Harnessing the endogenous Type I-C CRISPR-Cas system for genome editing in Bifidobacterium breve.

Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. The widespread presence of CRISPR-Cas systems in the B. breve genome makes endogenous CRISPR-based gene editing toolkits a promising tool. This study revealed that Type I-C CRISPR-Cas systems in B. breve can be divided into two groups based on the amino acid sequences encoded by cas gene clusters. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in five B. breve strains from both groups using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expands the toolkit for gene manipulation in B. breve and provides a new approach toward functional genome editing and analysis of B. breve strains.IMPORTANCEThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single-base substitution, gene insertion, and successive gene editing in B. breve. This study will facilitate discovery of functional genes and elucidation of molecular mechanisms of B. breve pertaining to health-associated benefits.

Oral Administration of Probiotic Bifidobacterium breve Ameliorates Tonic-Clonic Seizure in a Pentylenetetrazole-Induced Kindling Mouse Model via Integrin-Linked Kinase Signaling.

Epilepsy is a chronic neurological disorder characterized by recurrent seizures that affects over 70 million people worldwide. Although many antiepileptic drugs that block seizures are available, they have little effect on preventing and curing epilepsy, and their side effects sometimes lead to serious morbidity. Therefore, prophylactic agents with anticonvulsant properties and no adverse effects need to be identified. Recent studies on probiotic administration have reported a variety of beneficial effects on the central nervous system via the microbiota-gut-brain axis. In this study, we investigated the effects of the oral administration of Bifidobacterium breve strain A1 [MCC1274] (B. breve A1) on tonic-clonic seizure in a pentylenetetrazole (PTZ)-induced kindling mouse (KD mouse) model. We found that the oral administration of B. breve A1 every other day for 15 days significantly reduced the seizure score, which gradually increased with repetitive injections of PTZ in KD mice. The administration of B. breve A1, but not saline, to KD mice significantly increased the level of Akt Ser473 phosphorylation (p-Akt) in the hippocampus; this increase was maintained for a minimum of 24 h after PTZ administration. Treatment of B. breve A1-administered KD mice with the selective inhibitor of integrin-linked kinase (ILK) Cpd22 significantly increased the seizure score and blocked the antiepileptic effect of B. breve A1. Moreover, Cpd22 blocked the B. breve A1-induced increase in hippocampal p-Akt levels. These results suggest that the ILK-induced phosphorylation of Akt Ser473 in the hippocampus might be involved in the antiepileptic effect of B. breve A1.

Combination of Bifidobacterium breve and antibiotics against Clostridioides difficile: effect of the time interval of combination on antagonistic activity.

Co-administration of probiotics and antibiotics has been used to prevent or treat primary Clostridioides difficile (pCDI), and the closer the interval between the combination, the more effective it is, but the reason behind this is unknown. In this study, the cell-free culture supernatant (CFCS) of Bifidobacterium breve YH68 was used in combination with vancomycin (VAN) and metronidazole (MTR) to treat C. difficile cells. The growth and biofilm production of C. difficile under different co-administration time interval treatments were determined by optical density and crystalline violet staining, respectively. The toxin production of C. difficile was determined by enzyme immunoassay, and the relative expressions of C. difficile virulence genes tcdA and tcdB were determined by real-time qPCR method. Meanwhile, the types and contents of organic acids in YH68-CFCS were investigated by LC-MS/MS. The results showed that YH68-CFCS in combination with VAN or MTR significantly inhibited the growth, biofilm production, and toxin production of C. difficile in the effective time interval range (0-12 h) but did not affect the expression level of C. difficile virulence genes. In addition, the effective antibacterial component of YH68-CFCS is lactic acid (LA).

Hippocampal neurogenesis and Arc expression are enhanced in high-fat fed prepubertal female pigs by a diet including omega-3 fatty acids and Bifidobacterium breve CECT8242.

