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A simple cut and stretch assay to detect antimicrobial resistance genes on bacterial plasmids by single-molecule fluorescence microscopy

Goyal, Gaurav ; Ekedahl, Elina ; Nyblom, My ; Krog, Jens LU ; Fröbrant, Erik ; Brander, Magnus ; Sewunet, Tsegaye ; Tangkoskul, Teerawit ; Giske, Christian G. and Sandegren, Linus , et al. (2022) In Scientific Reports 12(1).
Abstract

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... (More)

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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publishing date
type
Contribution to journal
publication status
published
subject
in
Scientific Reports
volume
12
issue
1
article number
9301
publisher
Nature Publishing Group
external identifiers
  • scopus:85131634550
  • pmid:35660772
ISSN
2045-2322
DOI
10.1038/s41598-022-13315-w
language
English
LU publication?
yes
id
f2bf8ed9-6fce-4eb8-abdf-3e127dc8d197
date added to LUP
2022-10-06 15:15:29
date last changed
2024-06-13 19:58:46
@article{f2bf8ed9-6fce-4eb8-abdf-3e127dc8d197,
  abstract     = {{<p>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.</p>}},
  author       = {{Goyal, Gaurav and Ekedahl, Elina and Nyblom, My and Krog, Jens and Fröbrant, Erik and Brander, Magnus and Sewunet, Tsegaye and Tangkoskul, Teerawit and Giske, Christian G. and Sandegren, Linus and Thamlikitkul, Visanu and Ambjörnsson, Tobias and Westerlund, Fredrik}},
  issn         = {{2045-2322}},
  language     = {{eng}},
  number       = {{1}},
  publisher    = {{Nature Publishing Group}},
  series       = {{Scientific Reports}},
  title        = {{A simple cut and stretch assay to detect antimicrobial resistance genes on bacterial plasmids by single-molecule fluorescence microscopy}},
  url          = {{http://dx.doi.org/10.1038/s41598-022-13315-w}},
  doi          = {{10.1038/s41598-022-13315-w}},
  volume       = {{12}},
  year         = {{2022}},
}