Introduction

Escherichia coli (E. coli), a prominent commensal bacterium in gastrointestinal tract of animals and humans, acts not only as an important human pathogen associated with intestinal diseases but also a causative agent resulting in diarrhoea in animals1,2. Danofloxacin (DAN) is a fluoroquinolone active against a broad spectrum of bacteria3.

The MIC is the lowest concentration of antimicrobial agent that inhibits growth of the organism in the microdilution wells as detected by the unaided eye; the minimal bactericidal concentration (MBC) is the minimal concentration of drug needed to kill most (≥ 99.9%) of the viable organisms after incubation for a fixed length of time (generally 24 h); the mutant prevention concentration (MPC) is defined as the concentration preventing growth at a high (˃ 109 CFU/mL) inoculum using agar dilution methodology4.

Antibiotic resistance has become a global health threat for decades. It has been upgraded by major world health organizations to one of the top health concerns of the twenty-first century5. In the prevention and control of drug resistance, the most important is to develop new antibiotics and to design effective dosage schedules. It is regrettable that the speed of antibacterial drug development is far behind the speed of drug resistance. From 1985 to 2016, the number of antibiotic resistant bacteria infections is increasing whereas the development of new antibiotics is constantly decreasing6. Moreover, the development of drug resistance is unimaginable, and drug resistant bacteria appear in the early stage of clinical application of many new drugs7. Therefore, a rational dosage schedule must be designed to reduce the emergence of drug resistance. The design of rational dosage schedule is dependent on (i) the linkage of Pharmacokinetics (PK) data to ex vivo or in vivo pharmacodynamic (PD) data generated in animal models or in clinical trials and (ii) The killing mechanism of the drug against bacteria. The integrated PK-PD parameters the area under curve (AUC24h)/MIC and maximum concentration of drug (Cmax)/MIC are therefore closely related to a successful treatment outcome3.

However, few previous publications have described the PK of DAN in animal model, especially in piglets. The objectives of this study were (1) to implant an ultrafiltration probe into the ileum of pig to continuously collect intestinal fluid; (2) to establish plasma and ileum PK data for DAN in piglet after intramuscular (i.m.) treatment; (3) to determine integrated PK-PD parameters (AUC24h/MIC and Cmax/MIC) for DAN in vivo; (4) to establish the AUC/MIC ex vivo that produce bacteriostatic, bactericidal, and eradication by using the inhibitory effect sigmoid Emax equation; and (5) to calculate respectively different dosages based on AUC24h/MIC ratio that might provide a novel approach to the rational design of dosage schedules which provide maximal efficacy and minimizes opportunity for the emergence of resistant strains.

Materials and methods

Ethical statement

All of the experiments were conducted under the supervision of the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences in accordance with animal ethics guidelines and approved protocols. The Harbin Veterinary Research Institute Animal Ethics Committee approval number was SYXK (Hei) 2012-2067.

Pathogenicity test

The laboratory preserved strains (HP65, HP189, HP232 and HP501) were conveniently selected for pathogenicity test. Pathogenicity test was performed with 39 Kunming mice (weighted 32 ± 3 g, specific pathogen free grade) purchased from Experimental Animal Centre of Harbin Medical University. Every three mice were intraperitoneal administrated with 200µL E. coli suspension (107, 108, and 109 CFU/mL). The blank control group (n = 3) were intraperitoneal administrated with 200 µL sterile broth. The mice were observed every 3 h after the injection up to 72 h. If there were three mice dead in a group, the inoculant given to this group was considered to be of high pathogenicity.

Bacterial strain, antimicrobials, and chemicals

According to the results of pathogenicity test, an EPEC E. coli strain HP501 (O158) was selected for subsequent PD experiments. The reference strain E. coli ATCC25922 strain was purchased from the NATIONAL CENTER FOR MEDICAL CULTURE COLLECTIONS (Beijing, China). Mueller Hinton broth was purchased from Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China. DAN injection was obtained from Zhejiang Guobang Pharmaceutical Co., Ltd., China. The pure reference standard of DAN and ciprofloxacin were obtained from the Sigma-Aldrich, China. Atropine sulfate was supplied by the Shanghai Chemical Reagent Factory (Shanghai, China). Isoflurane was purchased from Hebei Yipin Pharmaceutical Co., Ltd., China and propofol injectable emulsion from Xi'an Libang Pharmaceutical Co., Ltd., China.

