Antibiotic Cycling Affects Resistance Evolution Independently of Collateral Sensitivity

Abstract

Antibiotic cycling has been proposed as a promising approach to slow down resistance evolution against currently employed antibiotics. It remains unclear, however, to which extent the decreased resistance evolution is the result of collateral sensitivity, an evolutionary trade-off where resistance to one antibiotic enhances the sensitivity to the second, or due to additional effects of the evolved genetic background, in which mutations accumulated during treatment with a first antibiotic alter the emergence and spread of resistance against a second antibiotic via other mechanisms. Also the influence of the antibiotic exposure patterns on the outcome of drug cycling is unknown. Here, we systematically assessed effects of the evolved genetic background by focusing on the first switch between two antibiotics against Salmonella Typhimurium, with cefotaxime fixed as the first and a broad variety of other drugs as the second antibiotic. By normalizing the antibiotic concentrations to eliminate effects of collateral sensitivity, we demonstrated a clear contribution of the evolved genetic background beyond collateral sensitivity, which either enhanced or reduced the adaptive potential depending on the specific drug combination. We further demonstrated that the gradient strength with which cefotaxime was applied affected both cefotaxime resistance evolution and adaptation to second antibiotics, an effect that was associated with higher levels of clonal interference and reduced cost of resistance in populations evolved under weaker cefotaxime gradients. Overall, our work highlights that drug cycling can affect resistance evolution independently of collateral sensitivity, in a manner that is contingent on the antibiotic exposure pattern.

Fitness Costs of Antibiotic Resistance Impede the Evolution of Resistance to Other Antibiotics

“…5X also evolved a higher MIC than the OM, which surpassed the maximum concentration of NIT encountered in the plates by about three-fold (OM, R14: 8 μg/mL; 5X, R8: 32 μg/mL). This “overshoot” in resistance has been reported before and has important consequences since bacteria encountering sub-lethal concentrations of antibiotics can evolve resistance beyond clinical breakpoints . Sequencing revealed that the OM did not acquire mutations in any genes implicated in NIT resistance, while the 5X evolved resistance via mutations in the nitroreductase enzymes classically known to confer NIT resistance.…”

“…We then repeated evolutions with the OM (n = 16), adjusting the NIT gradient to accommodate this increased sensitivity (maximum [NIT] = 10 μg/mL), as has been previously carried out to eliminate effects of collateral sensitivity on resistance evolution. 20 In contrast to the WT, movement of OM replicates through NIT SAGE plates showed large variation (Figure S2B). Cells were again confined to within ∼10 mm of the inoculation site after 24 h (Figure 2A,C), suggesting that the increased sensitivity to NIT was not the cause behind this impaired movement.…”

“…To link this reduction in movement through soft agar to fitness, we used the area under the curve (AUC) measurements to quantify bacterial growth. , AUC incorporates three fitness parameters: the lag phase duration, the exponential growth rate, and the yield (maximum cell density). The AUC of the WT population was ∼8 times higher than that of the OM, showing large growth deficits (Figure B).…”

“…To the best of our knowledge, collateral sensitivity to nitrofurantoin in CHL-resistant cells has not been reported before. We then repeated evolutions with the OM ( n = 16), adjusting the NIT gradient to accommodate this increased sensitivity (maximum [NIT] = 10 μg/mL), as has been previously carried out to eliminate effects of collateral sensitivity on resistance evolution . In contrast to the WT, movement of OM replicates through NIT SAGE plates showed large variation (Figure S2B).…”

“…17−19 Hypersensitive bacterial populations are often growthimpaired, and Brepoels et al recently showed that resistance evolution is impaired in hypersensitive bacterial populations independently of collateral sensitivity in certain drug sequences. 20,21 Here, we show that evolution of resistance to chloramphenicol (CHL) in Escherichia coli K-12 substrain MG1655 cripples its growth rate and movement through soft agar, which impedes its ability to evolve resistance to secondary antibiotics in antibiotic gradients independently of collateral sensitivity. We leveraged the high-throughput mutant generation capacity of the soft agar gradient evolution (SAGE) system to evolve 16 independent isogenic populations (referred to here as replicates) of WT and CHL-resistant E. coli (OM) separately to two different antibiotics, nitrofurantoin (NIT) and streptomycin (STR) in parallel.…”