Escherichia coli infections cause early mortality of commercial broiler chickens. Although enrofloxacin has long been used in poultry, the in vivo pharmacokinetic/pharmacodynamic (PK/PD) relationship of enrofloxacin against E. coli is unclear. The present study aimed to establish an in vivo PK/PD model of enrofloxacin against E. coli in seven-day-old chicks and to ascertain whether the selection of target organ for PD determination is critical for parameter magnitude calculation in enrofloxacin PK/PD modeling.Systemic E. coli in different organs varied, with the Emax ranging from − 4.4 to − 5.8 Log10 colony forming units (cfu)/mL or cfu/g. Both the surrogate AUC0–24/MIC of enrofloxacin or AUC0–24/MIC of the combination of enrofloxacin and ciprofloxacin correlated well with effectiveness in each organ. The AUC0–24/MIC ratio of the combination of enrofloxacin and ciprofloxacin producing bactericidal and elimination effects were 21.29 and 32.13 in blood; 41.68, and 58.52 in the liver; and 27.65 and 46.22 in the lung, respectively.The in vivo effectiveness of enrofloxacin against E. coli in different organs was not identical after administration of the same dosage. To describe the magnitude of PK/PD parameter exactly, bacterial loading reduction in different organs as PD endpoints should be evaluated and compared in PK/PD modeling. The selection of a target organ to evaluate PDs is critical for rational dosage recommendation.The in vivo effectiveness of enrofloxacin against  Escherichia coli [E. coli infections contribute significantly to the early mortality of commercial broiler chickens [E. coli challenge are rare [E. coli infections in the poultry industry.It is estimated that 50% of total poultry loses could be attributed to first week mortalities . Among thia coli . Systemichickens . Howeverare rare . Until nEnrofloxacin, a second-generation fluoroquinolone, is commonly used in chickens because of its favorable pharmacokinetic (PK) profile and its excellent activity against gram-negative aerobic bacteria and some gram-positive bacteria , 6. HoweE. coli, Pasteurella multocida, and Salmonella typhimurium [E. coli in chicks. In vivo PK/PD modeling has great advantages over ex vivo modeling in describing the PK/PD relationship [Optimizing the use of an antimicrobial should be based on a good understanding of its PK and pharmacodynamic (PD) relationship in target animals against specific bacterial species . Althoughimurium –20, to ttionship , especiationship . WhetherE. coli was developed using the reductions in the bacterial burden in the blood, liver, and lung as the PD endpoints; (3) whether the concentration of ciprofloxacin influence the in vivo PK/PD modeling of enrofloxacin was evaluated; and (4) the corresponding magnitude of PK/PD parameters for a certain efficacy were determined.In the present study, to further understand the PK/PD relationship of enrofloxacin, especially whether the selection of a target organ for PD determination is critical for parameter magnitude calculation in PK/PD modeling, broilers were used as an animal model. The following aspects were investigated: (1) The pharmacokinetics of enrofloxacin and its metabolism to ciprofloxacin were determined at three different dosage administrations; (2) the in vivo PK/PD modeling of enrofloxacin against E. coli O78 were the same (0.5 μg/mL). The corresponding MBC values were 0.5 and 1 μg/mL respectively. The MIC and MBC of enrofloxacin in serum were identical (0.5 μg/mL). The MPC of enrofloxacin was 3.2 μg/mL  for enrofloxacin and ciprofloxacin were about 3.3~ 3.4 h and 4.3~ 5 h, respectively, with peak concentrations (Cmax) of 0.16, 1.76, and 2.86 μg/mL for enrofloxacin at 1, 10, and 20 mg/kg, respectively; and of 0.03, 0.10, and 0.37 μg/mL at corresponding doses for ciprofloxacin. The Cmax of ciprofloxacin was much lower and emerged later than enrofloxacin. The AUC0–24 of enrofloxacin at 1, 10, and 20 mg/kg b.w. was 1.64, 17.95, and 30.07 h, respectively, and the corresponding values for ciprofloxacin were 0.61, 1.42, and 4.93 h, respectively. The AUC0–24 values of enrofloxacin were 2.6, 12.6, and 6.09 times higher than those of ciprofloxacin at doses of 1, 10, and 20 mg/kg b.w. respectively. Dose proportionality was observed for the AUC0–24 of enrofloxacin and ciprofloxacin in the range of 1–20 mg/kg with r2 of 0.9868 and 0.9035, respectively. Thus, the AUC0–24 of other doses between 1 and 20 mg/kg could be calculated.The serum drug concentration-time profiles of enrofloxacin and ciprofloxacin after enrofloxacin administration at three dosages are illustrated in Fig. 0–24/MIC ratios of enrofloxacin against E. coli O78 for doses of 1, 2, 5, 7.5, 10, 12.5, 15, and 20 mg/kg were 5.27, 8.26, 17.20, 24.66, 32.11, 39.57, 47.02, and 61.93 h respectively. The corresponding AUC0–24/MIC values of ciprofloxacin were 0.36, 0.82, 2.2, 3.35, 4.50, 5.65, 