In Vitro And In Vivo Studies Of Mutant Selection Window For Fosfomycin Against Escherichia Coli And Pseudomonas Aeruginosa

Objective:To test the mutant selection window(MSW) hypothesis in vitro with Escherichia coli and Pseudomonas Aeruginosa exposed to fosfomycin.To test the MSW hypothesis in the tissue-cage infection model with E. coli and P. aeruginosa exposed to fosfomycin and to obtain the pharmacokinetic/ pharmacodynamics(PK/PD) parameters that restrict the selection of fosfomycin-resistant mutants.To examine the apparent contradiction between in vitro and in vivo studies, the biological fitness of the in vitro-isolated mutants was determined as the growth rate in broth and tissue-cage fluid and competition experiments for resistance mutants and their parental strains.Meterials and Methods:E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were stored at the Anhui Center for Surveillance of Bacterial Resistance. Female New Zealand White rabbits weighing 2.5–3 kg each were supplied by the Anhui Medical University Experimental Animal Center.Mueller-Hinton(MH) agar dilution method was used to determine minimum inhibitoryconcentration(MIC) of fosfomycin against E. coli ATCC 25922 and P. aeruginosa ATCC 27853. The E. coli ATCC 25922 or P. aeruginosa ATCC 27853 was enriched the concentration to 3×1010 colony forming units per milliliter in broth. High-density cultures were prepared from overnight cultures grown in MH agar medium followed of incubation with shaking at 35℃. Inoculated drugs-impregnated plates were incubated for 72 h, and the MPCpr was recorded as the lowest concentration completely inhibiting bacterial growth at this time.The effect of fosfomycin concentration on colony-forming ability of resistant mutant subpopulations of E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were measured. Cells were applied to drug-containing agar plates and, at the same time, the cell density of the culture was determined retrospectively by applying serial dilutions to drug-free agar.In vitro bactericidal activities of fosfomycin against high-inoculum of E. coli and P. aeruginosa were assessed by modified time-kill assays. The drugs were added to the bacterial cultures at concentrations corresponding to 0.5×, 4×, 8×, 16×, and 32× MIC and 25 μg/m L of glucose-6-phosphate. Bacterial cultures and antibiotics were incubated at 35°C. Surviving bacteria were counted after 0, 4, 8, 12, 24 and 48 hours of incubation at 35 oC by subculturing 100 μl serial dilution of every flask on MH agar plates.Allelic diversity was examined with about 18 fosfomycin-resistant mutants, including 9 isolates of E. coli and 9 of P. aeruginosa selected from in vitro. Chromosomal DNA was isolated from each of them and the wild-type strain. The corresponding mur A、glp T、uhp T、uhp A、pts I and cya A genes were PCR amplified and sequenced.Local infection with E. coli and P. aeruginosa was established in rabbits, and theinfected animals were treated with various doses of three times a day fosfomycin for three or nine consecutive days to provide antibiotic concentrations below the minimum inhibitory concentration(MIC), between the MIC and the mutant prevention concentration(MPC), and above the MPC. The total drug concentration of fosfomycin was determined by gas chromatograph(GC). PK/PD indices were calculated according to a noncompartmental model by use of graphpad prism 5.01. All indices were determined after the sixth dose, at which time steady-state kinetics were reached.Quantification of the antimicrobial effect was monitored. In each animal experiment, surviving bacteria were counted every 24 h of incubation at 35 oC by subculturing 100 μl serial dilution of every plastic ball on MH agar plates. In order to account for antibiotic carryover, all samples were diluted sufficiently prior to plating, therefore reducing the antibiotic concentration below the MIC of the drug. The lower limit of accurate detection was 2 log10 CFU/m L.Loss of susceptibility was monitored in MH broth at the endpoint of the in vitro time-kill studies and in tissue-cage fluid obtained daily before fosfomycin administration, during antibacterial treatment, and 24 and 48 h after the termination of fosfomycin treatment. After counting cuf on drug-free plates to determine treatment efficacy, an attempt was made to scrape together all colonies from the most heavily populated plates. This material was resuspended in 5 ml normal saline to obtain an approximate cell density of 1×108 CFU/m L and replated on MH agar plates(supplemented with 25 μg/m L of glucose-6- phosphate) with and without 4× MIC of fosfomycin. Samples were considered to be enriched for drug-resistant mutants if the proportion of resistant CFU observed was ≥1 