Animals and implantation of an ultrafiltration probe

Six healthy males, castrated crossbred piglets (Duroc × Landrace), weighing 21–35 kg, were obtained from Harbin breed swine farm. They were housed in individual cages and allowed a 7 day acclimation period prior to the initiation of the study. Piglets had free access to water and fed antibiotic free feed twice a day.

Ultrafiltration probes were implanted into the ileum of piglets according to the previous study with modified8. Several BASi accessories were used for in vivo ultrafiltration sampling. These include: in vivo ultrafiltration sampling probes (MF-7023); flanged tubing connectors (MD-1510); vacuum vial needles (MD-1320) and the vacuum needle holder (MD-1322); vacutainers (MF-7024). Feed and water were withheld from the piglets for 12 h prior to surgery. Surgical procedures have been performed two days with three surgeries done each day. The piglets were pre-medicated with intramuscular administration of 0.02 mg/kg atropine sulfate prior to anesthesia. Approximately 15 min later, anesthesia was induced with 5 mg/kg propofol injectable emulsion intravenously through the ear vein. Each piglet was placed in left lateral recumbency on the surgical table and manually intubated orotracheally (Fig. 1a). General anesthesia was maintained by inhalation of 1.5–3.0% isoflurane in 100% oxygen on a circle circuit with mechanical ventilation. During surgery, the steers were given a continuous infusion of physiological saline at 10 mL/kg/h. The electrocardiogram, oxygen saturation, and non-invasive blood pressure via doppler were monitored throughout surgery (Fig. 1f).

Figure 1
figure 1

Implantation of ultrafiltration probes into the ileum of piglets. (A) each piglet was placed in left lateral recumbency on the surgical table and manually intubated orotracheally; (B) a vertical 10 cm skin incision was made approximately 10 cm above the midline of the abdomen and approximately 5 cm from the last rib; (C) A stab incision was made with a scalpel blade in the ileum, and an introducer needle was placed through the stab incision into the lumen of the ileum; (D, E), nodular suture of the ileum incision with 2–0 absorbable surgical suture; (F) the electrocardiogram, oxygen saturation, and non-invasive blood pressure via doppler were monitored throughout surgery; (G) the vacuum tube holder and needle are sutured to the skin.

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The abdominal hair was clipped and partially disinfected with iodophor. A vertical 10 cm skin incision was made approximately 10 cm above the midline of the abdomen and approximately 5 cm from the last rib (Fig. 1b). Sharp dissection continued through the external and internal abdominal oblique muscles, transverse abdominal muscle, and peritoneum to enter the peritoneal cavity. The cecum was identified and exteriorized caudally to expose the ileocecal fold8. The ileocecal fold was used to identify the ileum, the location of the probe was 10 cm away from the cecum. A stab incision was made with a scalpel blade in the ileum, and an introducer needle (MR-5313, BASi, USA) was placed through the stab incision into the lumen of the ileum (Fig. 1c). The collecting end of the ultrafiltration probe was inserted through the introducer needle aborally toward the cecum so that the entire collecting apparatus was within the lumen8. The introducer needle was fed off the probe, and nodular suture of the ileum incision with 2–0 absorbable surgical suture obtained from Shanghai Pudong Jinhuan Medical Supplies Co., Ltd. (Fig. 1d, e). The free ends of the probes were exteriorized to the skin incision approximately 25 cm above the midline of the abdomen by using the introducer needle to create a tunnel from the abdominal cavity through the muscle layers8. A skin incision with a blade further exteriorized the introducer needle so that the free end of the probe could be fed through to the outside of the body. The abdominal incision was closed in three layers. The first layer included the peritoneum, transverse abdominal, and internal abdominal oblique muscles. This layer was closed in a simple continuous pattern using 2–0 absorbable surgical suture. The external abdominal oblique muscle was also closed with 2–0 absorbable surgical suture in a simple continuous pattern. Skin layer sutured with 3–0 absorbable surgical suture. The vacuum tube holder and needle are sutured to the skin (Fig. 1g). After recovery from anesthesia, they were housed in individual cages and received drug treatment to prevent infection by intramuscular injection of penicillin (1,000,000 IU/kg) and application of iodophor over the wound twice a day for 3 days.