PURPOSE: Obesity during childhood has become a pandemic disease, mainly caused by a diet rich in sugars and fatty acids. Among other negative effects, these diets can induce cognitive impairment and reduce neuroplasticity. It is well known that omega-3 and probiotics have a beneficial impact on health and cognition, and we have hypothesized that a diet enriched with Bifidobacterium breve and omega-3 could potentiate neuroplasticity in prepubertal pigs on a high-fat diet. METHODS: Young female piglets were fed during 10 weeks with: standard diet (T1), high-fat (HF) diet (T2), HF diet including B. breve CECT8242 (T3) and HF diet including the probiotic and omega-3 fatty acids (T4). Using hippocampal sections, we analyzed by immunocytochemistry the levels of doublecortin (DCX) to study neurogenesis, and activity-regulated cytoskeleton-associated protein (Arc) as a synaptic plasticity related protein. RESULTS: No effect of T2 or T3 was observed, whereas T4 increased both DCX+ cells and Arc expression. Therefore, a diet enriched with supplements of B. breve and omega-3 increases neurogenesis and synaptic plasticity in prepubertal females on a HF diet from nine weeks of age to sexual maturity. Furthermore, the analysis of serum cholesterol and HDL indicate that neurogenesis was related to lipidic demand in piglets fed with control or HF diets, but the neurogenic effect induced by the T4 diet was exerted by mechanisms independent of this lipidic demand. CONCLUSION: Our results show that the T4 dietary treatment is effective in potentiating neural plasticity in the dorsal hippocampus of prepubertal females on a HF diet.

Bifidobacterium breve CBT BR3 is effective at relieving intestinal inflammation by augmenting goblet cell regeneration.

BACKGROUND AND AIM: Bifidobacterium breve was the first bacteria isolated in the feces of healthy infants and is a dominant species in the guts of breast-fed infants. Some strains of B. breve have been shown to be effective at relieving intestinal inflammation, but the modes of action have yet to be elucidated. In this study, we investigated the mechanisms of action of B. breve CBT BR3 isolated from South Korean infant feces in relieving colitis in vitro and in vivo. METHODS: Colitis was induced in mice with dextran sodium sulfate (DSS) and dinitrobenzene sulfonic acid (DNBS). Quantitative reverse-transcription polymerase chain reaction, in vitro FITC-dextran flux permeability assay, and aryl hydrocarbon receptor (AhR) luciferase assay are performed using Caco-2 cells and HT29-Lucia™ AhR cells. RESULTS: B. breve CBT BR3 was orally administered. B. breve CBT BR3 improved colitis symptoms in both DSS- and DNBS-induced colitis models. B. breve CBT BR3 increased the number of goblet cells per crypt. B. breve increased the mRNA expressions of Notch, Spdef, Muc5, and Il22. The mRNA expressions of Occludin, which encodes a membrane tight-junction protein, and Foxo3, which encodes a protein related to butyrate metabolism, were also increased in the DSS- and DNBS-induced colitis models. B. breve CBT BR3 protected inflammation-induced epithelial cell permeability and improved goblet cell function by inducing aryl hydrocarbon receptor in vitro. CONCLUSIONS: These results indicate that B. breve CBT BR3 is effective at relieving intestinal inflammation by augmenting goblet cell regeneration.

Structural characteristics and functional properties of a fucose containing prebiotic exopolysaccharide from Bifidobacterium breve NCIM 5671.

AIM: To evaluate the structure and functions of capsular exopolysaccharide (CPS) from Bifidobacterium breve NCIM 5671. METHODS AND RESULTS: A CPS produced by the probiotic bacteria B. breve NCIM 5671 was isolated and subjected to characterization through GC analysis, which indicated the presence of rhamnose, fucose, galactose, and glucose in a molar ratio of 3:1:5:3. The average molecular weight of the CPS was determined to be ∼8.5 × 105 Da. Further, NMR analysis revealed the probable CPS structure to be composed of major branched tetra- and penta-saccharide units alternately repeating and having both α- and ß-configuration sugar residues. CPS displayed an encouraging prebiotic score for some of the studied probiotic bacteria. Compared to standard inulin, CPS showed better resistance to digestibility against human GI tract in vitro. DPPH, total antioxidant, and ferric reducing assays carried out for CPS displayed decent antioxidant activity too. CONCLUSION: This study indicates that the CPS from B. breve NCIM 5671 has the potential to be utilized as a prebiotic food supplement. It is a high-molecular-weight (∼8.5 × 105 Da) capsular heteropolysaccharide containing rhamnose, fucose, galactose, and glucose.

