An Efficient Assay to Screen β-Lactamase Inhibitors from Plant Extracts

Abstract

β-Lactam-antibiotics (BLAs), the most widely used group of antibiotics, have significantly served the humanity, since their discovery. Some bacteria (e.g., methicillin resistant Staphylococcus aureus) have developed mutated penicillin-binding proteins/enzymes and thus developed resistance to almost all of the β-lactam-antibiotics. Thus rendering antibiotic industry; worth US $25 billion almost to death-bed. Addition of a β-lactamase (BLase) inhibitor to β-lactamantibiotics has been partially successful in alleviating this resistance. Though synthetic β- lactamase-inhibitors are at the scene, but search for new inhibitors from natural sources is a sound cry of the time. Preliminary screening of crude plant extracts is almost impossible using reported microbiological and/or instrumental techniques due to coloured nature of extracts, limited availability of purified β-lactamase and other problems. Hence a new, easy, efficient, economical and versatile assay was developed and practiced successfully in our laboratory. Transformed _{Escherichia coli} DH5α with pET21α, which has a gene for β-lactamase was used. Transformation was carried out by modified basic CaCl₂ method with plasmid DNA. Assay was performed in test tubes according to reported templates, modified and managed to nullify colour problems. About 250 plant extracts were screened and the new method was found satisfactory in all aspects. Hence, it is suitable for large-scale screening of crude and semi-purified extracts.