RESUMO
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.
Assuntos
Bifidobacterium breve , Sistemas CRISPR-Cas , Humanos , Edição de Genes/métodos , Bifidobacterium breve/genética , Ácido Linoleico , Transferases/genética , UracilaRESUMO
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).
Assuntos
Toxinas Bacterianas , Bifidobacterium breve , Clostridioides difficile , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Clostridioides difficile/genética , Enterotoxinas/genética , Enterotoxinas/metabolismo , Clostridioides , Cromatografia Líquida , Proteínas de Bactérias/metabolismo , Espectrometria de Massas em Tandem , Vancomicina/farmacologia , Metronidazol/farmacologia , Metronidazol/metabolismoRESUMO
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.
Assuntos
Bifidobacterium breve , Colite , Humanos , Animais , Camundongos , Células Caliciformes/metabolismo , Bifidobacterium breve/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Células CACO-2 , Colite/induzido quimicamente , Colite/terapia , Colite/metabolismo , Inflamação/terapia , Inflamação/metabolismo , RNA Mensageiro/genética , Regeneração , Sulfato de Dextrana , Mucosa Intestinal , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
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 â¼8.5 × 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 (â¼8.5 × 105 Da) capsular heteropolysaccharide containing rhamnose, fucose, galactose, and glucose.
Assuntos
Bifidobacterium breve , Prebióticos , Humanos , Fucose , Galactose , Ramnose , GlucoseRESUMO
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.
Assuntos
Bifidobacterium breve , Clostridioides difficile , Selênio , Antioxidantes , Bifidobacterium breve/metabolismo , Clostridioides , Glutationa Peroxidase/metabolismo , Ácido Láctico , Selênio/metabolismoRESUMO
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.
Assuntos
Bifidobacterium breve , Probióticos , Animais , Bifidobacterium , Peso ao Nascer , Criança , Humanos , Lactente , Recém-Nascido de Baixo Peso , Recém-Nascido , Probióticos/uso terapêutico , RatosRESUMO
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.
Assuntos
Bacteriemia , Bifidobacterium breve , Fasciite Necrosante , Probióticos , Antibacterianos/uso terapêutico , Bacteriemia/diagnóstico , Bacteriemia/tratamento farmacológico , Fasciite Necrosante/diagnóstico , Fasciite Necrosante/tratamento farmacológico , Humanos , LactenteRESUMO
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.
Assuntos
Bifidobacterium breve , Colite , Microbioma Gastrointestinal , Animais , Colite/induzido quimicamente , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Mucosa Intestinal , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Bifidobacterium breve , Clostridioides difficile , Probióticos , Animais , Bifidobacterium/genética , Clostridioides , Fezes , Camundongos , RNA Ribossômico 16SRESUMO
There has been a marked increase in life-threatening food allergy (FA). One hypothesis is that changes in bacterial communities may be key to FA. To better understand how gut microbiota regulates FA in humans, we established a mouse model with FA induced by ovalbumin. We found that the mice with FA had abnormal bacterial composition, accompanied by increased immunoglobulin G, immunoglobulin E, and interleukin-4/interferon-γ, and there existed a certain coherence between them. Interestingly, Bifidobacterium breve M-16V may alter the gut microbiota to alleviate the allergy symptoms by IL-33/ST2 signaling. Our results indicate that gut microbiota is essential for regulating FA to dietary antigens and demonstrate that intervention in bacterial community regulation may be therapeutically related to FA.
Assuntos
Hipersensibilidade Alimentar/tratamento farmacológico , Interleucina-33/metabolismo , Probióticos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Bifidobacterium breve/efeitos dos fármacos , Bifidobacterium breve/imunologia , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/imunologia , Camundongos Endogâmicos BALB C , Ovalbumina/farmacologia , Transdução de Sinais/imunologiaRESUMO
Yogurt is a popular product worldwide partly because of the health-promoting effects of the probiotics that it contains. Probiotics with high survivability constitute a promising direction for fortified yogurt products. This study aimed to prepare Bifidobacterium breve-loaded yogurt with the bacteria surviving transit to the lower part of small intestine or colon. Bifidobacterium breve beads were prepared through an ion-crosslinking method using low methoxyl pectin as the encapsulating material. Features such as encapsulation efficiency and stability during storage and passage through the simulated gastrointestinal tract were studied in vitro. A commercial starter was used for yogurt fermentation, and B. breve with or without encapsulation was added as a probiotic supplement with the starter or 3 to 4 h after fermentation. The effects of B. breve beads on yogurt characteristics were evaluated after different fermentation processes: BC, milk fermented with marketed yogurt starter; UBFF, unencapsulated B. breve added to fresh milk and then fermented; EBFF, encapsulated B. breve added to fresh milk and then fermented; UBAF, unencapsulated B. breve added after fermentation with the starter; and EBAF, encapsulated B. breve beads added 3 to 4 h after fermentation with the starter. Evaluation was based on texture, electronic nose, and electronic tongue analyses. The particle size analysis of B. breve beads showed that they were uniform, mostly spherical, 1 to 1.5 mm in diameter with encapsulating efficiency higher than 99%. Following treatment with the simulated gastrointestinal tract conditions, the number of B. breve decreased by 1.76 and 4.82 log cfu/g for B. breve beads and unencapsulated B. breve, respectively. The EBAF group showed the lowest viscosity (2,235.67 cP) at d 0, and the lower postfermentation degree was reflected by the slow increase in yogurt viscosity. All groups kept a relatively stable pH during storage. The cohesiveness values of the EBAF and UBAF groups were significantly higher than those of the other groups. The trends in texture changes within the BC, UBFF, and EBFF groups were similar, and the UBAF and EBAF groups showed similar trends. In conclusion, B. breve beads showed good stability in vitro and improved yogurt characteristics by increasing the survival rate of the encapsulated cells. Good compatibility of low methoxyl pectin beads with yogurt was also observed.
