Wastewater surveillance is increasingly used for antimicrobial resistance (AMR) monitoring in urban environments, but low-resource settings often lack a piped sewerage system. Instead, coprophagous flies—flies that ingest feces—may serve as composite samplers for monitoring fecal wastes present in terrestrial environments. We evaluated whether the class 1 integron-integrase gene intI1 was associated with genetic markers of AMR and fecal source tracking markers (FST) in coprophagous flies collected from latrine entrances and food preparation areas in low-income urban Maputo, Mozambique. We quantified intI1, an enteric 16S rRNA target (for normalization), three FST markers, and 30 ARG targets using qPCR. We normalized concentrations of intI1 and each target to enteric 16S rRNA. We fit linear mixed models with a random intercept for housing compound to estimate within-fly associations between log10 relative abundance of intI1 and log10 relative abundance of each target with and without adjustment for fly taxonomic group, capture location, and standardized fly mass. We also modeled per-fly unique ARG count (i.e., number of ARG targets detected) using Poisson regression. Of 188 flies assayed, 176 passed internal controls; intI1 and enteric 16S rRNA were detected in 95% and 96% of flies, respectively. Higher relative abundance of intI1 was positively associated with ARG and FST targets, with the strongest associations observed for sulfonamide-(sul1: β = 0.87; 95% CI: 0.81, 0.94; sul2: β = 0.81; 95% CI: 0.73, 0.89), tetracycline- (tetA: β = 0.78; 95% CI: 0.70, 0.85; tetB: β = 0.69; 95% CI: 0.60, 0.79), and trimethoprim-related (dfrA17: β = 0.78; 95% CI: 0.70, 0.86) genes. Associations with FST markers were weaker (i.e., human mtDNA: β = 0.46; 95% CI: 0.37, 0.55; human-associated Bacteroides: β = 0.34; 95% CI: 0.25, 0.43). Higher relative abundance of intI1 was also associated with a greater number of ARGs detected: each 10-fold increase in intI1 was associated with an 8% higher expected unique ARG count (aRR=1.08, 95% CI: 1.04–1.12). These findings support the need for further research across different settings exploring intI1 carried by coprophagous flies as a potential standardized screening target for AMR surveillance in unsewered terrestrial environments.

The authors have declared no competing interest.

NCT02362932

This study was funded by the Bill & Melinda Gates Foundation (www.gatesfoundation.org) Grant (OPP1137224 to JB) and by USDA NIFA (2025-68015-44818 to DC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Ethical approval for the MapSan Trial was obtained from the Mozambican Comité Nacional de Bioética para a Saúde (CNBS), Ministério da Saúde (333/CNBS/14) and the Georgia Institute of Technology Institutional Review Board (protocol #H15160). No additional permits were required for the work described in this study.

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Protocols and data used in this paper are available at a dedicated data repository at Open Science Framework (OSF.io). DOI: 10.17605/OSF.IO/BTZHE

https://osf.io/btzhe/.