Chemicals and reagents
The standard ceftiofur hydrochloride (det. purity: 97.9%) purchased from Dr. Ehrenstorfer (Augsburg, Germany) and 5% ceftiofur hydrochloride injection purchased from Pfizer Animal Health Co., Ltd., Canada, were used during the study. All the chemical reagents used were HPLC grade.
Bacterial strain isolation
E. coli ATCC 25922 was purchased from American Type Cell Culture and used as a quality control strain for drug susceptibility testing. Actinobacillus pleuropneumoniae serotype 1 (BW39) was used to determine the EOFF and COPD values. A total of 101 Actinobacillus pleuropneumoniae strains were donated by the International Research Center for Animal Diseases, China State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University. Thirty-four Actinobacillus pleuropneumoniae strains were isolated from the tissue of infected pigs. The strains were stored at -80°C prior to each experiment. Prior to testing, each isolate was subcultured at least three times in TSA and TSB containing 5% newborn calf serum and 1% NAD (Zhejiang Tianhang Biotechnology Co., Ltd.).
Determination of antimicrobial susceptibility and epidemiologic cutoff
The susceptibility of 135 Actinobacillus pleuropneumoniae strains to ceftiofur was determined by inoculating the strains on TSA agar plates supplemented with newborn calf serum and 1% NAD and incubating the plates at 37 °C in an atmosphere containing 5% CO2 for 24 h; the susceptibility was measured using the standard agar dilution method with concentrations of ceftiofur between 0.00375-32 μg/ml,, according to CLSI protocols. The MIC distribution was constructed and converted into a cumulative log-normal distribution. Then, nonlinear regression was employed to fit the cumulative log2-transformed MIC data to obtain a range of optimum wild-type MIC distributions, which contained the wild-type MIC in the range of 0.1% and 99.9%, and to calculate the probability of MIC data falling within the cutoff range. The optimum fit was defined as the fit where the difference between the estimate of the isolate number and the actual number was minimal. The cutoff value would encompass at least 95% of the wild-type isolates . A wild-type cutoff (COWT) was developed based on the MIC distribution following CLSI M37-A3. The COWT value was calculated by ECOFFinder software (J. Turnidge, Kahlmeter, and Kronvall 2006), which is available on the CLSI website (https://clsi.org/education/microbiology/ecoffinder/).
Pharmacodynamics of ceftiofur against BW39
MIC, MBC and MPC determination
The minimum inhibitory concentration (MIC) of ceftiofur against BW39 (serotype 1) was determined by the broth micro dilution method according to the CLSI (Clinical and Laboratory Standards Institute, 2015) and in serum, as vitro MIC and ex vivo MIC. The MIC was determined as the lowest ceftiofur concentration that visibly inhibited the growth of bacteria at the end of the 24 h incubation period. For the MBC of ceftiofur against BW39, 100 μL from each well was was subjected to 10-fold or more dilution with broth and serum, as vitro MBC and ex vivo MBC; 10 µL of each solution was spread on TSA agar plates and incubated at 37 °C for 24 h for colony forming unit (CFU) counting. The MBC was defined as the lowest drug concentration that resulted in a 99.9% reduction in the bacterial density. The mean was expressed as the final result.
The mutant prevention concentration (MPC) was determined by the agar method according to the procedures of Blondeau . Exponential growth phase bacteria were pelleted by centrifuging at 3000 rpm at 4 °C. The pellet was then diluted to 3 × 1010 CFU/mL with TSB medium. An aliquot of 100 μL of the 1010 CFU/mL bacterial suspension was cultured on TSA agar plates containing various concentrations of ceftiofur (0×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, 16×MIC, 32×MIC, and 64×MIC) obtained from a series of two-fold dilutions. Inoculated plates were incubated for 72 h, and colonies were counted every 24 h. All MPC determinations were performed in duplicate. The MPC was defined as the lowest ceftiofur concentration with no visible bacterial growth on agar plates after incubation for 72 h.
