Analysis of penicillin-binding protein genes of clinical isolates of Streptococcus pneumoniae with reduced susceptibility to amoxicillin

The recent emergence of pneumococcal isolates exhibiting an unusual resistance phenotype of higher amoxicillin MICs in relation to the penicillin MICs prompted an analysis of the pbp genes from three such strains isolated in France. For comparison, three amoxicillin-susceptible strains were included in the study. DNA sequence analysis of the pbp2x, pbp2b, and pbp1a genes revealed extensive sequence divergence in all six isolates compared to the sequences of the genes of penicillin-susceptible strain R6. With the exception of pbp2b, no amino acid mutations were unique to the resistant isolates. Transformation experiments with cloned pbp genes isolated from one of the resistant isolates demonstrated a stepwise development of amoxicillin resistance involving penicillin-binding proteins (PBPs) 2X, 2B, and 1A. Full resistance, equivalent to that of the donor strain, was achieved only when genomic DNA was transformed into R6(2x/2b/1a) mutants, suggesting that full resistance development in this isolate is mediated by a non-PBP determinant. Moreover, the recently identified murMN resistance determinant does not appear to have any impact on resistance in this isolate. This determinant (from the French isolate) was, however, able to transform an R6 mutant harboring pbp2x, pbp2b, and pbp1a genes from a Hungarian clone with an extremely high level of penicillin resistance so that it had increased levels of penicillin resistance. These results indicate that the development of high-level beta-lactam resistance is a complex process and that the involvement of MurMN in penicillin resistance appears to be dependent on specific mutations in PBPs 2X, 2B, and/or 1A. Furthermore, an additional (as yet unidentified) non-PBP-mediated resistance determinant is required for full resistance development in some pneumococci.