Characterization of β-lactamase activity using isothermal titration calorimetry

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 8; pp. 2031 - 2038
• Main Authors: Wang, Wen-Jing, Wang, Qian, Zhang, Ye, Lu, Rui, Zhang, Yi-Lin, Yang, Ke-Wu, Lei, Jin-E, He, Yuan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2017
• Subjects: Anti-Bacterial Agents - pharmacology; antibiotic resistance; Antimicrobial resistance; bacteria; benzylpenicillin; beta-lactamase; beta-Lactamase Inhibitors - pharmacology; beta-Lactamases - metabolism; calorimetry; Calorimetry - methods; cefazolin; enzymatic hydrolysis; enzyme kinetics; Escherichia coli; heat; hydrolysis; imipenem; Inhibitor; Isothermal titration calorimetry; Microbial Sensitivity Tests; screening; spectrophotometers; Spectrophotometry; Steady-state kinetics; Thermodynamics; titration; β-lactamase; Thermodynamics; Inhibitor; Isothermal titration calorimetry; Antimicrobial resistance; β-lactamase; Steady-state kinetics
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2017.04.011

Hydrolysis of β-lactam antibiotic by β-lactamase is the most common mechanism of β-lactam resistance in clinical isolates. Timely detection and characterization of β-lactamases are therefore of utmost biomedical importance. Conventional spectrophotometric method is time-consuming and cannot provide thermodynamic information on β-lactamases. A new assay was developed for the study of β-lactamase activity in protein solutions (Metallo-β-lactamase L1) and in clinical bacterial cells, based on heat-flow changes derived from enzymatic hydrolysis of β-lactams using isothermal titration calorimetry. (1) The thermokinetic parameters of three antibiotics (penicillin G, cefazolin and imipenem) and the inhibition constant of an azolylthioacetamide inhibitor were determined using the calorimetric assay. The results from the calorimetric assays were consistent with the data from the spectrophotometric assay. (2) The values of heat change in the calorimetric assay using two clinical Escherichia coli strains correlated well with their antibiotic susceptibility results from the broth dilution experiment. The subtypes of β-lactamase were also determined in the calorimetric assay. The ITC assay is a reliable and fast method to study β-lactamase enzyme kinetics and inhibition. It can also provide thermodynamic information on antibiotic hydrolysis, which has been taken advantage of in this work to study β-lactamase activity in two clinical Escherichia coli isolates. As the first calorimetric study of β-lactamase activity, it may provide a new assay to assist biomedical validation of new β-lactamase inhibitors, and also has potential applications on rapid antibiotic susceptibility testing and screening β-lactamase producing bacteria. [Display omitted] •A new calorimetric assay is proposed for the study of β-lactamase activity.•The ITC assay is reliable and fast to provide the thermokinetic and inhibition information of a purified β-lactamase.•It also shows great potential in detecting β-lactamase activity and discriminating its subtypes in clinical bacteria.