The In-Vitro Sensitivity Testing of Beta-Lactam Antibiotic Combinations

Abstract

Clavulanic acid is a beta-lactam that has a high affinity for beta-lactamase enzymes, but is also inherently stable to the majority of them. Combination of clavulanic acid with amoxycillin protects the latter from attack by beta-lactamase enzymes and allows it to achieve its antibacterial effect. The effect of clavulanic acid on the activity of amoxycillin was studied, initially against well-characterised strains of enterobacteria. Chessboard titrations, disc sensitivity testing, and minimum inhibitory concentration (MIC) determination were used. A turbidimetric system was also used which allowed some effects of the combination to be studied. From the results, it became apparent that apart from its role as a beta-lactamase inhibitor, clavulanic acid affects the activity of other beta-lactam antibiotics in a different manner. This interaction was seen with beta-lactamase negative organisms and took the form of a complimentation effect due to the fact that different penicillin binding proteins (PBPs) were being bound by clavulanic acid and its partner. Clavulanic acid binds to PBP2 and from the studies carried out, greatest complimentation occurred when it was combined with an antibiotic that bound to PBP1. It also became apparent that there were problems with testing the sensitivity of organisms to the combination of amoxycillin and clavulanic acid. In a second part of the study, the methods for determining the susceptibility of strains of bacteria to amoxycillin in the presence of clavulanic acid were examined. Conventional disc sensitivity testing resulted in a majority of bacterial strains being classified as of intermediate sensitivities, even when the MIC of amoxycillin in the presence of clavulanic acid suggested that the strains were sensitive. Breakpoint sensitivity testing overcame this problem and a recommendation is made that this should be the method of choice for testing this combination.