Bifidobacterium Pseudolongum Research Articles

Administration of selenomethionine in combination with serine benefits diabetes via gut microbiota.

Either selenium or serine could modulate glucose homeostasis, however, whether there are synergistic effects of selenium with serine on diabetes remains to be unknown. In the present study, eight male db/m mice were used as a control, and 24 male diabetic db/db mice were either orally gavaged with PBS, or with selenomethionine alone, or with both selenomethionine and serine, to investigate the effects of selenomethionine and serine on body weight and glucose level. Furthermore, intestinal microbiota composition was analyzed and fecal microbiota transplantation (FMT) was performed to explore whether microbes mediate the beneficial effects of selenomethionine and serine. The results showed that administration of selenomethionine decreased body weight, adipose tissue weight and serum glucose level in db/db diabetic mice. Importantly, administration of selenomethionine in combination with serine exerted better effects than selenomethionine alone did. Furthermore, a combined administration of selenomethionine and serine restored the microbial composition in diabetic mice. Corynebacterium glutamicum, Bifidobacterium pseudolongum, and Aerococcus urinaeequi were significantly decreased, whereas Lactobacillus murinus was increased in mice in the selenomethionine group and selenomethionine in combination with serine group, when compared with those in the db/db group. FMT decreased body weight and glucose level in db/db mice, further indicating that microbes play critical roles in the beneficial effects of selenomethionine and serine. Thus, we concluded that administration of selenomethionine in combination with serine benefits diabetes via gut microbes. Our results suggested that the synergic application of selenomethionine and serine could be potentially used for the treatment of diabetes.

Dietary Indian frankincense (Boswellia serrata) ameliorates murine allergic asthma through modulation of the gut microbiome

• Mice given an oral supplement of Boswellia serrate had suppressed asthma symptoms. • Treated mice had significant increases of Bifidobacterium in their guts. • The Bifidobacterium was further identified as B. pseudolongum . • Oral administration of isolated B. pseudolongum alleviated airway inflammation. Asthma is associated with significant morbidity. The gut microbiome has been shown to effect asthma development and exacerbation. In this study, we tested an oral supplement containing Boswellia serrata (Indian frankincense) tree resin on allergic pulmonary inflammation and gut microbiome. An OVA based allergic airway model was used and mice were orally gavaged 100 mg/kg of Boswellia serrata as a supplementation throughout asthma sensitization and challenge. Treated mice showed significant weight loss, lower total lung leukocytes, eosinophil and Th2 cytokines, improved histology scoring and reduced reactivity to methacholine challenge. Asthmatic mice without Boswellia serrata supplementation showed a decrease in overall bacterial diversity, while treated mice were protected against loss. Boswellia serrata treated mice had a significant increase in Bifidobacterium, which was identified as Bifidobacterium pseudolongum. Oral administration of B. pseudolongum also reduced airway inflammation, suggesting Boswellia serrata may work as an anti-asthma agent via increases in B. pseudolongum from prebiotic influences.

Yogurt Benefits Bone Mineralization in Ovariectomized Rats with Concomitant Modulation of the Gut Microbiome.

Evidence supports that gut-modulating foods potentially can suppress bone loss in postmenopausal women. This study aims to investigate the effect of milk calcium-enriched milk, yogurt, and yogurt-inulin combination on the gut-bone association. A 6-week intervention study is conducted in ovariectomized rats. Four pastes containing milk calcium-fortified milk (M-Ca), milk calcium-fortified yogurt (Y-Ca), inulin-fortified Y-Ca (Y-I-Ca), or an isoconcentration of calcium carbonate (Ca-N), and a calcium-deficient paste are provided. M-Ca does not influence bone mineral density and content (BMD and BMC), femur mechanical strength, or femoral microstructure compared to Ca-N, but Y-Ca increases spine BMD. The serum metabolome reveals that Y-Ca modulated glycine-related pathways with reduced glycine, serine, and threonine. No additive effects of yogurt and inulin are found on bone parameters. Correlation analysis shows that increased lactobacilli and reduced Clostridiaceae members in Y-Ca is associated with an increased spine BMD. Increases in Bifidobacterium pseudolongum, Turicibacter, Blautia, and Allobaculum and gut short-chain fatty acids in Y-I-Ca are not reflected in bone parameters. Yogurt as calcium vehicle contributes to increased spine BMD concomitant with changes in the gut microbiome and glycine-related pathways, while adding inulin to yogurt does not affect bone mineralization in ovariectomized rats.

