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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.
    Ahlstrom, Christina A. A.
    et al.
    US Geol Survey, Alaska Sci Ctr, Anchorage, AK 99508 USA..
    Woksepp, Hanna
    Kalmar Cty Reg, Dept Res, Kalmar, Sweden.;Linnaeus Univ, Dept Chem & Biomed Sci, Kalmar, Sweden..
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ramey, Andrew M. M.
    US Geol Survey, Alaska Sci Ctr, Anchorage, AK 99508 USA..
    Bonnedahl, Jonas
    Linköping Univ, Dept Biomed & Clin Sci, Linköping, Sweden.;Kalmar Cty Reg, Dept Infect Dis, Kalmar, Sweden..
    Exchange of Carbapenem-Resistant Escherichia coli Sequence Type 38 Intercontinentally and among Wild Bird, Human, and Environmental Niches2023In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 89, no 6Article in journal (Refereed)
    Abstract [en]

    Carbapenem-resistant Enterobacteriaceae (CRE) are a global threat to human health and are increasingly being isolated from nonclinical settings. OXA-48-producing Escherichia coli sequence type 38 (ST38) is the most frequently reported CRE type in wild birds and has been detected in gulls or storks in North America, Europe, Asia, and Africa. The epidemiology and evolution of CRE in wildlife and human niches, however, remains unclear. We compared wild bird origin E. coli ST38 genome sequences generated by our research group and publicly available genomic data derived from other hosts and environments to (i) understand the frequency of intercontinental dispersal of E. coli ST38 clones isolated from wild birds, (ii) more thoroughly measure the genomic relatedness of carbapenem-resistant isolates from gulls sampled in Turkey and Alaska, USA, using long-read whole-genome sequencing and assess the spatial dissemination of this clone among different hosts, and (iii) determine whether ST38 isolates from humans, environmental water, and wild birds have different core or accessory genomes (e.g., antimicrobial resistance genes, virulence genes, plasmids) which might elucidate bacterial or gene exchange among niches. Our results suggest that E. coli ST38 strains, including those resistant to carbapenems, are exchanged between humans and wild birds, rather than separately maintained populations within each niche. Furthermore, despite close genetic similarity among OXA-48-producing E. coli ST38 clones from gulls in Alaska and Turkey, intercontinental dispersal of ST38 clones among wild birds is uncommon. Interventions to mitigate the dissemination of antimicrobial resistance throughout the environment (e.g., as exemplified by the acquisition of carbapenem resistance by birds) may be warranted.

    IMPORTANCE Carbapenem-resistant bacteria are a threat to public health globally and have been found in the environment as well as the clinic. Some bacterial clones are associated with carbapenem resistance genes, such as Escherichia coli sequence type 38 (ST38) and the carbapenemase gene blaOXA-48. This is the most frequently reported carbapenem-resistant clone in wild birds, though it was unclear if it circulated within wild bird populations or was exchanged among other niches. The results from this study suggest that E. coli ST38 strains, including those resistant to carbapenems, are frequently exchanged among wild birds, humans, and the environment. Carbapenem-resistant E. coli ST38 clones in wild birds are likely acquired from the local environment and do not constitute an independent dissemination pathway within wild bird populations. Management actions aimed at preventing the environmental dissemination and acquisition of antimicrobial resistance by wild birds may be warranted.

  • 5.
    Ahlstrom, Christina A.
    et al.
    US Geol Survey, Alaska Sci Ctr, 4210 Univ Dr, Anchorage, AK 99508 USA..
    Woksepp, Hanna
    Kalmar Cty Hosp, Dept Dev & Publ Hlth, S-39185 Kalmar, Sweden.;Linnaeus Univ, Dept Med & Optometry, S-39185 Kalmar, Sweden..
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mohsin, Mashkoor
    Univ Agr Faisalabad, Inst Microbiol, Faisalabad 38040, Pakistan..
    Hasan, Badrul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Anim Bacteriol Sect, Anim Bacteriol Sect Microbial Sci Pests & Dis, Bundoora, Vic, Australia..
    Muzyka, Denys
    Inst Expt & Clin Vet Med, Natl Sci Ctr, UA-61023 Kharkiv, Ukraine..
    Hernandez, Jorge
    Kalmar Cty Hosp, Dept Clin Microbiol, SE-39185 Kalmar, Sweden..
    Aguirre, Filip
    Kalmar Cty Hosp, Dept Clin Microbiol, SE-39185 Kalmar, Sweden..
    Tok, Atalay
    Uppsala Univ, Dept Med Sci, Zoonosis Sci Ctr, SE-75185 Uppsala, Sweden..
    Soderman, Jan
    Linköping Univ, Dept Clin & Expt Med, Lab Med, Linköping, Sweden..
    Olsen, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Ramey, Andrew M.
    US Geol Survey, Alaska Sci Ctr, 4210 Univ Dr, Anchorage, AK 99508 USA..
    Bonnedahl, Jonas
    Linköping Univ, Dept Biomed & Clin Sci, S-58183 Linköping, Sweden.;Region Kalmar Cty, Dept Infect Dis, S-39185 Kalmar, Sweden..
    Genomically diverse carbapenem resistant Enterobacteriaceae fromwild birds provide insight into global patterns of spatiotemporal dissemination2022In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 824, article id 153632Article in journal (Refereed)
    Abstract [en]

