AUTHOR=Iannelli Francesco , Santoro Francesco , Santagati Maria , Docquier Jean-Denis , Lazzeri Elisa , Pastore Gabiria , Cassone Marco , Oggioni Marco R. , Rossolini Gian M. , Stefani Stefania , Pozzi Gianni 

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

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.01670

DOI=10.3389/fmicb.2018.01670

ISSN=1664-302X

ABSTRACT=<p>The <italic>mef</italic>(A) gene was originally identified as the resistance determinant responsible for type M resistance to macrolides, a phenotype frequently found in clinical isolates of <italic>Streptococcus pneumoniae</italic> and <italic>Streptococcus pyogenes</italic>. MefA was defined as a secondary transporter of the major facilitator superfamily driven by proton-motive force. However, when characterizing the <italic>mef</italic>(A)-carrying elements Tn<italic>1207.1</italic> and Φ1207.3, another macrolide resistance gene, <italic>msr</italic>(D), was found adjacent to <italic>mef</italic>(A). To define the respective contribution of <italic>mef</italic>(A) and <italic>msr</italic>(D) to macrolide resistance, three isogenic deletion mutants were constructed by transformation of a <italic>S. pneumoniae</italic> strain carrying Φ1207.3: (i) Δ<italic>mef</italic>(A)–Δ<italic>msr</italic>(D); (ii) Δ<italic>mef</italic>(A)–<italic>msr</italic>(D); and (iii) <italic>mef</italic>(A)–Δ<italic>msr</italic>(D). Susceptibility testing of mutants clearly showed that <italic>msr</italic>(D) is required for macrolide resistance, while deletion of <italic>mef</italic>(A) produced only a twofold reduction in the minimal inhibitory concentration (MIC) for erythromycin. The contribution of <italic>msr</italic>(D) to macrolide resistance was also studied in <italic>S. pyogenes</italic>, which is the original host of Φ1207.3. Two isogenic strains of <italic>S. pyogenes</italic> were constructed: (i) FR156, carrying Φ1207.3, and (ii) FR155, carrying Φ1207.3/Δ<italic>msr</italic>(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 <italic>msr</italic>(D) gene could restore macrolide resistance in Δ<italic>msr</italic>(D) mutants. Radiolabeled erythromycin was retained by strains lacking <italic>msr</italic>(D), while <italic>msr</italic>(D)-carrying strains showed erythromycin efflux. Deletion of <italic>mef</italic>(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 <italic>mef</italic>(A) encodes the transmembrane channel, and <italic>msr</italic>(D) the two ATP-binding domains.</p>