Bacterial growth and time-killing curve of BW39 in vitro and ex vivo
The BW39 isolate was selected to determine the growth curve and time-killing curve in TSB broth and serum. The in vitro and ex vivo growth curves of the BW39 isolate were established by plotting time versus log10 CFU/mL. An aliquot of 5 mL of BW39 grown to mid-log phase with a starting inoculum of 106 CFU/mL was added to 5 mL of TSB broth supplemented with serial concentrations of ceftiofur corresponding to 0×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, 16×MIC, and 32×MIC for the in vitro time-killing curve. In addition, an aliquot of 5 mL of BW39 grown to the mid-log phase with a starting inoculum of 106 CFU/mL was co-incubated with 5 mL sterilized blank serum added with ceftiofur corresponding to the concentration of ceftiofur in serum ,which was collected from healthy pigs at different time points (0, 0.33, 0.66, 1, 1.5, 2, 3, 5, 8, 12, 24, 36, 48, 72 and 96 h) after I.M. administration of a single injection of 5 mg/kg ceftiofur hydrochloride for the determination of the ex vivo time-killing curve. The tubes were incubated at 37 °C with 5% CO2. Each culture was serially diluted 10-fold with sterile saline, and 100 μL of each dilution was spread onto TSA agar plates at different time points (0, 2, 4, 6, 8, 12, and 24 h). Then, the bacterial count (CFU/mL) was determined after incubation for 24 h at 37 °C with 5% CO2. The limit of detection was 10 CFU/mL. The in vitro and ex vivo time-killing curves of ceftiofur against BW39 were established by plotting the time versus log10 CFU/mL. The experiment was tested in triplicate.
In vitro PAE determination
Approximately 1.8 mL exponential phase A.pleuropneumoniae BW39 (1.0 × 107 CFU/mL) was mixed with 0.2 mL ceftiofur, to generate final concentrations of 1 MIC, 2 MIC and 4 MIC. A 0.2 mL aliquot of physiological saline was used as control. Volumes were cultured in glass tubes and grown for 1h and 2 h to induce PAE production. 100 μL cultured medium was mixed with 0.9 mL TSB medium and cultured 24 h at 37 °C with 5% CO2. 100 μL samples were taken at 0, 1, 2, 4, 6, 8, and 12 h, and serially diluted 10-fold with sterile physiological saline to count cells. Each treatment was performed four times. Growth curve for A.pleuropneumoniae BW39, at different ceftiofur concentrations were established, and T (time required for bacterial numbers to be 10 times higher than 0 h in the test groups) and C (time required for the bacterial num- bers to be 10 times higher than 0 h in control groups) values were calculated. PAE was calculated as the differ- ence between T and C (PAE = T - C).
Pharmacokinetics of ceftiofur in plasma of pigs
Six 6-week-old healthy castrated crossbred piglets (Duroc×Landrace×Yorkshire) pigs with an average weight of 15±2 kg were purchased from the Livestock and Poultry Breeding Center of Hubei Province (Wuhan, China). The animals were acclimatized for a period of one week before the experiment. The temperature and relative humidity of the housing environment were kept at 18–25 °C and 45–65%, respectively. The piglets were maintained in accordance with the National Standards for Laboratory Animals of China (GB 14925–2010), and allowed adlibitum access to water and antibiotic-free food. The euthanasia procedure was carried out by pentobarbital sodium with intravenous injection when study was finished. All animal experiment procedures were approved by the Institutional Animal Care and Use Committee at Huazhong Agricultural University (HZAUSW-2016-007).
Six pigs were received ceftiofur hydrochloride injection at a dose of 5 mg/kg·b.w by intramuscular injection of neck. Blood samples (5 mL) from each pig of each group were gently collected from the jugular vein at 0, 0.33, 0.66, 1, 1.5, 2, 3, 5, 8, 12, 24, 36, 48, 72 and 96 h. Plasma samples were obtained by centrifuging the blood at 3500 rpm/min for 10 min, and the samples were stored at -20 °C prior to the analysis and analysis within 3 days after sampling.
