Pharmacodynamics of Ceftazidime plus the Serine β-Lactamase Inhibitor AM-112 against Escherichia coli Containing TEM-1 and CTX-M-1 β-Lactamases

ABSTRACT A strain of Escherichia coli containing TEM-1 and CTX-M-1 was tested in an in vitro pharmacokinetic model against ceftazidime with and without AM-112, a serine β-lactamase inhibitor. Ceftazidime alone was less effective than ceftazidime plus AM-112, and a single dose was more effective than three fractionated doses.

Ceftazidime was obtained from Eli Lilly & Co., Basingstoke, United Kingdom. AM-112 was supplied by SynProTec Ltd., Manchester, United Kingdom. Experiments were performed with E. coli containing a bla TEM-1 and CTX-M-1 gene, for which both ␤-lactamases are plasmid encoded, from the collection of the Department of Pathology and Microbiology, University of Bristol. L1 ␤-lactamase as previously described (8) was used to neutralize AM-112 and ceftazidime.
The ceftazidime MIC was determined by using British Society for Antimicrobial Chemotherapy guidelines (1), except that 10% Mueller-Hinton broth was used with fixed concentrations of 4 and 16 mg of AM-112/liter with an inoculum of approximately 10 6 CFU/ml.
The in vitro pharmacokinetic model as previously described (5) was used for all kill curve experiments. The 10 6 CFU/ml inoculum was prepared by adding 720 l of overnight broth culture of the test strain to the culture chamber and allowing the model to run for 45 min. The larger inoculum of 10 8 CFU/ml was prepared by inoculating the culture chamber with 2 ml of a 0.5 McFarland standard equivalent suspension of the test strain and allowing the model to run overnight (18 h) so that a steady state could be achieved.
The pharmacokinetics simulated for both ceftazidime and AM-112 were a maximum concentration of 200 mg of serum/ liter and a half-life of 2 h. Ceftazidime was added to the central chamber at time zero and at 8 and 16 h. AM-112 was added to the chamber either as one dose at time zero or fractionated into three 8-hourly doses. Samples were collected from the model throughout the 24-h time period (time zero and 1, 2, 3, 4, 5, 6, 7, 8, 16, and 24 h) for assessments of the viable counts and determinations of ceftazidime concentrations by bioassays as previously described (2). The measures of antibacterial effects calculated for each inoculum were the log changes in viable counts at 8, 16, and 24 hours; the median times for the initial inoculum to be reduced by 99.9%; and the areas under the bacterial kill curve from 0 to 24 h (AUBKC24). All experiments were performed in triplicate. Statistical analysis was done by analysis of variance (ANOVA), and model assumptions were assessed graphically. Results are presented as differences in mean responses, with 95% confidence intervals.
The ceftazidime MIC for the E. coli was 64 mg/liter alone and 0.25 mg/liter when AM-112 was present at a fixed concentration of either 4 or 16 mg/liter. Ceftazidime on its own produced a 3.0 to 3.5 log 10 reduction in viable count up to 16 h with the 10 6 -CFU/ml inoculum and  ministration of AM-112 resulted in a drop in viable count below the detection limit by 16 h; this drop occurred with once-a-day (OD) administration by 8 h (Fig. 1) (Table 1). Statistical comparisons of the two inocula and the three dosing regimens indicated that there were significant differences in the antibacterial effect between the two inocula and between the three dosing regimens, as measured by AUBKC24 and the changes in the viable counts at 24 h.
The magnitude of the differences between the three dosing regimens also differed between the two inocula (for AUBKC24, the P value was Ͻ0.001; for the changes in viable counts at 24 h, the P value was 0.01) ( Table 2). As all dosing regimens involving AM-112 resulted in pathogen clearance by 24 h, the differences in AUBKC24 noted in the OD versus TDS dosage comparisons are due to differences in the viable counts between 0 and 16 h.
These results build on those of Jamieson et al. (3), who showed that ceftazidime plus AM-112 at a ratio of 1:1 reduced the MICs for Enterobacteriaceae with extended-spectrum ␤-lactamases. The drug combination also reduced the 50% effective doses for ceftazidime in a murine intraperitoneal infection model with an Enterobacter cloacae-producing class C ␤-lactamase and Klebsiella pneumoniae-producing SHV-5.
Importantly, the effect of AM-112 is not greatly changed by bacterial inocula; in addition, our data indicate that administering AM-112 OD with the ceftazidime is more effective than giving the dose of AM-112 in three divided fractions. This finding is almost certainly related to the greater early AM-112 exposures with this regimen. Surprisingly, the optimal dosing regimens for ␤-lactam-␤-lactamase inhibitors based on pharmacodynamic principles are not firmly established (7). However, it is known that serine ␤-lactamase inhibitor concentrations can be low for periods between doses without reducing the antibacterial effect (9). In addition, it takes some time after ␤-lactamase inhibitor removal for the ␤-lactamase enzyme production of bacteria to return to baseline (6). These factors, combined with the AM-112 concentration-dependent reduction of the ceftazidime MIC, may explain the superiority of OD inhibitor administration (D. M. Livermore et al., 41st ICAAC).
In conclusion, the addition of AM-112 to ceftazidime at dynamic drug concentrations increases the antibacterial effect against an E. coli strain producing TEM-1 and CTX-M-1 enzymes. Dose fractionation with AM-112 shows that OD administration is best, indicating high early ␤-lactamase exposure; peak concentration may be an important pharmacokinetic driver for serine ␤-lactamase inhibitors.