log10 greater than the baseline proportion observed in untreated rabbits at day 0. The MICs of fosfomycin for bacteria isolated from plastic ball were determined prior to and after antimicrobial exposure by using the standard method described above.Fisher’s exact test was used for statistical analysis of the PK/PD data, with an infected but untreated set of rabbits as a control. P<0.05 was considered to be statistically significant.Selection of mutants resistant were obtained by plating the high inocula(~1010 CFU) of parental strains on MH agar plates containing 25 μg/m L of glucose-6-phosphate and 8× MIC of fosfomycin, and MICs were determined to confirm resistance. Flasks containing 50 ml of fresh MH broth were inoculated with 0.5 ml of an overnight broth culture of the pathogen to be tested. Subcultures were grown in MH broth and tissue-cage fluid in the absence of fosfomycin. Appropriate dilutions of cultures were plated on MH agar medium every 2 h. After overnight incubation at 35°C colonies were counted and the generation time was calculated.Five rabbits were inoculated with a 1:1 mix of fosfomycin-resistance mutants and their parental strains for a final inoculum size approximately of 5×105. Twenty-four hours after inoculation, the rabbits were treated with fosfomycin(300 mg/kg/day) or untreated. After 48 hours of inoculation, the bacterial counts for fosfomycin-resistance mutants and their parental strains were determined by using the plate colony-counting methods. The competition index(CI) was defined as the mutant/wild-type ratio.Results:The MIC and MIC99 of fosfomycin for E. coli 25922 were estimated at 2 and 1.2 μg/m L, and those for P. aeruginosa 27853 were 4 and 3.6 μg/m L, respectively. With E. coli and P. aeruginosa, the exact MPC of fosfomycin was estimated at 57.6 and 102.4 μg/m L, respectively.In the in vitro time-kill studies, fosfomycin did not exhibit rapid bactericidal activityagainst high-inoculum planktonic E. coli and P. aeruginosa. At antibiotic concentrations of 4 ×, 8× and 16 × MIC, there was a temporary inhibitory effect until 12 h and then both two isolates quickly re-grew; moreover, the mutants of E. coli and P. aeruginosa with increased MICs were readily selected at the endpoint of the time-kill studies. At both the lowest(0.5 × MIC) and the highest(32 × MIC) concentrations, no development of resistance against fosfomycin was observed.Eighteen independent mutants, incluing 9 E. coli and 9 P. aeruginosa were obtained in vitro, and all of them were resistant to fosfomycin with varying degrees. The analysis of the respective sequences showed that 8 of 9 E. coli mutants contained mutations in the glp T、uhp T、uhp A、pts I and cya A gene sequence, while 5 of 9 P. aeruginosa mutants contained mutations in the glp T gene sequenceIn the in vivo studies, against P. aeruginosa 27853, an initial bacterial reduction was observed at doses of fosfomycin ≥100 mg/kg/day but ≤900 mg/kg/day; however, bacterial re-growth occurred between day 2 and 3 of exposure to fosfomycin alone in the killing curves; When antibiotic concentrations were inside the selection window(group B, C, D and E), the population was enriched with resistant mutants on the plates with 4× the MIC of fosfomycin. The pharmacodynamic thresholds at which resistant mutants are not selected in vivo was estimated as time above the MPC of >70% or AUC24/MPC > 90 h, where AUC24 is the area under the drug concentration time curve in a 24-h interval. In contrast, growth inhibition of E. coli was observed for dose of fosfomycin ≥100 mg/kg/day, and no bacterial re-growth was observed during the late treatment and post-treatment for all doses of fosfomycin.Growth curves were determined in order to evaluate the biological fitness. With E. coli, resistant mutants showed a significant slowing in their rate of growth compared with the susceptible parental strains; both the in vitro and in vivo competition experimentshowed that the resistant E. coli mutants were suppressed. With P. aeruginosa, no significant difference in growth rate could be detected between the susceptible and resistant strains both in MH and tissue-cage fluid; both the in vitro and in vivo competition experiment rendered no differences between the resistant mutants and ATCC 27853.Conclusions:Agar plate determinations are fit the MSW for fosfomycin treatment of rabbits infected with P. aeruginosa.The emergence of fosfomycin-resistant E. coli mutants was detected in vitro but not in an animal model when drug-concentration fell into the MSW. Some specific factors, such as the biological fitness costs of resistance and the host immune response, may be the potential causes of the the apparent contradiction between in vitro and in vivo studies.