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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.
    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. 

  • 5. Brolund, Alma
    et al.
    Franzen, Oscar
    Melefors, Ojar
    Tegmark-Wisell, Karin
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Plasmidome-Analysis of ESBL-Producing Escherichia coli Using Conventional Typing and High-Throughput Sequencing2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 6, p. e65793-Article in journal (Refereed)
    Abstract [en]

    Infections caused by Extended spectrum beta-lactamase (ESBL)-producing E. coli are an emerging global problem, threatening the effectiveness of the extensively used beta-lactam antibiotics. ESBL dissemination is facilitated by plasmids, transposons, and other mobile elements. We have characterized the plasmid content of ESBL-producing E. coli from human urinary tract infections. Ten diverse isolates were selected; they had unrelated pulsed-field gel electrophoresis (PFGE) types (<90% similarity), were from geographically dispersed locations and had diverging antibiotic resistance profiles. Three isolates belonged to the globally disseminated sequence type ST131. ESBL-genes of the CTX-M-1 and CTX-M-9 phylogroups were identified in all ten isolates. The plasmid content (plasmidome) of each strain was analyzed using a combination of molecular methods and high-throughput sequencing. Hidden Markov Model-based analysis of unassembled sequencing reads was used to analyze the genetic diversity of the plasmid samples and to detect resistance genes. Each isolate contained between two and eight distinct plasmids, and at least 22 large plasmids were identified overall. The plasmids were variants of pUTI89, pKF3-70, pEK499, pKF3-140, pKF3-70, p1ESCUM, pEK204, pHK17a, p083CORR, R64, pLF82, pSFO157, and R721. In addition, small cryptic high copy-number plasmids were frequent, containing one to seven open reading frames per plasmid. Three clustered groups of such small cryptic plasmids could be distinguished based on sequence similarity. Extrachromosomal prophages were found in three isolates. Two of them resembled the E. coli P1 phage and one was previously unknown. The present study confirms plasmid multiplicity in multi-resistant E. coli. We conclude that high-throughput sequencing successfully provides information on the extrachromosomal gene content and can be used to generate a genetic fingerprint of possible use in epidemiology. This could be a valuable tool for tracing plasmids in outbreaks.

  • 6.
    Brolund, Alma
    et al.
    Publ Hlth Agcy Sweden, Dept Microbiol, Solna, Sweden.
    Rajer, Fredrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Giske, Christian G.
    Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Stockholm, Sweden;Karolinska Univ Hosp, Dept Clin Microbiol, Solna, Sweden.
    Melefors, Öjar
    Publ Hlth Agcy Sweden, Dept Microbiol, Solna, Sweden;Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Stockholm, Sweden.
    Titelman, Emilia
    South Stockholm Gen Hosp, Dept Infect Dis, Stockholm, Sweden.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dynamics of Resistance Plasmids in Extended-Spectrum-beta-Lactamase-Producing Enterobacteriaceae during Postinfection Colonization2019In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 63, no 4, article id e02201-18Article in journal (Refereed)
    Abstract [en]

    Extended-spectrum beta-lactamase-producing Enterobacteriaceae (EPE) are a major cause of bloodstream infections, and the colonization rate of EPE in the gut microbiota of individuals lacking prior hospitalization or comorbidities is increasing. In this study, we performed an in-depth investigation of the temporal dynamics of EPE and their plasmids during one year by collecting fecal samples from three patients initially seeking medical care for urinary tract infections. In two of the patients, the same strain that caused the urinary tract infection ( UTI) was found at all consecutive samplings from the gut microbiota, and no other EPEs were detected, while in the third patient the UTI strain was only found in the initial UTI sample. Instead, this patient presented a complex situation where a mixed microbiota of different EPE strain types, including three different E. coli ST131 variants, as well as different bacterial species, was identified over the course of the study. Different plasmid dynamics were displayed in each of the patients, including the spread of plasmids between different strain types over time and the transposition of bla(CTX-M-15) from the chromosome to a plasmid, followed by subsequent loss through homologous recombination. Small cryptic plasmids were found in all isolates from all patients, and they appear to move frequently between different strains in the microbiota. In conclusion, we could demonstrate an extensive variation of EPE strain types, plasmid composition, rearrangements, and horizontal gene transfer of genetic material illustrating the high dynamics nature and interactive environment of the gut microbiota during post-UTI carriage.

  • 7.
    Brolund, Alma
    et al.
    Karolinska Univ Hosp, Div Clin Microbiol, Dept Lab Med LABMED, Stockholm, Sweden..
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Characterization of ESBL disseminating plasmids2016In: INFECTIOUS DISEASES, ISSN 2374-4235, Vol. 48, no 1, p. 18-25Article, review/survey (Refereed)
    Abstract [en]

    Bacteria producing extended-spectrum -lactamases (ESBLs) constitute a globally increasing problem that contributes to treatment complications and elevated death rates. The extremely successful dissemination by ESBL-producing Enterobacteriaceae during the latest decades is a result of the combination of mobilization, evolution and horizontal spread of -lactamase genes on plasmids. In parallel, spread of these plasmids to particularly well-adapted bacterial clones (outbreak clones) has expanded. In this review we describe ESBL-producing bacteria and the genetic mechanisms for dissemination of ESBL resistance. We describe available methodology for studying plasmids and the importance of including plasmids in epidemiological typing as natural parts of the organisms. Plasmids play a fundamental role in how resistance arises and disseminates.