    Carbapenem resistant Enterobacteriaceae (CRE) are a threat to public health globally, yet the role of the environment in the epidemiology of CRE remains elusive. Given that wild birds can acquire CRE, likely from foraging in anthropogenically impacted areas, and may aid in the maintenance and dissemination of CRE in the environment, a spatiotemporal comparison of isolates from different regions and timepoints may be useful for elucidating epidemiological information. Thus, we characterized the genomic diversity of CRE from fecal samples opportunistically collected from gulls (Larus spp.) inhabiting Alaska (USA), Chile, Spain, Turkey, and Ukraine and from black kites (Milvus migrans) sampled in Pakistan and assessed evidence for spatiotemporal patterns of dissemination. Within and among sampling locations, a high diversity of carbapenemases was found, including Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), oxacillinase (OXA), and Verona integron Metallo beta-lactamase (VIM). Although the majority of genomic comparisons among samples did not provide evidence for spatial dissemination, we did find strong evidence for dissemination among Alaska, Spain, and Turkey. We also found strong evidence for temporal dissemination among samples collected in Alaska and Pakistan, though the majority of CRE clones were transitory and were not repeatedly detected among locations where samples were collected longitudinally. Carbapenemase-producing hypervirulent K. pneumoniae was isolated from gulls in Spain and Ukraine and some isolates harbored antimicrobial resistance genes conferring resistance to up to 10 different antibiotic classes, including colistin. Our results are consistent with local acquisition of CRE by wild birds with spatial dissemination influenced by intermediary transmission routes, likely involving humans. Furthermore, our results support the premise that anthropogenicallyassociated wild birds may be good sentinels for understanding the burden of clinically-relevant antimicrobial resistance in the local human population.

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

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

  • 7.
    Allander, Lisa
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Vickberg, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Lagerbäck, Pernilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Sandegren, Linus
    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, Infection medicine.
    Evaluation of In Vitro Activity of Double-Carbapenem Combinations against KPC-2-, OXA-48-and NDM-Producing Escherichia coli and Klebsiella pneumoniae2022In: Antibiotics, ISSN 0066-4774, E-ISSN 2079-6382, Vol. 11, no 11, article id 1646Article in journal (Refereed)
    Abstract [en]

    Double-carbapenem combinations have shown synergistic potential against carbapenemase-producing Enterobacterales, but data remain inconclusive. This study evaluated the activity of double-carbapenem combinations against 51 clinical KPC-2-, OXA-48-, NDM-1, and NDM-5-producing Escherichia coli and Klebsiella pneumoniae and against constructed E. coli strains harboring genes encoding KPC-2, OXA-48, or NDM-1 in an otherwise isogenic background. Two-drug combinations of ertapenem, meropenem, and doripenem were evaluated in 24 h time-lapse microscopy experiments with a subsequent spot assay and in static time-kill experiments. An enhanced effect in time-lapse microscopy experiments at 24 h and synergy in the spot assay was detected with one or more combinations against 4/14 KPC-2-, 17/17 OXA-48-, 2/17 NDM-, and 1/3 NDM-1+OXA-48-producing clinical isolates. Synergy rates were higher against meropenem- and doripenem-susceptible isolates and against OXA-48 producers. NDM production was associated with significantly lower synergy rates in E. coli. In time-kill experiments with constructed KPC-2-, OXA-48- and NDM-1-producing E. coli, 24 h synergy was not observed; however, synergy at earlier time points was found against the KPC-2- and OXA-48-producing constructs. Our findings indicate that the benefit of double-carbapenem combinations against carbapenemase-producing E. coli and K. pneumoniae is limited, especially against isolates that are resistant to the constituent antibiotics and produce NDM.

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  • 8.
    Atterby, Clara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Nykvist, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Natl Vet Inst, Uppsala, Sweden..
    Lustig, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Järhult, Josef D.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selection of Resistant Bacteria in Mallards Exposed to Subinhibitory Concentrations of Ciprofloxacin in Their Water Environment2021In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 65, no 3, article id e01858-20Article in journal (Refereed)
    Abstract [en]

    Emergence and selection of antibiotic resistance following exposure to high antibiotic concentrations have been repeatedly shown in clinical and agricultural settings, whereas the role of the weak selective pressures exerted by antibiotic levels below the MIC (sub-MIC) in aquatic environments due to anthropogenic contamination remains unclear. Here, we studied how exposure to sub-MIC levels of ciprofloxacin enriches for Escherichia coli with reduced susceptibility to ciprofloxacin using a mallard colonization model. Mallards were inoculated with two isogenic extended-spectrum-beta-lactamase (ESBL)-encoding E. coli strains, differing only by a gyrA mutation that results in increased MICs of ciprofloxacin, and exposed to different levels of ciprofloxacin in their swimming water. Changes in the ratios of mutant to parental strains excreted in feces over time and ESBL plasmid spread within the gut microbiota from individual birds were investigated. Results show that in vivo selection of gyrA mutants occurred in mallards during exposure to ciprofloxacin at concentrations previously found in aquatic environments. During colonization, resistance plasmids were readily transferred between strains in the intestines of the mallards, but conjugation frequencies were not affected by ciprofloxacin exposure. Our results highlight the potential for enrichment of resistant bacteria in wildlife and underline the importance of reducing antibiotic pollution in the environment.

