Dan I Andersson

Uppsala University, Uppsala, Uppsala, Sweden

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Publications (144)1006.92 Total impact

  • Marius Linkevicius · Jytte Mark Anderssen · Linus Sandegren · Dan I Andersson
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    ABSTRACT: Objectives: The objective of this study was to determine the fitness of Escherichia coli mutants with reduced susceptibility to tigecycline after exposure to adverse conditions in vitro and in vivo. Methods: Survival in response to low pH, bile salts, oxidative stress and human serum was examined for E. coli mutants with reduced susceptibility to tigecycline due to single mutations that caused increased efflux (marR, lon) or impaired LPS (rfaC, rfaE, lpcA). An in vitro competition assay was used to determine growth fitness defects. Competitive fitness was assessed using mouse infection models. MICs, exponential growth rates and expression levels of efflux-related genes were measured for genetically reconstructed double and triple mutants. Results: The LPS mutants were 48-85-fold more susceptible to bile salts compared with the ERN mutants and the WT. As shown by in vitro competitions, the fitness reduction was 0.3%-13% for ERN mutants and ∼24% for LPS mutants. During in vivo survival experiments, LPS mutants were outcompeted by the WT strain in the thigh infection model. Constructed double ERN and LPS mutants showed additive and synergistic increases in tigecycline MICs. Conclusions: Generally, reduced susceptibility to tigecycline caused a decrease in fitness under stressful in vitro and in vivo conditions with ERN mutants being fitter than LPS mutants. When combined, ERN mutations caused a synergistic increase in the MIC of tigecycline. These findings could explain why clinical resistance to tigecycline in E. coli is mainly associated with up-regulation of the AcrAB efflux system.
    No preview · Article · Feb 2016 · Journal of Antimicrobial Chemotherapy
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    Marius Linkevicius · Linus Sandegren · Dan I. Andersson
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    ABSTRACT: Tigecycline is a third-generation tetracycline active against multidrug-resistant bacterial pathogens. The objectives of our study were to examine the potential of the Tet(A), Tet(K), Tet(M) and Tet(X) tetracycline resistance proteins to acquire mutations causing tigecycline resistance and to determine how it affects resistance to earlier generations of tetracyclines. Mutations in all four tet genes caused significant increase in tigecycline MIC in Escherichia coli , and strains expressing mutant Tet(A) and Tet(X) variants reached clinically relevant MICs (2 mg/L and 3 mg/L, respectively). Mutations predominantly accumulated in transmembrane domains of the efflux pumps, most likely increasing the accommodation of tigecycline as a substrate. All selected Tet(M) mutants contained at least one mutation in functionally most important loop III of domain IV. Deletion of leucine 505 of this loop led to the highest increase of tigecycline MIC (0.5 mg/L) among Tet(M) mutants. It also caused collateral sensitivity to earlier generations of tetracyclines. A majority of the Tet(X) mutants showed increased activity against all three generations of tetracylines. All tested Tet proteins have the potential to acquire mutations leading to increased MICs of tigecycline. As tet genes are widely found in pathogenic bacteria and spread easily by horizontal gene transfer, resistance development by alteration of existing Tet proteins might compromise the future medical use of tigecycline. We predict that Tet(X) might become the most problematic future Tet determinant since its weak intrinsic tigecycline activity can be mutationally improved to reach clinically relevant levels without collateral loss in activity to other tetracyclines.
    Preview · Article · Nov 2015 · Antimicrobial Agents and Chemotherapy
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    Dataset: mn5096sup1
    Yang Chen · Joakim Näsvall · Shiying Wu · Dan I. Andersson · Maria Selmer