  • 8.
    Dvirnas, Albertas
    et al.
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Pichler, Christoffer
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Stewart, Callum L.
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Quaderi, Saair
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden.;Chalmers Univ Technol, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Nyberg, Lena K.
    Chalmers Univ Technol, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Muller, Vilhelm
    Chalmers Univ Technol, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Bikkarolla, Santosh Kumar
    Chalmers Univ Technol, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Kristiansson, Erik
    Univ Gothenburg, Chalmers Univ Technol, Dept Math Sci, Gothenburg, Sweden..
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Westerlund, Fredrik
    Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
    Ambjornsson, Tobias
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Facilitated sequence assembly using densely labeled optical DNA barcodes: A combinatorial auction approach2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 3, article id e0193900Article in journal (Refereed)
    Abstract [en]

    The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Piecing the contigs together is an especially difficult task for previously unsequenced DNA, and may not be feasible due to factors such as the lack of sufficient coverage or larger repetitive regions which generate gaps in the final sequence. Here we propose a new method for scaffolding such contigs. The proposed method uses densely labeled optical DNA barcodes from competitive binding experiments as scaffolds. On these scaffolds we position theoretical barcodes which are calculated from the contig sequences. This allows us to construct longer DNA sequences from the contig sequences. This proof-of-principle study extends previous studies which use sparsely labeled DNA barcodes for scaffolding purposes. Our method applies a probabilistic approach that allows us to discard "foreign" contigs from mixed samples with contigs from different types of DNA. We satisfy the contig non-overlap constraint by formulating the contig placement challenge as a combinatorial auction problem. Our exact algorithm for solving this problem reduces computational costs compared to previous methods in the combinatorial auction field. We demonstrate the usefulness of the proposed scaffolding method both for synthetic contigs and for contigs obtained using Illumina sequencing for a mixed sample with plasmid and chromosomal DNA.

  • 9. Frykholm, K.
    et al.
    Nyberg, L. K.
    Lagerstedt, E.
    Noble, C.
    Fritzsche, J.
    Karami, N.
    Ambjornsson, T.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Westerlund, F.
    Fast size-determination of intact bacterial plasmids using nanofluidic channels2015In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 15, no 13, p. 2739-2743Article in journal (Refereed)
    Abstract [en]

    We demonstrate how nanofluidic channels can be used as a tool to rapidly determine the number and sizes of plasmids in bacterial isolates. Each step can be automated at low cost, opening up opportunities for general use in microbiology labs.

  • 10.
    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.

  • 11.
    Gullberg, Erik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Ilbäck, Carolina
    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.
    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.
    Selection of Resistant Bacteria at Very Low Antibiotic Concentrations2011In: PLoS pathogens, ISSN 1553-7366, Vol. 7, no 7, p. e1002158-Article in journal (Refereed)
    Abstract [en]

    The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations.

  • 12.
    Hasan, Badrul
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
    Melhus, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Alam, Munirul
    Olsen, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    The Gull (Chroicocephalus brunnicephalus) as an Environmental Bioindicator and Reservoir for Antibiotic Resistance on the Coastlines of the Bay of Bengal2014In: Microbial Drug Resistance, ISSN 1076-6294, E-ISSN 1931-8448, Vol. 20, no 5, p. 466-471Article in journal (Refereed)
    Abstract [en]

    The presence and frequency of multiresistant bacteria in wild birds act as indicators of the environmental contamination of antibiotic resistance. To explore the rate of contamination mediated by Escherichia coli, 150 fecal samples from the brown-headed gull (Chroicocephalus brunnicephalus) and 8 water samples from the Bay of Bengal area were collected, cultured, and tested for antibiotic susceptibility. Special attention was paid to extended-spectrum beta-lactamase (ESBL)-producing isolates, which were further characterized genetically. Antibiotic resistance was found in 42.3% (36/85) of the E. coli isolates and multidrug resistance in 11.8%. Isolates from the area with a higher human activity were more resistant than those from an area with a lower level of activity. Most frequent was resistance to ampicillin (29.4%), followed by trimethoprim-sulfamethoxazole (24.7%) and quinolones (22.4%). Carriage of ESBL-producing E. coli was relatively high (17.3%) in the gulls, whereas no ESBL producers were found in the water. All ESBL-producing E. coli isolates, but one, carried blaCTX-M-15 or blaCTX-M-15-like genes. A blaCTX-M-14-like enzyme was found as an exception. Gulls from two different colonies shared E. coli clones and harbored the clinically relevant sequence types ST10, ST48, and ST131. The high frequency of antibiotic resistance and ESBL production among E. coli isolates from gulls indicates that the environmental contamination of antibiotic resistance has already gone far on the coastlines of the Bay of Bengal. Considering the limited control over the antibiotic consumption and waste from human activities in Bangladesh, there is no easy solution in sight.

