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  • 1.
    Adler, Marlen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Anjum, Mehreen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 5, p. 1188-1198Article in journal (Refereed)
    Abstract [en]

    The worldwide spread of ESBL-producing Enterobacteriaceae has led to an increased use of carbapenems, the group of beta-lactams with the broadest spectrum of activity. Bacterial resistance to carbapenems is mainly due to acquired carbapenemases or a combination of ESBL production and reduced drug influx via loss of outer-membrane porins. Here, we have studied the development of carbapenem resistance in Escherichia coli in the absence of beta-lactamases. We selected mutants with high-level carbapenem resistance through repeated serial passage in the presence of increasing concentrations of meropenem or ertapenem for similar to 60 generations. Isolated clones were whole-genome sequenced, and the order in which the identified mutations arose was determined in the passaged populations. Key mutations were reconstructed, and bacterial growth rates of populations and isolated clones and resistance levels to 23 antibiotics were measured. High-level resistance to carbapenems resulted from a combination of downstream effects of envZ mutation and target mutations in AcrAB-TolC-mediated drug export, together with PBP genes [mrdA (PBP2) after meropenem exposure or ftsI (PBP3) after ertapenem exposure]. Our results show that antibiotic resistance evolution can occur via several parallel pathways and that new mechanisms may appear after the most common pathways (i.e. beta-lactamases and loss of porins) have been eliminated. These findings suggest that strategies to target the most commonly observed resistance mechanisms might be hampered by the appearance of previously unknown parallel pathways to resistance.

  • 2.
    Adler, Marlen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Anjum, Mehreen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Influence of acquired β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli2013In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 1, p. 51-59Article in journal (Refereed)
    Abstract [en]

    Objectives: To investigate the influence of plasmid-borne β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli and the fitness costs associated with resistance. Methods: Stepwise selection of carbapenem-resistant mutants with or without the extended-spectrum β-lactamase (ESBL)-encoding plasmid pUUH239.2 was performed. Mutation rates and mutational pathways to resistance were determined. In vitro-selected and constructed mutants were characterized regarding the MICs of the carbapenems, porin expression profiles, growth rates and the presence of mutations in the porins ompC/ompF and their regulatory genes. The influence of the plasmid-encoded β-lactamases TEM-1, OXA-1 and CTX-M-15 on resistance development was determined. Results: Results show that E. coli readily developed reduced carbapenem susceptibility and clinical resistance levels by a combination of porin loss and increased β-lactamase expression, especially towards ertapenem. All tested β-lactamases (CTX-M-15, TEM-1 and OXA-1) contributed to reduced carbapenem susceptibility in the absence of porin expression. However, complete loss of porin expression conferred a 20% fitness cost on the bacterial growth rate. Increased β-lactamase expression through spontaneous gene amplification on the plasmid was a major resistance factor. Conclusions: Plasmid-encoded β-lactamases, including non-ESBL enzymes, have a strong influence on the frequency and resistance level of spontaneous carbapenem-resistant mutants. The fitness cost associated with the loss of OmpC/OmpF in E. coli most likely reduces the survivability of porin mutants and could explain why they have not emerged as a clinical problem in this species.

  • 3.
    Adler, Marlen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Anjum, Mehreen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Berg, Otto, G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    High Fitness Costs and Instability of Gene Duplications Reduce Rates of Evolution of New Genes by Duplication-Divergence Mechanisms2014In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 31, no 6, p. 1526-1535Article in journal (Refereed)
    Abstract [sv]

    An important mechanism for generation of new genes is by duplication-divergence of existing genes. Duplication-divergence includes several different sub-models, such as subfunctionalization where after accumulation of neutral mutations the original function is distributed between two partially functional and complementary genes, and neofunctionalization where a new function evolves in one of the duplicated copies while the old function is maintained in another copy. The likelihood of these mechanisms depends on the longevity of the duplicated state, which in turn depends on the fitness cost and genetic stability of the duplications. Here, we determined the fitness cost and stability of defined gene duplications/amplifications on a low copy number plasmid. Our experimental results show that the costs of carrying extra gene copies are substantial and that each additional kbp of DNA reduces fitness by approximately 0.15%. Furthermore, gene amplifications are highly unstable and rapidly segregate to lower copy numbers in absence of selection. Mathematical modelling shows that the fitness costs and instability strongly reduces the likelihood of both sub- and neofunctionalization, but that these effects can be off-set by positive selection for novel beneficial functions.

  • 4.
    Albrecht, Lisa M
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cross-Resistance to Antibiotics After Exposure to Qaternary Ammonium CompoundsManuscript (preprint) (Other academic)
    Abstract [en]

    Quaternary ammonium compounds (QACs) are common antimicrobials that are used in a variety of consumer products, such as lotions, sunscreen, hair conditioners and hand sanitizers, to inhibit bacterial growth. However, it has been noted that bacteria exposed to QACs can develop resistance, and additionally, resistance to QACs has been observed to provide cross-resistance to antibiotics. In order to identify genetic adaptations for this resistance pattern, we exposed E. coli to three different QACs at sub-MIC and above-MIC concentrations, and identified genetic changes by whole genome sequencing. We found that initial adaptation, at sub-MIC levels, happened through efflux mechanisms, and that subsequent genetic changes, during above-MIC exposure, involved genes associated with the cell membranes and with transcription/translation. We also found that these genetic changes provided cross-resistance to other QACs as well as to several antibiotics.

  • 5.
    Albrecht, Lisa M
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Potentiation of the Selective Effect of Antibiotics by Metal IonsManuscript (preprint) (Other academic)
    Abstract [en]

    Complex mixtures of antibiotics and metals are present in many environments ranging from municipal sewage to irrigation water and manure used as agricultural fertilizer. Such mixtures of drugs and metals exert unique selection pressures on local bacterial communities and could function as hotspots for enrichment of antibiotic resistance genes. The presence of metals in the environment has previously been linked to increases in tolerance to antibiotics. In this study, we investigated metal-potentiated selection of antibiotic resistant Salmonella enterica strains. Six environmentally relevant metals were examined in combinations with three different antibiotics. By performing competitions between an antibiotic resistant mutant and the isogenic wild type in each metal-antibiotic combination, we assessed the minimal selective concentration (MSC) of the antibiotic for the resistant strain. The metals silver, cadmium and mercury all exhibited potentiating effects, reducing the MSC of the antibiotic up to 5-fold as compared to in the absence of the metal. We further show that the potentiating metals increased permeability of the cellular outer membrane. These results demonstrate that the presence of a metal can decrease the concentration of an antibiotic required to select for an antibiotic resistant strain, and they indicate that this process involves metal-facilitated uptake of the antibiotic following damage to the outer membrane.

