A Combination of Structural, Genetic, Phenotypic and Enzymatic Analyses Reveals the Importance of a Predicted Fucosyltransferase to Protein O-Glycosylation in the Bacteroidetes.

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species-Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked ß1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.

Berberine inhibits dendritic cells differentiation in DSS-induced colitis by promoting Bacteroides fragilis.

BACKGROUNDS: Berberine (BBR), a compound long used in traditional Chinese medicine, has been reported to have therapeutic effects in treating ulcerative colitis (UC), attributed to its anti-inflammatory properties and restorative potential of tight junctions (TJs). However, the mechanism by which BBR affects intestinal bacteria and immunity is still unclear. METHODS: This study investigated the effects of BBR on intestinal bacteria and the inflammatory response in dextran sulfate sodium (DSS)-induced colitis mice. Immunohistochemistry (IHC) and electron microscopy were used to detect intestinal TJs. Microflora analysis was used to screen for bacteria regulated by BBR. RESULTS: The results showed that BBR had increased colonic epithelium zonula occludens proteins-1 (ZO-1) and occludin expression and reduced T-helper 17/T regulatory ratio in DSS-induced mice. Mechanically, BBR eliminated DSS-induced intestinal flora disturbances in mice, particularly increased Bacteroides fragilis (B. fragilis) in vivo and in vitro. B. fragilis decreased the interleukin-6 induced by dendritic cells through some heat-resistant component rather than nucleic acids or proteins. CONCLUSIONS: Overall, these data suggest that BBR had a moderating effect on DSS-induced colitis. This compound may regulate intestinal immune cell differentiation by affecting the growth of B. fragilis, providing new insights into the potential application of BBR in UC.

Comparative evaluation of agar dilution and broth microdilution by commercial and in-house plates for Bacteroides fragilis group: An economical and expeditious approach for resource-limited settings.

OBJECTIVE: To compare the performance of agar dilution and broth microdilution by commercial and in-house prepared plates for the Bacteroides fragilis group. The cost analysis was performed to demonstrate that in-house prepared BMD plates were a suitable alternative to agar dilution given the high cost and low feasibility of incorporating commercial BMD plates in routine, particularly in the tertiary care institutes of many low- and middle-income countries. METHODS: Thirty B. fragilis group isolates were tested against six antibiotics, frequently used as empirical therapy for anaerobic infections including metronidazole, clindamycin, imipenem, piperacillin-tazobactam, cefoxitin, and chloramphenicol. The running consumable expenditure for all methodologies was calculated. RESULTS: The results demonstrated essential and categorical agreement of >90% for all antibiotics except cefoxitin, which showed <90% categorical agreement. No major or very major errors were observed. We observed a high agreement and strong concordance for MIC values between both methods and inter-rate reliability of >0.9 by Cohen's kappa analysis, indicating almost perfect agreement between both methods using either of the plates. In contrast to agar dilution, a 20.5 fold cost reduction was seen in BMD using in-house plates and a 5.8 fold reduction using commercial plates to test a single isolate. However, when testing 30 isolates concurrently the cost significantly increased for commercial BMD plates by 8.4 folds, and only 1.03 fold cost reduction was seen with in-house BMD plates. CONCLUSION: BMD gives comparable results to agar dilution and can be considered a method of choice to test a small number of samples. The technique is an economical option when plates are standardized in-house and could be employed for susceptibility testing of the B. fragilis group.

Baicalin ameliorates Mycoplasma gallisepticum-induced inflammatory injury in the chicken lung through regulating the intestinal microbiota and phenylalanine metabolism.

