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  • 1.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Having your cake and eating it - Staphylococcus aureus small colony variants can evolve faster growth rate without losing their antibiotic resistance2017In: MICROBIAL CELL, ISSN 2311-2638, Vol. 4, no 8, p. 275-277Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus can produce small colony variants (SCVs) during infections. These cause significant clinical problems because they are difficult to detect in standard microbiological screening and are associated with persistent infections. The major causes of the SCV phenotype are mutations that inhibit respiration by inactivation of genes of the menadione or hemin biosynthesis pathways. This reduces the production of ATP required to support fast growth. Importantly, it also decreases cross-membrane potential in SCVs, resulting in decreased uptake of cationic compounds, with reduced susceptibility to aminoglycoside antibiotics as a consequence. Because SCVs are slow-growing (mutations in men genes are associated with growth rates in rich medium similar to 30% of the wild-type growth rate) bacterial cultures are very susceptible to rapid takeover by faster-growing mutants (revertants or suppressors). In the case of reversion, the resulting fast growth is obviously associated with the loss of antibiotic resistance. However, direct reversion is relatively rare due to the very small genetic target size for such mutations. We explored the phenotypic consequences of SCVs evolving faster growth by routes other than direct reversion, and in particular whether any of those routes allowed for the maintenance of antibiotic resistance. In a recent paper (mBio 8: e00358-17) we demonstrated the existence of several different routes of SCV evolution to faster growth, one of which maintained the antibiotic resistance phenotype. This discovery suggests that SCVs might be more adaptable and problematic that previously thought. They are capable of surviving as a slow-growing persistent form, before evolving into a significantly faster-growing form without sacrificing their antibiotic resistance phenotype.

  • 2.
    Cao, Sha
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandis, Gerrit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Alternative Evolutionary Pathways for Drug-Resistant Small Colony Variant Mutants in Staphylococcus aureus2017In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 3, article id e00358-17Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus is known to generate small colony variants (SCVs) that are resistant to aminoglycoside antibiotics and can cause persistent and recurrent infections. The SCV phenotype is unstable, and compensatory mutations lead to restored growth, usually with loss of resistance. However, the evolution of improved growth, by mechanisms that avoid loss of antibiotic resistance, is very poorly understood. By selection with serial passaging, we isolated and characterized different classes of extragenic suppressor mutations that compensate for the slow growth of small colony variants. Compensation occurs by two distinct bypass mechanisms: (i) translational suppression of the initial SCV mutation by mutant tRNAs, ribosomal protein S5, or release factor 2 and (ii) mutations that cause the constitutive activation of the SrrAB global transcriptional regulation system. Although compensation by translational suppression increases growth rate, it also reduces antibiotic susceptibility, thus restoring a pseudo-wild-type phenotype. In contrast, an evolutionary pathway that compensates for the SCV phenotype by activation of SrrAB increases growth rate without loss of antibiotic resistance. RNA sequence analysis revealed that mutations activating the SrrAB pathway cause upregulation of genes involved in peptide transport and in the fermentation pathways of pyruvate to generate ATP and NAD(+), thus explaining the increased growth. By increasing the growth rate of SCVs without the loss of aminoglycoside resistance, compensatory evolution via the SrrAB activation pathway represents a threat to effective antibiotic therapy of staphylococcal infections. IMPORTANCE Small colony variants (SCVs) of Staphylococcus aureus are a significant clinical problem, causing persistent and antibiotic-resistant infections. However, SCVs are unstable and can rapidly evolve growth-compensated mutants. Previous data suggested that growth compensation only occurred with the loss of antibiotic resistance. We have used selection with serial passaging to uncover four distinct pathways of growth compensation accessible to SCVs. Three of these paths (reversion, intragenic suppression, and translational suppression) increase growth at the expense of losing antibiotic resistance. The fourth path activates an alternative transcriptional program and allows the bacteria to produce the extra ATP required to support faster growth, without losing antibiotic resistance. The importance of this work is that it shows that drug-resistant SCVs can evolve faster growth without losing antibiotic resistance.