Effects of High-Fat Diet on the Gut Microbiota of Renalase Gene Knockout Mice

Metabolic diseases caused by gene and unhealthy living habits are increasing, which seriously threaten the life of people worldwide. Moreover, the microbiome has been shown to play an active role in the prevention and treatment of metabolic diseases. However, reliable evidence on renalase gene (Rnls), as a common gene related to metabolic diseases, is still lacking with regard to the influence on the microbiome. Hence, we investigated the effect of a normal diet (ND) and a high-fat diet (HFD) on the gut microbiota of Rnls knockout (Rnls−/−) and wild-type (Rnls+/+) mice. At the end of the 8-week experiment, DNA samples were extracted from fresh feces, and the composition of microbiota was profiled. The species in Rnls+/+-ND group were Bifidobacterium pseudolongum and Lactobacillus reuteri. Conversely, the species in Rnls−/−-ND group belonged to the genera Lactobacillus and Turicibacter. The HFD changed the ratio of Firmicutes/Bacteroidetes; while the bacteria in the Rnls+/+-HFD and Rnls−/−-HFD groups were different. Overall, this study not only revealed the composition of microbiota in Rnls−/− mice, but also indicated that Rnls and the bacteria related to Rnls may be new candidates in the prevention and diagnosis of metabolic diseases at an early stage.

Lactation time influences the composition of Bifidobacterium and Lactobacillus at species level in human breast milk.

Human breast milk is a source of microorganisms for infants that play an important role in building infant gut health and immunity. The bacterial composition in human breast milk is influenced by lactation time. This study aimed to investigate the influence of lactation time on bacteria in breast milk at the genus level and the species levels of Bifidobacterium and Lactobacillus on days 2-4, 8, 14, and 30. Eighteen individuals were recruited and 60 milk samples were collected. The 16S rRNA gene, and the bifidobacterial groEL and lactobacilli groEL genes were used for amplicon sequencing. The results revealed that the alpha diversities of colostrum and transition 1 (day 8) milk were lower than that of transition 2 (day 14) and mature milk. PCoA analysis showed that bacterial composition in colostrum and transition 1 milk differed from transition 2 and mature milk. A lower relative abundance of Blautia was found in colostrum and transition 1 milk compared with mature milk and lower abundances of Ruminococcus, Dorea, and Escherichia-Shigella were found in transition 1 compared with mature milk. Bifidobacterium ruminantium, Limosilactobacillus mucosae, and Ligilactobacillus ruminis were the predominant species across all four lactation stages, while Bifidobacterium bifidum was lower in transition 1, and Bifidobacterium pseudocatenulatum and Bifidobacterium pseudolongum were higher in transition 1 milk. This study indicated that the bacterial composition in colostrum was more similar to that of transition 1 milk, whereas the bacterial community in transition 2 milk was similar to that of mature milk which suggests that bacterial composition in human breast milk shows stage-specific signatures even within a short period at both genus level and Bifidobacterium and Lactobacillus species levels, providing insights into probiotic supplementation for the nursing mother.

Gut Microbiota Modulates the Protective Role of Ginsenoside Compound K Against Sodium Valproate-Induced Hepatotoxicity in Rat.

This study aimed to investigate the potential role of gut microbiota in the hepatotoxicity of sodium valproate (SVP) and the protective effect of ginsenoside compound K (G-CK) administration against SVP-induced hepatotoxicity in rats. Measurements of 16S rRNA showed that SVP supplementation led to a 140.749- and 248.900-fold increase in the relative abundance of Akkermansia muciniphila (A. muciniphila) and Bifidobacterium pseudolongum (B. pseudolongum), respectively (p < 0.05). The increase in A. muciniphila was almost completely reversed by G-CK treatment. The relative abundance of A. muciniphila was strongly positively correlated with aspartate transaminase (AST) and alanine aminotransferase (ALT) levels (r > 0.78, p < 0.05). The PICRUSt analysis showed that G-CK could inhibit the changes of seven pathways caused by SVP, of which four pathways, including the fatty acid biosynthesis, lipid biosynthesis, glycolysis/gluconeogenesis, and pyruvate metabolism, were found to be negatively correlated with AST and ALT levels (r ≥ 0.70, p < 0.01 or < 0.05). In addition, the glycolysis/gluconeogenesis and pyruvate metabolism were negatively correlated with the relative abundance of A. muciniphila (r > 0.65, p < 0.01 or < 0.05). This alteration of the gut microbiota composition that resulted in observed changes to the glycolysis/gluconeogenesis and pyruvate metabolism may be involved in both the hepatotoxicity of SVP and the protective effect of G-CK administration against SVP-induced hepatotoxicity. Our study provides new evidence linking the gut microbiota with SVP-induced hepatotoxicity.

