Antimicrobials and chemicals
Penicillin, as a sodium salt for injection, and cefquinome for injection were purchased from Hebei Yuanzheng Pharmaceutical Co., Ltd., P.R. China. Cefquinome standard was from China Institute of Veterinary Drugs Control, Beijing, P.R. China. Acetonitrile and formic acid (chromatography grade) were from Fisher Scientific.
MIC99, MIC, and MPC determination
E. coli strain ATCC 25922 stored at -70°C was grown in Mueller-Hinton broth or on Mueller-Hinton agar. MIC99 and MPC were determined as described elsewhere [15]. Briefly, for MIC99, bacterial cultures were grown overnight (≥10 h) in the constant temperature oscillation incubator at 37°C, 220 r/min, normal atmosphere, serially diluted, and approximately 106 cells were applied to agar plates containing various concentrations of cefquinome. After incubation at 37°C for 16-18 h, bacterial colonies were counted, and the fraction relative to the bacterial inoculum was calculated. Drug concentration that inhibited growth by 99% was defined as MIC99. For MPC, above 1010 cells were applied to multiple cefquinome-containing agar plates. After incubation at 37°C for a total of 96 h and the examination of the appearance of colonies every 24 h, MPC was recorded as the lowest antibiotic concentration at which no colonies grew on an agar plate. The MIC99 and MPC were determined in the five independent experiments.
Tissue-cage infection model
Healthy castrated cross-bred piglets (Duroc × Landrace × Yorkshire),weight ranging from 25 to 30 kg, were housed individually and fed antibiotic-free food twice a day. Water was available ad libitum. The experimental protocol was approved by the Committee on the Ethics of animals of South China Agricultural University (Approval number 2013-01; 15 March 2013).
The tissue-cages were made in-house from platinum-cured medical grade silicone tubing (Medical silicon, SF Medical; Beijing Jingcheng Chuangye Medical Instrument Co., Ltd., Beijing, P.R. China) and modified slightly from similar cages described by Sidhu et al. [16]. Briefly, the dimensions of the tissue-cages were of 65 mm length, 18 mm external diameter and 13 mm internal diameter. Each cage had 24 identical holes and each hole has a surface area of 9.6 mm2; the total exchange surface area was 2.3 cm2.
Two tissue-cages were implanted subcutaneously in each animal, one on either side of the neck approximately equidistant from the jugular vein and spinal cord under aseptic conditions. Surgical insertion was carried out under deep sedation (pentobarbital sodium) and local infiltration anaesthesia (procainamide hydrochloride injection) in piglets. After surgery, the piglets were treated with intramuscular penicillin (160 000 IU/kg) twice a day for 3-5 days to prevent infection. The non-steroidal anti-inflammatory drug (NSAID) was provided for analgesia in post-operation simultaneously. By 4 weeks after implantation, each tissue-cage had become sealed with a thin layer of connective tissue and had been filled with clear, yellowish tissue-cage fluid. Above 1010 CFU of exponentially growing E. coli ATCC 25922 culture was concentrated in 1 mL of saline and injected into each tissue-cage. Two days after infection, 0.5 mL of tissue-cage fluid was withdrawn from each cage for a viable-bacteria count. Piglets having above 108 CFU/mL viable bacterial cells in tissue-cage fluid were treated with various doses and intervals of cefquinome.
