Therapeutic potential of heat-killed Enterococcus faecalis (EC-12) para-probiotic in reversing maternal separation-triggered social deficit in mice

Early life stress due to maternal separation (MS) disrupts the gut–brain axis (GBA), leading to long-term neurobiological and behavioral deficits, particularly social behavior impairments. Although various probiotics have shown therapeutic potential, the efficacy of heat-killed Enterococcus faecalis EC-12 (EC-12) as a para-probiotic remains largely unknown. This study aimed to evaluate the therapeutic potential of EC-12 in reversing MS-induced behavioral and molecular abnormalities in mice. Male C57BL/6 J mice were subjected to MS from postnatal days (PD) 3–20 and then divided into four groups with or without EC-12 supplementation. After five weeks of EC-12 administration, behavioral assessments were conducted, followed by prefrontal cortex transcriptomic analysis, cecal content microbiomic analysis, and serum and cecal content metabolomic analyses. EC-12 supplementation significantly restored social interaction behavior in mice with MS without affecting anxiety- or depression-like phenotypes. Transcriptome analysis revealed significant upregulation of vasopressin-related genes (Trpv4 and Ecrg4), with vasopressin secretion being the most enriched biological pathway. EC-12 also increased the levels of L-tryptophan and L-tyrosine and upregulated Tph2 gene expression. The gut microbiota composition was substantially reshaped in MS-EC-12 mice, with enrichment of beneficial genera (Blautia, Dubosiella, Romboutsia), and fecal metabolomics revealed increased levels of phenylacetic acid, glycerol 3-phosphate, and pectin, with significant activation of glycerolipid metabolism. These findings suggest that heat-killed EC-12 can mitigate early life stress-induced social deficits through microbiota- and metabolite-mediated modulation of GBA and may serve as a promising para-probiotic intervention for neurodevelopmental disorders.