Exploring the effect of viable but non-culturable (VBNC) state Salmonella Enteritidis on gut microbiome imbalance in an infectious colitis model

• The impacts of VBNC S. Enteritidis on intestinal inflammation and gut microbiome. • Histopathological alterations and elevated inflammatory cytokines were observed. • Firmicutes/Bacteroidota ratio in VBNC S. Enteritidis group was highest. • VBNC S. Enteritidis exacerbated colitis in mice. Salmonella Enteritidis ( S. Enteritidis ), one of the most frequent food infections, causes ulcerative colitis and then a viable but nonculturable (VBNC) state with chlorine treatment, in which the bacteria do not grow in culture medium but are still potentially virulent. To investigate the impact of VBNC-state S. Enteritidis on the intestinal inflammation and pathogenic gut microbiome changes, we established a dextran sulfate sodium (DSS), VBNC+DSS and CVCC+DSS ( S. Enteritidis in the logarithmic growth phase) induced mice colitis model. Histopathological analysis, immunohistochemistry, and immunofluorescence staining were used to evaluated the effects of VBNC-state S. Enteritidis on the intestinal inflammation in DSS-induced colitis mice. Next, we employed 16S rRNA sequencing and diversity analysis to characterize the heterogeneity of gut microbiota. The results demonstrated that VBNC-state S. Enteritidis exacerbated colitis severity, as evidenced by colonic shortening, histopathological alterations, and elevated levels of pro-inflammatory cytokines (LPS, TNF-α, and IL-6). VBNC-state S. Enteritidis severely compromised intestinal barrier function, characterized by a marked reduction in goblet cell density, diminished mucus layer thickness, and downregulated expression of critical junctional proteins (MUC2 and occludin). Notably, we observed that a significantly elevated Firmicutes/Bacteroidota (F/B) ratio in VBNC+DSS group, which might be a pathogenic factor for ulcerative colitis. In addition, the relative abundance of pathogenic bacteria of Samonella enterica, Escherichia flexneri, Enterococcus faecalis and Klebsiella pneumoniae showed marked increase in the VBNC+DSS groups. The results of PICRUSt and KEGG pathway analysis in VBNC+DSS group showed higher abundance of flagellar assembly and bacterial secretion system. This study provided critical insights into the microbiota dynamics and immune responses associated with VBNC-state S. Enteritidis exposure in the context of DSS-induced colitis.