Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.
Journal article, 2016

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.

Author

Vilhelm Müller

Chalmers, Biology and Biological Engineering, Chemical Biology

Fredrika Rajer

Uppsala University

Karolin Frykholm

Chalmers, Biology and Biological Engineering, Chemical Biology

Lena Nyberg

Chalmers, Biology and Biological Engineering, Chemical Biology

Mahmood Saair Quaderi

Chalmers, Biology and Biological Engineering, Chemical Biology

Lund University

Joachim Fritzsche

Chalmers, Physics, Chemical Physics

Erik Kristiansson

University of Gothenburg

Chalmers, Mathematical Sciences, Applied Mathematics and Statistics

T. Ambjornsson

Lund University

L. Sandegren

Uppsala University

Fredrik Westerlund

Chalmers, Biology and Biological Engineering, Chemical Biology

Scientific Reports

2045-2322 (ISSN)

Vol. 6 37938- 37938

Driving Forces

Sustainable development

Innovation and entrepreneurship

Areas of Advance

Nanoscience and Nanotechnology (2010-2017)

Life Science Engineering (2010-2018)

Subject Categories

Biomedical Laboratory Science/Technology

Microbiology in the medical area

Chemical Sciences

DOI

10.1038/srep37938

PubMed

27905467

More information

Latest update

11/20/2018