Baicalin shows excellent protective effects against Mycoplasma gallisepticum (MG) induced inflammatory injury as discussed in our previous studies. However, the physiological effects of baicalin are notable in contrast to its low bioavailability, and the critical mechanism for the protective effects of baicalin against MG infection is still unclear. The main objective of this study was to investigate whether baicalin alleviates MG-induced lung inflammatory injury through regulating gut microbiota. Using an MG infection model, results showed that baicalin treatment significantly reduced MG colonization and ameliorated the abnormal pathological changes in the lung. Baicalin treatment also reduced the level of proinflammatory cytokines and suppressed proinflammatory protein expression. Notably, MG infection changed the gut microbiota composition, however, the abnormal gut microbiota composition was partially alleviated by baicalin treatment. Baicalin significantly enriched the commensal bacterium Bacteroides fragilis, and gavaged with Bacteroides fragilis alleviating MG infection-induced inflammatory injury in the lung. In addition, baicalin reversed peripheral accumulation of phenylalanine induced by MG infection. Importantly, increased phenylalanine induced excessive necroptosis through the modulation of gga-miR-190a-3p-Fas-associated protein with death domain (FADD) axis in HD11 macrophages. Together, our findings highlighted the role of gut microbiota and phenylalanine metabolism in MG infection and confirmed that baicalin could effectively inhibit MG-induced inflammatory injury in the lung by remodeling the gut microbiota and phenylalanine metabolism.

The microbiome, genetics, and gastrointestinal neoplasms: the evolving field of molecular pathological epidemiology to analyze the tumor-immune-microbiome interaction.

Metagenomic studies using next-generation sequencing technologies have revealed rich human intestinal microbiome, which likely influence host immunity and health conditions including cancer. Evidence indicates a biological link between altered microbiome and cancers in the digestive system. Escherichia coli and Bacteroides fragilis have been found to be enriched in colorectal mucosal tissues from patients with familial adenomatous polyposis that is caused by germline APC mutations. In addition, recent studies have found enrichment of certain oral bacteria, viruses, and fungi in tumor tissue and fecal specimens from patients with gastrointestinal cancer. An integrative approach is required to elucidate the role of microorganisms in the pathogenic process of gastrointestinal cancers, which develop through the accumulation of somatic genetic and epigenetic alterations in neoplastic cells, influenced by host genetic variations, immunity, microbiome, and environmental exposures. The transdisciplinary field of molecular pathological epidemiology (MPE) offers research frameworks to link germline genetics and environmental factors (including diet, lifestyle, and pharmacological factors) to pathologic phenotypes. The integration of microbiology into the MPE model (microbiology-MPE) can contribute to better understanding of the interactive role of environment, tumor cells, immune cells, and microbiome in various diseases. We review major clinical and experimental studies on the microbiome, and describe emerging evidence from the microbiology-MPE research in gastrointestinal cancers. Together with basic experimental research, this new research paradigm can help us to develop new prevention and treatment strategies for gastrointestinal cancers through targeting of the microbiome.

Zerumbone Restores Gut Microbiota Composition in ETBF Colonized AOM/DSS Mice.

Colorectal cancer (CRC) is the leading cause of common malignant neoplasm worldwide. Many studies have analyzed compositions of gut microbiota associated with various diseases such as inflammatory bowel diseases (IBD) and colon cancer. One of the most representative bacteria involved in CRC is enterotoxigenic Bacteroides fragilis (ETBF), a species belonging to phylum Bacteroidetes. We used ETBF colonized mice with azoxymethane (AOM)/dextran sulphate sodium (DSS) and zerumbone, a compound with anti-bacterial effect, to determine whether zerumbone could restore intestinal microbiota composition. Four experimental groups of mice were used: sham, ETBF colonized AOM/DSS group, ETBF colonized AOM/DSS group zerumbone 60 mg kg-1 (ETBF/AOM/ DSS + Z (60)), and only zerumbone (60 mg kg-1)-treated group. We performed reversible dye terminators-based analysis of 16S rRNA gene region V3-V4 for group comparison. Microbiota compositions of ETBF/AOM/DSS + Z (60) group and ETBF colonized AOM/DSS group not given zerumbone were significantly different. There were more Bacteroides in ETBF/AOM/DSS + Z (60) group than those in ETBF colonized AOM/DSS group, suggesting that B. fragilis could be a normal flora activated by zerumbone. In addition, based on linear discriminant analysis of effect size (LEfSe) analysis, microbial diversity decreased significantly in the ETBF colonized AOM/DSS group. However, after given zerumbone, the taxonomic relative abundance was increased. These findings suggest that zerumbone not only influenced the microbial diversity and richness, but also could be helpful for enhancing the balance of gut microbial composition. In this work, we demonstrate that zerumbone could restore the composition of intestinal microbiota.

A Fragile Balance: The Important Role of the Intestinal Microbiota in the Prevention and Management of Colorectal Cancer.