  • 3.
    De Rosa, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lu, Lu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Zamaratski, Edouard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Szałaj, Natalia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wadensten, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lenhammar, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gising, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Roos, Annette K.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Karlen, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB)2017In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 25, no 3, p. 897-911Article in journal (Refereed)
    Abstract [en]

    Type I signal peptidases are potential targets for the development of new antibacterial agents. Here we report finding potent inhibitors of E. coli type I signal peptidase (LepB), by optimizing a previously reported hit compound, decanoyl-PTANA-CHO, through modifications at the N- and C-termini. Good improvements of inhibitory potency were obtained, with IC50s in the low nanomolar range. The best inhibitors also showed good antimicrobial activity, with MICs in the low μg/mL range for several bacterial species. The selection of resistant mutants provided strong support for LepB as the target of these compounds. The cytotoxicity and hemolytic profiles of these compounds are not optimal but the finding that minor structural changes cause the large effects on these properties suggests that there is potential for optimization in future studies.

  • 4.
    Garoff, Linnéa
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Praski Alzrigat, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Effect of aminoacyl-tRNA synthetase mutations on susceptibility to ciprofloxacin in Escherichia coli2018In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 73, no 12, p. 3285-3292Article in journal (Refereed)
    Abstract [en]

    Background: Chromosomal mutations that reduce ciprofloxacin susceptibility in Escherichia coli characteristically map to drug target genes (gyrAB and parCE), and genes encoding regulators of the AcrAB-TolC efflux pump. Mutations in RNA polymerase can also reduce susceptibility, by up-regulating the MdtK efflux pump.

    Objectives: We asked whether mutations in additional chromosomal gene classes could reduce susceptibility to ciprofloxacin.

    Methods: Experimental evolution, complemented by WGS analysis, was used to select and identify mutations that reduce susceptibility to ciprofloxacin. Transcriptome analysis, genetic reconstructions, susceptibility measurements and competition assays were used to identify significant genes and explore the mechanism of resistance.

    Results: Mutations in three different aminoacyl-tRNA synthetase genes (leuS, aspS and thrS) were shown to re- duce susceptibility to ciprofloxacin. For two of the genes (leuS and aspS) the mechanism was partially dependent on RelA activity. Two independently selected mutations in leuS (Asp162Asn and Ser496Pro) were studied in most detail, revealing that they induce transcriptome changes similar to a stringent response, including up-regulation of three efflux-associated loci (mdtK, acrZ and ydhJK). Genetic analysis showed that reduced susceptibility depended on the activity of these loci. Broader antimicrobial susceptibility testing showed that the leuS mutations also reduce susceptibility to additional classes of antibiotics chloramphenicol, rifampicin, mecillinam, ampicillin and trimethoprim).

    Conclusions: The identification of mutations in multiple tRNA synthetase genes that reduce susceptibility to ciprofloxacin and other antibiotics reveals the existence of a large mutational target that could contribute to re- sistance development by up-regulation of an array of efflux pumps.

  • 5.
    Huseby, Douglas L
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Methods to determine mutational trajectories after experimental evolution of antibiotic resistance.2018In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 1736, p. 95-103Article in journal (Refereed)
    Abstract [en]

    The evolution of bacterial resistance to antibiotics by mutation within the genome (as distinct from horizontal gene transfer of new material into a genome) could occur in a single step but is usually a multistep process. Resistance evolution can be studied in laboratory environments by serial passage of bacteria in liquid culture or on agar, with selection at constant, or varying, concentrations of drug. Whole genome sequencing can be used to make an initial analysis of the evolved mutants. The trajectory of evolution can be determined by sequence analysis of strains from intermediate steps in the evolution, complemented by phenotypic analysis of genetically reconstructed isogenic strains that recapitulate the intermediate steps in the evolution.

