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Rapid detection of antimicrobial resistance by microbial ribosome immunoprecipitation

A drug resistance, microbial technology, applied in the field of biomedicine and diagnostics, can solve undisclosed or proposed problems, and achieve the effect of increased test sensitivity, reduced variability and degradation rate, increased variability and degradation rate

Pending Publication Date: 2021-02-05
AUTONOMOUS UNIVERSITY OF BARCELONA
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of them disclose or suggest the use of the disclosed assays to detect resistance-associated microbial nucleic acids

Method used

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  • Rapid detection of antimicrobial resistance by microbial ribosome immunoprecipitation
  • Rapid detection of antimicrobial resistance by microbial ribosome immunoprecipitation
  • Rapid detection of antimicrobial resistance by microbial ribosome immunoprecipitation

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0092] Example 1: Selective detection of antibiotic resistance in transformed bacterial cultures

[0093] To assess the efficiency of immunoprecipitation of bacterial ribosome-associated transcripts, Escherichia coli cultures transformed with plasmids conferring resistance to the antibiotics ampicillin (AmpR positive) or kanamycin (KanR positive), respectively, were genotyped. Control settings. Ribosomal immunoprecipitates were tested with ampicillin and kanamycin primers to identify the presence or absence of antibiotic resistance genes. Experiments were performed using two technical replicates and five biological replicates (standard error mean SEM).

[0094] Enriched antibiotic resistance genes in transformed E. coli cultures can be specifically identified by qRT-PCR by ribosomal immunoprecipitation using antibodies against bacterial ribosomal subunits (see figure 1 ).

example 2

[0095] Example 2: Selective increase of AmpR expression in ampicillin-transformed E. coli cultures

[0096] To assess whether this method allows for the discrimination of increases in active versus inactive translated genes, E. coli cultures were transformed with plasmids containing resistance cassettes against two antibiotics (ampicillin and neomycin), while only applying ampicillin selective pressure.

[0097] Using this method, it is possible to detect a selective increase in the expression of the ampR gene, especially during exponential growth, while substantially not affecting neoR (see figure 2 ), thus highlighting the ability of the method to provide a higher detection range for transcriptionally active genes.

example 3

[0098] Example 3: Monitoring of antibiotic-induced dynamic expression changes in mixed cultures

[0099] To test the ability of this method to determine dynamic changes in selective pressure and transcriptional state, we used mixed E. coli cultures containing both kanamycin- and ampicillin-resistant cells and subjected them to different antibiotic stresses (ampicillin-resistant Xilin for 2.5h, and then switch to kanamycin for up to 24h). Subsequent qRT-PCR results for the ampR and kanR genes allowed us to closely monitor the associated changes in gene expression of these AMR genes in the recovered immunoprecipitated transcripts (see image 3 ), thus highlighting the diagnostic potential of this method.

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Abstract

The present invention provides a method for determining the resistance of a microorganism to a drug by detecting at least a transcript of a drug resistance gene from a microorganism in a biological sample, the method comprising the steps of: (i) lysing the cells by means of a chemical or a mechanical method, thereby obtaining a lysate and cell debris; (ii) obtaining a ribosome-antibody complex from the lysate using an antibody or a fragment thereof which binds specifically to a microorganism-ribosomal protein; (iii) purifying the mRNA associated to the ribosome-antibody complex by means of a nucleic acid extraction method; and (iv) submitting the resulting mRNA to a specific gene detection method, thereby identifying the at least one drug resistance gene transcript of the biological sample. Provided methods and kits allow determining the resistance to antibiotics of a biological sample in a rapid and reliable manner, thereby minimizing the risk of AMR and allowing the definition of thetherapeutic potential of a selected antibiotic.

Description

technical field [0001] The present invention relates to the fields of biomedicine and diagnostics, in particular to a method for determining the resistance of biological samples to antibiotics in a rapid and reliable manner, thereby minimizing the risk of AMR and allowing the definition of the therapeutic potential of selected antibiotics and kit Background technique [0002] The advent of antibiotics represented a worldwide social and medical revolution. Thanks to the discovery of penicillin, in 1928, a previously fatal infection became a trivial ailment. However, the misuse and abuse of antimicrobial drugs has led to the selection of pathogenic bacterial strains that exhibit antimicrobial resistance (AMR). [0003] AMR has become a worldwide epidemic, with some estimates suggesting that by 2050, 10 million people worldwide will die each year due to bacterial antibiotic resistance. Currently, there are more than 23.000 deaths per year due to AMR in Europe and a similar n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/18G01N33/68
CPCG01N33/6803G01N33/6842C12Q1/18C12Q1/689C12Q1/6804C12Q1/6851C12Q2600/158
Inventor 艾伯特·昆塔纳·罗梅洛埃里森达·桑兹·伊格莱西亚斯
Owner AUTONOMOUS UNIVERSITY OF BARCELONA
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