Amoxicillin Resistance: An In Vivo Study on the Effects of an Approved Formulation on Antibiotic Resistance in Broiler Chickens

Abstract

Background: Antimicrobial resistance (AMR) is a growing global concern in poultry production, where antibiotic use can disrupt gut microbiota and enrich antimicrobial resistance genes (ARGs). Objectives: This study aimed to assess the in vivo effects of a veterinary-approved amoxicillin formulation on gut microbiome composition and ARG profiles in broiler chickens. Methods: A total of 120 Ross-308 broiler chickens were randomly allocated into 12 experimental groups (n = 10 per group), with three replicates per treatment. Birds received either full-dose (1×), a subtherapeutic quarter-dose (¼×) of amoxicillin, a placebo (starch), or no treatment. Cloacal swabs were collected on days 0, 14, and 28 for shotgun metagenomic sequencing. One-way ANOVA was used to evaluate treatment effects on body weight, with significant differences observed from day 14 onward (p < 0.0001). Results: The ¼× dose caused a more pronounced microbiome shift than the 1× dose, with a marked reduction in Pseudomonadota and increase in Bacillota and Bacteroidota. ARG abundance declined in the ¼× group (from 1386 to 1012). While TEM-type ESBL genes were ubiquitous, CTX-M-1 emerged only after ¼× treatment. Worryingly, 20 types of vancomycin resistance genes were detected across all samples. Plasmid-borne ARGs and mobile genetic elements decreased in the ¼× group. Conclusions: Even subtherapeutic antibiotic exposure significantly reshapes the gut microbiota composition and ARG landscape, highlighting the need for refined risk assessments and microbiome-conscious antimicrobial policies in poultry farming.