In vitro adhesion, pilus expression, and in vivo amelioration of antibiotic-induced microbiota disturbance by Bifidobacterium spp. strains from fecal donors.

Fecal microbiota transplantation (FMT) is used routinely to treat recurrent Clostridioides difficile infection (rCDI) and investigated as a treatment for numerous conditions associated with gut microbiota alterations. Metagenomic analyses have indicated that recipient colonization by donor bacteria may be associated with favorable clinical outcomes. Bifidobacteria are abundant gut commensals associated with health. We have previously demonstrated that Bifidobacterium strains transferred in FMT can colonize recipients in long term, at least for a year, and recovered such strains by cultivation. This study addressed in vitro adhesion and pilus gene expression of long-term colonizing Bifidobacterium strains from FMT donors as well as in vivo colonization and capability to ameliorate antibiotic-induced microbiota disturbance. RNA-Seq differential gene expression analysis showed that the strongly adherent B. longum strains DY_pv11 and DX_pv23 expressed tight adherence and sortase-dependent pilus genes, respectively. Two B. longum strains, adherent DX_pv23 and poorly adhering DX_pv18, were selected to address in vivo colonization and efficacy to restore antibiotic-disturbed microbiota in C57BL/6 murine model. DX_pv23 colonized mice transiently with a rate comparable to that of the B. animalis BB-12 used as a reference. Although long-term colonization was not observed with any of the three strains, 16S rRNA gene profiling revealed that oral administration of DX_pv23 enhanced the recovery of antibiotic-disturbed microbiota to the original configuration significantly better than the other strains. The findings suggest that selected strains from FMT donors, such as DX_pv23 in this study, may have therapeutic potential by in vitro expression of colonization factors and boosting endogenous gut microbiota.

Development of a time-dependent oral colon delivery system of anaerobic Odoribacter splanchnicus for bacteriotherapy.

Odoribacter (O.) splanchnicus is an anaerobic member of the human intestinal microbiota. Its decrease in abundance has been associated with inflammatory bowel disease (IBD), non-alcoholic fatty liver, and cystic fibrosis. Considering the anti-inflammatory properties of O. splanchnicus and its possible use for IBD, intestinal isolate O. splanchnicus 57 was here formulated for oral colonic release based on a time-dependent strategy. Freeze-drying protocol was determined to ensure O. splanchnicus 57 viability during the process. Disintegrating tablets, containing the freeze-dried O. splanchnicus 57, were manufactured by direct compression and coated by powder-layering technique with hydroxypropyl methylcellulose (Methocel™ E50) in a tangential-spray fluid bed. Eudragit® L was then applied by spray-coating in a top-spray fluid bed. Double-coated tablets were tested for release, showing gastric resistance properties and, as desired, lag phases of reproducible duration prior to release in phosphate buffer pH 6.8. The cell viability and anti-inflammatory activity of the strain were assessed after the main manufacturing steps. While freeze-drying did not affect bacterial viability, the tableting and coating processes were more stressful. Nonetheless, O. splanchnicus 57 cells survived manufacturing and the final formulations had 106-107 CFU/g of viable cells. The strain kept its anti-inflammatory properties after tableting and coating, reducing Escherichia coli lipopolysaccharide-induced interleukin-8 cytokine release from HT-29 cells. Overall, O. splanchnicus 57 strain was formulated successfully for oral colon delivery, opening new ways to formulate pure cultures of single anaerobic strains or mixtures for oral delivery.

Novel strain of Pseudoruminococcus massiliensis possesses traits important in gut adaptation and host-microbe interactions.