  • 9.
    Atterby, Clara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nykvist, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Lustig, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Järhult, Josef D.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Spread of resistance plasmids and selection of resistant bacteria among Mallards exposed to sub-inhibitory concentrations of antibiotics in their water environmentManuscript (preprint) (Other academic)
  • 10.
    Bikkarolla, Santosh K.
    et al.
    Chalmers Univ Technol, Dept Biol & Biol Engn, Gothenburg, Sweden.
    Nordberg, Viveka
    Karolinska Univ Hosp, Dept Neonatol, Stockholm, Sweden;Karolinska Inst, Div Pediat, Dept Clin Sci Intervent & Technol, Stockholm, Sweden.
    Rajer, Fredrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mueller, Vilhelm
    Chalmers Univ Technol, Dept Biol & Biol Engn, Gothenburg, Sweden.
    Kabir, Muhammad Humaun
    Karolinska Inst, Dept Lab Med, Stockholm, Sweden.
    Sriram, K. K.
    Chalmers Univ Technol, Dept Biol & Biol Engn, Gothenburg, Sweden.
    Dvirnas, Albertas
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden.
    Ambjornsson, Tobias
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden.
    Giske, Christian G.
    Karolinska Inst, Dept Lab Med, Stockholm, Sweden;Karolinska Univ Hosp, Dept Clin Microbiol, Stockholm, Sweden.
    Naver, Lars
    Karolinska Univ Hosp, Dept Neonatol, Stockholm, Sweden;Karolinska Inst, Div Pediat, Dept Clin Sci Intervent & Technol, Stockholm, Sweden.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Westerlund, Fredrik
    Chalmers Univ Technol, Dept Biol & Biol Engn, Gothenburg, Sweden.
    Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak2019In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 4, article id e00347-19Article in journal (Refereed)
    Abstract [en]

    The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epidemiological tracing, since tracing is usually based on bacterial clonality. We have developed a method, based on optical DNA mapping combined with Cas9-assisted identification of resistance genes, which is used here to characterize plasmids during an extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae outbreak at a Swedish neonatal intensive care unit. The outbreak included 17 neonates initially colonized with ESBL-producing Klebsiella pneumoniae (ESBL-KP), some of which were found to carry additional ESBL-producing Escherichia coli (ESBL-EC) in follow-up samples. We demonstrate that all ESBL-KP isolates contained two plasmids with the bla(CTX-M-15) gene located on the smaller one (similar to 80 kbp). The same ESBL-KP clone was present in follow-up samples for up to 2 years in some patients, and the plasmid carrying the bla(CTX-M-15) gene was stable throughout this time period. However, extensive genetic rearrangements within the second plasmid were observed in the optical DNA maps for several of the ESBL-KP isolates. Optical mapping also demonstrated that even though other bacterial clones and species carrying bla(CTX-M) group 1 genes were found in some neonates, no transfer of resistance plasmids had occurred. The data instead pointed toward unrelated acquisition of ESBL-producing Enterobacteriaceae (EPE). In addition to revealing important information about the specific outbreak, the method presented is a promising tool for surveillance and infection control in clinical settings. IMPORTANCE This study presents how a novel method, based on visualizing single plasmids using sequence-specific fluorescent labeling, could be used to analyze the genetic dynamics of an outbreak of resistant bacteria in a neonatal intensive care unit at a Swedish hospital. Plasmids are a central reason for the rapid global spread of bacterial resistance to antibiotics. In a single experimental procedure, this method replaces many traditional plasmid analysis techniques that together provide limited details and are slow to perform. The method is much faster than long-read whole-genome sequencing and offers direct genetic comparison of patient samples. We could conclude that no transfer of resistance plasmids had occurred between different bacteria during the outbreak and that secondary cases of ESBL-producing Enterobacteriaceae carriage were instead likely due to influx of new strains. We believe that the method offers potential in improving surveillance and infection control of resistant bacteria in hospitals.

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  • 11. 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, 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.

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  • 12.
    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.

  • 13.
    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.

  • 14.
    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, 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.

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  • 15.
    Dvirnas, Albertas
    et al.
    Department of Astronomy and Theoretical Physics, Lund University, Lund, Sweden..
    Stewart, Callum
    Department of Astronomy and Theoretical Physics, Lund University, Lund, Sweden..
    Müller, Vilhelm
    Division of Chemical Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden..
    Bikkarolla, Santosh Kumar
    Division of Chemical Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden..
    Frykholm, Karolin
    Division of Chemical Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden..
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kristiansson, Erik
    Department of Mathematical Sciences, Chalmers University of Technology and the University of Gothenburg, Gothenburg, Sweden..
    Westerlund, Fredrik
    Division of Chemical Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden..
    Ambjörnsson, Tobias
    Department of Astronomy and Theoretical Physics, Lund University, Lund, Sweden..
    Detection of structural variations in densely-labelled optical DNA barcodes: A hidden Markov model approach2021In: PLOS ONE, E-ISSN 1932-6203, Vol. 16, no 11, p. e0259670-e0259670Article in journal (Refereed)
    Abstract [en]

