Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping

Research output: Contribution to journalArticle

Abstract

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.

Details

Authors
  • Vilhelm Müller
  • Fredrika Rajer
  • Karolin Frykholm
  • Lena K. Nyberg
  • Saair Quaderi
  • Joachim Fritzsche
  • Erik Kristiansson
  • Tobias Ambjörnsson
  • Linus Sandegren
  • Fredrik Westerlund
Organisations
External organisations
  • Chalmers University of Technology
  • Uppsala University
  • University of Gothenburg
  • Lund University
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Medical Genetics
Original languageEnglish
Article number37938
JournalScientific Reports
Volume6
Publication statusPublished - 2016 Dec 1
Publication categoryResearch
Peer-reviewedYes