Screening and characterization of a ß-xylosidase from Bifidobacterium breve K-110 and its application in the biotransformation of the total flavonoids of epimedium to icariin with α-l-rhamnosidase.

Among the flavonoids of epimedium, epimedin B, epimedin C, and icariin are considered to be representative components and their structures are quite similar. Besides sharing the same backbone, the main difference is the sugar groups attached at the positions of C-3 and C-7. Despite their structural similarities, their potencies differ significantly, and only icariin is currently included in the Chinese Pharmacopoeia as a quality marker (Q-marker) for epimedium flavonoids. Furthermore, icariin has the functions of anti-aging, anti-inflammation, antioxidation, anti-osteoporosis, and ameliorating fibrosis. We used bioinformatics to look for the GH43 family ß-xylosidase genes BbXyl from Bifidobacterium breve K-110, which has a length of 1347 bp and codes for 448 amino acids. This will allow us to convert epimedin B and epimedin C into icariin in a specific way. The expression level of recombinant BbXyl in TB medium containing 1 % inulin as carbon source, with an inducer concentration of 0.05 mmol/L and a temperature of 28 °C, was 86.4 U/mL. Previous studies found that the α-l-rhamnosidase BtRha could convert epoetin C to produce icariin, so we combined BbXyl and BtRha to catalyze the conversion of epimedium total flavonoids in vitro and in vivo to obtain the product icariin. Under optimal conditions, in vitro hydrolysis of 5 g/L of total flavonoids of epimedium eventually yielded a concentration of icariin of 678.1 µmol/L. To explore the conversion of total flavonoids of epimedium in vivo. Under the optimal conditions, the yield of icariin reached 97.27 µmol/L when the total flavonoid concentration of epimedium was 1 g/L. This study is the first to screen xylosidases for the targeted conversion of epimedin B to produce icariin, and the first to report that epimedin B and epimedin C in the raw epimedium flavonoids can convert efficiently to icariin by a collaborative of ß-xylosidase and α-l-rhamnosidase.

Bifidobacterium breve CCFM1025 attenuates major depression disorder via regulating gut microbiome and tryptophan metabolism: A randomized clinical trial.

OBJECTIVE: Psychobiotics, as a novel class of probiotics mainly acting on the gut-brain axis, have shown promising prospects in treating psychiatric disorders. Bifidobacterium breve CCFM1025 was validated to have an antidepressant-like effect in mice. This study aims to assess its psychotropic potential in managing major depression disorder (MDD) and unravel the underlying mechanisms. METHODS: Clinical Trial Registration: https://www.chictr.org.cn/index.aspx (identifier: NO. ChiCTR2100046321). Patients (n = 45) diagnosed with MDD were randomly assigned to the Placebo (n = 25) and CCFM1025 (n = 20) groups. The freeze-dried CCFM1025 in a dose of viable bacteria of 1010 CFU was given to MDD patients daily for four weeks, while the placebo group was given maltodextrin. Changes from baseline in psychometric and gastrointestinal symptoms were evaluated using Hamilton Depression Rating scale-24 Items (HDRS-24), Montgomery-Asberg Depression Rating Scale (MADRS), Brief Psychiatric Rating Scale (BPRS), and Gastrointestinal Symptom Rating Scale (GSRS). Serum measures were also determined, i.e., cortisol, TNF-α, and IL-ß. Serotonin turnover in the circulation, gut microbiome composition, and tryptophan metabolites were further investigated for clarifying the probiotics' mechanisms of action. RESULTS: CCFM1025 showed a better antidepressant-like effect than placebo, based on the HDRS-24 (placebo: M = 6.44, SD = 5.44; CCFM1025: M = 10.40, SD = 6.85; t(43) = 2.163, P = 0.036, d = 0.640) and MADRS (placebo: M = 4.92, SD = 7.15; CCFM1025: M = 9.60, SD = 7.37; t(43) = 2.152, P = 0.037, d = 0.645) evaluation. The factor analysis of BPRS and GSRS suggested that patients' emotional and gastrointestinal problems may be affected by the serotonergic system. Specifically, CCFM1025 could significantly and to a larger extend reduce the serum serotonin turnover compared with the placebo (placebo: M = -0.01, SD = 0.41; CCFM1025: M = 0.27, SD = 0.40; t(43) = 2.267, P = 0.029, d = 0.681). It may be due to changes in gut microbiome and gut tryptophan metabolism under the probiotic treatment, such as changes in alpha diversity, tryptophan, and indoles derivatives. CONCLUSION: B. breve CCFM1025 is a promising candidate psychobiotic strain that attenuates depression and associated gastrointestinal disorders. The mechanisms may be relevant to the changes in the gut microbiome and tryptophan metabolism. These findings support the future clinical applications of psychobiotics in the treatment of psychiatric disorders.

