title: The secondary resistome of multidrug-resistant Klebsiella pneumoniae.
abstract: Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of individual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of K. pneumoniae (ST258). We constructed a high-density transposon mutant library of >430,000 unique Tn5 insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Transposon Directed Insertion-site Sequencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential for growth during exposure to colistin (n = 35), imipenem (n = 1) or ciprofloxacin (n = 1) in addition to known resistance determinants, collectively termed the "secondary resistome". As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin resistance (dedA) restored colistin susceptibility by reducing the minimum inhibitory concentration from 8 to 0.5 μg/ml, 4-fold below the susceptibility breakpoint (S ≤ 2 μg/ml). This finding suggests that the secondary resistome is a potential target for developing antimicrobial "helper" drugs that restore the efficacy of existing antimicrobials.
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark.|Department of Biomedical Sciences, Ross University School of Veterinary Medicine, Basseterre, St Kitts, West Indies.
authors.forename: Bimal
authors.initials: B
authors.lastname: Jana
authors.affiliation: Pathogen Genomics, Wellcome Trust Sanger Institute, Cambridge, UK.
authors.forename: Amy K
authors.initials: AK
authors.lastname: Cain
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, USA.
authors.forename: William T
authors.initials: WT
authors.lastname: Doerrler
authors.affiliation: Pathogen Genomics, Wellcome Trust Sanger Institute, Cambridge, UK.
authors.forename: Christine J
authors.initials: CJ
authors.lastname: Boinett
authors.affiliation: Pathogen Genomics, Wellcome Trust Sanger Institute, Cambridge, UK.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Fookes
authors.affiliation: Pathogen Genomics, Wellcome Trust Sanger Institute, Cambridge, UK.
authors.forename: Julian
authors.initials: J
authors.lastname: Parkhill
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark.|Department of Biomedical Sciences, Ross University School of Veterinary Medicine, Basseterre, St Kitts, West Indies.
authors.forename: Luca
authors.initials: L
authors.lastname: Guardabassi
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002874:Chromosome Mapping; D004251:DNA Transposable Elements; D024901:Drug Resistance, Multiple, Bacterial; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D006801:Humans; D007710:Klebsiella Infections / Q000382:microbiology*; D007711:Klebsiella pneumoniae / Q000187:drug effects* / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D016254:Mutagenesis, Insertional; D009154:Mutation; D000073336:Whole Genome Sequencing
chemical_list: D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements