A237T as a modulating mutation in naturally occurring extended-spectrum TEM-type beta-lactamases

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Beta-lactamases are enzymes that confer bacterial resistance to beta-lactam antibiotics.
• Antibiotic resistance evolves through the accumulation of genetic mutations.
• Understanding the evolutionary mechanisms of beta-lactamase resistance is crucial for developing new therapeutic strategies.

Purpose of the Study:

• To investigate the impact of the A237T mutation on TEM-1 beta-lactamase activity and substrate specificity.
• To determine the effect of combining the A237T mutation with existing TEM-10 mutations (R164S and E240K).
• To explore the role of seemingly neutral mutations in the evolution of antibiotic resistance.

Main Methods:

• Site-directed mutagenesis was used to introduce the A237T substitution into TEM-1 beta-lactamase.
• Minimum Inhibitory Concentrations (MICs) were determined for various beta-lactam antibiotics (cephalothin, cefotaxime, ceftazidime, aztreonam) against Escherichia coli expressing different TEM-1 variants.
• Comparative analysis of MIC values was performed to assess the effect of individual and combined mutations.

Main Results:

• The A237T mutation alone caused a slight increase in cephalothin MIC but did not affect cefotaxime, ceftazidime, or aztreonam activity.
• When combined with TEM-10 mutations (R164S/E240K), the A237T mutation significantly altered substrate preference.
• Specifically, ceftazidime and aztreonam MICs decreased substantially, while the cefotaxime MIC increased, indicating a shift in resistance profile.

Conclusions:

• The A237T mutation acts as a modulating mutation, influencing the substrate specificity of TEM-1 beta-lactamase.
• Acquisition of apparently neutral or deleterious mutations can lead to highly effective resistance mechanisms against multiple beta-lactams.
• Consideration of modulating mutations is essential for a comprehensive understanding of beta-lactamase evolution and the development of strategies to combat antibiotic resistance.