The mechanisms responsible for macrolide resistance in Streptococcus pneumoniae mutants, selected from susceptible strains by serial passage in azithromycin, were investigated. These mutants were resistant to 14-and 15-membered macrolides, but resistance could not be explained by any clinically relevant resistance determinant [mef(A), erm(A), erm(B), erm(C), erm(TR), msr(A), mph(A), mph(B), mph(C), ere(A), ere(B)]. An investigation into the sequences of 23S rRNAs in the mutant and parental strains revealed individual changes of C2611A, C2611G, A2058G, and A2059G (Escherichia coli numbering) in four mutants. Mutations at these residues in domain V of 23S rRNA have been noted to confer erythromycin resistance in other species. Not all four 23S rRNA alleles have to contain the mutation to confer resistance. Some of the mutations also confer coresistance to streptogramin B (C2611A, C2611G, and A2058G), 16-membered macrolides (all changes), and clindamycin (A2058G and A2059G). Interestingly, none of these mutations confer high-level resistance to telithromycin (HMR-3647). Further, two of the mutants which had no changes in their 23S rRNA sequences had changes in a highly conserved stretch of amino acids ( 63 KPWRQKGTGRAR 74 ) in ribosomal protein L4. One mutant contained a single amino acid change (G69C), while the other mutant had a 6-base insert, resulting in two amino acids (S and Q) being inserted between amino acids Q67 and K68. To our knowledge, this is the first description of mutations in 23S rRNA genes or ribosomal proteins in macrolide-resistant S. pneumoniae strains.

Resistance to macrolides in pneumococci is generally mediated by methylation of 23S rRNA via erm(B) methylase which can confer a macrolide (M)-, lincosamide (L)-, and streptogramin B (S B )-resistant (MLS B ) phenotype or by drug efflux via mef(A) which confers resistance to 14-and 15-membered macrolides only. We studied 20 strains with unusual ML or MS B phenotypes which did not harbor erm(B) or mef(A). The strains had been isolated from patients in Eastern Europe and North America from 1992 to 1998. These isolates were found to contain mutations in genes for either 23S rRNA or ribosomal proteins. Three strains from the United States with an ML phenotype, each representing a different clone, were characterized as having an A2059G (Escherichia coli numbering) change in three of the four 23S rRNA alleles. Susceptibility to macrolides and lincosamides decreased as the number of alleles in isogenic strains containing A2059G increased. Sixteen MS B strains from Eastern Europe were found to contain a 3-amino-acid substitution ( 69 GTG 71 to TPS) in a highly conserved region of the ribosomal protein L4 ( 63 KPWRQKGTGRAR 74 ). These strains formed several distinct clonal types. The single MS B strain from Canada contained a 6-amino-acid L4 insertion ( 69 GTGREKGT-GRAR), which impacted growth rate and also conferred a 500-fold increase in MIC on the ketolide telithromycin. These macrolide resistance mechanisms from clinical isolates are similar to those recently described for laboratory-derived mutants.

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 g/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 g/ml) and telithromycin (MIC, 4 g/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.Resistance to macrolides is increasingly reported in clinical isolates of Streptococcus pneumoniae worldwide (10, 14). Resistance to macrolides was primarily related to modification of the ribosomal targets of these antibiotics. This mechanism relies on N-6 dimethylation of a specific adenine residue in 23S rRNA which confers cross-resistance to macrolides, lincosamides, and streptogramins B, the so-called MLS B phenotype, and is encoded in pneumococci by genes belonging to the erm(B) or erm(A) class (11,18,26). Subsequently, target modification was reported in the majority if not all macrolideresistant pneumococci (11,26). More recently, a mechanism of resistance by active efflux of erythromycin due to the mefE gene, renamed mef(A) (18), was reported in S. pneumoniae and appeared to be predominant in the United States and Canada, with prevalences ranging from 41 to 85% (9,19,20). The efflux phenotype, which is also called the M phenotype, is characterized by resistance to 14-membered-ring (erythromycin, clarithromycin, and roxithromycin) and 15-membered-ring (azithromycin) macrolides only. Ribosomal mutation has been reported only recently in a few clinical isolates of S. pneumoniae (5,21,22). The changes were clustered in a highly conserved sequence of L4 and in nucleotide residues of domain V of 23S rRNA which have a key role in macrolide binding. A recent study by Davies et al. (4) showed that mutants can readily be selected in pneumococci in the presence of any of several MLS B antibiotics in vit...

Ceftobiprole exhibited tight binding to PBP2a in methicillin-resistant Staphylococcus aureus, PBP2x in penicillin-resistant Streptococcus pneumoniae, and PBP3 and other essential penicillin-binding proteins in methicillin-susceptible S. aureus, Escherichia coli, and Pseudomonas aeruginosa. Ceftobiprole also bound well to PBP2 in the latter organisms, contributing to the broad-spectrum antibacterial activity against gram-negative and gram-positive bacteria.