Antimicrobial Growth Promoters Altered the Function but Not the Structure of Enteric Bacterial Communities in Broiler Chicks ± Microbiota Transplantation

{"Background": "The use of antimicrobial growth promoters (AGPs) in livestock is restricted in many jurisdictions due to concerns about antimicrobial resistance in zoonotic bacteria. Understanding how AGPs work could help develop effective non-antibiotic alternatives. One proposed mechanism is modulation of enteric bacterial communities, but the dynamic intestinal environment and high inter-animal variability make this difficult to study. Ceragenins are synthetic non-peptide mimics of cathelicidin antimicrobial peptides that resist enzymatic degradation and can be delivered orally. This study investigated whether the conventional AGP virginiamycin and the ceragenin CSA-44 modify the structure and function of the intestinal microbiota to enhance broiler chick growth, and whether early microbiota transplantation promotes a homogeneous, diverse enteric microbiota.", "Methods": "Ross 308FF broiler chicks were obtained from a local hatchery and maintained in sterile individually ventilated cages in containment mode. On day 1 post-hatch, chicks received either an oral gavage of cecal digesta slurry from healthy adult donor chickens (MT+) or sterile Columbia Broth-glycerol (MT\u2212). On day 3, chicks were assigned to one of three diets: unamended starter ration (control), starter ration containing 20 mg/kg virginiamycin, or starter ration containing 620 mg/kg CSA-44. Feed consumption and bird weight were recorded daily. On days 10 and 15 post-hatch, birds were euthanized and cecal digesta was collected. Bacterial DNA was extracted and the V4 region of the 16S rRNA gene was sequenced using Illumina MiSeq. Metabolomic analysis was performed using 1H-NMR spectroscopy. Short-chain fatty acids (SCFAs) in feces were quantified by gas chromatography on days 5, 10, and 15. Statistical analyses included mixed models in SAS, alpha-diversity (Shannon index) and beta-diversity (Bray-Curtis) evaluated by Kruskal-Wallis and PERMANOVA, differential abundance by ANCOM, and metabolomics by univariate and multivariate analyses using MATLAB and MetaboanalystR.", "Key Results": "Neither virginiamycin nor CSA-44 influenced feed consumption (virginiamycin: p \u2265 0.067), weight gain, or feed conversion ratio relative to control, except that CSA-44-treated birds consumed more feed (p < 0.001) but had reduced weight gain when receiving the microbiota transplant (p \u2264 0.051). Microbiota transplantation significantly altered cecal bacterial community composition and diversity (p \u2264 0.002). MT+ birds showed higher alpha-diversity (Shannon index \u22486.5, comparable to adult donors) and distinct beta-diversity compared to MT\u2212 birds. In MT\u2212 birds, cecal communities were dominated by Lachnospiraceae (60%), Ruminococcaceae (12%), and Lactobacillaceae (10%), while MT+ birds also harbored Bacteroidaceae, Rikenellaceae, Sutterellaceae, and Atopobiaceae. Alpha- and beta-diversity differed between day 10 and day 15 in MT\u2212 birds (p \u2264 0.035) but not in MT+ birds (p \u2265 0.092), indicating early stabilization of the transplanted microbiota. Neither virginiamycin nor CSA-44 affected bacterial diversity in MT+ birds (p \u2265 0.084). In MT\u2212 birds, CSA-44 reduced alpha-diversity at day 15 (p = 0.033), attributed to lower abundance of Butyricicoccaceae. Only acetic acid was detected above the limit of detection in feces, with no treatment differences (p \u2265 0.097). Metabolomic analysis revealed that microbiota transplantation altered the cecal metabolome: at day 10, MT+ control birds had higher concentrations of 2-isopropylmalic acid, alanine, and isoleucine (p \u2264 0.045); at day 15, higher glucose-6-phosphate (p = 0.013) and lower glucose and glucose-1-phosphate (p \u2264 0.050). Virginiamycin altered the metabolome in MT\u2212 birds: at day 10, higher phenylalanine and tyrosine, lower \u03b1-ketobutyrate and glutamate (p \u2264 0.050); at day 15, higher maltose (p \u2264 0.033), lower 2-isopropylmalic acid, glutamate, tryptamine, and tyrosine (p \u2264 0.047). In MT+ birds, virginiamycin reduced 2-isopropylmalic acid, glucose, and valine at day 10 (p \u2264 0.034), and at day 15 increased valine (p = 0.028) while decreasing \u03b1-ketobutyrate, glutamate, glucose, and isoleucine (p \u2264 0.037). CSA-44 in MT\u2212 birds at day 10 increased isobutyrate and 2-isopropylmalic acid, decreased glutamate and isoleucine (p \u2264 0.044); at day 15 increased isoleucine and valine, decreased glucose-1-phosphate and formate (p \u2264 0.048). In MT+ birds, CSA-44 at day 10 increased \u03b1-ketobutyrate, decreased glucose and glutamate (p \u2264 0.048); at day 15 increased 2-isopropylmalic acid (p = 0.010), decreased glucose-1-phosphate and fructose (p \u2264 0.048).", "Clinical Implications": "The study demonstrates that AGPs and the ceragenin CSA-44 alter the function of the enteric microbiota (as measured by metabolomics) without changing bacterial community structure, suggesting that metabolic modulation\u2014particularly of glycolysis and branched-chain amino acid synthesis pathways\u2014may be the mechanism by which AGPs impart health benefits. Microbiota transplantation successfully established a stable, diverse cecal community that may enhance bird health through upregulation of BCAA synthesis. These findings indicate that evaluating AGP alternatives must include comprehensive assessment of host-microbiota functional interactions, not just compositional changes. The study was limited by its short duration (15 days vs. typical 35\u201345 day production cycles), low replicate number (n=8), and lack of immunological stress, which may have prevented detection of growth promotion effects. Future research should examine AGP effects over longer periods, in production settings, and under immune stress conditions."}