Expression of the mef (E) Gene Encoding the Macrolide Efflux Pump Protein Increases in Streptococcus pneumoniae with Increasing Resistance to Macrolides

Active macrolide efflux is a major mechanism of macrolide resistance inStreptococcus pneumoniae in many parts of the world, especially North America. In Canada, this active macrolide efflux inS. pneumoniae is predominantly due to acquisition of themef (E) gene. In the present study, we assessed themef (E) gene sequence as well asmef (E) expression in variety of low- and high-level macrolide-resistant, clindamycin-susceptible (M-phenotype)S. pneumoniae isolates (erythromycin MICs, 1 to 32 μg/ml; clindamycin MICs, ≤0.25 μg/ml). Southern blot hybridization withmef (E) probe and EcoRI digestion and relative real-time reverse transcription-PCR were performed to study themef (E) gene copy number and expression. Induction ofmef (E) expression was analyzed by Etest susceptibility testing pre- and postincubation with subinhibitory concentrations of erythromycin, clarithromycin, azithromycin, telithromycin, and clindamycin. The macrolide efflux gene,mef (E), was shown to be a single-copy gene in all 23 clinicalS. pneumoniae isolates tested, and expression post-macrolide induction increased 4-, 6-, 20-, and 200-fold in isolates with increasing macrolide resistance (erythromycin MICs 2, 4, 8, and 32 μg/ml, respectively). Sequencing analysis of the macrolide efflux genetic assembly (mega) revealed thatmef (E) had a 16-bp deletion 153 bp upstream of the putative start codon in all 23 isolates. A 119-bp intergenic region betweenmef (E) andmel was sequenced, and a 99-bp deletion was found in 11 of the 23 M-phenotypeS. pneumoniae isolates compared to the published mega sequence. However, themef (E) gene was fully conserved among both high- and low-level macrolide-resistant isolates. In conclusion, increased expression ofmef (E) is associated with higher levels of macrolide resistance in macrolide-resistantS. pneumoniae.