HypermutatorPseudomonas aeruginosaexploits multiple genetic pathways to develop multidrug resistance during long-term infections in the airways of cystic fibrosis patients
Authors: Colque C.A. , Albarracín Orio A.G. , Feliziani S. , Marvig R.L. , Tobares A.R. , Johansen H.K. , Molin S. , Smania A.M. 
Published: October 2019

ABSTRACTPseudomonas aeruginosaexploits intrinsic and acquired resistance mechanisms to resist almost every antibiotic used in chemotherapy. Antimicrobial resistance inP. aeruginosaisolated from cystic fibrosis (CF) patients is further enhanced by the occurrence of hypermutator strains, a hallmark of chronic CF infections. However, the within-patient genetic diversity ofP. aeruginosapopulations related to antibiotic resistance remains unexplored. Here, we show the evolution of the mutational resistome profile of aP. aeruginosahypermutator 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 be positively selected, driven by antibiotic treatment. Antibiotic resistance increased as infection progressed towards the establishment of a population constituted by genotypically diversified coexisting sub-lineages, all of which converged to multi-drug resistance. These sub-lineages emerged by parallel evolution through distinct evolutionary pathways, which affected genes of the same functional categories. Interestingly,ampCandfstI, encoding the β-lactamase and penicillin-binding protein 3, respectively, were found 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 expandingP. aeruginosastrategies for persistence.IMPORTANCEBy increasing mutation rates, hypermutators boost antibiotic resistance evolution by enabling bacterial pathogens to fully exploit their genetic potential and achieve resistance mechanisms for almost every known antimicrobial agent. Here, we show how co-existing clones from aP. aeruginosahypermutator lineage that evolved during 20 years of chronic infection and antibiotic chemotherapy, converged to multidrug resistance by targeting genes from alternative genetic pathways that are part of the broadP. aeruginosaresistome. Within this complex assembly of combinatorial genetic changes, in some specific cases, multiple mutations are needed in the same gene to reach a fine tuned resistance phenotype. Hypermutability enables this genetic edition towards higher resistance profiles by recurrently targeting these genes, thus promoting new epistatic relationships and the emergence of innovative resistance-conferring alleles. Our findings help to understand this link between hypermutability and antibiotic resistance, a key challenge for the design of new therapeutic strategies.