PBP2a mutations causing high-level Ceftaroline resistance in clinical methicillin-resistant Staphylococcus aureus isolates

Abstract

Ceftaroline is the first member of a novel class of cephalosporins approved for use in the United States. Although prior studies have identified eight ceftaroline-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolates in Europe and Asia with MICs ranging from 4 to 8 mg/liter, high-level resistance to ceftaroline (>32 mg/liter) has not been described in MRSA strains isolated in the United States. We isolated a ceftaroline-resistant (MIC > 32 mg/liter) MRSA strain from the blood of a cystic fibrosis patient and five MRSA strains from the respiratory tract of this patient. Whole-genome sequencing identified two amino acid-altering mutations uniquely present in the ceftaroline-binding pocket of the transpeptidase region of penicillin-binding protein 2a (PBP2a) in ceftaroline-resistant isolates. Biochemical analyses and the study of isogenic mutant strains confirmed that these changes caused ceftaroline resistance. Thus, we identified the molecular mechanism of ceftaroline resistance in the first MRSA strain with high-level ceftaroline resistance isolated in the United States.

FIG 1

Active site of acyl-PBP2a from MRSA with bound ceftaroline and the two contiguous amino acids that are altered (Y446N and E447K) in the resistant organisms. This view of the PBP2a transpeptidase active site shows the Y446 (red) and E447 (green) amino acids highlighted in front of a bound molecule of ceftaroline (yellow). The transpeptidase portion of the molecule is colored blue, and the allosteric portion of PBP2a is colored violet. The structure has PDB accession number 3ZG0 and was published by Otero et al. (25).

FIG 2

Natural history of six MRSA clinical isolates collected over 154 days with their PBP2a active-site amino acid mutations and ceftaroline MICs. S, sputum samples; B, blood isolate. The highly resistant isolates have MICs of >32 mg/liter, which are marked in red. Init, initial.

FIG 3

Bocillin competition assay to determine the IC₅₀ of ceftaroline for the PBP2a wild type and mutants. The y axis represents the residual fluorescence intensity of Bocillin, while the x axis represents the logarithmic ceftaroline concentration (in molar). The Y446N mutant did not show any decrease in fluorescence intensity even with the highest concentration of inhibitor tested, and thus, the results for that mutant are not represented here.