Dynamics of Mutations during Development of Resistance by Pseudomonas aeruginosa against Five Antibiotics

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes considerable morbidity and mortality, specifically during intensive care. Antibiotic-resistant variants of this organism are more difficult to treat and cause substantial extra costs compared to susceptible strains. In the laboratory, P. aeruginosa rapidly developed resistance to five medically relevant antibiotics upon exposure to stepwise increasing concentrations. At several time points during the acquisition of resistance, samples were taken for whole-genome sequencing. The increase in the MIC of ciprofloxacin was linked to specific mutations in gyrA, parC, and gyrB, appearing sequentially. In the case of tobramycin, mutations in fusA, HP02880, rplB, and capD were induced. The MICs of the beta-lactam compounds meropenem and ceftazidime and the combination of piperacillin and tazobactam correlated linearly with beta-lactamase activity but not always with individual mutations. The genes that were mutated during the development of beta-lactam resistance differed for each antibiotic. A quantitative relationship between the frequency of mutations and the increase in resistance could not be established for any of the antibiotics. When the adapted strains are grown in the absence of the antibiotic, some mutations remained and others were reversed, but this reversal did not necessarily lower the MIC. The increased MIC came at the cost of moderately reduced cellular functions or a somewhat lower growth rate. In all cases except ciprofloxacin, the increase in resistance seems to be the result of complex interactions among several cellular systems rather than individual mutations.

FIG 1

Antibiotic resistance development by P. aeruginosa expressed as 2-fold increases (Incr.) in MICs as a function of time (days) in minimal medium with stepwise increasing concentrations of the antibiotics indicated. Piper, piperacillin; Tazo, tazobactam.

FIG 2

Increase (Incr.) in the MIC by factors of 2 and frequencies of mutations observed in the replicates on which WGS was performed as a function of time (days) in the presence of the antibiotic indicated and after its removal from the growth medium. All mutations found at frequencies exceeding 0.5 were also discovered in the second replicate by PCR, except for the parC mutations in response to ciprofloxacin, where P85L in the first was replaced by S87N in the second. Piper, piperacillin; Tazo, tazobactam.

FIG 3

(A) β-Lactamase activities measured in cells adapted to the beta-lactam antibiotics indicated and after subsequent growth for 15 days in the absence of the drugs. The unit of β-lactamase activity is nanomoles of nitrocefin hydrolyzed per minute per milligram of protein. (B) Increase (Incr.) in the MIC as a function of β-lactamase activity. The correlation between β-lactamase activity and MIC is statistically significant for the two antibiotics indicated with asterisks. Piper, piperacillin; tazo, tazobactam.

FIG 4

μ_max and maintenance energy of strains adapted to the antibiotics indicated immediately after growth in the presence of the drugs (after exposure) and after 14 days of subsequent growth in their absence (after removal). An asterisk indicates that the growth rate was significantly lower than that of the wild type, while double asterisks indicate that the growth rates before and after growth in the absence of the antibiotic differed significantly. Panels: A, μ_max in duplications (Piper, piperacillin; tazo, tazobactam); B, estimation of the maintenance energy by extrapolation to D (specific growth rate in duplication per h) = 0 of the specific glucose (gluc) consumption measured at several dilution rates; C, μ_max of wild-type P. aeruginosa and cells adapted to tobramycin (TBM) in the presence or absence of the drug as a function of pH.