Objectives: Effluents contain a diverse abundance of antibiotic resistance genes that augment the resistome of receiving aquatic environments. However, uncertainty remains regarding their temporal persistence, transcription and response to anthropogenic factors, such as antibiotic usage. We present a spatiotemporal study within a river catchment (River Cam, UK) that aims to determine the contribution of antibiotic resistance genecontaining effluents originating from sites of varying antibiotic usage to the receiving environment. Methods: Gene abundance in effluents (municipal hospital and dairy farm) was compared against background samples of the receiving aquatic environment (i.e. the catchment source) to determine the resistome contribution of effluents. We used metagenomics and metatranscriptomics to correlate DNA and RNA abundance and identified differentially regulated gene transcripts. Results: We found thatmean antibiotic resistance gene and transcript abundances were correlated for both hospital (ρ=0.9, two-tailed P < 0.0001) and farm (ρ=0.5, two-tailed P < 0.0001) effluents and that two β-lactam resistance genes (blaGES and blaOXA)were overexpressed in all hospital effluent samples. High β-lactam resistance gene transcript abundancewas related to hospital antibiotic usage over timeand hospital effluents contained antibiotic residues. Conclusions: We conclude that effluents contribute high levels of antibiotic resistance genes to the aquatic environment; these genes are expressed at significant levels and are possibly related to the level of antibiotic usage at the effluent source.