenes</i>. MefA was defined as a secondary transporter of the major facilitator superfamily driven by proton-motive force. However, when characterizing the <i>mef</i>(A)-carrying elements Tn<i>1207.1</i> and Φ1207.3, another macrolide resistance gene, <i>msr</i>(D), was found adjacent to <i>mef</i>(A). To define the respective contribution of <i>mef</i>(A) and <i>msr</i>(D) to macrolide resistance, three isogenic deletion mutants were constructed by transformation of a <i>S. pneumoniae</i> strain carrying Φ1207.3: (i) Δ<i>mef</i>(A)-Δ<i>msr</i>(D); (ii) Δ<i>mef</i>(A)-<i>msr</i>(D); and (iii) <i>mef</i>(A)-Δ<i>msr</i>(D). Susceptibility testing of mutants clearly showed that <i>msr</i>(D) is required for macrolide resistance, while deletion of <i>mef</i>(A) produced only a twofold reduction in the minimal inhibitory concentration (MIC) for erythromycin. The contribution of <i>msr</i>(D) to macrolide resistance was also studied in <i>S. pyogenes</i>, which is the original host of Φ1207.3. Two isogenic strains of <i>S. pyogenes</i> were constructed: (i) FR156, carrying Φ1207.3, and (ii) FR155, carrying Φ1207.3/Δ<i>msr</i>(D). FR155 was susceptible to erythromycin, whereas FR156 was resistant, with an MIC value of 8 μg/ml. Complementation experiments showed that reintroduction of the <i>msr</i>(D) gene could restore macrolide resistance in Δ<i>msr</i>(D) mutants. Radiolabeled erythromycin was retained by strains lacking <i>msr</i>(D), while <i>msr</i>(D)-carrying strains showed erythromycin efflux. Deletion of <i>mef</i>(A) did not affect erythromycin efflux. This data suggest that type M resistance to macrolides in streptococci is due to an efflux transport system of the ATP-binding cassette (ABC) superfamily, in which <i>mef</i>(A) encodes the transmembrane channel, and <i>msr</i>(D) the two ATP-binding domains.