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Kill Rate and Evaluation of Ex Vivo PK/PD Integration of Cefquinome Against Actinobacillus pleuropneumoniae

We wished to study the detailed and precise antibacterial activity of cefquinome against Actinobacillus pleuropneumoniae (APP) in vitro and ex vivo. We analyzed the relationships between kill rate and cefquinome concentration in broth and between pharmacokinetic/pharmacodynamic (PK/PD) parameters and antibacterial effect in serum and tissue cage fluid (TCF) of piglets. Cefquinome exhibited time-dependent antibacterial activity against APP according to the kill rate. The maximum kill rate was 0.48 log10 CFU/mL/h at the 0-9-h period in broth. In the ex vivo PK/PD study, the maximum concentration (Cmax), time to reach the maximum concentration (Tmax), terminal half-life (T1/2β), and area under the concentration time curve (AUCinfinity) were 5.65 μg/ml, 0.58 h, 2.24 h, and 18.48 μg·h/ml in serum and 1.13 μg/ml, 2.60 h, 12.22 h, and 20.83 μg·h/ml in TCF, respectively. The values of area under the curve during 24 h/minimum inhibitory concentration (AUC24h/MIC) for bacteriostatic, bactericidal, and bacterial eradication effects were 18.94, 246.8, and 1013.23 h in serum and 4.20, 65.81, and 391.35 h in TCF, respectively. Our findings will provide a valuable basis for optimization of dosage regimens when applying cefquinome to treat APP infection.


INTRODUCTION
Porcine contagious pleuropneumonia (PCP), caused by Actinobacillus pleuropneumoniae (APP), is a serious respiratory system diseases in pigs (1,2). The classical symptoms of acute PCP are hemorrhagic necrotizing pneumonia and fibrinous pneumonia with high morbidity and mortality (3,4). Long-term infected pigs would show weight decline, low feed conversion rate, and delayed sale time. Therefore, PCP has caused considerable economic losses to farmers and severely restricted the development of the pig industry. However, because the serotype of A. pleuropneumoniae was more than those of 19 species and there was no effectively and commonly used vaccine to protect pigs at present (5)(6)(7). Hence, antimicrobial therapy is still a rapid and efficacious method for PCP treatment, such as cephalosporins, fluoroquinolones, and macrolides (8)(9)(10)(11)(12)(13)(14). However, with the abundant unreasonable application of drugs, multidrug-resistant pathogens were gradually selected and spread all over the world. Cephalosporins had been broadly applied in veterinary and human clinical therapy, and the resistant gene can spread from animal bacteria to human bacteria. Therefore, the resistance of veterinary bacteria to cephalosporins may not only reduce the antibacterial effect but also seriously threaten the health and life of humans (15,16). One of the major resistant mechanisms is the production of β-lactamase under selective pressure of antibiotics applied in irrational application. Therefore, a rational dosage regimen is an important method to prevent the emergence and spread of resistant bacteria. In view of these points, dosage regimens should be optimized not only to reach a clinical efficacy but also to prevent the emergence and spread of resistant bacteria.
The pharmacokinetic/pharmacodynamic (PK/PD) integration model can comprehensively study the relationship between PK/PD parameters and antibacterial effect and has been widely applied to design rational dosage regimens in clinical trials. At present, the common PK/PD model can classify to in vitro, in vivo, and ex vivo models. Each model has its own advantage. Compared to in vitro and in vivo models, the ex vivo model needs few animals, low cost, and less harm, and the results can partially reflect the influence of the host. The tissue cage (TC) model was an ideal method for ex vivo PK/PD integration and has been applied in several animals to study the ex vivo PK/PD for respiratory pathogens, such as Pasteurella multocida, Mannheimia haemolytica, and A. pleuropneumoniae (17)(18)(19).
Cefquinome is a broad-spectrum fourth-generation cephalosporin used only in animals. It has been approved for the treatment of several diseases caused by A. pleuropneumoniae, Klebsiella pneumoniae, and Streptococcus suis (8,20,21). One report (22) has applied the TC model to study the ex vivo PK/PD integration of cefquinome against Escherichia coli. As far as we know, the ex vivo PK/PD integration of cefquinome against APP has not been reported. Therefore, in this study, we will apply a piglet TC model to study the ex vivo PK/PD integration of cefquinome against APP. Meanwhile, a kill rate-based timekill curve will be conducted to study the relationships between cefquinome concentrations and kill rates in vitro. We wished that these findings could precisely clarify the antibacterial activities of cefquinome against APP to design a rational dosage regimen.