Effects of paternal high-fat diet and maternal rearing environment on the gut microbiota and behavior

Exposing a male rat to an obesogenic high-fat diet (HFD) influences attractiveness to potential female mates, the subsequent interaction of female mates with infant offspring, and the development of stress-related behavioral and neural responses in offspring. To examine the stomach and fecal microbiome’s potential roles, fecal samples from 44 offspring and stomach samples from offspring and their fathers were collected and bacterial community composition was studied by 16 small subunit ribosomal RNA (16S rRNA) gene sequencing. Paternal diet (control, high-fat), maternal housing conditions (standard or semi-naturalistic housing), and maternal care (quality of nursing and other maternal behaviors) affected the within-subjects alpha-diversity of the offspring stomach and fecal microbiomes. We provide evidence from beta-diversity analyses that paternal diet and maternal behavior induced community-wide shifts to the adult offspring gut microbiome. Additionally, we show that paternal HFD significantly altered the adult offspring Firmicutes to Bacteroidetes ratio, an indicator of obesogenic potential in the gut microbiome. Additional machine-learning analyses indicated that microbial species driving these differences converged on Bifidobacterium pseudolongum. These results suggest that differences in early-life care induced by paternal diet and maternal care significantly influence the microbiota composition of offspring through the microbiota-gut-brain axis, having implications for adult stress reactivity.

Addition of Prebiotics to the Ketogenic Diet Improves Metabolic Profile but Does Not Affect Seizures in a Rodent Model of Infantile Spasms Syndrome.

The ketogenic diet (KD) is an effective treatment for infantile spasms syndrome (IS). However, the KD has implications for somatic growth, development, and the gut microbiota. The impact of incorporating a prebiotic fiber (PRE, oligofructose-enriched inulin, 0.8 g/dL) into a KD diet on spasms, developmental milestones, fecal gut microbiota, metabolites, and hippocampal mitochondrial metabolism were examined. Following IS induction, animals were randomized to KD or KD + PRE diets. A third group without IS and suckled by dams was included as a normally developing reference group (R). PRE inclusion decreased ketones and increased circulating glucose levels but had no impact on spasms. In the liver, PRE increased triglyceride concentrations, decreased carnitine levels, and downregulated genes encoding enzymes responsible for ketogenesis. In the hippocampus, PRE increased glutathione levels but did not affect the maximal respiratory capacity of mitochondria. Analysis of the gut microbiota showed that KD + PRE increased microbial richness and the relative abundance of Bifidobacterium pseudolongum and Lactobacillus johnsonii. No differences in developmental milestones (i.e., surface righting, negative geotaxis, and open field behavior) were observed between KD and KD + PRE, except for ultrasonic vocalizations that were more frequent in KD + PRE. In summary, PRE did not impact spasms or developmental outcomes, but was effective in improving both metabolic parameters and gut microbiota diversity.

Arginine-mediated gut microbiome remodeling promotes host pulmonary immune defense against nontuberculous mycobacterial infection

