Optimal regimens regimens based on PK/PD cutoff evaluation of ceftiofur against Actinobacillus pleuropneumoniae in swine

doi:10.21203/rs.3.rs-22198/v2

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Optimal regimens regimens based on PK/PD cutoff evaluation of ceftiofur against Actinobacillus pleuropneumoniae in swine

https://doi.org/10.21203/rs.3.rs-22198/v2

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published 29 Sep, 2020

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Background Actinobacillus pleuropneumoniae , formerly known as Haemophilus pleuropneumoniae , can cause pleuropneumoniae in pigs of different ages, leading to significant mortality. Ceftiofur was the first cephalosporin antibiotic used in animals that was effective against gram-negative and gram-positive bacteria. This study aimed to determine epidemiologic cutoff value (ECV), and pharmacodynamic-pharmacokinetic cutoff. Establishing Clinical breakpoint of ceftiofur against Actinobacillus pleuropneumoniae based on the pharmacodynamic-pharmacokinetic cutoff.

Results The epidemiologic cutoff value was 0.125 μg/mL. The results of the pharmacodynamic study showed that the MICs of BW39 were 0.5 μg/mL and 1 μg/mL under in vitro and ex-vivo conditions, respectively, and the minimal bactericidal concentrations (MBCs) under in vitro and ex vivo conditions were both 1 μg/mL. The time-killing profiles of ceftiofur against BW39 were time-dependent with a partly concentration-dependent pattern. According to the inhibitory sigmoid E max model, the AUC 24 h /MIC values for the bacteriostatic, bactericidal, and elimination effects in serum were 45.73, 63.83, and 69.04 h for healthy pigs, respectively. Based on the Monte Carlo simulation, the CO PD was calculated as 2 μg/mL, and the optimized dosage regimen of ceftiofur against Actinobacillus pleuropneumoniae to achieve bacteriostatic, bactericidal, and elimination effects over 24 h was 2.13, 2.97, and 3.42 mg/kg for the 50% target attainment rate (TAR) and 2.47, 3.21, and 3.70 mg/kg for the 90% TAR, respectively.

Conclusions In conclusion, we reveal the EOFF and PK/PD cutoff values of ceftiofur against A. pleuropneumoniae in piglets. However, with the paucity of clinical data for ceftiofur to establish a clinical cutoff against A. pleuropneumoniae, the PK/PD cutoff value of 2 μg/mL will be recommended as surrogate. According to the PK/PD data and the MIC distribution in China, the single bactericidal dose was 3.21 mg/kg for the 90% target, which would be more able to cure Actinobacillus pleuropneumoniae and avoid the emergence of resistance for clinical ceftiofur use of ceftiofur in piglet.

Large Animal Medicine

Ceftiofur

Actinobacillus pleuropneumoniae

epidemiologic cutoff value

pharmacokinetic/pharmacodynamics cutoff

dosage

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae is a highly contagious respiratory disease that causes hemorrhage, purulent and fibrous pleuropneumonia. The disease is widely distributed and causes severe losses to the pig industry. The morbidity of the disease can be as high as 100%, but usually varies from 30 to 50%, with a case fatality rate as high as 50% [1]. There are currently 18 serotypes of Actinobacillus pleuropneumoniae [2, 3], and the prevalent serotypes vary in different countries and regions worldwide.

Ceftiofur was the first third-generation cephalosporin antibiotic used in animals. It is effective for gram-negative bacteria and gram-positive bacteria and has a good clinical therapeutic effect against animal respiratory diseases [4]. In 1988, the United States Food and Drug Administration (FDA) first approved ceftiofur sodium for use in bovine respiratory bacterial diseases. The FDA has approved cefiofur hydrochloride, ceftiofur sodium and ceftiofur crystal-free acid for the treatment of respiratory diseases of pigs, cattle, horses, goats and sheep[5-7].