Infant Formulas With Postbiotics: An Updated Systematic Review.

OBJECTIVES: Infant formulas (IF) with postbiotics, defined as inanimate microorganisms and/or their components that confer a health benefit on the host, are available. We systematically updated evidence on the safety and health effects of administering iF with postbiotics (with or without other modifications) compared with standard IF. METHODS: The Cochrane Library, MEDLINE, and EMBASE databases were searched to December 2021. RESULTS: Eleven randomized controlled trials were included. Using the Cochrane Risk of Bias Tool 2, for the primary outcomes, 5 trials had an overall high risk of bias, and 6 trials had some concerns of bias. Most data were available on IF fermented with Bifidobacterium breve C50 and Streptococcus thermophilus (BB/ST). These formulas, compared with the standard IF, were safe and well tolerated. Postbiotic formulas with additional modifications (ie, formula fermented with BB/ST & prebiotics, partly fermented formula with BB/ST and prebiotics with or without modified milk fat, partly fermented antiregurgitation formula with BB/ST and prebiotics) were generally safe and well tolerated but did not offer clear benefits replicated in other studies. Only limited data were available on formula fermented with Lactobacillus paracasei CBA L74. CONCLUSIONS: IF with postbiotics evaluated so far are safe and well tolerated by infants who cannot be breastfed. No firm conclusion can, however, be reached regarding the clinical effects and benefit of one formula over another. It seems reasonable to discuss with healthcare providers current evidence regarding specific modifications in infant formulas and let them decide whether the expected benefits meet expectations and are worth the cost.

Antagonistic activity of selenium-enriched Bifidobacterium breve against Clostridioides difficile.

Probiotics have the potential to be used in the prevention of Clostridioides difficile infection (CDI). In this study, selenium (Se)-enriched Bifidobacterium breve YH68-Se was obtained under optimal culture conditions with single-factor and response surface optimization. The overall environmental resistance of YH68-Se was superior to that of the parental strain YH68, mainly reflected in the substantial improvement of antioxidant activity and gastrointestinal tolerance. YH68-Se dramatically inhibited C. difficile growth, spore, biofilm, toxin production, and virulence gene expression, rapidly disrupted C. difficile cell membrane permeability and integrity, and altered the membrane proton motive force (PMF), induced a large outflow of intracellular substances and eventually caused bacterial death. The main factor inducing this process originated from the lactic acid (LD) in YH68-Se. In addition, the LD production of YH68 increased with increasing selenite concentration and was accompanied by enhanced activities of thioredoxin reductase (TrxR), glutathione peroxidase (GSH-Px), and increased concentration of autoinducer-2 (AI-2), which may be the crucial factors contributing to the outstanding probiotic properties of YH68-Se and their potent antagonism of C. difficile. KEY POINTS: • Compared with the parental strain B. breve YH68, the environmental resistance of YH68-Se was improved. • YH68-Se was able to produce more lactic acid, which suppressed the important physiological activities of C. difficile and rapidly disrupted their cell membrane structures. • Sodium selenite in the suitable concentration range gradually increases the yield of lactic acid and phenylacetic acid, increased the concentration of autoinducer-2, and enhanced the activities of antioxidant enzymes TrxR and GSH-Px in YH68.