    Full-text · Dataset · Nov 2015
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    Yang Chen · Joakim Näsvall · Shiying Wu · Dan I. Andersson · Maria Selmer
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    ABSTRACT: Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). Here, the first crystal structure of an ANT(3′′)(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 2.5 Å 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.
    Full-text · Article · Nov 2015 · Acta Crystallographica Section D Biological Crystallography
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    Hervé Nicoloff · Dan I Andersson
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    ABSTRACT: Objectives Indirect resistance (IR), the ability of an antibiotic-resistant population of bacteria to protect a susceptible population, has been previously observed for β-lactamase-producing bacteria and associated with antimicrobial treatment failures. Here, we determined whether other resistance determinants could cause IR in the presence of five other classes of antibiotics. Methods A test was designed to detect IR and 14 antibiotic resistance genes were tested in the presence of 13 antibiotics from six classes. A bioassay was used to measure the ability of resistance-causing enzymes to decrease the concentration of active antibiotics in the medium. Results We confirmed IR in the presence of β-lactam antibiotics (ampicillin and mecillinam) when TEM-1A was expressed. We found that bacteria expressing antibiotic-modifying or -degrading enzymes Ere(A), Tet(X2) or CatA1 caused IR in the presence of macrolides (erythromycin and clarithromycin), tetracyclines (tetracycline and tigecycline) and chloramphenicol, respectively. IR was not observed with resistance determinants that did not modify or destroy antibiotics or with enzymes modifying aminoglycosides or degrading fosfomycin. IR was dependent on the resistance enzymes decreasing the concentration of active antibiotics in the medium, hence allowing nearby susceptible bacteria to resume growth once the antibiotic concentration fell below their MIC. Conclusions IR was not limited to β-lactamase-producing bacteria, but was also caused by resistant bacteria carrying cytoplasmic antibiotic-modifying or -degrading enzymes that catalyse energy-consuming reactions requiring complex cellular cofactors. Our results suggest that IR is common and further emphasizes that coinfecting agents and the human microflora can have a negative impact during antimicrobial therapy.
    Preview · Article · Oct 2015 · Journal of Antimicrobial Chemotherapy
  • Michael Knopp · Dan I Andersson
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    ABSTRACT: The fitness cost of antibiotic resistance is a key parameter in determining the evolutionary success of resistant bacteria. Studies of the effect of antibiotic resistance on bacterial fitness are heavily biased towards target alterations. Here we investigated how the costs in form of a severely impaired growth rate associated with resistance due to absence of two major outer membrane porins can be genetically compensated. We performed an evolution experiment with 16 lineages of a double mutant of E. coli with the ompCF genes deleted, and reduced fitness and increased resistance to different classes of antibiotics, including the carbapenems ertapenem and meropenem. After serial passage for only 250 generations, the relative growth rate increased from 0.85 up to 0.99 (susceptible wild type set to 1.0). Compensation of the costs followed two different adaptive pathways where upregulation of expression of alternative porins bypassed the need for functional OmpCF porins. The first compensatory mechanism involved mutations in the phoR and pstS genes, causing constitutive high-level expression of the PhoE porin. The second mechanism involved mutations in the hfq and chiX genes that disrupted Hfq-dependent small RNA regulation, causing overexpression of the ChiP porin. While susceptibility was restored in compensated mutants with PhoE overexpression, evolved mutants with high ChiP expression maintained the resistance phenotype. Our findings may explain why porin-composition often is altered in resistant clinical isolates and provide new insights into how bypass mechanisms may allow genetic adaptation to a common multi-drug resistance mechanism.
    No preview · Article · Sep 2015 · Molecular Biology and Evolution
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    ABSTRACT: 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.
    No preview · Article · Sep 2015 · Journal of Antimicrobial Chemotherapy
  • Diarmaid Hughes · Dan I Andersson
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    ABSTRACT: 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.
    No preview · Article · Jul 2015 · Nature Reviews Genetics
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    Dan I. Andersson · Jon Jerlström-Hultqvist · Joakim Näsvall
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    ABSTRACT: 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. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.
    Full-text · Article · Jun 2015 · Cold Spring Harbor perspectives in biology
  • Dan I Andersson
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    ABSTRACT: 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 to use for risk assessment and 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, since 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. Copyright © 2015. Published by Elsevier Ltd.
    No preview · Article · May 2015 · Clinical Microbiology and Infection