    Large-scale genomic alterations play an important role in disease, gene expression, andchromosome evolution. Optical DNA mapping (ODM), commonly categorized into sparsely-labelled ODM and densely-labelled ODM, provides sequence-specific continuous intensityprofiles (DNA barcodes) along single DNA molecules and is a technique well-suited fordetecting such alterations. For sparsely-labelled barcodes, the possibility to detect largegenomic alterations has been investigated extensively, while densely-labelled barcodeshave not received as much attention. In this work, we introduce HMMSV, a hidden Markovmodel (HMM) based algorithm for detecting structural variations (SVs) directly in densely-labelled barcodes without access to sequence information. We evaluate our approach usingsimulated data-sets with 5 different types of SVs, and combinations thereof, and demon-strate that the method reaches a true positive rate greater than 80% for randomly generatedbarcodes with single variations of size 25 kilobases (kb). Increasing the length of the SV fur-ther leads to larger true positive rates. For a real data-set with experimental barcodes onbacterial plasmids, we successfully detect matching barcode pairs and SVs without any par-ticular assumption of the types of SVs present. Instead, our method effectively goes throughall possible combinations of SVs. Since ODM works on length scales typically not reachablewith other techniques, our methodology is a promising tool for identifying arbitrary combina-tions of genomic alterations.

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  • 16. 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.

  • 17.
    Garmendia, Eva
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Disciplinary Domain of Medicine and Pharmacy, research centers etc., Uppsala Antibiotic Center.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Disciplinary Domain of Medicine and Pharmacy, research centers etc., Uppsala Antibiotic Center.
    Pränting, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ReAct.
    Hoegberg, Alexandra
    Uppsala Monitoring Centre.
    Making sense of antibiotic resistance: Communicate for change2021Report (Other (popular science, discussion, etc.))
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  • 18.
    Goyal, Gaurav
    et al.
    Chalmers Univ Technol, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Ekedahl, Elina
    Chalmers Univ Technol, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Nyblom, My
    Chalmers Univ Technol, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Krog, Jens
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Fröbrant, Erik
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Brander, Magnus
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Sewunet, Tsegaye
    Karolinska Inst, Dept Lab Med, Stockholm, Sweden..
    Tangkoskul, Teerawit
    Mahidol Univ, Fac Med, Siriraj Hosp, Bangkok, Thailand..
    Giske, Christian G.
    Karolinska Inst, Dept Lab Med, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Microbiol, Stockholm, Sweden..
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Thamlikitkul, Visanu
    Mahidol Univ, Fac Med, Siriraj Hosp, Bangkok, Thailand..
    Ambjörnsson, Tobias
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Westerlund, Fredrik
    Chalmers Univ Technol, Dept Biol & Biol Engn, Gothenburg, Sweden..
    A simple cut and stretch assay to detect antimicrobial resistance genes on bacterial plasmids by single-molecule fluorescence microscopy2022In: Scientific Reports, E-ISSN 2045-2322, Vol. 12, no 1, article id 9301Article in journal (Refereed)
    Abstract [en]

    Antimicrobial resistance (AMR) is a fast-growing threat to global health. The genes conferring AMR to bacteria are often located on plasmids, circular extrachromosomal DNA molecules that can be transferred between bacterial strains and species. Therefore, effective methods to characterize bacterial plasmids and detect the presence of resistance genes can assist in managing AMR, for example, during outbreaks in hospitals. However, existing methods for plasmid analysis either provide limited information or are expensive and challenging to implement in low-resource settings. Herein, we present a simple assay based on CRISPR/Cas9 excision and DNA combing to detect antimicrobial resistance genes on bacterial plasmids. Cas9 recognizes the gene of interest and makes a double-stranded DNA cut, causing the circular plasmid to linearize. The change in plasmid configuration from circular to linear, and hence the presence of the AMR gene, is detected by stretching the plasmids on a glass surface and visualizing by fluorescence microscopy. This single-molecule imaging based assay is inexpensive, fast, and in addition to detecting the presence of AMR genes, it provides detailed information on the number and size of plasmids in the sample. We demonstrate the detection of several β-lactamase-encoding genes on plasmids isolated from clinical samples. Furthermore, we demonstrate that the assay can be performed using standard microbiology and clinical laboratory equipment, making it suitable for low-resource settings.

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  • 19.
    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.

  • 20.
    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.

  • 21.
    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.

  • 22.
    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.

  • 23.
    Hjort, Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jurén, Pontus
    Public Health Agency of Sweden, Solna, Sweden.
    Toro, Juan Carlos
    Public Health Agency of Sweden, Solna, Sweden.
    Hoffner, Sven
    Department of Global Public Health, Karolinska Institutet, Stockholm, Sweden.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dynamics of Extensive Drug Resistance Evolution of Mycobacterium tuberculosis in a Single Patient During 9 Years of Disease and Treatment2020In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 225, no 6, p. 1011-1020Article in journal (Refereed)
    Abstract [en]

    Mycobacterium tuberculosis is one of the hardest to treat bacterial pathogens with a high capacity to develop antibiotic resistance by mutations. Here we have performed whole-genome sequencing of consecutive M. tuberculosis isolates obtained during 9 years from a patient with pulmonary tuberculosis. The infecting strain was isoniazid resistant and during treatment it stepwise accumulated resistance mutations to 8 additional antibiotics. Heteroresistance was common and subpopulations with up to 3 different resistance mutations to the same drug coexisted. Sweeps of different resistant clones dominated the population at different time points, always coupled to resistance mutations coinciding with changes in the treatment regimens. Resistance mutations were predominant and no hitch-hiking, compensatory, or virulence-increasing mutations were detected, showing that the dominant selection pressure was antibiotic treatment. The results highlight the dynamic nature of M. tuberculosis infection, population structure, and resistance evolution and the importance of rapid antibiotic susceptibility tests to battle this pathogen.