BACKGROUND: Colorectal cancer is the second leading cause of cancer-related death worldwide. In recent years, researchers have focussed on the role of the intestinal microbiota in both the prevention and the treatment of colorectal cancer. SUMMARY: The evidence in the literature supports that there is a fragile balance between different species of bacteria in the human gut. A disturbance of this balance towards increased levels of the bacteria Fusobacterium nucleatum and Bacteroides fragilis is associated with an increased risk of colorectal cancer. The mechanisms involved include the release of toxins which activate inflammation and the regulation of specific miRNAs (with an increase in the expression of oncogenic miRNAs and a decrease in the expression of tumour suppressor miRNAs), thereby increasing cell proliferation and leading to tumorigenesis. On the other hand, Lactobacillus and Bifidobacterium have a protective effect against the development of colorectal cancer through mechanisms that involve an increase in the levels of anticarcinogenic metabolites such as butyrate and a decrease in the activity of proinflammatory pathways. Even though preliminary studies support that the use of probiotics in the prevention and management of colorectal cancer is promising, more research is needed in this field. Key Message: The association between the intestinal microbiota, diet and colorectal cancer remains an active area of research with expected future applications in the use of probiotics for the prevention and management of this significant disease.

Spatially distinct physiology of Bacteroides fragilis within the proximal colon of gnotobiotic mice.

A complex microbiota inhabits various microenvironments of the gut, with some symbiotic bacteria having evolved traits to invade the epithelial mucus layer and reside deep within the intestinal tissue of animals. Whether these distinct bacterial communities across gut biogeographies exhibit divergent behaviours is largely unknown. Global transcriptomic analysis to investigate microbial physiology in specific mucosal niches has been hampered technically by an overabundance of host RNA. Here, we employed hybrid selection RNA sequencing (hsRNA-Seq) to enable detailed spatial transcriptomic profiling of a prominent human commensal as it colonizes the colonic lumen, mucus or epithelial tissue of mice. Compared to conventional RNA-Seq, hsRNA-Seq increased reads mapping to the Bacteroides fragilis genome by 48- and 154-fold in mucus and tissue, respectively, allowing for high-fidelity comparisons across biogeographic sites. Near the epithelium, B. fragilis upregulated numerous genes involved in protein synthesis, indicating that bacteria inhabiting the mucosal niche are metabolically active. Further, a specific sulfatase (BF3086) and glycosyl hydrolase (BF3134) were highly induced in mucus and tissue compared to bacteria in the lumen. In-frame deletion of these genes impaired in vitro growth on mucus as a carbon source, as well as mucosal colonization of mice. Mutants in either B. fragilis gene displayed a fitness defect in competing for colonization against bacterial challenge, revealing the importance of site-specific gene expression for robust host-microbial symbiosis. As a versatile tool, hsRNA-Seq can be deployed to explore the in vivo spatial physiology of numerous bacterial pathogens or commensals.

Antibacterial and antibiofilm effects of α-humulene against Bacteroides fragilis.

The rapid increase in antibiotic resistance has prompted the discovery of drugs that reduce antibiotic resistance or new drugs that are an alternative to antibiotics. Plant extracts have health benefits and may also exhibit antibacterial and antibiofilm activities against pathogens. This study determined the antibacterial and antibiofilm effects of α-humulene extracted from plants against enterotoxigenic Bacteroides fragilis, which causes inflammatory bowel disease. The minimum inhibitory concentration and biofilm inhibitory concentration of α-humulene for B. fragilis were 2 µg/mL, and the biofilm eradication concentration was in the range of 8-32 µg/mL. The XTT reduction assay confirmed that the cellular metabolic activity in biofilm rarely occurred at the concentration of 8-16 µg/mL. In addition, biofilm inhibition by α-humulene was also detected via confocal laser scanning microcopy. Quantitative real-time polymerase chain reaction (qPCR) was also used to investigate the effect of α-humulene on the expression of resistance-nodulation-cell division type multidrug efflux pump genes (bmeB1 and bmeB3). According to the results of qPCR, α-humulene significantly reduced the expression of bmeB1 and bmeB3 genes. This study demonstrates the potential therapeutic application of α-humulene for inhibiting the growth of B. fragilis cells and biofilms, and it expands the knowledge about biofilm medicine.

