Multi-dimensional dynamic assessment revealed the antibiotic-induced pseudo-germ-free model in SD rats stabilized within 4 days.

Pseudo-germ-free (PGF) animal models are typically established after 7 days of antibiotic treatment, mostly validated using 16S rRNA sequencing. However, this method cannot distinguish live from dead bacteria and may overestimate the required depletion time. We investigated whether PGF model establishment in Sprague-Dawley (SD) rats could be completed in shorter than 7 days using an antibiotic cocktail. Gut microbiota was dynamically monitored with antibiotic cocktail treatment on days 0 to 7 by aerobic/anaerobic bacterial culture, Gram staining, and 16S rRNA sequencing, alongside body weight, food intake, and water intake. Body weight continued to increase in the Test group, while feed and water intake decreased only transiently during the first 2 days and returned to normal from day 3. No visible aerobic or anaerobic bacterial growth after 4 days and persisting through day 7. 16S rRNA sequencing showed that shared amplicon sequence variants (ASVs) with the Control group dropped from 379 on day 0 to 27 on day 4, stabilizing at 19-31 ASVs thereafter. Alpha diversity indices showed no significant changes after day 3 (p > 0.05). Beta diversity showed the peak period of microbiota restructuring on days 2-3 (R 2 = 0.558, p = 0.001), while the effect size between adjacent time points dropped to an extremely low level after day 4 (R 2 ≤ 0.025). LEfSe identified Escherichia-Shigella as the dominant genus. The multi-dimensional data demonstrated that 4 days of antibiotic cocktail treatment were sufficient to establish a stable PGF state in SD rats, aligning with 3R principles.