Bifidobacterium breve during infancy attenuates mobility in low birthweight rats.

BACKGROUND: Children with low birthweight (LBW) have a higher risk for developing attention-deficit/hyperactivity disorder, for which no prophylactic measure exists. The gut microbiota in infants with LBW is different from that in infants with normal birthweight and is associated with attention-deficit/hyperactivity disorder. Oral supplementation with Bifidobacterium has several health benefits, such as suppressing inflammation. METHODS: We examined the effect of gavage supplementation with Bifidobacterium breve M-16V from postnatal days 1-21 in a rat model of intrauterine hypoperfusion. RESULTS: The open-field test at 5 weeks of age (equivalent to human pubertal age) showed that rats in the LBW-vehicle group were marginally hyperactive compared with rats in the sham group, while rats in the LBW-B.breve group were significantly hypoactive compared with rats in the LBW-vehicle group. The gut microbiota in the LBW-vehicle group exhibited a profile significantly different from that in the sham group, whereas the gut microbiota in the LBW-B.breve group did not exhibit a significant difference from that in the sham group. Anatomical/histological evaluation at 6 weeks of age demonstrated that the brain weight and the cerebral areas on coronal sections were reduced in the LBW groups compared with the sham group. Probiotic supplementation did not ameliorate these morphological brain anomalies in LBW animals. The percentage of Iba-1+ cells in the brain was not different among the LBW-B.breve, LBW-vehicle, and sham groups. CONCLUSION: Bifidobacterium breve supplementation during early life is suggested to have the potential to help children with LBW attenuate hypermobility in adolescence.

First case of necrotizing fasciitis and bacteremia caused by Bifidobacteriumbreve.

We report the first case of necrotizing fasciitis and bacteremia caused by Bifidobacterium breve. Some Bifidobacterium breve strains are known as probiotic bacterium. However, it causes bacteremia in infants and immunocompromised patients. Our patient developed necrotizing fasciitis which was thought to have been infected from chronic diabetic foot ulcers. Bifidobacterium breve was isolated from the patient's blood and soft tissue sample. The patient underwent amputation and intravenous antibiotics administration.

Probiotic Bifidobacterium breve prevents DOCA-salt hypertension.

Many probiotics that affect gut microbial ecology have been shown to produce beneficial effects on renin-angiotensin-dependent rodent models and human hypertension. We hypothesized that Bifidobacterium breve CECT7263 (BFM) would attenuate hypertension in deoxycorticosterone acetate (DOCA)-salt rats, a renin-independent model of hypertension. Rats were randomly divided into five groups: control, DOCA-salt, treated DOCA-salt-BFM, treated DOCA-salt-butyrate, and treated DOCA-salt-acetate, for 5 weeks. BFM prevented the increase in systolic blood pressure, cardiac weight, and renal damage induced by DOCA-salt. BFM increased acetate-producing bacterial population and gut acetate levels, improved colonic integrity, normalized endotoxemia, plasma trimethylamine (TMA) levels, and restored the Th17 and Treg content in mesenteric lymph nodes and aorta. Furthermore, BFM improved nitric oxide-dependent vasorelaxation induced by acetylcholine in aortic rings and reduced NADPH oxidase activity in DOCA-salt animals. These protective effects were mimicked by acetate, but not by butyrate supplementation. These data demonstrate that BFM induces changes in gut microbiota linked with attenuation of endothelial dysfunction and increase in blood pressure in this low-renin form of hypertension. These beneficial effects seem to be mediated by increased acetate and reduced TMA production by gut microbiota, thus, improving gut integrity and restoring Th17/Tregs polarization and endotoxemia.

Heat-sterilized Bifidobacterium breve prevents depression-like behavior and interleukin-1ß expression in mice exposed to chronic social defeat stress.