Sample collection and HPLC analysis

DAN was intramuscularly injected at 2.5 mg/kg body weight in each piglet after 72 h probe insertion. Blood samples (5 mL) from the brachiocephalic vein were collected into EDTA dipotassium salt tubes at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 26, 30, and 48 h after drug administration3. To collect ultrafiltrate from the gastrointestinal tract, a 3 mL vacutainer (Fig. 1d) was then inserted into the needle of the vacuum vial needle holder. Approximately 300–400 µL of ultrafiltrate is collected each hour, and the probes will continuously collect ultrafiltrate. The timed interval samples were collected at 0, 1, 2, 4, 6, 8, 12, 24, 26, 30, and 48 h after drug administration by changing the vacutainer tubes. Plasma was separated by centrifugation at 3000 g for 10 min and then the supernatant was transferred into a fresh tube. All plasma and ultrafiltrate samples stored at − 80 °C till analysis.

The method for the analysis of DAN concentration was modified from that described by Schrickx and Garcia9,10. The HPLC system Waters 2695 was connected to a Waters 2475 fluorescence detector (λex = 280 nm and λem = 450 nm) with a mixture of acetonitrile and aqueous solution (15:85, v/v) as the mobile phase. The aqueous solutions were prepared by dissolving potassium dihydrogenophosphate (0.020 M), phosphoric acid (0.006 M), and tetraethylammonium bromide (0.012 M) in water. The pH of the mobile phase was adjusted to 3.0 by addition of 2 N NaOH. The flow rate was set at 1.0 mL/min; A Waters C18 reverse phase column C18 (250 mm × 4.6 mm I.D.; particle size, 5 μm) was used to perform HPLC at 30 °C; and the injection volume was 10 μL.

Samples were thawed at room temperature, and 10 μL of 50 μg/mL ciprofloxacin was added to plasma (490 μL) and ileum ultrafiltrate dilution (50 μL ultrafiltrate and 440 μL mobile phase) as the internal standard. After adding 3 mL of acetonitrile, the plasma samples were shaken at 220 oscillations/min for 15 min and centrifuged at 12,000 g for 10 min. The organic layer was transferred into a fresh tube and dried at 40 °C under nitrogen stream. The residue was dissolved in the mobile phase (0.5 mL), and 10 µL injected for HPLC analysis. The ultrafiltrate sample was diluted 10 times with mobile phase and analyzed directly by HPLC without extraction after filtered.

The limit of detection (LOD) was 0.005 µg/mL and the limit of quantification (LOQ) was 0.01 µg/mL, respectively. Standard curves were linear from 0.01 to 1.5 µg/mL in plasma (R2 = 0.9999) and ileum ultrafiltrate (R2 = 0.9998). The intra-day and inter-day variation for determination in plasma ranged from 0.39 to 1.95% and 0.18 to 4.74%. The intra-day and inter-day variation for determination in ileum ultrafiltrate ranged from 0.47 to 1.42% and 0.26 to 1.86%, respectively. The recovery of DAN in plasma ranged from 88.50 to 104.80%.

Pharmacokinetic analysis

PK parameters for DAN concentration–time data for plasma, ileum ultrafiltrate were calculated by using the WinNonlin software (version 5.2.1, Pharsight Corporation, USA). Minimum Akaike Information Criterion (AIC) was applied to discriminate the model with the best fit11. The data for plasma of all the six piglets were best fitted to a two-compartmental model. The data for ileum ultrafiltrate of six piglets were best described by a non-compartment model.

Pharmacodynamics analysis

Determination of MIC, MBC, and MPC for E. coli in vitro

The MIC was determined by broth microdilution testing in accordance with the guidelines in CLSI12. The MBC was performed according to the guidelines in CLSI13. The MPC was conducted based on the previous study14.

