The increasing emergence of new bacterial β-lactamases that can efficiently hydrolyze β-lactam antibiotics to clinically inactive carboxylic acids has created an intractable problem in the treatment of bacterial infections, and it is highly desirable to develop a useful tool that can rapidly screen bacteria for β-lactamases against a variety of antibiotic candidates in a high-throughput manner. This paper describes the use of a fluorescein-labeled β-lactamase mutant (E166Cf) as a convenient fluorescent tool to screen β-lactamases, including the Bacillus cereus β-lactamase I (PenPC), B. cereus β-lactamase II, Bacillus licheniformis PenP, Escherichia coli TEM-1, and Enterobacter cloacae P99 against various β-lactam antibiotics (penicillin G, penicillin V, ampicillin, cefuroxime, cefoxitin, moxalactam, cephaloridine), using a 96-well microplate reader. The E166Cf mutant was constructed by replacing Glu166 on the flexible Ω-loop, which is close to the enzyme's active site, with a cysteine residue on a class A β-lactamase (B. cereus PenPC) and subsequently labeling the mutant with thiol-reactive fluorescein-5-maleimide. Such modifications significantly impaired the hydrolytic activity of the E166Cf mutant compared to that of the wild-type enzyme. The fluorescence intensity of the E166Cf mutant increases in the presence of β-lactam antibiotics. For antibiotics that are resistant to hydrolysis by the E166Cf mutant (cefuroxime, cefoxitin, moxalactam), the fluorescence signal slowly increases until it reaches a plateau. For antibiotics that can be slowly hydrolyzed by the E166Cf mutant (penicillin G, penicillin V, ampicillin), the fluorescence signal rapidly increases to the plateau and then declines after a prolonged incubation. The E166Cf mutant retains its characteristic pattern of fluorescence signals in the presence of both bacterial β-lactamases and β-lactamase-resistant antibiotics. In contrast, in the presence of both bacterial β-lactamases and β-lactamase-sensitive antibiotics, the fluorescence signals of the E166Cf mutant were decreased. The fluorescence signals from the E166Cf mutant allow an unambiguous differentiation of β-lactamase-resistant antibiotics from β-lactamase-sensitive ones in the screening of bacterial β-lactamases against a panel of antibiotic candidates. This simple method may provide an alternative tool in choosing potent β-lactam antibiotics for treatment of bacterial infections.
ASJC Scopus subject areas
- Analytical Chemistry