Fecal microbiota transplantation (FMT) is an efficient treatment for recurrent Clostridioides difficile infection and currently investigated as a treatment for other intestinal and systemic diseases. Better understanding of the species potentially transferred in FMT is needed. We isolated from a healthy fecal donor a novel strain E10-96H of Pseudoruminococcus massiliensis, a recently described strictly anaerobic species currently represented only by the type strain. The whole genome sequence of E10-96H had over 98% similarity with the type strain. E10-96H carries 20 glycoside hydrolase encoding genes, degrades starch in vitro and thus may contribute to fiber degradation, cross-feeding of other species and butyrate production in the intestinal ecosystem. The strain carries pilus-like structures, harbors pilin genes in its genome and adheres to enterocytes in vitro but does not provoke a proinflammatory response. P. massiliensis seems to have commensal behavior with the host epithelium, and its role in intestinal ecology should be studied further.

Novel Odoribacter splanchnicus Strain and Its Outer Membrane Vesicles Exert Immunoregulatory Effects in vitro.

Odoribacter splanchnicus, belonging to the order Bacteroidales, is a common, short-chain fatty acid producing member of the human intestinal microbiota. A decreased abundance of Odoribacter has been linked to different microbiota-associated diseases, such as non-alcoholic fatty liver disease, cystic fibrosis and inflammatory bowel disease (IBD). The type strain of O. splanchnicus has been genome-sequenced, but otherwise very little is known about this anaerobic bacterium. The species surfaces in many microbiota studies and, consequently, comprehension on its interactions with the host is needed. In this study, we isolated a novel strain of O. splanchnicus from a healthy fecal donor, identified it by genome sequencing and addressed its adhesive, epithelium reinforcing and immunoregulatory properties. Our results show that O. splanchnicus strain 57 is non-adherent to enterocytes or mucus, does not reinforce nor compromise Caco-2 monolayer integrity and most likely harbors penta-acylated, less endotoxic lipid A as part of its lipopolysaccharide (LPS) structure based on the lack of gene lpxM and in vitro results on low-level NF-κB activity. The studies by transmission electron microscopy revealed that O. splanchnicus produces outer membrane vesicles (OMV). O. splanchnicus cells, culture supernatant i.e., spent medium or OMVs did not induce interleukin-8 (IL-8) response in HT-29 enterocyte cells suggesting a very low proinflammatory capacity. On the contrary, the treatment of HT-29 cells with O. splanchnicus cells, spent medium or OMVs prior to exposure to Escherichia coli LPS elicited a significant decrease in IL-8 production as compared to E. coli LPS treatment alone. Moreover, O. splanchnicus spent supernatant induced IL-10 production by immune cells, suggesting anti-inflammatory activity. Our in vitro findings indicate that O. splanchnicus and its effector molecules transported in OMVs could potentially exert anti-inflammatory action in the gut epithelium. Taken together, O. splanchnicus seems to be a commensal with a primarily beneficial interaction with the host.

Colonic Mucosal Microbiota and Association of Bacterial Taxa with the Expression of Host Antimicrobial Peptides in Pediatric Ulcerative Colitis.

Inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn's disease (CD), are chronic debilitating disorders of unknown etiology. Over 200 genetic risk loci are associated with IBD, highlighting a key role for immunological and epithelial barrier functions. Environmental factors account for the growing incidence of IBD, and microbiota are considered as an important contributor. Microbiota dysbiosis can lead to a loss of tolerogenic immune effects and initiate or exacerbate inflammation. We aimed to study colonic mucosal microbiota and the expression of selected host genes in pediatric UC. We used high-throughput 16S rDNA sequencing to profile microbiota in colonic biopsies of pediatric UC patients (n = 26) and non-IBD controls (n = 27). The expression of 13 genes, including five for antimicrobial peptides, in parallel biopsies was assessed with qRT-PCR. The composition of microbiota between UC and non-IBD differed significantly (PCoA, p = 0.001). UC children had a decrease in Bacteroidetes and an increase in several family-level taxa including Peptostreptococcaceae and Enterobacteriaceae, which correlated negatively with the expression of antimicrobial peptides REG3G and DEFB1, respectively. Enterobacteriaceae correlated positively with the expression siderophore binding protein LCN2 and Betaproteobacteria negatively with DEFB4A expression. The results indicate that reciprocal interaction of epithelial microbiota and defense mechanisms play a role in UC.

