Fecal microbiota transplantation (FMT) has been successfully applied on clinical aspects, but its clinical outcomes remain unpredictable due to inconsistent donor screening protocols across hospitals, institutions, and countries. Hence, a retrospective analysis of metagenomic data from published studies on FMT donors via a unified bioinformatics workflow might contribute to the understanding of the safety considerations for donor screening and the fecal microbial profiles of qualified donors. In this study, we reanalyzed metagenomic data of 475 screened donor fecal samples from 24 studies spanning China, the USA, Canada, New Zealand, and the Netherlands. The genomic safety risks were evaluated by profiling antibiotic resistance genes (ARGs) and virulence factors (VFs), the results of which showed that no major toxin-associated virulence genes, such as Shiga toxin, Shiga-like toxin, or botulinum neurotoxin (BoNTs) genes harbored in the detected Escherichia coli, Clostridium butyricum, and Streptococcus pneumoniae, but several high-risk ARGs remained insufficiently addressed. The distribution of ARG-harboring bacteria in eligible FMT donors was country-specific. The alpha-diversity and microbial community structure were comparable between donor fecal samples from China and the USA. Interestingly, the core microbiome in fecal samples from Canada, the Netherlands, and New Zealand formed a single guild, while that from China and the USA formed two guilds, with predominantly positive intra-guild and negative inter-guild correlations, indicating that the co-abundance patterns of the core microbiome were conserved among certain countries. Furthermore, an exploratory retrospective classifier was developed based on core microbiome profiles to distinguish eligible FMT donors from general healthy individuals. These results provide evidence for integrating metagenomic sequencing into future FMT donor screening strategies.

Postbiotics derived from lactic acid bacteria (LAB) have attracted growing interest as stable and potentially safer alternatives to probiotics for use in foods and health-related products. Comprehensive safety evaluation remains essential before their broader application. In this study, we assessed the safety profiles of RHT3201, a postbiotic preparation derived from Lacticaseibacillus rhamnosus IDCC 3201, through genomic, genotoxic, acute oral, and subchronic oral toxicity studies. Whole-genome analysis showed that IDCC 3201 lacks antimicrobial resistance genes and exhibits no hemolytic activity, supporting the genomic safety of the source strain. RHT3201 showed no genotoxic potential in either in vitro or in vivo assays, as evidenced by no structural or numerical chromosomal aberrations at concentrations up to 5,000 μg/ml in CHL/IU cells and no increase in micronucleated polychromatic erythrocytes, with no suppression of bone marrow erythropoiesis by oral administration of RHT3201 at doses up to 15,000 mg/kg/day using a mouse model. In rats, single oral doses of up to 15,000 mg/kg caused no mortality, treatment-related clinical signs, or gross pathological abnormalities, indicating an approximate lethal dose greater than 15,000 mg/kg. In a 90-day repeated-dose oral toxicity study, no adverse treatment-related effects were observed at doses up to 5,000 mg/kg/day. Mild liver and thyroid histopathological findings were considered adaptive and reversible. Accordingly, the no-observed-adverse-effect level was determined to be 5,000 mg/kg/day. Taken together, these findings support the safety of RHT3201 as a LAB-derived postbiotic ingredient.