Benzoic acid as a dietary supplement mitigates inflammation and intestinal injury in acute enterotoxigenic <i>Escherichia coli</i>-infected mice without adverse effects in healthy mice

Benzoic acid is a naturally occurring compound found in fruits and is also commercially synthesized as an additive in the food, feed, and pharmaceutical industries. This study investigated the effects of benzoic acid as a dietary supplement on inflammation and intestinal injury in acute <i>Escherichia coli</i> (ETEC)-infected or healthy mice. Thirty-six BALB/c mice were divided into three groups, with 12 mice in each group for a 16-day feeding trial. In group 1, mice were fed a basal diet, six mice were sacrificed, and six mice were intraperitoneally injected with phosphate-buffered saline on day 15. Groups 2 and 3 were fed a basal diet and a diet containing 0.6% benzoic acid, respectively. Half of the mice in each group were sacrificed, while the others were intraperitoneally injected with ETEC on day 15. The results indicated that benzoic acid had no adverse effects on healthy mice regarding growth, organ indices, inflammation, intestinal injury parameters, and cecal short-chain fatty acid levels. Importantly, benzoic acid reduced inflammation in ETEC-infected mice, as evidenced by decreased serum IL-1β, TNF-α, and INF-γ levels, along with increased jejunal <i>TLR-2</i> and <i>MyD88</i> mRNA expression. Besides, benzoic acid mitigated intestinal injury in ETEC-infected mice by increasing the jejunal villus height (VH) and the ratio of VH to crypt depth, elevating jejunal <i>Occludin</i> mRNA levels, decreasing serum D-lactate and diamine oxidase levels, and increasing the cecal acetic acid level. 16s rRNA sequencing revealed that benzoic acid altered the β-diversity of ETEC-infected mice and increased the abundances of <i>Erysipelotrichaceae</i>, <i>Faecalibaculum</i>, and <i>Turicibacter</i> in their gut microbiota. Spearman correlation analysis further indicated that the protective effects of benzoic acid against ETEC infection were closely linked to specific gut microbiota, namely <i>Erysipelotrichaceae</i>, <i>Faecalibaculum</i>, <i>Bifidobacterium</i>, and <i>Limosilactobacillus</i>. Collectively, these findings suggest that benzoic acid could serve as a safe dietary supplement for healthy mice and may alleviate inflammation and intestinal injury in mice with acute ETEC infection.