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Introduction: This study investigated the separate impacts of diet and pre-operative antibiotics on gut microbiome and colonic anastomotic healing using a mouse model., Methods: Male C57BL/6J mice were fed either low-fat-high-fibre (SD) or high-fat-low-fiber (WD) groups for 6 weeks, then further received either pre-operative antibiotics or a control sham before a colonic anastomotic procedure was performed. After 7 days, the anastomosis was assessed and microbiota composition and biodiversity were analyzed in anastomotic tissue and stool., Results: WD-fed mice had shorter survival (5.2 ​± ​2.3 vs. 6.9 ​± ​2.3 days, p ​= ​0.022), increased weight loss (5.55 ​± ​3.80g vs. 2.65 ​± ​2.36g, p ​= ​0.03), and reduced biodiversity compared to SD-fed mice. Pre-operative antibiotics improved anastomotic healing scores (1.33 ​± ​0.65 vs. 2.08 ​± ​0.79, p ​= ​0.02) and reduced Enterococcus faecalis growth in tissue and stool (p ​= ​0.02, p ​= ​0.02). Improved anastomotic healing correlated with lower Enterococcus abundance (p ​= ​0.04) and higher collagen III and IV levels (p ​= ​0.01, 0.04) in anastomotic tissue., Conclusion: SD promotes enhanced post-operative recovery and increased microbiome biodiversity, while pre-operative antibiotics enhance anastomotic healing by suppressing Enterococcus faecalis growth, mitigating collagen III/IV degradation., (Copyright © 2024 Elsevier Inc. All rights reserved.)