A CRISPR/Cas12a-Based System for Sensitive Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales

TL;DR

A new CRISPR-based test detects antibiotic-resistant genes in bacteria with high sensitivity and specificity, which could help combat antibiotic resistance.

Contribution

A PCR-coupled CRISPR/Cas12a fluorescence assay for sensitive and specific detection of NDM-producing genes in bacteria.

Findings

The CRISPR/Cas12a system achieved a detection limit of 2.7 × 10^0 CFU/mL for blaNDM-1 carrying E. coli.

The assay outperformed conventional PCR and real-time PCR in detecting AMR bacteria in food samples.

The method can distinguish single-nucleotide polymorphisms and is highly specific to NDM-producing genes.

Abstract

Antimicrobial-resistant (AMR) bacteria pose a significant global health threat, and bacteria that produce New Delhi metallo-β-lactamase (NDM) are particularly concerning due to their resistance to most β-lactam antibiotics, including carbapenems. The emergence and spread of NDM-producing genes in food-producing animals highlight the need for a fast and accurate method for detecting AMR bacteria. We therefore propose a PCR-coupled CRISPR/Cas12a-based fluorescence assay that can detect NDM-producing genes (blaNDM) in bacteria. Thanks to its designed gRNA, this CRISPR/Cas12a system was able to simultaneously cleave PCR amplicons and ssDNA-FQ reporters, generating fluorescence signals. Our method was found to be highly specific when tested against other foodborne pathogens that do not carry blaNDM and also demonstrated an excellent capability to distinguish single-nucleotide polymorphism. In the case of blaNDM-1 carrying E. coli, the assay performed exceptionally well, with a detection limit of 2.7 × 100 CFU/mL: 100 times better than conventional PCR with gel electrophoresis. Moreover, the developed assay detected AMR bacteria in food samples and exhibited enhanced performance compared to previously published real-time PCR assays. Thus, this novel PCR-coupled CRISPR/Cas12a-based fluorescence assay has considerable potential to improve current approaches to AMR gene detection and thereby contribute to mitigating the global threat of AMR.

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