  • 6.
    Albrecht, Lisa M
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mutation in the Copper-Induced sil Operon Enables High-Level Silver Resistance and Silver Facilitated Co-Selection of Multidrug Resistance PlasmidManuscript (preprint) (Other academic)
    Abstract [en]

    Human activities are responsible for an accumulation of metals in health care and agricultural environments, and plasmid-encoded metal tolerance operons enable bacteria to rapidly adapt to metal exposure under such conditions. While the mechanisms of action of many metal resistance systems have been described, there is still limited understanding of their role in co-selection of antibiotic resistance in metal-containing environments. Whether plasmid-encoded metal resistance genes confer significant selective advantages is of interest as it has implications for plasmid enrichment and the spread of plasmid-borne antibiotic resistance genes. To increase our understanding of plasmid-mediated metal resistance, we studied the sil operon and its phenotypes in E. coli during growth in the absence and presence of silver and copper. We found that the sil operon provides resistance to both silver and copper. However, it is induced by copper only, and constitutive expression due to point mutations in the two-component silS gene provides high-level silver resistance. Furthermore, we showed that a high-level silver resistant mutant could be enriched in the presence of silver. This enrichment entailed co-selection of the multidrug resistance plasmid pUUH239.2. Our results show that a copper resistance operon can provide high-level silver resistance following a single point mutation, and that the silver resistance phenotype subsequently can co-select for antibiotic resistance in the presence of silver. 

  • 7.
    Ancillotti, Mirko
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics.
    Eriksson, Stefan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics. Uppsala University, Disciplinary Domain of Humanities and Social Sciences, Faculty of Theology, Department of Theology.
    Veldwijk, Jorien
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics. Institute of Health Policy and Management, Erasmus University, Bayle (J) building - Campus Woudestein, Burgemeester Oudlaan 50, 3062 PA Rotterdam, The Netherlands.
    Nihlén Fahlquist, Jessica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Godskesen, Tove
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics. Department of Health Care Sciences, Ersta Sköndal Bräcke University College, Ersta Sköndal Bräcke högskola, Stigbergsgatan 30, 100 61 Stockholm, Sweden.
    Public awareness and individual responsibility needed for judicious use of antibiotics: a qualitative study of public beliefs and perceptions2018In: BMC Public Health, ISSN 1471-2458, E-ISSN 1471-2458, Vol. 18, no 1, article id 1153Article in journal (Refereed)
    Abstract [en]

    Background

    High consumption of antibiotics has been identified as an important driver for the increasing antibiotic resistance, considered to be one of the greatest threats to public health globally. Simply informing the public about this consequence is insufficient to induce behavioral change. This study explored beliefs and perceptions among Swedes, with the aim of identifying factors promoting and hindering a judicious approach to antibiotics use. The study focused primarily on the medical use of antibiotics, also considering other aspects connected with antibiotic resistance, such as travelling and food consumption.

    Methods

    Data were collected through focus group discussions at the end of 2016. Twenty-three Swedes were recruited using an area-based approach and purposive sampling, aiming for as heterogeneous groups as possible regarding gender (13 women, 10 men), age (range 20–81, mean 38), and education level. Interview transcripts were analyzed using qualitative content analysis. The Health Belief Model was used as a theoretical framework.

    Results

    Antibiotic resistance was identified by participants as a health threat with the potential for terrible consequences. The severity of the problem was perceived more strongly than the actual likelihood of being affected by it. Metaphors such as climate change were abundantly employed to describe antibiotic resistance as a slowly emerging problem. There was a tension between individual (egoistic) and collective (altruistic) reasons for engaging in judicious behavior. The individual effort needed and antibiotics overprescribing were considered major barriers to such behavior. In their discussions, participants stressed the need for empowerment, achieved through good health communication from authorities and family physicians.

    Conclusions

    Knowledge about antibiotic consumption and resistance, as well as values such as altruism and trust in the health care system, has significant influence on both perceptions of individual responsibility and on behavior. This suggests that these factors should be emphasized in health education and health promotion. To instead frame antibiotic resistance as a slowly emerging disaster, risks diminish the public perception of being susceptible to it.

  • 8.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Evolving promiscuously2011In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 4, p. 1199-1200Article in journal (Other academic)
  • 9.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Improving predictions of the risk of resistance development against new and old antibiotics2015In: Clinical Microbiology and Infection, ISSN 1198-743X, E-ISSN 1469-0691, Vol. 21, no 10, p. 894-898Article, review/survey (Refereed)
    Abstract [en]

    The methods used today by academic researchers and the pharmaceutical industry to assess the risk of emergence of resistance, for example during development of new antibiotics or when assessing an old antibiotic, are sub-optimal. Even though easy to perform, the presently used serial passage procedures, minimal prevention concentration measurements and determination of mutation rates in vitro are generally providing inadequate knowledge for risk assessment and making decisions to continue/discontinue drug development. These methods need to be complemented and replaced with more relevant methods such as determination of whether resistance genes already pre-exist in various metagenomes, and the likelihood that these genes can transfer into the relevant pathogens and be stably maintained. Furthermore, to determine the risk of emergence of mutationally conferred resistance the fitness effect of the resistance mechanism is key, as this parameter will determine the ability of the resistant mutants to be maintained and enriched in the host after they have emerged. This information combined with knowledge of bacterial population sizes and growth and killing dynamics at relevant infection sites should allow for better forecasting of the risk of resistance emerging in clinical settings.

  • 10.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Shrinking Bacterial Genomes: Former skeptics recognize that the genomes of microbial parasites and symbionts are subject to dynamic downsizing2008In: Microbe, ISSN 1558-7452, Vol. 3, no 3, p. 124-130Article in journal (Refereed)
  • 11.
    Andersson, Dan I
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The biological cost of mutational antibiotic resistance: any practical conclusions?2006In: Curr Opin Microbiol, ISSN 1369-5274, Vol. 9, no 5, p. 461-5Article in journal (Other scientific)
  • 12.
    Andersson, Dan I.
    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.
    Antibiotic resistance and its cost: is it possible to reverse resistance?2010In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 8, no 4, p. 260-271Article, review/survey (Refereed)
    Abstract [en]

    Most antibiotic resistance mechanisms are associated with a fitness cost that is typically observed as a reduced bacterial growth rate. The magnitude of this cost is the main biological parameter that influences the rate of development of resistance, the stability of the resistance and the rate at which the resistance might decrease if antibiotic use were reduced. These findings suggest that the fitness costs of resistance will allow susceptible bacteria to outcompete resistant bacteria if the selective pressure from antibiotics is reduced. Unfortunately, the available data suggest that the rate of reversibility will be slow at the community level. Here, we review the factors that influence the fitness costs of antibiotic resistance, the ways by which bacteria can reduce these costs and the possibility of exploiting them.

  • 13.
    Andersson, Dan I.
    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 Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Effects of antibiotic resistance on bacterial fitness, virulence and transmission2008In: Evolutionary biology of bacterial and fungal pathogens / [ed] Baquero, Fernando Nombela, Cesar Cassell, Gail H., Washington DC: ASM Press , 2008, p. 307-318Chapter in book (Refereed)
  • 14.
    Andersson, Dan I
    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 Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Evolution of antibiotic resistance at non-lethal drug concentrations.2012In: Drug resistance updates, ISSN 1368-7646, E-ISSN 1532-2084, Vol. 15, no 3, p. 162-172Article in journal (Refereed)
    Abstract [en]

    Human use of antimicrobials in the clinic, community and agricultural systems has driven selection for resistance in bacteria. Resistance can be selected at antibiotic concentrations that are either lethal or non-lethal, and here we argue that selection and enrichment for antibiotic resistant bacteria is often a consequence of weak, non-lethal selective pressures - caused by low levels of antibiotics - that operates on small differences in relative bacterial fitness. Such conditions may occur during antibiotic therapy or in anthropogenically drug-polluted natural environments. Non-lethal selection increases rates of mutant appearance and promotes enrichment of highly fit mutants and stable mutators.

