Identification of a novel plasmid-encoded dihydrofolate reductase mediating high-level resistance to trimethoprim

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Trimethoprim is a crucial antibiotic for treating bacterial infections.
• Antibiotic resistance, particularly to trimethoprim, poses a significant global health threat.
• Plasmid-mediated resistance mechanisms are a major driver of antimicrobial resistance spread.

Purpose of the Study:

• To characterize a novel trimethoprim resistance determinant found in a Proteus mirabilis isolate.
• To investigate the genetic basis and biochemical properties of this resistance mechanism.
• To understand the implications of this novel enzyme for trimethoprim efficacy.

Main Methods:

• Isolation and characterization of a Proteus mirabilis strain from South Africa.
• Plasmid DNA extraction and analysis to identify the resistance gene.
• Molecular cloning and expression of the dihydrofolate reductase gene.
• Biochemical assays to determine enzyme kinetics and inhibition profiles.

Main Results:

• A 79kb transferable plasmid (pUK672) conferring high-level trimethoprim resistance (MIC > 2048 mg/l) was identified.
• A novel dihydrofolate reductase enzyme, designated type VI, was responsible for trimethoprim resistance.
• The type VI enzyme is small (10,000 daltons), unstable in vitro, and highly resistant to trimethoprim and methotrexate.
• The enzyme's properties are distinct from previously described plasmid-encoded dihydrofolate reductases.

Conclusions:

• A new plasmid-mediated trimethoprim resistance mechanism mediated by the type VI dihydrofolate reductase has been identified in Proteus mirabilis.
• The unique biochemical characteristics of this enzyme may present challenges for existing diagnostic and therapeutic strategies.
• Further research is warranted to understand the prevalence and spread of this novel resistance mechanism.