In vitro time-kill curves

For the in vitro time-kill curves, 9.9 mL Mueller Hinton (MH) broth with concentrations of DAN ranging from 1/2 to 32 times of the MICs (0.25–16 µg/mL) for E. coli HP501 (O158, MIC = 0.5 µg/mL) and 0.1 mL bacteria (0.5 Mc Farland standards, approximately 108 CFU/mL) were co-incubated at 37 °C, which were tested separately. The inoculum sizes of the organisms used to generate the time-kill curves were approximately 106 CFU/mL. An aliquot of 100 µL from each tube was placed in 0.9 mL of MH broth (1/10 dilution, 10–1). Then, 100 µL of MH broth were serially diluted in 0.9 mL to give dilutions of 10–2, 10–3, 10–4, 10–5, 10–6, 10–7, 10–8, 10–9 and 10–10. Five 20 µL drops of each dilution were dropped onto MH broth plates for determination of the CFU at 0, 1, 2, 4, 6, 8, 12 and 24 h. Finally, bacteria were counted after the plates were incubated at 37 °C for 24 h.

PK-PD integration for DAN in ileum ultrafiltrate

After i.m. dosing of DAN, the ex vivo AUC at 24 h (AUC24h)/MIC ratio and C/MIC were determined for ileum ultrafiltrate by using in vitro MIC data and in vivo PK parameters. The relationship between the AUC24h/MIC, Cmax/MIC and the log10 difference between the initial bacterial count (CFU/mL) and the bacterial count after 24 h of incubation was established for ultrafiltrate by using the inhibitory effect sigmoid Emax equation 15. This model is described by the following equation:

$$E={E}_{max}-\frac{\left({E}_{max}-{E}_{0}\right)\cdot {C}^{N}}{{EC}_{50}^{N}+{C}^{N}},$$

where E is the antibacterial effect determined as the change in the bacterial count (log10 CFU/mL) in the ultrafiltrate sample after 24 h of incubation compared to the initial log10 CFU/mL; E0 and Emax are the changes in log10 difference in bacterial count between 0 and 24 h in the control sample and in the DAN containing samples, respectively; EC50 is the AUC24h/MIC or C/MIC producing 50% of the maximal antibacterial effect; C is the AUC24h/MIC or C/MIC in the effect compartment; N is the Hill coefficient, which describes the steepness of the (AUC24h/MIC)-effect curve or C/MIC-effect curve. DAN has inhibitory effect on bacterial growth. The Emax and E0 were obtained from the ex vivo PD test. These PD parameters EC50, C, N were calculated by using the WinNonlin software (version 5.2.1, Pharsight Corporation, USA).

The values of PK/PD parameters AUC24h/MIC and Cmax/MIC required to achieving the three levels of antibacterial effect of DAN in ileum ultrafiltrate: (1) E = 0, bacteriostatic action (no change in bacterial count after 24 h of incubation); (2) E = -1, bactericidal action (a 99.9% decrease in the bacterial count); (3) E = − 3, bacterial elimination (the lowest AUC24/MIC that produced a reduction in the count to 10 CFU/mL).

Calculation of the administered dosage

The dose was calculated by the using the following formula15:

$$\mathrm{Dose}=\frac{\left(AUC/MIC\right)\times CL}{fu\times F}\times MIC,$$

where AUC/MIC is the targeted endpoint for optimal efficacy; CL is clearance per day; MIC is minimum inhibitory concentration ; fu is free fraction of drug in ileum ultrafiltrate (from 0 to 1); F is the bioavailability factor (from 0 to 1). In this study, fu is 1 because the ileum ultrafiltrate collected by the ultrafiltration probe contained only the free state of DAN.

The dose was calculated respectively by the formula above, based on the PK/PD parameters AUC24h/MIC and Cmax/MIC required to achieve the bacteriostatic action, bactericidal action and bacterial elimination.

Statistical analyses

Statistical analysis was undertaken by analysis of variance using GraphPad Software Prism 6 (version 6.01). Differences between the means of the plasma group and ileum ultrafiltrate group were assessed using two-tailed paired Student’s t test. All data were presented as the mean ± SD, where P < 0.05 was considered a statistically significant difference.

