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

The (A) gene was originally identified as the resistance determinant responsible for type M resistance to macrolides, a phenotype frequently found in clinical isolates of and . MefA was defined as a secondary transporter of the major facilitator superfamily driven by proton-motive force. However, when characterizing the (A)-carrying elements Tn and Φ1207.3, another macrolide resistance gene, (D), was found adjacent to (A). To define the respective contribution of (A) and (D) to macrolide resistance, three isogenic deletion mutants were constructed by transformation of a strain carrying Φ1207.3: (i) Δ(A)-Δ(D); (ii) Δ(A)-(D); and (iii) (A)-Δ(D). Susceptibility testing of mutants clearly showed that (D) is required for macrolide resistance, while deletion of (A) produced only a twofold reduction in the minimal inhibitory concentration (MIC) for erythromycin. The contribution of (D) to macrolide resistance was also studied in , which is the original host of Φ1207.3. Two isogenic strains of were constructed: (i) FR156, carrying Φ1207.3, and (ii) FR155, carrying Φ1207.3/Δ(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 (D) gene could restore macrolide resistance in Δ(D) mutants. Radiolabeled erythromycin was retained by strains lacking (D), while (D)-carrying strains showed erythromycin efflux. Deletion of (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 (A) encodes the transmembrane channel, and (D) the two ATP-binding domains.