Clinical breakpoint are MIC values, which are used in clinical microbiology laboratories to categorize microorganisms as clinically[8]. Establishing clinical breakpoint contains three parts: (i) an epidemiological cut-off value (ECOFF), described by EUCAST as the ECOFF, it defines the upper end(>95%) of the wild type MIC distribution. (ii) a MIC to define a PK/PD cut-off, named by EUCAST as the PK/PD breakpoint. It is calculated from selected PK/PD index in target population(iii) clinical cut-off described by EUCAST in human medicine, it is related to clinical outcomes( cure vs non-cure) and requires specific investigations during prospective clinical trials[9]. Although the CLSI has established the breakpoint of ceftiofur against Actinobacillus pleuropneumoniae, considering that the use of antibiotics and drug resistance in China is totally different, it is necessary to formulate the relevant criteria for ceftiofur against Actinobacillus pleuropneumoniae in China based on the local situation.

The abuse of antibiotics and unreasonable doses of antimicrobial agents are the main factors that contribute to the development of resistance[10, 11]. Existing data have suggested that the resistance rate of Actinobacillus pleuropneumoniae to tetracyclines and penicillin reached 90% and showed different degrees of resistance to macrolides and aminoglycosides. However, Actinobacillus pleuropneumoniae is still highly sensitive to ceftiofur [12-14] and Clinical breakpoint should be define for each animal species and for the relevant bacterial target pathogens. Therefore, we need to use scientific methods to determine the reasonable dosage of ceftiofur and establish drug resistance criteria to protect the clinical efficacy of ceftiofur. Previous study [15]has shown the PK data of ceftiofur sodium and ceftiofur hydrochloride and the recommended dosage of ceftiofur for the treatment of swine respiratory disease in the U.S. and in Europe ranged from 3 to 5 mg of ceftiofur equivalents (CE)/kg body weight. PK/PD integration modeling data can provide an optimal drug dosage strategy, reducing resistance development, which is a key method to evaluate the clinically relevant relationship between time, drug concentration, and effect. However, there are no data supporting the recommended dosage of ceftiofur based on the PD of ceftiofur against Actinobacillus pleuropneumoniae in China.

The purposes of this investigation were to (i) study the pharmacokinetic/pharmacodynamic of ceftiofur against Actinobacillus pleuropneumoniae, (ii) formulate a rational dosage strategy and review the preceding recommended dosage based on PK/PD modeling for ceftiofur against Actinobacillus pleuropneumoniae, and (iii) establish a clinical breakpoint based on pharmacokinetic/pharmacodynamic cutoff values.

MIC distribution and epidemiologic cutoff value

The MIC distribution of ceftiofur against Actinobacillus pleuropneumoniae is shown in Figure 1. The MIC values ranged from 0.0075 to 4 μg/mL. The MIC₅₀ and MIC₉₀ were calculated to be 0.015 and 0.5 μg/mL, respectively. we selected the serotype 1 strain App BW39 whose MIC was similar to MIC90(0.5 μg/mL) to analyzed for PD study in broth and serum. The ECV using the freeware statistic software “ECOFFinder” was 0.125 μg/mL, encompassing 99.9% of the wild-type isolates.

MICs, MBCs, MPC and PAE

The MIC values of ceftiofur against App BW39 were 0.5 μg/mL and 1 μg/mL in vitro and ex vivo, respectively, and the MBC values were both 2 μg/mL in vitro and ex vivo. The MBC/MIC ratios were 2 and 1 in vitro and ex vivo, respectively, indicating that ceftiofur is a bactericidal drug. The MPC value of ceftiofur against App BW39 was 1.6 μg/mL, and the MSW was 1-1.6 μg/mL. The post antibiotic effect (PAE) values after exposure to the concentrations of 1MIC, 2MIC and 4MIC were 0.33, 0.41 and 0.66h for 1h exposure and 0.73, 0.92 and 1.17 h for 2 h exposure, respectively (Table 1). The result suggested PAE had a positive correlation with exposure time. PAE was also prolonged with the increase of drug concentration, but the degree of enhancement was weak.