Potential prebiotic effect of a long-chain dextran produced by Weissella cibaria: an in vitro evaluation.

Dextrans are homopolysaccharides of D-glucose units produced by lactic acid bacteria. They have several technological applications and potential utilisation in positively modulating gut microbiota is attracting increasing attention. Whereas the prebiotic activity of low polymerisation degree (DP) dextrans has been established, high DP dextrans still deserve deeper investigation. In the present study, a long linear chain dextran produced by Weissella cibaria was compared to inulin with regards to the growth of specific health-related taxa and to the production of organic acids in pH-controlled batch cultures of intestinal microbiota. qPCR quantification of Lactobacillus, Bifidobacterium, Prevotella, Bacteroides fragilis, and Faecalibacterium prausnitzii revealed differences in their relative abundance, depending on the carbon source, that reflected the pattern of fermentation products determined by HPLC. Dextran mainly enhanced the relative amount of Prevotella and Bacteroides, consistently with a favourable acetate-propionate ratio suggesting a promising utilisation as functional ingredient in the food industry.

Clostridioides difficile LuxS mediates inter-bacterial interactions within biofilms.

The anaerobic gut pathogen, Clostridioides difficile, forms adherent biofilms that may play an important role in recurrent C. difficile infections. The mechanisms underlying C. difficile community formation and inter-bacterial interactions are nevertheless poorly understood. C. difficile produces AI-2, a quorum sensing molecule that modulates biofilm formation across many bacterial species. We found that a strain defective in LuxS, the enzyme that mediates AI-2 production, is defective in biofilm development in vitro. Transcriptomic analyses of biofilms formed by wild type (WT) and luxS mutant (luxS) strains revealed a downregulation of prophage loci in the luxS mutant biofilms compared to the WT. Detection of phages and eDNA within biofilms may suggest that DNA release by phage-mediated cell lysis contributes to C. difficile biofilm formation. In order to understand if LuxS mediates C. difficile crosstalk with other gut species, C. difficile interactions with a common gut bacterium, Bacteroides fragilis, were studied. We demonstrate that C. difficile growth is significantly reduced when co-cultured with B. fragilis in mixed biofilms. Interestingly, the absence of C. difficile LuxS alleviates the B. fragilis-mediated growth inhibition. Dual species RNA-sequencing analyses from single and mixed biofilms revealed differential modulation of distinct metabolic pathways for C. difficile WT, luxS and B. fragilis upon co-culture, indicating that AI-2 may be involved in induction of selective metabolic responses in B. fragilis. Overall, our data suggest that C. difficile LuxS/AI-2 utilises different mechanisms to mediate formation of single and mixed species communities.

Binary interactions between the yeast Candida albicans and two gut-associated Bacteroides species.

The yeast Candida albicans forms part of the natural gut microbiota of healthy human individuals and its interactions with other microbial symbionts can impact host well-being. We therefore studied binary interactions between potentially pathogenic representatives of the gut-associated bacterial genus Bacteroides and C. albicans using anaerobic bacteria/yeast co-cultures prepared with a quarter-strength brain heart infusion (» BHI; 9.25 g/l) broth. We found that, except for minor changes observed in the cell numbers of one out of four C. albicans strains tested, yeast growth was largely unaffected by the presence of the bacteria. In contrast, growth of Bacteroides fragilis NCTC 9343 and Bacteroides vulgatus ATCC 8482 was significantly enhanced in the presence of C. albicans. Supplementation of Bacteroides monocultures with dead Candida albicans CAB 392 cells, containing intact outer cell wall mannan layers, resulted in increased bacterial concentrations. Subsequent culturing of the Bacteroides strains in a liquid minimal medium supplemented with candidal mannan demonstrated that B. vulgatus ATCC 8482, unlike B. fragilis NCTC 9343, utilized the mannan. Furthermore, by reducing the initial oxygen levels in monocultures prepared with » BHI broth, bacterial numbers were significantly enhanced compared to in monocultures prepared with » BHI broth not supplemented with the reducing agent l-cysteine hydrochloride. This suggests that C. albicans can stimulate Bacteroides growth via aerobic respiration and/or antioxidant production. The cell-free supernatant of 24-h-old C. albicans CAB 392 monocultures was also found to increase Bacteroides growth and chloramphenicol sensitivity.

