Fecal microbiota transplantation has consolidated itself as one of the most effective interventions in modern medicine for recurrent Clostridioides difficile infection, with success rates exceeding 90 percent in landmark trials. Increasingly, FMT is being explored for a wide spectrum of disorders linked to intestinal dysbiosis, including inflammatory, metabolic, and immune-mediated diseases. Yet unlike conventional pharmaceuticals composed of defined molecular entities, FMT involves the transfer of a complex and dynamic biological ecosystem whose composition remains only partially characterized. This intrinsic complexity complicates standardization, traceability, and quality control — fundamental requirements for the development of safe and scalable therapeutic products. Stool banks have emerged as essential infrastructures to centralize donor recruitment, ensure standardized screening, and support traceability, but their implementation remains heterogeneous across countries, reflecting differences in regulatory environments, technological capacity, and clinical practice.

The stringency of donor selection is the study’s first striking finding. The team, led by Lucas de Figueiredo Soveral and Carlos Rodrigo Zárate-Bladés at the Federal University of Santa Catarina, recruited 100 candidates through public calls between July and September 2023. Each volunteer completed a structured clinical screening questionnaire developed from a comparative analysis of major international guidelines, including the European consensus conference, the International Consensus Conference on stool banking, the Australian consensus statements, the Netherlands Working Group protocols, and a Brazilian FMT center at the Federal University of Minas Gerais. Exclusion criteria covered recent antibiotic use, chronic gastrointestinal diseases, metabolic or autoimmune disorders, recent infections, high-risk behaviors, and abnormal bowel habits, alongside questions about illicit drug use, chronic medication, and occupational exposure to hazardous substances. Only six candidates passed the clinical stage. After comprehensive laboratory screening — complete blood counts, serum electrolytes, renal and hepatic function markers, nutritional parameters, and serological tests for HIV, hepatitis A, B, and C, HTLV, cytomegalovirus, and Epstein-Barr virus — two more candidates were eliminated. One was excluded after multiplex PCR detected toxigenic C. difficile in the stool; another did not complete the process. Just four donors made it into the biobank, a four percent approval rate that mirrors the restrictive approval rates reported by international biobanking initiatives and underscores how demanding safe donor selection truly is.

Eligible donors received standardized collection kits with sterile containers and single-use stool collection devices, and samples were transported in insulated coolers so that no more than six hours elapsed between stool passage and laboratory processing. Under a laminar flow cabinet, fecal material was homogenized in sterile phosphate-buffered saline, filtered to remove particulate debris, aliquoted into 10 milliliter fractions, and stored at minus 80 degrees Celsius with continuous monitoring and full documentation for traceability. This operational framework, the authors argue, is particularly relevant for low- and middle-income countries, where microbiota-based therapies often face infrastructural and regulatory barriers and where workflows must be feasible within existing public healthcare systems.

Then came the genomic deep dive. The team extracted high-molecular-weight DNA from each donor’s fecal sample and performed shotgun metagenomic sequencing on Oxford Nanopore’s MinION platform, generating 7.09 gigabases of data across 5.44 million reads with an N50 of 2.55 kilobases. Resistance determinants were identified directly from sequencing reads using the Resistance Gene Identifier against the Comprehensive Antibiotic Resistance Database, retaining only Perfect and Strict hits to minimize false-positive assignments. The analysis revealed 180 different resistance-related gene entries across the four donors, with detection frequencies ranging from 87 genes in one sample to 137 in another. Only 58 entries were consistently detected in all samples, while 122 showed partial occurrence — a shared core resistome alongside marked interindividual variability. Among the consensus genes, just eleven were canonical antibiotic resistance genes, including APH(3′)-IIIa, CfxA2, CfxA3, ErmF, mefA, AlaS, and five tetracycline resistance determinants: tet(32), tet(40), tet(O), tet(Q), and tet(W).

The taxonomic associations of these dominant genes tell a coherent story about which gut bacteria serve as resistance reservoirs. tet(Q) was predominantly associated with Bacteroides fragilis, while tet(W) was mainly linked to Bifidobacterium longum. tet(O) was primarily attributed to Campylobacter jejuni, ErmF was also largely assigned to Bacteroides fragilis, and CfxA2 was associated with Prevotella intermedia. The proportional abundance of these genes varied dramatically between donors: in one sample, tet(Q) alone accounted for 34.3 percent of resistance gene abundance, while in another its dominance fell to 4.6 percent with increased contributions from tet(W) and the aminoglycoside-modifying gene ANT(6)-Ib. Across all donors, the resistome was dominated by aminoglycoside resistance (32.9 percent) and tetracycline resistance (28.9 percent), with multidrug resistance determinants adding another 19.3 percent. Functionally, resistance was mediated overwhelmingly through target alteration (58.2 percent) and target protection (26.8 percent), with enzymatic inactivation contributing 8.4 percent and efflux systems just 2.9 percent — indicating that resistance in these healthy guts is primarily about modifying or shielding antibiotic targets rather than degrading drugs or pumping them out.