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  • 24.
    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.

  • 25.
    KK, Sriram
    et al.
    Division of Chemical Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96 Gothenburg, Sweden..
    Lin, Yii-Lih
    Division of Chemical Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96 Gothenburg, Sweden..
    Sewunet, Tsegaye
    Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, 141 52 Stockholm, Sweden..
    Wrande, Marie
    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.
    Giske, Christian
    Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, 141 52 Stockholm, Sweden. Clinical Microbiology, Karolinska University Hospital, 171 76 Stockholm, Sweden..
    Westerlund, Fredrik
    Division of Chemical Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96 Gothenburg, Sweden..
    A Parallelized Nanofluidic Device for High-Throughput Optical DNA Mapping of Bacterial Plasmids2021In: Micromachines, E-ISSN 2072-666X, Vol. 12, no 10, article id 1234Article in journal (Refereed)
    Abstract [en]

    Optical DNA mapping (ODM) has developed into an important technique for DNA analysis, where single DNA molecules are sequence-specifically labeled and stretched, for example, in nanofluidic channels. We have developed an ODM assay to analyze bacterial plasmids-circular extrachromosomal DNA that often carry genes that make bacteria resistant to antibiotics. As for most techniques, the next important step is to increase throughput and automation. In this work, we designed and fabricated a nanofluidic device that, together with a simple automation routine, allows parallel analysis of up to 10 samples at the same time. Using plasmids encoding extended-spectrum beta-lactamases (ESBL), isolated from Escherichia coli and Klebsiella pneumoniae, we demonstrate the multiplexing capabilities of the device when it comes to both many samples in parallel and different resistance genes. As a final example, we combined the device with a novel protocol for rapid cultivation and extraction of plasmids from fecal samples collected from patients. This combined protocol will make it possible to analyze many patient samples in one device already on the day the sample is collected, which is an important step forward for the ODM analysis of plasmids in clinical diagnostics.

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  • 26. 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.

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  • 27.
    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, 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.

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  • 28.
    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.

  • 29.
    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.

  • 30.
    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.

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  • 31.
    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.

  • 32.
    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, 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.

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  • 33.
    Muller, Vilhelm
    et al.
    Chalmers Univ Technol, Dept Biol & Biol Engn, S-41296 Gothenburg, Sweden..
    Nyblom, My
    Chalmers Univ Technol, Dept Biol & Biol Engn, S-41296 Gothenburg, Sweden..
    Johnning, Anna
    Chalmers Univ Technol, Dept Math Sci, S-41296 Gothenburg, Sweden.;Univ Gothenburg, S-41296 Gothenburg, Sweden.;Fraunhofer Chalmers Ctr, Syst & Data Anal, S-41288 Gothenburg, Sweden.;Univ Gothenburg, Ctr Antibiot Resistance Res, CARe, S-40530 Gothenburg, Sweden..
    Wrande, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dvirnas, Albertas
    Lund Univ, Dept Astron & Theoret Phys, S-22362 Lund, Sweden..
    Sriram, K. K.
    Chalmers Univ Technol, Dept Biol & Biol Engn, S-41296 Gothenburg, Sweden..
    Giske, Christian G.
    Karolinska Inst, Dept Lab Med, S-14186 Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Microbiol, S-17176 Stockholm, Sweden..
    Ambjornsson, Tobias
    Lund Univ, Dept Astron & Theoret Phys, S-22362 Lund, Sweden..
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kristiansson, Erik
    Chalmers Univ Technol, Dept Math Sci, S-41296 Gothenburg, Sweden.;Univ Gothenburg, Ctr Antibiot Resistance Res, CARe, S-40530 Gothenburg, Sweden..
    Westerlund, Fredrik
    Chalmers Univ Technol, Dept Biol & Biol Engn, S-41296 Gothenburg, Sweden..
    Cultivation-Free Typing of Bacteria Using Optical DNA Mapping2020In: ACS - Infectious Diseases, E-ISSN 2373-8227, Vol. 6, no 5, p. 1076-1084Article in journal (Refereed)
    Abstract [en]

    A variety of pathogenic bacteria can infect humans, and rapid species identification is crucial for the correct treatment. However, the identification process can often be time-consuming and depend on the cultivation of the bacterial pathogen(s). Here, we present a stand-alone, enzyme-free, optical DNA mapping assay capable of species identification by matching the intensity profiles of large DNA molecules to a database of fully assembled bacterial genomes (>10 000). The assay includes a new data analysis strategy as well as a general DNA extraction protocol for both Gram-negative and Gram-positive bacteria. We demonstrate that the assay is capable of identifying bacteria directly from uncultured clinical urine samples, as well as in mixtures, with the potential to be discriminative even at the subspecies level. We foresee that the assay has applications both within research laboratories and in clinical settings, where the time-consuming step of cultivation can be minimized or even completely avoided.