  • 13.
    Hasan, Badrul
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Melhus, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Drobni, Mirva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Hernandez, Jorge
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Waldenström, Jonas
    Alam, Munirul
    Olsen, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Antimicrobial Drug-Resistant Escherichia coli in Wild Birds and Free-range Poultry, Bangladesh2012In: Emerging Infectious Diseases, ISSN 1080-6040, E-ISSN 1080-6059, Vol. 18, no 12, p. 2055-2058Article in journal (Refereed)
    Abstract [en]

    Multidrug resistance was found in 22.7% of Escherichia coli isolates from bird samples in Bangladesh; 30% produced extended-spectrum β-lactamases, including clones of CTX-M genes among wild and domestic birds. Unrestricted use of antimicrobial drugs in feed for domestic birds and the spread of resistance genes to the large bird reservoir in Bangladesh are growing problems.

  • 14.
    Hou, Zining
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    An, Yu
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hjort, Klas
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wu, Zhigang
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Time lapse investigation of antibiotic susceptibility using a microfluidic linear gradient 3D culture device2014In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, no 17, p. 3409-3418Article in journal (Refereed)
    Abstract [en]

    This study reports a novel approach to quantitatively investigate the antibacterial effect of antibiotics on bacteria using a three-dimensional microfluidic culture device. In particular, our approach is suitable for studying the pharmacodynamics effects of antibiotics on bacterial cells temporally and with a continuous range of concentrations in a single experiment. The responses of bacterial cells to a linear concentration gradient of antibiotics were observed using time-lapse photography, by encapsulating bacterial cells in an agarose-based gel located in a commercially available microfluidics chamber. This approach generates dynamic information with high resolution, in a single operation, e. g., growth curves and antibiotic pharmacodynamics, in a well-controlled environment. No pre-labelling of the cells is needed and therefore any bacterial sample can be tested in this setup. It also provides static information comparable to that of standard techniques for measuring minimum inhibitory concentration (MIC). Five antibiotics with different mechanisms were analysed against wild-type Escherichia coli, Staphylococcus aureus and Salmonella Typhimurium. The entire process, including data analysis, took 2.5-4 h and from the same analysis, high-resolution growth curves were obtained. As a proof of principle, a pharmacodynamic model of streptomycin against Salmonella Typhimurium was built based on the maximal effect model, which agreed well with the experimental results. Our approach has the potential to be a simple and flexible solution to study responding behaviours of microbial cells under different selection pressures both temporally and in a range of concentrations.

  • 15. Koskiniemi, Sanna
    et al.
    Garza-Sanchez, Fernando
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Webb, Julia S.
    Braaten, Bruce A.
    Poole, Stephen J.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hayes, Christopher S.
    Low, David A.
    Selection of Orphan Rhs Toxin Expression in Evolved Salmonella enterica Serovar Typhimurium2014In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, no 3, p. e1004255-Article in journal (Refereed)
    Abstract [en]

    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.

  • 16.
    Koskiniemi, Sanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gibbons, Henry S.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Anwar, Naeem
    Ouellette, Gary
    Broomall, Stacey
    Karavis, Mark
    McGregor, Paul
    Liem, Alvin
    Fochler, Ed
    McNew, Lauren
    Rosenzweig, Carolyn Nicole
    Rhen, Mikael
    Skowronski, Evan W.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pathoadaptive Mutations in Salmonella enterica Isolated after Serial Passage in Mice2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 7, p. e70147-Article in journal (Refereed)
    Abstract [en]

    How pathogenic bacteria adapt and evolve in the complex and variable environment of the host remains a largely unresolved question. Here we have used whole genome sequencing of Salmonella enterica serovar Typhimurium LT2 populations serially passaged in mice to identify mutations that adapt bacteria to systemic growth in mice. We found unique pathoadaptive mutations in two global regulators, phoQ and stpA, which increase the competitive indexes of the bacteria 3- to 5-fold. Also, all mouse-adapted lineages had changed the orientation of the hin invertable element, resulting in production of a FliC type of flagellum. Competition experiments in mice with locked flagellum mutants showed that strains expressing the FliC type of flagellum had a 5-fold increase in competitive index as compared to those expressing FljB type flagellum. Combination of the flagellum cassette inversion with the stpA mutation increased competitive indexes up to 20-fold. These experiments show that Salmonella can rapidly adapt to a mouse environment by acquiring a few mutations of moderate individual effect that when combined confer substantial increases in growth.

  • 17.
    Linkevicius, Marius
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Anderssen, Jytte Mark
    Statens Serum Institut.
    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.
    Fitness of Escherichia coli mutants with reduced susceptibility to tigecycline2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 5, p. 1307-1313Article in journal (Refereed)
    Abstract [en]

    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. 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. 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 similar to 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. 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.