Major depressive disorder (MDD) is a common and serious psychiatric disease that involves brain inflammation. Bifidobacterium breve is commonly used as a probiotic and was shown to improve colitis and allergic diseases by suppressing the inflammatory response. Heat-sterilized B. breve has beneficial effects on inflammation. We hypothesize, therefore, that this probiotic might reduce depression symptoms. We tested this is a mouse model of social defeat stress. C57BL/6J mice exposed to chronic social defeat stress (CSDS) for five consecutive days developed a mild depression-like behavior characterized by a social interaction impairment. CSDS also altered the gut microbiota composition, such as increased abundance of Bacilli, Bacteroidia, Mollicutes, and Verrucomicrobiae classes and decreased Erysipelotrichi class. The prophylactic effect of heat-sterilized B. breve as a functional food ingredient was evaluated on the depression-like behavior in mice. The supplementation started two weeks before and lasted two weeks after the last exposure to CSDS. Two weeks after CSDS, the mice showed deficits in social interaction and increased levels of inflammatory cytokines, including interleukin-1ß (IL-1ß) in the prefrontal cortex (PFC) and hippocampus (HIP). Heat-sterilized B. breve supplementation significantly prevented social interaction impairment, suppressed IL-1ß increase in the PFC and HIP, and modulated the alteration of the gut microbiota composition induced by CSDS. These findings suggest that heat-sterilized B. breve prevents depression-like behavior and IL-1ß expression induced by CSDS through modulation of the gut microbiota composition in mice. Therefore, heat-sterilized B. breve used as an ingredient of functional food might prevent MDD.

In vitro validation studies for adhesion factor and adhesion efficiency of probiotic Bacillus licheniformis MCC 2514 and Bifidobacterium breve NCIM 5671 on HT-29 cell lines.

Probiotic bacterial adhesion to the epithelial cell is a composite process and in vivo adhesion studies can be strengthened with the improved in vitro models for preliminary screening of potentially adherent strains. With this rationale, the study aimed is the first report to demonstrate the colonizing efficiency of probiotic Bacillus licheniformis MCC 2514 in comparison to Bifidobacterium breve NCIM 5671on HT-29 cell line. B. licheniformis (54.28 ± 0.99%) and Bif. breve (70.23 ± 0.85%) adhered in a higher percentage on fibronectin and mucin, respectively. However, the adhesion was higher for B. licheniformis when compared to Bif. breve. In adhesion score, B. licheniformis obtained about 138.85 ± 12.32, whereas Bif. breve got the score of 43.05 ± 9.12. The same trend continued in the adhesion percentage study, where B. licheniformis adhered 75.5 ± 5.2%, higher than Bif. breve which adhered 32.66 ± 3.2%. In invasion assay, both the bacteria significantly decreased the colonization of the pathogen Kocuria rhizophila ATCC 9341 about 97.32 ± 0.81% in the competitive assay, 97.87 ± 0.73% in exclusion assay and 82.19 ± 2.51% in displacement assay. The cytotoxicity effects of the test bacterial strains against HT-29 cell line through MTT assay determined no viability loss in the treated cells. Therefore, the data obtained from the in vitro studies showed that both B. licheniformis and Bif. breve had shown significantly good invasion on pathogen and adhesion capacity on HT-29 cell line.

Bifidobacterium breve BBG-001 and intestinal barrier function in preterm babies: Exploratory Studies from the PiPS Trial.

