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Hughes, Diarmaid
Publications (10 of 115) Show all publications
Cao, S., Huseby, D. L., Brandis, G. & Hughes, D. (2017). Alternative Evolutionary Pathways for Drug-Resistant Small Colony Variant Mutants in Staphylococcus aureus. mBio, 8(3), Article ID e00358-17.
Open this publication in new window or tab >>Alternative Evolutionary Pathways for Drug-Resistant Small Colony Variant Mutants in Staphylococcus aureus
2017 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 3, article id e00358-17Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
AMER SOC MICROBIOLOGY, 2017
Keyword
ATP, experimental evolution, growth compensation, hemin biosynthesis, menaquinone, SrrAB, transcriptional regulation, translational suppression
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-330055 (URN)10.1128/mBio.00358-17 (DOI)000404733300009 ()
Available from: 2017-11-16 Created: 2017-11-16 Last updated: 2017-11-16Bibliographically approved
Banin, E., Hughes, D. & Kuipers, O. P. (2017). Bacterial pathogens, antibiotics and antibiotic resistance: Editorial. FEMS Microbiology Reviews, 41(3), 450-452, Article ID fux016.
Open this publication in new window or tab >>Bacterial pathogens, antibiotics and antibiotic resistance: Editorial
2017 (English)In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 41, no 3, p. 450-452, article id fux016Article in journal, Editorial material (Other academic) Published
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-332902 (URN)10.1093/femsre/fux016 (DOI)000402064900012 ()28486583 (PubMedID)
Available from: 2017-11-06 Created: 2017-11-06 Last updated: 2017-11-06Bibliographically approved
Nielsen, E. I., Khan, D. D., Cao, S., Lustig, U., Hughes, D., Andersson, D. I. & Friberg, L. E. (2017). Can a pharmacokinetic/pharmacodynamic (PKPD) model be predictive across bacterial densities and strains?: External evaluation of a PKPD model describing longitudinal in vitro data. Journal of Antimicrobial Chemotherapy, 72(11), 3108-3116
Open this publication in new window or tab >>Can a pharmacokinetic/pharmacodynamic (PKPD) model be predictive across bacterial densities and strains?: External evaluation of a PKPD model describing longitudinal in vitro data
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2017 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 72, no 11, p. 3108-3116Article in journal (Refereed) Published
Abstract [en]

Background: Pharmacokinetic/pharmacodynamic (PKPD) models developed based on data from in vitro time-kill experiments have been suggested to contribute to more efficient drug development programmes and better dosing strategies for antibiotics. However, for satisfactory predictions such models would have to show good extrapolation properties. Objectives: To evaluate if a previously described mechanism-based PKPD model was able also to predict drug efficacy for higher bacterial densities and across bacterial strains. Methods: A PKPD model describing the efficacy of ciprofloxacin on Escherichia coli was evaluated. The predictive performance of the model was evaluated across several experimental conditions with respect to: (i) bacterial start inoculum ranging from the standard of similar to 10(6) cfu/mL up to late stationary-phase cultures; and (ii) efficacy for seven additional strains (three laboratory and four clinical strains), not included during the model development process, based only on information regarding their MIC. Model predictions were performed according to the intended experimental protocol and later compared with observed bacterial counts. Results: The mechanism-based PKPD model structure developed based on data from standard start inoculum experiments was able to accurately describe the inoculum effect. The model successfully predicted the time course of drug efficacy for additional laboratory and clinical strains based on only the MIC values. The model structure was further developed to better describe the stationary phase data. Conclusions: This study supports the use of mechanism-based PKPD models based on preclinical data for predictions of untested scenarios.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS, 2017
National Category
Pharmaceutical Sciences Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-340705 (URN)10.1093/jac/dkx269 (DOI)000413464200019 ()28961946 (PubMedID)
Funder
Swedish Foundation for Strategic Research
Available from: 2018-02-21 Created: 2018-02-21 Last updated: 2018-02-21Bibliographically approved
Pietsch, F., Bergman, J. M., Brandis, G., Marcusson, L. L., Zorzet, A., Huseby, D. L. & Hughes, D. (2017). Ciprofloxacin selects for RNA polymerase mutations with pleiotropic antibiotic resistance effects. Journal of Antimicrobial Chemotherapy, 72(1), 75-84
Open this publication in new window or tab >>Ciprofloxacin selects for RNA polymerase mutations with pleiotropic antibiotic resistance effects
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2017 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 72, no 1, p. 75-84Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS, 2017
National Category
Infectious Medicine Microbiology in the medical area Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-319816 (URN)10.1093/jac/dkw364 (DOI)000394038700010 ()27621175 (PubMedID)
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research Knut and Alice Wallenberg Foundation
Available from: 2017-04-10 Created: 2017-04-10 Last updated: 2018-01-13Bibliographically approved
De Rosa, M., Lu, L., Zamaratski, E., Szałaj, N., Cao, S., Wadensten, H., . . . Karlen, A. (2017). Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB). Bioorganic & Medicinal Chemistry, 25(3), 897-911
Open this publication in new window or tab >>Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB)
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2017 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 25, no 3, p. 897-911Article in journal (Refereed) Published
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.

