CRISPR-AMRtracker: a novel toolkit to monitor the antimicrobial resistance gene transfer in fecal microbiota

Abstract The spread of antibiotic resistance genes (ARGs), especially those on plasmids, poses a major risk to global health. However, the extent and frequency of ARG transfer in microbial communities among human, animal, and environmental sectors is not well understood due to a lack of effective tracking tools. We have developed a new fluorescent tracing tool, CRISPR-AMRtracker, to study ARG transfer. It combines CRISPR/Cas9 fluorescence tagging, fluorescence-activated cell sorting, 16S rRNA gene sequencing, and microbial community analysis. The tool integrates a fluorescent tag immediately downstream ARGs, allowing for gene transfer tracking without affecting host cell antibiotic susceptibility, fitness, conjugation, or transposition. Our experiments show that sfGFP-tagged plasmid-borne mcr-1 can transfer across different bacteria species in feces, including Escherichia , Shigella , Lactobacillus , Staphylococcus , Enterococcus , and Bacillus . CRISPR-AMRtracker offers a powerful tool for monitoring ARG transfer in microbiota communities and can inform strategies to combat the threat of antibiotic resistance.