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  • 34.
    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, 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.

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  • 35.
    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, 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.

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  • 36.
    Nyberg, Lena K.
    et al.
    Chalmers, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Quaderi, Saair
    Chalmers, Dept Biol & Biol Engn, Gothenburg, Sweden.;Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Emilsson, Gustav
    Chalmers, Dept Biol & Biol Engn, Gothenburg, Sweden.;Chalmers, Dept Appl Phys, Gothenburg, Sweden..
    Karami, Nahid
    Univ Gothenburg, Sahlgrenska Acad, Dept Infect Dis, Gothenburg, Sweden..
    Lagerstedt, Erik
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Muller, Vilhelm
    Chalmers, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Noble, Charleston
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Hammarberg, Susanna
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Nilsson, Adam N.
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Sjoberg, Fei
    Univ Gothenburg, Sahlgrenska Acad, Dept Infect Dis, Gothenburg, Sweden..
    Fritzsche, Joachim
    Chalmers, Dept Appl Phys, Gothenburg, Sweden..
    Kristiansson, Erik
    Univ Gothenburg, Dept Math Sci, Chalmers Univ Technol, Gothenburg, Sweden..
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ambjörnsson, Tobias
    Lund Univ, Dept Astron & Theoret Phys, Lund, Sweden..
    Westerlund, Fredrik
    Chalmers, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules2016In: Scientific Reports, E-ISSN 2045-2322, Vol. 6, article id 30410Article in journal (Refereed)
    Abstract [en]

    The rapid spread of antibiotic resistance - currently one of the greatest threats to human health according to WHO - is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics.

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  • 37.
    Nyblom, My
    et al.
    Chalmers Univ Technol, Dept Life Sci, S-41296 Gothenburg, Sweden..
    Johnning, Anna
    Chalmers Univ Technol & Univ Gothenburg, Dept Math Sci, S-41296 Gothenburg, Sweden.;Fraunhofer Chalmers Ctr, Dept Syst & Data Anal, S-41288 Gothenburg, Sweden.;Ctr Antibiot Resistance Res Gothenburg CARe, S-40530 Gothenburg, Sweden..
    Frykholm, Karolin
    Chalmers Univ Technol, Dept Life Sci, S-41296 Gothenburg, Sweden..
    Wrande, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mueller, Vilhelm
    Chalmers Univ Technol, Dept Life Sci, S-41296 Gothenburg, Sweden..
    Goyal, Gaurav
    Chalmers Univ Technol, Dept Life Sci, S-41296 Gothenburg, Sweden..
    Robertsson, Miriam
    Chalmers Univ Technol, Dept Life Sci, S-41296 Gothenburg, Sweden..
    Dvirnas, Albertas
    Lund Univ, Dept Astron & Theoret Phys, S-22362 Lund, Sweden..
    Sewunet, Tsegaye
    Karolinska Inst, Dept Lab Med, S-14186 Stockholm, Sweden..
    Sriram, K. K.
    Chalmers Univ Technol, Dept Life Sci, S-41296 Gothenburg, Sweden..
    Ambjornsson, Tobias
    Lund Univ, Dept Astron & Theoret Phys, S-22362 Lund, Sweden..
    Giske, Christian G.
    Karolinska Inst, Dept Lab Med, S-14186 Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Microbiol, S-17176 Stockholm, Sweden..
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kristiansson, Erik
    Chalmers Univ Technol & Univ Gothenburg, Dept Math Sci, S-41296 Gothenburg, Sweden.;Ctr Antibiot Resistance Res Gothenburg CARe, S-40530 Gothenburg, Sweden..
    Westerlund, Fredrik
    Chalmers Univ Technol, Dept Life Sci, S-41296 Gothenburg, Sweden.;Ctr Antibiot Resistance Res Gothenburg CARe, S-40530 Gothenburg, Sweden..
    Strain-level bacterial typing directly from patient samples using optical DNA mapping2023In: Communications Medicine, E-ISSN 2730-664X, Vol. 3, no 1, article id 31Article in journal (Refereed)
    Abstract [en]

    Background: Identification of pathogens is crucial to efficiently treat and prevent bacterial infections. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions.

    Methods: In this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation. For the typing, different taxonomical resolutions were examined and cultivated pure Escherichia coli and Klebsiella pneumoniae samples were used for parameter optimization. Finally, the method was applied to mixed bacterial samples and uncultured urine samples from patients with urinary tract infections.

    Results: We demonstrate that optical DNA mapping of single DNA molecules can identify Escherichia coli and Klebsiella pneumoniae at the strain level directly from patient samples. At a taxonomic resolution corresponding to E. coli sequence type 131 and K. pneumoniae clonal complex 258 forming distinct groups, the average true positive prediction rates are 94% and 89%, respectively. The single-molecule aspect of the method enables us to identify multiple E. coli strains in polymicrobial samples. Furthermore, by targeting plasmid-borne antibiotic resistance genes with Cas9 restriction, we simultaneously identify the strain or subtype and characterize the corresponding plasmids.