  • 18.
    Linkevicius, Marius
    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 I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mechanisms and fitness costs of tigecycline resistance in Escherichia coli2013In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 12, p. 2809-2819Article in journal (Refereed)
    Abstract [en]

    Objectives: To stepwise select tigecycline-resistant Escherichia coli mutants in vitro, determine the mutation rates, identify the resistance mechanisms, determine the resistance level and cross-resistance to other antibiotic classes, evaluate the fitness costs of tigecycline resistance mechanisms and investigate if the same in vitro-identified target genes were mutated in clinical isolates.

    Methods: Spontaneous mutants with reduced susceptibility to tigecycline were selected on agar plates supplemented with tigecycline. Resistance levels and cross-resistance were evaluated by performing MIC assays and determining mutation rates using Luria-Delbruck fluctuation tests. Mutant fitness was estimated by measuring exponential growth rates, lag phase and total yield. Illumina whole-genome sequencing was used to identify mutations increasing MICs of tigecycline.

    Results: Spontaneous mutants with reduced susceptibility to tigecycline were selected at a rate of similar to 10-8 to 10-6 per cell per generation; however, the clinical MIC breakpoint was not reached. The resistance level of tigecycline was low and some of the mutants had elevated MICs of hydrophobic drugs (chloramphenicol, erythromycin and novobiocin) or decreased MICs of SOS response inducers (ciprofloxacin and nitrofurantoin). Mutations were identified in efflux regulatory network genes (lon, acrR and marR) or lipopolysaccharide core biosynthesis pathway genes (lpcA, rfaE, rfaD, rfaC and rfaF). Mutations in the same target genes were found in clinical isolates.

    Conclusions: Tigecycline selects for low-level resistance mutations with relatively high mutation rates and the majority of them come with a substantial fitness cost. Further in vivo experiments are needed to evaluate how these mutations affect bacterial virulence and ability to establish a successful infection.

  • 19.
    Linkevicius, Marius
    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 I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Potential of tetracycline resistance proteins to evolve tigecycline resistance2016In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 60, no 2, p. 789-796Article in journal (Refereed)
    Abstract [en]

    Tigecycline is a glycylcycline antibiotic 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 this affects resistance to earlier classes of tetracyclines. Mutations in all four tet genes caused a significant increase in the tigecycline MIC in Escherichia coli, and strains expressing mutant Tet(A) and Tet(X) variants reached clinically relevant MICs (2 mg/liter and 3 mg/liter, 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 the functionally most important loop III of domain IV. Deletion of leucine 505 of this loop led to the highest increase of the tigecycline MIC (0.5 mg/liter) among Tet(M) mutants. It also caused collateral sensitivity to earlier classes of tetracyclines. A majority of the Tet(X) mutants showed increased activity against all three classes 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.

  • 20.
    Lytsy, Birgitta
    et al.
    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.
    Tano, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Torell, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Melhus, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    The first major extended-spectrum beta-lactamase outbreak in Scandinavia was caused by clonal spread of a multiresistant Klebsiella pneumoniae producing CTX-M-152008In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 116, no 4, p. 302-8Article in journal (Refereed)
    Abstract [en]

    Between May and December 2005, 64 multidrug-resistant isolates of Klebsiella pneumoniae were detected from patients admitted to Uppsala University Hospital. This represented a dramatic increase in ESBL-producing K. pneumoniae compared to previous years. To investigate the epidemiology and to characterize the resistance mechanisms of the isolates, a study was initiated. Antibiotic susceptibility was determined by means of the Etest and the disc diffusion method. Extended-spectrum beta-lactamase (ESBL) production was identified by clavulanic acid synergy test and confirmed with PCR amplification followed by DNA sequencing. DNA profiles of the isolates were examined with pulsed-field gel electrophoresis (PFGE). All isolates were resistant or exhibited reduced susceptibility to cefadroxil, cefuroxime, cefotaxime, ceftazidime, aztreonam, piperacillin/tazobactam, ciprofloxacin, tobramycin, and trimethoprim-sulfamethoxazole. They produced ESBL of the CTX-M-15 type, and the involvement of a single K. pneumoniae clone was shown. This is the first major clonal outbreak of multiresistant ESBL-producing K. pneumoniae in Scandinavia. The outbreak demonstrates the epidemic potential of enterobacteria containing ESBLs of the CTX-M type, even in a country with a relatively low selective pressure and a low prevalence of multiresistant bacteria.

  • 21.
    Mueller, Vilhelm
    et al.
    Chalmers, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Rajer, Fredrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Frykholm, Karolin
    Chalmers, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Nyberg, Lena K.
    Chalmers, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Quaderi, Saair
    Chalmers, Dept Biol & Biol Engn, Gothenburg, Sweden.;Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Fritzsche, Joachim
    Chalmers, Dept Appl Phys, Gothenburg, Sweden..
    Kristiansson, Erik
    Univ Gothenburg, Chalmers Univ Technol, Dept Math Sci, Gothenburg, Sweden.;Univ Gothenburg, Ctr Antibiot Resistance Res CARe, Gothenburg, Sweden..
    Ambjornsson, Tobias
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Westerlund, Fredrik
    Chalmers, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 37938Article in journal (Refereed)
    Abstract [en]

    Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples.