  • 6.
    Huseby, Douglas L.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pietsch, Franziska
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandis, Gerrit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Garoff, Linnéa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tegehall, Angelica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mutation supply and relative fitness shape the genotypes of ciprofloxacin-resistant Escherichia coli2017In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 34, no 5, p. 1029-1039Article in journal (Refereed)
    Abstract [en]

    Ciprofloxacin is an important antibacterial drug targeting Type II topoisomerases, highly active against Gram-negatives including Escherichia coli. The evolution of resistance to ciprofloxacin in E. coli always requires multiple genetic changes, usually including mutations affecting two different drug target genes, gyrA and parC. Resistant mutants selected in vitro or in vivo can have many different mutations in target genes and efflux regulator genes that contribute to resistance. Among resistant clinical isolates the genotype, gyrA S83L D87N, parC S80I is significantly overrepresented suggesting that it has a selective advantage. However, the evolutionary or functional significance of this high frequency resistance genotype is not fully understood. By combining experimental data and mathematical modeling, we addressed the reasons for the predominance of this specific genotype. The experimental data were used to model trajectories of mutational resistance evolution under different conditions of drug exposure and population bottlenecks. We identified the order in which specific mutations are selected in the clinical genotype, showed that the high frequency genotype could be selected over a range of drug selective pressures, and was strongly influenced by the relative fitness of alternative mutations and factors affecting mutation supply. Our data map for the first time the fitness landscape that constrains the evolutionary trajectories taken during the development of clinical resistance to ciprofloxacin and explain the predominance of the most frequently selected genotype. This study provides strong support for the use of in vitro competition assays as a tool to trace evolutionary trajectories, not only in the antibiotic resistance field.

  • 7.
    Jeannot, Frédéric
    et al.
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Taillier, Thomas
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Despeyroux, Pierre
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Renard, Stéphane
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Rey, Astrid
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Mourez, Michaël
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Poeverlein, Christoph
    Sanofi Aventis Deutschland GmbH, Integrated Drug Discovery, R&D, Ind Pk Hoechst, D-65926 Frankfurt, Germany.
    Khichane, Imène
    Sanofi R&D, Analyt Sci, LGCR, 13 Quai Jules Guesde, F-94400 Vitry Sur Seine, France.
    Perrin, Marc-Antoine
    Sanofi R&D, Analyt Sci, LGCR, 13 Quai Jules Guesde, F-94400 Vitry Sur Seine, France.
    Versluys, Stéphanie
    Evotec France, 195 Route Espagne,BP 13669, F-31036 Toulouse 1, France.
    Stavenger, Robert A.
    GlaxoSmithKline, Antibacterial DPU, 1250 Collegeville Rd, Collegeville, PA 19426 USA.
    Huang, Jianzhong
    GlaxoSmithKline, Antibacterial DPU, 1250 Collegeville Rd, Collegeville, PA 19426 USA.
    Germe, Thomas
    John Innes Ctr, Dept Biol Chem, Norwich Res Pk, Norwich NR4 7UH, Norfolk, England.
    Maxwell, Anthony
    John Innes Ctr, Dept Biol Chem, Norwich Res Pk, Norwich NR4 7UH, Norfolk, England.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bacqué, Eric
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Imidazopyrazinones (IPYs): Non-Quinolone Bacterial Topoisomerase Inhibitors Showing Partial Cross-Resistance with Quinolones2018In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 61, no 8, p. 3565-3581Article in journal (Refereed)
    Abstract [en]

    In our quest for new antibiotics able to address the growing threat of multidrug resistant infections caused by Gram-negative bacteria, we have investigated an unprecedented series of non-quinolone bacterial topoisomerase inhibitors from the Sanofi patrimony, named IPYs for imidazopyrazinones, as part of the Innovative Medicines Initiative (IMI) European Gram Negative Antibacterial Engine (ENABLE) organization. Hybridization of these historical compounds with the quinazolinediones, a known series of topoisomerase inhibitors, led us to a novel series of tricyclic IPYs that demonstrated potential for broad spectrum activity, in vivo efficacy, and a good developability profile, although later profiling revealed a genotoxicity risk. Resistance studies revealed partial cross-resistance with fluoroquinolones (FQs) suggesting that IPYs bind to the same region of bacterial topoisomerases as FQs and interact with at least some of the keys residues involved in FQ binding.