ABSTRACT Nontuberculous mycobacterial pulmonary diseases (NTM-PDs) are emerging as global health threats with issues of antibiotic resistance. Accumulating evidence suggests that the gut–lung axis may provide novel candidates for host-directed therapeutics against various infectious diseases. However, little is known about the gut–lung axis in the context of host protective immunity to identify new therapeutics for NTM-PDs. This study was performed to identify gut microbes and metabolites capable of conferring pulmonary immunity to NTM-PDs. Using metabolomics analysis of sera from NTM-PD patients and mouse models, we showed that the levels of l-arginine were decreased in sera from NTM-PD patients and NTM-infected mice. Oral administration of l-arginine significantly enhanced pulmonary antimicrobial activities with the expansion of IFN-γ-producing effector T cells and a shift to microbicidal (M1) macrophages in the lungs of NTM-PD model mice. Mice that received fecal microbiota transplants from l-arginine-treated mice showed increased protective host defense in the lungs against NTM-PD, whereas l-arginine-induced pulmonary host defense was attenuated in mice treated with antibiotics. Using 16S rRNA sequencing, we further showed that l-arginine administration resulted in enrichment of the gut microbiota composition with Bifidobacterium species. Notably, oral treatment with either Bifidobacterium pseudolongum or inosine enhanced antimicrobial pulmonary immune defense against NTM infection, even with multidrug-resistant clinical NTM strains. Our findings indicate that l-arginine-induced gut microbiota remodeling with enrichment of B. pseudolongum boosts pulmonary immune defense against NTM infection by driving the protective gut–lung axis in vivo.

Effects of fructooligosaccharides (FOS) on the composition of cecal and fecal microbiota and the quantitative detection of FOS-metabolizing bacteria using species-specific primers.

Fructooligosaccharides (FOS) are a kind of prebiotic. Previous studies concerning the effect of FOS on intestinal microbiota have focused on Bifidobacterium and Lactobacillus. However, the presence of other FOS-utilizing bacteria makes it necessary to investigate the quantitative changes in these bacterial species in the intestine after FOS intake. In this study, the composition of cecal and fecal microbiota was analyzed using MiSeq sequencing, and the abundance of FOS-utilizing bacteria was detected using quantitative polymerase chain reaction after the oral administration of FOS. Species-specific primers for FOS-utilizing bacteria were designed with superior amplification efficiency for quantification. After FOS intervention, the relative abundance of Bifidobacterium pseudolongum in feces increased to 17.37% and the abundance reached 2.28 × 1010 CFU g-1 . The abundance of Bifidobacterium longum and Bifidobacterium breve did not change significantly. Whereas the abundance of Ligilactobacillus murinus decreased, that of Lactiplantibacillus plantarum, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus remained at approximately 104 CFU g-1 . Species-specific primers for FOS-utilizing bacteria were successfully developed, and we confirmed that FOS significantly increased the relative abundance and the abundance of B.pseudolongum in mice, while decreasing the proportion of Lactobacillus. The detection of these species using 16S ribosomal DNA sequencing and quantitative polymerase chain reaction showed the same results. Further investigations are needed to reveal the response of the intestinal microbiota to different FOS compositions. These techniques will contribute to future studies about the composition and dynamics of the intestinal microflora. © 2022 Society of Chemical Industry.

Indole-3-Acetic Acid Alters Intestinal Microbiota and Alleviates Ankylosing Spondylitis in Mice.

Ankylosing spondylitis (AS) is a systemic, chronic, and inflammatory autoimmune disease associated with the disorder of intestinal microbiota. Unfortunately, effective therapies for AS are lacking. Recent evidence has indicated that indole-3-acetic acid (IAA), an important microbial tryptophan metabolite, can modulate intestinal homeostasis and suppress inflammatory responses. However, reports have not examined the in vivo protective effects of IAA against AS. In this study, we investigated the protective effects and underlying mechanisms through which IAA acts against AS. We constructed a proteoglycan (PG)-induced AS mouse model and administered IAA (50 mg/kg body weight) by intraperitoneal injection daily for 4 weeks. The effects of IAA on AS mice were evaluated by examining disease severity, intestinal barrier function, aryl hydrocarbon receptor (AhR) pathway, T-helper 17 (Th17)/T regulatory (Treg) balance, and inflammatory cytokine levels. The intestinal microbiota compositions were profiled through whole-genome sequencing. We observed that IAA decreased the incidence and severity of AS in mice, inhibited the production of pro-inflammatory cytokines (tumor necrosis factor α [TNF-α], interleukin [IL]-6, IL-17A, and IL-23), promoted the production of the anti-inflammatory cytokine IL-10, and reduced the ratios of pro-/anti- inflammatory cytokines. IAA ameliorated pathological changes in the ileum and improved intestinal mucosal barrier function. IAA also activated the AhR pathway, upregulated the transcription factor forehead box protein P3 (FoxP3) and increased Treg cells, and downregulated the transcription factors retinoic acid receptor–related orphan receptor gamma t (RORγt) and signal transducer and activator of transcription 3 (STAT3) and decreased Th17 cells. Furthermore, IAA altered the composition of the intestinal microbiota composition by increasing Bacteroides and decreasing Proteobacteria and Firmicutes, in addition to increasing the abundances of Bifidobacterium pseudolongum and Mucispirillum schaedleri. In conclusion, IAA exerted several protective effects against PG-induced AS in mice, which was mediated by the restoration of balance among the intestinal microbial community, activating the AhR pathway, and inhibiting inflammation. IAA might represent a novel therapeutic approach for AS.