    Conclusion: The optical DNA mapping method is accurate and directly applicable to polymicrobial and clinical samples without cultivation. Hence, it has the potential to rapidly provide comprehensive diagnostics information, thereby optimizing early antibiotic treatment and opening up for future precision medicine management.

    Plain language summary: For bacterial infections, it is important to rapidly and accurately identify and characterize the type of bacteria involved so that optimal antibiotic treatment can be given quickly to the patient. However, current diagnostic methods are sometimes slow and cannot be used for mixtures of bacteria. We have, therefore, developed a method to identify bacteria directly from patient samples. The method was tested on two common species of disease-causing bacteria – Escherichia coli and Klebsiella pneumoniae – and it could correctly identify the bacterial strain or subtype in both urine samples and mixtures. Hence, the method has the potential to provide fast diagnostic information for choosing the most suited antibiotic, thereby reducing the risk of death and suffering.

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  • 38.
    Olsson, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Wistrand-Yuen, Pikkei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Nielsen, Elisabet I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Friberg, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lagerbäck, Pernilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Tängdén, Thomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Efficacy of Antibiotic Combinations against Multidrug-Resistant Pseudomonas aeruginosa in Automated Time-Lapse Microscopy and Static Time-Kill Experiments2020In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 64, no 6, article id e02111-19Article in journal (Refereed)
    Abstract [en]

    Antibiotic combination therapy is used for severe infections caused by multidrug-resistant (MDR) Gram-negative bacteria, yet data regarding which combinations are most effective are lacking. This study aimed to evaluate the in vitro efficacy of polymyxin B in combination with 13 other antibiotics against four clinical strains of MDR Pseudomonas aeruginosa. We evaluated the interactions of polymyxin B in combination with amikacin, aztreonam, cefepime, chloramphenicol, ciprofloxacin, fosfomycin, linezolid, meropenem, minocycline, rifampin, temocillin, thiamphenicol, or trimethoprim by automated time-lapse microscopy using predefined cutoff values indicating inhibition of growth (<= 10(6) CFU/ml) at 24 h. Promising combinations were subsequently evaluated in static time-kill experiments. All strains were intermediate or resistant to polymyxin B, antipseudomonal beta-lactams, ciprofloxacin, and amikacin. Genes encoding beta-lactamases (e.g., bla(PAO) and bla(OXA-50)) and mutations associated with permeability and efflux were detected in all strains. In the time-lapse microscopy experiments, positive interactions were found with 39 of 52 antibiotic combination/bacterial strain setups. Enhanced activity was found against all four strains with polymyxin B used in combination with aztreonam, cefepime, fosfomycin, minocycline, thiamphenicol, and trimethoprim. Time-kill experiments showed additive or synergistic activity with 27 of the 39 tested polymyxin B combinations, most frequently with aztreonam, cefepime, and meropenem. Positive interactions were frequently found with the tested combinations, against strains that harbored several resistance mechanisms to the single drugs, and with antibiotics that are normally not active against P. aeruginosa. Further study is needed to explore the clinical utility of these combinations.

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  • 39.
    Palica, Katarzyna
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Vorácová, Manuela
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Skagseth, Susann
    The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science and Technology, UiT The Arctic University of Norway, N-9037 Tromsø, Norway.
    Andersson Rasmussen, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Allander, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hubert, Madlen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Schrøder Leiros, Hanna-Kirstirep
    The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science and Technology, UiT The Arctic University of Norway, N-9037 Tromsø, Norway.
    Andersson, Hanna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Erdélyi, Máté
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Metallo-β-Lactamase Inhibitor Phosphonamidate Monoesters2022In: ACS Omega, E-ISSN 2470-1343, Vol. 7, no 5, p. 4550-4562Article in journal (Refereed)
    Abstract [en]

    Being the second leading cause of death and the leading cause of disability-adjusted life years worldwide, infectious diseases remain─contrary to earlier predictions─a major consideration for the public health of the 21st century. Resistance development of microbes to antimicrobial drugs constitutes a large part of this devastating problem. The most widely spread mechanism of bacterial resistance operates through the degradation of existing β-lactam antibiotics. Inhibition of metallo-β-lactamases is expected to allow the continued use of existing antibiotics, whose applicability is becoming ever more limited. Herein, we describe the synthesis, the metallo-β-lactamase inhibition activity, the cytotoxicity studies, and the NMR spectroscopic determination of the protein binding site of phosphonamidate monoesters. The expression of single- and double-labeled NDM-1 and its backbone NMR assignment are also disclosed, providing helpful information for future development of NDM-1 inhibitors. We show phosphonamidates to have the potential to become a new generation of antibiotic therapeutics to combat metallo-β-lactamase-resistant bacteria.