Results

Pathogenicity test

The results of E. coli virulence test for mice were presented in Table 1. Mice in each group showed different degrees of death when different concentrations of E. coli were inoculated into the peritoneal cavity of mice. The strain HP501 among them had the strongest lethal ability to mice. Therefore, an EPEC E. coli strain HP501 (O158) was selected for subsequent PD experiments.

Table 1 The results of E. coli virulence test for mice.
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Pharmacokinetics analysis

Semi-logarithmic plot of concentration–time profile of plasma at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h (A) and ileum ultrafiltrate at 0, 1, 2, 4, 6, 8, 12, 24, 26, 30 and 48 h (B) after DAN i.m. administration at a dose regimen of 2.5 mg/kg in piglets are presented in Fig. 2. In Table 2, the DAN concentration in plasma reached its maximum at 1.10 ± 0.03 h, but it was 6.00 ± 0.00 h in the ileum ultrafiltrate. The mean Cmax of the ileum is 13.59 times that of plasma. The elimination half-life (T1/2) in the ileum ultrafiltrate (6.84 ± 1.49 h) was shorter than those in plasma (7.58 ± 3.20 h).

Figure 2
figure 2

Semi-logarithmic plot of concentration–time profile of plasma at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h (A) and ileum ultrafiltrate at 0, 1, 2, 4, 6, 8, 12, 24, 26, 30 and 48 h (B) after DAN i.m. administration at a dose regimen of 2.5 mg/kg in piglets. Values are means ± SD (n = 6).

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Table 2 PK parameters for DAN in plasma and ultrafiltrate after i.m. administration at a dose of 2.5 mg/kg (n = 6).
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Pharmacodynamics analysis

MIC, MBC, and MPC against E. coli in vitro

The MIC of DAN in MH broth against E. coli (O158) was 0.5 µg/mL and its corresponding MBC values was 0.5 µg/mL. The MPC of DAN in MH broth was 4 µg/mL (Table 3).

Table 3 MIC, MBC, and MPC (µg/mL) of DAN against E. coli HP501 (O158).
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In vitro time-kill curves

The in vitro time-kill curves were showed in Fig. 3. The bactericidal activity increased with increasing concentration of DAN in MH broth medium. The bacteria were all killed after 8 h of co-cultured with MH broth which containing DAN concentration was 16 times or 32 times the MIC. This result indicates that the killing mechanism of DAN against E. coli is concentration-dependent.

Figure 3
figure 3

The in vitro time killing curve of DAN against E. coli HP501 (O158, MIC = 0.5 μg/mL).

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PK-PD integration for DAN in ileum ultrafiltrate

The PK-PD integration parameters for the in vivo PK data and the MICs determined in vitro for ileum ultrafiltrate are presented in Table 4. The mean AUC/MIC and AUC/MPC were 170.64 ± 17.14 h and 21.33 ± 2.14 h for E. coli HP501 (O158) in ileum ultrafiltrate after i.m. administration of DAN at 2.5 mg/kg. The time for which the concentration in ultrafiltrate remained above the MIC and MBC were 29.76 ± 0.68 h and 19.32 ± 0.19 h, respectively. The mean Cmax/MIC ratio and Cmax/MPC were 13.32 ± 1.84 and 1.67 ± 0.23, respectively.

Table 4 PK/PD integration parameters for DAN in ileum ultrafiltrate after i.m. administration at a dose of 2.5 mg/kg. (n = 6).
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AUC24h/MIC and C/MIC values were generated according to the PK parameters in vivo in ultrafiltrate and the MIC in MH broth. The relationship between ex vivo antimicrobial efficacy and PK/PD surrogate markers (AUC24h/MIC or Cmax/MIC) is presented in Fig. 4 by the inhibitory effect sigmoid Emax equation. Both the AUC24h/MIC and C/MIC had a good correlation with antimicrobial efficacy (R2 = 0.99). The estimated Emax (Log10 CFU/mL), E0 (Log10 CFU/mL), EC50 (h), and N (slope) are showed in Table 5. According to the inhibitory effect sigmoid Emax equation:\(E=2.32-\frac{6.67\cdot {C}^{4.51}}{{114.78}^{4.51}+{C}^{4.51}}\), the calculated mean AUC24h/MIC, is the C in the inhibitory effect sigmoid Emax equation, for ileum ultrafiltrate that produced bacteriostasis, bactericidal activity, and elimination of bacteria were 99.85, 155.57, and 218.02 h, respectively (Table 5); the calculated mean Cmax/MIC for ileum ultrafiltrate that produced bacteriostasis, bactericidal activity, and elimination of bacteria were 4.14, 6.49, and 9.05, respectively (Table 5).