Pharmacokinetic measurements
Eighteen piglets were randomly allocated to 7 administration groups and treated at 0.1, 0.2, 0.4, 0.8, or 1.0 mg/kg of body weight once a day (24 h interval) or 0.2 and 0.4 mg/kg of body weight twice a day (12 h interval). 0.1, 0.8, 1.0 mg/kg groups had 2 piglets and 4 tissue cages of each group. 0.2, 0.4 mg/kg (12 h and 24 h interval) groups had 3 piglets and 6 tissue cages of each group, which had one more piglet compared to 0.1, 0.8, 1.0 mg/kg groups respectively because these two dosages easily induced resistant mutation. And this series of dosages were determined by recommended dose which was 2 mg cefquinome/kg body weight in intramuscularly once daily for 3-5 days [13] and pre-experiments data (not provided). Cefquinome were administrated intramuscularly (intragluteal muscles) for consecutive 5 times beginning on the 3rd day after infection with E. coli ATCC 25922 for every piglet in administration groups. The control group, three piglets, received sterile physiological saline (1 mL) simultaneously in the same way. Tissue-cage fluid (0.5 mL) was collected from the cage at 1, 3, 6, 9, 12, and 24 h after each administration in group with 24 h interval. For groups with 12 h interval, samples were collected at 1, 3, 6, 9, 12 h after each administration. Fluid samples were clarified by centrifugation at 3 000 × g for 10 min and stored at -20°C.
The concentrations of cefquinome was determined using an Agilent 1200 series high performance liquid chromatography and an Agilent 6400 triple quadrupole mass spectrometer equipped with an electrospray ionization source (HPLC-MS/MS, Agilent Technologies, USA). The chromatographic separation was achieved on a Phenonenex BDS C18 column (150 mm × 2 mm; internal diameter, 5 μm, Phenomenex Technologies) at 40°C with a thermostat column oven (Agilent 1200 series, Agilent Technologies). The mobile phase consisted of solution A (water with 0.1% formic acid, V/V) and solution B (acetonitrile) at 0.25 mL/min flow rate. The gradient elution was: 0-1 min, 5% B; 1-5.5 min, 60% B; 5.5-10 min, 5% B. The injection volume was 5 μL.
A calibrated curve was constructed by adding a known amount of cefquinome to blank tissue-cage fluid over concentrations ranged from 0.001 μg/mL to 1 μg/mL. The lower limit of quantification (LLOQ) of cefquinome was 5 ng/mL. The recoveries of cefquinome in tissue-cage fluid were 94.2 ± 7.34% (mean ± standard deviation, SD, n = 5). The coefficients of variability (CV%) were all < 10% for both intra-assay and inter-assay variation.
Pharmacokinetic/pharmacodynamic (PK/PD) indices such as T>MIC99, T>MPC, AUC/MIC99, AUC/MPC, Cmax/MIC99, Cmax/MPC were calculated according to a noncompartmental analysis using WinNonlin programme (version 6.1, Pharsight Corporation, Mountain View, CA, USA). The liner trapezoidal rule was used to calculate the area under the concentration-time curve (AUC). All PK/PD indices calculations referred to the 12 h and 24 h dosing interval immediately following the fifth injection after finishing administration using the cefquinome concentrations in tissue cage fluid.
Loss of susceptibility to cefquinome
Potential loss of susceptibility was monitored in tissue-cage fluid (0.5 mL/cage) obtained daily before and during the cefquinome treatment (after every administration) and 24 and 48 h after the termination of treatment. To amplify cultures, half of each sample was incubated overnight in drug-free Mueller-Hinton broth, and then the MIC was determined with the CLSI [17] agar dilution method. The other half of each sample was serially diluted with sterile physiological saline and applied to agar either lacking drug or containing cefquinome at 1 × MIC of the starting culture. After incubation at 37°C for 24-48 h, colonies were calculated, and the fraction of mutants in the population was calculated.
Resistant mutants (growing on 1 × the MIC of cefquinome-containing agar) were also chosen randomly from samples that had cefquinome concentrations predominantly in the lower, middle, or upper part of the selection window with 12 h or 24 h interval administration. Single colonies of these mutants were passaged 5 times on drug-free agar, and the MIC to cefquinome was then determined.
Statistical analysis
Fisher’s exact test was used for statistical analysis of the PK/PD data, with an infected but untreated set of piglets (3 piglets, 6 tissue-cages) as a control. P < 0.05 was considered to be statistically significant.