Assessment of Differences in Antimicrobial Effect Determined with Two In Vitro Pharmacodynamic Models: Impact of Surface Area to Volume Ratio

Roger L. White, Pharm.D., Charles R. Bonapace, Pharm.D., Lawrence V. Friedrich, Pharm.D., Michael J. Rybak, Pharm.D., Diane M. Cappelletty, Pharm.D., Renee-Claude Mercier, Pharm.D., Heather H. Houlihan, Pharm.D., Jeffrey R. Aeschlimann, Pharm.D., John A. Bosso, Pharm.D.

Pharmacotherapy. 2003;23(5) 

In This Article


Laboratory-grade ceftazidime pentahydrate powder (GlaxoSmithKline Pharmaceuticals, Research Triangle Park, NC) was used for bioassay standard curves, and pharmaceutical-grade ceftazidime powder (GlaxoSmithKline Pharmaceuticals) was used for in vitro model comparisons. All antibiotic solutions were prepared immediately before use according to the manufacturer's recommendations. Pseudomonas aeruginosa American-Type Culture Collection (ATCC) 27853 was the test organism.

The minimum inhibitory concentration (MIC) of ceftazidime was determined in triplicate on 15-cm Mueller-Hinton II agar plates (BBL, Cockeysville, MD) using E test strips (AB Biodisk, Solna, Sweden) according to the manufacturer's recommendations after 18 hours of incubation at 35°C. The modal MIC was used for all data analyses.

Model experiments were performed in two different laboratories. Each in vitro system was a two-compartment model. The peripheral compartment was represented by either a disposable dialyzer unit or a membrane composed of the same semipermeable cellulose ester material (Figures 1 and 2). Since the same peripheral compartment membrane material was used in each model, the pore size was the same for both models (0.004 µm). However, the surface area of the peripheral compartment:volume of the peripheral compartment ratios (SA:Vs) were 5.34 cm-1 and 4.80 cm-1 for models A and B, respectively. Each model experiment was performed in duplicate. A separate growth-control model experiment was performed simultaneously. All experiments were performed at 37°C, model A in a microbiologic warm room, model B using a hot plate and water bath. Cation-adjusted Mueller-Hinton broth (calcium and magnesium concentrations 25 and 12.5 mg/L, respectively) without antibiotic was infused by peristaltic pump (Masterflex; Cole-Parmer Institute Co., Chicago, IL) through the central compartment of each model for 1 hour at a flow rate equivalent to the clearance of ceftazidime necessary to achieve a half-life of approximately 2 hours. The peripheral compartment was then inoculated with P. aeruginosa ATCC 27853 in logarithmic growth phase to achieve a final inoculum of approximately 5 x 105 colony-forming units (cfu)/ml.

Schematic of in vitro model A.

Schematic of in vitro model B.

A bolus dose of ceftazidime was administered into the central compartment to achieve the target concentration of 20 µg/ml (4 x MIC of the test organism). A continuous infusion of antibiotic-containing cation-adjusted Mueller-Hinton broth, designed to maintain this concentration, was infused through the central compartment of each model at the same flow rate as above. Samples (0.2 ml) to determine ceftazidime concentrations were obtained from central and peripheral compartments at 0, 0.5, 2, 4, 18, and 24 hours. Samples (0.3 ml) for bacterial enumeration were obtained from the peripheral compartment at these same time points.

At each time point, the sample withdrawn from the peripheral compartment of each model was diluted 10-fold with sterile normal saline as necessary for accurate enumeration of colony counts. For model A, samples were placed onto 10-cm Mueller-Hinton II agar plates using a spiral plater (Spiral Systems, Cincinnati, OH). For model B, samples were pipetted onto 15-cm Mueller-Hinton II agar plates. All plates were incubated for 24 hours at 35°C, and surviving colonies enumerated. The limit of quantification with these methods was 400 cfu/ml.

Immediately after collection, samples to determine ceftazidime concentrations were stored at -70°C until analyzed. Concentrations from each model were quantified by bioassay in triplicate on 15-cm Mueller-Hinton II agar plates by one laboratory. Twenty microliters of standard solutions and unknown samples were placed on blank disks and incubated at 35°C for 18 hours. Zones of inhibition were measured to the nearest 0.1 mm using a calibrated viewer (Transidyne General Corp., Ann Arbor, MI). The indicator organism was B. subtilis ATCC 6633, and the linear assay range was 4-48 µg/ml (R2 = 0.9852, CV ≤ 2.5%).

The mean cfu/ml from the peripheral compartment and mean ceftazidime concentrations from the central and peripheral compartments were used in all calculations. The percentage of ceftazidime penetration into the peripheral compartment of each model was calculated at each time point as follows:

% penetration = ConcP

where ConcP and ConcC represent concentrations of ceftazidime in the peripheral and central compartments, respectively. The area under the concentration-time curves for the central (AUCC) and peripheral compartments (AUCP) from 0-4 and 0-24 hours were calculated for each model using the linear trapezoidal rule. Using these AUCs, the percentage of ceftazidime penetration into the peripheral compartment from 0-4 and 0-24 hours was calculated as follows:

% penetration = AUCP

To assess antimicrobial effect, the log10 area under the kill curve (log10 AUKC) and log10 area under the growth control curve (log10 AUGC) were calculated using the linear trapezoidal rule from the log10 cfu/ml at each time point of the cfu/ml versus time curve. The percent effect was calculated as follows:

% effect = (log10 AUGC - log10 AUKC)
log10 AUGC

In addition, the percent differences in AUCP, penetration, and effects at 0-4 and 0-24 hours between models A and B were calculated.


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