Hybrid genome assemblies of Bacteroidota isolates uncover mobile genetic elements, antimicrobial resistance genes, and novel antimicrobial peptides

ABSTRACT Members of the phylum Bacteroidota dominate the mammalian gut microbiota and represent promising candidates for next-generation probiotics. However, the resolution of complete Bacteroidota genomes is often hindered by extensive repetitive sequences and mobile genetic elements, a technical challenge further exacerbated by high intraspecies diversity and strain-level variants that drive assembly fragmentation and preclude the recovery of contiguous scaffolds. We recently isolated 18 strains that belonged to the Bacteroidales order of Bacteroidota from mouse ceca, human feces, and tortoise stool, which we now sequenced using both Illumina and Oxford Nanopore platforms. We developed a specialized assembly workflow to reconstruct high-resolution genomes and characterize their extrachromosomal landscapes. The hybrid pipeline yielded 2 fully closed, single-contig chromosomes, 10 almost closed genomes, and 6 draft assemblies. Genomic integrity was exceptionally high, with completeness scores ranging from 99.6% to 100% and contamination levels below 2%. A total of 12 circular plasmids were detected across 10 isolates; eight isolates harbored a single plasmid, while two isolates (241163E and 241171E) each carried two distinct circular plasmids. Seven of these plasmids were experimentally confirmed through isolation and sequencing, and five were classified as viral contigs. Functional annotation revealed a diverse repertoire of antimicrobial resistance genes and identified several predicted antimicrobial peptides in the assemblies through machine-learning analysis, of which two selected ones were experimentally verified. Overall, the resulting assemblies provide a reliable foundation for tracking mobile genetic elements, assessing resistance potential, and mining bioactive peptides in Bacteroidota species intended for therapeutic development. IMPORTANCE Bacteria in the phylum Bacteroidota are essential to mammalian gut health and are currently being developed as advanced probiotics. However, their complex DNA often prevents scientists from mapping their complete genetic blueprints, which can leave hidden traits like antibiotic resistance undetected. In this study, we combined two advanced DNA sequencing methods to create highly accurate and complete genetic maps for 18 Bacteroidota isolates from humans, mice, and tortoise. We successfully uncovered small, elusive DNA rings (potential plasmids) and confirmed their physical presence in the laboratory. Furthermore, we identified genes responsible for antibiotic resistance and used artificial intelligence to discover new antimicrobial peptides. This research provides a crucial foundation for the scientific community to accurately track mobile genes, rigorously evaluate the safety of potential gut therapeutics, and discover novel molecules for medical applications.