Time-killing curves

The time-killing curves of ceftiofur against BW39 in vitro and ex vivo are shown in Figures 2 and 3. When the drug concentration was 1/2 MIC-1 MIC, bacterial growth was not inhibited. Concentrations between 1 MIC and 2 MIC showed a slight inhibitory effect on the bacterial growth. Ceftiofur achieved a maximum bactericidal effect from 4 MIC-8 MIC. The ex vivo killing curve showed that the plasma collected between 0.33 and 2 h achieved the maximum bactericidal effect at the highest ceftiofur concentration. When the concentration was below the MIC at 96 h, ceftiofur no longer inhibited the growth of bacteria.

Pharmacokinetic analysis of ceftiofur in plasma

LOD and LOQ of HPLC methods for detecting DFC were 0.03 μg/mL and 0.08 μg/mL,respectively. The linear range of the standard and working curve of DFC ranged from 0.1 to 50 μg/mL, with a coefficient of determination (R²) of 0.9999 and 0.9987, respectively The recovery of ceftiofur in plasma ranged from 82.95% to 84.38%. The pharmacokinetic parameters calculated using non-compartment model analysis after I.M. (5 mg/kg BW) administration in healthy pigs are shown in Table 2. DFC concentrations were measured at different times shown in Figure 4.

PK/PD integration and analysis

The PK/PD indices were determined by integrating the in vivo PK data and the ex vivo time-killing curve. The ratios of C max/MIC, AUC_{24 h}/MIC, and T > MIC were 22.33±1.98, 358.84±57.42 h, and 72.2±7.32 h, respectively. The values of the PK/PD index versus antibacterial effects in serum were simulated by the sigmoid E_max model . The model parameters of Hill coefficient, N, E max and AUC 24 h/MIC of the different levels are presented in Table 3. The values of the AUC24 h/MIC ratio required for bacteriostatic activity (E = 0), bactericidal activity (E = −3), and bacterial elimination (E = −4) were 45.73, 63.83, and 69.04 h, respectively.”

Dose estimation

According to the PK-PD integration model, the PK-PD index, AUC 24 h/MIC, was altered based on different outcomes in healthy swine. The prediction of ceftiofur curing A. pleuropneumoniae diseases for the 50% and 90% targets was calculated by the Monte Carlo simulation and dosage equation, which are shown in Figure 5. The doses predicted to exhibit bacteriostatic, bactericidal, and elimination effects for A. pleuropneumoniae over 24 h were 2.13, 2.97, and 3.42 mg/kg for the 50% target attainment rate (TAR) and 2.47, 3.21, and 3.70 mg/kg for the 90% TAR, respectively, based on Crystal Ball software.

CO_PD determination with Monte Carlo Simulation

According to the MIC distribution, AUC_{24 h} derived from healthy pigs and the PK-PD target (AUC_{24 h}/MIC)_ex of 63.83 h, 10,000 subjects were simulated by Monte Carlo simulation. The PTA (possibility target achievement) values were calculated as 88.94%, 100% and 100% when the MICs (PK-PD cut- off) were defined as 4, 2 and 1 μg/ml, respectively (Table 4) . Therefore, the CO_PD for ceftiofur against Actinobacillus pleuropneumoniae was defined as 2 μg/mL, for the current dose of 5mg/kg by IM route.

Ceftiofur, whether administered as hydrochloride or sodium salt, was metabolized rapidly to desfuroylceftiofur. The plasma half-life of ceftiofur sodium after intravenous dosing in swine was approximately 10 min [16]. The previous study[17] has tested the susceptibility of ceftiofur and its metabolite desfuroylceftiofur against Actinobacillus pleuropneumoniae(n=50),the MIC90 of ceftiofur is 0.0078 μg/mL，and 0.015 μg/mL for desfuroylceftiofur, which suggests ceftiofur can be predictive of in vitro activity of ceftiofur and its major metabolite, desfuroylceftiofur. In our study, we determined the vitro susceptibility of ceftiofur, instead of desfuroylceftiofur, against 135 Actinobacillus pleuropneumoniae strains. The MIC values ranged from 0.0075μg/mL to 4 μg/mL, and the MIC₅₀ and MIC₉₀ values were 0.015 μg/mL and 0.5 μg/mL, respectively, which were slightly different from the results of ceftiofur against 50 Actinobacillus pleuropneumoniae strains (MIC ranged from 0.0039-0.0015 μg/mL) [17].

