PBP 2a mutations producing very-high-level resistance to beta-lactams

Area of Science:

• Microbiology
• Antibiotic Resistance
• Molecular Biology

Background:

• Methicillin-resistant Staphylococcus aureus (MRSA) resistance to beta-lactam antibiotics is primarily mediated by penicillin-binding protein 2a (PBP 2a), encoded by the mecA gene.
• Novel beta-lactams with high affinity for PBP 2a are being developed to combat MRSA infections.

Purpose of the Study:

• To investigate the potential for MRSA to develop resistance to novel, high-affinity beta-lactams.
• To identify the specific mutations in PBP 2a that contribute to high-level resistance.

Main Methods:

• MRSA strain COL was subjected to prolonged exposure to an investigational carbapenem (L-695,256) to select for resistant mutants.
• Genetic analysis of resistant mutants, including sequencing of the mecA gene and PBP 2a.
• Introduction of various PBP 2a constructs with specific mutations into methicillin-susceptible Staphylococcus aureus strains to assess their impact on resistance levels.

Main Results:

• A highly resistant mutant, COL52, was generated, exhibiting PBP 2a with a two-amino-acid deletion and three substitution mutations.
• Specific amino acid substitutions (E→K at position 237 and V→E at position 470) were found to be critical for high-level resistance.
• The highest level of resistance was observed when all four identified mutations were present in PBP 2a.

Conclusions:

• Emergence of PBP 2a-mediated resistance to high-affinity beta-lactams in MRSA is likely to necessitate multiple mutations within the mecA gene.
• Chromosomal mutations appear to play a minimal role in the development of this specific resistance mechanism.