A Novel Plate Assay System for Screening of Antimicrobial Agents and β-Lactamase Inhibitors from Natural Sources

Abstract

β-Lactam class of antibiotics like amoxicillin, ticarcillin, and piperacillin is still used widely especially in developing countries against gram-positive as well as gram-negative organisms. It is used mainly for the treatment of respiratory infections and urinary infections. Resistance to β-lactam antibiotics is often attained by synthesizing β-lactamase enzymes, which act by attacking the β-lactam ring. To overcome this resistance, β-lactam classes of antibiotics are generally prescribed in combination with β-lactamase inhibitors like tazobactam, clavulanic acid, or sulbactam. There are reports of new strains with β-lactamase enzymes resistant to these inhibitors. One of the major sources of novel therapeutic agents is from the natural sources like plants, algae and other microorganisms. The existing methods of β-lactamase assays are based on calorimetric method and may not be always suitable for coloured organic solvent extracts of plants. Also, the varying pH of many of the extracts often becomes a problem in conducting these assays. Here, we report a novel agar plate assay method by which it is possible for screening antimicrobial agents as well as β-lactamase inhibitors simultaneously with a single assay. The method is based on Kirby-Bauer disc diffusion assay using Mueller-Hinton agar. The β-lactamase-producing organism is swabbed on two plates, one with a β-lactam antibiotic and the other without the antibiotic. Paper discs are impregnated with the extract in investigation and applied on the two plates. If it is showing zone of inhibition in both the plates, it can be due to a possible antimicrobial agent, but if it is showing zone of inhibition in only the plate with β-lactam antibiotic, then this may be due to the presence of a possible β-lactam inhibitor. This system was tested good using five different MRSA strains, three different β-lactamase inhibitors and diethyl ether extract of Holoptelea integrifolia.