BACKGROUND: Uncertainty remains about the role of probiotics to prevent necrotising enterocolitis (NEC) some of which arises from the variety of probiotic interventions used in different trials, many with no prior evidence of potential efficacy. Mechanistic studies of intestinal barrier function embedded in a large probiotic trial could provide evidence about which properties of probiotics might be important for NEC prevention thus facilitating identification of strains with therapeutic potential. METHODS: Intestinal permeability, stool microbiota, SCFAs and mucosal inflammation were assessed from the second postnatal week in babies enrolled to a randomised controlled trial of B. breve BBG-001 (the PiPS trial). Results were compared by allocation and by stool colonisation with the probiotic. RESULTS: Ninety-four preterm babies were recruited across six nested studies. B. breve BBG-001 content was higher by allocation and colonisation; Enterobacteriaceae and acetic acid levels were higher by colonisation. No measure of intestinal barrier function showed differences. The PiPS trial found no evidence of efficacy to reduce NEC. CONCLUSIONS: That the negative results of the PiPS trial were associated with failure of this probiotic to modify intestinal barrier function supports the possibility that the tests described here have the potential to identify strains to progress to large clinical trials. IMPACT: Uncertainty about the therapeutic role of probiotics to prevent necrotising enterocolitis is in part due to the wide range of bacterial strains with no previous evidence of efficacy used in clinical trials. We hypothesised that mechanistic studies embedded in a probiotic trial would provide evidence about which properties of probiotics might be important for NEC prevention. The finding that the probiotic strain tested, Bifidobacterium breve BBG-001, showed neither effects on intestinal barrier function nor clinical efficacy supports the possibility that these tests have the potential to identify strains to progress to large clinical trials.

Alleviation effects of Bifidobacterium breve on DSS-induced colitis depends on intestinal tract barrier maintenance and gut microbiota modulation.

PURPOSE: The study aimed to investigate the discrepancy and potential mechanisms of different CLA-producing B. breve on dextran sulphate sodium (DSS)-induced colitis. METHODS: Colitis was induced in C57BL/6 J mice using DSS. Disease activity index (DAI), histopathological changes, epithelial barrier integrity and epithelial apoptosis were determined. Gut microbiota were gauged to evaluate the systemic effects of CLA-producing B. breve. RESULTS: Oral administration of different B. breve showed different effects, in which B. breve M1 and B. breve M2 alleviated the inflammation induced by DSS as well as significantly increased the concentration of mucin2 (MUC2) and goblet cells, but neither B. breve M3 nor B. breve M4 had those protective effects. Meanwhile, B. breve M1 and B. breve M2 treatments significantly up-regulated the tight junction (TJ) proteins and ameliorated the epithelial apoptosis lead by DSS challenge. Moreover, inflammatory cytokines (TNF-α, IL-6) were modulated by B. breve M1 and B. breve M2, neither B. breve M3 nor B. breve M4. Furthermore, B. breve M1 and B. breve M2 reduced the abundance of Bacteroides and increased the abundance of Odoribacter, then rebalanced the damaged gut microbiota. Colonic CLA concentrations in mice fed with B. breve M1, B. breve M2, B. breve M3 and B. breve M4 decreased successively, which showed significant positive correlation with the effectiveness of relieving colitis. CONCLUSIONS: Bifidobacterium breve M1 and B. breve M2 alleviated DSS-induced colitis by producing CLA, inhibiting the inflammatory cytokines, maintaining of the intestinal epithelial barrier and regulating the gut microbiota.

Evaluation of the therapeutic effect and dose-effect of Bifidobacterium breve on the primary Clostridioides difficile infected mice.

Probiotics are widely used as an adjuvant agent for the prevention of primary Clostridioides difficile infection (pCDI) and are less commonly used in the treatment of pCDI. Here, the different doses of Bifidobacterium breve YH68 were used to treat the pCDI mouse model and the actual therapeutic effect was evaluated. Fecal samples of pCDI mice were collected from the pre-infection, post-infection, and post-treatment stages. Simultaneous 16S rRNA amplicon sequencing and non-targeted metabolite assays were performed on these mouse feces, followed by correlation analysis. We found that high doses of B. breve YH68 exerted prominent therapeutic effects and no side effects in pCDI mice, resulted in a high survival rate, accompanied by a dose-effect relationship. YH68 enhanced the levels of caffeine, butyric acid, secondary bile acids in the feces of pCDI mice and significantly upregulated the abundance of genera associated with these metabolites, including Akkermansia, Coprococcus, Oscillospira, and Ruminococcus. Meanwhile, YH68 downregulated the levels of cortisol and phytosphingosine, and these metabolites were positively correlated with the abundance of the Klebsiella and Pseudomonas genera. These findings indicated that YH68 has outstanding therapeutic effects on the pCDI mouse model and is expected to be a potential new option for clinical pCDI therapy.Key points• Bifidobacterium breve YH68 has therapeutic effects on the pCDI mice and was accompanied by a dose-effect relationship.• Bifidobacterium breve YH68 enhanced the levels of caffeine, butyric acid, secondary bile acids in the feces of pCDI mice after treatment, as well as upregulated the abundance of beneficial microbes.• Bifidobacterium breve YH68 decreased the levels of cortisol and phytosphingosine and downregulated the abundance of harmful microbes.