The epidemiologic cutoff was established based on the pretest of drug susceptibility and then analyzed by statistical methods. At present, CLSI and EUCAST guidelines mainly involve analysis by nonlinear regression, which was proposed by Turnidge [18]. EUCAST based on the nonlinear regression complied the ECOFFinder software[19] the principle of which is the same as Turnidge’s method. In this study, the epidemiologic cutoff was 0.125 µg/L according to the ECOFFinder results.

There are currently 18 serotypes of Actinobacillus pleuropneumoniae [2, 3]. The prevalent serotypes are different all over the world. The main prevalent serotypes in Canada are serotypes 1, 3 and 5, those in Europe are serotypes 1, 2, 5, 7 and 9 [20], and those in the United States are serotypes 1, 5 and 7 [21]. However, serotypes 1, 3 ,5 and 7 were the main prevalent serotypes in China[22]. Moreover, serotype 1 was more virulent than other serotypes[23, 24] Therefore, A strain App BW39, Whose MIC was similar to MIC90(0.5 μg/mL) and serotype was serotype 1, was analyzed for PD study in broth and serum . According to the MIC and MBC values in vitro and ex vivo, the MBC/MIC ratio was 2 and 1, respectively. Therefore, ceftiofur is a bactericidal drug with an MBC/MIC ratio < 4 [25]. The results of in vitro and ex vivo killing curves suggested, when the drug concentration was 1/2 MIC-1 MIC, bacterial growth was not inhibited. the concentration from 4 MIC-8 MIC achieved the maximum bactericidal effect. with the concentration increasing, the bactericidal effectr increased and achieved the maximum bactericidal effect at 4MIC-8MIC. These findings suggest that ceftiofur has a time-dependent nhibition activity same with a partly concentration-dependent pattern against Actinobacillus pleuropneumoniae both in vitro and ex vivo[26].

To establish a pharmacokinetic/pharmacodynamics cutoff, we needed to obtain the PK-PD parameters, MIC distribution and pharmacodynamic targets. The most appropriate PK/PD index to determine the antibacterial efficacy and predict the therapeutic efficacy for β-lactam antibiotics is the percentage of duration that the concentration of free drug is greater than the MIC (%T > MIC) [27]. The killing curve shows that ceftiofur has a time-dependent inhibition activity same with a partly concentration-dependent pattern against Actinobacillus pleuropneumoniae. Moreover, PK/PD index depends on the shape of the plasma exposure curve which may differ widely between the many modalities of Antimicrobial drug administration, such as antimicrobial drug was incorporated in food or drinking water or administrated by oral. VetCAST explore alternatives, in particular the possible use of AUC/MIC as a default index. The index of the AUC/MIC ratio was also used to describe the character of the antibacterial activity of time-dependent killing with prolonged persistent efficacy [28]. In our manuscript, The PK/PD index (AUC/MIC), using an inhibitory sigmoidal Emax model, showed a favorable correlation (r2 = 0.9967) with the predicted antibacterial efficacy. For these reasons, the AUC/MIC parameter was regarded as the most appropriate PK/PD index to describe the antibacterial activity of ceftiofur in serum. An AUC/MIC ratio > 125 h is generally considered the best activity indicator [29, 30]. However, the target parameters might be different for different drugs, target organisms or bacteria[31]. For example, the AUC_{24 h}/MIC values for three levels of preventive, therapeutic, and bacterial eradication were 28.40, 29.51 and 29.13 h, respectively, in previous study on PK/PD modeling of Ceftiofur Sodium against Haemophilus parasuis infection in pigs[26]. In another study, the AUC_{24 h}/MIC values for three levels of preventive, therapeutic, and bacterial eradication were 24.6, 43.8 and 58.4 h, respectively [32]. Prior to our study, no PK-PD integration modeling analyses were performed for ceftiofur against Actinobacillus pleuropneumoniae. In our study, the inhibitory sigmoidal E_maxmodel was employed for the simulation of the PK/PD integration model to obtain the AUC_{24 h}/MIC value. The ex vivo values of AUC_{24 h}/MIC of ceftiofur against bacteriostatic, bactericidal and eradication were 45.73, 63.83 and 69.04 h, respectively. Through Monte Carlo simulations, when the MIC values were 2 μg/mL and 4 μg/mL, the probability of target attainment was 100% and 88.94%, respectively. Therefore, the CO_PD was calculated as 2 μg/mL, for the current dose of 5 mg/kg by IM route.

