Determining the horizontal transfer of antibiotic resistance genes: using high-throughput fluorescence-based sorting approaches v1

Despite the significant role of plasmids play in the global spread of antimicrobial resistance (AMR), current methods to study the transfer of antibiotic resistance genes (ARGs) mainly rely on bacterial cultivation or sequencing techniques, leaving a knowledge gap in understanding ARGs dissemination transfer within natural microbial communities. To address this, new tools allowing real-time tracking of the spread of ARGs are essential for comprehensive environmental risk assessments. Herein, we present a culture-independent protocol for examining the horizontal transfer of ARGs across diverse bacterial populations. This method utilizes CRISPR-based editing to fluorescently label wild-type AMR plasmids, facilitating their identification and sorting via Fluorescence-activated cell sorting (FACS). As an illustrative example, we detail a step-by-step protocol targeting gfp-tagged AMR plasmid, followed by conjugation procedures and FACS-based selection of green fluorescent protein (GFP)-expressing transconjugants. This fluorescence-based approach applied to real-life bacterial populations can be uniquely deployed to examine One Health risk factors such climate changes and environmental pollution.