  • 22.
    Nahid, Fouzia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Quaid I Azam Univ, Dept Microbiol, Islamabad, Pakistan.
    Zahra, Rabaab
    Quaid I Azam Univ, Dept Microbiol, Islamabad, Pakistan..
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    A bla(OXA-181)-harbouring multi-resistant ST147 Klebsiella pneumoniae isolate from Pakistan that represent an intermediate stage towards pan-drug resistance2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 12, article id e0189438Article in journal (Refereed)
    Abstract [en]

    Carbapenem resistant Klebsiella pneumoniae (CR-KP) infections are an ever-increasing global issue, especially in the Indian subcontinent. Here we report genetic insight into a bla(OXA-181) harbouring Klebsiella pneumoniae, belonging to the pandemic lineage ST147, that represents an intermediate stage towards pan-drug resistance. The CR-KP isolate DA48896 was isolated from a patient from Pakistan and was susceptible only to tigecycline and colistin. It harboured bla(OXA-181) and was assigned to sequence type ST147. Analysis from whole genome sequencing revealed a very high sequence similarity to the previously sequenced pan-resistant K. pneumoniae isolate MS6671 from the United Arab Emirates. The two isolates are very closely related with only 46 chromosomal nucleotide differences, 14 indels and differences in plasmid content. Both carry a substantial number of plasmidborne and chromosomally encoded resistance determinants. Interestingly, the two differences in susceptibility between the isolates could be attributed to DA48896 lacking an insertion of bla(OXA-181) into the mgrB gene that results in colistin resistance in MS6671 and SNPs affecting AcrAB efflux pump expression likely to result in tigecycline resistance. These differences between the otherwise very similar isolates indicate that strong selection has occurred for resistance towards these last-resort drugs and illustrates the trajectory of resistance evolution of OXA-181-producing versions of the ST147 international risk clone.

  • 23.
    Ny, Sofia
    et al.
    Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Alfred Nobels Alle 10, S-14152 Stockholm, Sweden;Publ Hlth Agcy Sweden, Nobels Vag 18, S-17182 Stockholm, Sweden.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Salemi, Marco
    Univ Florida, Dept Pathol, Emerging Pathogens Inst, POB 100009, Gainesville, FL 32610 USA.
    Giske, Christian G.
    Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Alfred Nobels Alle 10, S-14152 Stockholm, Sweden.
    Genome and plasmid diversity of Extended-Spectrum beta-Lactamase-producing Escherichia coli ST131-tracking phylogenetic trajectories with Bayesian inference2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 10291Article in journal (Refereed)
    Abstract [en]

    Clonal lineages of ESBL (Extended-Spectrum beta-Lactamase)-producing E. coli belonging to sequence type 131 (ST131) have disseminated globally during the last 30 years, leading to an increased prevalence of resistance to fluoroquinolones and extended-spectrum cephalosporins in clinical isolates of E. coli. We aimed to study if Swedish ESBL-producing ST131 isolates originated from single or multiple introductions to the population by assessing the amount of genetic variation, on chromosomal and plasmid level, between Swedish and international E. coli ST131. Bayesian inference of Swedish E. coli ST131 isolates (n = 29), sequenced using PacBio RSII, together with an international ST131 dataset showed that the Swedish isolates were part of the international ST131 A, C1 and C2 clades. Highly conserved plasmids were identified in three clusters although they were separated by several years, which indicates a strong co-evolution between some ST131 lineages and specific plasmids. In conclusion, the tight clonal relationship observed within the ST131 clades, together with highly conserved plasmids, challenges investigation of strain transmission events. A combination of few SNPs on a genome-wide scale and an epidemiological temporospatial link, are needed to track the spread of the ST131 subclones.

  • 24.
    Rajer, Fredrika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Allander, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Philip, Karlsson
    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.
    Evolutionary trajectories towards high-level β-lactam/β-lactamase inhibitor resistance of a multi-resistance plasmid carrying multiple β-lactamasesManuscript (preprint) (Other academic)
  • 25.
    Rajer, Fredrika
    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.
    The role of antibiotic resistance genes in the fitness cost of multi-resistance plasmidsManuscript (preprint) (Other academic)
  • 26.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Low sub-minimal inhibitory concentrations of antibiotics generate new types of resistance2019In: Sustainable Chemistry and Pharmacy, ISSN 2352-5541, Vol. 11, p. 46-48Article in journal (Refereed)
    Abstract [en]

    Antibiotic resistance is a major threat to modern medicine. Routes of transmission of resistant bacteria are complex and include spread between humans, between humans and animals, between animals and to humans and animals via the environment. Recent findings have shown that resistant bacteria can be selectively enriched even at antibiotic concentrations several hundred-fold lower than previously expected, such as those found in sewage water. In addition, these low concentrations can select for high level resistant bacteria with very low fitness cost in contrast to resistant bacteria selected at high concentrations such as during antibiotic treatment of patients. This calls for action to determine what concentrations and combinations of antibiotics that can be considered safe in waste water and ensure proper measures to reduce the antropogenic contamination with antibiotics.