Keyword
Antibacterials, Escherichia coli, Oligopeptides, Solid-phase peptide synthesis, Type I signal peptidase
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-314110 (URN)10.1016/j.bmc.2016.12.003 (DOI)000394201900009 ()28038943 (PubMedID)
Funder
Swedish Research Council, 521-2014-6711 521-2013-2904 521-2013-3105 621-2014-6215Swedish Foundation for Strategic Research , RIF14-0078Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

Maria De Rosa and Lu Lu contributed equally to this work.

Available from: 2017-01-27 Created: 2017-01-27 Last updated: 2018-01-13Bibliographically approved
Petursdottir, D. H., Nordlander, S., Qazi, K. R., Carvalho-Queiroz, C., Osman, O. A., Hell, E., . . . Sverremark-Ekstrom, E. (2017). Early-Life Human Microbiota Associated With Childhood Allergy Promotes the T Helper 17 Axis in Mice. Frontiers in Immunology, 8, Article ID 1699.
Open this publication in new window or tab >>Early-Life Human Microbiota Associated With Childhood Allergy Promotes the T Helper 17 Axis in Mice
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2017 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 8, article id 1699Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2017
Keyword
infant microbiota, allergic heredity, immune development, germ-free, T helper 17-responsesIN
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-343555 (URN)10.3389/fimmu.2017.01699 (DOI)000416803000001 ()
Funder
Swedish Research Council, 2013-02904Swedish Heart Lung FoundationTorsten Söderbergs stiftelseStiftelsen Olle Engkvist ByggmästareCarl Tryggers foundation Swedish Research Council, 2016-04449
Available from: 2018-03-02 Created: 2018-03-02 Last updated: 2018-03-02Bibliographically approved
Hughes, D. & Andersson, D. I. (2017). Environmental and genetic modulation of the phenotypic expression of antibiotic resistance. FEMS Microbiology Reviews, 41(3), 374-391, Article ID fux004.
Open this publication in new window or tab >>Environmental and genetic modulation of the phenotypic expression of antibiotic resistance
2017 (English)In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 41, no 3, p. 374-391, article id fux004Article, review/survey (Refereed) Published
Abstract [en]

Antibiotic resistance can be acquired by mutation or horizontal transfer of a resistance gene, and generally an acquired mechanism results in a predictable increase in phenotypic resistance. However, recent findings suggest that the environment and/or the genetic context can modify the phenotypic expression of specific resistance genes/mutations. An important implication from these findings is that a given genotype does not always result in the expected phenotype. This dissociation of genotype and phenotype has important consequences for clinical bacteriology and for our ability to predict resistance phenotypes from genetics and DNA sequences. A related problem concerns the degree to which the genes/mutations currently identified in vitro can fully explain the in vivo resistance phenotype, or whether there is a significant additional amount of presently unknown mutations/genes (genetic 'dark matter') that could contribute to resistance in clinical isolates. Finally, a very important question is whether/how we can identify the genetic features that contribute to making a successful pathogen, and predict why some resistant clones are very successful and spread globally? In this review, we describe different environmental and genetic factors that influence phenotypic expression of antibiotic resistance genes/mutations and how this information is needed to understand why particular resistant clones spread worldwide and to what extent we can use DNA sequences to predict evolutionary success.

