Rapid functional definition of extended spectrum β-lactamase activity in bacterial cultures via competitive inhibition of fluorescent substrate cleavage.
The functional definition of extended-spectrum β-lactamase (ESBL) activity is a clinical challenge. Here we report a rapid and convenient assay of β-lactamase activity through the competitive inhibition of fluorescent substrate hydrolysis that provides a read-out nearly 40× more rapidly than conventional techniques for functional definition. A panel of β-lactam antibiotics was used for competition against β-lactamase enzyme-activated photosensitizer (β-LEAP) yielding a competitive index (C(i)) in 30 min. Significant differences in the relative C(i) values of the panel of β-lactams were determined in vitro for Bacillus cereus penicillinase. Additionally, the relative C(i) values for whole bacterial cell suspensions of B. cereus 5/β were compared with the relative minimal inhibitory concentration (MIC) values and a correlation coefficient of 0.899 was determined. We further demonstrated the ability of β-LEAP to probe the capacity of ceftazidime to inhibit the enzyme activity of a panel of ESBL-producing Escherichia coli. The bacteria were assayed for susceptibility to ceftazidime and the relative MIC values were compared with the relative C(i) values for ceftazidime yielding a correlation coefficient of 0.984. This work demonstrates for the first time the whole cell assay of the competitive inhibition of β-lactamase enzyme activity and derivation of associated constants.
Sallum
Ulysses W
UW
Wellman Center for Photomedicine, Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA.
Zheng
Xiang
X
Verma
Sarika
S
Hasan
Tayyaba
T
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001409:Bacillus cereus / Q000187:drug effects / Q000201:enzymology; D001667:Binding, Competitive; D002442:Ceftazidime / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes; D008826:Microbial Sensitivity Tests; D017319:Photosensitizing Agents; D013379:Substrate Specificity; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D017319:Photosensitizing Agents; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; D002442:Ceftazidime