All detectable high-molecular-mass penicillin-binding proteins are modified in a high-level beta-lactam-resistant clinical isolate of Streptococcus mitis

Abstract

All detectable high-molecular-mass penicillin-binding proteins (HMM PBPs) are altered in a clinical isolate of Streptococcus mitis for which the beta-lactam MICs are increased from those previously reported in our region (cefotaxime MIC, 64 microg/ml). These proteins were hardly detected at concentrations that saturate all PBPs in clinical isolates and showed, after densitometric analysis, 50-fold-lower radiotracer binding. Resistance was related to mosaic structure in all HMM PBP-coding genes, where critical region replacement was complemented not only by substitutions already reported for the closely related Streptococcus pneumoniae but also by other specific replacements that are presumably close to the active-site serine. Mosaic structure was also presumed in a pbp1a-sensitive strain used for comparison, confirming that these structures do not unambiguously imply, by themselves, detectable critical changes in the kinetic properties of these proteins.

FIG. 1

Radioactive detection of PBPs from strains 127R and 209S. Membranes containing approximately 200 μg of proteins were labeled with the indicated concentration of ¹²⁵I-penicillin X and were incubated for 15 min at 37°C. Penicillin concentrations are given in micrograms per milliliter.

FIG. 2

Protein sequence alignments from sensitive and resistant strains of S. mitis. All amino acid sequences belong to different strains of S. mitis and are named under their accession number (GenBank). 127R results are from this study. Shown in boldface are conserved motifs and selected amino acid positions where we detected variation along the complete protein sequence of sensitive strains 209S (this study) and S. mitis NCTC 10712 (there were no amino acid differences between them). ^R, β-lactam-resistant strain; ^S, β-lactam-sensitive strain; ∗∗, changes not previously reported in pneumococci; #, changes found only in strain 127R. The indicated numbers correspond to positions in PBPs from S. pneumoniae R6.