Determination of MIC and Time-Kill Curves of Cefquinome Against APP in TSB
APP was incubated in TSB and MHA with 4% newborn bovine serum and 1% NAD (1 mg/ml). The MIC of cefquinome against APP was tested by microdilution according to the reference methods of the Clinical and Laboratory Standards Institute (23). Briefly, after the APP was incubated for 8 h in a constant temperature shaker (200 rpm, 37 • C), and the exponential phase bacterium suspension was diluted to a final concentration of 5 × 10 5 colony-forming units (CFU)/mL by 10-fold dilution. The cefquinome suspension ranging from 0.0078 to 4 mg/l was prepared after serial twofold dilutions with TSB, and 100 µl was added to a 96-well plate. After 100 µl bacterial sample was added to each well, the plate was cultured for 18-20 h in a humidified incubator (37 • C, 5% CO 2 ). The MIC was determined as the minimal concentration of cefquinome without visible turbidity. All experiments were repeated thrice.

Calculation and Integration of Kill Rate and Concentration
The antibacterial effect was described as the maximum change of APP number (Log 10 CFU/mL) over each time period. The kill rate was the antibacterial effect divided by the corresponding time period. For kill curves, the kill rate was represented by the slope of the curve at a given time period (0-1, 1-3, 3-6, 6-9, 0-3, 0-6, and 0-9 h). Seven kill rates were obtained from different cefquinome concentrations. Finally, the kill rate (in the same period) and cefquinome concentration relationship were analyzed by using the sigmoid E max model (WinNonlin 5.2.1, Pharsight, MO, USA). The equation was described as follows: where E is the kill rate; E max is the maximum kill rate in TSB with cefquinome during each period; E 0 is the kill rate in control TSB; C e is the cefquinome concentration; N is the Hill coefficient that describes the steepness of the curve for the kill rate and cefquinome concentration; and EC 50 is the cefquinome concentration producing 50% of the maximum kill rate. The coefficient of determination (R 2 ) represented the relationship between kill rate and cefquinome concentration and can be acquired after sigmoid E max simulation. The higher the value of R 2 , the higher the level of correlation between kill rate and concentration.

Animals and TC Implantation
Six healthy piglets (Duroc × Landrace × Yorkshire, three females and three males, ∼25 kg) were purchased from the Guangzhou Fine Breed Swine Farm (Guangzhou, China). They were housed in separate cages and fed antibiotic-free fodder twice daily, while water was provided ad libitum. TCs were the same as those employed in previous study (22). The brief operation to implant TC was as follows. Firstly, a deep general anesthesia was induced by application of pentobarbital sodium into the neck by intramuscular injection. Then, the local infiltration anesthesia was induced by subcutaneous injection of procainamide hydrochloride. Thirdly, two TCs (sterilized by 75% ethyl alcohol and ultraviolet light) were implanted subcutaneously to each side of the neck equidistant from the jugular vein and spinal cord in each piglet. At last, penicillin (1000,000 IU/kg) and tetracycline ointment were used twice daily for 3 days to prevent infections. After 4-5 weeks, the TC was filled with TCF. After bacteriological examination, the TC without bacteria was used for the following experiment. The experimental protocol was approved (2016016) by the Committee on the Ethical Use of Animals of South China Agricultural University (Guangzhou, China).
Determination of the cefquinome concentration was undertaken by high-performance liquid chromatography tandem mass spectrometry, as described in our previous study (8). PK data were analyzed based on the non-compartment model by WinNonlin 5.2.1.
The MIC test methods for serum and TCF samples were the same as in the TSB sample. For ex vivo kill curves, 5 µl exponential phase bacterial suspension (∼10 9 CFU/ml) was added to sterilized tubes containing 0.5 ml TCF samples (for serum, 10 µl suspension was added to 1 ml serum sample) to ensure that the final bacterial concentration was about 5 × 10 7 CFU/ml and cultured in a humidified incubator (37 • C, 5% CO 2 ). Then, 50 µl suspension sample was removed at 0, 1, 3, 6, 9, 12, and 24 h for APP counting. The ex vivo antibacterial timekill curves were depicted the same as described in TSB. The antibacterial effect was described as the maximum change of APP number (Log 10 CFU/mL) over 24 h.

