BIOLUMINESCENCE BIOASSAY FOR BETA-LACTAMASE INHIBITORS SCREENING

The ß-lactam family is one of the most widely used groups of antibiotics. Production of ß-lactamases is the most common and important mechanism of resistance found in Gram-negative bacteria acting against the ß- lactam antibiotic. In order to preserve the utility of ß-lactam antibiotics, two strategies were advanced: (a) The discovery or design of ß-lactam antibiotics that are able to evade enzymatic inactivation conferred by ß-lactamases, and (b) inhibition of ß-lactamases so that ß- lactam can reach the penicillin binding proteins (PBPs), the target of ß-lactam antibiotics. Currently, there are a limited numbers of drugs that contain combinations of ß- lactam antibiotic with ß-lactamases inhibitors including "Augmentin" that contains a combination of amoxicillin and Potassium clavulanate.

The developed assay use bacterial indicator strains that containing bioluminescent plasmid and ß-lactam multi resistant plasmid. The bioluminescent plasmid is caring promoter which activates due genotoxic damage, or due cytotoxic substances, and metabolic changes.

In this study, we examined the bacterial response to different ß-lactam Antibiotics including Piperacillin (ureidopenicillin class), Ampicillin (aminopenicillin family) and Amoxicillin (aminopenicillin family). After finding the highest concentration of antibiotic that each bacterial species is resistant to, we added commercial ß-lactamase inhibitors: Tazobactam, Sulbactam and potassium clavulanate, respectively and studied effect of these compounds (antibiotics and inhibitors) on the bacteria. Our findings demonstrate that the bacteria respond to the combined antibiotics/ß-lactamase inhibitors.

After the proof of concept stage, the next aims of this study will be to screen ß-lactam inhibitors from environment isolates of bacteria and fungi.