Development of a CRISPR-Cas12a Assay for Detection of the PnimBG Mutation Associated with Heme-Dependent Metronidazole Resistance in Clostridioides difficile

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 (PnimBG). We aimed to develop a CRISPR-Cas12a-based assay for rapid detection of the PnimBG 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 PnimBG (MTZ-resistant) and PnimBT (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 PnimBG and PnimBT alleles in C. difficile. In a collection of clinical isolates, the assay correctly identified all 11 MTZ-susceptible isolates carrying the PnimBT allele and all 10 MTZ-resistant isolates carrying the PnimBG 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 PnimBG mutation associated with heme-dependent MTZ resistance in C. difficile. This approach could complement conventional susceptibility testing for MTZ resistance detection.