Ex vivo PK/PD Integration
For PK/PD integration, the AUC 24h /MIC (AUC 24h was the cefquinome concentration of each sample multiplied by 24 h) was applied to evaluate the ex vivo antibacterial effect by the sigmoid E max model. The model equation was described as follows: where I is the antibacterial effect; I 0 is the maximum change in Log 10 CFU/mL of the control sample (drug free) after incubation for 24 h; I max is the maximum reduction of bacterial count (Log 10 CFU/mL) in TCF or serum samples (contain cefquinome) after incubation for 24 h; C e is the value of AUC 24h /MIC; IC 50 is the value of AUC 24h /MIC needed to reach 50% I max ; and N is the Hill coefficient that describes the steepness of the curve for AUC 24h /MIC and antibacterial effect.
The antibacterial effect of cefquinome was quantified for three levels according to the value of I. I = 0 was defined as a "bacteriostatic" effect. I = −3 was considered a "bactericidal" effect. I = −4 was defined as a "bacterial-eradication" effect.

Statistical Analysis
The one-way ANOVA test (SPSS, version 22, IBM) was applied to analyze the difference of cefquinome PK indices among sera and TCF of each piglet. p < 0.05 indicated significant difference.

MIC and Kill Curves of Cefquinome Against APP in TSB
The MIC of cefquinome against APP was 0.008 µg/ml in TSB. The time-kill curves are depicted in Figure 1. The reduction of the APP population ranged from 1.02 to 6.32 Log 10 CFU/mL at different cefquinome concentrations (1 to 64 × MIC) during 0 to 24 h. At 0.5 × MIC, a slight increase (1.91 Log 10 CFU/mL) in APP number was observed. At 1 × MIC and 2 × MIC, the total reduction of APP was 1.02 Log 10 CFU/mL and 1.08 Log 10 CFU/mL, respectively, and a regrowth of APP was observed after 9 h. At 4 × MIC, a bactericidal effect (−3.16 Log 10 CFU/mL) was achieved without regrowth. At 8 × MIC, a bacterial-eradication effect (−4.43 Log 10 CFU/mL) was reached. At 16 × MIC, the antibacterial effect reached a maximum value (−6.29 Log 10 CFU/mL) and no longer increased with drug concentration added.

Integration of the Kill Rate and Cefquinome Concentration
Because the APP population was lower than the limitation of detection after 9 h during 16 to 64 × MIC, we only calculated and analyzed the kill rate during 0 to 9 h for precise PK/PD integration ( Table 1). At each time period, the kill rate increased upon addition of cefquinome during low drug concentrations (0-8 × MIC) and did not obviously increase and showed no significant difference during high drug concentrations (8-64 × MIC). At each drug concentration, the kill rate decreased gradually along with the prolongation of the incubation time. Therefore, a long interaction time was needed to produce bactericidal effect (6 h for 8 × MIC, 12 h for 4 × MIC).
After analyzing the relationship between cefquinome concentration and kill rate during 0 to 9 h by the sigmoid E max model, the main parameters and correlations (R 2 ) are as listed in Table 2. R 2 ranged from 0.9183 to 0.9955. The fittest relationship was at the 0-9-h time period (R 2 = 0.9955) (Figure 2). The maximum kill rate was 0.48 Log 10 CFU/mL/h. The kill rate increased rapidly with increasing drug concentration from 0.5 × MIC to 8 × MIC and then increased slowly from 8 × MIC to 16 × MIC (Figure 2). The kill rate was stable and virtually unchanged when the cefquinome concentration >16 × MIC.

