The Studies Of Aminoglycoside Antibiotics To Mutant Selection Window For Pseudomonas Aeruginosa

[Objective] To establish a method to measure mutant prevention concentration (MPC) in vitro, and to measure the MPCs and mutant selection window of amino glycoside antibiotics, tobramycin, amikacin and gentamycin ,for Pseudomonas aeruginosa strain ATCC27853. Combined with pharmacokinetic parameters, mutant selection window (MSW) and the capacity of aminoglycoside antibiotics for restricting the selection of next-step Pseudomonas aeruginosa resistant mutants were evaluated. In the meantime, the effects of drug concentration on the recovery of resistant colonies and detection of amicoglycosides modification enzyme (AMES) gene were observed. On the base of in vitro studies, the MSW hypothesis in vivo was studied by using tobramycin- Pseudomonas aeruginosa strain ATCC27853 as an example ,and the pharmacokinetic/pharmadynamic (PK/PD) parameters that can predict the selection of mutants in vivo were studied. So as to provide scientific evidences for the new clinical medication based on MSW theory, to restrain the resistance of. Pseudomonas aeruginosa to aminoglycoside antibiotics.[ Methods ]The cells of 10¹⁰ colony form units per milliliter Pseudomonas aeruginosa were enriched in broth, and the minimal inhibitory concentration(MIC), MIC for 99% of input cells (MIC99), provisional MPC(MPCpr) and MPC of aminoglycoside antibiotics against Pseudomonas aeruginosa ATCC27853 strains were determined by agar plates dilution method. It is through amplifying and sequencing amicoglycosides modification enzyme (AMES) gene by PCR, to know the correlation between drug concentration and the detection of AMES gene. The rabbit tissue cage model of Pseudomonas aeruginosa strain ATCC27853 infection was established, and 1mg/kg 3mg/kg 20mg/kg 40mg/kg 60mg/kg of tobramycin were given through intramuscular injection. The tobramycin concentrations in tissue cage fluid were placed below MIC99, between the MIC99 and MPC and above MPC in the treatment period, and the concentration partly overlapped the MSW. Five doses of tobramycinwere given once daily for five days to create a gradient arrange of different drug exposures. Untreated rabbits were given saline following the same dosing schedule as controls. Measuring the MICs and the resistance mutant frequency assessed the emergence of resistance under the dosing regiment studied. The concentrations of tobramycin in the tissue cage fluid were analysis by high-performance liquid chromatographic method or improved bioanalysis. The correlation between drug concentration and drug resistance was analysized.[Results] The MPCs of tobramycin,amikacin and gentamycin for Pseudomonas aeruginosa strain ATCC27853 were 8μg/ml, 12.5μg/ml, and 10.5μg/m, and the MPC/MIC₉₉(SI) were 32, 12 and 20 respectively.The concentration of aminoglycosides antibiotics (tobramycin and gentamycin) has strong effects on the recovery of resistant colonies and detection of AMES gene of Pseudomonas aeruginosa strain.Resistant mutants of strain ATCC 27853 were selectively enriched when tobramycin concentration in tissue cage fluid was inside MSW, and no enrichment of mutants occurred when tobramycin concentration was outside MSW ;When the tobramycin concentration was in the lower part of MSW, the resistant mutants were selectively enriched most easily ; When the concentration of tobramycin was kept above MPC(T>MPC) for more than 23.21% of the treatment duration, the mutants selection of strain ATCC27853 in vivo can be prevented.. When the maximal concentration of tobramycin in tissue cage fluid (Cmax) was above MIC₉₉ but lower than MPC, MIC increased if the time of concentration was kept within MSW for longer than 96.7% of the whole treatment span, the resistant mutants will be selectively enrichment in vivo. . When Cmax was lower than MIC₉₉, resistance will not occur ; No susceptibility to drug resistance were observed when Cmax/MIC value was larger than 26,or AUC0-24/MIC value was higher than 320,or AUC0-24/MPC was higher than 20.[ Conclusion ](1) As far as Pseudomonas aeruginosa strain ATCC27853 is concerned, tobramycin is more powerful than gentamycin to restrict the next-step resistance mutations (2) Aminoglycosides concentration has strong effects on the recovery ofresistant colonies and detection of AMES gene. (3) In the in vivo study, the enrichment of resistant mutants will not occur when tobramycin concentration is above MPC or below MIC₉₉. Only when Cmax is lower than MPC and, most of the time, between MSW range, drug resistance is apt to be induced. When concentrations in the tissue cage fluid of rabbits was kept above MPC, tobramycin can restrict selective enrichment of mutants. When the concentrations of tobramycin in the tissue cage fluid was below MIC₉₉, no mutants were selectively enriched. (5) Resistant mutants of strain ATCC27853 were selectively enriched when tobramycin concentration in tissue cage fluid was inside MSW, and especially when tobramycin concentration was placed in the lower part of MSW. (6) In consider of pharmacokinetic/pharmacodynamic parameters, T>MIC₉₉ T>MPC Tmsw and Cmax/MIC may be the better items to predict the occurrence of drug resistance to aminoglycosides antibiotics in the treatment.