The selection of the test strain
In this study, 210 E.coli strains isolated from pigs with common colibacillosis were used to determine the MIC90 value. The MIC values were determined by an agar dilution method as a preliminary screening, according to Clinical and Laboratory Standards Institute (CLSI) reference methods [15]. Strains whose MIC values equal to the MIC90 value of 210 strains were selected for pathogenicity test.
Pathogenicity test (the license number: SCXK 2011–0015) was carried out with 30 Kunming mice (SPF grade, specific pathogen free grade) purchased from animal experimental center of southern medical university. Every three mice were infected by intraperitoneal injection of 0.2 mL E.coli saline suspension (1 × 109 colony-forming unit, CFU/mL). The blank control group was given the same amount of saline. The mice were observed every 3 h after the injection until 72 h. If there were two or three mice dead in a group, then the inoculant given to this group was considered to be of high pathogenicity. The dead mice were dissected, and gram staining, microscopy and biochemical identification were carried out for the bacteria isolated from liver to determine whether the acquisition was the same as the inoculant.
The bacteria strain that was considered to be of high pathogenicity was selected out. In the present study, the bacterium was inoculated into the tissue-cage. Since cefquinome acts on the bacterium in the tissue-cage fluid, we need to measure the MIC of cefquinome against the selected strain in the tissue-cage fluid. Due to the tissue-cage fluid was limited, we determined the MIC once again by a micro dilution assay, and the matrix was the tissue-cage fluid.
The target strain was sent to Guangzhou King med Center for Clinical Laboratory Co., Ltd. for further bacterial identification, and the detection method was GB/T4789.6-2003.
Antimicrobials, chemicals
Cefquinome Sulfate Injection (Cobactan, the Batch Number A621B01) was purchased from Merck&Co. INC. Cefquinome standard was purchased from China Institute of Veterinary Drugs Control, (Beijing, P.R. China). Pentobarbital sodium was from Jian Yang Biotechnology Co., Ltd. Procainamide hydrochloride was purchased from Xin zheng Co., Ltd., Tianjin Pharmaceutical Group.
Animals and surgical procedures
Sixteen healthy castrated cross-bred piglets (Duroc × Landrace × Yorkshire) were used, weighing 25-30 kg. They were housed in a ventilated barn individually. Each animal was fed antibiotic-free food (guangchubao premix feed for pig, Guangzhou Zhongwang Feed Company) twice a day and water was available ad libitum. The tissue cages were prepared from medical grade silicone rubber tubing and modified slightly from similar cages described by Sidhu [16]. The length of the tissue cage was 65 mm, the internal and external diameter were 13 mm and 18 mm, respectively. Each cage had 24 identical holes and the surface area of each hole was 9.6 mm2, therefore the total exchange surface was 2.3 cm2. Two sterile tissue cages were implanted subcutaneously in each animal, one on each side of the neck approximately equidistant from the jugular vein and spinal cord. Surgical insertion was carried out under deep general anesthesia induced with pentobarbital sodium and local infiltration anesthesia by the injection of procainamide hydrochloride. Animals were allowed to recover from surgery for 3–4 weeks to permit wound healing and the growth of granulation tissue into and around the cages. After the surgery, piglets were treated with intramuscular penicillin twice a day for 3 days to prevent infection. The compound aminopyrine injection was administrated for post-operative analgesia simultaneously. About 3–4 weeks after implantation, the surface of the cage becomes encapsulated by connective tissue, and the interior is filled with tissue-cage fluid. All procedures involving animals were conducted under the close supervision and guidance of an experienced veterinary surgeon. The experimental protocol was approved by the Committee on the Ethics of animals of South China Agricultural University (Approval number 2014–04; 10 February 2014).
Animal infection and treatment
Before the infection experiment, 0.5 mL tissue-cage fluid was sampled from the tissue cage to confirm the tissue-cage fluid was sterile. The animal with bacterial present in the tissue-cage fluid before inoculation of E.coli was excluded from the study. In the current study, no piglets were excluded from this study due to a prior infection within the tissue cage. The tissue cages were inoculated with 1 mL of E.coli saline suspension (1.4 × 108 CFU/mL). The piglets remained infected without treatment for two days.
Animals were divided randomly into 8 groups (2 piglets, 4 tissue cages/group). Cefquinome sulfate injection was administrated intramuscularly at different dosage for different group after bacterial infection. Seven doses (0.2, 0.4, 0.6, 0.8, 1, 2, 4 mg/kg) of cefquinome were given (twice a day for 3 days) to create a range of different drug exposures. The control group received sterile physiological saline (1 mL) simultaneously in the same way.
