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The imbalance of Th17 and Treg cell differentiation, intestinal flora imbalance, and intestinal mucosal barrier damage may be important links in the occurrence and development of inflammatory bowel disease (IBD) since Th17 and Treg differentiation are affected by the intestinal flora. This study aimed to explore the effect of Escherichia coli (E. coli) LF82 on the differentiation of Th17 and Treg cells and the role of the intestinal flora in mouse colitis. The effects of E. coli LF82 infection on intestinal inflammation were evaluated by analyzing the disease activity index, histology, myeloperoxidase activity, FITC-D fluorescence value, and claudin-1 and ZO-1 expression. The effects of E. coli LF82 on the Th17/Treg balance and intestinal flora were analyzed by flow cytometry and 16S rDNA sequencing. Inflammatory markers, changes in the intestinal flora, and Th17/Treg cells were then detected after transplanting fecal bacteria from normal mice into colitis mice infected by E. coli LF82. We found that E. coli LF82 infection can aggravate the intestinal inflammation of mice colitis, destroy their intestinal mucosal barrier, increase intestinal mucosal permeability, and aggravate the imbalance of Th17/Treg differentiation and the disorder of intestinal flora. After improving the intestinal flora imbalance by fecal bacteria transplantation, intestinal inflammation and intestinal mucosal barrier damage were reduced, and the differentiation balance of Th17 and Treg cells was restored. This study showed that E. coli LF82 infection aggravates intestinal inflammation and intestinal mucosal barrier damage in colitis by affecting the intestinal flora composition and indirectly regulating the Th17 and Treg cell differentiation balance. [ABSTRACT FROM AUTHOR]