6.80, and 13.24 h, respectively.The AUC0–24/MIC correlated well with effectiveness in each organ, with r2 values greater than 0.85. The in vivo effectiveness of enrofloxacin against E. coli in different organs varied, with Emax ranging from − 4.4 to − 5.8 Log10 cfu/mL. Using the PK of enrofloxacin for simulation, then the AUC0–24/MIC values of enrofloxacin for the bactericidal effect in the blood, liver, and lung were 19.32, 32.15, and 23.41, respectively. The slopes were 2.58, 2.36, and 3.01 for the blood, liver, and lung, respectively  of enrofloxacin in different organs and PK/PD indices of enrofloxacin, or the combination of enrofloxacin and ciprofloxacin, are shown in Fig. E. coli when simulating colibacillosis, such as intramuscular (IM) and oral administration [7 cfu/mL E. coli in chickens. However, the infection dose was lower than that used in a previous report [E. coli culture containing 1.2 × 109 cfu/mL. The differences in infection dose might be explained in two ways: The broilers used by Sang were 39 days old, whereas ours were 7 days old, and the inoculation method was different, oral gavage in Sang’s report vs. IM injection in this study.There are several infection routes for stration , 23. Thes report , where c1/2β) values in this study (9.78–11.4 h) were similar to those in previous reports [0–24 value (17.95 h) in this study was much lower than that reported in previously  [0–24 was observed for enrofloxacin and ciprofloxacin. This phenomenon was similar to that reported in previous studies [The PKs of enrofloxacin have been investigated in goats, pigs, calves, horses, and sheep –28. It h reports . After oekala’s) , 32. Theekala’s) . With anekala’s) . However studies , 33.E. coli in chicks. One of the best PK/PD parameter for fluoroquinolones is AUC0–24/MIC [0–24/MIC for enrofloxacin or the combination of enrofloxacin and ciprofloxacin correlated well with effectiveness in each organ. It seems that the metabolism of ciprofloxacin has little influence on the PK/PD modeling of enrofloxacin. However, whether the active metabolite plays a role in emerging resistance or has an impact on dosing optimization needs further investigation [0–24/MIC for the bactericidal effect were 19.3–32.15 in the different organs using only the concentration of enrofloxacin for simulation, and 21.29–41.68 when using the combined concentrations of enrofloxacin and ciprofloxacin for simulation. The values were much lower than those of enrofloxacin against E. coli or Salmonella typhimurium in the intestinal content of infected chicken calculated from ex vivo PK/PD modeling  [0–24/MIC calculation; therefore, the value of AUC0–24/MIC in the intestines may be higher than that in serum to achieve the same effect. Second, as reported previously, the discrepancy between ex vivo PK/PD modeling and in vivo PK/PD modeling was obvious [To the best of our knowledge, there has been no in vivo PK/PD modeling study of enrofloxacin against 0–24/MIC –36, and tigation , becausetigation –40. The ctively) , 16. Sev0–24/MIC, to attain the same effect was different. The AUC0–24/MIC value for the bactericidal effect in the liver was higher than that in lung, and twice than that in blood. This phenomenon was also observed in our previous study. The AUC0–24/MIC values of danofloxacin against Salmonella typhimurium for the same effect in different organs also showed marked differences [Using the bacterial burden reduction in each organ as PD endpoints, the value of the PK/PD parameter, AUCferences . Similarferences , 41. Theferences , 42–47. The results obtained using this model need to be validated by clinical trials in relevant animal species. However, it is still a critical step to increase our understanding of PK/PD relationships for antimicrobials. To simulate the clinical use of enrofloxacin, enrofloxacin was administrated via gavage in this study; however, the most recent basic principles of the prudent use of antimicrobials do not support the group oral administration of fluoroquinolones in animals.E. coli in seven-day-old chicks was established using bacteria loading reduction in several organs as PD endpoints. The in vivo effectiveness of enrofloxacin against E. coli in different organs varied with Emax ranging from − 4.4 to − 5.8 Log10 cfu/mL. Both the surrogate AUC0–24/MIC of enrofloxacin or the combination AUC0–24/MIC of enrofloxacin and ciprofloxacin correlated well with effectiveness in each organ. The combined AUC0–24/MIC ratios producing bactericidal and elimination effects were 21.29 and 32.13 in blood; 41.68 and 58.52 in liver; and 27.65 and 46.22 in lung, respectively. The magnitude of PK/PD index was lowest for the same effect in blood, but highest in the liver, indicating that at the same dosage, the in vivo effectiveness of enrofloxacin against E. coli in different organs was not identical. This study emphasized the importance