Using Shotgun Sequencing to Describe the Changes Induced by In-Feed Zinc Oxide and Apramycin in the Microbiomes of Pigs One Week Postweaning

Weaning is a critical period for piglets, during which potentially harmful bacteria such as Escherichia coli can increase in abundance in the intestine, creating digestive problems and diarrhea. In-feed antibiotics, the most frequent administration route for antibiotics in livestock, and therapeutic doses of zinc oxide (ZnO) help to control diarrhea but prompt secondary problems such as antimicrobial resistance and soil pollution from heavy metals. ABSTRACT Postweaning diarrhea (PWD) is a relevant problem associated with early weaning on pig farms. For decades, in-feed antibiotics and therapeutic zinc oxide (ZnO) have been widely used to prevent PWD in piglets. The European Union is banning both strategies in 2022 due to antimicrobial resistance and environmental contamination concerns, respectively. Understanding the effects of these products on the pig microbiome is crucial for correcting potential microbial disbalances that would prompt PWD. Using shotgun sequencing, three trials were carried out to explore the impact of in-feed apramycin and ZnO, combined with different farm hygiene protocols, on the fecal microbiomes of piglets 7 days postweaning. In trial 1, 28-day-old piglets were allocated to one of three groups: control diet (Ct), Ct + ZnO (Zn), and Ct + apramycin (Ab). In trials 2 and 3, piglets were allocated to the same treatments, but the trials also included different cleaning protocols, achieving different hygiene levels. In-feed treatments impacted the richness, diversity, and relative abundance of the piglets’ microbiome more than hygiene. Pigs in the Ct group showed higher species richness than pigs in the Ab and Zn groups. A clustering analysis evidenced a link between Enterobacteriaceae in the Ct group; Lactobacillaceae and Veillonellaceae mainly in the Ct group; and Bacteroidaceae, Ruminococcaceae, Oscillospiraceae, Acidaminococcaceae, and Lactobacillaceae in the Ab and Zn groups. Functional data analysis revealed a higher abundance of virulence genes in the Ct group microbiomes and heavy metal and antimicrobial resistance-related functions in the Zn treatment group. The results demonstrate that alternatives to Ab and ZnO should balance the microbial abundance and stimulate the growth of commensals to outcompete potential pathogens. IMPORTANCE Weaning is a critical period for piglets, during which potentially harmful bacteria such as Escherichia coli can increase in abundance in the intestine, creating digestive problems and diarrhea. In-feed antibiotics, the most frequent administration route for antibiotics in livestock, and therapeutic doses of zinc oxide (ZnO) help to control diarrhea but prompt secondary problems such as antimicrobial resistance and soil pollution from heavy metals. Understanding how these strategies impact the gut microbiota is crucial for establishing health biomarkers and designing successful replacement strategies. Using shotgun sequencing, this study compares the microbiota of pigs after early weaning when treated with in-feed antibiotics, ZnO, or treatment-free diets to describe differences that could define the susceptibility to infections, providing the basis for future research on improving intestinal resilience through microbiota-based strategies.