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  • 40.
    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 Sciences, Infection medicine.
    Karlsson, Philip A.
    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 toward High-Level β-Lactam/β-Lactamase Inhibitor Resistance in the Presence of Multiple β-Lactamases2022In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 66, no 6, article id e00290-22Article in journal (Refereed)
    Abstract [en]

    β-Lactam antibiotics are the first choice for the treatment of most bacterial infections. However, the increased prevalence of β-lactamases, in particular extended-spectrum β-lactamases, in pathogenic bacteria has severely limited the possibility of using β-lactam treatments. Combining β-lactam antibiotics with β-lactamase inhibitors can restore treatment efficacy by negating the effect of the β-lactamase and has become increasingly important against infections caused by β-lactamase-producing strains. Not surprisingly, bacteria with resistance to even these combinations have been found in patients. Studies on the development of bacterial resistance to β-lactam/β-lactamase inhibitor combinations have focused mainly on the effects of single, chromosomal or plasmid-borne, β-lactamases. However, clinical isolates often carry more than one β-lactamase in addition to multiple other resistance genes. Here, we investigate how the evolutionary trajectories of the development of resistance to three commonly used β-lactam/β-lactamase inhibitor combinations, ampicillin-sulbactam, piperacillin-tazobactam, and ceftazidime-avibactam, were affected by the presence of three common β-lactamases, TEM-1, CTX-M-15, and OXA-1. First-step resistance was due mainly to extensive gene amplifications of one or several of the β-lactamase genes where the amplification pattern directly depended on the respective drug combination. Amplifications also served as a stepping-stone for high-level resistance in combination with additional mutations that reduced drug influx or mutations in the β-lactamase gene blaCTX-M-15. This illustrates that the evolutionary trajectories of resistance to β-lactam/β-lactamase inhibitor combinations are strongly influenced by the frequent and transient nature of gene amplifications and how the presence of multiple β-lactamases shapes the evolution to higher-level resistance.

  • 41.
    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)
  • 42.
    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 Multiresistance Plasmids2022In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 13, no 1, article id e03552-21Article in journal (Refereed)
    Abstract [en]

    By providing the bacterial cell with protection against several antibiotics at once, multiresistance plasmids have an evolutionary advantage in situations where antibiotic treatments are common, such as in hospital environments. However, resistance plasmids can also impose fitness costs on the bacterium in the absence of antibiotics, something that may limit their evolutionary success. The underlying mechanisms and the possible contribution of resistance genes to such costs are still largely not understood. Here, we have specifically investigated the contribution of plasmid-borne resistance genes to the reduced fitness of the bacterial cell. The pUUH239.2 plasmid carries 13 genes linked to antibiotic resistance and reduces bacterial fitness by 2.9% per generation. This cost is fully ameliorated by the removal of the resistance cassette. While most of the plasmid-borne resistance genes individually were cost-free, even when overexpressed, two specific gene clusters were responsible for the entire cost of the plasmid: the extended-spectrum-beta-lactamase gene bla(CTX-M-15 )and the tetracycline resistance determinants tetAR. The bla(CTX-M-15 ) cost was linked to the signal peptide that exports the beta-lactamase into the periplasm, and replacement with an alternative signal peptide abolished the cost. Both the tetracycline pump TetA and its repressor TetR conferred a cost on the host cell, and the reciprocal expression of these genes is likely fine-tuned to balance the respective costs. These findings highlight that the cost of clinical multiresistance plasmids can be largely due to particular resistance genes and their interaction with other cellular systems, while other resistance genes and the plasmid backbone can be cost-free.

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  • 43.
    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.

  • 44.
    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.

  • 45.
    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.

  • 46.
    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.

  • 47.
    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.

  • 48.
    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.

  • 49.
    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.

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  • 50.
    Sánchez Martín, Darío
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering, Nanotechnology and Functional Materials.
    Li, Tingting
    Wrande, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. 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.
    Tian, Bo
    Strömme, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering, Nanotechnology and Functional Materials.
    Zardán Gómez de la Torre, Teresa
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering, Nanotechnology and Functional Materials.
    Reduced amplification by phi29 DNA polymerase in the presence of unbound oligos during reaction in RCA2024In: Biosensors and Bioelectronics: X, ISSN 2590-1370, Vol. 17, article id 100456Article in journal (Refereed)
    Abstract [en]

    Synthetic single-stranded oligonucleotides play crucial roles in DNA amplification reactions for various applications, such as serving as primers, enabling magnetic separation, and generating dsDNA for subsequent digestion. Typically, these oligos are added in excess to ensure rapid binding to their intended targets. However, while performing rolling circle amplification (RCA) using phi29 DNA polymerase, we observed a decrease in amplification efficiency when oligos were present in the reaction. This phenomenon was consistently observed in two separate laboratories, prompting this study to delve into the root causes responsible for the decline in RCA efficiency. The lowered efficiency was consistent regardless of the manufacturer or any mutations in the phi29 polymerase. We identified several variables that influenced RCA efficiency, mainly the length of the oligos used and the presence of modifications, particularly those obstructing 3’ end digestion. This strongly suggests that the exonuclease domain of phi29 DNA polymerase is responsible for the competition-based inhibition. Our investigation shows that even picomole quantities of oligos can significantly reduce total DNA production during the phi29 DNA polymerase-mediated amplification process. Conversely, the addition of oligos to the reaction did not impede the efficiency of Bst 3.0 polymerase, likely due to the lack of an exonuclease domain of said polymerase. While increasing the quantity of phi29 DNA polymerase in the reaction partially alleviated the adverse effects of excess oligos, we believe it is crucial to carefully optimize the oligo quantities to achieve maximum amplification of the desired targets.

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