Detection and isolation of typical gut indigenous bacteria in ICR mice fed wheat bran and wheat straw fibre

Wheat bran (WB) and wheat straw fibre (WSF) are by-products of the wheat flour industry. To prove the existence of indigenous gut bacteria responsible for WB and WSF, the Institute of Cancer Research (ICR) mice were fed a diet containing no fibre (CS), 10% WB, or 5% WSF for 14 d. The caecal microbiome was analysed by 16S rDNA (V4 region) amplicon sequencing. Typical colonies were isolated and estimated by 16S rRNA gene BLASTn analysis. The predominant amplicon sequence variants in all diet groups belonged to Bifidobacterium pseudolongum- and Faecalibaculum rodentium-like bacteria. Lactobacillus johnsonii- and Limosilactobacillus reuteri-like bacteria were high in the WB group compared with those in the CS group. Lactobacillus johnsonii Wheat-1 and L. reuteri Wheat-12 strains could be isolated. Lactobacillus johnsonii Wheat-1 exhibited good fermentation activity in 10% (w/v) WB suspension. Superoxide anion radical scavenging capacity of the WB suspension was significantly increased by the fermentation.

Trifolium pratense ethanolic extract alters the gut microbiota composition and regulates serum lipid profile in the ovariectomized rats

BackgroundTrifolium pratense (red clover) ethanolic extract (TPEE) has been used as a popular over-the-counter remedy for the management of menopausal symptoms. Prolonged consumption of herbal extract has been shown to regulate the composition of gut microbiota. This study was designed to elucidate the influence of TPEE on the gut microbiota composition in the ovariectomized (OVX) rats.MethodsOVX rats were treated with TPEE at 125, 250, 500 mg/kg/day, or controls (pomegranate extract, 500 mg/kg/day; estradiol, 25 μg/kg/day) for 12 weeks. Gut microbiota analysis was conducted by extracting the microbial DNA from fecal samples and microbiome taxonomic profiling was carried out by using next-generation sequencing. The levels of serum biomarkers were analyzed using enzyme-linked immunosorbent assay (ELISA) kit. The prediction of functional biomarker of microbiota was performed using PICRUSt to investigate the potential pathways associated with gut health and serum lipid profile regulation. To study the correlation between gut microbiota composition and serum lipid levels, Spearman’s correlation coefficients were defined and analyzed. Additionally, gas chromatography–mass spectrometry analysis was conducted to uncover additional physiologically active ingredients.ResultsTPEE-treated OVX rats showed significant reduction in serum triglycerides (TG), total cholesterols (TCHOL), and LDL/VLDL levels but increase in HDL level. The alteration in the pathways involve in metabolism was the most common among the other KEGG categories. Particularly, TPEE also significantly reduced the relative abundance of sequences read associated with inflammatory bowel disease (IBD) and the peroxisome proliferator-activated receptor (PPAR) signalling pathway. TPEE intervention was seen to reduce the Firmicutes to Bacteroidetes (F/B) ratio in the OVX rats, denoting a reduction in microbial dysbiosis in the OVX rats. Correlation analysis at the phylum level revealed that Bacteriodetes and Proteobacteria were strongly correlated with serum TG, TCHOL and HDL levels. At the species level, Bifidobacterium pseudolongum group was seen to positively correlate with serum HDL level and negatively correlated with serum AST, ALT, LDL/VLDL, TCHOL, and TG levels.ConclusionsTPEE treatment showed therapeutic benefits by improving the intestinal microbiota composition which strongly correlated with the serum lipid and cholesterol levels in the OVX rats.

