Metagenomic analyses of house fly microbial communities reveals their role as sources, sentinels and disseminators of pathogens and antimicrobial resistance

House flies (Musca domestica L.), breed within and feed upon microbe-rich substrates, serving as reservoirs and mechanical vectors for diverse bacterial pathogens, including antimicrobial-resistant (AMR) strains. While flies pose a direct threat to human and animal health by acquiring and disseminating harmful bacteria, this same behavior positions them as potential sentinels for monitoring microbes in the environment. We have been performing metagenomic sequencing to identify and quantify microbes carried by house flies; however, there was no reproducible, easy to use tool which could profile the microbiome, identify pathogens, and detect AMR genes (ARGs) within the sample. Therefore, we created BALROG (Bacterial AntimicrobiaL Resistance annOtation of metaGenomes), a high-throughput bioinformatic workflow pipeline which uses advanced algorithms to rapidly identify and characterize pathogens and ARGs harbored within house fly bacterial communities. BALROG can be used to analyze both short and long read metagenomic data, highlighting its utility in surveillance of microbial threats carried by flies. BALROG determined that flies from urban and agricultural environments carried bacterial pathogens of both livestock and humans and a wide array of ARGs, with many present on mobile elements like plasmids. >Our approach comprises an early warning system for tracking the incidence, prevalence and dissemination of antibiotic-resistant and pathogenic bacteria in agricultural and human settings, which will improve risk assessment and disease management strategies.