Characterization of R factor beta-lactamases by the acidimetric method

Area of Science:

• Microbiology
• Biochemistry

Background:

• R factors confer antibiotic resistance in bacteria.
• Beta-lactamases are enzymes that hydrolyze beta-lactam antibiotics, leading to resistance.
• Ampicillin resistance in Escherichia coli and Klebsiella is often mediated by R factors.

Purpose of the Study:

• To characterize the properties and regulation of beta-lactamases produced by R factors.
• To develop a sensitive and efficient assay for R factor beta-lactamases.

Main Methods:

• Characterization of beta-lactamase activity using an acidimetric method with phenol red indicator.
• Optimization of the assay sensitivity using phosphate buffer.
• Distinguishing beta-lactamase classes based on substrate profile, heat inactivation, and K(m) values.
• Investigating the regulatory mechanisms of beta-lactamase production.

Main Results:

• A sensitive acidimetric assay for beta-lactamase activity was developed, capable of detecting as little as 0.05 unit.
• Two distinct classes of beta-lactamases were identified based on enzymatic properties.
• Most R factor beta-lactamases studied were subject to cyclic adenosine monophosphate-mediated catabolite repression.

Conclusions:

• The developed acidimetric assay is a rapid, convenient, and sensitive method for studying R factor beta-lactamases.
• Beta-lactamases from ampicillin-resistant strains exhibit diverse properties and are regulated by catabolite repression.