Identification of antibiotic resistance genes in fecal microbiota selected donors during the establishment of a biobank in the south of Brazil.

Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection (rCDI) and is increasingly explored for other dysbiosis-related disorders. However, its implementation as a regulated therapeutic strategy requires robust donor screening, biosafety frameworks, and standardized processing workflows. Here, we describe the establishment of the first fecal microbiota biobank in southern Brazil and evaluate the incorporation of metagenomic sequencing as a complementary layer of donor characterization and safety assessment. A structured donor selection pipeline based on international guidelines was implemented, integrating clinical screening, biochemical and serological testing, and microbiological analyses. Of 100 screened candidates, only four donors met all eligibility criteria and were included in the biobank, highlighting the stringency of the selection process. Shotgun metagenomic sequencing revealed a diverse resistome across all donors, including a shared core set of resistance genes alongside marked interindividual variability. Dominant resistance determinants included tetracycline-associated genes, ermF, CfxA-type β-lactamases, and aminoglycoside-modifying enzymes, which were associated with specific gut taxa. Genomic context analysis further identified four ARG-containing contigs with features compatible with potential mobility, although active horizontal gene transfer was beyond the scope of this study. Notably, tet(Q) and ermF were predominantly associated with Bacteroides fragilis, supporting its role as a reservoir of resistance determinants in the gut microbiota. These findings support the use of metagenomic profiling as a complementary approach to donor characterization and risk assessment rather than as a standalone criterion for donor exclusion. Overall, this study provides a framework for microbiota biobank implementation and highlights the potential of metagenomic analysis to complement conventional biosafety screening in FMT.