Unraveling Genomic Diversity in Pseudomonas aeruginosa Cystic Fibrosis Lung Infection and Its Impact on Antimicrobial Resistance

Pseudomonas aeruginosa is the predominant bacterial pathogen in the cystic fibrosis (CF) lung, establishing a chronic, drug-resistant infection often via a single adapting and diversifying strain. In this study, we collected a bio-repository of 300 isolates of P. aeruginosa from four CF patients to unravel the extent of within-patient genomic diversity in CF lung infection; elucidate the evolutionary processes by which this diversity is generated; and understand how diversification impacts antimicrobial resistance. We found that populations of P. aeruginosa in the CF lung displayed patient specific genomic diversity ranging from 1 to 199 median pairwise SNPs, and 31 to 4,592 total SNPs within a population. Diversification was driven by a combination of de novo mutations, recombination, and variations in gene content, with the highest levels of population diversity associated with the presence of DNA mismatch repair mutants. Populations with higher levels of genomic diversity displayed increased variation in antibiotic resistance phenotypes; however, even populations with lower genomic diversity displayed substantial diversity in resistance profiles. Intriguingly, we found evidence that mutations in DNA mismatch repair can facilitate the evolution of seemingly undesirable traits in vivo , such as increased sensitivity to certain antimicrobials. Overall, our findings highlight that (i) P. aeruginosa genomic population diversity in chronic CF lung infection is highly variable between patients; (ii) hypermutator populations can evolve increased sensitivity to antimicrobials even under apparent antibiotic selection; and that (iii) current methods of single-isolate sampling and susceptibility testing should be re-evaluated in the study of chronic P. aeruginosa infection. SIGNIFICANCE Chronic lung infection with Pseudomonas aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Upon infection, P. aeruginosa rapidly acquires mutations, which can result in diverse, treatment-resistant populations. However, within-patient diversity is largely overlooked by clinicians and researchers. We sequenced the genomes of 300 CF lung-derived isolates of P. aeruginosa to investigate genomic diversity and its role in the evolution of antimicrobial resistance (AMR). Genomic diversity was variable, with higher levels of diversity correlating with increased variation in AMR. In two patients, mutations in DNA mismatch repair genes drove the evolution of increased susceptibility to multiple antimicrobials. Based on these findings, we advocate for deeper population sampling to improve our understanding of CF lung infection. ### Competing Interest Statement The authors have declared no competing interest.