  • 15.
    Andersson, Dan I.
    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.
    Gene amplification and adaptive evolution in bacteria2009In: Annual Review of Genetics, ISSN 0066-4197, E-ISSN 1545-2948, Vol. 43, p. 167-195Article, review/survey (Refereed)
    Abstract [en]

    Gene duplication-amplification (GDA) processes are highly relevant biologically because they generate extensive and reversible genetic variation on which adaptive evolution can act. Whenever cellular growth is restricted, escape from these growth restrictions often occurs by GDA events that resolve the selective problem. In addition, GDA may facilitate subsequent genetic change by allowing a population to grow and increase in number, thereby increasing the probability for subsequent adaptive mutations to occur in the amplified genes or in unrelated genes. Mathematical modeling of the effect of GDA on the rate of adaptive evolution shows that GDA will facilitate adaptation, especially when the supply of mutations in the population is rate-limiting. GDA can form via several mechanisms, both RecA-dependent and RecA-independent, including rolling-circle amplification and nonequal crossing over between sister chromatids. Due to the high intrinsic instability and fitness costs associated with GDAs, they are generally transient in nature, and consequently their evolutionary and medical importance is often underestimated.

  • 16.
    Andersson, Dan I.
    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 Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Microbiological effects of sublethal levels of antibiotics2014In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 12, no 7, p. 465-478Article, review/survey (Refereed)
    Abstract [en]

    The widespread use of antibiotics results in the generation of antibiotic concentration gradients in humans, livestock and the environment. Thus, bacteria are frequently exposed to non-lethal (that is, subinhibitory) concentrations of drugs, and recent evidence suggests that this is likely to have an important role in the evolution of antibiotic resistance. In this Review, we discuss the ecology of antibiotics and the ability of subinhibitory concentrations to select for bacterial resistance. We also consider the effects of low-level drug exposure on bacterial physiology, including the generation of genetic and phenotypic variability, as well as the ability of antibiotics to function as signalling molecules. Together, these effects accelerate the emergence and spread of antibiotic-resistant bacteria among humans and animals.

  • 17.
    Andersson, Dan I
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Muller's ratchet decreases fitness of a DNA-based microbe1996In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 93, no 2, p. 906-907Article in journal (Refereed)
    Abstract [en]

    Muller proposed that an asexual organism will inevitably accumulate deleterious mutations, resulting in an increase of the mutational load and an inexorable, ratchet-like, loss of the least mutated class [Muller, H.J. (1964) Mutat. Res. 1, 2-9]. The operation of Muller's ratchet on real populations has been experimentally demonstrated only in RNA viruses. However, these cases are exceptional in that the mutation rates of the RNA viruses are extremely high. We have examined whether Muller's ratchet operates in Salmonella typhimurium, a DNA-based organism with a more typical genomic mutation rate. Cells were grown asexually under conditions expected to result in high genetic drift, and the increase in mutational load was determined. S. typhimurium accumulated mutations under these conditions such that after 1700 generations, 1% of the 444 lineages tested had suffered an obvious loss of fitness, as determined by decreased growth rate. These results suggest that in the absence of sex and with high genetic drift, genetic mechanisms, such as back or compensatory mutations, cannot compensate for the accumulation of deleterious mutations. In addition, we measured the appearance of auxotrophs, which allowed us to calculate an average spontaneous mutation rate of approximately 0.3-1.5 x 10(-9) mutations per base pair per generation. This rate is measured for the largest genetic target studied so far, a collection of about 200 genes.

  • 18.
    Andersson, Dan I.
    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 Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Persistence of antibiotic resistance in bacterial populations2011In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 35, no 5, p. 901-911Article, review/survey (Refereed)
    Abstract [en]

    Unfortunately for mankind, it is very likely that the antibiotic resistance problem we have generated during the last 60 years due to the extensive use and misuse of antibiotics is here to stay for the foreseeable future. This view is based on theoretical arguments, mathematical modeling, experiments and clinical interventions, suggesting that even if we could reduce antibiotic use, resistant clones would remain persistent and only slowly (if at all) be outcompeted by their susceptible relatives. In this review, we discuss the multitude of mechanisms and processes that are involved in causing the persistence of chromosomal and plasmid-borne resistance determinants and how we might use them to our advantage to increase the likelihood of reversing the problem. Of particular interest is the recent demonstration that a very low antibiotic concentration can be enriching for resistant bacteria and the implication that antibiotic release into the environment could contribute to the selection for resistance.

  • 19.
    Andersson, Dan I
    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 Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selection and Transmission of Antibiotic-Resistant Bacteria2017In: Microbiology Spectrum, ISSN 2165-0497, Vol. 5, no 4, article id UNSP MTBP-0013-2016Article in journal (Refereed)
    Abstract [en]

    Ever since antibiotics were introduced into human and veterinary medicine to treat and prevent bacterial infections there has been a steady selection and increase in the frequency of antibiotic resistant bacteria. To be able to reduce the rate of resistance evolution, we need to understand how various biotic and abiotic factors interact to drive the complex processes of resistance emergence and transmission. We describe several of the fundamental factors that underlay resistance evolution, including rates and niches of emergence and persistence of resistant bacteria, time- and space-gradients of various selective agents, and rates and routes of transmission of resistant bacteria between humans, animals and other environments. Furthermore, we discuss the options available to reduce the rate of resistance evolution and/or transmission and their advantages and disadvantages.

  • 20.
    Andersson, Dan I.
    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 Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kubicek-Sutherland, Jessica Z.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mechanisms and consequences of bacterial resistance to antimicrobial peptides2016In: Drug resistance updates, ISSN 1368-7646, E-ISSN 1532-2084, Vol. 26, p. 43-57Article, review/survey (Refereed)
    Abstract [en]

    Cationic antimicrobial peptides (AMPs) are an intrinsic part of the human innate immune system. Over 100 different human AMPs are known to exhibit broad-spectrum antibacterial activity. Because of the increased frequency of resistance to conventional antibiotics there is an interest in developing AMPs as an alternative antibacterial therapy. Several cationic peptides that are derivatives of AMPs from the human innate immune system are currently in clinical development. There are also ongoing clinical studies aimed at modulating the expression of AMPs to boost the human innate immune response. In this review we discuss the potential problems associated with these therapeutic approaches. There is considerable experimental data describing mechanisms by which bacteria can develop resistance to AMPs. As for any type of drug resistance, the rate by which AMP resistance would emerge and spread in a population of bacteria in a natural setting will be determined by a complex interplay of several different factors, including the mutation supply rate, the fitness of the resistant mutant at different AMP concentrations, and the strength of the selective pressure. Several studies have already shown that AMP-resistant bacterial mutants display broad cross-resistance to a variety of AMPs with different structures and modes of action. Therefore, routine clinical administration of AMPs to treat bacterial infections may select for resistant bacterial pathogens capable of better evading the innate immune system. The ramifications of therapeutic levels of exposure on the development of AMP resistance and bacterial pathogenesis are not yet understood. This is something that needs to be carefully studied and monitored if AMPs are used in clinical settings.

