Within-patient evolution of <i>Pseudomonas aeruginosa</i> populations during antimicrobial treatment

<i>Pseudomonas aeruginosa</i> infections are notoriously difficult to treat and are associated with high rates of morbidity and mortality. While the genetic basis of resistance in <i>P. aeruginosa</i> is well documented <i>in vitro</i>, less is known about how resistance evolves within patients during antibiotic therapy. Standard approaches based on analysis of clonal isolates may miss within-patient diversity, potentially overlooking low-frequency mutations that contribute to treatment failure. In this study, we compared single-colony isolate whole-genome sequencing with culture-enriched metagenomic sequencing to monitor the evolution of <i>P. aeruginosa</i> populations in patients receiving antibiotic therapy. The culture-enriched metagenomic approach enabled the detection of emerging resistance mutations, such as low-frequency variants in <i>ampC</i> and <i>ftsI</i>, before these variants rose to fixation. It also revealed genetically resistant subpopulations missed by isolate sequencing alone. Overall, our findings highlight the value of population-based metagenomic sequencing in capturing bacterial adaptation during infection and underscore its potential to improve resistance surveillance and guide personalized antimicrobial therapy.