Fructooligosaccharides (FOS) significantly increased the relative abundance of intestinal B. pseudolongum in mice with different genotypes

Fructooligosaccharides (FOS) promote the proliferation of Bifidobacterium, especially Bifidobacterium pseudolongum in C57BL/6J mice. However, the response of intestinal microbes to FOS is influenced by host genotypes. Therefore, we compared the intestinal microbiota of four commonly used mice before and after FOS intervention, including C57BL/6J, BALB/c, Institute Cancer Research (ICR), and Kunming (KM) mice. The intestinal microbiota of the four genotypes exhibited similarities in composition but differences in relative abundance. Bifidobacterium was significantly increased to different degrees in the four genotypes of mice after FOS intervention, and Akkermansia and Bacteroides were also significantly increased in BALB/c and KM mice. Lactobacillus and Alistipes levels were unchanged or decreased. Within the genus Bifidobacterium, B. pseudolongum was the dominant species in the four genotypes of mice and proliferated significantly after FOS intervention, with dramatic proliferation in C57BL/6J mice (9.49%). Furthermore, eight strains of B. pseudolongum were screened from the feces of mice with four genotypes, and there was a great difference in the ability and manner of utilizing FOS among the strains. The strains from C57BL/6J mice exhibited the strongest utilization of 1-kestose (GF2), whereas other strains could utilize both GF2 and nistose (GF3) weakly. The gut microbial analysis of mice with different genotypes complemented our previous studies. The results provided the background strains of the different mouse genotypes and suggested a correlation between the utilization ability and the response of the strains to FOS. Further studies on the utilization ability of strains and competition in the intestine will contribute to the understanding of the mechanisms of the intestinal microbial response to diet.

A Distinct Gut Microbial Ecology is Associated with Enhanced Chemosensitivity Following Neutrophil Attenuation in Pancreatic Cancer

Background: Infiltration of innate immune cells, such as neutrophils, in the tumor microenvironment (TME) are associated with worse oncologic outcomes in pancreatic ductal adenocarcinoma (PDAC). Given recent evidence highlighting the complex intersection between chemosensitivity and the gut microbiome in solid tumors, we sought to investigate if the enhanced chemosensitivity observed with neutrophil attenuation in PDAC is associated with a distinct gut microbial ecology. Methods: C57BL/6 mice were orthotopically injected with syngeneic KrasG12D/+;Trp53fl/+;Pdx-Cre(KPC) PDAC cells, and randomized into four treatment groups after 10d tumor growth (n=8-10/arm): vehicle-control, anti-Ly6G antibody treatment alone with neutrophil-attenuating—but not depleting—dosing (25 μg/dose) q3d, gemcitabine(100 mg/kg) and paclitaxel(10 mg/kg) qwk(G/P), and G/P+anti-Ly6G. Fecal samples from mice (n=3/group) were collected 10d after treatment and submitted for shotgun metagenomic sequencing. Results: Bray-Curtis principal coordinate analysis of microbial β-diversity revealed significant differences in microbial composition with neutrophil attenuation and chemotherapy. Abundance of 37 species were differentially significant between G/P+anti-Ly6G and G/P-treated cohorts (p-adj<0.05). Seven Alistipes spp. (class Bacteroidea)—previously associated with chemoresistance in solid cancers—were significantly reduced in the gut microbiome of G/P+anti-Ly6G-treated mice. Bifidobacterium pseudolongum, previously associated with PDAC tumor progression, was the most significantly reduced bacterial species in the gut microbiome of G/P+anti-Ly6G-treated mice when compared with G/P alone-group. Conclusion: Our data unearth potential crosstalk between a distinct but narrow gut microbial ecology and anti-tumor adaptive immunity in response to neutrophil attenuation that is associated with improved chemosensitivity in PDAC. Therapeutic manipulation of this microbial program may mitigate chemoresistance and improve outcomes in PDAC patients.