The PK of healthy pigs corresponded to a non-compartmental model. The peak concentration reached 22.33±3.17 at 0.66-2h, the elimination half-time was 19.51±2.76 h, and AUC was 358.84±91.87μg·h/mL, which was similar to previous study[3]. The PK of ceftiofur may differ from that of diseased pigs. Previous study has demonstrated that infected pigs may have higher values of Vz/F and CL/F, but lower values of AUC,

MRT and t1/2z compared with non-infected pigs.

PD-PD integration modeling can be used to select rational dose regimes in veterinary medicine[30]. Based on the dose estimation equation and Monte Carlo simulation, the ‘fu’ in the equation was the free fraction of the drug in plasma. Ceftiofur had a high protein binding rate, which was 90% in cow and adult cattle [33, 34],The EMA[35] reported that the protein binding of ceftiofur and its metabolite DFC in swine was 0.7, which was applied in this research. According to the MIC distribution in China, the predicted daily doses for the 50% and 90% targets of ceftiofur with bactericidal activity against Actinobacillus pleuropneumoniae were 2.97 and 3.21 mg/kg body weight, respectively. The Monte Carlo simulation combined with the dosage equation and taking into consideration the PK/PD integration could set the target percentage to simulate models for all data in relation to incidence in swine [36, 37].

CBP is used to define susceptibility and resistance. In general, the determination of CBP should take into account the ECOFF, COPD and clinical cutoff values [24, 25]. Under the clinically recommended dose(5mg/kg), the ceftiofur CO_PDvalue (2 μg/mL) against A. pleuropleumoniae was higher than the ECOFF value (0.125 μg/mL). It probably means that the current dose (5.0 mg/ kg) could guarantee clinical efficacy to treat the wild-type populations of A. pleuropneumoniae. In fact, our calculated accurate dose of ceftiofur hydrochloride for a PTA≥90% was 3.21mg/kg. Previous study[3] has shown the similarity of the pharmacokinetic parameters of the sodium and hydrochloride formulations of ceftiofur, similar therapeutic efficacy therefore can be inferred for the two products. So, Considering the MIC distribution in China, the dose of ceftiofur could be 3.21mg/kg, However, owing to the paucity of relevant data to bridge the relationship between MIC and clinical cure, it is practically difficult to determine a clinical cutoff in veterinary medicine [24, 26]. In our study, CO_PD values contains the ECOFF values, and the CO_PD(2 μg/mL) will therefore be recommended as surrogate. Although the ECOFF or PK/PD cutoff could not replace the CBP, it still provides a useful interpretative criterion.

In conclusion, we reveal the EOFF and PK/PD cutoff values of ceftiofur against A. pleuropneumoniae in piglets. However, with the paucity of clinical data for ceftiofur to establish a clinical cutoff against A. pleuropneumoniae, the PK/PD cutoff value of 2 μg/mL will be recommended as surrogate. Moreover, according to the PK/PD data and the MIC distribution in China, the single bactericidal dose was 3.21 mg/kg for the 90% target, which would be more able to cure Actinobacillus pleuropneumoniae and avoid the emergence of resistance for clinical ceftiofur use of ceftiofur in piglet.

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 CO_PD 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[38] 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 [39]. A wild-type cutoff (CO_WT) was developed based on the MIC distribution following CLSI M37-A3. The CO_WT 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 [40]. Exponential growth phase bacteria were pelleted by centrifuging at 3000 rpm at 4 °C. The pellet was then diluted to 3 × 10¹⁰CFU/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 × 10⁷CFU/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

Animals

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).

Samples collection

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.

