Genomic evidence for flies as carriers of zoonotic pathogens on dairy farms

Abstract

Dairy farms are major reservoirs of zoonotic bacterial pathogens, which harbor antimicrobial resistance genes (ARGs), and raise critical questions about their dissemination on and off the farm environment. Here, we investigated the role of coprophagous muscid flies (Diptera: Muscidae) as carriers of zoonotic pathogens and antimicrobial resistance. We collected cow manure and flies on a dairy farm and used shotgun metagenomics to identify the presence of clinically relevant bacteria, virulence factors, and ARGs in both environments. Our results reveal that, although the fly microbiome is largely composed of manure-associated taxa, they also harbor specific insect-associated bacteria, which may be involved in nutrient provisioning to the host. Furthermore, we identifed shared ARGs, virulence factors, and zoonotic pathogens enriched within the fly gastrointestinal tract (GIT). Our study illustrates the potential flow of pathogenic microorganisms from manure to coprophagous flies, suggesting that flies may pose an important zoonotic threat on dairy farms.

Fig. 1. Comparison of highly abundant MAGs identified from fly and manure samples.

Chord diagrams depict the 50 most abundant MAGs found in flies (A) and manure (B) with their corresponding abundances in both manure (blue arc) and fly GITs (red arc) shown by connecting lines/chords. The thickness of each line/chord is proportional to the corresponding MAG’s abundance, which was calculated as the centered log ratio normalized proportion of the total counts successfully mapped to the MAG divided by the estimated number of genomes in each sample (see methods). MAGs with <20% prevalence across all samples were removed from the dataset prior to CLR transformation and downstream analysis.

Fig. 2. Functional analysis of the most abundant MAGs.

A Comparison of shared and unique KEGG orthologs (KOs) in the 21 representative fly-selected, manure-selected, and common MAGs. Genus identification of each MAG is shown below the Venn-diagram, with parentheses indicating when multiple MAGs were assigned to the same genus. Daggers (†) indicate MAGs without a specific genus identification. B Bar graph showing the distribution of clusters of orthologous groups of proteins (COGs) associated with the common transmissible KOs (top bar, orange), unique KOs to flies (middle bar, red), and unique KOs to manure (bottom bar, blue). Lines indicate comparisons where the proportion of counts is significantly different (Fisher’s exact test, p < 0.05). C Overview of complete KEGG metabolic pathways in the fly-selected, manure-selected, and common MAG groups. The ternary plot shows the relative proportion of MAGs in each group encoding complete metabolic pathways. The position of the pathway on the ternary plot represents the prevalence in each group, with pathways equally common between groups in the center. The color of each point indicates the metabolic class the pathway belongs to.

Fig. 3. Distribution of virulence factors (VFs) and antimicrobial resistance genes (ARGs) in manure (blue) and fly (red) metagenomes.

A Venn diagrams showing in descending order all common and unique virulence (left) and antimicrobial resistance (right) features, features that were present in at least 50% of fly and cow samples (50% prevalence), and features corresponding to genes for which the reads covered ≥90% of the gene length and were present in at least half of the samples (≥50% prevalence & ≥90% coverage). Left circle (blue) corresponds to the manure microbiomes; right circle (red) corresponds to the fly GIT microbiomes. B Box plot showing the relative abundances (centered normalized log ratios) of 6 VFs with ≥50% prevalence and ≥90% coverage in fly samples. C Box plot showing the relative abundance of corresponding ARGs with at least 50% prevalence and 90% coverage in either fly or manure samples. Top bars (red) depict fly abundances; bottom bars (blue) depict manure abundances. VFs were identified through mapping against the VFdb and ARGs were identified through mapping against the Resfinder database. Asterisks indicate comparisons with a significantly different abundance between fly and manure samples (p < 0.05, Wilcoxon signed rank test).

Fig. 4. PCA plots of fly and cow samples identified as containing pathogen sequences.

Cow and fly samples were plotted on a PCA based on the dissimilarity of the recovered Bos taurus COI sequences. Fly samples are represented by red dots and manure samples are represented by blue dots. Samples with at least 10% total coverage against the respective reference pathogen genome are labeled. Corresponding coverage and CLR transformed abundance for each pathogen are shown in Supplementary Fig. 8.