Phylogenetic intermixing reveals stable fly-mediated circulation of mastitis-associated bacteria in dairy settings

Stomoxys flies are common blood-feeding pests on dairy farms and are suspected carriers of pathogenic bacteria due to their close association with 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 acquisition of mastitis-causing bacteria from manure by Stomoxys or the functional diversity of Stomoxys-associated taxa. In this study, we address these key knowledge gaps 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 containing known mastitis agents 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 may serve as important vehicles of pathogen dissemination across the dairy farm environment.