  • 27.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selection of antibiotic resistance at very low antibiotic concentrations2014In: Upsala Journal of Medical Sciences, ISSN 0300-9734, E-ISSN 2000-1967, Vol. 119, no 2, p. 103-107Article, review/survey (Refereed)
    Abstract [en]

    Human use of antibiotics has driven the selective enrichment of pathogenic bacteria resistant to clinically used drugs. Traditionally, the selection of resistance has been considered to occur mainly at high, therapeutic levels of antibiotics, but we are now beginning to understand better the importance of selection of resistance at low levels of antibiotics. The concentration of an antibiotic varies in different body compartments during treatment, and low concentrations of antibiotics are found in sewage water, soils, and many water environments due to natural production and contamination from human activities. Selection of resistance at non-lethal antibiotic concentrations (below the wild-type minimum inhibitory concentration) occurs due to differences in growth rate at the particular antibiotic concentration between cells with different tolerance levels to the antibiotic. The minimum selective concentration for a particular antibiotic is reached when its reducing effect on growth of the susceptible strain balances the reducing effect (fitness cost) of the resistance determinant in the resistant strain. Recent studies have shown that resistant bacteria can be selected at concentrations several hundred-fold below the lethal concentrations for susceptible cells. Resistant mutants selected at low antibiotic concentrations are generally more fit than those selected at high concentrations but can still be highly resistant. The characteristics of selection at low antibiotic concentrations, the potential clinical problems of this mode of selection, and potential solutions will be discussed.

  • 28.
    Sandegren, Linus
    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.
    Bacterial gene amplification: implications for the evolution of antibiotic resistance2009In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 7, no 8, p. 578-588Article, review/survey (Refereed)
    Abstract [en]

    Recent data suggest that, in response to the presence of antibiotics, gene duplication and amplification (GDA) constitutes an important adaptive mechanism in bacteria. For example, resistance to sulphonamide, trimethoprim and beta-lactams can be conferred by increased gene dosage through GDA of antibiotic hydrolytic enzymes, target enzymes or efflux pumps. Furthermore, most types of antibiotic resistance mechanism are deleterious in the absence of antibiotics, and these fitness costs can be ameliorated by increased gene dosage of limiting functions. In this Review, we highlight the dynamic properties of gene amplifications and describe how they can facilitate adaptive evolution in response to toxic drugs.

  • 29.
    Sandegren, Linus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Groenheit, Ramona
    Koivula, Tuija
    Ghebremichael, Solomon
    Advani, Abdolreza
    Castro, Elsie
    Pennhag, Alexandra
    Hoffner, Sven
    Mazurek, Jolanta
    Pawlowski, Andrzej
    Kan, Boris
    Bruchfeld, Judith
    Melefors, Öjar
    Källenius, Gunilla
    Genomic Stability over 9 Years of an Isoniazid Resistant Mycobacterium tuberculosis Outbreak Strain in Sweden2011In: PLoS ONE, ISSN 1932-6203, Vol. 6, no 1, p. e16647-Article in journal (Refereed)
    Abstract [en]

    In molecular epidemiological studies of drug resistant Mycobacterium tuberculosis (TB) in Sweden a large outbreak of an isoniazid resistant strain was identified, involving 115 patients, mainly from the Horn of Africa. During the outbreak period, the genomic pattern of the outbreak strain has stayed virtually unchanged with regard to drug resistance, IS6110 restriction fragment length polymorphism and spoligotyping patterns. Here we present the complete genome sequence analyses of the index isolate and two isolates sampled nine years after the index case as well as experimental data on the virulence of this outbreak strain. Even though the strain has been present in the community for nine years and passaged between patients at least five times in-between the isolates, we only found four single nucleotide polymorphisms in one of the later isolates and a small (4 amino acids) deletion in the other compared to the index isolate. In contrast to many other evolutionarily successful outbreak lineages (e. g. the Beijing lineage) this outbreak strain appears to be genetically very stable yet evolutionarily successful in a low endemic country such as Sweden. These findings further illustrate that the rate of genomic variation in TB can be highly strain dependent, something that can have important implications for epidemiological studies as well as development of resistance.

  • 30.
    Sandegren, Linus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lindqvist, Anton
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kahlmeter, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nitrofurantoin resistance mechanism and fitness cost in Escherichia coli2008In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 62, no 3, p. 495-503Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES

    The biological fitness cost of antibiotic resistance is a key parameter in determining the rate of appearance and spread of antibiotic-resistant bacteria. We identified mutations conferring nitrofurantoin resistance and examined their effect on the fitness of clinical Escherichia coli isolates.

    METHODS

    By plating bacterial cells on agar plates containing nitrofurantoin, spontaneous nitrofurantoin-resistant E. coli mutants were isolated. The fitness of susceptible and resistant strains was measured as growth rate in the presence and absence of nitrofurantoin in rich culture medium. Time-kill kinetics of the resistant mutants was compared with the susceptible strains. Resistance mutations were identified by DNA sequencing.