Figure 4
figure 4

A, B: Plots of antibacterial effect [E, log10 (CFU/mL)] versus respectively AUC24h/MIC and Cmax/MIC of DAN in ileum ultrafiltrate in the ex vivo PK/PD model against E. coli with an inoculum size of 1.49 × 106 CFU/mL, based on inhibitory effect sigmoid Emax model. Where, R2 is the correlation coefficient; MIC: minimum inhibitory concentration; AUC24h: area under the concentration–time curve from 0 to 24 h after DAN administration; Cmax: the maximum concentration during the dosage interval. The circles represent the values observed for individual animals, and the curve is the line of best fit.

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Table 5 The values of PK/PD parameters AUC24h/MIC and Cmax/MIC required to achieving various levels of antibacterial effect of DAN.
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Calculation of the administered dosage

Three different dosages (1.49 mg/kg, 2.42 mg/kg, and 3.24 mg/kg) were calculated respectively based on AUC24h/MIC ratio for bacteriostatic, bactericidal and bacterial eradication in Table 6.

Table 6 Different dosages were calculated respectively based on AUC24h/MIC ratio for bacteriostatic, bactericidal and bacterial eradication.
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Discussion

DAN is a fluoroquinolones antibacterial that has wide spectrum of antimicrobial activity against most gram-negative bacteria and some gram-positive bacteria3. Improper use of antibiotics that inappropriate dosing, dosing interval, dosing time, dosing route, dosing conditions lead to poor treatment effect and serious bacterial resistance16. To date, PK-PD modeling has been widely used to improve dosing regimens of approved drugs and individualized treatment17. Therefore, it was investigated that the PK, PD, and PK-PD integration of DAN in piglet model against E. coli in this study.

PK data of DAN in plasma has been described in several species, including cattle, cows, ewes, sheep, goat, camel, brown trout, rabbits, chicken, turkeys, ducks, donkeys, horses and pigs3. After i.m. administration of DAN at a dose of 2.5 mg/kg body weight in piglets, the PK parameters including that the time to reach to maximum concentration (Tmax) in plasma, the peak drug concentration (Cmax), the area under the curve (AUC0-t), and the elimination half-life (T1/2β) do not differ to a large extent from previous studies18,19,20.

Previous publications were mainly focused on PK data of DAN in plasma but ignored the concentrations of antimicrobial agents in their target sites. Therefore, the PK data in both plasma and ileum were analyzed in our study. Luminal gastrointestinal tract (GIT) fluid could be collected by a wide variety of approaches. For instance, euthanasia of multiple animals to collect GIT contents at multiple time points, requiring a large number of trial animals and yielding a relatively low number of animals per time point21. Several cannulas and tubes have been used to collect GIT fluid from ileum, rumen and duodenal cannulas in animals15,22. These cannulas must be anchored to the abdominal wall which results in disrupting normal intestinal motility, carrying inherent risks of leakage, dislodgement, and peritonitis and significant welfare concerns. In addition, antimicrobial drugs such as fluoroquinolones can be extensively bound to the intestinal contents, but intestinal concentrations should take into account only the unbound and active drug for subsequent PD studies. Ultrafiltration probe technology can solve this problem well in consideration of animal welfare. Ultrafiltration probes have been used to collect interstitial fluid (ISF) in a number of species8,23,24. In this study, ultrafiltration sampling probes were successfully placed in the ileum of piglets with the aid of anesthetic, and ileum ultrafiltrate contained only free drugs was collected at different time points. These free drug concentrations could finally give a value of an exposure parameter for the digestive tract (AUCileum) that, when combined with the relevant MIC in ileum ultrafiltrate, would predict the efficacy of the drug dosing regimen on the intestinal bacterial population.

