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Traditional studies on the evolution of antibiotic resistance development use approaches that can range from laboratory-based experimental studies, to epidemiological surveillance, to sequencing of clinical isolates. However, evolutionary trajectories also depend on the environment in which selection takes place, compelling the need to more deeply investigate the impact of environmental complexities and their dynamics over time. Herein, we explored the within-patient adaptive long-term evolution of a Pseudomonas aeruginosa hypermutator lineage in the airways of a cystic fibrosis (CF) patient by performing a chronological tracking of mutations that occurred in different subpopulations; our results demonstrated parallel evolution events in the chromosomally encoded class C β-lactamase (blaPDC). These multiple mutations within blaPDC shaped diverse coexisting alleles, whose frequency dynamics responded to the changing antibiotic selective pressures for more than 26 years of chronic infection. Importantly, the combination of the cumulative mutations in blaPDC provided structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrated selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. A “gain of function” of collateral resistance toward ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed, and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the evolutionary trajectories paved by bacteria toward a multidrug-resistant phenotype, driven by decades of antibiotic treatment in the natural CF environmental setting. IMPORTANCE Antibiotics are becoming increasingly ineffective to treat bacterial infections. It has been consequently predicted that infectious diseases will become the biggest challenge to human health in the near future. Pseudomonas aeruginosa is considered a paradigm in antimicrobial resistance as it exploits intrinsic and acquired resistance mechanisms to resist virtually all antibiotics known. AmpC β-lactamase is the main mechanism driving resistance in this notorious pathogen to β-lactams, one of the most widely used classes of antibiotics for cystic fibrosis infections. Here, we focus on the β-lactamase gene as a model resistance determinant and unveil the trajectory P. aeruginosa undertakes on the path toward a multidrug-resistant phenotype during the course of two and a half decades of chronic infection in the airways of a cystic fibrosis patient. Integrating genetic and biochemical studies in the natural environment where evolution occurs, we provide a unique perspective on this challenging landscape, addressing fundamental molecular mechanisms of resistance.

Bacteria with increased mutation rates (mutators) are common in chronic infections and are associated with poorer clinical outcomes, especially in the case of Pseudomonas aeruginosa infecting cystic fibrosis (CF) patients. There is, however, considerable between-patient variation in both P. aeruginosa mutator frequency and the composition of co-infecting pathogen communities. We investigated whether community context might affect selection of mutators. Using an in vitro CF model community, we show that P. aeruginosa mutators were favoured in the absence of other species but not in their presence. This was because there were trade-offs between adaptation to the biotic and abiotic environments (for example, loss of quorum sensing and associated toxin production was beneficial in the latter but not the former in our in vitro model community) limiting the evolvability advantage of an elevated mutation rate. Consistent with a role of co-infecting pathogens selecting against P. aeruginosa mutators in vivo, we show that the mutation frequency of P. aeruginosa population was negatively correlated with the frequency and diversity of co-infecting bacteria in CF infections. Our results suggest that co-infecting taxa can select against P. aeruginosa mutators, which may have potentially beneficial clinical consequences.

Intense genome sequencing of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) airways has shown inefficient eradication of the infecting bacteria, as well as previously undocumented patient-to-patient transmission of adapted clones. However, genome sequencing has limited potential as a predictor of chronic infection and of the adaptive state during infection, and thus there is increasing interest in linking phenotypic traits to the genome sequences. Phenotypic information ranges from genome-wide transcriptomic analysis of patient samples to determination of more specific traits associated with metabolic changes, stress responses, antibiotic resistance and tolerance, biofilm formation and slow growth. Environmental conditions in the CF lung shape both genetic and phenotypic changes of P. aeruginosa during infection. In this Review, we discuss the adaptive and evolutionary trajectories that lead to early diversification and late convergence, which enable P. aeruginosa to succeed in this niche, and we point out how knowledge of these biological features may be used to guide diagnosis and therapy.

Antibiotic resistance has become a serious threat to human health (WHO Antibacterial Agents in Clinical Development: an Analysis of the Antibacterial Clinical Development Pipeline, Including Tuberculosis. Geneva: World Health Organization; 2017), and the ability to predict antibiotic resistance from genome sequencing has become a focal point for the medical community. With this genocentric prediction in mind, we were intrigued about two particular findings for a collection of clinical Pseudomonas aeruginosa isolates (Marvig et al. Nature Genetics 2015;47:57–64; Frimodt-Møller et al. Scientific Reports 2018;8:12512; Bartell et al. Nature Communications 2019;10:629): (i) 15 out of 52 genes found to be frequently targeted by adaptive mutations during the initial infection stage of cystic fibrosis airways (‘candidate pathoadaptive genes’) (Marvig et al. Nature Genetics 2015;47:57–64) were associated with antibiotic resistance (López-Causapé et al. Fronters in Microbiology 2018;9:685; López-Causapé et al. Antimicrobal Agents and Chemotherapy 2018;62:e02583-17); (ii) there was a parallel lack of resistance development and linkage to the genetic changes in these antibiotic-resistance-associated genes (Frimodt-Møller et al. Scientific Reports 2018;8:12512; Bartell et al. Nature Communications 2019;10:629). In this review, we highlight alternative selective forces that potentially enhance the infection success of P. aeruginosa and focus on the linkage to the 15 pathoadaptive antibiotic-resistance-associated genes, thereby showing the problems we may face when using only genomic information to predict and inform about relevant antibiotic treatment.

Whenever Pseudomonas aeruginosa is cultured from cystic fibrosis (CF) patient airways, the primary goal is eradication by antibiotic therapy. Success is defined by ≥6 months of negative bacterial airway cultures. However, we suspect that P. aeruginosa persists in airways without clinical detection for long periods. Out of 298 P. aeruginosa-infected Copenhagen CF patients, we identified 80 with complete P. aeruginosa monitoring records and measured their maximum P. aeruginosa-free eradication periods (MEP). Isolates from 72 patients were whole-genome sequenced (n=567) and clone typed. Select isolate relatedness was examined through phylogenetic analysis and phenotypic multivariate modelling. 69 (86%) patients exhibited eradication in the monitoring period (2002–2018). Sequenced isolates bridged the MEP of 42 patients, and the same clone type persisted over the MEP in 18 (43%) patients. Patients with failed eradication were on average treated more intensively with antibiotics, but this may be linked to their more severe pre-MEP infection trajectories. Of the 42 patients, 26 also had sinus surgery; the majority (n=15) showed MEPs adjacent to surgery, and only five had persisting clone types. Importantly, combined phylogenetic–phenomic evaluation suggests that persisting clone types are a result of re-emergence of the same strain rather than re-infection from the environment, and similar relatedness is exhibited by paired lower and upper airway samples and in transmission cases. In conclusion, nearly half of CF patients with supposed eradication may not truly be cleared of their original bacteria according to omics-based monitoring. This distinct cohort that is persistently infected would probably benefit from tailored antibiotic therapy., For 80 cystic fibrosis patients, this study used omics and positive culture history of P. aeruginosa infections to show that strains routinely persist over lengthy Pseudomonas-free periods. The authors recommend using genomic data in “eradication” metrics. https://bit.ly/2H318Ca