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    Elisabeth Thulin · Martin Sundqvist · Dan I Andersson
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    ABSTRACT: Mecillinam is a β-lactam antibiotic that is mainly used for treatment of uncomplicated urinary tract infections. The objectives of this study were to identify mutations that confer mecillinam resistance in laboratory-isolated mutants and clinical isolates of Escherichia coli and to determine why mecillinam resistance remains rare clinically even though resistance is easily selected in the laboratory. In laboratory selections, mutation frequencies to mecillinam resistance varied between 8x10(-8) - 2x10(-5) per cell depending on the concentration of mecillinam used during selection. Several genes have been demonstrated to give mecillinam resistance, but here eight novel genes, previously unknown to be involved in mecillinam resistance were identified. These genes encode functions involved in the respiratory chain, the ribosome, cystein biosynthesis, tRNA synthesis and pyrophosphate metabolism. The clinical isolates exhibited significantly higher fitness and a different mutation spectrum than the laboratory-isolated mutants. The cysB gene was mutated (inactivated) in all the clinical isolates, in contrast to the laboratory-isolated mutants where mainly other types of more costly mutations were found. Our results suggest that the mutation frequency to mecillinam resistance is high because of the large mutational target (at least 38 genes). However, the majority of these resistant mutants have a low growth rate, reducing the probability that they are stably maintained in the bladder. Inactivation of the cysB gene, and a resulting loss of biosynthesis of cysteine, is the major mechanism of mecillinam resistance in clinical isolates of E. coli. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    Preview · Article · Jan 2015 · Antimicrobial Agents and Chemotherapy
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    ABSTRACT: To assure correct antibiotic treatment and reduce unnecessary use of antibiotics there is an urgent need for new rapid methods for species identification and determination of antibiotic susceptibility in infectious pathogenic bacteria. We have developed a general method to rapidly identify the bacterial species causing an infection and determine their antibiotic susceptibility profiles. An initial short cultivation step in the absence and presence of different antibiotics was combined with a sensitive species-specific padlock probe detection of the bacterial target DNA to allow determination of growth (i.e. resistance) and no growth (i.e. susceptibility). A proof-of-concept was established for urinary tract infections where we applied the method to determine the antibiotic susceptibility profile of E. coli for two drugs with 100% accuracy in 3.5 hours. The short assay time from sample to readout enables fast appropriate treatment with effective drugs, and minimizes the need of prescribing broad-spectrum antibiotics due to unknown resistance profiles of the treated infection. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Preview · Article · Nov 2014 · Journal of Clinical Microbiology
  • Hava Lofton · Naeem Anwar · Mikeal Rhen · Dan I. Andersson
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    ABSTRACT: Abstract OBJECTIVES: To examine the effects of mutations in the waaY, phoP and pmrB genes, which confer resistance to antimicrobial peptides (AMPs), on fitness of Salmonella Typhimurium. METHODS: Survival during low pH, oxidative stress, stationary-phase incubation, exposure to serum and bile and growth in mice and laboratory media were determined by time-kills, disc inhibition assays, competition experiments and optical density measurements. RESULTS: Individual mutations in the waaY gene (involved in LPS core biosynthesis) and in the phoP and pmrB genes (part of two different two-component regulatory systems, phoPQ and pmrAB) conferred no or minor effects on bacterial survival during stressful in vitro conditions or in mice. In contrast, a waaY-phoP-pmrB triple mutant was compromised under most assay conditions. CONCLUSIONS: Results from this study show that AMP resistance can be cost-free, as assessed by several assays that attempt to mimic the conditions a bacterium might encounter within a host. Our findings imply that future therapeutic use of AMPs could select for fit mutants with cross-resistance to human defence peptides and that potential resistance development in response to therapeutic use of AMPs needs to be carefully monitored.
    No preview · Article · Sep 2014 · Journal of Antimicrobial Chemotherapy
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    ABSTRACT: 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.
    Full-text · Article · Aug 2014 · mBio
  • Dan I Andersson · Diarmaid Hughes
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    ABSTRACT: 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.
    No preview · Article · May 2014 · Nature Reviews Microbiology
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    Song Sun · Maria Selmer · Dan I Andersson
