Development and qualification of a pharmacodynamic model for the pronounced inoculum effect of ceftazidime against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic, gram-negative pathogen responsible for high morbidity and mortality (19). P. aeruginosa has multiple mechanisms of resistance to antibiotics, including efflux pumps, the enzymatic degradation of antibiotics by, e.g., beta-lactamases, and target structure alteration (19, 25, 45). Due to its remarkable ability to resist killing by antibiotics (45), many P. aeruginosa isolates from nosocomial infections are multidrug resistant. The proportion of ceftazidime-resistant P. aeruginosa isolates from intensive care units increased from approximately 14% in 1997 to 24% in 2003 in the United States (23). Infections with a high bacterial density at the initiation of antibiotic therapy may present a therapeutic problem, including a higher risk for the emergence of resistance due to the larger number of bacteria present and the higher probability of having at least one resistant bacterial cell within a large initial inoculum (CFUo) (32). The probability of the emergence of resistance may be substantially increased at high CFUo, as the amplification of resistant subpopulations has been demonstrated to occur secondarily to low-intensity antimicrobial exposure (60). The inoculum effect was first described by Kirby (34) in vitro for penicillin activity against staphylococci. Subsequently, many studies assessed the effect of CFUo on the MIC (6, 16). Mouton et al. (47, 48) derived the relationship between MIC and CFUo if the growth rate and maximal killing rate constant are independent of the CFUo. This effect of the CFUo on the MIC needs to be distinguished from the inoculum effect in our time-kill study, since we assessed the effect of the CFUo on the rate of bacterial killing. Importantly, high CFUo are associated with increased mortality and attenuate antibiotic effects in animal infection models (6, 20, 57). Potential mechanistic explanations for the inoculum effect of beta-lactams include the breakdown of beta-lactams by beta-lactamases, cell-to-cell communication, and the differential expression of penicillin-binding proteins (PBPs) at a high bacterial density (14, 57). In P. aeruginosa, cell-to-cell communication is known to be mediated by the release of freely diffusible signal molecules such as two N-acylhomoserine lactones and the Pseudomonas quinolone signal molecule (2-heptyl-3-hydroxy-4-quinolone) (29, 54). High concentrations of the N-butyryl-l-homoserine lactone signal molecule induce the expression of MexAB-OprM in P. aeruginosa (40, 53), for which ceftazidime is a substrate (5, 19, 41), and the mexAB-oprM operon has its highest expression during the mid-stationary-growth phase (40). These signal molecules are known to affect the expression of several hundred genes in P. aeruginosa (54). Mathematical models that can describe a slower bacterial killing rate at high CFUo have not been published. A pharmacokinetic/pharmacodynamic (PKPD) model that can describe the inoculum effect of P. aeruginosa may support the optimization of dosage regimens and generate hypotheses on how to minimize the emergence of resistance for new and established antibiotics. The objectives of the current study were to (i) study the effect of CFUo on the rate and extent of the killing of P. aeruginosa PAO1 by ceftazidime in vitro, (ii) develop a mechanism-based, mathematical model that can accommodate a range of CFUo, and (iii) qualify this model by integrating literature results for in vitro PD models with P. aeruginosa PAO1 and P. aeruginosa ATCC 27853 for ceftazidime monotherapy. (This work was presented in part at the 2008 Annual Meeting of the Population Approach Group in Australia & New Zealand, Dunedin, New Zealand, and at the 2007 Annual Meeting of the American Association of Pharmaceutical Scientists, San Diego, CA.)