title: Genetic adaptation of Pseudomonas aeruginosa during chronic lung infection of patients with cystic fibrosis: strong and weak mutators with heterogeneous genetic backgrounds emerge in mucA and/or lasR mutants.
abstract: During the chronic lung infection of patients with cystic fibrosis (CF), Pseudomonas aeruginosa can survive for long periods due to adaptive evolution mediated by genetic variation. Hypermutability is considered to play an important role in this adaptive evolution and it has been demonstrated that mutator populations are amplified in the CF lung by hitchhiking with adaptive mutations. Two of the genes that are frequently mutated in isolates from chronic infection are mucA and lasR. Loss-of-function mutations in these genes determine the phenotypic switch to mucoidy and loss of quorum sensing, which are considered hallmarks of chronic virulence. The aims of our study were to investigate (1) the genetic background of the P. aeruginosa subpopulations with non-mutator, weak or strong mutator phenotype and their dynamics during the chronic lung infection, and (2) the time sequence in which the hypermutable, mucoid and quorum-sensing-negative phenotypes emerge during chronic lung infection. For these purposes the sequences of mutS, mutL, uvrD, mutT, mutY and mutM anti-mutator genes as well as of mucA and lasR were analysed in 70 sequential P. aeruginosa isolates obtained from the respiratory secretions of 10 CF patients (one to three isolates per time point). Analysis of the genetic background of the mutator phenotype showed that mutS was the most commonly affected gene followed by mutL in isolates with strong mutator phenotype. The mutT, mutY, mutM genes were affected in isolates with low fold-changes in the mutation frequencies compared to the reference strain PAO1. Isolates with non-mutator, weak or strong mutator phenotype were represented at all time points showing co-existence of these subpopulations, which suggests parallel evolution of the various mutators in the different focal niches of infection in the CF lung. Mutations in mucA and lasR occurred earlier than mutations in the anti-mutator genes, showing that hypermutability is not a prerequisite for the acquisition of mucoidy and loss of quorum sensing, considered hallmarks of chronic virulence. Significantly higher mutation rates and MICs of ceftazidime, meropenem and ciprofloxacin were found for isolates collected late (more than 10 years) during the chronic lung infection compared to isolates collected earlier, which suggests an amplification of the mutator subpopulation by hitchhiking with development of antibiotic resistance. Similar evolutionary pathways concordant with adaptive radiation were observed in different clonal lineages of P. aeruginosa from CF patients.
authors.affiliation: Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Denmark.
authors.forename: Oana
authors.initials: O
authors.lastname: Ciofu
authors.affiliation: Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Denmark.
authors.forename: Lotte F
authors.initials: LF
authors.lastname: Mandsberg
authors.affiliation: Department of Clinical Microbiology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.|Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bjarnsholt
authors.affiliation: Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Denmark.
authors.forename: Tina
authors.initials: T
authors.lastname: Wassermann
authors.affiliation: Department of Clinical Microbiology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.|Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Denmark.
authors.forename: Niels
authors.initials: N
authors.lastname: Høiby
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002908:Chronic Disease; D003550:Cystic Fibrosis / Q000150:complications*; D006801:Humans; D008171:Lung Diseases / Q000382:microbiology*; D009154:Mutation; D011552:Pseudomonas Infections / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000502:physiology; D053038:Quorum Sensing; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C080365:LasR protein, Pseudomonas aeruginosa; C107895:MucA protein, Pseudomonas; D015534:Trans-Activators