  • 8.
    Juhas, Mario
    et al.
    Univ Zurich, Inst Med Microbiol, Gloriastr 30, CH-8006 Zurich, Switzerland.
    Widlake, Emma
    Cardiff Univ, Sch Med, Div Infect & Immun, Cardiff CF14 4XN, S Glam, Wales.
    Teo, Jeanette
    Natl Univ Singapore Hosp, Dept Lab Med, 5 Lower Kent Ridge Rd, Singapore 119074, Singapore.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tyrrell, Jonathan M.
    Cardiff Univ, Sch Med, Div Infect & Immun, Cardiff CF14 4XN, S Glam, Wales.
    Polikanov, Yury S.
    Univ Illinois, Coll Liberal Arts & Sci, Dept Biol Sci, 900 South Ashland Ave,MBRB 4170, Chicago, IL 60607 USA.
    Ercan, Onur
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Petersson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Aboklaish, Ali F.
    Cardiff Univ, Sch Med, Div Infect & Immun, Cardiff CF14 4XN, S Glam, Wales.
    Rominski, Anna
    Univ Zurich, Inst Med Microbiol, Gloriastr 30, CH-8006 Zurich, Switzerland.
    Crich, David
    Wayne State Univ, Dept Chem, 5101 Cass Ave, Detroit, MI 48202 USA.
    Bottger, Erik C.
    Univ Zurich, Inst Med Microbiol, Gloriastr 30, CH-8006 Zurich, Switzerland.
    Walsh, Timothy R.
    Cardiff Univ, Sch Med, Div Infect & Immun, Cardiff CF14 4XN, S Glam, Wales.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hobbie, Sven N.
    Univ Zurich, Inst Med Microbiol, Gloriastr 30, CH-8006 Zurich, Switzerland.
    In vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii2019In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 74, no 4, p. 944-952Article in journal (Refereed)
    Abstract [en]

    Objectives: Widespread antimicrobial resistance often limits the availability of therapeutic options to only a few last-resort drugs that are themselves challenged by emerging resistance and adverse side effects. Apramycin, an aminoglycoside antibiotic, has a unique chemical structure that evades almost all resistance mechanisms including the RNA methyltransferases frequently encountered in carbapenemase-producing clinical isolates. This study evaluates the in vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii, and provides a rationale for its superior antibacterial activity in the presence of aminoglycoside resistance determinants.

    Methods: A thorough antibacterial assessment of apramycin with 1232 clinical isolates from Europe, Asia, Africa and South America was performed by standard CLSI broth microdilution testing. WGS and susceptibility testing with an engineered panel of aminoglycoside resistance-conferring determinants were used to provide a mechanistic rationale for the breadth of apramycin activity.

    Results: MIC distributions and MIC90 values demonstrated broad antibacterial activity of apramycin against Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Morganella morganii, Citrobacter freundii, Providencia spp., Proteus mirabilis, Serratia marcescens and A. baumannii. Genotypic analysis revealed the variety of aminoglycoside-modifying enzymes and rRNA methyltransferases that rendered a remarkable proportion of clinical isolates resistant to standard-of-care aminoglycosides, but not to apramycin. Screening a panel of engineered strains each with a single well-defined resistance mechanism further demonstrated a lack of cross-resistance to gentamicin, amikacin, tobramycin and plazomicin.

    Conclusions: Its superior breadth of activity renders apramycin a promising drug candidate for the treatment of systemic Gram-negative infections that are resistant to treatment with other aminoglycoside antibiotics.

