ABSTRACTMacrolide resistance in Streptococcus pneumoniaedue to efflux has emerged as an important worldwide clinical problem over the past decade. Efflux is mediated by the genes of the genetic element mega (macrolide efflux genetic assembly) and related elements, such as Tn1207.1. These elements contain two adjacent genes, mef(mefEor mefA) and the closely related melgene (msrAhomolog), encoding a proton motive force pump and a putative ATP-binding cassette transporter homolog, and are transcribed as an operon (M. Del Grosso et al., J. Clin. Microbiol. 40:774-778, 2004; K. Gay and D. S. Stephens, J. Infect. Dis. 184:56-65, 2001; and M. Santagati et al., Antimicrob. Agents Chemother. 44:2585-2587, 2000). Previous studies have shown that Mef is required for macrolide resistance in S. pneumoniae; however, the contribution of Mel has not been fully determined. Independent deletions were constructed in mefEand melin the serotype 14 macrolide-resistant strains GA16638 (erythromycin [Em] MIC, 8 to 16 μg/ml) and GA17719 (Em MIC, 2 to 4 μg/ml), which contain allelic variations in the mega element. The MICs to erythromycin were significantly reduced for the independent deletion mutants of both mefEand melcompared to those of the parent strains and further reduced threefold to fourfold to Em MICs of <0.15 μg/ml with mefE meldouble mutants. Using quantitative reverse transcription-PCR, the expression of mefEin the meldeletion mutants was increased more than 10-fold. However, in the mefEdeletion mutants, the expression of meldid not differ significantly from the parent strains. The expression of both mefEand melwas inducible by erythromycin. These data indicate a requirement for both Mef and Mel in the novel efflux-mediated macrolide resistance system in S. pneumoniaeand other gram-positive bacteria and that the system is inducible by macrolides.