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Sandegren, Linus
Publications (10 of 37) Show all publications
Brolund, A., Rajer, F., Giske, C. G., Melefors, Ö., Titelman, E. & Sandegren, L. (2019). Dynamics of Resistance Plasmids in Extended-Spectrum-beta-Lactamase-Producing Enterobacteriaceae during Postinfection Colonization. Antimicrobial Agents and Chemotherapy, 63(4), Article ID e02201-18.
Open this publication in new window or tab >>Dynamics of Resistance Plasmids in Extended-Spectrum-beta-Lactamase-Producing Enterobacteriaceae during Postinfection Colonization
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2019 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 63, no 4, article id e02201-18Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
AMER SOC MICROBIOLOGY, 2019
Keywords
antibiotic resistance, colonization, ESBL, Enterobacteriaceae, plasmid
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-381565 (URN)10.1128/AAC.02201-18 (DOI)000462474100047 ()30745391 (PubMedID)
Funder
Swedish Research Council, K2013-99X-22208-01-5Carl Tryggers foundation , CTS11:403Magnus Bergvall Foundation
Note

De två första författarna delar förstaförfattarskapet.

Available from: 2019-04-16 Created: 2019-04-16 Last updated: 2019-08-06Bibliographically approved
Ny, S., Sandegren, L., Salemi, M. & Giske, C. G. (2019). Genome and plasmid diversity of Extended-Spectrum beta-Lactamase-producing Escherichia coli ST131-tracking phylogenetic trajectories with Bayesian inference. Scientific Reports, 9, Article ID 10291.
Open this publication in new window or tab >>Genome and plasmid diversity of Extended-Spectrum beta-Lactamase-producing Escherichia coli ST131-tracking phylogenetic trajectories with Bayesian inference
2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 10291Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Infectious Medicine Microbiology
Identifiers
urn:nbn:se:uu:diva-390778 (URN)10.1038/s41598-019-46580-3 (DOI)000475559200020 ()31312006 (PubMedID)
Available from: 2019-08-16 Created: 2019-08-16 Last updated: 2019-08-16Bibliographically approved
Sandegren, L. (2019). Low sub-minimal inhibitory concentrations of antibiotics generate new types of resistance. Sustainable Chemistry and Pharmacy, 11, 46-48
Open this publication in new window or tab >>Low sub-minimal inhibitory concentrations of antibiotics generate new types of resistance
2019 (English)In: Sustainable Chemistry and Pharmacy, ISSN 2352-5541, Vol. 11, p. 46-48Article in journal (Refereed) Published
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.

Keywords
Antibiotic resistance, Selection, Sewage, Contamination
National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-375793 (URN)10.1016/j.scp.2018.12.006 (DOI)000455920800007 ()
Funder
Swedish Research Council Formas, 2016-00642
Available from: 2019-02-18 Created: 2019-02-18 Last updated: 2019-02-18Bibliographically approved
Bikkarolla, S. K., Nordberg, V., Rajer, F., Mueller, V., Kabir, M. H., Sriram, K. K., . . . Westerlund, F. (2019). Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak. mBio, 10(4), Article ID e00347-19.
Open this publication in new window or tab >>Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak
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2019 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 4, article id e00347-19Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
AMER SOC MICROBIOLOGY, 2019
Keywords
CRISPR/Cas9, optical DNA mapping, antibiotic resistance, intensive care unit, plasmids
National Category
Microbiology in the medical area Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-393529 (URN)10.1128/mBio.00347-19 (DOI)000481617000002 ()31289171 (PubMedID)
Funder
Swedish Research Council, K2013-99X-22208-01-5
Available from: 2019-09-24 Created: 2019-09-24 Last updated: 2019-09-24Bibliographically approved
Dvirnas, A., Pichler, C., Stewart, C. L., Quaderi, S., Nyberg, L. K., Muller, V., . . . Ambjornsson, T. (2018). Facilitated sequence assembly using densely labeled optical DNA barcodes: A combinatorial auction approach. PLoS ONE, 13(3), Article ID e0193900.
Open this publication in new window or tab >>Facilitated sequence assembly using densely labeled optical DNA barcodes: A combinatorial auction approach
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2018 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 3, article id e0193900Article in journal (Refereed) Published
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.

