Characterization of the resistome in manure, soil and wastewater from dairy and beef production systems

Abstract

It has been proposed that livestock production effluents such as wastewater, airborne dust and manure increase the density of antimicrobial resistant bacteria and genes in the environment. The public health risk posed by this proposed outcome has been difficult to quantify using traditional microbiological approaches. We utilized shotgun metagenomics to provide a first description of the resistome of North American dairy and beef production effluents, and identify factors that significantly impact this resistome. We identified 34 mechanisms of antimicrobial drug resistance within 34 soil, manure and wastewater samples from feedlot, ranch and dairy operations. The majority of resistance-associated sequences found in all samples belonged to tetracycline resistance mechanisms. We found that the ranch samples contained significantly fewer resistance mechanisms than dairy and feedlot samples, and that the resistome of dairy operations differed significantly from that of feedlots. The resistome in soil, manure and wastewater differed, suggesting that management of these effluents should be tailored appropriately. By providing a baseline of the cattle production waste resistome, this study represents a solid foundation for future efforts to characterize and quantify the public health risk posed by livestock effluents.

Figure 1. A comparison of fecal samples collected from calves and adult cattle.

(A) NMDS ordination of fecal sample resistomes from calf vs. adult cattle. (B) Proportion of all aligned reads that aligned to ARGs within different resistance classes, in adult cattle versus calf feces. MDR = Multi-drug resistant mechanisms; MLS = Macrolide-lincosamide-streptogramin.

Figure 2. The calf fecal resistome is significantly more diverse and rich than the adult fecal resistome.

Number of fecal samples containing alignments within each mechanism and class of resistance, separated by calves vs. adults, and conventionally raised vs. organically raised calves.

Figure 3. Beef and dairy systems have different resistomes.

NMDS ordination at the mechanism level of (A) (adult) fecal, soil and wastewater samples and (B) only adult fecal samples were both significantly different based on system, e.g., beef vs. dairy (ANOSIM P < 0.05).

Figure 4. Soil samples are significantly more diverse and rich than wastewater.

Dotplots showing Shannon’s diversity and richness at the mechanism and class levels, separated by system (beef vs. dairy) and colored by sample matrix, i.e., feces (black), soil (red) and wastewater (blue). Bolded text within each panel indicates which matrices differed based on Nemenyi post-hoc pairwise comparisons (WW = wastewater). Diversity and richness were not significantly different between beef and dairy at any level.

Figure 5. Specific resistance classes drive separation of soil from fecal and wastewater resistomes.

NMDS ordination of fecal (black), soil (red) and wastewater (green) samples based on normalized counts of alignments aggregated at the resistance class level. Biplot coordinates of resistance classes are labeled with the class name, and show that aminoglycoside, phenicol and spectinomycin resistances differentiate the soil from the fecal and wastewater resistomes.

Figure 6. Binary heatmap of resistance mechanisms and classes identified in fecal, soil and wastewater samples collected from a US and a Canadian (CA) feedlot.

Black = absent, red = present.