The global emergence and dissemination of antimicrobial resistance (AMR) pose a serious threat to human health. From a One Health perspective, human, animal, plant, and environmental microbiomes are an interconnected ecological continuum. Environmental microbiomes serve as reservoirs of antibiotic resistance genes (ARGs) and conduits for their exchange. Environmental matrices create interfaces where resistant bacteria, mobile genetic elements, phages, selective pressures, and anthropogenic drivers interact. Because resistance is expressed and ARGs are transferred at the single-cell level, understanding environmental AMR requires identifying viable and active hosts and resolving how bacteria, plasmids, and phages mediate horizontal gene transfer.

Although cultivation, qPCR, and metagenomics have advanced environmental AMR research, important limitations remain. Most environmental microbes are still uncultured, so culture-based methods miss many active antibiotic-resistant bacteria (ARB). DNA-based approaches mainly describe resistance potential and often cannot determine whether ARGs originate from extracellular DNA, dead cells, or viable resistant bacteria. Single-cell methods are therefore needed to resolve active resistant hosts, link resistance genes to metabolic activity, and trace transmission routes.

This JoVE Topical Collection invites clear, visual, and reproducible protocols for single-cell methods that identify, assess, and track environmental AMR. Topics include single-cell Raman spectroscopy, stable isotope probing, fluorescence labeling and imaging, single-cell transcriptomics and genomics, microfluidics, cell sorting, and integrated phenotype-genotype workflows. We welcome submissions that develop or apply these approaches in environmental AMR research, including studies of soils, sediments, wastewater, surface waters, biofilms, agricultural systems, and related settings. By advancing such methods, this collection aims to support risk assessment, resistance tracking, and practical strategies for environmental AMR surveillance and ecological control.