Bacteriological examination of tissue-cage fluid
A 0.5 mL sample of tissue-cage fluid was removed from the tissue cage prior to every drug administration during the whole treatment and 12 h, 24 h and 36 h after the last treatment. Within 1 h after sampling, 100 μL of the tissue-cage fluid was serially 10-fold diluted in sterile physiological saline. From each dilution, 0.1 mL was plated onto MacConkey Agar Plate for overnight incubation at 37 °C for manual colony counts. The theoretical limit of detection of this procedure was 20 CFU/mL. The efficacy of cefquinome was calculated as the decrease in the amount of the bacteria over 3 days of treatment compared with the bacteria levels before the treatment.
The emergence of resistance under the dosing regimens in this study was assessed by measuring the MIC for E.coli recovered from tissue-cage fluid over the 3-day treatment period and 24 h posttreatment observation period. The MIC of cefquinome for the test strain was determined in duplicate using the micro dilution technique established by the CLSI [15].
Pharmacokinetic sampling and drug analysis
The tissue-cage fluid sample (0.5 mL) was collected from the inserted tissue cages with a syringe at 1, 3, 6, 9 and 12 h after each dosing. Samples were clarified by centrifugation at 3000 g for 10 min and stored at −20 °C until analyzed.
Cefquinome concentrations in tissue-cage fluid were analyzed by an Agilent 1200 series high performance liquid chromatography and an Agilent 6400 triple quadrupole mass spectrometer equipped with an electrospray ionization source (high performance liquid chromatography tandem mass spectrometry, HPLC-MS/MS, Agilent Technologies, and 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. All tissue-cage fluid samples were allowed to thaw at room temperature prior to analysis and then aliquots 200 μL tissue-cage fluid were added to a 1.5 mL micro centrifuge tube. Protein precipitation was accomplished by adding the same volume acetonitrile to the samples. After vorexing for 30s, and centrifuging at 3000 g for 10 min. 200 μL clear supernatant were pipetted into a fresh vial and 800 μL of water were added. After vortex-mixing for 15 s, the samples were filtered through a 0.22 μm nylon syringe filter (JinTeng Experiment Equipment Company) and then injected into an auto sampler vial. Cefquinome quantification in the tissue-cage fluid was linear within a range of 25–5000 ng/mL and the correlation coefficieent was >0.99. The lower limit of quantitation was 5 ng/mL. The recoveries of cefquinome in tissue-cage fluid were 90.2 ± 3.17 % (mean ± standard deviation, n = 5). The coefficients of variability (CV %) were all < 8 % for both intra-assay and inter-assay variation.
Pharmacokinetics/pharmacodynamics (PK/PD) integration and modeling
The PK/PD parameters, i.e., %T > MIC, AUC0–12/MIC (AUC, area under the curve) and Cmax/MIC (Cmax, the maximum concentration) were calculated for each tissue cage, from 0 to 12 h after every drug administration. This is due to the dosage regimens. In this study, we treated the animals with continuous multiple dosing (twice a day for 3 days), with the dosing interval of 12 h. %T > MIC (the percentage of time that drug concentration remains above the MIC) and Cmax (the maximum concentration measured in the tissue-cage fluid) were taken directly from the concentration-time profiles. AUC0–12, the area under the concentration-time curve, was calculated by the trapezoidal rule. The dose–response effect of the drug was analyzed by fitting the %T > MIC, AUC0–12/MIC and Cmax/MIC ratio versus the reduction of bacteria count with a inhibitory form of the sigmoid Emax model, through which we can further elucidate the PK/PD indices required for various degrees of antibacterial efficacy. The equation used to characterize the PK/PD parameters and log10CFU/mL could be described as the following:
$$ E={E}_{max}-\frac{\left({E}_{max}-{E}_0\right)\times {C}_e^N}{C_e^N+E{C}_{50}^N} $$
Where E is the antibacterial effect, defined as the change of bacterial count (log10 CFU/mL) after every drug administration (during 12 h treatment period); E
max
is the change of bacterial count in control sample (absence of cefquinome) after 12 h incubation; E
0
is the maximum antibacterial effect, determined as the maximum change of bacterial count every 12 h; C
e
is the PK/PD parameter being examined (e.g., %T > MIC, AUC0–12/MIC, or Cmax/MIC); EC
50
is the value of PK/PD index of drug producing 50 % of the maximum antibacterial effect, and N is the Hill coefficient that describes the steepness of the effect curve. These PD indices were calculated by running the WinNonlin (Pharsight Corporation, Mountain View, CA, USA) sigmoid E
max model to fit the experimental data collected. Nonlinear regression analysis was used to determine which PK/PD index best correlated with the bacterial reduction. Spearman’s rank correlation coefficient was calculated to evaluate the relationship between drug efficacy and the PK/PD indices.