Even though the use of antibiotics for food-producing animals may contribute to the emergence of antimicrobial resistance, antibiotics are still used as growth promoters. Due to consumer and regulatory pressures, the use of alternatives to antibiotics as growth promoters is increasing, thus more information is needed on their capability to disseminate antimicrobial resistance compared to antibiotics. We investigated the impacts of carbadox (antibiotic), copper sulfate and zinc oxide (metals) and mushroom powder (natural product) on the pig fecal resistome and microbiome. Antibiotic resistance gene (ARG) and mobile genetic element (MGE) abundances were measured using a high-throughput qPCR array with 382 primer pairs. Bacterial community composition was determined by 16S rRNA gene sequencing. More ARGs co-occurred with MGEs in the growth promoter group samples than in the control group samples. Community composition could not be linked to resistome in the growth promoter group samples, indicating a potential decoupling of ARGs and phylogeny. Additionally, machine-learning methods aided in defining the community and resistome differences in response to treatments. Since increased ARG mobility potential was the primary response to the dietary additives used in this study, we suggest that ARG mobility should be considered when designing antimicrobial use policies and antimicrobial resistance surveillances.

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16The use of antimicrobials in animal production has been shown to increase the abundance of 17 antibiotic resistance genes (ARGs) in microbiomes and it is recommended by international health 18 organizations that the use of antimicrobial growth promoters would be restricted. Consequently, 19 the use alternative growth promoters is increasing, however, their influence on the collection of 20ARGs (the resistome) in the animal microbiome is understudied. We investigated the impact of 21 different growth promoters on the pig fecal resistome and microbiome. The growth promoters 22were carbadox (antibiotic), copper sulfate and zinc oxide (metal) or mushroom powder (natural 23 product). Six pens of seven weanling piglets per treatment were used in a growth trial and after 24 33 days, fecal samples were taken from one median weight female and male pig per pen. 25Samples from the same pen were pooled, and DNA was isolated. The community composition 26 was investigated by 16S rRNA gene sequencing and relative ARG and mobile genetic element 27 (MGE) abundances were measured using qPCR array with 382 primers. Only modest shifts were 28 observed in community structure and resistome in response to growth promoters, but more ARGs 29were co-occurring with MGEs in growth promoter group samples than in the control group 30 samples. The taxonomic structure could not be linked to resistome structure in the growth 31 promoter groups. The ARG-MGE co-occurrence patterns suggest that replacing the use of 32 antibiotics with alternative growth promoters might be an insufficient antibiotic resistance 33 mitigation strategy and active selection against ARGs will require a more comprehensive 34 approach. 35

One hundred sixty crossbred pigs (Duroc × (York × Landrace)) weaned at 18.8 d of age and weighing an average of 13.1 lb were used in a 35-day growth trial to evaluate Cordyceps mushroom powder as potential alternative to carbadox in nursery pig diets. Pigs were divided by weight, sex, litter, and assigned to body weight (BW) blocks. Within BW blocks, sex ratios were constant in each pen. Each pen within a BW block was randomly assigned a dietary treatment. Growth performance was analyzed using BW, average daily gain (ADG), average daily feed intake (ADFI), and feed conversion as feed-to-gain (F:G). Pigs were blocked by weight with 5 or 6 pigs per pen and there were 6 pens per treatment. There were 5 diets used in the study: a negative diet or a positive control (carbadox, 50 g/ton); 300 or 600 ppm mushroom powder, and a step-down treatment (900, 900, 450, 300, and 150 ppm mushroom powder during weeks 1, 2, 3, 4, and 5, respectively). At various points of the study, pigs fed the 300 ppm and the step-down mushroom powder treatments tended to have improved (P < 0.10) growth performance compared with those fed the negative control diet. During Phase 4 of the study, pigs fed carbadox had greater ADG (P < 0.02) and improved feed efficiency (P < 0.09) over pigs fed the negative control diet. However, overall data showed that there were no statistical differences among treatments (P > 0.05). In summary, pigs fed 300 ppm mushroom powder or the step-down treatment showed comparable results to pigs fed carbadox. However, future research is needed under a greater disease pressure to show mushroom powder's full potential as an alternative to antibiotics.