Abstract PR03: Immuno-reactive cancer organoid models to examine microbiome metabolite effects on immune checkpoint blockade efficacy

Abstract Introduction: As the number of available immunotherapies for solid tumors increase, their prevalence in the clinic continues to rise as well. While the results are promising and immunotherapies have benefits over traditional chemotherapeutics, a sizable percentage of patients are non-responders to all types of immunotherapy as a treatment option. These differences in sensitivity can be either innate or acquired. Yet, there has been limited 3D in vitro models to assess tumor immune-reactivity. These systems are ideal for isolating specific molecular mechanisms that dictate cell behavior and interactions. Our goal was to create an organoid model containing cancer cells paired with cytotoxic T-cells to model immune checkpoint blockade (ICB) efficacy. This model could then be used to examine novel microbiome-ICB interactions shown by recent research to alter therapeutic response levels in patients. Methods: We created tumor organoids consisting of matched tumor and immune cells, embedded in extracellular matrix (ECM)-like hydrogels. Organoids were treated with therapeutic equivalent doses of anti-PD-1 and anti-CTLA-4 or single dose of anti-CD-47. The organoids were also exposed to physiologic concentrations of metabolites 3-indolepropionic acid derived from the bacterial species Clostridium sporogenes, hippurate derived from Clostridiales, Faecalbacterium prausnitzii, and Eubacterium, pyocyanin derived from Pseudomonas aeruginosa, butyrate derived from Faecalbacterium prausnitzii, and inosine derived from Bifidobacterium pseudolongum. Each of these bacterial species and the associated metabolite represent a likely effector of host immune function described in literature and therefore a potential effector of ICB response. Organoids were analyzed with cell viability assays, flow cytometry, RT-qPCR, and immunohistochemistry staining to determine the effects of the metabolites on ICB response. Results and Discussion: We showed that ICB therapy stimulated internally localizing T-cells, inducing T-cell-mediated tumor cell killing. ICB treated samples resulted significant loss of viability with corroborating readings from the other methods of characterization. RT-qPCR and flow cytometry demonstrated the cellular changes due to bacterial metabolite co-administration. These results include increased expression of CD-8 T-cell co-receptor, increased cytokine production, and increased effector T-cell viability. Conclusion: We have created an ex-vivo tumor immune-reactive organoid model for studying immunotherapy. We are working to elucidate the effects of microenvironment factors, such as microbiome metabolites, and observe their impacts on immunotherapy efficacy to better understand what conditions are conducive or detrimental to successful ICB treatment. This abstract is also being presented as Poster P002. Citation Format: Ethan Shelkey, Yong Lu, David Soto-Pantoja, Shay Soker. Immuno-reactive cancer organoid models to examine microbiome metabolite effects on immune checkpoint blockade efficacy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr PR03.

Supplemental Clostridium butyricum MIYAIRI 588 Affects Intestinal Bacterial Composition of Finishing Pigs.

Animal gastrointestinal tracts are populated by highly diverse and complex microbiotas. The gut microbiota influences the bioavailability of dietary components and is closely associated with physiological processes in the host. Clostridium butyricum reportedly improves growth performance and affects the gut microbiota and immune functions in post-weaning piglets. However, the effects of C. butyricum on finishing pigs remain unclear. Therefore, we herein investigated the effects of C. butyricum MIYAIRI 588 (CBM588) on the gut microbiota of finishing pigs. 16S rRNA gene sequencing was performed using fecal samples and ileal, cecal, and colonic contents collected after slaughtering. The α-diversity of the small intestinal microbiota was lower than that of the large intestinal microbiota, whereas β-diversity showed different patterns depending on sample collection sites. The administration of CBM588 did not significantly affect the α- or β-diversity of the microbiotas of fecal and intestinal content samples regardless of the collection site. However, a linear discriminant ana­lysis Effect Size revealed that the relative abundance of Lactobacillaceae at the family level, Bifidobacterium at the order level, and Lactobacillus ruminis and Bifidobacterium pseudolongum at the species level were higher in the fecal samples and cecal and colonic contents of the treatment group than in those of the control group. Therefore, the administration of CBM588 to finishing pigs affected the composition of the gut microbiota and increased the abundance of bacteria that are beneficial to the host. These results provide important insights into the effects of probiotic administration on relatively stable gut microbial ecosystems.

Adjunctive Probiotic Lactobacillus rhamnosus Probio-M9 Administration Enhances the Effect of Anti-PD-1 Antitumor Therapy via Restoring Antibiotic-Disrupted Gut Microbiota.

