Diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin, and telithromycin in Streptococcus pneumoniae

Abstract

Mechanisms of resistance were studied in 22 macrolide-resistant mutants selected in vitro from 5 parental strains of macrolide-susceptible Streptococcus pneumoniae by serial passage in various macrolides (T. A. Davies, B. E. Dewasse, M. R. Jacobs, and P. C. Appelbaum, Antimicrob. Agents Chemother., 44:414-417, 2000). Portions of genes encoding ribosomal proteins L22 and L4 and 23S rRNA (domains II and V) were amplified by PCR and analyzed by single-strand conformational polymorphism analysis to screen for mutations. The DNA sequences of amplicons from mutants that differed from those of parental strains by their electrophoretic migration profiles were determined. In six mutants, point mutations were detected in the L22 gene (G95D, P99Q, A93E, P91S, and G83E). The only mutant selected by telithromycin (for which the MIC increased from 0.008 to 0.25 microg/ml) contained a combination of three mutations in the L22 gene (A93E, P91S, and G83E). L22 mutations were combined with an L4 mutation (G71R) in one strain and with a 23S rRNA mutation (C2611A) in another strain. Nine other strains selected by various macrolides had A2058G (n = 1), A2058U (n = 2), A2059G (n = 1), C2610U (n = 1), and C2611U (n = 4) mutations (Escherichia coli numbering) in domain V of 23S rRNA. One mutant selected by clarithromycin and resistant to all macrolides tested (MIC, >32 microg/ml) and telithromycin (MIC, 4 microg/ml) had a single base deletion (A752) in domain II. In six remaining mutants, no mutations in L22, L4, or 23S rRNA could be detected.

FIG. 1.

Analysis of DNAs from parental strains and mutants by PCR-SSCP. Two portions of the rrl gene corresponding to 23S rRNA domain V (from nucleotides 1990 to 2134 [upper left] and from nucleotides 2331 to 2769 [upper middle]), a portion of the rrl gene for domain II of 23S rRNA (upper right), a part of the L4 gene, rplD (lower left), and the entire L22 gene, rplV (lower right), were amplified by PCR. Examples of the migration patterns obtained for these genes after separation of the denaturated PCR product by nondenaturant polyacrylamide gel electrophoresis are shown. The DNAs from the parent strains are designated by a number; the DNAs from the corresponding mutants are designated by the same number followed by the initial of the selector antibiotic: A, azithromycin; C, clarithromycin; E, erythromycin; R, roxithromycin; P, pristinamycin; T, telithromycin. Migration patterns for mutants 5A in the upper left gel, mutants 1C, 3C, 2A, 4C, and 4A in the upper middle gel, mutant 2C in the upper right gel, mutant 1A in the lower left gel, and mutants 1A, 1T, 3E, 3R, 4E, and 5R in the lower right gel (where C, A, T, E, and R correspond to clarithromycin, azithromycin, telithromycin, erythromycin, and roxithromycin, respectively) differ from the patterns of the parent DNAs.