Assessment of Minocycline and Polymyxin B Combination against Acinetobacter baumannii

Bacterial isolates and in vitro susceptibility.Four strains of A. baumannii (1 laboratory isolate and 3 clinical isolates) with a broad range of minocycline susceptibility were used (Table 1). All strains belonged to the Acinetobacter calcoaceticus-A. baumannii complex based on the API 20 NE system (bioMérieux Vitek, Marcy l'Etoile, France). Identification of A. baumannii was confirmed by detection of the OXA51 gene by PCR (14). MICs were determined by broth dilution in triplicate according to the CLSI in the presence and absence of phenylanine-arginine β-naphthylamide (PAβN) (100 μg/ml), an efflux pump inhibitor (15). In order to exclude the inhibitory effect of PAβN, the bacterial burden of each positive control was determined by quantitative culture.

Resistance mechanisms.Colony quantitative PCR (qPCR) was carried out to determine the presence of the tetA, tetB, tetM, and tet39 genes in A. baumannii strains. Colonies of overnight-streaked strains on LB agar plates were chosen, resuspended, heated, and used as PCR templates. The qPCR was run using Sybr green Select master mix (ABI) in an ABI 7000 sequence detection system. The threshold cycle (C_T) values were normalized with the housekeeping gene recA of the same strain. The difference (ΔC_T) was used as a logarithmic power (base = 2) to calculate the relative signal of the gene. The transcription levels of the known efflux genes, adeB, adeJ, and adeG, were determined by reverse transcription (RT)-qPCR. Cells were grown in cation-adjusted Mueller-Hinton broth (Ca-MHB) and centrifuged, and total RNA was isolated (Ambion RiboPure-Bacteria RNA isolation kit [ABI]). The RT reaction was performed using the TaqMan reverse transcriptase reagent kit (ABI) with a mixture of primers. The results (C_Ts) were normalized with the housekeeping gene rpoB.

Time-kill studies.Time-kill studies were performed using approximately 1 × 10⁶ CFU/ml of each strain with clinically relevant minocycline concentrations (2 μg/ml and 8 μg/ml), with and without a subinhibitory polymyxin B concentration (0.5 μg/ml). The time-kill study methods have been described previously (16). Briefly, an overnight culture of the isolate was diluted with prewarmed Ca-MHB and incubated further at 35°C until reaching log-phase growth. The bacterial suspension was further diluted with Ca-MHB according to absorbance at 630 nm. Fifteen milliliters of the suspension was transferred to sterile flasks, containing a specific amount of drug to achieve the desired final concentration, and placed in a shaker bath. Serial samples (0.5 ml) were taken in duplicate at baseline and at 1, 2, 4, 8, 12, and 24 h, centrifuged (10,000 × g for 15 min), and reconstituted with sterile normal saline to their original volumes to minimize any drug carryover effect. Bacterial populations were quantified by plating the 10× serial dilutions onto Mueller-Hinton agar plates and incubated at 35°C for 18 to 24 h. The bacterial density from each sample was determined by visual inspection of CFU. The limit of detection for the time-kill studies was 100 CFU/ml. These are based on plating 200 μl of an undiluted sample and visual inspection with ≥20 CFU per plate. Experiments were performed at least in triplicate on different days.

Intracellular concentration.The inoculum was prepared as detailed above. The intracellular minocycline concentration was determined with or without polymyxin B in triplicate. Five milliliters of the bacterial suspension was exposed to minocycline (8 μg/ml) with or without polymyxin B (0.5 μg/ml). After incubation in a water shaker bath at 35°C for 30 min, a sample (500 μl) was taken to determine bacterial density by quantitative culture. The remaining bacterial suspension was centrifuged (3,220 × g at 4°C for 10 min). The pellet was washed twice with 50 mM phosphate-buffered saline (PBS) (pH 7.0) and reconstituted in 100 μl water. The samples were transferred to new tubes, and 400 μl acetonitrile with doxycycline (internal standard) was added to extract minocycline from bacterial cells. The tubes were mixed by vortexing for 15 s and centrifuged for 15 min at 18, 000 × g, and supernatants were evaporated to dryness under a stream of ambient air. After reconstituting with 1 ml of 50% methanol and centrifuging for 15 min at 18,000 × g, the minocycline concentration was determined by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method as detailed below. The final intracellular drug concentration in each sample was normalized by the viable bacterial burden after drug exposure. These experiments were repeated 3 times on different days.

Neutropenic murine pneumonia model.The in vivo antibacterial activity of minocycline in combination with polymyxin B was examined using a neutropenic murine pneumonia model. This infection model has been described elsewhere (16, 17). Briefly, transient neutropenia was induced in female Swiss Webster mice weighing between 20 and 25 g (Harlan, Indianapolis, IN). The mice were given food/water ad libitum, and were administered two intraperitoneal doses of cyclophosphamide (150 mg/kg at 4 days prior to infection and 100 mg/kg at 1 day prior to infection). The experimental protocol was approved by the University of Houston Institutional Animal Care and Use Committee.