Emerging evidence supports that the efficacy of immune checkpoint blockade (ICB) therapy is associated with the host’s gut microbiota, as prior antibiotic intake often leads to poor outcome and low responsiveness toward ICB treatment. Therefore, we hypothesized that the efficacy of ICB therapy like anti-programmed cell death protein-1 (PD-1) treatment required an intact host gut microbiota, and it was established that probiotics could enhance the recovery of gut microbiota disruption by external stimuli. Thus, the present study aimed to evaluate the effect of the probiotics, Lactobacillus rhamnosus Probio-M9, on recovering antibiotic-disrupted gut microbiota and its impact on the outcome of ICB therapy in tumor-bearing mice. We first disrupted the mouse microbiota by antibiotics and then remediated the gut microbiota by probiotics or naturally. Tumor transplantation was then performed, followed by anti-PD-1-based antitumor therapy. Changes in the fecal metagenomes and the tumor suppression effect were monitored during different stages of the experiment. Our results showed that Probio-M9 synergized with ICB therapy, significantly improving tumor inhibition compared with groups not receiving the probiotic treatment (P < 0.05 at most time points). The synergistic effect was accompanied by effective restoration of antibiotic-disrupted fecal microbiome that was characterized by a drastically reduced Shannon diversity value and shifted composition of dominating taxa. Moreover, probiotic administration significantly increased the relative abundance of beneficial bacteria (e.g., Bifidobacterium pseudolongum, Parabacteroides distasonis, and some Bacteroides species; 0.0001 < P < 0.05). The gut microbiome changes were accompanied by mild reshaping of the functional metagenomes characterized by enrichment in sugar degradation and vitamin and amino acid synthesis pathways. Collectively, this study supported that probiotic administration could enhance the efficacy and responsiveness of anti-PD-1-based immunotherapy, and Probio-M9 could be a potential candidate of microbe-based synergistic tumor therapeutics. The preclinical data obtained here would support the design of future human clinical trials for further consolidating the current findings and for safety assessment of probiotic adjunctive treatment in ICB therapy.

Administration of Aspergillus oryzae suppresses DSS-induced colitis

Aspergillus oryzae, a filamentous fungus, has long been used for the production of traditional Japanese foods. Here, we analyzed how A. oryzae administration affects the intestinal environment in mice. The results of 16S rRNA gene sequencing of the gut microbiota indicated that after the administration of heat-killed A. oryzae spores, the relative abundance of an anti-inflammatory Bifidobacterium pseudolongum strain became 2.0-fold greater than that of the control. Next, we examined the effect of A. oryzae spore administration on the development of colitis induced by dextran sodium sulfate in mice; we found that colitis was alleviated by not only heat-killed A. oryzae spores, but also the cell wall extracted from the spores. Our findings suggest that A. oryzae holds considerable potential for commercial application in the production of both traditional Japanese fermented foods and new foods with prebiotic functions.

Bifidobacteria exhibited stronger ability to utilize fructooligosaccharides, compared with other bacteria in the mouse intestine.

Fructooligosaccharides (FOS) have been identified as important prebiotics. Previous studies have found that they can significantly promote the proliferation of Bifidobacterium pseudolongum in the mouse intestine. However, it is still unclear which other bacteria in the mouse intestine can utilize FOS, and the differences in the ability to utilize FOS. In this study, the bacteria capable of utilizing FOS were isolated from mice feces and their ability to utilize FOS was compared. Draft genome sequencing was also applied to explain the differences in FOS utilization at the gene levels. A total of 15 species were isolated from mouse feces and 13 species were able to utilize fructofuranosylnystose (GF2). Eleven species could utilize nistose (GF3), but not Enterococcus hirae and Lactobacillus reuteri. In contrast, 1-kestose (GF4) was hardly utilized. The enzyme activity determination and draft genome sequencing-based analyses revealed that all isolated species used the phosphotransferase system or permease system to transport FOS into the cells before hydrolysis by β-fructofuranosidase. Although β-fructofuranosidase exists in all strains, there are big differences in the corresponding coding genes between bifidobacteria and non-bifidobacteria. Compared with the other isolates, Bifidobacterium species exhibited higher enzyme activity and shorter generation time, leading to a stronger ability to utilize FOS. © 2021 Society of Chemical Industry.