  • 21.
    Andersson, Dan I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jerlström-Hultqvist, Jon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Näsvall, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Evolution of New Functions De Novo and from Preexisting Genes2015In: Cold Spring Harbor Perspectives in Biology, ISSN 1943-0264, E-ISSN 1943-0264, Vol. 7, no 6, article id a017996Article, review/survey (Refereed)
    Abstract [en]

    How the enormous structural and functional diversity of new genes and proteins was generated (estimated to be 10^10-€“10^12 different proteins in all organisms on earth [Choi I-G, Kim S-H. 2006. Evolution of protein structural classes and protein sequence families. Proc Natl Acad Sci 103: 14056–14061] is a central biological question that has a long and rich history. Extensive work during the last 80 years have shown that new genes that play important roles in lineage-specific phenotypes and adaptation can originate through a multitude of different mechanisms, including duplication, lateral gene transfer, gene fusion/fission, and de novo origination. In this review, we focus on two main processes as generators of new functions: evolution of new genes by duplication and divergence of pre-existing genes and de novo gene origination in which a whole protein-coding gene evolves from a noncoding sequence.

  • 22.
    Andersson, Dan I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Koskiniemi, Sanna
    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.
    Biological roles of translesion synthesis DNA polymerases in eubacteria2010In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 77, no 3, p. 540-548Article, review/survey (Refereed)
    Abstract [en]

    Biological systems are strongly selected to maintain the integrity of their genomes by prevention and repair of external and internal DNA damages. However, some types of DNA lesions persist and might block the replication apparatus. The universal existence of specialized translesion synthesis DNA polymerases (TLS polymerases) that can bypass such lesions in DNA implies that replication blockage is a general biological problem. We suggest that the primary function for which translesion synthesis polymerases are selected is to rescue cells from replication arrest at lesions in DNA, a situation that, if not amended, is likely to cause an immediate and severe reduction in cell fitness and survival. We will argue that the mutagenesis observed during translesion synthesis is an unavoidable secondary consequence of this primary function and not, as has been suggested, an evolved mechanism to increase mutation rates in response to various stresses. Finally, we will discuss recent data on additional roles for translesion synthesis polymerases in the formation of spontaneous deletions and in transcription-coupled TLS, where the coupling of transcription to TLS is proposed to allow the rescue of the transcription machinery arrested at DNA lesions.

  • 23.
    Andersson, Dan I
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Maisner Patin, Sophie
    Nilsson, Annika
    Kugelberg, Elisabeth
    The Biological Cost of Antibiotic resistance2007In: Enzyme-Mediated Resistance to Antibiotics: Mechanism, Dissemination, an Prospects for Inhibition, 2007, p. 339-348Chapter in book (Refereed)
  • 24.
    Andersson, Dan I
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nicoloff, Hervé
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mechanisms and clinical relevance of bacterial heteroresistance2019In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 17, no 8, p. 479-496Article, review/survey (Refereed)
    Abstract [en]

    Antibiotic heteroresistance is a phenotype in which a bacterial isolate contains subpopulations of cells that show a substantial reduction in antibiotic susceptibility compared with the main population. Recent work indicates that heteroresistance is very common for several different bacterial species and antibiotic classes. The resistance phenotype is often unstable, and in the absence of antibiotic pressure it rapidly reverts to susceptibility. A common mechanistic explanation for the instability is the occurrence of genetically unstable tandem amplifications of genes that cause resistance. Due to their instability, low frequency and transient character, it is challenging to detect and study these subpopulations, which often leads to difficulties in unambiguously classifying bacteria as susceptible or resistant. Finally, in vitro experiments, mathematical modelling, animal infection models and clinical studies show that the resistant subpopulations can be enriched during antibiotic exposure, and increasing evidence suggests that heteroresistance can lead to treatment failure.

  • 25.
    Atterby, Clara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nykvist, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Lustig, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Järhult, Josef D.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Spread of resistance plasmids and selection of resistant bacteria among Mallards exposed to sub-inhibitory concentrations of antibiotics in their water environmentManuscript (preprint) (Other academic)
  • 26.
    Balaban, Nathalie Q.
    et al.
    Hebrew Univ Jerusalem, Racah Inst Phys, Jerusalem, Israel.
    Helaine, Sophie
    Imperial Coll London, MRC Ctr Mol Bacteriol & Infect, London, England.
    Lewis, Kim
    Northeastern Univ, Dept Biol, Boston, MA 02115 USA.
    Ackermann, Martin
    Swiss Fed Inst Technol, Inst Biogeochem & Pollutant Dynam, Zurich, Switzerland;Eawag, Dept Environm Microbiol, Dubendorf, Switzerland.
    Aldridge, Bree
    Tufts Univ, Sch Med, Dept Mol Biol & Microbiol, Boston, MA 02111 USA.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brynildsen, Mark P.
    Princeton Univ, Dept Chem & Biol Engn, Princeton, NJ 08544 USA.
    Bumann, Dirk
    Univ Basel, Biozentrum, Focal Area Infect Biol, Basel, Switzerland.
    Camilli, Andrew
    Tufts Univ, Sch Med, Dept Mol Biol & Microbiol, Boston, MA 02111 USA.
    Collins, James J.
    MIT, Dept Biol Engn, Inst Med Engn & Sci, 77 Massachusetts Ave, Cambridge, MA 02139 USA;MIT, Synthet Biol Ctr, 77 Massachusetts Ave, Cambridge, MA 02139 USA;Harvard Univ, Wyss Inst Biol Inspired Engn, Boston, MA 02115 USA;Broad Inst MIT & Harvard, Cambridge, MA USA.
    Dehio, Christoph
    Univ Basel, Biozentrum, Focal Area Infect Biol, Basel, Switzerland.
    Fortune, Sarah
    Harvard TH Chan Sch Publ Hlth, Dept Immunol & Infect Dis, Boston, MA USA.
    Ghigo, Jean-Marc
    Inst Pasteur, Genet Biofilms Lab, Paris, France.
    Hardt, Wolf-Dietrich
    Swiss Fed Inst Technol, Inst Microbiol, Zurich, Switzerland.
    Harms, Alexander
    Univ Basel, Biozentrum, Focal Area Infect Biol, Basel, Switzerland.
    Heinemann, Matthias
    Univ Groningen, Groningen Biomol Sci & Biotechnol Inst, Mol Syst Biol, Groningen, Netherlands.
    Hung, Deborah T.
    Broad Inst MIT & Harvard, Cambridge, MA USA.
    Jenal, Urs
    Univ Basel, Biozentrum, Focal Area Infect Biol, Basel, Switzerland.
    Levin, Bruce R.
    Emory Univ, Dept Biol, Atlanta, GA 30322 USA.
    Michiels, Jan
    Univ Leuven, KU Leuven, Ctr Microbiol, Leuven, Belgium.
    Storz, Gisela
    Eunice Kennedy Shriver Natl Inst Child Hlth & Hum, Div Mol & Cellular Biol, Bethesda, MD USA.
    Tan, Man-Wah
    Genentech Inc, Infect Dis Dept, San Francisco, CA USA.
    Tenson, Tanel
    Univ Tartu, Inst Technol, Tartu, Estonia.
    Van Melderen, Laurence
    Univ Libre Bruxelles, Fac Sci, Brussels, Belgium.
    Zinkernagel, Annelies
    Univ Zurich, Univ Hosp Zurich, Div Infect Dis, Zurich, Switzerland.
    Definitions and guidelines for research on antibiotic persistence2019In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 17, no 7, p. 441-448Article, review/survey (Refereed)
    Abstract [en]

    Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that the guidelines outlined in this article will pave the way for better characterization of antibiotic persistence and for understanding its relevance to clinical outcomes.

  • 27.
    Baltekin, Özden
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Boucharin, Alexis
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tano, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Antibiotic susceptibility testing in less than 30 min using direct single-cell imaging2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 34, p. 9170-9175Article in journal (Refereed)
    Abstract [en]

    The emergence and spread of antibiotic-resistant bacteria are aggravated by incorrect prescription and use of antibiotics. A core problem is that there is no sufficiently fast diagnostic test to guide correct antibiotic prescription at the point of care. Here, we investigate if it is possible to develop a point-of-care susceptibility test for urinary tract infection, a disease that 100 million women suffer from annually and that exhibits widespread antibiotic resistance. We capture bacterial cells directly from samples with low bacterial counts (10(4) cfu/mL) using a custom-designed microfluidic chip and monitor their individual growth rates using microscopy. By averaging the growth rate response to an antibiotic over many individual cells, we can push the detection time to the biological response time of the bacteria. We find that it is possible to detect changes in growth rate in response to each of nine antibiotics that are used to treat urinary tract infections in minutes. In a test of 49 clinical uropathogenic Escherichia coli (UPEC) isolates, all were correctly classified as susceptible or resistant to ciprofloxacin in less than 10 min. The total time for antibiotic susceptibility testing, from loading of sample to diagnostic readout, is less than 30 min, which allows the development of a point-of-care test that can guide correct treatment of urinary tract infection.

  • 28. Baquero, María-Rosario
    et al.
    Nilsson, Annika I
    Turrientes, María del Carmen
    Sandvang, Dorthe
    Galán, Juan Carlos
    Martínez, Jose Luís
    Frimodt-Møller, Niels
    Baquero, Fernando
    Andersson, Dan I
    Karolinska Institute, Solna, Sweden & Swedish Institute for Infectious Disease Control.
    Polymorphic mutation frequencies in Escherichia coli: emergence of weak mutators in clinical isolates2004In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 16, p. 5538-5542Article in journal (Refereed)
    Abstract [en]

    Polymorphisms in the rifampin resistance mutation frequency (f) were studied in 696 Escherichia coli strains from Spain, Sweden, and Denmark. Of the 696 strains, 23% were weakly hypermutable (4 x 10(-8) < or = f < 4 x 10(-7)), and 0.7% were strongly hypermutable (f > or = 4 x 10(-7)). Weak mutators were apparently more frequent in southern Europe and in blood isolates (38%) than in urinary tract isolates (25%) and feces of healthy volunteers (11%).

  • 29. Bergthorsson, Ulfar
    et al.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Roth, John R.
    Ohno's dilemma: evolution of new genes under continuous selection2007In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 104, no 43, p. 17004-17009Article in journal (Refereed)
    Abstract [en]

    New genes with novel functions arise by duplication and divergence, but the process poses a problem. After duplication, an extra gene copy must rise to sufficiently high frequency in the population and remain free of common inactivating lesions long enough to acquire the rare mutations that provide a new selectable function. Maintaining a duplicated gene by selection for the original function would restrict the freedom to diverge. (We refer to this problem as Ohno's dilemma). A model is described by which selection continuously favors both maintenance of the duplicate copy and divergence of that copy from the parent gene. Before duplication, the original gene has a trace side activity (the innovation) in addition to its original function. When an altered ecological niche makes the minor innovation valuable, selection favors increases in its level (the amplification), which is most frequently conferred by increased dosage of the parent gene. Selection for the amplified minor function maintains the extra copies and raises the frequency of the amplification in the population. The same selection favors mutational improvement of any of the extra copies, which are not constrained to maintain their original function (the divergence). The rate of mutations (per genome) that improve the new function is increased by the multiplicity of target copies within a genome. Improvement of some copies relaxes selection on others and allows their loss by mutation (becoming pseudogenes). Ultimately one of the extra copies is able to provide all of the new activity.

  • 30. Björkholm, Britta
    et al.
    Sjölund, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Falk, Per G.
    Berg, Otto G.
    Engstrand, Lars
    Andersson, Dan I.
    Mutation frequency and biological cost of resistance in Helicobacter pylori.2001In: Proc Natl Acad Sci U S A, Vol. 98, no 25, p. 14607-12Article in journal (Refereed)
  • 31. Bottger, Erik C
    et al.
    Pletschette, Michel
    Andersson, Dan
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Drug resistance and fitness in Mycobacterium tuberculosis infection.2005In: J Infect Dis, ISSN 0022-1899, Vol. 191, no 5, p. 823-4; author reply 824Article in journal (Refereed)
  • 32.
    Chen, Yang
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Näsvall, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wu, Shiying
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3'')(9) adenyltransferase2015In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 71, p. 2267-2277Article in journal (Refereed)
    Abstract [en]

    Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleo\-tidyltransferases (ANTs). Here, the first crystal structure of an ANT(3$^\prime$$^\prime$)(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.

  • 33.
    Freihofer, Pietro
    et al.
    Univ Zurich, Inst Med Microbiol, CH-8006 Zurich, Switzerland..
    Akbergenov, Rashid
    Univ Zurich, Inst Med Microbiol, CH-8006 Zurich, Switzerland..
    Teo, Youjin
    Univ Zurich, Inst Med Microbiol, CH-8006 Zurich, Switzerland..
    Juskeviciene, Reda
    Univ Zurich, Inst Med Microbiol, CH-8006 Zurich, Switzerland..
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bottger, Erik C.
    Univ Zurich, Inst Med Microbiol, CH-8006 Zurich, Switzerland..
    Nonmutational compensation of the fitness cost of antibiotic resistance in mycobacteria by overexpression of tlyA rRNA methylase2016In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 22, no 12, p. 1836-1843Article in journal (Refereed)
    Abstract [en]