Keyword
persisters, pan genome, epistasis, successful clones, heteroresistance, virulence
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-332901 (URN)10.1093/femsre/fux004 (DOI)000402064900008 ()28333270 (PubMedID)
Available from: 2017-11-06 Created: 2017-11-06 Last updated: 2017-11-06Bibliographically approved
Hughes, D. & Andersson, D. I. (2017). Evolutionary Trajectories to Antibiotic Resistance (71ed.). Annual Review of Microbiology, 71, 579-596
Open this publication in new window or tab >>Evolutionary Trajectories to Antibiotic Resistance
2017 (English)In: Annual Review of Microbiology, ISSN 0066-4227, E-ISSN 1545-3251, Vol. 71, p. 579-596Article, review/survey (Refereed) Published
Abstract [en]

The ability to predict the evolutionary trajectories of antibiotic resistance would be of great value in tailoring dosing regimens of antibiotics so as to maximize the duration of their usefulness. Useful prediction of resistance evolution requires information about (a) the mutation supply rate, (b) the level of resistance conferred by the resistance mechanism, (c) the fitness of the antibiotic-resistant mutant bacteria as a function of drug concentration, and (d) the strength of selective pressures. In addition, processes including epistatic interactions and compensatory evolution, coselection of drug resistances, and population bottlenecks and clonal interference can strongly influence resistance evolution and thereby complicate attempts at prediction. Currently, the very limited quantitative data on most of these parameters severely limit attempts to accurately predict trajectories of resistance evolution.

Keyword
epistasis, mutation rates, population bottlenecks, relative fitness, coselection, selection pressure
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-347307 (URN)10.1146/annurev-micro-090816-093813 (DOI)000411800600030 ()28697667 (PubMedID)978-0-8243-1171-1 (ISBN)
Available from: 2018-03-29 Created: 2018-03-29 Last updated: 2018-03-29Bibliographically approved
Praski Alzrigat, L., Huseby, D. L., Brandis, G. & Hughes, D. (2017). 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, Soxs. Journal of Antimicrobial Chemotherapy, 2(11), 3016-3024
Open this publication in new window or tab >>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, Soxs
2017 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 2, no 11, p. 3016-3024Article in journal (Refereed) Published
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.

National Category
Microbiology in the medical area
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-320003 (URN)10.1093/jac/dkx270 (DOI)000413464200006 ()28962020 (PubMedID)
Funder
Swedish Research Council, 2013-02904Swedish Research Council, 2016-04449
Available from: 2017-04-21 Created: 2017-04-21 Last updated: 2018-02-02Bibliographically approved
Kacar, B., Garmendia, E., Tuncbag, N., Andersson, D. I. & Hughes, D. (2017). Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs. mBio, 8(4), Article ID e01276-17.
Open this publication in new window or tab >>Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs
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2017 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 4, article id e01276-17Article in journal (Refereed) Published
Abstract [en]

Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreign tuf genes are transferable to the E. coli genome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coli genome. However, when the tufB gene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tuf with its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tuf partially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.IMPORTANCE Horizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacing tuf with ancestral homologs and homologs from distantly related organisms, we investigated the constraints on HGT of a highly conserved gene with multiple interaction partners. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 bya. Only variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth, demonstrating the limited functional interchangeability of E. coli tuf with its homologs. Our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.

Keyword
EF-Tu, ancient genes, horizontal gene transfer, proteobacteria, tuf
National Category
Microbiology
Research subject
Biology with specialization in Microbiology; Biology with specialization in Molecular Biology; Biology with specialization in Evolutionary Genetics
Identifiers
urn:nbn:se:uu:diva-330501 (URN)10.1128/mBio.01276-17 (DOI)000409384300045 ()28851849 (PubMedID)
Available from: 2017-10-02 Created: 2017-10-02 Last updated: 2017-12-19Bibliographically approved
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