Adaptive Evolution within Gut Microbiomes of Healthy People.

Natural selection shapes bacterial evolution in all environments. However, the extent to which commensal bacteria diversify and adapt within the human gut remains unclear. Here, we combine culture-based population genomics and metagenomics to investigate the within-microbiome evolution of Bacteroides fragilis. We find that intra-individual B. fragilis populations contain substantial de novo nucleotide and mobile element diversity, preserving years of within-person history. This history reveals multiple signatures of within-person adaptation, including parallel evolution in sixteen genes. Many of these genes are implicated in cell-envelope biosynthesis and polysaccharide utilization. Tracking evolutionary trajectories using near-daily metagenomic sampling, we find evidence for years-long coexistence in one subject despite adaptive dynamics. We used public metagenomes to investigate one adaptive mutation common in our cohort and found that it emerges frequently in Western, but not Chinese, microbiomes. Collectively, these results demonstrate that B. fragilis adapts within individual microbiomes, pointing to factors that promote long-term gut colonization.

Antibiotic Korormicin A Kills Bacteria by Producing Reactive Oxygen Species.

Korormicin is an antibiotic produced by some pseudoalteromonads which selectively kills Gram-negative bacteria that express the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR.) We show that although korormicin is an inhibitor of Na+-NQR, the antibiotic action is not a direct result of inhibiting enzyme activity. Instead, perturbation of electron transfer inside the enzyme promotes a reaction between O2 and one or more redox cofactors in the enzyme (likely the flavin adenine dinucleotide [FAD] and 2Fe-2S center), leading to the production of reactive oxygen species (ROS). All Pseudoalteromonas contain the nqr operon in their genomes, including Pseudoalteromonas strain J010, which produces korormicin. We present activity data indicating that this strain expresses an active Na+-NQR and that this enzyme is not susceptible to korormicin inhibition. On the basis of our DNA sequence data, we show that the Na+-NQR of Pseudoalteromonas J010 carries an amino acid substitution (NqrB-G141A; Vibrio cholerae numbering) that in other Na+-NQRs confers resistance against korormicin. This is likely the reason that a functional Na+-NQR is able to exist in a bacterium that produces a compound that typically inhibits this enzyme and causes cell death. Korormicin is an effective antibiotic against such pathogens as Vibrio cholerae, Aliivibrio fischeri, and Pseudomonas aeruginosa but has no effect on Bacteroides fragilis and Bacteroides thetaiotaomicron, microorganisms that are important members of the human intestinal microflora.IMPORTANCE As multidrug antibiotic resistance in pathogenic bacteria continues to rise, there is a critical need for novel antimicrobial agents. An essential requirement for a useful antibiotic is that it selectively targets bacteria without significant effects on the eukaryotic hosts. Korormicin is an excellent candidate in this respect because it targets a unique respiratory enzyme found only in prokaryotes, the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR). Korormicin is synthesized by some species of the marine bacterium Pseudoalteromonas and is a potent and specific inhibitor of Na+-NQR, an enzyme that is essential for the survival and proliferation of many Gram-negative human pathogens, including Vibrio cholerae and Pseudomonas aeruginosa, among others. Here, we identified how korormicin selectively kills these bacteria. The binding of korormicin to Na+-NQR promotes the formation of reactive oxygen species generated by the reaction of the FAD and the 2Fe-2S center cofactors with O2.

The Protective Role of Bacteroides fragilis in a Murine Model of Colitis-Associated Colorectal Cancer.

