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.

Aims The aim of this study was to determine the effects of unsaturated fatty acids on clinical plasmids. Methods and Results Two unsaturated fatty acids, linoleic acid (LA) and α‐linolenic acid (ALA) at final concentration 0, 0·03, 0·3 and 3 mmol l−1, respectively, were used to assess the effects on conjugative transfer of a mcr‐1‐harbouring plasmid pCSZ4 (IncX4) in conjugation experiment. The inhibitory mechanisms were analysed by molecular docking and the gene expression of virB11 was quantitated by qRT‐PCR. Target plasmid diversity was carried out by TrwD/VirB11 homology protein sequence prediction analysis. Our results showed that LA and ALA inhibit plasmid pCSZ4 transfer by binding to the amino acid residues (Phe124 and Thr125) of VirB11 with dose‐dependent effects. The expression levels of virB11 gene were also significantly inhibited by LA and ALA treatment. Protein homology analysis revealed a wide distribution of TrwD/VirB11‐like genes among over 37 classes of plasmids originated from both Gram‐negative and Gram‐positive bacteria. Conclusions This study demonstrates representing a diversity of plasmids that may be potentially inhibited by unsaturated fatty acids. Significance and Impact of the Study Our work reported here provides additional support for application of curbing the spread of multiple plasmids by unsaturated fatty acids.