    Several studies over the last few decades have shown that antibiotic resistance mechanisms frequently confer a fitness cost and that these costs can be genetically ameliorated by intra- or extragenic second-site mutations, often without loss of resistance. Another, much less studied potential mechanism by which the fitness cost of antibiotic resistance could be reduced is via a regulatory response where the deleterious effect of the resistance mechanism is lowered by a physiological alteration that buffers the mutational effect. In mycobacteria, resistance to the clinically used tuberactinomycin antibiotic capreomycin involves loss-of-function mutations in rRNA methylase TIyA or point mutations in 16S rRNA (in particular the A1408G mutation). Both of these alterations result in resistance by reducing drug binding to the ribosome. Here we show that alterations of tlyA gene expression affect both antibiotic drug susceptibility and fitness cost of drug resistance. In particular, we demonstrate that the common resistance mutation A1408G is accompanied by a physiological change that involves increased expression of the tlyA gene. This gene encodes an enzyme that methylates neighboring 16S rRNA position C1409, and as a result of increased TIyA expression the fitness cost of the A1408G mutation is significantly reduced. Our findings suggest that in mycobacteria, a nonmutational mechanism (i.e., gene regulatory) can restore fitness to genetically resistant bacteria. Our results also point to a new and clinically relevant treatment strategy to combat evolution of resistance in multidrug-resistant tuberculosis. Thus, by utilizing antagonistic antibiotic interactions, resistance evolution could be reduced.

  • 34.
    Ge, Xueliang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Oliveira, Ana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Gutiérrez-de-Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Inhibition of translation termination by small molecules targeting ribosomal release factors2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 15424Article in journal (Refereed)
    Abstract [en]

    The bacterial ribosome is an important drug target for antibiotics that can inhibit different stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifically target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they differ considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identification of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without affecting peptide bond formation.

  • 35. Geli, Patricia
    et al.
    Andersson, Mikael
    Svensson, Åke
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    A multi-type branching model with varying environment for bacterial dynamics with postantibiotic effect2009In: Journal of Theoretical Biology, ISSN 0022-5193, E-ISSN 1095-8541, Vol. 256, no 1, p. 58-64Article in journal (Refereed)
    Abstract [en]

    A multi-type branching process with varying environment was used to construct a pharmacokinetic/pharmacodynamic (PK/PD) model that captures the postantibiotic effect (PAE) seen in bacterial populations after exposure of antibiotics. This phenomenon of continued inhibition of bacterial growth even after removal of the antibiotic from the growth medium is of high relevance in the context of optimizing dosing regimens. The clinical implication of long PAEs lies in the interesting possibility of increasing the intervals between drug administrations. The model structure is generalizable to most types of antibiotics and is useful both as a theoretical framework for understanding the time properties of PAE and to explore optimal antibiotic dosing regimens. Data from an in vitro study with Escherichia coli exposed to different dosing regimens of cefotaxime were used to evaluate the model.

  • 36.
    Gullberg, Erik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Albrecht, Lisa M
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Karlsson, Christoffer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selection of a multidrug resistance plasmid by sublethal levels of antibiotics and heavy metals2014In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 5, no 5, p. e01918-14-Article in journal (Refereed)
    Abstract [en]

    How sublethal levels of antibiotics and heavy metals select for clinically important multidrug resistance plasmids is largely unknown. Carriage of plasmids generally confers substantial fitness costs, implying that for the plasmid-carrying bacteria to be maintained in the population, the plasmid cost needs to be balanced by a selective pressure conferred by, for example, antibiotics or heavy metals. We studied the effects of low levels of antibiotics and heavy metals on the selective maintenance of a 220-kbp extended-spectrum β-lactamase (ESBL) plasmid identified in a hospital outbreak of Klebsiella pneumoniae and Escherichia coli. The concentrations of antibiotics and heavy metals required to maintain plasmid-carrying bacteria, the minimal selective concentrations (MSCs), were in all cases below (almost up to 140-fold) the MIC of the plasmid-free susceptible bacteria. This finding indicates that the very low antibiotic and heavy metal levels found in polluted environments and in treated humans and animals might be sufficiently high to maintain multiresistance plasmids. When resistance genes were moved from the plasmid to the chromosome, the MSC decreased, showing that MSC for a specific resistance conditionally depends on genetic context. This finding suggests that a cost-free resistance could be maintained in a population by an infinitesimally low concentration of antibiotic. By studying the effect of combinations of several compounds, it was observed that for certain combinations of drugs each new compound added lowered the minimal selective concentration of the others. This combination effect could be a significant factor in the selection of multidrug resistance plasmids/bacterial clones in complex multidrug environments.

    IMPORTANCE: Antibiotic resistance is in many pathogenic bacteria caused by genes that are carried on large conjugative plasmids. These plasmids typically contain multiple antibiotic resistance genes as well as genes that confer resistance to biocides and heavy metals. In this report, we show that very low concentrations of single antibiotics and heavy metals or combinations of compounds can select for a large plasmid that carries resistance to aminoglycosides, β-lactams, tetracycline, macrolides, trimethoprim, sulfonamide, silver, copper, and arsenic. Our findings suggest that the low levels of antibiotics and heavy metals present in polluted external environments and in treated animals and humans could allow for selection and enrichment of bacteria with multiresistance plasmids and thereby contribute to the emergence, maintenance, and transmission of antibiotic-resistant disease-causing bacteria.

  • 37.
    Gumpert, Heidi
    et al.
    Tech Univ Denmark, Dept Syst Biol, Lyngby, Denmark.;Univ Copenhagen, Hvidovre Hosp, Dept Clin Microbiol, Hvidovre, Denmark..
    Kubicek-Sutherland, Jessica Z.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Porse, Andreas
    Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, Lyngby, Denmark..
    Karami, Nahid
    Univ Gothenburg, Sahlgrenska Acad, Inst Biomed, Dept Infect Dis, Gothenburg, Sweden..
    Munck, Christian
    Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, Lyngby, Denmark..
    Linkevicius, Marius
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Adlerberth, Ingegerd
    Univ Gothenburg, Sahlgrenska Acad, Inst Biomed, Dept Infect Dis, Gothenburg, Sweden..
    Wold, Agnes E.
    Univ Gothenburg, Sahlgrenska Acad, Inst Biomed, Dept Infect Dis, Gothenburg, Sweden..
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sommer, Morten O. A.
    Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, Lyngby, Denmark..
    Transfer and Persistence of a Multi-Drug Resistance Plasmid in situ of the Infant Gut Microbiotain the Absence of Antibiotic Treatment2017In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 1852Article in journal (Refereed)
    Abstract [en]

    The microbial ecosystem residing in the human gut is believed to play an important role in horizontal exchange of virulence and antibiotic resistance genes that threatens human health. While the diversity of gut-microorganisms and their genetic content has been studied extensively, high-resolution insight into the plasticity, and selective forces shaping individual genomes is scarce. In a longitudinal study, we followed the dynamics of co-existing Escherichia coli lineages in an infant not receiving antibiotics. Using whole genome sequencing, we observed large genomic deletions, bacteriophage infections, as well as the loss and acquisition of plasmids in these lineages during their colonization of the human gut. In particular, we captured the exchange of multidrug resistance genes, and identified a clinically relevant conjugative plasmid mediating the transfer. This resistant transconjugant lineage was maintained for months, demonstrating that antibiotic resistance genes can disseminate and persist in the gut microbiome; even in absence of antibiotic selection. Furthermore, through in vivo competition assays, we suggest that the resistant transconjugant can persist through a fitness advantage in the mouse gut in spite of a fitness cost in vitro. Our findings highlight the dynamic nature of the human gut microbiota and provide the first genomic description of antibiotic resistance gene transfer between bacteria in the unperturbed human gut. These results exemplify that conjugative plasmids, harboring resistance determinants, can transfer and persists in the gut in the absence of antibiotic treatment.

