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ABSTRACT Tetracyclines serve as broad-spectrum antibiotics to treat bacterial infections. The discovery of new tetracycline resistance genes has led to new questions about the underlying mechanisms of resistance, gene transfer, and their relevance to human health. We tracked changes in the abundance of a 55-kbp conjugative transposon (CTn214) carrying tetQ, a tetracycline resistance gene, within a Bacteroides fragilis metagenome-assembled genome derived from shotgun sequencing of microbial DNA extracted from the ileal pouch of a patient with ulcerative colitis. The mapping of metagenomic reads to CTn214 revealed the multi-copy nature of a 17,044-nt region containing tetQ in samples collected during inflammation and uninflamed visits. B. fragilis cultivars isolated from the same patient during periods of inflammation harbored CTn214 integrated into the chromosome or both a circular, multi-copy, extrachromosomal region of the CTn214 containing tetQ and the corresponding integrated form. The tetracycline-dependent mechanism for the transmission of CTn214 is nearly identical to a common conjugative transposon found in the genome of B. fragilis (CTnDOT), but the autonomously amplified nature of a circular 17,044-nt region of CTn214 that codes for tetQ and the integration of the same sequence in the linear chromosome within the same cell is a novel observation. Genome and transcriptome sequencing of B. fragilis cultivars grown under different concentrations of tetracycline and ciprofloxacin indicates that tetQ in strains containing the circular form remains actively expressed regardless of treatment, while the expression of tetQ in strains containing the linear form increases only in the presence of tetracycline.IMPORTANCEThe exchange of antibiotic production and resistance genes between microorganisms can lead to the emergence of new pathogens. In this study, short-read mapping of metagenomic samples taken over time from the illeal pouch of a patient with ulcerative colitis to a Bacteroides fragilis metagenome-assembled genome revealed two distinct genomic arrangements of a novel conjugative transposon, CTn214, that encodes tetracycline resistance. The autonomous amplification of a plasmid-like circular form from CTn214 that includes tetQ potentially provides consistent ribosome protection against tetracycline. This mode of antibiotic resistance offers a novel mechanism for understanding the emergence of pathobionts like B. fragilis and their persistence for extended periods of time in patients with inflammatory bowel disease.

ABSTRACTBacteroides fragilis comprises 1%–5% of the gut microbiota in healthy humans but can expand to >50% of the population in ulcerative colitis (UC) patients experiencing inflammation. The mechanisms underlying such microbial blooms are poorly understood, but the gut of UC patients has physicochemical features that differ from healthy patients and likely impact microbial physiology. For example, levels of the secondary bile acid deoxycholate (DC) are highly reduced in the ileoanal J-pouch of UC colectomy patients. We isolated a B. fragilis strain from a UC patient with pouch inflammation (i.e., pouchitis) and developed it as a genetic model system to identify genes and pathways that are regulated by DC and that impact B. fragilis fitness in DC and crude bile. Treatment of B. fragilis with a physiologically relevant concentration of DC reduced cell growth and remodeled transcription of one-quarter of the genome. DC strongly induced expression of chaperones and select transcriptional regulators and efflux systems, and down-regulated protein synthesis genes. Using a barcoded collection of ≈50,000 unique insertional mutants, we further defined B. fragilis genes that contribute to fitness in media containing DC or crude bile. Genes impacting cell envelope functions including cardiolipin synthesis, cell surface glycosylation, and systems implicated in sodium-dependent bioenergetics were major bile acid fitness factors. As expected, there was limited overlap between transcriptionally regulated genes and genes that impacted fitness in bile when disrupted. Our study provides a genome-scale view of a B. fragilis bile response and genetic determinants of its fitness in DC and crude bile.IMPORTANCEThe Gram-negative bacterium Bacteroides fragilis is a common member of the human gut microbiota that colonizes multiple host niches and can influence human physiology through a variety of mechanisms. Identification of genes that enable B. fragilis to grow across a range of host environments has been impeded in part by the relatively limited genetic tractability of this species. We have developed a high-throughput genetic resource for a B. fragilis strain isolated from a UC pouchitis patient. Bile acids limit microbial growth and are altered in abundance in UC pouches, where B. fragilis often blooms. Using this resource, we uncovered pathways and processes that impact B. fragilis fitness in bile and that may contribute to population expansions during bouts of gut inflammation.