| Objective: To determine the minimal inhibitory concentration (MIC) of tigecycline and other antibiotics against KPC-producing Klebsiella pneumoniae, and to assess the clinical efficacy of tigecycline and minocycline in treatment of infections cuaused by this organism with a pharmacokinetics/pharmacodynamics (PK/PD) model using Monte-Carlo stimulation. To investigate the in vitro synergistic effects of tigecycline or minocycline combined with aminoglycosides and to determine the mutant prevention concentration(MPC) and mutant selection window (MSW) for evaluating the ability of tigecycline combined with other antibiotics on the prevention of resistance. To verify that combination therapy can restrict the emergence of resistance in vivo using a rat tissue cage infection model. To investigate the mechanism of tigecycline combined with aminoglycosides on the prevention of resistance in KPC-producing Klebsiella pneumonia by strand-specific transcriptome sequencing and other assays.Methods: (1) The MICs of tigecycline and other antibotics against 68 KPC-producing Klebsiella pnumonia strains were determined by agar dilution method. The optimal PK/PD parameter for evaluating tigecycline and minocycline was the ratio of steady-state total drug area under the curve to MIC (AUC/MIC). The AUC/MIC was calculated and Monte-Carlo simulation with presumed breakpoints of efficacy derived from clinical studies was performed. Then, the probability of target attainment (PTA) cumulative fraction of response (CFR) were calculated. (2) A checkerboard method that adhered to the recommendations of Clinical Laboratory Standards Institute (CLSI) was applied to assess the synergism effect of tigecycline and minocycline combined with amioglycosides, and the Fraction Inhibitory Concentration Index (FICI) was calculated according to the results.Time-kill assayes were further performed to verify the results. The MPCs of tigecycline.colistin and amikacin alone and in combination were measured through the two-fold agar dilution method, and then the MSWs were calculated and analysed. (3) Local tissue cage infection model with KPC-producing Klebsiella pneumonia was established in rats, and the infected animals were treated various doses of tigeycline, minocycline alone and in combination for 7 consecutive days. Then the the quantity of bacteria, the frequency of resistant mutants. and the changes of MIC were determined. and the bacterial growth curve of resistant mutants were determined. (4) Resistant mutant strains were screened by strand-specific transcriptome sequencing for analyzing the difference of expression under various antimicrobial pressures. The tetracycline and amino glycoside-resistant genes were screened by PCR and the expression of acrB and its regulatory gene ramA of tigecycline and minocycline resistance mutant strains were determined by RT-PCR. The changes of bacterial cell membrane potentials of aminoglycoside-resistant mutant strains were determined by flow cytometry assay.Results: (1) Among 68 KPC-producing Klebsiella pneumonia strains, all were resistant to meropenem, ceftazidime, cefotaxime and aztreonam; the sensitivity of colistin was 97%;great difference exsiting in tigecycline sensitivity according to different breakpoints, and the MIC50 and MIC90 were 1 mg/L and 2 mg/L. respectively. The sensitivity of minocycline was 50%. The sensitivity of amikacin was higher than that of gentamicin(51.5% VS 32.4%). The Monte-Carlo simulation showed that. regardless of the PK/PD breakpoints for AUC0-24h/MIC> 6.96, or/AUC0-24h /MIC> 0.9, the recommended dose or double dose of tigecycline could not approach the clinical goal (CFR > 90%). and so is minocycline. (2) For aminoglycoside sensitive strains, the FICI of > 50% strains was < 0.5 in tigecycline or minocycline combined with amikacin or gentamicin, and the FICI of the other strains ranged between 0.5 to 1; For aminoglycoside highly resistant strains, no synergies were observed between tigecycline or minocycline and aminoglycosides, with the FICI > 1. The time-kill curve assays further validated the results of the checkerboard method. The MPC value of tigecycline, colistin and amikacin for KPC-producing Klebsiella pneumoniae ranged from 4 to 32 mg/L, 64 to > 128 mg/L, and 32 to 128 mg/L,respectively. The mean MSW value of tigecycline combined with amikacin for four strains was 12.0, which was significantly lower than that of tigecycline combined with polymyxin(200.1) or polymyxin combined with amikacin (372.4). Stong correlation were found between MSWs of combination therapy and those of monotherapy. The resistance mutation frequency in 2MIC concentration of colistin combined with 2 MIC tigecycline or amikacin is only 10-100 times lower than the single drug, but drug mutation frequency in 1 MIC tigecycline combined with 1 MIC amikacin resistance could reduce by 1000-10000 times compared with that in the single drug. and 2MIC concentration of the two drugs combined could completely inhibit the growth of resistant mutant bacteria. (3) In the tissue cage infection model, significant difference of bacteria count was found between the control group and the antibiotic group, and the decrease of the bacterial count in the combined group was the highest. which was statistically different from that of the monotherapy groups. The frequency of resistant mutants between tigecycline or amikacin group and the control group was not statistically significant before administration. After 7 days’administration of tigecycline alone, the frequency of resistant mutants increased from 10-6 to 10-2 at 1MIC concentration tigeycline plates, and the frequency of resistant mutants at 2MIC concentration increased to about 10-3. In the combination group, only one rat had a resistance mutation rate of 2.12 × 10-3 at 1MIC concentration. and no colony growth in the other rats. (4) No let genes involving tetracycline resistances were detected in Kp46,minocycline and tigecycline resistant mutants. aac(3)-Ⅱ aac(6’)-Ⅰ and ant(3")-Ⅰ involving aminoglycoside resistance were detected in Kp46 and amikacin resistant mutants.Transcriptome sequencing revealed that the amikacin resistance may be associated with the up-expression of genes encoding elongation factor, polyamine and putrescinet, and the down-expression of genes involving ubiquinone oxidoreductase and K+-H+ reverse transport; and the gentamicin resistance may be associated with the up-expression of genes involving Cpx system and the down-expression of genes involving encoding cytochrome.The expression of 30S ribosomal protein S22 was also significantly decreased in the gentamicin-resistant strains, which may also be associated with gentamicin resistance.Compared with clinically sensitive strains, the membrane potential of aminoglycoside resistant mutants was significantly decreased. The overexpression of acrB and its regulator ramA was associated with tigecycline and minocycline resistance. The average relative expression levels of ramA and acrB in tigecycline-resistant mutants increased by 70.4 and 6.8 times, respectively, and the average relative expression levels of ramA and acrB in minocycline-resistant mutants increased by 11.6 and 3.6 times, respectively.Conclusions: (1) In this study, KPC-producing Klebsiella pneumoniae merely showed relatively higher sensitivity to colistin. tigecycline. minocycline and amikacin, and the MIC values of tigecycline and minocycline were significantly higher than those of strains without KPC- producing. Monte-Carlo simulation with clinical PK/PD parameters showed that the overall efficacy of tigecycline or ininocycline monotherapy for KPC-producing Klebsiella pneumoniae infections was not ideal. (2) Tigecycline or minocycline combined with aminoglycosides on both sensitive strains showed great synergistic activity. The MPCs of colistin, tigecycline and amikacin were very high. Tigecycline and amikacin combination could effectively restrict the emergence of resistance. (3) tigecycline and amikacin monotherapy could lead to enrichment of drug-resistant mutant strains in vivo.The efficacy of combination therapy was better than single drugs, and frequency of resistance was significantly reduced, suggesting that combined therapy can reduce bacterial resistance. (4) The mechanism of tigecycline combined with aminoglycosides to inhibit the occurrence of resistance may be related to the different requirements of cell membrane potential in tigecycline- and aminoglycoside- resistant mutants. |
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