Sample analysis

DFC concentration were measured by HPLC described by previous study[26] 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, ƒC_max/MIC, and ƒAUC_{0–24 h}/MIC) for an antibiotic [41]. 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; E_max 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 E_max 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; (AUC_{24 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 (CO_PD)

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 (AUC_24h/MIC) when each possible MIC and the target AUC_24h/MIC achieving a bactericidal action (E = −3). The AUC_24h was assumed to be normally. CO_PD was defined as the maximal MIC value at which the corresponding PTA was ≥ 90% [42].

APP: Actinobacillus pleuropneumoniae,

AST:Antimicrobial susceptibility testing

AUC/MIC:The area under the concentration-time curve to MIC ratio

CLSI: Clinical & Laboratory Standards Institute

CO_PD :PK/PD(pharmacokinetic/pharmacodynamic) cutoff value (named by EUCAST as the PK/PD breakpoint)

DFC: Desfuroylceftiofur;

EMA: European Medicines Agency

ECOFF: Epidemiological cutoff value [synonym of wild-type cutoff value (COWT)]

EUCAST: European Committee on AST

HPLC: High performance liquid

LOD: Limited of Detection

LOQ: Limited of Quantitation

MBC: Minimal bactericidal concentration

MIC: Minimal inhibitory concentration

NAD: Nicotinamide Adenine Dinucleotide
PAE: Post-antibiotic effect

PK/PD: Pharmacokinetic/pharmacodynamic

PTA: Probability of target attainment

TSA: Tryptic Soy Agar

TSB: Tryptic Soy Broth

Ethics approval and consent to participate

All animal studies were carried out in accordance with the National Standards for Laboratory Animals of China (GB 14925–2010). The protocol of in vivo experiment and strain isolation procedures from animals were approved by the Institutional Animal Care and Use Committee at Huazhong Agricultural University (HZAUSW-2016-007).

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed in this study are available from the corresponding author on reasonable request.

Competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest

Funding

The authors thank the financial supporting from National key research and development program in China (2016YFD0501310;2018YFD0500301). The funding body had no role in the design of the study, collection, analysis, and interpretation of data and in writing the manuscript.

Author contributions

LLH, DS, DMC conceived this work. DS, HH, SYX designed the experiment. DS and MK performed the experiments. DS wrote the manuscript. LLH and DS improved the language. All authors reviewed the manuscript.

Acknowledgments

Not applicable.

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Table 1 Post antibiotic effect (PAE) after 1h and 2h

Ceftiofur (μg/mL)	1h (h)	2h (h)
MIC	0.33	0.73
2MIC	0.41	0.92
4MIC	0.66	1.17

Table 2 Pharmacokinetic parameters of Ceftiofur after I.M administration (5 mg/kg) in healthy pigs

Parameters	Units	Healthy
AUC_0-24h	μg·h/mL	358.84±91.87
AUC_0-∞	μg·h/mL	372.05±97.35
C_max	μg/mL	22.33±3.17
T_max	hr	0.66-2
T1/2	h	19.51±2.76
Ke	h-1	0.04±0.01
CL/F	mL/kg/h	282.49±52.91
MRT_0-t	h	22.82±1.83
MRT_0-∞	h	26.33±2.94

Table 3 PK/PD analysis of data acquired from ex vivo time-killing curve of ceftiofur against a representative strain of APP BW39 in serums

Parameter	Unit	healthy
E_max	Log₁₀ CFU/ml	3.68
E₀	Log₁₀ CFU/ml	-6.88
E_max-E₀	Log₁₀ CFU/ml	10.56
EC₅₀	h	21.97
N	-	1.78
AUC 24h /MIC for bacteriostatic	h	45.73
AUC 24h /MIC for bactericidal	h	63.83
AUC 24h /MIC for elimination	h	69.04

Table 4 The AUC₂₄/MIC values calculated with Monte Carlo simulation for PTA

Dose

(mg/kg)

AUC

(μg·h/mL)

MIC(μg/mL)

358.84±91.87

100%

88.94%

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Optimal regimens regimens based on PK/PD cutoff evaluation of ceftiofur against Actinobacillus pleuropneumoniae in swine

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