Efficacy of Humanized Carbapenem and Ceftazidime Regimens against Enterobacteriaceae Producing OXA-48 Carbapenemase in a Murine Infection Model

Antimicrobial test agents.

Commercially available ertapenem (Invanz, Merck & Co., Inc., Whitehouse Station, NJ), doripenem (Doribax; Janssen Pharmaceuticals, Inc., Raritan, NJ), ceftazidime (Fortaz; GlaxoSmithKline, Philadelphia, PA), and levofloxacin (Sagent Pharmaceuticals, Schaumburg, IL) were obtained from the pharmacy department at Hartford Hospital. Each antibiotic was reconstituted according to the manufacturer's prescribing information and further diluted in normal saline to the concentrations required for dosing. Prior to each experiment, dosing solutions were prepared and stored under refrigeration until the time of use and were discarded after 24 h.

Bacterial isolates and susceptibility testing.

An isogenic pair consisting of a wild-type Klebsiella pneumoniae strain (KP 454) and an OXA-48 strain derived by conjugation using antibiotic-containing plates, as well as two clinical OXA-48-producing K. pneumoniae isolates (KP 452 and KP 453), were provided by P. Nordmann, Hospital Bicêtre (Bicêtre, France). Ertapenem and doripenem MICs for these isolates were determined via Etest (AB bioMérieux, Solna, Sweden) according to the manufacturer's instructions, while MICs for ceftazidime and levofloxacin were determined using broth microdilution consistent with the Clinical and Laboratory Standards Institute (CLSI) methodology (8). Pseudomonas aeruginosa ATCC 27853 was used as the quality-control strain for the two agents, with MICs determined by broth microdilution. MIC determinations were conducted in triplicate and are reported as the modal MIC. Four additional clinical OXA-48-producing Enterobacteriaceae isolates (KP 464, KP 465, EC 418, CF 32) were kindly provided by C. Lascols, International Health Management Associates, Inc. (Schaumburg, IL) (9). MICs for these isolates to all four test agents were determined using broth microdilution as outlined above. Isolates were screened for the presence of KPC, OXA-48-like, VIM, IMP, and NDM carbapenemases, as well as for extended-spectrum β-lactamases (TEM, SHV, and CTX-M) and AmpC β-lactamases (CMY, DHA, FOX, MOX, ACC, and ACT) as previously described (9). All isolates were stored in double-strength skim milk (Remel, Lenexa, KS) at −80°C. Prior to all in vivo experiment, each isolate was subcultured twice onto Trypticase soy agar with 5% sheep blood (BD Biosciences, Sparks, MD) and incubated for 18 to 24 h at 35°C.

Immunocompetent thigh infection model.

This study was reviewed and approved by the Institutional Animal Care and Use Committee at Hartford Hospital, Hartford, CT. Pathogen-free ICR mice weighing 20 to 22 g were acquired from Harlan Laboratories (Indianapolis, IN). All animals were maintained in accordance with the National Research Council's recommendations and were provided food and water ad libitum. An intraperitoneal injection of uranyl nitrate (5 mg/kg of body weight) was given 3 days prior to inoculation for each experiment to slow antibiotic clearance and produce a predictable level of renal impairment (10). Two hours prior to the initiation of antimicrobial therapy, both thighs of each animal were inoculated intramuscularly with 0.1 ml of an inoculum solution containing 10⁸ CFU/ml of the test isolate in normal saline.

Neutropenic thigh infection model.

In order to best characterize the achievable efficacy of each agent tested, the isogenic pair (wild-type and OXA-48-producing strains) and three clinical OXA-48-producing strains were also evaluated in a neutropenic infection model for comparison. All mice utilized in the neutropenic studies underwent the same procedures as described for the immunocompetent model but were also given intraperitoneal injections of 100 and 150 mg cyclophosphamide/kg (Baxter, Deerfield, IL) 1 and 4 days prior to inoculation, respectively, to induce neutropenia (10). In addition, the inoculum solution used to produce thigh infection in the neutropenic model was reduced to 10⁷ CFU/ml.

In vivo efficacy.

