Pseudomonas aeruginosa exploits intrinsic and acquired resistance mechanisms to resist almost every antibiotic used in chemotherapy. Antimicrobial resistance in P. aeruginosa isolates recovered from cystic fibrosis (CF) patients is further enhanced by the occurrence of hypermutator strains, a hallmark of chronic infections in CF patients. However, the within-patient genetic diversity of P. aeruginosa populations related to antibiotic resistance remains unexplored. Here, we show the evolution of the mutational resistome profile of a P. aeruginosa hypermutator lineage by performing longitudinal and transversal analyses of isolates collected from a CF patient throughout 20 years of chronic infection. Our results show the accumulation of thousands of mutations, with an overall evolutionary history characterized by purifying selection. However, mutations in antibiotic resistance genes appear to have been positively selected, driven by antibiotic treatment. Antibiotic resistance increased as infection progressed toward the establishment of a population constituted by genotypically diversified coexisting sublineages, all of which converged to multidrug resistance. These sublineages emerged by parallel evolution through distinct evolutionary pathways, which affected genes of the same functional categories. Interestingly, ampC and ftsI, encoding the β-lactamase and penicillin-binding protein 3, respectively, were found to be among the most frequently mutated genes. In fact, both genes were targeted by multiple independent mutational events, which led to a wide diversity of coexisting alleles underlying β-lactam resistance. Our findings indicate that hypermutators, apart from boosting antibiotic resistance evolution by simultaneously targeting several genes, favor the emergence of adaptive innovative alleles by clustering beneficial/compensatory mutations in the same gene, hence expanding P. aeruginosa strategies for persistence.

21 cystic fibrosis 22 WORD COUNT ABSTRACT: Abstract, 249 words. Importance, 147 words 23 WORD COUNT TEXT: 4995 words.24 2 25 ABSTRACT 26 Pseudomonas aeruginosa exploits intrinsic and acquired resistance mechanisms to resist almost 27 every antibiotic used in chemotherapy. Antimicrobial resistance in P. aeruginosa isolated from 28 cystic fibrosis (CF) patients is further enhanced by the occurrence of hypermutator strains, a 29 hallmark of chronic CF infections. However, the within-patient genetic diversity of P. aeruginosa 30 populations related to antibiotic resistance remains unexplored. Here, we show the evolution of 31 the mutational resistome profile of a P. aeruginosa hypermutator lineage by performing 32 longitudinal and transversal analyses of isolates collected from a CF patient throughout 20 years 33 of chronic infection. Our results show the accumulation of thousands of mutations with an overall 34 evolutionary history characterized by purifying selection. However, mutations in antibiotic 35resistance genes appear to be positively selected, driven by antibiotic treatment. Antibiotic 36 resistance increased as infection progressed towards the establishment of a population constituted 37 by genotypically diversified coexisting sub-lineages, all of which converged to multi-drug 38 resistance. These sub-lineages emerged by parallel evolution through distinct evolutionary 39 pathways, which affected genes of the same functional categories. Interestingly, ampC and fstI, 40 encoding the β-lactamase and penicillin-binding protein 3, respectively, were found among the 41 most frequently mutated genes. In fact, both genes were targeted by multiple independent 42 mutational events, which led to a wide diversity of coexisting alleles underlying β-lactam 43 resistance. Our findings indicate that hypermutators, apart from boosting antibiotic resistance 44 evolution by simultaneously targeting several genes, favor the emergence of adaptive innovative 45 alleles by clustering beneficial/compensatory mutations in the same gene, hence expanding P. 46 aeruginosa strategies for persistence. 47 IMPORTANCE 483 By increasing mutation rates, hypermutators boost antibiotic resistance evolution by enabling 49 bacterial pathogens to fully exploit their genetic potential and achieve resistance mechanisms for 50 almost every known antimicrobial agent. Here, we show how co-existing clones from a P. 51 aeruginosa hypermutator lineage that evolved during 20 years of chronic infection and antibiotic 52 chemotherapy, converged to multidrug resistance by targeting genes from alternative genetic 53 pathways that are part of the broad P. aeruginosa resistome. Within this complex assembly of 54 combinatorial genetic changes, in some specific cases, multiple mutations are needed in the same 55 gene to reach a fine tuned resistance phenotype. Hypermutability enables this genetic edition 56 towards higher resistance profiles by recurrently targeting these genes, thus promoting new 57 epistatic relationships and the emergence of innovativ...