A study described the PK variables of DAN in gastrointestinal content from ileum, jejunum, caecum and colon in healthy and in salmonella typhimurium infected pigs25. Lindecrona et al. administered DAN intravenously and found maximum concentration (4.46 µg/mL) at 2 h. While, in our study, piglets were administered intramuscularly and Cmax value (6.66 ± 0.92 µg/mL) was observed at 6 h. The difference in time to reach the Cmax value of DAN may be due the different administration routes. The AUCileum/AUCplasma ratio and MRT were 19.89 and 14.16 ± 1.52 h, higher than 10.97 and 9.86 h as reported by Lindecrona et al.. DAN concentrations in the gastrointestinal tract is much higher than the corresponding plasma concentrations in both the study in ours and Lindecrona et al.’ s, indicating that intestinal drug concentration could better predict the intestinal exposure of DAN than the plasma concentration.

There are no previous studies reported that the AUC/MPC for DAN against E. coli. In this study, the AUC/MPC ratio is 21.33 ± 2.14 (Table 4), which similar to the previous study (18.8 h) performed enrofloxacin against E. coli15. Previous study illustrated that ciprofloxacin treatment at AUC/MPC ratios of 22 could prevent the resistant mutants of E. coli with an inoculum sizes of 1010 CFU/mL26. The Cmax/MPC ratio (1.67 ± 0.23) obtained in our study was close to that reported in previous study (1.4) for enrofloxacin against E. coli.

Rational use of antibiotics requires extensive knowledge of the infectious bacteria and the PK and PD of the drugs used27. The efficacy of antimicrobial agents against bacterial pathogens is classically predicted from PK/PD studies. In general, to be efficacious against the pathogens responsible for an infection, drug dosing regimens should ensure that the optimal value of the PK/PD index correlated with the antibacterial activity of the drug is achieved28. For fluoroquinolones, a Cmax/MIC ≥ 10 or an AUC24h/MIC ≥ 125 is the PK/PD index that reach antimicrobial efficacy3,29,30. This finding was also true in this study (Table 5), both in vitro kill curves indicated that the higher the concentration of DAN, the better the bactericidal effect. Moreover, both the AUC24h/MIC and Cmax/MIC had a good correlation with antimicrobial efficacy (R2 = 0.99) by the inhibitory effect sigmoid Emax equation. The calculated mean AUC24h/MIC for ileum ultrafiltrate that achieved bacteriostatic, bactericidal, and eradication were 99.85, 155.57, and 218.02 h. These results are consistent with other studies which AUC24h/MIC ratios for the quinolones should be at least 125 for optimal bactericidal efficacy against gram-negative pathogens31.

Previous studies have found that the increase in antibiotic resistance in the intestinal flora was mainly due to the abuse of antimicrobials in unsuitable routes of administration32,33. Therefore, three different doses (1.49 mg/kg, 2.42 mg/kg and 3.24 mg/kg) were calculated respectively based on the PK/PD index (AUC24h/MIC ratio) for bacteriostatic, bactericidal and bacterial eradication in the present study. However, it is necessary to design experiments with either naturally diseased animals or disease models to establish the lowest AUC/MICs that lead to the bacterial elimination in vivo3. Such experiments will further provide a more rational basis for the selection of optimal dosage schedules for DAN.

Conclusion

In this study, ultrafiltration probes were successfully placed in the ileum of piglets to collect ileum ultrafiltrate. The mean PK/PD index (AUC24h/MIC) for ileum ultrafiltrate that achieved bacteriostatic, bactericidal, and eradication were 99.85, 155.57, and 218.02 h, respectively. Three different dosages (1.49 mg/kg, 2.42 mg/kg, and 3.24 mg/kg) were calculated respectively based on AUC24h/MIC ratio above, which might provide a novel approach to the rational design of dosage schedules.