HypermutatorPseudomonas aeruginosaexploits multiple genetic pathways to develop multidrug resistance during long-term infections in the airways of cystic fibrosis patients

Abstract

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...

From genotype to phenotype: adaptations of Pseudomonas aeruginosa to the cystic fibrosis environment

“…Chromosomal inversions were also shown to disrupt the reading frame of mutS and induce hypermutability in clinical strains from the C lineage [39]. Hypermutable isolates, thus, accumulate a mean of 16-fold more mutations, with a median of 48 SNPs per year (range of 2 to more than 350 SNPs per year) [7,8,15,17,45].…”

“…Indeed, it has been shown that antibiotic exposure promotes the emergence of hypermutability in P. aeruginosa, then favouring acquisition of antibiotic resistance [45,[48][49][50][51]. However, Mehta and colleagues also observed that some hypermutable lineages would spontaneously decline and disappear from the evolving population [49].…”

Spatiotemporal manipulation of the mismatch repair system ofPseudomonas putidaaccelerates phenotype emergence

“…Since mutation rates must be precisely controlled to avoid extensive accumulation of deleterious mutations and to prevent genomic instability, the overexpression of mutator alleles should be driven from tightly-regulated expression systems (which is always challenging, irrespective of the bacterial host 74 ) or during short periods of time. Thus, the easy-to-cure mutator plasmids developed in this study, which can be rapidly removed from isolated clones displaying the phenotype of interest, offer a clear advantage over conventional mutator strains—where the mutator phenotype is elicited by genomic (hence, essentially irreversible) modifications, as epitomized by the emergence of mutator phenotypes of P. aeruginosa in clinically-relevant setups 75-77 . In contrast with the results of the ChnR/P chnB -dependent module, the thermoinducible mutator devices allowed for a tightly- regulated expression of mutL E36K .…”

Evolution and adaptation ofPseudomonas aeruginosain the paranasal sinuses of people with cystic fibrosis

“…Although the accumulation of beneficial traits along with many neutral or deleterious ones may be the consequence of a higher mutation rate, the mutator phenotype itself is unseen by selection. Rather, mutators evolve by the indirect effects of selection on linked traits, such as loss of acute virulence in CF, increased biofilm formation, metabolic adaptations, and antibiotic resistance ( 51, 53, 58, 83–85 ). The probability of this linkage increases in small populations under strong selection that are limited for beneficial genetic variation or under conditions when effects of drift relative to selection are amplified ( 86 ).…”