DFC concentration were measured by HPLC described by previous study to represent ceftiofur plasma concentration, as ceftiofur is rapidly metabolized to DFC in piglets.
Extraction: 0.5 mL plasma was mixed with 7 mL of 0.4% DTE-borate buffer. The mixture was incubated for 15 min at 50 °C in a water bath, with a 10 s vortexing every 3 min. Samples were then centrifuged after cooling to 25 °C, and the supernatant was collected.
Solid phase extraction: An Agilent HLB column (60 mg/3 cc) was activated and equilibrated consecutively with 3 mL methanol and ultrapure water. Extracted ma- terials were added to the HLB column and a flow rate set at 1 mL/min. The column was then eluted with 5 mL methanol, after which the eluate was concentrated by nitrogenblow at 35°C. The concentrated solution was vortexed with 0.5 mL ultrapure water, sonicated for 5 min. The DFC standard was added to 0.5 mL plasma (to achieve final concentrations of 0.1, 0.25, 0.5,1.0, 2.5, 5.0, 10.0, 20.0 and 50 μg/mL) and prepared with same process as samples from test groups.
The quantitative analysis of ceftiofur in plasma was performed with a Water 2695 series HPLC with a UV detector at a wavelength of 266 nm. A ZORBAX Stable Bond-C18 column (250 mm× 4.6 mm, i.d. 5 μm, Agilent) was used to achieve chromatographic separation. The mobile phase consisted of 0.1% trifluoroacetic acid (phase A) and acetonitrile (phase B) at a flow rate of 1 mL/min at 30 °C with isometric elution conditions (86:14, v/v).
The concentration-time data for ceftiofur in plasma samples harvested from healthy pigs were analyzed by WinNonlin 5.2.1 software (Pharsight Corporation, Mountain View, CA, USA) to obtain the pharmacokinetic parameters.
Pharmacokinetic/pharmacodynamic integration and dose estimations
There are three standard indices (ƒ%T > MIC, ƒCmax/MIC, and ƒAUC0–24 h/MIC) for an antibiotic . The inhibitory sigmoid Emax model (Hill equation) was analyzed the integration of AUC24h/MIC ratio in vitro and bacteria count change (CFU/ml) in serum during 24 hr incubation. The model equation was described as follows:
where E is the antibacterial effect measured as the change in the bacterial count (log10 CFU/mL) in plasma sample after 24 h of incubation compared with the initial incubation, E0 is the change in log10 difference in bacterial count in the control sample after 24 h of incubation; Emax is the maximum antibacterial effect determined as the difference in log10 CFU/mL in the sample after the incubation, EC50 is the PK/PD parameter value producing 50% of the maximum antibacterial effect; C is the PK/PD parameter value in the effect compartment (the ex vivo site, that is plasma); and N is the Hill coefficient, which describes the steepness of the PK/PD parameter-effect curve.
PK/PD parameter values corresponding to the E value (derived from the sigmoid Emax equation) in plasma were used to deduce an optimal dose regimen. The potential optimal dosage was calculated using the following equation:
where MIC is the ex vivo minimum inhibitory concentration; (AUC24 h/MIC)ex is the target end point for optimal efficacy; CL is the clearance; fu is the free fraction of ceftiofur in plasma; and F is the bioavailability of ceftiofur.
The distribution probabilities for predicted daily dosage were performed to achieve simulated 50% and 90% TAR under 1,000 trials with Crystal Ball software (version 11.1.2, Oracle, United States).
Monte Carlo simulation and the pharmacokinetic/pharmacodynamics cutoff (COPD)
A Monte Carlo simulation (MCS) with 10,000 iterations was conducted using Crystal Ball software (version 7.2.2) (Oracle, United States) based on PK parameters and calculated PK/PD targets (AUC24h/MIC) when each possible MIC and the target AUC24h/MIC achieving a bactericidal action (E = −3). The AUC24h was assumed to be normally. COPD was defined as the maximal MIC value at which the corresponding PTA was ≥ 90% .