National Category
Bioinformatics (Computational Biology)
Identifiers
urn:nbn:se:uu:diva-350618 (URN)10.1371/journal.pone.0193900 (DOI)000427030800026 ()29522539 (PubMedID)
Funder
Swedish Research Council, 2014-4305Swedish Research Council, K2013-99X-22208-01-5Åke Wiberg FoundationEU, Horizon 2020Knut and Alice Wallenberg Foundation
Available from: 2018-05-22 Created: 2018-05-22 Last updated: 2018-05-22Bibliographically approved
Sandegren, L., Stedt, J., Lustig, U., Bonnedahl, J., Andersson, D. I. & Järhult, J. D. (2018). Long-term carriage and rapid transmission of extended spectrum beta-lactamase-producing E. coli within a flock of Mallards in the absence of antibiotic selection. Environmental Microbiology Reports, 10(5), 576-582
Open this publication in new window or tab >>Long-term carriage and rapid transmission of extended spectrum beta-lactamase-producing E. coli within a flock of Mallards in the absence of antibiotic selection
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2018 (English)In: Environmental Microbiology Reports, ISSN 1758-2229, E-ISSN 1758-2229, Vol. 10, no 5, p. 576-582Article in journal (Refereed) Published
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.

National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-358301 (URN)10.1111/1758-2229.12681 (DOI)000446986500008 ()30043488 (PubMedID)
Funder
Swedish Research Council Formas, 211-2013-1320Swedish Research Council Formas, 2016-00642Swedish Research Council, K2013-99X-22208-01-5Swedish Research Council, 2016-02606EU, European Research Council
Available from: 2018-08-27 Created: 2018-08-27 Last updated: 2018-12-05Bibliographically approved
Nahid, F., Zahra, R. & Sandegren, L. (2017). A bla(OXA-181)-harbouring multi-resistant ST147 Klebsiella pneumoniae isolate from Pakistan that represent an intermediate stage towards pan-drug resistance. PLoS ONE, 12(12), Article ID e0189438.
Open this publication in new window or tab >>A bla(OXA-181)-harbouring multi-resistant ST147 Klebsiella pneumoniae isolate from Pakistan that represent an intermediate stage towards pan-drug resistance
2017 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 12, article id e0189438Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2017
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-340250 (URN)10.1371/journal.pone.0189438 (DOI)000417469900058 ()
Funder
Swedish Research Council, K2013-99X-22208-01-5Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2018-01-31 Created: 2018-01-31 Last updated: 2018-01-31Bibliographically approved
Brolund, A. & Sandegren, L. (2016). Characterization of ESBL disseminating plasmids. INFECTIOUS DISEASES, 48(1), 18-25
Open this publication in new window or tab >>Characterization of ESBL disseminating plasmids
2016 (English)In: INFECTIOUS DISEASES, ISSN 2374-4235, Vol. 48, no 1, p. 18-25Article, review/survey (Refereed) Published
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.

Keywords
Enterobacteriaceae, resistance, extended-spectrum -lactamase, plasmid typing, epidemiological typing
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-299123 (URN)10.3109/23744235.2015.1062536 (DOI)000373618300002 ()26135711 (PubMedID)
Available from: 2016-07-14 Created: 2016-07-14 Last updated: 2016-07-14Bibliographically approved
Adler, M., Anjum, M., Andersson, D. I. & Sandegren, L. (2016). Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli. Journal of Antimicrobial Chemotherapy, 71(5), 1188-1198
Open this publication in new window or tab >>Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli
2016 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 5, p. 1188-1198Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology Microbiology
Identifiers
urn:nbn:se:uu:diva-221428 (URN)10.1093/jac/dkv475 (DOI)000376291300008 ()26869688 (PubMedID)
Funder
Swedish Research Council Formas, 2013-5476-25194-9EU, European Research Council, 282004
Available from: 2014-03-31 Created: 2014-03-31 Last updated: 2017-12-05Bibliographically approved
Mueller, V., Rajer, F., Frykholm, K., Nyberg, L. K., Quaderi, S., Fritzsche, J., . . . Westerlund, F. (2016). Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping. Scientific Reports, 6, Article ID 37938.
Open this publication in new window or tab >>Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 37938Article in journal (Refereed) Published
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.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-311501 (URN)10.1038/srep37938 (DOI)000388980000001 ()27905467 (PubMedID)
Funder
EU, Horizon 2020, 634890Torsten Söderbergs stiftelseÅke Wiberg FoundationSwedish Research Council, 2014-4305 K2013-99X-22208-01-5
Available from: 2016-12-28 Created: 2016-12-28 Last updated: 2018-01-13Bibliographically approved
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