| 1. |
- Johnning, Anna, 1985, et al.
(författare)
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The resistomes of six carbapenem-resistant pathogens - a critical genotype-phenotype analysis
- 2018
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Ingår i: Microbial Genomics. - : Microbiology Society. - 2057-5858. ; 4:11
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Tidskriftsartikel (refereegranskat)abstract
- Carbapenem resistance is a rapidly growing threat to our ability to treat refractory bacterial infections. To understand how carbapenem resistance is mobilized and spread between pathogens, it is important to study the genetic context of the underlying resistance mechanisms. In this study, the resistomes of six clinical carbapenem-resistant isolates of five different species - Acinetobacter baumannii, Escherichia colt, two Klebsiella pneumoniae, Proteus mirabilis and Pseudomonas aeruginosa - were characterized using whole genome sequencing. All Enterobacteriaceae isolates and the A. baumannii isolate had acquired a large number of antimicrobial resistance genes (7-18 different genes per isolate), including the following encoding carbapenemases: bla(KPC-2), bla(OXA-48), bla(OXA-72), bla(NDM-1), bla(NDm-7) and bla(VIM-1). In addition, a novel version of bla(SHv) was discovered. Four new resistance plasmids were identified and their fully assembled sequences were verified using optical DNA mapping. Most of the resistance genes were colocalized on these and other plasmids, suggesting a risk for coselection. In contrast, five out of six carbapenemase genes were present on plasmids with no or few other resistance genes. The expected level of resistance - based on acquired resistance determinants - was concordant with measured levels in most cases. There were, however, several important discrepancies for four of the six isolates concerning multiple classes of antibiotics. In conclusion, our results further elucidate the diversity of carbapenemases, their mechanisms of horizontal transfer and possible patterns of co-selection. The study also emphasizes the difficulty of using whole genome sequencing for antimicrobial susceptibility testing of pathogens with complex genotypes.
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| 2. |
- Karami, Nahid, 1959, et al.
(författare)
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Identity of bla ctx-m carrying plasmids in sequential esbl-e. Coli isolates from patients with recurrent urinary tract infections
- 2021
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Ingår i: Microorganisms. - : MDPI AG. - 2076-2607. ; 9:6
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Tidskriftsartikel (refereegranskat)abstract
- Plasmid-mediated multidrug resistance in E. coli is becoming increasingly prevalent. Considering this global threat to human health, it is important to understand how plasmid-mediated resistance spreads. From a cohort of 123 patients with recurrent urinary tract infections (RUTI) due to extended spectrum beta-lactamase (ESBL)-producing Escherichia coli (ESBL E. coli), only five events with a change of ESBL E. coli strain between RUTI episodes were identified. Their blaCTX-M encoding plasmids were compared within each pair of isolates using optical DNA mapping (ODM) and PCR-based replicon typing. Despite similar blaCTX-M genes and replicon types, ODM detected only one case with identical plasmids in the sequential ESBL E. coli strains, indicating that plasmid transfer could have occurred. For comparison, plasmids from seven patients with the same ESBL E. coli strain reoccurring in both episodes were analyzed. These plasmids (encoding blaCTX-M-3, blaCTX-M-14, and blaCTX-M-15 ) were unaltered for up to six months between recurrent infections. Thus, transmission of blaCTX-M plasmids appears to be a rare event during the course of RUTI. Despite the limited number (n = 23) of plasmids investigated, similar blaCTX-M-15 plasmids in unrelated isolates from different patients were detected, suggesting that some successful plasmids could be associated with specific strains, or are more easily transmitted.
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| 3. |
- Lindberg, David, 1986, et al.
(författare)
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Binding of Thioflavin-T to Amyloid Fibrils Leads to Fluorescence Self-Quenching and Fibril Compaction
- 2017
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 56:16, s. 2170-2174
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Tidskriftsartikel (refereegranskat)abstract
- Thioflavin-T binds to and detects amyloid fibrils via fluorescence enhancement. Using a combination of linear dichroism and fluorescence spectroscopies, we report that the relation between the emission intensity and binding of thioflavin-T to insulin fibrils is nonlinear and discuss this in relation to its use in kinetic assays. We demonstrate, from fluorescence lifetime recordings, that the nonlinearity is due to thioflavin-T being sensitive to self-quenching. In addition, thioflavin-T can induce fibril compaction but not alter fibril structure. Our work underscores the photophysical complexity of thioflavin-T and the necessity of calibrating the linear range of its emission response for quantitative in vitro studies.
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| 4. |
- Müller, Vilhelm, 1990, et al.
(författare)
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Cultivation-Free Typing of Bacteria Using Optical DNA Mapping
- 2020
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Ingår i: Acs Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 6:5, s. 1076-1084
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Tidskriftsartikel (refereegranskat)abstract
- 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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| 5. |
- Nordell, Pär, 1978, et al.
(författare)
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DNA Polymorphism as an Origin of Adenine-Thymine Tract Length-Dependent Threading Intercalation Rate
- 2008
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 130:44, s. 14651-14658
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Tidskriftsartikel (refereegranskat)abstract
- Binuclear ruthenium complexes that bind DNA by threading intercalation have recently been found to exhibit an exceptional kinetic selectivity for long polymeric adenine-thymine (AT) DNA. A series of oligonucleotide hairpin duplexes containing a central tract of 6-44 alternating AT base pairs have here been used to investigate the nature of the recognition mechanism. We find that, above a threshold AT tract length corresponding to one helix turn of B-DNA, a dramatic increase in threading intercalation rate occurs. In contrast, such length dependence is not observed for rates of unthreading. Intercalation by any mechanism that depends on the open end of the hairpin was found not to be important in the series of oligonucleotides used, as verified by including in the study a hairpin duplex cyclized by a copper-catalyzed "click" reaction. Our observations are interpreted in terms of a conformational pre-equilibrium, determined by the length of the AT tract. We finally find that mismatches or loops in the oligonucleotide facilitate the threading process, of interest for the development of mismatch-recognizing probes. © 2008 American Chemical Society.