  • 38.
    Gustafsson, Ingegerd
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Cars, Otto
    Andersson, Dan I.
    Fitness of antibiotic resistant Staphylococcus epidermidis assessed by competition on skin of human volunteersManuscript (Other academic)
  • 39.
    Gustafsson, Ingegerd
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Sjölund, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Torell, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Johannesson, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Engstrand, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Andersson, Dan I.
    Bacteria with increased mutation frequency and antibiotic resistance are enriched in the commensal flora of patients with high antibiotic usage2003In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 52, no 4, p. 645-650Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: We examined how prolonged antibiotic treatment affected the resistance and mutation frequency of human microflora isolated from intestine (Escherichia coli, enterococci spp.), pharynx (alpha-streptococci) and nostril (coagulase-negative staphylococci, CoNS).

    METHODS: Samples were collected from patients at the Center of Cystic Fibrosis (n=18) and the haematology ward (n=18) of the University Hospital, Uppsala, Sweden. The individually used amount of antibiotics for 1 year was recorded as the defined daily dose (DDD). Primary health care patients (n=30), with no antibiotic treatment for 1 year before sampling, were used as controls. Three isolates of each bacterium from each patient were examined. Antibiotic susceptibilities were determined by disc diffusion. Mutation frequencies to rifampicin resistance were measured on 30 independent cultures of each bacterial species from each individual by plating on rifampicin agar plates. For alpha-streptococci the mutation frequency to streptomycin resistance was also determined.

    RESULTS: Isolates from patients with high antibiotic use showed a pronounced shift towards increased resistance and a small but significant increase in the mutation frequency compared with isolates from the controls. For E. coli, enterococci and CoNS the increase in geometric mean mutation frequency in the patient group was 3-, 1.8- and 1.5-fold, respectively (P values 0.0001, 0.016 and 0.012). For alpha-streptococci there was a significant difference in geometric mean mutation frequency between patient and control groups for streptomycin resistance (P=0.024) but not for rifampicin resistance (P=0.74).

    CONCLUSIONS: High antibiotic use selected for commensals with highly increased resistance and a slight increase in mutation frequency.

  • 40.
    Hjort, Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nicoloff, Hervé
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Unstable tandem gene amplification generates heteroresistance (variation in resistance within a population) to colistin in Salmonella enterica2016In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 102, no 2, p. 274-289Article in journal (Refereed)
    Abstract [en]

    Heteroresistance, a phenomenon where subpopulations of a bacterial isolate exhibit different susceptibilities to an antibiotic, is a growing clinical problem where the underlying genetic mechanisms in most cases remain unknown. We isolated colistin resistant mutants in Escherichia coli and Salmonella enterica serovar Typhimurium at different concentrations of colistin. Genetic analysis showed that genetically stable pmrAB point mutations were responsible for colistin resistance during selection at high drug concentrations for both species and at low concentrations for E. coli. In contrast, for S. Typhimurium mutants selected at low colistin concentrations, amplification of different large chromosomal regions conferred a heteroresistant phenotype. All amplifications included the pmrD gene, which encodes a positive regulator that up-regulates proteins that modify lipid A, and as a result increase colistin resistance. Inactivation and over-expression of the pmrD gene prevented and conferred resistance, respectively, demonstrating that the PmrD protein is required and sufficient to confer resistance. The heteroresistance phenotype is explained by the variable gene dosage of pmrD in a population, where sub-populations with different copy number of the pmrD gene show different levels of colistin resistance. We propose that variability in gene copy number of resistance genes can explain the heteroresistance observed in clinically isolated pathogenic bacteria.

  • 41.
    Hughes, Diarmaid
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Adventures with mutation and selection in beehive and cow country2010In: The Lure of Bacterial Genetics: a Tribute to John Roth / [ed] Maloy, S., Hughes, K.T. and Casadesus, J, Washington, DC: ASM Press , 2010, p. 245-253Chapter in book (Other academic)
  • 42.
    Hughes, Diarmaid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Environmental and genetic modulation of the phenotypic expression of antibiotic resistance2017In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 41, no 3, p. 374-391, article id fux004Article, review/survey (Refereed)
    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.

  • 43.
    Hughes, Diarmaid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Evolutionary consequences of drug resistance: shared principles across diverse targets and organisms2015In: Nature reviews genetics, ISSN 1471-0056, E-ISSN 1471-0064, Vol. 16, no 8, p. 459-471Article, review/survey (Refereed)
    Abstract [en]

    Drug therapy has a crucial role in the treatment of viral, bacterial, fungal and protozoan infections, as well as the control of human cancer. The success of therapy is being threatened by the increasing prevalence of resistance. We examine and compare mechanisms of drug resistance in these diverse biological systems (using HIV and Plasmodium falciparum as examples of viral and protozoan pathogens, respectively) and discuss how factors-such as mutation rates, fitness effects of resistance, epistasis and clonal interference-influence the evolutionary trajectories of drug-resistant clones. We describe commonalities and differences related to resistance development that could guide strategies to improve therapeutic effectiveness and the development of a new generation of drugs.

  • 44.
    Hughes, Diarmaid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Evolutionary Trajectories to Antibiotic Resistance2017In: Annual Review of Microbiology, ISSN 0066-4227, E-ISSN 1545-3251, Vol. 71, p. 579-596Article, review/survey (Refereed)
    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.

  • 45.
    Hughes, Diarmaid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selection of resistance at lethal and non-lethal antibiotic concentrations2012In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 15, no 5, p. 555-560Article in journal (Refereed)
    Abstract [en]

    Much of what we currently know about the genetics and evolution of antibiotic-resistance is based on selections with lethal drug concentrations that allow the detection of rare mutants with strong phenotypes. These data may be misleading with regard to the evolution of antibiotic resistance in natural environments, because bacteria are frequently exposed to concentration gradients of antibiotics. A significant part of antibiotic-resistance evolution may occur when bacteria are exposed to non-lethal concentrations of drug. High-resolution competition assays show that resistance mutations are rapidly enriched, and selected de novo, at very low antibiotic concentrations. Genomic analysis is providing a better understanding of how frequent and small-effect mutations selected at very low antibiotic concentrations contribute to the step-wise development of antibiotic resistance.

