Abstract
The mef(A) gene was originally identified as the resistance determinant responsible for type M resistance to macrolides, a phenotype frequently found in clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes. MefA was defined as a secondary transporter of the major facilitator superfamily driven by proton-motive force. However, when characterizing the mef(A)-carrying elements Tn1207.1 and Φ1207.3, another macrolide resistance gene, msr(D), was found adjacent to mef(A). To define the respective contribution of mef(A) and msr(D) to macrolide resistance, three isogenic deletion mutants were constructed by transformation of a S. pneumoniae strain carrying Φ1207.3: (i) Δmef(A)–Δmsr(D); (ii) Δmef(A)–msr(D); and (iii) mef(A)–Δmsr(D). Susceptibility testing of mutants clearly showed that msr(D) is required for macrolide resistance, while deletion of mef(A) produced only a twofold reduction in the minimal inhibitory concentration (MIC) for erythromycin. The contribution of msr(D) to macrolide resistance was also studied in S. pyogenes, which is the original host of Φ1207.3. Two isogenic strains of S. pyogenes were constructed: (i) FR156, carrying Φ1207.3, and (ii) FR155, carrying Φ1207.3/Δmsr(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 msr(D) gene could restore macrolide resistance in Δmsr(D) mutants. Radiolabeled erythromycin was retained by strains lacking msr(D), while msr(D)-carrying strains showed erythromycin efflux. Deletion of mef(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 mef(A) encodes the transmembrane channel, and msr(D) the two ATP-binding domains.
Highlights
Macrolides are antibiotic compounds composed of, or-membered lactones to which amino and/or neutral sugars are linked (Roberts et al, 1999; Roberts, 2008)
To allow construction of mutants deleted for the macrolide resistance genes object of this study, and to facilitate further analysis, it was essential to work in a S. pneumoniae strain which is readily transformable and whose genomic sequence is available (Hoskins et al, 2001)
We constructed isogenic deletion mutants in S. pneumoniae and S. pyogenes to define the respective contribution of mef (A) and msr(D) to macrolide resistance
Summary
Macrolides are antibiotic compounds composed of (erythromycin and clarithromycin), (azithromycin), or (josamycin, spiramycin, tylosin)-membered lactones to which amino and/or neutral sugars are linked (Roberts et al, 1999; Roberts, 2008). The macrolide efflux mef (A) gene was originally described in Streptococcus pyogenes (Clancy et al, 1996) while the allelic variant mef (E) was first described in Streptococcus pneumoniae (Tait-Kamradt et al, 1997). These variants are highly homologous (about 90% nucleotide identity) and are grouped in the same mef (A) class of macrolide resistance genes (Roberts et al, 1999). In S. pyogenes, mef (A) is carried by 1207.3, a 52,491-bp prophage which we found in the erythromycin-resistant clinical strain 2812A, transferable to a variety of streptococcal species and whose left 7244-bp sequence is 100% identical to pneumococcal Tn1207.1 (Santagati et al, 2003; Pozzi et al, 2004; Iannelli et al, 2014a). Results of functional studies were in accordance with bioinformatics analysis predicting that the tandem mef (A)-msr(D) gene pair encodes an efflux transport system of the ATP-binding cassette (ABC) superfamily
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