NutriBrain: protocol for a randomised, double-blind, controlled trial to evaluate the effects of a nutritional product on brain integrity in preterm infants.

BACKGROUND: The gut microbiota and the brain are connected through different mechanisms. Bacterial colonisation of the gut plays a substantial role in normal brain development, providing opportunities for nutritional neuroprotective interventions that target the gut microbiome. Preterm infants are at risk for brain injury, especially white matter injury, mediated by inflammation and infection. Probiotics, prebiotics and L-glutamine are nutritional components that have individually already demonstrated beneficial effects in preterm infants, mostly by reducing infections or modulating the inflammatory response. The NutriBrain study aims to evaluate the benefits of a combination of probiotics, prebiotics and L-glutamine on white matter microstructure integrity (i.e., development of white matter tracts) at term equivalent age in very and extremely preterm born infants. METHODS: This study is a double-blind, randomised, controlled, parallel-group, single-center study. Eighty-eight infants born between 24 + 0 and < 30 + 0 weeks gestational age and less than 72 h old will be randomised after parental informed consent to receive either active study product or placebo. Active study product consists of a combination of Bifidobacterium breve M-16V, short-chain galacto-oligosaccharides, long-chain fructo-oligosaccharides and L-glutamine and will be given enterally in addition to regular infant feeding from 48 to 72 h after birth until 36 weeks postmenstrual age. The primary study outcome of white matter microstructure integrity will be measured as fractional anisotropy, assessed using magnetic resonance diffusion tensor imaging at term equivalent age and analysed using Tract-Based Spatial Statistics. Secondary outcomes are white matter injury, brain tissue volumes and cortical morphology, serious neonatal infections, serum inflammatory markers and neurodevelopmental outcome. DISCUSSION: This study will be the first to evaluate the effect of a combination of probiotics, prebiotics and L-glutamine on brain development in preterm infants. It may give new insights in the development and function of the gut microbiota and immune system in relation to brain development and provide a new, safe treatment possibility to improve brain development in the care for preterm infants. TRIAL REGISTRATION: ISRCTN, ISRCTN96620855 . Date assigned: 10/10/2017.

Gut Microbiota Profile in Children with IgE-Mediated Cow's Milk Allergy and Cow's Milk Sensitization and Probiotic Intestinal Persistence Evaluation.

Food allergy (FA) and, in particular, IgE-mediated cow's milk allergy is associated with compositional and functional changes of gut microbiota. In this study, we compared the gut microbiota of cow's milk allergic (CMA) infants with that of cow's milk sensitized (CMS) infants and Healthy controls. The effect of the intake of a mixture of Bifidobacterium longum subsp. longum BB536, Bifidobacterium breve M-16V and Bifidobacterium longum subsp. infantis M-63 on gut microbiota modulation of CMA infants and probiotic persistence was also investigated. Gut microbiota of CMA infants resulted to be characterized by a dysbiotic status with a prevalence of some bacteria as Haemophilus, Klebsiella, Prevotella, Actinobacillus and Streptococcus. Among the three strains administered, B.longum subsp. infantis colonized the gastrointestinal tract and persisted in the gut microbiota of infants with CMA for 60 days. This colonization was associated with perturbations of the gut microbiota, specifically with the increase of Akkermansia and Ruminococcus. Multi-strain probiotic formulations can be studied for their persistence in the intestine by monitoring specific bacterial probes persistence and exploiting microbiota profiling modulation before the evaluation of their therapeutic effects.