Open AccessAmino Acid Mutations Essential to Production of an Altered PBP 2X Conferring High-Level β-Lactam Resistance in a Clinical Isolate of Streptococcus pneumoniae
Author(s) -
Anthony M. Smith,
Keith P. Klugman
Publication year - 2005
Publication title -
antimicrobial agents and chemotherapy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.07
H-Index - 259
eISSN - 1070-6283
pISSN - 0066-4804
DOI - 10.1128/aac.49.11.4622-4627.2005
Subject(s) - cefotaxime , penicillin , penicillin binding proteins , streptococcus pneumoniae , microbiology and biotechnology , biology , streptococcaceae , cephalosporin , antibiotics , mutagenesis , mutation , genetics , gene
Altered penicillin-binding protein 2X (PBP 2X) is a primary β-lactam antibiotic resistance determinant and is essential to the development of penicillin and cephalosporin resistance in the pneumococcus. We have studied the importance for resistance of 23 amino acid substitutions located in the transpeptidase domain (TD) of PBP 2X from an isolate with high-level resistance, isolate 3191 (penicillin MIC, 16 μg/ml; cefotaxime MIC, 4 μg/ml). Strain R62X/2B/1A/mur (for which the MICs are as described for isolate 3191) was constructed by transforming laboratory strain R6 with all the necessary resistance determinants (altered PBPs 2X, 2B, and 1A and altered MurM) from isolate 3191. Site-directed mutagenesis was used to reverse amino acid substitutions in altered PBP 2X, followed by investigation of the impact of these reversions on resistance levels in R62X/2B/1A/mur . Of the 23 substitutions located in the TD of PBP 2X, reversals at six positions decreased the resistance levels in R62X/2B/1A/mur . Reversal of the Thr338Pro and Ile371Thr substitutions individually decreased the penicillin and cefotaxime MICs to 2 and 1 μg/ml, respectively, and individually displayed the greatest impact on resistance. To a lesser extent, reversal of the Leu364Phe, Ala369Val, Arg384Gly, and Tyr595Phe substitutions individually also decreased the penicillin and cefotaxime MICs. Reversal at all six positions collectively decreased both the penicillin and the cefotaxime MICs of R62X/2B/1A/mur to 0.06 μg/ml. This study confirms the essential role of altered PBP 2X as a resistance determinant. Our data reveal that, for isolate 3191, the six amino acid substitutions described above are collectively essential to the production of an altered PBP 2X required for high-level resistance to penicillin and cefotaxime.