The most technically consequential finding concerns genomic context. Because the presence of a resistance gene alone does not indicate its potential for transmission, the team assembled sequencing reads into contigs and screened them for plasmid associations and mobility-related features. Four ARG-containing contigs showed features compatible with potential mobility. In one donor, a tet(Q) gene sat on a circular contig classified as a plasmid with a score of 0.9949, containing a plasmid replication gene repA and an IS66 family transposase located roughly 1.1 kilobases downstream of the coding region. In another donor, an APH(3′)-IIIa gene occupied a circular plasmid contig carrying recombination proteins and a MOBV relaxase, while a separate tet(Q) gene lay about 5.4 kilobases upstream of a region containing multiple conjugative transfer genes — traO, traN, traM, traJ, traG, and traD — plus a relaxase. In a third donor, tet(Q) and ermF occurred together on a chromosomal contig with an IS30 family transposase immediately upstream of ermF, sharing architectural features with the CTnDOT family of Bacteroides conjugative transposons, which carries both determinants and contains mobilization regions. The authors emphasize that active horizontal gene transfer was beyond the scope of the study, but the genomic environments they documented are strikingly compatible with dissemination.

These findings carry real clinical weight. The concern is not hypothetical: in a seminal 2019 case, an extended-spectrum beta-lactamase-producing Escherichia coli was traced from donor material to an FMT recipient who developed bacteremia and died. Conversely, FMT has also been shown to promote sustained decolonization of multidrug-resistant organisms, replacing baseline ESBL-producing bacteria with susceptible strains. These contrasting outcomes suggest that the clinical implications of resistance genes in FMT depend on the broader donor-recipient context — the bacterial host, the genomic context, and especially the recipient’s clinical condition and anticipated antimicrobial exposure. The Brazilian team’s position is nuanced: metagenomic profiling should serve as a complementary layer of donor characterization and risk assessment rather than as a standalone exclusion criterion. When clinically relevant determinants are identified, decision makers could pursue phenotypic antimicrobial susceptibility testing or deeper genomic characterization, potentially drawing on existing World Health Organization guidance for surveillance of carbapenem-resistant Gram-negative bacteria in healthcare settings.

The study also acknowledges its limitations honestly. Each sequenced donor represented a single biological sample, so differences in gene occurrence should be interpreted as descriptive and exploratory; no inferential statistics were performed. ARG detection was based on sequence matching without phenotypic susceptibility testing or functional characterization, and the Nanopore platform’s read accuracy — between 79.6 and 84.7 percent — may influence sequence alignment and gene assignment. The screening workflow also lacked laboratory-based toxicological analyses for environmental contaminants, pharmaceutical residues, pesticides, and heavy metals, although exposure risks were assessed through the clinical questionnaire. Future studies with larger donor cohorts, complementary sequencing approaches, and longitudinal donor-recipient analyses integrating microbiome profiles with clinical outcomes will be needed to define the clinical relevance of resistome profiling.

Beyond donor screening, alternative FMT formulations may offer complementary biosafety strategies. Sterile fecal filtrates and defined microbial consortia — such as the oral microbiome therapeutic SER-109 — do not rely on transferring viable bacterial communities and may reduce some risks of introducing donor-derived organisms, with early clinical evidence suggesting retained therapeutic efficacy pending validation in larger settings. For now, the Florianópolis biobank stands as a proof of concept that rigorous conventional screening and metagenomic resistome analysis can coexist within a single operational framework, even in resource-constrained environments. As FMT expands toward increasingly vulnerable patient populations, the invisible resistance genes carried by every healthy donor — and the mobile elements that might shuttle them between gut bacteria — are becoming a central frontier in the safety of microbiota-based medicine.

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

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

Article References: Soveral, L. D. F., Holanda, L. R. D. L., Assunção, I. B. F., Gomes, L. G., de Souza, I. B., Rodrigues, G. D. S., Vanny, P. A., Bruna-Romero, O., Palmeiro, J. K., Scheffer, M. C., Sincero, T. C. M., & Zárate-Bladés, C. R. (2026). Identification of antibiotic resistance genes in fecal microbiota selected donors during the establishment of a biobank in the south of Brazil. Frontiers in Antibiotics, 5, Article 1905540. https://doi.org/10.3389/frabi.2026.1905540

Image Credits: AI Generated

DOI: 10.3389/frabi.2026.1905540

Keywords: Identification, antibiotic, resistance, genes, fecal, microbiota, selected, donors, during, establishment, biobank, south