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    ABSTRACT: Penicillin-binding proteins (PBPs) are enzymes responsible for the polymerization of the glycan strand and the cross-linking between glycan chains as well as the target proteins for β-lactam antibiotics. Mutational alterations in PBPs can confer resistance either by reducing binding of the antibiotic to the active site or by evolving a β-lactamase activity that degrades the antibiotic. As no systematic studies have been performed to examine the potential of all PBPs present in one bacterial species to evolve increased resistance against β-lactam antibiotics, we explored the ability of fifteen different defined or putative PBPs in Salmonella enterica to acquire increased resistance against penicillin G. We could after mutagenesis and selection in presence of penicillin G isolate mutants with amino-acid substitutions in the PBPs, FtsI, DacB and DacC (corresponding to PBP3, PBP4 and PBP6) with increased resistance against β-lactam antibiotics. Our results suggest that: (i) most evolved PBPs became 'generalists" with increased resistance against several different classes of β-lactam antibiotics, (ii) synergistic interactions between mutations conferring antibiotic resistance are common and (iii) the mechanism of resistance of these mutants could be to make the active site more accessible for water allowing hydrolysis or less binding to β-lactam antibiotics.
    Full-text · Article · May 2014 · PLoS ONE
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    ABSTRACT: 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.
    No preview · Article · Mar 2014 · Molecular Biology and Evolution
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    Full-text · Dataset · Mar 2014
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    ABSTRACT: Clonally derived bacterial populations exhibit significant genotypic and phenotypic diversity that contribute to fitness in rapidly changing environments. Here, we show that serial passage of Salmonella enterica serovar Typhimurium LT2 (StLT2) in broth, or within a mouse host, results in selection of an evolved population that inhibits the growth of ancestral cells by direct contact. Cells within each evolved population gain the ability to express and deploy a cryptic "orphan" toxin encoded within the rearrangement hotspot (rhs) locus. The Rhs orphan toxin is encoded by a gene fragment located downstream of the "main" rhs gene in the ancestral strain StLT2. The Rhs orphan coding sequence is linked to an immunity gene, which encodes an immunity protein that specifically blocks Rhs orphan toxin activity. Expression of the Rhs orphan immunity protein protects ancestral cells from the evolved lineages, indicating that orphan toxin activity is responsible for the observed growth inhibition. Because the Rhs orphan toxin is encoded by a fragmented reading frame, it lacks translation initiation and protein export signals. We provide evidence that evolved cells undergo recombination between the main rhs gene and the rhs orphan toxin gene fragment, yielding a fusion that enables expression and delivery of the orphan toxin. In this manner, rhs locus rearrangement provides a selective advantage to a subpopulation of cells. These observations suggest that rhs genes play important roles in intra-species competition and bacterial evolution.
    Full-text · Article · Mar 2014 · PLoS Genetics
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    ABSTRACT: Genes introduced by horizontal gene transfer (HGT) from other species constitute a significant portion of many bacterial genomes and the evolutionary dynamics of HGTs are important for understanding the spread of antibiotic resistance and the emergence of new pathogenic strains of bacteria. The fitness effects of the transferred genes largely determine the fixation rates and the amount of neutral diversity of newly acquired genes in bacterial populations. Comparative analysis of bacterial genomes provides insight into what genes are commonly transferred, but direct experimental tests of the fitness constraints on HGT are scarce. Here, we address this paucity of experimental studies by introducing 98 random DNA fragments varying in size from 0.45 to 5 kb from Bacteroides, Proteus and human intestinal phage into a defined position in the Salmonella chromosome and measuring the effects on fitness. Using highly sensitive competition assays, we found that 8 inserts were deleterious with selection coefficients (s) ranging from ≈ -0.007 to -0.02 and ninety did not have significant fitness effects. When inducing transcription from a PBAD promoter located at one end of the inserts, 16 transfers were deleterious and 82 were not significantly different from the control. In conclusion, a major fraction of the inserts had minor effects on fitness implying that extra DNA transferred by HGT, even though it does not confer an immediate selective advantage, could be maintained at selection-transfer balance and serve as raw material for the evolution of novel beneficial functions.
    No preview · Article · Feb 2014 · Molecular Biology and Evolution

Publication Stats

7k Citations
1,006.92 Total Impact Points


  • 1991-2015
    • Uppsala University
      • • Department of Medical Biochemistry and Microbiology
      • • Department of Cell and Molecular Biology
      Uppsala, Uppsala, Sweden
  • 2004-2007
    • Karolinska Institutet
      • Department of Microbiology, Tumor and Cell Biology (MTC)
      Solna, Stockholm, Sweden
    • Hospital Universitario Ramón y Cajal
      • Departamento de Microbiologia y Parasitología
      Madrid, Madrid, Spain
  • 1999-2006
    • Swedish Institute for Communicable Disease Control
      Tukholma, Stockholm, Sweden
  • 2005
    • KTH Royal Institute of Technology
      • School of Biotechnology (BIO)
      Tukholma, Stockholm, Sweden
  • 2003
    • Uppsala University Hospital
      • Department of Infectious Diseases
      Uppsala, Uppsala, Sweden
  • 1990
    • University of Utah
      • Department of Biology
      Salt Lake City, Utah, United States