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Cut and stretch assay reveals resistance genes

Snij- en rektest onthult resistentiegenenScientific Reports (2022). DOI: 10.1038/s41598-022-13315-w” width=”800″ height=”530″/>

Experimental workflow. (a) Cas9 gRNA is added to the plasmid sample. If the AMR gene is present, Cas9 makes a double-stranded excision, resulting in plasmid linearization. (b) Glass coverslips are functionalized with a mixture of silanes with amine and vinyl terminal groups. DNA molecules are stained with YOYO-1 and stretched by capillary force between the silanized coverslip and a glass slide. (c) Image processing to detect molecules. A Laplace or Gaussian (LoG) filter is used for edge detection and molecular length, width, eccentricity and straightness filters are used to accept or reject the detected molecules. Examples of unaccepted molecules are marked with yellow (too wide), blue (too curved), or green arrows (overlapping molecules), respectively. (d) Representative images of molecules observed during imaging. Molecules i-iv (red bar) are rejected and molecules vx (green bar) are accepted. (e) Intensity profile for molecules in (d) along the yellow dotted line. The intensity is higher when two strands of the circular plasmid are located together. Scale bar 15 µm. (f) length vs. intensity plot for a sample carrying a single plasmid. Data for Cas9 targeting the blaCTX-M gene are shown as red dots and control as black dots. (g) Length histograms of the data in (f). Credit: Scientific Reports (2022). DOI: 10.1038/s41598-022-13315-w

Which genes for antimicrobial resistance are present in bacteria, for example in a hospital ward? For laboratories with limited financial resources, characterizing bacterial DNA is difficult because it often requires expensive equipment. Chalmers researchers have now developed a method that can detect specific bacterial genes encoding resistance using standard microscopes, which are already being used to diagnose tuberculosis in low-income countries.

Antimicrobial resistance is one of the biggest health threats worldwide, as common infections no longer respond to antibiotics. This can lead to serious illness and death, for example in neonatal sepsis, ie severe bacterial blood infections in newborn infants.

The genes that confer resistance to bacteria, for example by breaking down antibiotics, are often found on plasmids, the circular DNA molecules that do not belong to the chromosomal bacterial DNA. Plasmids can transfer between bacterial strains and species and thus can spread rapidly in a bacterial population.

Microscope already present in many laboratories

“Effective and simple methods are needed to characterize bacterial plasmids and detect resistance genes when an infection spreads in hospitals. This is a problem for resource-constrained laboratories, as existing methods require expensive equipment,” said Fredrik Westerlund, Professor of Chemical Biology at Chalmers.

Thanks to a tuberculosis diagnosis program, many laboratories in low- and middle-income countries already have standard fluorescence microscopes. This was the starting point for Fredrik Westerlund’s research group. They based their newly developed method on these microscopes, which are present in the hospital laboratory of their collaboration partners in Dar es Salam, Tanzania.

Linear DNA molecule can be detected

To find specific genes, the researchers use so-called gene scissors, CRISPR-Cas9, which can recognize and cut DNA strands in any predetermined order, so unique that specific genes can be found.

“If a resistance gene is present on the plasmid, it is cut by Cas9. The DNA is then stretched on a glass slide and imaged by fluorescence microscopy, and the linear molecule can be detected. The images for analysis can be acquired by an ordinary smartphone, which you simply attach to the eyepiece of the microscope,” says Gaurav Goyal, a postdoc in the research group.

‘Any microbiology laboratory can perform this plasmid analysis’

Gaurav Goyal explains that the method is currently intended for epidemiological studies – to characterize bacterial plasmids and understand the spread of antibiotic resistance. For example, it may be relevant to investigate how many newborns in a hospital ward are carriers of bacteria with resistance genes. In the long run, it could also be used for diagnosis.

“We have started developing the method for labs with limited resources, but any microbiology lab can perform this plasmid analysis – and get relevant results. In addition to finding resistance genes on plasmids, the method can also be used to measure the size and number of to determine the plasmids in a sample. Our method is simple and faster than other methods, which can also be useful in modern microbiology laboratories in high-income countries,” says Fredrik Westerlund.

The research was published in Scientific Reports


Drug resistance molecule can spread through bacterial ‘communities’


More information:
Gaurav Goyal et al, A simple cut and stretch test to detect antimicrobial resistance genes on bacterial plasmids by single-molecule fluorescence microscopy, Scientific Reports (2022). DOI: 10.1038/s41598-022-13315-w

Provided by Chalmers University of Technology


Quote: Cut and stretch assay reveals resistance genes (2022, June 20) retrieved June 20, 2022 from https://phys.org/news/2022-06-assay-reveals-resistance-genes.html

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