  • 9.
    Pantel, Lucile
    et al.
    Nosopharm, Espace Innovat 2, 110 Allee Charles Babbage, F-30000 Nimes, France.
    Florin, Tanja
    Univ Illinois, Ctr Biomol Sci, Chicago, IL 60607 USA.
    Dobosz-Bartoszek, Malgorzata
    Univ Illinois, Dept Biol Sci, Chicago, IL 60607 USA.
    Racine, Emilie
    Nosopharm, Espace Innovat 2, 110 Allee Charles Babbage, F-30000 Nimes, France.
    Sarciaux, Matthieu
    Nosopharm, Espace Innovat 2, 110 Allee Charles Babbage, F-30000 Nimes, France.
    Serri, Marine
    Nosopharm, Espace Innovat 2, 110 Allee Charles Babbage, F-30000 Nimes, France.
    Houard, Jessica
    Nosopharm, Espace Innovat 2, 110 Allee Charles Babbage, F-30000 Nimes, France.
    Campagne, Jean-Marc
    CNRS, ENSCM, Inst Charles Gerhardt Montpellier, UMR 5253,UM, Montpellier, France.
    de Figueiredo, Renata Marcia
    CNRS, ENSCM, Inst Charles Gerhardt Montpellier, UMR 5253,UM, Montpellier, France.
    Midrier, Camille
    CNRS, ENSCM, Inst Charles Gerhardt Montpellier, UMR 5253,UM, Montpellier, France.
    Gaudriault, Sophie
    Univ Montpellier, INRA, DGIMI, Montpellier, France.
    Givaudan, Alain
    Univ Montpellier, INRA, DGIMI, Montpellier, France.
    Lanois, Anne
    Univ Montpellier, INRA, DGIMI, Montpellier, France.
    Forst, Steve
    Univ Wisconsin, Dept Biol Sci, POB 413, Milwaukee, WI 53201 USA.
    Aumelas, Andre
    Nosopharm, Espace Innovat 2, 110 Allee Charles Babbage, F-30000 Nimes, France.
    Cotteaux-Lautard, Christelle
    Aix Marseille Univ, IRBA, Fac Med, TMCD2,UMR MD1, Marseille, France.
    Bolla, Jean-Michel
    Aix Marseille Univ, IRBA, Fac Med, TMCD2,UMR MD1, Marseille, France.
    Lundberg, Carina Vingsbo
    Statens Serum Inst, Bacteria Parasites & Fungi, DK-2300 Copenhagen, Denmark.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Villain-Guillot, Philippe
    Nosopharm, Espace Innovat 2, 110 Allee Charles Babbage, F-30000 Nimes, France.
    Mankin, Alexander S.
    Univ Illinois, Ctr Biomol Sci, Chicago, IL 60607 USA.
    Polikanov, Yury S.
    Univ Illinois, Dept Biol Sci, Chicago, IL 60607 USA;Univ Illinois, Dept Med Chem & Pharmacognosy, Chicago, IL 60607 USA.
    Gualtieri, Maxime
    Nosopharm, Espace Innovat 2, 110 Allee Charles Babbage, F-30000 Nimes, France.
    Odilorhabdins, Antibacterial Agents that Cause Miscoding by Binding at a New Ribosomal Site2018In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 70, no 1, p. 83-94Article in journal (Refereed)
    Abstract [en]

    Growing resistance of pathogenic bacteria and shortage of antibiotic discovery platforms challenge the use of antibiotics in the clinic. This threat calls for exploration of unconventional sources of antibiotics and identification of inhibitors able to eradicate resistant bacteria. Here we describe a different class of antibiotics, odilorhabdins (ODLs), produced by the enzymes of the non-ribosomal peptide synthetase gene cluster of the nematode-symbiotic bacterium Xenorhabdus nematophila. ODLs show activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae, and can eradicate infections in animal models. We demonstrate that the bactericidal ODLs interfere with protein synthesis. Genetic and structural analyses reveal that ODLs bind to the small ribosomal subunit at a site not exploited by current antibiotics. ODLs induce miscoding and promote hungry codon readthrough, amino acid misincorporation, and premature stop codon bypass. We propose that ODLs' miscoding activity reflects their ability to increase the affinity of non-cognate aminoacyl-tRNAs to the ribosome.