PK of Cefquinome and Ex vivo Antibacterial Activity in Serum and TCF
The concentration-time curves of cefquinome are depicted in Figure 3A (serum) and Figure 3B (TCF). The main PK parameters are listed in Table 3. The mean values of the maximum concentration (C max ) were 5.65 ± 1.10 µg/ml and 1.13 ± 0.06 µg/ml, the time needed to reach the maximum concentration (T max ) were 0.58 ± 0.20 h and 2.6 ± 0.8 h, the area under the concentration-time curves (AUC infinity ) were 18.48 ± 3.28 µg h/ml and 20.83 ± 0.57 µg h/ml, and the terminal halflives (T 1/2β ) were 2.24 ± 0.13 h and 12.22 ± 0.85 h in serum and TCF, respectively. The MICs of cefquinome against APP were both 0.016 µg/ml in serum and TCF. The ex vivo time-kill curves of cefquinome against APP in serum and TCF are depicted in Figures 4, 5 respectively. For serum samples, a bacteriostatic effect (1.30 Log 10 CFU/ml reduction) could be achieved for samples collected at 12 h and APP was regrown after 3 h. The bactericidal effect (3.89 and 3.90 Log 10 CFU/ml reduction) was observed for samples collected at 6 and 8 h. The bacterial-eradication effect (4.02 to 4.60 Log 10 CFU/ml reduction) was produced for samples collected at 0.083 to 4 h. For TCF, samples collected at 0.5 to 24 h exerted a bacterial-eradication effect (4.06 to 4.30 Log 10 CFU/ml reduction) without regrowth after 24 h of incubation. Bactericidal activity (3.58 Log 10 CFU/ml reduction) was observed for the sample collected at 0.25 h. The bacteriostatic effect (0.15 to 2.54 Log 10 CFU/ml reduction) was observed for samples collected at 48 to 96 h.

Ex vivo PK/PD Integration and Analyses
The parameters integrated by the PK/PD model are presented in Table 4. A specimen plot of AUC 24h /MIC-effect (Log 10 CFU/mL) is depicted in Figure 6A (serum) and Figure 6B