    RESULTS

    Spontaneous resistant mutants of initially susceptible clinical E. coli appeared with a rate of 10(-7)/cell/generation, and these mutants showed a reduction in the growth rate compared with the susceptible parent strain. Similarly, comparison of a set of susceptible and resistant clinical isolates of E. coli showed that the average growth rate of the resistant mutants was approximately 6% lower than the susceptible strains. Furthermore, the bacterial growth rate in the presence of nitrofurantoin at therapeutic levels was greatly reduced even for nitrofurantoin-resistant mutants. The resistance-conferring mutations were identified in the nsfA and nfsB genes that encode oxygen-insensitive nitroreductases.

    CONCLUSIONS

    Nitrofurantoin resistance confers a reduction in fitness in E. coli in the absence of antibiotic. In the presence of therapeutic levels of nitrofurantoin, even resistant mutants are so disturbed in growth that they are probably unable to become enriched and establish an infection.

  • 31.
    Sandegren, Linus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Linkevicius, Marius
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lytsy, Birgitta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Åsa, Melhus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Transfer of an Escherichia coli ST131 multiresistance cassette has created a Klebsiella pneumoniae-specific plasmid associated with a major nosocomial outbreak2012In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 67, no 1, p. 74-83Article in journal (Refereed)
    Abstract [en]

    Objectives:

    To characterize the complete sequence, horizontal spread and stability of the CTX-M-15-encoding multiresistance plasmid of a Klebsiella pneumoniae strain involved in a large nosocomial outbreak.

    Methods:

    The 220 kbp plasmid pUUH239.2 was completely sequenced using 454 technology. The conjugational host range, conjugation frequencies, plasmid stability and fitness cost of plasmid carriage were studied in vitro. Conjugational spread during the outbreak was assessed retrospectively by multiplex PCR screening, restriction fragment length polymorphism and PFGE.

    Results:

    Plasmid pUUH239.2 encodes resistance to β-lactams (blaCTX-M-15, blaTEM-1 and blaOXA-1), aminoglycosides [aac-(6′)-1b-cr and aadA2], tetracyclines [tet(A) and tetR], trimethoprim (dhfrXII), sulphonamides (sul1) quaternary ammonium compounds (qacEΔ1), macrolides [mph(A)-mxr-mphR(A)] and heavy metal ions (silver, copper and arsenic). The plasmid consists of a backbone, highly similar to the K. pneumoniae plasmid pKPN3, and a 41 kbp resistance region, highly similar to the resistance regions of plasmids pEK499 and pC15-1a previously isolated from Escherichia coli strains belonging to the outbreak lineage ST131 (where ST stands for sequence type). The pUUH239.2 plasmid is stable in K. pneumoniae but unstable in E. coli and confers a fitness cost when introduced into a naive host cell. Transfer of pUUH239.2 from the outbreak K. pneumoniae clone to the E. coli of the patients’ intestinal floras has occurred on multiple occasions during the outbreak.

    Conclusions:

    The plasmid pUUH239.2 is a composite of the pKPN3 K. pneumoniae plasmid backbone and the blaCTX-M-15-encoding multiresistance cassette associated with the internationally recognized outbreak strain E. coli ST131. The resulting plasmid differs in stability between K. pneumoniae and E. coli, and this has probably limited the spread of this plasmid during the outbreak.

  • 32.
    Sandegren, Linus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Stedt, Johan
    Centre for Ecology and Evolution in Microbial Model Systems, School of Natural Sciences, Linnaeus University, Kalmar Sweden.
    Lustig, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bonnedahl, Jonas
    Centre for Ecology and Evolution in Microbial Model Systems, School of Natural Sciences, Linnaeus University, Kalmar Sweden.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Järhult, Josef D.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Long-term carriage and rapid transmission of extended spectrum beta-lactamase-producing E. coli within a flock of Mallards in the absence of antibiotic selection2018In: Environmental Microbiology Reports, ISSN 1758-2229, E-ISSN 1758-2229, Vol. 10, no 5, p. 576-582Article in journal (Refereed)
    Abstract [en]

    Wild birds have been suggested as transmitters and reservoirs for antibiotic resistant bacteria. We performed an experimental study investigating carriage time and interindividual transmission of extended spectrum beta‐lactamase‐ (ESBL‐)producing Escherichia coli in Mallards (Anas platyrhynchos) to assess if the birds carry the bacteria long enough to transfer them geographically during migration. Mallards were inoculated intraoesophageally with four different strains of ESBL‐producing E. coli and kept together in a flock. The ESBL‐strains belonged to sequence types previously shown to spread between birds and humans. Culturing from faecal samples showed presence of ESBL‐producing E. coli the entire 29 day experimental period. An extensive and rapid transmission of the different ESBL‐strains between individuals (including non‐inoculated controls) was observed. In necropsy samples, we detected ESBL‐strains in the cecum even in faeces‐negative birds, indicating that this part of the intestine could function as a reservoir of resistant bacteria. We demonstrate that birds can carry ESBL‐producing E. coli for long enough times to travel far during migration and the extensive interindividual transmission suggests spread between individuals in a dense bird population as a mechanism that allow persistence of resistant bacteria.

