Development of a CRISPR-Cas12a Assay for Detection of the P nimB G Mutation Associated with Heme-Dependent Metronidazole Resistance in Clostridioides difficile
Year: 2026
Type: preprint
Abstract: Objective Heme-dependent metronidazole (MTZ) resistance has emerged in Clostridioides difficile and is primarily mediated by a T-to-G single-nucleotide polymorphism (SNP) in the promoter of nimB (P nimB G ). We aimed to develop a CRISPR-Cas12a-based assay for rapid detection of the P nimB G mutation associated with MTZ resistance. Methods A series of primers and CRISPR RNAs (crRNAs) were designed and optimized to identify the most effective combination for discriminating between P nimB G (MTZ-resistant) and P nimB T (MTZ-susceptible) alleles. Detection was evaluated by visual fluorescence using blue-light and UV imaging systems, as well as by quantitative fluorescence measurements. Results The CRISPR-Cas12a assay accurately differentiated P nimB G and P nimB T alleles in C. difficile . In a collection of clinical isolates, the assay correctly identified all 11 MTZ-susceptible isolates carrying the P nimB T allele and all 10 MTZ-resistant isolates carrying the P nimB G allele. Fluorescence signals were readily detected both visually and quantitatively. Furthermore, colony PCR using crude colony lysates yielded results comparable to those obtained with purified genomic DNA, enabling rapid colony-based detection without conventional DNA extraction. Conclusions The CRISPR-Cas12a assay enables rapid molecular detection of the P nimB G mutation associated with heme-dependent MTZ resistance in C. difficile . This approach could complement conventional susceptibility testing for MTZ resistance detection.
Authors: Nicola Szadna, Cayden Massy, Abiola O. Olaitan
Institution: University of Waterloo
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W7220912526