DISCUSSION
Actinobacillus pleuropneumoniae is a serious pathogenic bacterium which can cause respiratory diseases of pigs (24,25). Antibacterials were an important therapeutic method. However, with unreasonable application of a large number of drugs, multidrug-resistant pathogens gradually appeared which may reduce the antibacterial effect and threaten human health.  To solve drug resistance, development of new drugs and optimization of drug dosage regimens were two effective methods. However, the development rate of new drugs cannot keep up with the emergence of drug resistance. Therefore, it is a practical method to optimize the drug schemes.
For dosage regimen design, the commonly applied PD parameter was MIC. However, because of the limitation of the test method, there are some deficiencies. One deficiency is that the MIC was tested in static drug concentrations during a period which is known to have an "all or nothing" value. Hence, the MIC reflects only the final antibacterial effect, which does not clarify the dynamic process of antibacterial activity at different periods. The other deficiency is that the MIC is commonly examined in an artificial medium, which does not consider the interaction of body substances (e.g., neutrophils, cytokines, immune factors) (26,27). Therefore, for a detailed understanding of antibacterial activity, a kill rate-based time-kill curve has been applied for in vitro PK/PD integration. This method can provide more valuable data by reflecting the dynamic interaction from multidimensional aspects of drugs against pathogens. The kill rate also has been applied to classify drugs as time-dependent or concentration-dependent (28). Therefore, in the present study, we applied this model to study the relationship between concentrations and kill rate of cefquinome against APP during different time periods.
Our in vitro PK/PD results show that the kill rates of cefquinome against APP were relatively low. Even when the drug concentration reached 64 × MIC, the maximum kill rate was 0.59 Log 10 CFU/mL/h. The kill rate reached a maximum value when the drug concentration reached 8 × MIC and did not increase with the drug concentration added. We also found that the antibacterial activity of cefquinome against APP was according to time-dependent drug characteristics. That is to say, a more  than alone. In our study, a sigmoid E max model was applied to analyze the relationship between cefquinome concentrations and kill rate. As far as we know, one report (32) also applied this method to study the antibacterial activity of doxycycline against Mycoplasma gallisepticum. Their results showed that doxycycline exhibited time-dependent antibacterial activity and the maximum kill rate was 0.11 h −1 .
The PK properties of cefquinome in piglet serum and TCF were reported (22) after intramuscular administration at 2 mg/kg. The reported C max , T max , AUC, and T 1/2β were 6.15 and 1.15 µg/ml, 0.34 and 3.00 h, 21.40 and 17.79 µg h/ml, and 2.30 and 11.81 h, in serum and TCF, respectively, and were similar to our values.
For PK/PD integration, the commonly applied PK/PD indices included C max /MIC, AUC 24h /MIC, and %T > MIC (the percentage of time of drug concentration above MIC after each dosing interval). Generally, the fittest PK/PD parameter for time-dependent drugs was %T > MIC after different dosage treatments in dynamic models. Several reports had studied the PK/PD integration of cefquinome against other pathogens, such as Streptococcus suis, Haemophilus parasuis, Escherichia coli, and Staphylococcus aureus (21,(33)(34)(35), and they had calculated the different values of %T > MIC for achieving different antibacterial effect to guide the designation of dose regimens. However, in our present study, we applied AUC 24h /MIC for the ex vivo PK/PD integration of cefquinome against APP. Because the different values of %T > MIC cannot be acquired in static drug concentrations, AUC 24h /MIC had been widely applied for ex vivo PK/PD analysis in several kinds of drugs and bacteria. Aliabadi  At present, the PK/PD model can generally be classified to in vitro, ex vivo, and in vivo models. Each of these models has its own advantages and disadvantages. In the present study, we conducted in vitro and ex vivo PK/PD research. Compared to the in vivo study, these two studies can exhibit three different antibacterial characteristics. First, the in vitro study can precisely and directly reflect the antibacterial activity of drug-pathogen interactions. In addition, the in vitro study was more fit to identify the characteristics of drugs to time or concentration-dependent antibacterial activity than the in vivo model. Further, the ex vivo study could in detail reflect the difference of protein binding, nutrient substance, immune factors, and cytokines in body fluid (such as serum, plasma, TCF, and inflammatory exudate). For the in vivo study, the antibacterial effect is the final result of drug, pathogen, and host interactions which cannot describe the separately interactive process of drugs and host. Therefore, it is maybe more rational and effective to comprehensively consider the data from in vitro, ex vivo, and in vivo studies during design dosage regimens.
Although we had successfully studied the in vitro and ex vivo PK/PD integration of cefquinome against APP, there were still some limitations. One is that the TCF was collected between skin and muscle. Therefore, the substance and cefquinome PKs were different from the target organs (lung) of respiratory pathogens. The other limitation is the difference between health and infected body conditions. Actually, the immune system will be activated and produce a lot of immune factors by pathogen infection. In addition, to calculate the recommended dosage regimens, an MIC 90 was needed. Because the number of APP was not enough, we did not calculate the recommended dosages in this study. Therefore, in our further studies, we will optimize the ex vivo PK/PD model from these aspects, such as a microdialysis to collect lung fluids.

CONCLUSIONS
Cefquinome exhibited time-dependent antibacterial activity against APP based on the analyses of kill rate. The maximum kill rate was 0.48 Log 10 CFU/ml/h at 0-9 h in TSB.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Committee on the Ethics of Animals of South China Agricultural University.

AUTHOR CONTRIBUTIONS
LZ and HW contributed to the methodology, software use, validation, data analysis, writing, and project administration. HX, HD, GZ, and JH contributed to study supervision. LZ and HX contributed to funding acquisition. All authors contributed to the article and approved the submitted version.