Within-Patient Evolution of Pseudomonas aeruginosa Populations During Antimicrobial Treatment

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections pose a major challenge to effective treatment. Understanding genomic adaptations during antimicrobial therapy in patients infected with this pathogen is crucial for preventing therapeutic failure. Here we investigated the population diversity and evolution of P. aeruginosa collected longitudinally from six patients who evolved MDR infections. Serial P. aeruginosa clinical isolates (n=63) and culture-enriched metagenomic populations (n=39) were collected and subjected to whole-genome sequencing. The resulting data were used to characterize and compare the species composition, multi-locus sequence types (STs), and resistance-associated mutations present within each sample type. Single-isolate sequencing showed that each patient was infected with a single P. aeruginosa strain that accumulated mutations and became increasingly antibiotic-resistant over time. Mutations in genes associated with β-lactam resistance, including ampC, ftsI , and oprD, arose over time and corresponded with changes in antimicrobial susceptibility in single isolates. Species profiling of culture-enriched metagenomic populations revealed that all samples contained P. aeruginosa , but several also contained additional Gram-negative pathogens. Metagenomic analysis of culture-enriched populations identified resistance-associated mutations at variable frequencies, many of which were not identified in single isolates from the same sample. In some cases, resistance-associated mutations initially detected at low frequency rose to fixation after antimicrobial treatment. Overall, this study shows that population-based metagenomic sequencing effectively captures within-patient genomic diversity of P. aeruginosa during antimicrobial therapy, and could aid in the detection and interpretation of resistance-associated mutations in this pathogen.