  • 46. Huitric, E.
    et al.
    Verhasselt, P.
    Koul, A.
    Andries, K.
    Hoffner, S.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Rates and Mechanisms of Resistance Development in Mycobacterium tuberculosis to a Novel Diarylquinoline ATP Synthase Inhibitor2010In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 54, no 3, p. 1022-1028Article in journal (Refereed)
    Abstract [en]

    R207910 (also known as TMC207) is an investigational drug currently in clinical studies for the treatment of multidrug-resistant (MDR) tuberculosis. It has a high degree of antimycobacterial activity and is equally effective against drug-susceptible and MDR Mycobacterium tuberculosis isolates. In the present study, we characterized the development of resistance to R207910 in vitro. Ninety-seven independent R207910-resistant mutants were selected from seven different clinical isolates of M. tuberculosis (three drug-susceptible and four MDR isolates) at 10x, 30x, and 100x the MIC. At a concentration of 0.3 mg/liter (10x the MIC), the mutation rates ranged from 4.7 x 10(-7) to 8.9 x 10(-9) mutations per cell per division, and at 1.0 mg/liter (30x the MIC) the mutation rate ranged from 3.9 x 10(-8) to 2.4 x 10(-9). No resistant mutants were obtained at 3 mg/liter (100x the MIC). The level of resistance ranged from 0.12 to 3.84 mg/liter for the mutants identified; these concentrations represent 4- to 128-fold increases in the MICs. For 53 of the resistant mutants, the atpE gene, which encodes a transmembrane and oligomeric C subunit of the ATP synthase and which was previously shown to be involved in resistance, was sequenced. For 15/53 mutants, five different point mutations resulting in five different amino acid substitutions were identified in the atpE gene. For 38/53 mutants, no atpE mutations were found and sequencing of the complete F0 ATP synthase operon (atpB, atpE, and atpF genes) and the F1 ATP synthase operon (atpH, atpA, atpG, atpD, and atpC genes) from three mutants revealed no mutations, indicating other, alternative resistance mechanisms. Competition assays showed no measurable reduction in the fitness of the mutants compared to that of the isogenic wild types.

  • 47.
    Jerlström-Hultqvist, Joel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
    Warsi, Omar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Söderholm, Annika
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Knopp, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eckhard, Ulrich
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Vorontsov, Egor
    Proteomics Core Facility at Sahlgrenska Academy, Gothenburg University, Gothenburg, Sweden.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    A bacteriophage enzyme induces bacterial metabolic perturbation that confers a novel promiscuous function2018In: Nature Ecology & Evolution, E-ISSN 2397-334X, Vol. 2, no 8, p. 1321-1330Article in journal (Refereed)
    Abstract [en]

    One key concept in the evolution of new functions is the ability of enzymes to perform promiscuous side-reactions that serve as a source of novelty that may become beneficial under certain conditions. Here, we identify a mechanism where a bacteriophage-encoded enzyme introduces novelty by inducing expression of a promiscuous bacterial enzyme. By screening for bacteriophage DNA that rescued an auxotrophic Escherichia coli mutant carrying a deletion of the ilvA gene, we show that bacteriophage-encoded S-adenosylmethionine (SAM) hydrolases reduce SAM levels. Through this perturbation of bacterial metabolism, expression of the promiscuous bacterial enzyme MetB is increased, which in turn complements the absence of IlvA. These results demonstrate how foreign DNA can increase the metabolic capacity of bacteria, not only by transfer of bona fide new genes, but also by bringing cryptic bacterial functions to light via perturbations of cellular physiology.

  • 48. Kacar, Betül
    et al.
    Garmendia, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tuncbag, Nurcan
    Andersson, Dan I
    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.
    Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs2017In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 4, article id e01276-17Article in journal (Refereed)
    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.

  • 49.
    Khan, David D.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lagerbäck, Pernilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lustig, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nielsen, Elisabet I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Friberg, Lena E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    A mechanism-based pharmacokinetic/pharmacodynamic model allows prediction of antibiotic killing from MIC values for WT and mutants2015In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 70, no 11, p. 3051-3060Article in journal (Refereed)
    Abstract [en]

    Objectives: In silico pharmacokinetic/pharmacodynamic (PK/PD) models can be developed based on data from in vitro time-kill experiments and can provide valuable information to guide dosing of antibiotics. The aim was to develop a mechanism-based in silico model that can describe in vitro time-kill experiments of Escherichia coli MG1655 WT and six isogenic mutants exposed to ciprofloxacin and to identify relationships that may be used to simplify future characterizations in a similar setting. Methods: In this study, we developed a mechanism-based PK/PD model describing killing kinetics for E. coli following exposure to ciprofloxacin. WT and six well-characterized mutants, with one to four clinically relevant resistance mutations each, were exposed to a wide range of static ciprofloxacin concentrations. Results: The developed model includes susceptible growing bacteria, less susceptible (pre-existing resistant) growing bacteria, non-susceptible non-growing bacteria and non-colony-forming non-growing bacteria. The non-colony-forming state was likely due to formation of filaments and was needed to describe data close to the MIC. A common model structure with different potency for bacterial killing (EC50) for each strain successfully characterized the time-kill curves for both WT and the six E. coli mutants. Conclusions: The model-derived mutant-specific EC50 estimates were highly correlated (r(2) = 0.99) with the experimentally determined MICs, implying that the in vitro time-kill profile of a mutant strain is reasonably well predictable by the MIC alone based on the model.

  • 50.
    Khan, David
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lagerbäck, Pernilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Malmberg, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Kristoffersson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Gullberg, Erik
    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.
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Andersson, Dan I.
    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.
    Nielsen, Elisabet I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Friberg, Lena E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Predicting mutant selection in competition experiments with ciprofloxacin-exposed Escherichia coli2018In: International Journal of Antimicrobial Agents, ISSN 0924-8579, E-ISSN 1872-7913, Vol. 51, no 3, p. 399-406, article id S0924-8579(17)30392-8Article in journal (Refereed)
    Abstract [en]

    Predicting competition between antibiotic-susceptible wild-type (WT) and less susceptible mutant (MT) bacteria is valuable for understanding how drug concentrations influence the emergence of resistance. Pharmacokinetic/pharmacodynamic (PK/PD) models predicting the rate and extent of takeover of resistant bacteria during different antibiotic pressures can thus be a valuable tool in improving treatment regimens. The aim of this study was to evaluate a previously developed mechanism-based PK/PD model for its ability to predict in vitro mixed-population experiments with competition between Escherichia coli (E. coli) WT and three well-defined E. coli resistant MTs when exposed to ciprofloxacin. Model predictions for each bacterial strain and ciprofloxacin concentration were made for in vitro static and dynamic time–kill experiments measuring CFU (colony forming units)/mL up to 24 h with concentrations close to or below the minimum inhibitory concentration (MIC), as well as for serial passage experiments with concentrations well below the MIC measuring ratios between the two strains with flow cytometry. The model was found to reasonably well predict the initial bacterial growth and killing of most static and dynamic time–kill competition experiments without need for parameter re-estimation. With parameter re-estimation of growth rates, an adequate fit was also obtained for the 6-day serial passage competition experiments. No bacterial interaction in growth was observed. This study demonstrates the predictive capacity of a PK/PD model and further supports the application of PK/PD modelling for prediction of bacterial kill in different settings, including resistance selection.

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