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| 6. |
- Nordell, Pär, 1978, et al.
(författare)
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Finding at-DNA--kinetic recognition of long adenine-thymine stretches by metal-ligand complexes.
- 2008
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Ingår i: Nucleic acids symposium series (2004). - 1746-8272. ; :52, s. 131-132
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Tidskriftsartikel (refereegranskat)abstract
- High selectivity for long AT sequences can be attained by kinetically controlled DNA threading intercalation by binuclear ruthenium(II) complexes. The rate of intercalation is strongly correlated to the number of consecutive AT basepairs, being up to 2500 times faster with an AT polymer compared to mixed-sequence DNA.
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| 7. |
- Nyblom, My, 1995, et al.
(författare)
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Bacterial identification by optical mapping of genomic DNA in nanofluidic channels
- 2019
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Ingår i: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - 9781733419000 ; , s. 821-822
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Konferensbidrag (refereegranskat)abstract
- A variety of pathogenic bacteria can infect humans and the increase in bacteria resistant to common antibiotics is a large threat to human health worldwide. This work presents a method, based on optical DNA mapping (ODM) in nanofluidic channels, that can detect the type of bacterial present in a sample by matching the obtained maps of large DNA molecules to a database of fully assembled bacterial genomes. The extraction and labelling protocol has been designed to work for both Gram-positive and Gram-negative bacteria, not requiring any prior knowledge about the sample content.
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| 8. |
- Nyblom, My, 1995, et al.
(författare)
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Strain-level bacterial typing directly from patient samples using optical DNA mapping
- 2023
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Ingår i: COMMUNICATIONS MEDICINE. - : Springer Science and Business Media LLC. - 2730-664X. ; 3:31
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Tidskriftsartikel (refereegranskat)abstract
- 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. Nyblom, Johnning et al. develop an optical DNA mapping approach for bacterial strain typing of patient samples. They demonstrate rapid identification of clinically relevant E. coli and K. pneumoniae strains, without the need for cultivation. BackgroundIdentification 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.MethodsIn 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.
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| 9. |
- Feng, Bobo, 1987, et al.
(författare)
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Hydrophobic catalysis and a potential biological role of DNA unstacking induced by environment effects
- 2019
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 116:35, s. 17169-17174
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Tidskriftsartikel (refereegranskat)abstract
- Hydrophobic base stacking is a major contributor to DNA double-helix stability. We report the discovery of specific unstacking effects in certain semihydrophobic environments. Water-miscible ethylene glycol ethers are found to modify structure, dynamics, and reactivity of DNA by mechanisms possibly related to a biologically relevant hydrophobic catalysis. Spectroscopic data and optical tweezers experiments show that base-stacking energies are reduced while base-pair hydrogen bonds are strengthened. We propose that a modulated chemical potential of water can promote “longitudinal breathing” and the formation of unstacked holes while base unpairing is suppressed. Flow linear dichroism in 20% diglyme indicates a 20 to 30% decrease in persistence length of DNA, supported by an increased flexibility in single-molecule nanochannel experiments in poly(ethylene glycol). A limited (3 to 6%) hyperchromicity but unaffected circular dichroism is consistent with transient unstacking events while maintaining an overall average B-DNA conformation. Further information about unstacking dynamics is obtained from the binding kinetics of large thread-intercalating ruthenium complexes, indicating that the hydrophobic effect provides a 10 to 100 times increased DNA unstacking frequency and an “open hole” population on the order of 10−2 compared to 10−4 in normal aqueous solution. Spontaneous DNA strand exchange catalyzed by poly(ethylene glycol) makes us propose that hydrophobic residues in the L2 loop of recombination enzymes RecA and Rad51 may assist gene recombination via modulation of water activity near the DNA helix by hydrophobic interactions, in the manner described here. We speculate that such hydrophobic interactions may have catalytic roles also in other biological contexts, such as in polymerases.
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| 10. |
- Grunwald, Assaf, et al.
(författare)
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Bacteriophage strain typing by rapid single molecule analysis.
- 2015
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 43:18, s. 117-117
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Tidskriftsartikel (refereegranskat)abstract
- Rapid characterization of unknown biological samples is under the focus of many current studies. Here we report a method for screening of biological samples by optical mapping of their DNA. We use a novel, one-step chemo-enzymatic reaction to covalently bind fluorophores to DNA at the four-base recognition sites of a DNA methyltransferase. Due to the diffraction limit of light, the dense distribution of labels results in a continuous fluorescent signal along the DNA. The amplitude modulations (AM) of the fluorescence intensity along the stretched DNA molecules exhibit a unique molecular fingerprint that can be used for identification. We show that this labelling scheme is highly informative, allowing accurate genotyping. We demonstrate the method by labelling the genomes of λ and T7 bacteriophages, resulting in a consistent, unique AM profile for each genome. These profiles are also successfully used for identification of the phages from a background phage library. Our method may provide a facile route for screening and typing of various organisms and has potential applications in metagenomics studies of various ecosystems.
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