In Vitro And In Vivo Studies Of Mutant Selection Window For Staphylococcus Aureus

[Objective 1 To establish a method to measure mutant prevention concentration(MPC) in vitro, and to measure the MPCs and mutamt selection window of fluoroquinolones for Staphylococcus aureus strain ATCC25923, isogeneous mutants of strain ATCC25923 and clinical isolates. Combined with pharmacokinetic parameters, mutant selection window(MSW) and the capacity of fluoroquinolones for restricting the selection of next-step staphylococcus aureus resistant mutants were evaluated. In the meantime, the effects of drug concentration on the recovery of resistant colonies and allelic diversity were observed. On the base of in vitro studies, the MSW hypothesis in vivo was studied by using levofloxacin- Staphylococcus aureus strain ATCC25923 as an example , and the pharmacokinetic/pharmadynamic (PK/PD) parameters that can predict the selection of mutants in vivo were studied. Finally, the MSW hypothesis was evaluated at clinical patients by study on the conversion from rifampicin-susceptible to rifampicin-resistant phenotype of Staphylococcus aureus colonized in the noses of tuberculosis patients before and after standard anti-tuberculosis therapy.[Methods] The celles of 10¹⁰ colony form units per milliliter staphylococcus aureus were enriched in broth, and the minimal inhibitory concentration(MIC), MIC for 99% of input cells (MIC₉₉), provisional MPC(MPCpr) and MPC of fluoroquinolones against different strains were determined by agar plates dilution method. The QRDR of grlA and gyrA of mutants were amplified by PCR and sequenced, and the correlation between drug concentration and the selection of resistant allelic was studied. The rabbit tissue cage medel of Staphylococcus aureus strain ATCC25923 infection was established, and different doses levofloxacin were given through a no. 14 French urethral catheter passed orally into the stamach of each rabbit. The levofloxacin concentrations in tissue cage fluid were placed below MIC₉₉,between the MIC₉₉ and MPC and above MPC in the most treatment period, and the the concentration partly overlaped the MSW. Five doses of levofloxacin were given once daily for five days to create a rang of different drug exposures. Untreated rabbits were given saline followingthe same dosing schedule as controls. The emergence of resistance under the dosing regiment studied was assessed by measuring the MICs and the resistance mutant frequence. The concentrations of levofloxacin in the tissue cage fluid were analysized by high-performance liquid chromatographic method. The correlation between drug concentration and drug resistance was analysized. The MSW hypothesis was also studied at clinical patients. The tuberculosis patients whose noses colonized rifampicin-susceptible Staphylococcus aureus were selected, and the rifampicin-susceptibility of the Staphylococcus aureus colonized in the noses of tuberculosis patients was tested during standard anti-tuberculosis therapy including rifampicin. The strains of Staphylococcus aureus converted from rifampicin-susceptible to rifampicin-resistant phenotype were seperated ,and the rifampicin-susceptible isolates obtained before treatment were compared by PFGE and spa typing with rifampicin-resistant isolates after treatment. They were considered clonally identical if no difference in the band pattern and spa repeat sequence observed. [Results] Part 1 (1) The MPCs of moxifioxacin,gatifloxacin, pasufloxacin and ciprofloxacin for staphylococcus aureus strain ATCC25923 were 0.18μg/ml, 0.3μg/ml, 0.75μg/ml and 1.8μg/ml, and the MPC/MIC99 were 9.0, 7.5, 8.0 and 10.6 respectively. (2) Ciprofloxacin and gatifloxacin concentration have strong effects on the recovery of resistant colonies and selection of resistant alleles. (3) The MPCs of moxifloxacin and gatifloxacin for the first-step isogenous mutants were lower significantly than those of ciprofloxacin ,levofloxacin and pasufloxacin. The MSWs of 5 fluoroquinolones for the first-stepmutants were wider than those for strain ATCC25923, and the MSW of pasufloxacin was the widest. The second-step mutants were still susceptible to moxifloxacin and gatifloxacin, but the MPCs ranged from 8 to 16ug/ml. (4) The MPCs for 90% of the isolates(MPC_pr90) of moxifloxacin, gatifloxacin, pasufloxacin and ciprofloxacin for 42 ciprofloxacin-susceptible staphylococcus aureus clinical isolates were 0.5μg/ml, 0.5μg/ml, 4μg/ml and 8μg/ml ,and the MPC_pr90/MIC₉₀ were 4, 4, 16 and 16 respectively. The MPCs for 90% of the isolates(MPCpr₉₀) of moxifloxacin, gatifloxacin for 12 ciprofloxacin-resistant staphylococcus aureus clinical isolates were 16 and 16μg/ml, the MPCpr₉₀/MIC₉₀ were 8 and 8 respectively. Part 2 (1) Resistant mutants of strain ATCC 25923 were selectively enriched whenlevofloxacin concentration in tissue cage fluid was inside MSW, and no enrichment of mutants occurred when levofloxacin concentration was placed outside MSW. (2) When the levofloxacin concentration was in the lower part of MSW, the resistant mutants were selectively enriched most easily. (3) When the time of concentration above MPC(T> MPC) was longer than 16.7%, levofloxacin can prevent the mutants selection of strain ATCC25923 in vivo.When the maximal concentration of levofloxacin in tissue cage fluid (Cmax) was above MIC99 but lower than MPC, MIC increased if the time of concentration within MIC99 and MPC(Tmsw) was longer than 16.7%.When Cmax was lower than MIC₉₉, resistance cann't occur. (4) No changes in susceptibility were seen when Cmax/MIC value was larger than 8,or AUCoWMIC value was higher than 250,or AUCo-24/MPCwas higher than 40.Part 3 (1) The carriage rate of Staphylococcus aureus in the noses of tuberculosis patients was 16.9%. In the 58 enrolled patients, the clearance rate was 91.4% after 4 weeks rifampicin treatment. The isolates still colonized in 5 patients were rifampicin-resistant Staphylococcus aureus isolates. The rate of resistant occurrence was 6.8%. The results of spa typing and PFGE certified that the strains obtained before and after rifampicin treatment were clonally identical. (2) The carriage rate of Staphylococcus aureus in the noses of diabetes mellitus was 20.5%, and no resistant-strain was occured.Part 4: The Staphylococcus aureus isolates colonized in the noses were susceptible to most antimicrobial agent except penicillin and erythromycin.[Conclusion] (1) For Staphylococcus aureus strain ATCC 25923, isogeneous mutants of strain ATCC25923 and clinical isolates,the capacity of moxifloxacin and gatifloxacin for restricting the selection of next-step resistant mutants were stronger than that of pasufloxacin ,levofloxacin and ciprofloxacin. (2) Ciprofloxacin and gatifloxacin concentrations have strong effect on the recovery of resistant colonies and selection of resistant alleles. (3) Combined with pharmacokinetic parameters, moxifloxacin and gatifloxacin cann't restrict the next-step selective enrichment of resistant mutants among ciprofloxacin- or levofloxacin-resistant staphylococcus aureus. (4) When concentrations in the tissue cage fluid of rabbits was kept above MPC, levofloxacin can restrict selective enrichment of mutants. When the concentrations of levofloxacin in the...