Bactericidal interactions of a beta-lactam and beta-lactamase inhibitors in experimental Pseudomonas aeruginosa endocarditis caused by a constitutive overproducer of type Id beta-lactamase

Abstract

We investigated the in vitro and in vivo effects of a combination of a beta-lactam (ceftazidime) and a beta-lactamase inhibitor (dicloxacillin) to synergistically kill a ceftazidime-resistant variant, Pseudomonas aeruginosa PA-48, which overproduces type Id cephalosporinase constitutively. In vitro, dicloxacillin plus ceftazidime exerted bactericidal synergy at approximately 10(5) CFU/ml of inoculum (but not at approximately 10(7)-CFU inoculum), whereas other beta-lactamase inhibitors (sulbactam, clavulanic acid) showed no enhanced killing of PA-48 when combined with ceftazidime at clinically achievable levels for each agent. Dicloxacillin was a potent competitive inhibitor of the extracted Id cephalosporinase from strain PA-48 in short-term comixture studies (less than 10 min [Ki = 2 nM]); in contrast, longer-term comixture studies (90 min) indicated that dicloxacillin functions as a competitive substrate for the enzyme. Growth of PA-48 cells in the presence of dicloxacillin (12.5 to 100 micrograms/ml) had no significant effect on the production rates or functional activity of the Id enzyme. In experimental aortic valve endocarditis due to the ceftazidime-resistant variant (PA-48), rabbits received either no therapy, ceftazidime (25 mg/kg intramuscularly, every 4 h), or ceftazidime plus dicloxacillin (200 mg/kg intramuscularly, every 4 h). The combination regimen reduced mean bacterial densities of PA-48 within cardiac vegetations significantly below those in the other groups at both days 3 and 6 of treatment (P less than 0.005). However, mean vegetation bacterial densities remained greater than 6 log10 CFU/g in the combined treatment group. This modest in vivo synergistic effect (as compared to striking in vitro synergy at approximately 10(5)-CFU inoculum) most likely reflects the high densities of PA-48 achieved in vivo within cardiac vegetations (greater than 8 log10 CFU/g).