Bile acid fitness determinants of a Bacteroides fragilis isolate from a human pouchitis patient.

IMPORTANCE: The 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.

Bile acid fitness determinants of a Bacteroides fragilis isolate from a human pouchitis patient.

Bacteroides 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 downregulated 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.

Phage-resistant mutations impact bacteria susceptibility to future phage infections and antibiotic response.

Bacteriophage (phage) therapy in combination with antibiotic treatment serves as a potential strategy to overcome the continued rise in antibiotic resistance across bacterial pathogens. Understanding the impacts of evolutionary and ecological processes to the phage-antibiotic-resistance dynamic could advance the development of such combinatorial therapy. We tested whether the acquisition of mutations conferring phage resistance may have antagonistically pleiotropic consequences for antibiotic resistance. First, to determine the robustness of phage resistance across different phage strains, we infected resistant Escherichia coli cultures with phage that were not previously encountered. We found that phage-resistant E. coli mutants that gained resistance to a single phage strain maintain resistance to other phages with overlapping adsorption methods. Mutations underlying the phage-resistant phenotype affects lipopolysaccharide (LPS) structure and/or synthesis. Because LPS is implicated in both phage infection and antibiotic response, we then determined whether phage-resistant trade-offs exist when challenged with different classes of antibiotics. We found that only 1 out of the 4 phage-resistant E. coli mutants yielded trade-offs between phage and antibiotic resistance. Surprisingly, when challenged with novobiocin, we uncovered evidence of synergistic pleiotropy for some mutants allowing for greater antibiotic resistance, even though antibiotic resistance was never selected for. Our results highlight the importance of understanding the role of selective pressures and pleiotropic interactions in the bacterial response to phage-antibiotic combinatorial therapy.