  • 33.
    Sütterlin, Susanne
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Edquist, Petra
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Adler, Marlen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tängdén, Thomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Drobni, Mirva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
    Olsen, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Melhus, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Silver resistance genes are overrepresented among Escherichia coli isolates with CTX-M production2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 22, p. 6863-6869Article in journal (Refereed)
    Abstract [en]

    Members of the Enterobacteriaceae with extended-spectrum beta-lactamases (ESBLs) of the CTX-M type have disseminated rapidly in recent years and have become a threat to public health. In parallel with the CTX-M type expansion, the consumption and widespread use of silver-containing products has increased. To determine the carriage rates of silver resistance genes in different Escherichia coli populations, the presence of three silver resistance genes (silE, silP, and silS) and genes encoding CTX-M-, TEM-, and SHV-type enzymes were explored in E. coli isolates of human (n = 105) and avian (n = 111) origin. The antibiotic profiles were also determined. Isolates harboring CTX-M genes were further characterized, and phenotypic silver resistance was examined. The silE gene was present in 13 of the isolates. All of them were of human origin. Eleven of these isolates harbored ESBLs of the CTX-M type (P = 0.007), and eight of them were typed as CTX-M-15 and three as CTX-M-14. None of the silE-positive isolates was related to the O25b-ST131 clone, but 10 out of 13 belonged to the ST10 or ST58 complexes. Phenotypic silver resistance (silver nitrate MIC > 512 mg/liter) was observed after silver exposure in 12 of them, and a concomitant reduced susceptibility to piperacillin-tazobactam developed in three. In conclusion, 12% of the human E. coli isolates but none of the avian isolates harbored silver resistance genes. This indicates another route for or level of silver exposure for humans than that caused by common environmental contamination. Since silE-positive isolates were significantly more often found in CTX-M-positive isolates, it is possible that silver may exert a selective pressure on CTX-M-producing E. coli isolates.

  • 34.
    Tängdén, Thomas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Adler, Marlen
    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.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Löwdin, Elisabeth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Frequent emergence of porin-deficient subpopulations with reduced carbapenem susceptibility in ESBL-producing Escherichia coli during exposure to ertapenem in an in vitro pharmacokinetic model2013In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 6, p. 1319-1326Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES:

    Ertapenem resistance is increasing in Enterobacteriaceae. The production of extended-spectrum β-lactamases (ESBLs) and reduced expression of outer membrane porins are major mechanisms of resistance in ertapenem-resistant Klebsiella pneumoniae. Less is known of ertapenem resistance in Escherichia coli. The aim of this study was to explore the impact of ESBL production in E. coli on the antibacterial activity of ertapenem.

    METHODS:

    Two E. coli strains, with and without ESBL production, were exposed to ertapenem in vitro for 48 h at concentrations simulating human pharmacokinetics with conventional and higher dosages.

    RESULTS:

    Isolates with non-susceptibility to ertapenem (MICs 0.75-1.5 mg/L) were detected after five of nine time-kill experiments with the ESBL-producing strain. All of these isolates had ompR mutations, which reduce the expression of outer membrane porins OmpF and OmpC. Higher dosage did not prevent selection of porin-deficient subpopulations. No mutants were detected after experiments with the non-ESBL-producing strain. Compared with other experiments, experiments with ompR mutants detected in endpoint samples showed significantly less bacterial killing after the second dose of ertapenem. Impaired antibacterial activity against E. coli with ESBL production and ompR mutation was also demonstrated in time-kill experiments with static antibiotic concentrations.

    CONCLUSIONS:

    The combination of ESBL production and porin loss in E. coli can result in reduced susceptibility to ertapenem. Porin-deficient subpopulations frequently emerged in ESBL-producing E. coli during exposure to ertapenem at concentrations simulating human pharmacokinetics. Inappropriate use of ertapenem should be avoided to minimize the risk of selection of ESBL-producing bacteria with reduced susceptibility to carbapenems.

  • 35.
    Zining, Hou
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Yu, An
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hjort, Klas
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wu, Zhigang
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    A microfluidic approach for dynamic investigation of the antibiotic susceptibility of bacteria2013In: MME 2013 24th Micromechanics and Microsystems Europe Conference, 2013Conference paper (Refereed)
    Abstract [en]

    The possibility to study the effect on cell growth of a chemical is a valued tool for re- searchers in different areas, such as antibiotic resis- tance, cancer research and metabolic pathways. Traditional approaches need long time and no dynamic information is given. Microfluidics offers short diffusion lengths and steeper gradients for studies of antibiotic susceptibility, which could improve throughput greatly. By combining with time-lapse micrography, information on the dynam- ics may provide additional understanding. A micro- fluidic 3D cell culture chip was used to determine the MIC (minimum inhibitory concentration) and the dynamics of sub-MIC of E. coli against ampicillin and spectinomycin. And the same works have been done in Staphylococcus aureus and Salmonella. It is the first time to reveal this dynamic behaviour of bacteria against antibiotics quantitatively in a micro- fludic device. It is anticipated that it could be ex- tended to many other similar investigations in bio- logical and relevant pharmaceutical or clinical applications.

1 - 35 of 35
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