Comparative genomics reveals phylogenetic intermixing of Stomoxys fly, manure, and bovine mastitis-associated bacteria in dairy settings

Abstract Stomoxys flies are highly ubiquitous blood-feeding pests on dairy farms and are suspected carriers of pathogenic bacteria due to their close association with both manure reservoirs and cattle hosts. While prior studies using amplicon sequencing and culture dependent methodologies have been used to characterize the composition of the Stomoxys microbiota, little is known about strain level genomic and functional diversity of Stomoxys-associated bacteria. In this study, we address this key gap in knowledge by using whole genome sequencing to provide the first comparative genomic analysis of Stomoxys-derived Escherichia coli, Klebsiella pneumoniae, and Staphylococcaceae isolates. Our results show that fly and manure isolates collected from the same farm system are phylogenetically interspersed, with subsequent pairwise genome alignments revealing near identical strains and plasmids shared between the two sources. We further identified a phylogenetic clade of Mammaliicoccus sciuri, which contains known mastitis agents that were associated with both flies and manure. Functional analysis revealed that this clade was highly enriched in xylose metabolism genes that were rare across other M. sciuri lineages, suggesting potential niche differentiation within the genus. Collectively, our results provide strong evidence for the acquisition of fecal-associated bacterial isolates by adult Stomoxys flies, confirming the link between biting muscid flies and manure habitats. The intermixing of fly and manure isolates in clinically relevant taxonomic groups strongly suggests that flies serve as carriers of opportunistic mastitis-causing or other fecal-borne pathogens, and that flies may serve as important vehicles of pathogen dissemination across the dairy farm environment. Author summary Blood-feeding Stomoxys flies are a major pest species in dairy operations and have been implicated as carriers of fecal-borne pathogens responsible for bovine mastitis and other important cattle diseases. In a previous study, we generated a large collection of bacterial isolates from manure and Stomoxys flies collected from dairy barns. Most bacterial isolates derived from flies were identified as taxa associated with bovine mastitis, including Escherichia coli, Klebsiella pneumoniae, and non-aureus Staphylococcus; however, it is unknown if these represent bacterial strains acquired by flies from manure. Here, we examined this possibility directly by performing whole genome sequencing of a subset of potential mastitis pathogens derived primarily from flies and manure and supplemented this analysis with publicly available genomes on NCBI (including mastitis and additional fly and manure strains). Our results reveal significant interrelations between fly, manure, and clinically derived mastitis strains, suggesting that Stomoxys flies participate in the carriage and dissemination of bovine mastitis pathogens acquired from manure. Additionally, this work highlights the functional diversity of bacterial pathogens within the barn environment and greatly expands the available genomic data for understudied mastitis pathogens such as Mammaliicoccus sciuri. Competing Interest Statement The authors have declared no competing interest.