Human-simulated regimens of 2 g doripenem intravenously (i.v.) every 8 h as a 4-h infusion, 1 g ertapenem i.v. every 24 h, 2 g ceftazidime i.v. every 8 h as a 2-h infusion, and 500 mg levofloxacin every 24 h were previously developed and validated by our group to achieve the human pharmacodynamic profile associated with each of these dosing regimens and were utilized throughout the course of the in vivo experiments (11–14). Beginning 2 h after inoculation, groups of three mice were administered a human simulated regimen of doripenem, ertapenem, ceftazidime, or levofloxacin for each test isolate. All doses for the humanized regimens were administered as 0.2-ml subcutaneous injections; dosing regimens were repeated as necessary to complete 24 h of therapy. For each isolate evaluated, groups of three control animals were given normal saline in the same volume, route, and frequency as the most frequently administered antibiotic regimen over 24 h. One group of untreated control mice was harvested at 0 h, while the control group receiving normal saline and all treated mice were harvested at 24 h. Mice that failed to survive to 24 h were harvested at the time of expiration and were included in the final data analysis. Harvesting consisted of euthanization by CO₂ exposure followed by cervical dislocation. Thighs were removed from sacrificed animals, homogenized individually in normal saline, serially diluted, and plated onto Trypticase soy agar with 5% sheep blood for determination of bacterial density. Efficacy was calculated as the change in bacterial density (in log₁₀ CFU) in treated mice after 24 h from the starting bacterial densities in 0-h control animals for all experiments conducted in neutropenic mice. In order to control for the effect of the host and associated variability in bacterial density of growth controls between isolates in the immunocompetent model, efficacy was calculated as the change in bacterial density in treated mice after 24 h compared to that in the 24-h immunocompetent control mice (6, 15).

Bacterial isolates.

A wild-type K. pneumoniae strain (KP 454) and its derived isogenic OXA-48 strain, in addition to six clinical OXA-48-producing Enterobacteriaceae isolates, were utilized in the in vivo experiments. Phenotypic profiles for the antimicrobials evaluated and the known genotypic profiles for these isolates are described in Table 1 (9).

In vivo efficacy.

The isogenic wild-type and OXA-48-producing strains as well as all six clinical OXA-48-producing isolates were evaluated in the immunocompetent model. The mean (±standard deviation) bacterial density for control mice at 0 h was 7.05 ± 0.30 log₁₀ CFU/ml in immunocompetent mice and was maintained at a mean of 6.41 ± 0.62 log₁₀ CFU/ml after 24 h. The efficacy results of the immunocompetent studies are shown in Fig. 1. Bacterial reductions ≥1 log CFU were observed with all agents tested against the wild-type strain. Consistent with phenotypic profile, ceftazidime and levofloxacin also exhibited efficacy against the isogenic OXA-48 strain, whereas doripenem did not, despite having an MIC of 0.38 μg/ml; no activity was observed with ertapenem as anticipated given an MIC of 3 μg/ml. Furthermore, ≥1 log CFU bacterial reductions were also achieved with the humanized ceftazidime regimen for the two clinical isolates with MICs of ≤1 μg/ml (100% ƒT>MIC [time that the free drug concentration remains above the MIC during the dosing interval]) and one isolate with an MIC of 64 μg/ml (0% ƒT>MIC); observed reductions were less for the remaining three clinical strains, all with MICs of ≥64 μg/ml. The simulated levofloxacin regimen produced a greater-than-2-log CFU reduction for the clinical isolate with an MIC of ≤0.03 μg/ml (area under the curve [AUC]/MIC ≥ 1,467), while little to no efficacy was demonstrated for the five isolates with MICs of ≥4 μg/ml (AUC/MIC ≤ 11). Consistent with an MIC of ≥1 μg/ml (≤30% ƒT>MIC), the simulated dose of ertapenem did not result in substantial bacterial reductions for any of the clinical strains. While the observed efficacy for ceftazidime, levofloxacin, and ertapenem against the six clinical isolates correlated well with phenotypic profiles and pharmacodynamic targets, variable levels of bacterial reductions (range, 0.4 to 1.3 log CFU) were noted with the humanized high-dose, prolonged-infusion doripenem regimen for the six clinical strains with MICs of ≤8 μg/ml (ƒT>MIC ≥ 70%).

In the neutropenic model, the mean bacterial density for control mice at 0 h was 5.85 ± 0.46 log₁₀ CFU/ml and increased to a mean of 8.23 ± 0.53 log₁₀ CFU/ml after 24 h. The efficacy results of the neutropenic studies are shown in Fig. 2. Similar trends in efficacy with respect to phenotypic profiles and pharmacodynamic targets were noted for ceftazidime, levofloxacin, and ertapenem between immunocompetent and neutropenic studies. Efficacy was observed with doripenem against the isogenic and three clinical OXA-48-producing strains evaluated in the neutropenic model, all with MICs of ≤3 μg/ml (ƒT>MIC of ≥85% for all isolates). However, the magnitude of bacterial reduction was much less than what was observed with doripenem against the wild-type strain.