Minocycline pharmacokinetic study.The single-dose pharmacokinetics of minocycline in infected mice were explored. Three mice were sacrificed serially at 0.5, 1, 2, 5, and 8 h after an intraperitoneal injection (50 mg/kg), and blood samples were collected by cardiac puncture. Blood samples were allowed to clot on ice and centrifuged at 10,000 × g and 4°C for 15 min. Supernatants (100 μl) were mixed with 100 μl water and 400 μl acetonitrile (with 4 μg/ml doxycycline as an internal standard). Samples were vortexed for 15 min and centrifuged for 15 min at 18,000 × g, and supernatants were evaporated to dryness under a stream of ambient air. After reconstituting with 100 μl of 50% methanol and centrifuging for 15 min at 18,000 × g, the minocycline concentration was determined by a validated ultraperformance liquid chromatography (UPLC)-UV method, as detailed below. The serum concentrations of minocycline observed at each time point were averaged. A one-compartment model with linear absorption was fit to the average drug serum concentration-time profile. The fit of the model was assessed by the coefficient of determination. Using the best-fit parameters, the area under the concentration-time curve from 0 h to infinity (AUC_0–∞) was calculated (dose divided by total clearance). All modeling procedures were performed using the ADAPT II program (18).

Polymyxin B pharmacokinetic study.The single-dose pharmacokinetics of polymyxin B in infected mice were explored in a manner similar to that described above. Three mice were sacrificed serially at 0.5, 1, 2, and 4 h after an intraperitoneal injection (10 mg/kg). To explore the impact of renal function on drug elimination, an additional group of animals were given an intraperitoneal dose of uranyl nitrate (5 mg/kg) on day −2. Renal impairment was confirmed in selected animals on the day of infection by measuring serum creatinine. The polymyxin B concentration in serum was determined by a validated UPLC-MS/MS method (19).

Drug assays.The minocycline concentration in mouse serum was determined by a validated UPLC-UV method. Since a lower concentration was anticipated, a more sensitive LC-MS/MS method was used to determine the minocycline concentration in bacterial cells.

Both the UPLC-UV and LC-MS/MS assays shared the same chromatographic method, which consisted of a Waters Acquity UPLC and a Waters BEH C₁₈ column (1.7 μm by 2.1 by 50 mm). Mobile phases A and B were water and acetonitrile, respectively, both containing 0.1% formic acid. The injection volume was 5 μl. The analytes were separated by gradient elution at 45°C. The gradient was as follows: 0 to 0.5 min, 98% A; 0.5 to 0.7 min, 98 to 84% A; 0.7 to 1.2 min, 84 to 76% A; 1.2 to 1.7 min, 76 to 70% A; 1.7 to 2.1 min, 70 to 50% A; 2.1 to 2.5 min, 50 to 5% A; 2.5 to 3.0 min, 5% A; 3.0 to 3.2 min, 5 to 98% A; 3.2 to 5 min, 98% A.

The UV detection was performed on a Waters diode-arrayed detector at 350 nm. The intraday and interday variabilities of the UPLC-UV method were <0.82% and <4.39%, respectively. The linear range of detection was 0.03 to 64 μg/ml. The mass spectrometry analysis was performed on API5500 Qtrap triple-quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, Foster City, CA, USA) equipped with Turbo-Ion-Spray source. The multiple-reaction monitoring (MRM) scan type in positive mode was used. The transitions of m/z 458.3 to 441.2 and m/z 445.2 to 154.2 were used for quantifying minocycline and doxycycline, respectively. The intraday variability of the LC-MS/MS system was <7.5%, while the interday variability was <11.4%. The linear range of detection was 0.24 to 500 ng/ml.

In vivo antibacterial effect.The neutropenic mice were anesthetized and inoculated with approximately 1 × 10⁷ bacterial cells under largnygoscopic guidance. The inocula used were guided by pilot inoculum ranging studies to result in >50% mortality of animals at between 48 and 96 h postinfection. Approximately 2 h after infection, the mice were randomly divided into 4 treatment groups: (i) placebo, (ii) minocycline, (iii) polymyxin B, and (iv) a combination of minocycline and polymyxin B. The daily doses of minocycline and polymyxin B used were guided by the pharmacokinetic studies detailed above in order to mimic clinically relevant exposures of these agents. Experiments were performed in duplicate.

The bacterial burden in lung tissue was used to verify the initial inocula at baseline (n = 2), at 24 h (n = 3), and upon death or up to 96 h (n = 10 for each treatment group), as described elsewhere (20). Briefly, lung tissues were aseptically collected and homogenized in 10 ml sterile saline. The samples were centrifuged at 4,000 × g and 4°C for 15 min. The pellets were then resuspended in normal saline to 10× their original volumes to minimize any carryover effect from the drugs. The samples were diluted using 10× serial dilutions, quantitatively cultured on Mueller-Hinton agar plates, and then incubated for 24 h. The bacterial burden in lung tissues was calculated via visual inspection of colony growth and compared using Student's t test. All animals were observed every 8 h; at each inspection time, any animals that appeared to be moribund were humanely euthanized. Death was recorded as it occurred, at the next inspection time. Any mice remaining at the end of the 96-hour experiment were euthanized by CO₂ asphyxiation. Bacterial burdens observed at the same time frame were compared using Student's t test. Survival over time was assessed using Kaplan-Meier survival analysis and the log rank test.