Many patients with chronic inflammation of the gut, such as that observed in inflammatory bowel disease (IBD), develop colorectal cancer (CRC). Recent studies have reported that the development of IBD and CRC partly results from an imbalanced composition of intestinal microbiota and that intestinal inflammation in these diseases can be modulated by the microbiota. The human commensal Bacteroides fragilis is best exemplified playing a protective role against the development of experimental colitis in several animal disease models. In this study, we found that gut inflammation caused by dextran sulfate sodium (DSS) treatment was inhibited by B. fragilis colonization in mice. Further, we reveal a protective role of B. fragilis treatment against colon tumorigenesis using an azoxymethane (AOM)/DSS-induced model of colitis-associated colon cancer in mice and demonstrate that the decreased tumorigenesis by B. fragilis administration is accompanied by inhibited expression of C-C chemokine receptor 5 (CCR5) in the gut. We show direct evidence that the inhibition of tumor formation provided by B. fragilis in colitis-associated CRC animals was dependent on the production of polysaccharide A (PSA) from B. fragilis and that Toll-like receptor 2 (TLR2) signaling was responsible for the protective function of B. fragilisIMPORTANCE The incidence of colorectal cancer (CRC) is rapidly growing worldwide, and there is therefore a greater emphasis on studies of the treatment or prevention of CRC pathogenesis. Recent studies suggested that consideration of the microbiota is unavoidable to understand inflammation and tumorigenesis in the gastrointestinal tract. We demonstrate, using a mouse model of colitis-associated CRC, that human commensal B. fragilis protects against colon tumorigenesis. The protective role against tumor formation provided by B. fragilis is associated with inhibition of expression of the chemokine receptor CCR5 in the colon. The molecular mechanism for protection against CRC provided by B. fragilis is dependent on polysaccharide A production and is mediated by TLR2 signaling. Our results suggest that the commensal microorganism B. fragilis can be used to prevent inflammation-associated CRC development and may provide an effective therapeutic strategy for CRC.

Antimicrobial susceptibility testing of Bacteroides fragilis using the MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA).

This study evaluated the MBT-ASTRA for antimicrobial susceptibility testing of Bacteroides fragilis with different classes of antibiotics. MALDI-TOF MS peak AUCs from suspensions with B. fragilis with and without an antibiotic were used to calculate the relative growth (AUC "with antibiotic" divided by "without antibiotic"). Antimicrobial susceptibility testing of B. fragilis ATCC 25285 (susceptible) and B. fragilis O18 (resistant) was demonstrated with a clear difference of the relative growth between susceptible and resistant. The MBT-ASTRA needs further development and assessment but could be a relatively easy and inexpensive method for rapid antimicrobial susceptibility testing in specific cases of infection with B. fragilis.

Gut Symbiont Bacteroides fragilis Secretes a Eukaryotic-Like Ubiquitin Protein That Mediates Intraspecies Antagonism.

Human gut Bacteroides species produce different types of toxins that antagonize closely related members of the gut microbiota. Some are toxic effectors delivered by type VI secretion systems, and others are non-contact-dependent secreted antimicrobial proteins. Many strains of Bacteroides fragilis secrete antimicrobial molecules, but only one of these toxins has been described to date (Bacteroidales secreted antimicrobial protein 1 [BSAP-1]). In this study, we describe a novel secreted protein produced by B. fragilis strain 638R that mediated intraspecies antagonism. Using transposon mutagenesis and deletion mutation, we identified a gene encoding a eukaryotic-like ubiquitin protein (BfUbb) necessary for toxin activity against a subset of B. fragilis strains. The addition of ubb into a heterologous background strain conferred toxic activity on that strain. We found this gene to be one of the most highly expressed in the B. fragilis genome. The mature protein is 84% similar to human ubiquitin but has an N-terminal signal peptidase I (SpI) signal sequence and is secreted extracellularly. We found that the mature 76-amino-acid synthetic protein has very potent activity, confirming that BfUbb mediates the activity. Analyses of human gut metagenomic data sets revealed that ubb is present in 12% of the metagenomes that have evidence of B. fragilis As 638R produces both BSAP-1 and BfUbb, we performed a comprehensive analysis of the toxin activity of BSAP-1 and BfUbb against a set of 40 B. fragilis strains, revealing that 75% of B. fragilis strains are targeted by one or the other of these two secreted proteins of strain 638R.IMPORTANCE We are just beginning to understand some of the important interactions that occur between microbes of the human gut microbiota that dictate the composition and abundance of its constituent members. The ability of one member to produce molecules that directly kill a coresident member has been shown among minor gut species and is just starting to be studied in the abundant Bacteroides species. Here, we show that some strains of Bacteroides fragilis have acquired a gene encoding a secreted eukaryotic-like ubiquitin protein with potent inhibitory activity against other B. fragilis stains. This is the first bacterially encoded ubiquitin-like molecule shown to function like a bacterial toxin. This molecule is an example of a gut symbiont acquiring and adapting a eukaryotic molecule likely to increase its competitiveness in the mammalian gut. Understanding antagonistic factors produced by abundant gut symbionts is an important prerequisite to properly engineer strains to colonize the gut for health benefits.