  • 10.
    Petursdottir, Dagbjort H.
    et al.
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Nordlander, Sofia
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Qazi, Khaleda Rahman
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Carvalho-Queiroz, Claudia
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Osman, Omneya Ahmed
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Hell, Eva
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Bjorkander, Sophia
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Haileselassie, Yeneneh
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Navis, Marit
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Kokkinou, Efthymia
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Lio, Ivan Zong Long
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Hennemann, Julia
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Brodin, Bjorn
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Huseby, Douglas L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nilsson, Caroline
    Karolinska Inst, Sodersjukhuset, Dept Clin Sci & Educ, Stockholm, Sweden.;Sachs Childrens Hosp, Stockholm, Sweden..
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Udekwu, Klas I.
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Sverremark-Ekstrom, Eva
    Stockholm Univ, Arrhenius Labs Nat Sci, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Early-Life Human Microbiota Associated With Childhood Allergy Promotes the T Helper 17 Axis in Mice2017In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 8, article id 1699Article in journal (Refereed)
    Abstract [en]

    The intestinal microbiota influences immune maturation during childhood, and is implicated in early-life allergy development. However, to directly study intestinal microbes and gut immune responses in infants is difficult. To investigate how different types of early-life gut microbiota affect immune development, we collected fecal samples from children with different allergic heredity (AH) and inoculated germ-free mice. Immune responses and microbiota composition were evaluated in the offspring of these mice. Microbial composition in the small intestine, the cecum and the colon were determined by 16S rRNA sequencing. The intestinal microbiota differed markedly between the groups of mice, but only exposure to microbiota associated with AH and known future allergy in children resulted in a T helper 17 (Th17)-signature, both systemically and in the gut mucosa in the mouse offspring. These Th17 responses could be signs of a particular microbiota and a shift in immune development, ultimately resulting in an increased risk of allergy.

  • 11.
    Pietsch, Franziska
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bergman, Jessica M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandis, Gerrit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Marcusson, Linda L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Zorzet, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ciprofloxacin selects for RNA polymerase mutations with pleiotropic antibiotic resistance effects2017In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 72, no 1, p. 75-84Article in journal (Refereed)
    Abstract [en]

    Objectives: Resistance to the fluoroquinolone drug ciprofloxacin is commonly linked to mutations that alter the drug target or increase drug efflux via the major AcrAB-TolC transporter. Very little is known about other mutations that might also reduce susceptibility to ciprofloxacin. We discovered that an Escherichia coli strain experimentally evolved for resistance to ciprofloxacin had acquired a mutation in rpoB, the gene coding for the beta-subunit of RNA polymerase. The aim of this work was to determine whether this mutation, and other mutations in rpoB, contribute to ciprofloxacin resistance and, if so, by which mechanism. Methods: Independent lineages of E. coli were evolved in the presence of ciprofloxacin and clones from endpoint cultures were screened for mutations in rpoB. Ciprofloxacin-selected rpoB mutations were identified and characterized in terms of effects on susceptibility and mode of action. Results: Mutations in rpoB were selected at a high frequency in 3 out of 10 evolved lineages, in each case arising after the occurrence of mutations affecting topoisomerases and drug efflux. All ciprofloxacin-selected rpoB mutations had a high fitness cost in the absence of drug, but conferred a competitive advantage in the presence of ciprofloxacin. RNA sequencing and quantitative RT-PCR analysis showed that expression of mdtK, encoding a multidrug efflux transporter, was significantly increased by the ciprofloxacin-selected rpoB mutations. The susceptibility phenotype was shown to depend on the presence of an active mdtK and a mutant rpoB allele. Conclusions: These data identify mutations in RNA polymerase as novel contributors to the evolution of resistance to ciprofloxacin and show that the phenotype is mediated by increased MdtK-dependent drug efflux.

