Evolution of Habitat-Dependent Antibiotic Resistance in Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen that usually causes difficult-to-treat infections due to its low intrinsic antibiotic susceptibility and outstanding capacity for becoming resistant to antibiotics. In addition, it has a remarkable metabolic versatility, being able to grow in different habitats, from natural niches to different and changing inpatient environments. Study of the environmental conditions that shape genetic and phenotypic changes of P. aeruginosa toward antibiotic resistance supposes a novelty, since experimental evolution assays are usually performed with well-defined antibiotics in regular laboratory growth media. Therefore, in this work we address the extent to which the nutrients’ availability may constrain the evolution of antibiotic resistance. We determined that P. aeruginosa genetic trajectories toward resistance to tobramycin, ceftazidime, and ceftazidime-avibactam are different when evolving in laboratory rich medium, urine, or synthetic sputum. Furthermore, our study, linking genotype with phenotype, showed a clear impact of each analyzed environment on both the fitness and resistance level associated with particular resistance mutations. This indicates that the phenotype associated with specific resistance mutations is variable and dependent on the bacterial metabolic state in each particular habitat. Our results support that the design of evolution-based strategies to tackle P. aeruginosa infections should be based on robust patterns of evolution identified within each particular infection and body location. IMPORTANCE Predicting evolution toward antibiotic resistance (AR) and its associated trade-offs, such as collateral sensitivity, is important to design evolution-based strategies to tackle AR. However, the effect of nutrients' availability on such evolution, particularly those that can be found under in vivo infection conditions, has been barely addressed. We analyzed the evolutionary patterns of P. aeruginosa in the presence of antibiotics in different media, including urine and synthetic sputum, whose compositions are similar to the ones in infections, finding that AR evolution differs, depending on growth conditions. Furthermore, the representative mutants isolated under each condition tested render different AR levels and fitness costs, depending on nutrients’ availability, supporting the idea that environmental constraints shape the phenotypes associated with specific AR mutations. Consequently, the selection of AR mutations that render similar phenotypes is environment dependent. The analysis of evolution patterns toward AR requires studying growth conditions mimicking those that bacteria face during in vivo evolution.