Background

Forensic microbiology has been explored for body fluid identification and source attribution, yet its potential for characterizing recent behavior exposures remains insufficiently studied. Diet is a major modulator of the gut microbiome and may provide time-sensitive microbial signals complementary to static human genomic identity. This study investigated whether short-term dietary intervention induces reproducible gut microbial changes that can serve as candidate biomarkers of recent dietary exposure.

Methods

A crossover dietary intervention was conducted in 73 healthy adults. Participants underwent a 10-day ketogenic (KD) and a 10-day balanced diet (BD) phase, separated by a 10-day washout period. Blood and fecal samples were collected at four time points, before and after each dietary phase. Clinical indices, shotgun metagenomic sequencing, and metabolomic profiling were used to characterize host and microbial responses.
Results: Both dietary interventions altered host physiological parameters, with 36 blood indices changed after KD and 25 after BD (P < 0.05). Total cholesterol and apolipoprotein B showed opposite regulatory trends between two diets,, with significant diet-by-time interaction (P < 0.05), indicating distinct effects on lipid metabolism. At the microbial level, KD produced a more pronounced shift in overall gut community structure, whereas BD induced comparatively modest changes. Feature selection analyses identified discriminatory microbial markers for each dietary state.
Six species, were consistently depleted after KD and constituted a core negative biomarker set, whereas BD showed a more bidirectional pattern, with enrichment of three species and depletion of one.Co-occurrence network analysis further revealed distinct ecological responses to the two diets. KD was associated with a more modular and differentiated microbial organization, whereas BD maintained tighter cooperative interactions among fiber-associated fermentative taxa while limiting potentially harmful bacteria. Functional profiling showed that KD significantly altered 10 KEGG Level 3 pathways (4 up, 6 down), mainly involving carbohydrate utilization and related metabolic adaptation, whereas BD exerted a more focused functional effect, potentially linked to microbial micronutrient metabolism. Correlation analysis connected post-KD host physiological changes with coordinated shifts in microbial taxa, functional pathways, and glycoside hydrolase profiles, supporting a linked diet–microbiota–host response.

Conclusion

Short-term dietary intervention induces reproducible taxonomic and functional changes in the gut microbiome within days. These microbial signatures may serve as candidate dynamic biomarkers of recent dietary exposure and provide behavior-associated information that complements conventional DNA-based forensic identification. Their forensic utility, however, requires further validation under real-world conditions, including assessments of temporal resolution, inter-individual variability, and robustness in complex sample contexts.

Authors

• Jing Han (Hebei Medical University, China)
• Shujie Dou (Hebei Medical University, China)
• Guanju Ma (Hebei Medical University, China)
• Lihuan Zhu (Hebei Medical University, China)
• Yusi Zhu (Hebei Medical University, China)
• Shujin Li (Hebei Medical University, China)