In vitro pharmacodynamic syudies of B-lactam-B-lactamase inhibitor combinations against B-lactamase producing clinically important gram-negative bacteria

Abstract

Combinations of the beta-lactam and beta-lactamase inhibitor are antibiotics extensively used in clinic for the treatment of infectious disease caused by the beta-lactamase producing bacteria. The mode of action of beta-lactamase inhibitor is regarded as irreversible, suicide inhibitors of the target enzyme resulting in persistent activity of beta-lactams to inhibit bacterial cell wall synthesis, which leads to cell death. The present study aimed to evaluate the synergistic interaction between beta-lactams and beta-lactamase inhibitors on clinically important beta-lactamase producing gram-negative bacteria by checkerboard technique and time kill method. Clavulanic acid at 2 microgram/ml demonstrated synergy to amoxicillin against Moraxella catarrhalis and Haemophilus influenzae by reduction MIC of amoxicillin to 64 times. Similarly tazobactam at 4 .microgram/ml reduce the MIC of piperacillin against Klebsiella pneumoniae and Pseudomonas aeruginosa to 64 and 4 times, respectively. The MIC of cefoperazone against P. aeruginosa was decreased 8 times when being combined with sulbactam 8 microgram/ml whereas Acinetobacter baumannii required sulbactam 32 microgram/ml to reduce the MIC of cefoperazone. Additionally, beta-lactams (amoxicillin, piperacillin, cefoperazone) at 2 MIC in concomitant with beta-lactamase inhibitors at average concentration (clavulanic acid at 2 microgram/ml, tazobactam at 4 microgram/ml, sulbactam at 8 mg/ml) demonstrated the antibacterial properties and synergistic activity by decreasing colony forming unit more than 100 fold comparing with the most active single drug except for A. baumannii that required sulbactam at least 32 microgram/ml to show those properties. Regarding to post beta-lactamase inhibitor effect (PLIE), amoxicillin-clavulanic acid and piperacillin-tazobactam manifested the time period of PLIE that correlated to concentration of beta-lactamase inhibitors against H. influenzae and P. aeruginosa, respectively. Furthermore it found that one of three beta-lactamase inhibitors, clavulanic acid, demonstrated beta-lactamase induction effect by inducing Enterobacter cloacae to produce beta-lactamase that destroyed cefuroxime as tested by double disks as well as agar dilution methods. The MIC of cefuroxime was increased from 6 mg/ml to 32 microgram/ml on exposure to clavulanic acid 10 microgram/ml. The results obtained suggest that the concentration of beta-lactamase inhibitors and beta-lactams under studies are appropriate for clinical application.