The Fecal Resistome of Beef Cattle from Conventional Grain-fed and Grass-fed Systems in the Western United States

Antibiotics have been used in the beef industry to improve animal health. However, this can potentially contribute to developing antimicrobial resistance (AMR) in the bacteria shedding from animal guts in feces. Antimicrobial resistance bacteria (ARB) and their AMR genes (ARGs) may be transmitted to humans via beef contaminated by animal feces during livestock production and slaughtering. However, whether various grass and grain beef feeding systems impact ARB or ARGs in cattle feces is currently unknown. Therefore, the objective of this study was to characterize and compare the fecal resistome of cattle raised in various grass and grain-feeding systems in the Western United States. Fecal samples were collected from individual cattle at 14 months of age as a baseline and collected again from the same animals two days before harvest. Groups included: 1) Conventional grain-fed (CON, n = 10), 2) Grass-fed for 20 months (20GF, n = 10), 3) Grass-fed and then grain-finished for 45 days (GR45, n = 10), 4) Grass-fed for 25 months (25GF, n = 10). Beef cattle raised in grass-fed systems did not receive any antibiotics, while some cattle from CON and GR45 received therapeutic antibiotics, and those finished in the feedlot received monensin in their feedlot rations. Total microbial DNA was extracted from samples and sequenced using the Illumina NovaSeq 6000 platform (250bp paired-end, Illumina, Inc., San Diego, CA, USA) for resistome analysis. In total 598 ARGs were identified, and 448 were kept after the filtration. Regarding the Chao 1 diversity, the 25GF group had the smallest value compared to that of the other three groups (P < 0.05) at the harvest time. Shannon’s diversity suggested that the richness and evenness of ARGs are greater than CON and GR45 compared with 20GF and 25GF (P < 0.05). The beta diversity for GR45 and CON differed concerning the abundance of ARGs and the predominant ARG classes, as compared to the samples collected from cattle within grass-feeding systems at the harvest time and all the samples at the baseline (Stress = 0.07, R = 0.2111, P = 0.018), indicating the composition of fecal resistome were different between cattle under grain-finished groups and cattle under grass-feeding systems. The number of transferable ARGs, which can transfer from one environment to another by horizontal gene transfer, detected in CON, 20GF, GR45, and 25GF was 54, 29, 42, and 24, respectively. Among them, 14, 5, 2, and 2 unique transferable ARGs were identified, respectively. A total of 753 genes associated with resistance to biocides and metals (BMRGs) were identified, with 170 BMRGs found on plasmids and transposons, which were retained for further analysis after filtration. A smaller Shannon index of transferable BMRGs was observed for GR45 compared to 20GF and 25GF (P < 0.05). The results from the trial suggested that CON and GR45, which employed therapeutic and prophylactic antibiotics, enriched the diversity of ARGs, including transferable ARGs, in animals' feces. In grass-feeding systems where antibiotics were not administered, animals' feces exhibited greater diversity in transferable BMRGs, potentially creating selective pressure and promoting the development of ARGs. The enrichment in either ARG diversity or BMRG diversity of animal fecal resistome increases the spread of ARB in the production environment, which may eventually increase the risk of AMR in humans