of target organ selection for PD evaluation in PK/PD modeling.In conclusion, an in vivo PK/PD model of enrofloxacin against E. coli O78 strain, which was used in our previous study, was isolated from a broiler showing colibacillosis [E. coli strain ATCC 25922 was purchased from the Chinese Veterinary Culture Collection. The enrofloxacin reference standard and ciprofloxacin (98% purity) were purchased from Solarbio Life Sciences Co. Ltd. . Acetonitrile (ACN) and methanol (MeOH) were purchased from TEDIA . All reagents used in this experiment were of high performance liquid chromatography (HPLC) grade. The culture medium used in this experiment was purchased from Hope Biol-Technology Co. Ltd. . One-day-old healthy broilers (n = 324), obtained from the Jiangsu Institute of Poultry Sciences , were used in this study. These broilers were mixed sex (50:50), weighting from 35.2 to 45.6 g. A six-day acclimation period was set for animals before the study. The animals were maintained according to the National Standards for Laboratory Animals of China (GB 14925–2010). This study was approved by the Animal Experiments Ethics Committee at Yangzhou University (SYXK (Su) IACUC 2017–0045).The clinical cillosis , 48. TheE. coli O78 and ATCC 25922 were determined using the micro-dilution method, according to the Clinical and Laboratory Standards Institute (CLSI) reference method [10 cfu of E. coli and then incubated at 37 °C for 72 h. The MPC was determined as the lowest drug concentration that prevented bacterial growth.The MICs of enrofloxacin and ciprofloxacin against e method . The MICe method . To detee method . BrieflyE. coli culture containing ~ 107 cfu/mL through intramuscular injection (IM) in the chest muscle. The clinical symptoms and pathological changes were observed. Then, animals were sacrificed by a lethal intravenous injection of beuthanasia (0.3 mL/kg) after anesthesia with ketamine-Xylazine. The bacterial load in blood, liver, and lung were determined by the agar plate dilution method at 24 h post inoculation.Preliminary experiments were conducted to confirm the inoculation amount. After a 6-day acclimation period, broilers were inoculated with 0.5 mL of g to obtain serum. The serum was stored at − 20 °C until analysis. The concentrations of the drug in the serum were determined using HPLC with a fluorescence detector, as described previously, with some modifications [g for 10 min. The supernatant was transferred to a clean tube, dried under nitrogen, and re-dissolved with 0.1 mL 17% ACN. The sample was filtered through a 0.22-μm membrane before injecting into the HPLC apparatus. The recovery rate was between 80.2 and 91.3%, and the intra and inter coefficient of variation was less than 7%. The serum concentration–time data of enrofloxacin and ciprofloxacin for each animal was used to calculate the PK parameters in broilers using the WinNonlin software .Enrofloxacin was administered orally to 280 infected broilers at doses of 1, 10, and 20 mg/kg body weight (b.w.). Ten blood samples were collected at each time point . After sampling, animals were narcotized with ketamine-Xylazine and sacrificed by a lethal intravenous injection of beuthanasia (0.3 mL/kg). After incubation at room temperature, samples were centrifuged for 10 min at 3000×ications , 52. Brin = 54) were randomly divided into nine groups (n = 6 in each group) and treated with enrofloxacin for 3 successive days at doses ranging from 0 to 20 mg/kg b.w. . At 24 h after the last dose, the broilers were humanely killed through a lethal intravenous injection of beuthanasia (0.3 mL/kg) after anesthesia with ketamine-Xylazine to collect blood, liver, and lung samples. The bacterial loading in each organ was determined via plating dilutions onto MH agar and counting the colonies after incubation at 37 °C for 24 h. The effectiveness of enrofloxacin was expressed as the bacterial reduction after treatment compared with that before treatment in each organ.Infected broilers  was used to simulate the relationship between AUC0–24/MIC of enrofloxacin, or the combination AUC0–24/MIC of enrofloxacin and ciprofloxacin and in vivo effectiveness. The equation for this model was as follows:The best PK/PD parameter for fluoroquinolones is AUC/MIC or C0 is the change in log10 cfu/mL or log10 cfu/g in the control sample (absence of drug). Emax is the difference in effect between the greatest amount of growth  and the greatest amount of killing. Ce is the tested AUC0–24/MIC ratio; EC50 is the AUC0–24/MIC value that reached 50% of the Emax; and N is the Hill coefficient that describes the steepness of the AUC0–24/MIC-effect curve [In the above formula, Ect curve . The in P < 0.05 was considered statistically significant.The PK data, PK/PD data, and PK/PD curve fitting were analyzed using the WinNonlin software . T-tests were conducted for other data using SPSS software .