Evernimicin (SCH27899) inhibits a novel ribosome target site: Analysis of 23S ribosomal DNA mutants

Spontaneous mutants of susceptible clinical and laboratory isolates of Stre ptococcus pneumoniae exhibiting reduced susceptibility to evernimicin (SCH2 7899; MIG, 0.5 to 4.0 mg/liter) were selected on plates containing evernimi cin. Four isolates that did not harbor mutations in rplP (which encodes rib osomal protein L16) were further analyzed. Whole chromosomal DNA or PCR pro ducts of the 23S ribosomal DNA (rDNA) operons from these mutants could be u sed to transform the susceptible S. pneumoniae strain R6 to resistance at f requencies of 10(-5) and 10(-4), respectively, rates 10- to 100-fold lower than that for a single-allele chromosomal marker. The transformants appeare d slowly (48 to 72 h) on selective medium, and primary transformants passag ed on nonselective medium produced single colonies that displayed heterogen eous susceptibilities to evernimicin. A single passage on selective medium of colonies derived from a single primary transformant homogenized the resi stance phenotype. Sequence analysis of the 23S rDNA and rRNA from the resis tant mutants revealed single, unique mutations in each isolate at the equiv alent Escherichia coil positions 2469 (A --> C), 2480 (C --> T), 2535 (G -- > A), and 2536 (G --> C). The mutations map within two different stems of t he peptidyltransferase region of domain V. Because multiple copies of rDNA are present in the chromosome, gene conversion between mutant and wild-type 23S rDNA alleles may be necessary for stable resistance. Additionally, non e of the characterized mutants showed cross-resistance to any of a spectrum of protein synthesis inhibitors, suggesting that the target site of everni micin may be unique.