  • 12.
    Praski Alzrigat, Lisa
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandis, Gerrit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fitness cost constrains the spectrum of marR mutations in ciprofloxacin-resistant Escherichia coli: Multiple Antibiotic-Resistance, Gram-Negative Bacteria, Multidrug Efflux Pump, Urinary-Tract-Infections, Fluoroquinolone Resistance, Quinolone Resistance, Mechanisms, Expression, Sequence, Soxs2017In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 2, no 11, p. 3016-3024Article in journal (Refereed)
    Abstract [en]

    Objectives: To determine whether the spectrum of mutations in marR in ciprofloxacin-resistant clinical isolates of Escherichia coli shows evidence of selection bias, either to reduce fitness costs, or to increase drug resistance. MarR is a repressor protein that regulates, via MarA, expression of the Mar regulon, including the multidrug efflux pump AcrAB-TolC. Methods: Isogenic strains carrying 36 different marR alleles identified in resistant clinical isolates, or selected for resistance in vitro, were constructed. Drug susceptibility and relative fitness in growth competition assays were measured for all strains. The expression level of marA, and of various efflux pump components, as a function of specific mutations in marR, was measured by qPCR. Results: The spectrum of genetic alterations in marR in clinical isolates is strongly biased against inactivating mutations. In general, the alleles found in clinical isolates conferred a lower level of resistance and imposed a lower growth fitness cost than mutations selected in vitro. The level of expression of MarA correlated well with the MIC of ciprofloxacin. This supports the functional connection between mutations in marR and reduced susceptibility to ciprofloxacin. Conclusions: Mutations in marR selected in ciprofloxacin-resistant clinical isolates are strongly biased against inactivating mutations. Selection favours mutant alleles that have the lowest fitness costs, even though these cause only modest reductions in drug susceptibility. This suggests that selection for high relative fitness is more important than selection for increased resistance in determining which alleles of marR will be selected in resistant clinical isolates.

  • 13. Vestö, Kim
    et al.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Snygg, Lina
    Wang, Helen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Karolinska Inst, Dept Microbiol Tumor & Cell Biol.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Rhen, Mikael
    Muramyl Endopeptidase Spr Contributes to Intrinsic Vancomycin Resistance in Salmonella enterica Serovar Typhimurium2018In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 2941Article in journal (Refereed)
    Abstract [en]

    The impermeability barrier provided by the outer membrane of enteric bacteria, a feature lacking in Gram-positive bacteria, plays a major role in maintaining resistance to numerous antimicrobial compounds and antibiotics. Here we demonstrate that mutational inactivation of spr, coding for a muramyl endopeptidase, significantly sensitizes Salmonella enterica serovar Typhimurium to vancomycin without any accompanying apparent growth defect or outer membrane destabilization. A similar phenotype was not achieved by deleting the genes coding for muramyl endopeptidases MepA, PbpG, NlpC, YedA, or YhdO. The spr mutant showed increased autolytic behavior in response to not only vancomycin, but also to penicillin G, an antibiotic for which the mutant displayed a wild-type MIC. A screen for suppressor mutations of the spr mutant phenotype revealed that deletion of tsp (prc), encoding a periplasmic carboxypeptidase involved in processing Spr and PBP3, restored intrinsic resistance to vancomycin and reversed the autolytic phenotype of the spr mutant. Our data suggest that Spr contributes to intrinsic antibiotic resistance in S. Typhimurium without directly affecting the outer membrane permeability barrier. Furthermore, our data suggests that compounds targeting specific cell wall endopeptidases might have the potential to expand the activity spectrum of traditional Gram-positive antibiotics.

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