Properties of IRT-14 (TEM-45), a newly characterized mutant of TEM-type beta-lactamases

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Beta-lactamase enzymes are a primary mechanism of bacterial resistance to penicillin-class antibiotics.
• Escherichia coli is a common pathogen, and the emergence of antibiotic resistance is a significant public health concern.
• TEM-type beta-lactamases are prevalent and contribute to resistance against a wide range of beta-lactam antibiotics.

Purpose of the Study:

• To characterize a novel TEM-type beta-lactamase, designated IRT-14 (TEM-45), isolated from a clinical Escherichia coli strain.
• To investigate the molecular basis of resistance conferred by IRT-14, including its kinetic properties and susceptibility to beta-lactamase inhibitors.
• To identify the genetic mutations responsible for the altered enzyme characteristics and increased production.

Main Methods:

• Isolation and characterization of the novel beta-lactamase IRT-14 from clinical Escherichia coli P37.
• Determination of minimum inhibitory concentrations (MICs) for various beta-lactam antibiotics and combinations with inhibitors.
• Purification of the enzyme to homogeneity and determination of kinetic parameters (Kcat, Km, Kcat/Km) for different substrates.
• Analysis of inhibitory concentrations (IC50) for beta-lactamase inhibitors.
• Identification of mutations in the promoter region (G-162-->T) and coding sequence (Met-69-->Leu, Arg-275-->Gln) of the blaTEM gene.

Main Results:

• IRT-14 confers high-level resistance to broad-spectrum penicillins (e.g., amoxicillin MIC of 4,096 µg/ml).
• The enzyme exhibits reduced sensitivity to beta-lactamase inhibitors like clavulanic acid and tazobactam.
• Kinetic analysis revealed reduced affinity for broad-spectrum penicillins and cephalosporins.
• Increased enzyme production due to a G-to-T substitution in the promoter region (G-162-->T) contributes to resistance.
• Specific amino acid substitutions (Met-69-->Leu, Arg-275-->Gln) in the enzyme structure are associated with altered substrate affinity and inhibitor resistance.

Conclusions:

• IRT-14 is a novel TEM-type beta-lactamase that significantly contributes to antibiotic resistance in Escherichia coli.
• The resistance mechanism involves both increased enzyme expression and structural modifications affecting substrate binding and inhibitor susceptibility.
• Understanding these mutations is crucial for developing strategies to combat emerging antibiotic resistance.