In Vitro Activities of Pexiganan and 10 Comparator Antimicrobials against 502 Anaerobic Isolates Recovered from Skin and Skin Structure Infections.

Pexiganan, a cationic peptide, exhibited a broad range of anti-anaerobic antimicrobial activity. The MIC90s of studied isolates were as follows: Bacteroides fragilis, 16 µg/ml; other B. fragilis group spp., 4 µg/ml; Prevotella and Fusobacterium spp., 32 µg/ml; Porphyromonas spp., 64 µg/ml; Propionibacterium acnes, 4 µg/ml; Eggerthella lenta and Peptostreptococcus anaerobius, 32 µg/ml; other Gram-positive rods and cocci, 4 µg/ml; Clostridium perfringens, 128 µg/ml; and other clostridia, 256 µg/ml. Pexiganan cream shows potential as adjunctive therapy for skin and skin structure infections (SSSIs) involving anaerobes.

Strong antimicrobial activity of xanthohumol and other derivatives from hops (Humulus lupulus L.) on gut anaerobic bacteria.

Anaerobic bacteria, such as Bacteroides fragilis or Clostridium perfringens, are part of indigenous human flora. However, Clostridium difficile represents also an important causative agent of nosocomial infectious antibiotic-associated diarrhoea. Treatment of C. difficile infection is problematic, making it imperative to search for new compounds with antimicrobial properties. Hops (Humulus lupulus L.) contain substances with antibacterial properties. We tested antimicrobial activity of purified hop constituents humulone, lupulone and xanthohumol against anaerobic bacteria. The antimicrobial activity was established against B. fragilis, C. perfringens and C. difficile strains according to standard testing protocols (CLSI, EUCAST), and the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBC) were calculated. All C. difficile strains were toxigenic and clinically relevant, as they were isolated from patients with diarrhoea. Strongest antimicrobial effects were observed with xanthohumol showing MIC and MBC values of 15-107 µg/mL, which are close to those of conventional antibiotics in the strains of bacteria with increased resistance. Slightly higher MIC and MBC values were obtained with lupulone followed by higher values of humulone. Our study, thus, shows a potential of purified hop compounds, especially xanthohumol, as alternatives for treatment of infections caused by select anaerobic bacteria, namely nosocomial diarrhoea caused by resistant strains.

The sialate O-acetylesterase EstA from gut Bacteroidetes species enables sialidase-mediated cross-species foraging of 9-O-acetylated sialoglycans.

The gut harbors many symbiotic, commensal, and pathogenic microbes that break down and metabolize host carbohydrates. Sialic acids are prominent outermost carbohydrates on host glycoproteins called mucins and protect underlying glycan chains from enzymatic degradation. Sialidases produced by some members of the colonic microbiota can promote the expansion of several potential pathogens (e.g. Clostridium difficile, Salmonella, and Escherichia coli) that do not produce sialidases. O-Acetyl ester modifications of sialic acids help resist the action of many sialidases and are present at high levels in the mammalian colon. However, some gut bacteria, in turn, produce sialylate-O-acetylesterases to remove them. Here, we investigated O-acetyl ester removal and sialic acid degradation by Bacteroidetes sialate-O-acetylesterases and sialidases, respectively, and subsequent utilization of host sialic acids by both commensal and pathogenic E. coli strains. In vitro foraging studies demonstrated that sialidase-dependent E. coli growth on mucin is enabled by Bacteroides EstA, a sialate O-acetylesterase acting on glycosidically linked sialylate-O-acetylesterase substrates, particularly at neutral pH. Biochemical studies suggested that spontaneous migration of O-acetyl esters on the sialic acid side chain, which can occur at colonic pH, may serve as a switch controlling EstA-assisted sialic acid liberation. Specifically, EstA did not act on O-acetyl esters in their initial 7-position. However, following migration to the 9-position, glycans with O-acetyl esters became susceptible to the sequential actions of bacterial esterases and sialidases. We conclude that EstA specifically unlocks the nutritive potential of 9-O-acetylated mucus sialic acids for foraging by bacteria that otherwise are prevented from accessing this carbon source.