Isolation of Anti-Inflammatory and Epithelium Reinforcing Bacteroides and Parabacteroides Spp. from A Healthy Fecal Donor.

Altered intestinal microbiota is associated with systemic and intestinal diseases, such as inflammatory bowel disease (IBD). Dysbiotic microbiota with enhanced proinflammatory capacity is characterized by depletion of anaerobic commensals, increased proportion of facultatively anaerobic bacteria, as well as reduced diversity and stability. In this study, we developed a high-throughput in vitro screening assay to isolate intestinal commensal bacteria with anti-inflammatory capacity from a healthy fecal microbiota transplantation donor. Freshly isolated gut bacteria were screened for their capacity to attenuate Escherichia coli lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) release from HT-29 cells. The screen yielded a number of Bacteroides and Parabacteroides isolates, which were identified as P. distasonis, B. caccae, B. intestinalis, B. uniformis, B. fragilis, B. vulgatus and B. ovatus using whole genome sequencing. We observed that a cell-cell contact with the epithelium was not necessary to alleviate in vitro inflammation as spent culture media from the isolates were also effective and the anti-inflammatory action did not correlate with the enterocyte adherence capacity of the isolates. The anti-inflammatory isolates also exerted enterocyte monolayer reinforcing action and lacked essential genes to synthetize hexa-acylated, proinflammatory lipid A, part of LPS. Yet, the anti-inflammatory effector molecules remain to be identified. The Bacteroides strains isolated and characterized in this study have potential to be used as so-called next-generation probiotics.

Universal membrane-labeling combined with expression of Katushka far-red fluorescent protein enables non-invasive dynamic and longitudinal quantitative 3D dual-color fluorescent imaging of multiple bacterial strains in mouse intestine.

BACKGROUND: The human gastrointestinal (GI) tract microbiota has been a subject of intense research throughout the 3rd Millennium. Now that a general picture about microbiota composition in health and disease is emerging, questions about factors determining development of microbiotas with specific community structures will be addressed. To this end, usage of murine models for colonization studies remains crucial. Optical in vivo imaging of either bioluminescent or fluorescent bacteria is the basis for non-invasive detection of intestinal colonization of bacteria. Although recent advances in in vivo fluorescence imaging have overcome many limitations encountered in bioluminescent imaging of intestinal bacteria, such as requirement for live cells, high signal attenuation and 2D imaging, the method is still restricted to bacteria for which molecular cloning tools are available. RESULTS: Here, we present usage of a lipophilic fluorescent dye together with Katushka far-red fluorescent protein to establish a dual-color in vivo imaging system to monitor GI transit of different bacterial strains, suitable also for strains resistant to genetic labeling. Using this system, we were able to distinguish two different E. coli strains simultaneously and show their unique transit patterns. Combined with fluorescence molecular tomography, these distinct strains could be spatially and temporally resolved and quantified in 3D. CONCLUSIONS: Developed novel method for labeling microbes and identify their passage both temporally and spatially in vivo makes now possible to monitor all culturable bacterial strains, also those that are resistant to conventional genetic labeling.

The Potential of Gut Commensals in Reinforcing Intestinal Barrier Function and Alleviating Inflammation.

The intestinal microbiota, composed of pro- and anti-inflammatory microbes, has an essential role in maintaining gut homeostasis and functionality. An overly hygienic lifestyle, consumption of processed and fiber-poor foods, or antibiotics are major factors modulating the microbiota and possibly leading to longstanding dysbiosis. Dysbiotic microbiota is characterized to have altered composition, reduced diversity and stability, as well as increased levels of lipopolysaccharide-containing, proinflammatory bacteria. Specific commensal species as novel probiotics, so-called next-generation probiotics, could restore the intestinal health by means of attenuating inflammation and strengthening the epithelial barrier. In this review we summarize the latest findings considering the beneficial effects of the promising commensals across all major intestinal phyla. These include the already well-known bifidobacteria, which use extracellular structures or secreted substances to promote intestinal health. Faecalibacterium prausnitzii, Roseburia intestinalis, and Eubacterium hallii metabolize dietary fibers as major short-chain fatty acid producers providing energy sources for enterocytes and achieving anti-inflammatory effects in the gut. Akkermansia muciniphila exerts beneficial action in metabolic diseases and fortifies the barrier function. The health-promoting effects of Bacteroides species are relatively recently discovered with the findings of excreted immunomodulatory molecules. These promising, unconventional probiotics could be a part of biotherapeutic strategies in the future.

