Development of Antibiotic Resistance during Simulated Treatment of Pseudomonas aeruginosa in Chemostats

Abstract

During treatment of infections with antibiotics in critically ill patients in the intensive care resistance often develops. This study aims to establish whether under those conditions this resistance can develop de novo or that genetic exchange between bacteria is by necessity involved. Chemostat cultures of Pseudomonas aeruginosa were exposed to treatment regimes with ceftazidime and meropenem that simulated conditions expected in patient plasma. Development of antibiotic resistance was monitored and mutations in resistance genes were searched for by sequencing PCR products. Even at the highest concentrations that can be expected in patients, sufficient bacteria survived in clumps of filamentous cells to recover and grow out after 3 to 5 days. At the end of a 7 days simulated treatment, the minimal inhibitory concentration (MIC) had increased by a factor between 10 and 10,000 depending on the antibiotic and the treatment protocol. The fitness costs of resistance were minimal. In the resistant strains, only three mutations were observed in genes associated with beta-lactam resistance. The development of resistance often observed during patient treatment can be explained by de novo acquisition of resistance and genetic exchange of resistance genes is not by necessity involved. As far as conclusions based on an in vitro study using P. aeruginosa and only two antibiotics can be generalized, it seems that development of resistance can be minimized by treating with antibiotics in the highest concentration the patient can endure for the shortest time needed to eliminate the infection.

Fig 1. Comparison of the computer-simulated 5, 50, and 95 percentile concentrations of ceftazidime and meropenem as a function of time in critically ill patients and the concentrations achieved in the chemostat.

Fig 2. Density of a culture of P. aeruginosa exposed to ceftazidime and meropenem in the chemostat (D = 0.3 h^-1) at concentrations simulating the 5, 50 and 95 percentile of the concentration-time profiles as they are expected in critically ill patients.

Cultures were in steady state in the absence of antibiotics before day 0.

Fig 3. Comparison of cell morphology of regular growing culture and a culture growing at the 50 percentile concentration of ceftazidime during the initial exposure.

Exposure to other concentrations of ceftazidime and all experimental concentrations of meropenem yielded a similar morphology.

Fig 4. Minimum inhibitory concentrations (MIC) of P. aeruginosa culture as a function of time (days) during exposure to the 5, 50, and 95 percentile of concentration-time profiles as they are to be expected in critically ill patients of ceftazidime (left panel) and meropenem (right panel) in chemostats (D = 0.3 h^-1).

Fig 5. Fraction of cells from chemostat cultures that can grow on plates containing the 5, 50, 95 percentile of the ceftazidime steady-state concentration and the meropenem trough concentration (8 hour after drug administration) as these are to be expected in critically ill patients, as a function of time (days) during growth at the indicated antibiotic levels.

Fig 6. Maximum growth rate (μ_max) in the absence of antibiotics of cells taken from chemostat cultures exposed to the 95 percentile of concentration-time profiles as they are to be expected in critically ill patients of ceftazidime or meropenem as a function of time (days).

For each time point, the growth rate of four independent samples was measured.