Shotgun metagenomics reveals the microbiome and resistome of water harvesting ponds used by Kenyan rural smallholders

Abstract Water harvesting ponds are essential to smallholder farming across sub-Saharan Africa, yet their role in antimicrobial resistance (AMR) transmission remains unclear. Using shotgun metagenomics, we characterised the microbiome and resistome of 16 rural Kenyan ponds, detecting 582 antibiotic resistance gene (ARG) subtypes across 27 classes. Five ARG types (bacitracin, multidrug, polymyxin, beta-lactam and rifamycin) accounted for most ARGs (90.6%). Genome-resolved analyses recovered 1,542 metagenome-assembled genomes, including non-tuberculous Mycolicibacterium carrying rifamycin resistance ( rbpA ) and virulence factors including type VII secretion systems, dormancy regulators, and antigen 85 complex. ARG–mobile genetic element co-localisation was rare, and resistome risk scores were moderate (~22.4), indicating limited horizontal transfer potential compared to agricultural or hospital effluents. These ponds act as moderate, persistent environmental AMR reservoirs linked to farming practices. Strengthened antimicrobial stewardship, improved manure management and vegetative buffer zones could help mitigate AMR dissemination and support safer rural water systems under a One Health framework.