Mucosal Prevalence and Interactions with the Epithelium Indicate Commensalism of Sutterella spp.

Sutterella species have been frequently associated with human diseases, such as autism, Down syndrome, and inflammatory bowel disease (IBD), but the impact of these bacteria on health still remains unclear. Especially the interactions of Sutterella spp. with the host are largely unknown, despite of the species being highly prevalent. In this study, we addressed the interaction of three known species of Sutterella with the intestinal epithelium and examined their adhesion properties, the effect on intestinal barrier function and the pro-inflammatory capacity in vitro. We also studied the relative abundance and prevalence of the genus Sutterella and Sutterella wadsworthensis in intestinal biopsies of healthy individuals and patients with celiac disease (CeD) or IBD. Our results show that Sutterella spp. are abundant in the duodenum of healthy adults with a decreasing gradient toward the colon. No difference was detected in the prevalence of Sutterella between the pediatric IBD or CeD patients and the healthy controls. Sutterella parvirubra adhered better than the two other Sutterella spp. to differentiated Caco-2 cells and was capable of decreasing the adherence of S. wadsworthensis, which preferably bound to mucus and human extracellular matrix proteins. Furthermore, only S. wadsworthensis induced an interleukin-8 production in enterocytes, which could be due to different lipopolysaccharide structures between the species. However, its pro-inflammatory activity was modest as compared to non-pathogenic Escherichia coli. Sutterella spp. had no effect on the enterocyte monolayer integrity in vitro. Our findings indicate that the members of genus Sutterella are widely prevalent commensals with mild pro-inflammatory capacity in the human gastrointestinal tract and do not contribute significantly to the disrupted epithelial homeostasis associated with microbiota dysbiosis and increase of Proteobacteria. The ability of Sutterella spp. to adhere to intestinal epithelial cells indicate that they may have an immunomodulatory role.

BopA does not have a major role in the adhesion of Bifidobacterium bifidum to intestinal epithelial cells, extracellular matrix proteins, and mucus.

The ability of bifidobacteria to adhere to the intestine of the human host is considered to be important for efficient colonization and achieving probiotic effects. Bifidobacterium bifidum strains DSM20456 and MIMBb75 adhere well to the human intestinal cell lines Caco-2 and HT-29. The surface lipoprotein BopA was previously described to be involved in mediating adherence of B. bifidum to epithelial cells, but thioacylated, purified BopA inhibited the adhesion of B. bifidum to epithelial cells in competitive adhesion assays only at very high concentrations, indicating an unspecific effect. In this study, the role of BopA in the adhesion of B. bifidum was readdressed. The gene encoding BopA was cloned and expressed without its lipobox and hydrophobic signal peptide in Escherichia coli, and an antiserum against the recombinant BopA was produced. The antiserum was used to demonstrate the abundant localization of BopA on the cell surface of B. bifidum. However, blocking of B. bifidum BopA with specific antiserum did not reduce adhesion of bacteria to epithelial cell lines, arguing that BopA is not an adhesin. Also, adhesion of B. bifidum to human colonic mucin and fibronectin was found to be BopA independent. The recombinant BopA bound only moderately to human epithelial cells and colonic mucus, and it failed to bind to fibronectin. Thus, our results contrast the earlier findings on the major role of BopA in adhesion, indicating that the strong adhesion of B. bifidum to epithelial cell lines is BopA independent.