| In order to explore the relationship between different antibiotic dosing regimens and selective enrichment of resistant strains, tissue-cage infection model was established in rabbits to study cefquinome pharmacokinetics/pharmacodynamics parameters associated with the change of Staphylococcus aureus(S. aureus) susceptibility, and it is hoped that this model would provide a clear evidence that the resistant isolates would be selected predominantly when drug concentrations maintained inside a concentration window, so as to provide a reasonable dosing regimen for preventing the emergence of resistant bacteria.After a Wiffle ball was surgical implanted into each rabbit, above 1010 CFU of S.aureus ATCC29213 culture were injected into each Wiffle ball, and then tissue-cage fluid was withdrawn from each Wiffle ball for a viable-bacteria count. For having above 108CFU/m L viable bacterial cells in tissue-cage fluid, tissue-cage infection model was established successfully. According to the results of preliminary experiments, rabbits were administered cefquinome at doses of 4, 8, 16, 32 mg/kg of body weight once a day for 5days or 4, 8, 16, 24 mg/kg of body weight twice a day for 2.5 days, 0.5 m L sample of tissue-cage fluid was collected from the Wiffle ball at 2, 4, 6, 8, 10, 12, 24 h after each dosing with 24 h intervals. For groups with 12 h intervals, samples were collected at 2, 4, 6,8, 10, 12 h after each dosing. Multiple samples(1 m L) from each rabbit were collected from the Wiffle ball daily before, during the treatment(after every administration), and 24 h and48h once daily or 12 h and 24 h twice daily after the termination of treatment, wherein0.4m L tissue-cage fluid was used for MIC determination, 0.6m L tissue-cage fluid was used for determining the change of bacteria numbers and the mutant fraction.The cefquinome MIC, MIC99 and MPC by an agar dilution assay for S. aureus were0.5 μg/ m L, 0.3 μg/m L and 1.6 μg/m L, respectively. The cefquinome MIC in the blank tissue-cage fluid by a micro dilution assay for S. aureus was 0.5 μg/m L. Cefquinomeconcentrations in the tissue-cage fluid were determined by using HPLC-MS/MS method.The PK/PD parameters were calculated using Win Nonlin 5.2 programme. The mean values of AUC0-12 h and AUC0-24 h of cefquinome in the tissue-cage fluid following multiple dosing ranged from 5.590 to 69.782 μg·h/m L and from 6.874 to 55.558 μg·h/m L, respectively. The maximum concentrations ranged from 0.308 to 2.477 μg/m L and from 0.513 to 3.030μg/m L, respectively. Compared to the control groups, at dose of cefquinome above 4 mg/kg,growth inhibition of S. aureus was observed, indicating cefquinome in the therapeutic range had a profound effect on local colonization with S. aureus. But, loss of bacterial susceptibility occurred when %T> MIC99 ≥ 70% or %T>MPC< 58%. In addition, other PK/PD parameters also shown statistically significant correlations with the selection of resistance. When AUC/MIC99 fell between 61.33 h and 185.19 h, AUC/MPC fell between11.50 h and 34.72 h, Cmax/MIC99 fell between 2.27 and 10.1, and Cmax/MPC fell between0.43 and 1.89, the resistant strains were all observed.Eight resistant strains which were chosen randomly from the 1× MIC of cefquinome-containing MH agar and one original susceptible strain(bacteria were injected into Wiffle ball in the beginning) were characterized by PFGE method. The PFGE patterns were analyzed with Bio Numerics software using the Dice similarity coefficient with a cut-off at 90% of the similarity values to indicate identical PFGE types. The result showed that the original susceptible strain(S. aureus strain ATCC 29213) and the resistant strains were identical PFGE types. Clearly, the resistant strains originated from the initial population.These findings demonstrate that the MSW exists in vivo for cefquinome against S.aureus. Low-level cefquinome-resistant S. aureus were selected, which was evidenced by PFGE. The selection of resistant bacteria for cefquinome arose from both susceptible bacteria being killed and resistant bacteria re-growth. Keeping drug concentrations above the MPC for ≥ 58% of administration interval provides a strategy to keep effective antibacterial activity and minimize the emergence of resistance to cefquinome. |
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