Assuntos
Bifidobacterium breve/metabolismo , Probióticos/metabolismo , Iogurte/microbiologia , Animais , Colo/microbiologia , Fermentação , Qualidade dos Alimentos , Armazenamento de Alimentos , Concentração de Íons de Hidrogênio , Intestino Grosso/microbiologia , Leite/microbiologia , Pectinas , ViscosidadeRESUMO
Bifidobacteria are mutualistic intestinal bacteria, and their presence in the human gut has been associated with health-promoting activities. The presence of antibiotic resistance genes in this genus is controversial, since, although bifidobacteria are nonpathogenic microorganisms, they could serve as reservoirs of resistance determinants for intestinal pathogens. However, until now, few antibiotic resistance determinants have been functionally characterized in this genus. In this work, we show that Bifidobacterium breve CECT7263 displays atypical resistance to erythromycin and clindamycin. In order to delimit the genomic region responsible for the observed resistance phenotype, a library of genomic DNA was constructed and a fragment of 5.8 kb containing a gene homologous to rRNA methylase genes was able to confer erythromycin resistance in Escherichia coli This genomic region seems to be very uncommon, and homologs of the gene have been detected in only one strain of Bifidobacterium longum and two other strains of B. breve In this context, analysis of shotgun metagenomics data sets revealed that the gene is also uncommon in the microbiomes of adults and infants. The structural gene and its upstream region were cloned into a B. breve-sensitive strain, which became resistant after acquiring the genetic material. In vitro conjugation experiments did not allow us to detect gene transfer to other recipients. Nevertheless, prediction of genes potentially acquired through horizontal gene transfer events revealed that the gene is located in a putative genomic island.IMPORTANCEBifidobacterium breve is a very common human intestinal bacterium. Often described as a pioneer microorganism in the establishment of early-life intestinal microbiota, its presence has been associated with several beneficial effects for the host, including immune stimulation and protection against infections. Therefore, some strains of this species are considered probiotics. In relation to this, because probiotic bacteria are used for human and animal consumption, one of the safety concerns over these bacteria is the presence of antibiotic resistance genes, since the human gut is a densely populated habitat that could favor the transfer of genetic material to potential pathogens. In this study, we analyzed the genetic basis responsible for the erythromycin and clindamycin resistance phenotype of B. breve CECT7263. We were able to identify and characterize a novel gene homologous to rRNA methylase genes which confers erythromycin and clindamycin resistance. This gene seems to be very uncommon in other bifidobacteria and in the gut microbiomes of both adults and infants. Even though conjugation experiments showed the absence of transferability under in vitro conditions, it has been predicted to be located in a putative genomic island recently acquired by specific bifidobacterial strains.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Bifidobacterium breve/efeitos dos fármacos , Bifidobacterium breve/enzimologia , Clindamicina/farmacologia , Eritromicina/farmacologia , Metiltransferases/metabolismo , Proteínas de Bactérias/genética , Bifidobacterium breve/genética , Farmacorresistência Bacteriana , Microbioma Gastrointestinal , Transferência Genética Horizontal , Humanos , Intestinos/microbiologia , Metiltransferases/genética , FilogeniaRESUMO
BACKGROUND: Fermented milk products containing Bifidobacterium breve strain Yakult (BFM) may improve clinical status in ulcerative colitis (UC) patients. AIMS: To assess efficacy of BFM in maintaining remission in Japanese patients with quiescent UC. METHODS: This double-blind study (B-FLORA) enrolled 195 patients with quiescent UC, randomized to receive one pack of BFM fermented milk per day [Bifidobacterium breve strain Yakult (10 billion bacteria) and Lactobacillus acidophilus (1 billion bacteria)] (n = 98) or matching placebo (n = 97) for 48 weeks. The primary efficacy endpoint was relapse-free survival (relapse: rectal bleeding score ≥ 2 on Sutherland disease activity index scale for 3 consecutive days and/or initiation of remission induction therapy for worsening of UC). RESULTS: An interim analysis was conducted after inclusion and follow-up of one-third of patients for the first phase of the study (n = 195). Relapse-free survival was not significantly different between the BFM and placebo groups (P = 0.643; hazard ratio 1.16; 95% CI 0.63-2.14, log-rank test), nor was the incidence of relapse. Therefore, the study was discontinued for lack of efficacy. An exploratory analysis of fecal samples from a subgroup of patients revealed no effects of either study beverage on intestinal microbiota, but there was a significant decrease in Bifidobacterium species before relapse, regardless of treatment group. Three mild adverse events occurred for which a causal relationship with the study beverage could not be ruled out (placebo: abdominal bloating and stress in one patient; BFM: body odor in one patient). CONCLUSIONS: BFM had no effect on time to relapse in UC patients compared with placebo. STUDY REGISTRATION: UMIN000007593.
Assuntos
Bifidobacterium breve/fisiologia , Colite Ulcerativa/terapia , Produtos Fermentados do Leite/microbiologia , Microbioma Gastrointestinal , Intestinos/microbiologia , Lactobacillus acidophilus/fisiologia , Adulto , Idoso , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/microbiologia , Produtos Fermentados do Leite/efeitos adversos , Intervalo Livre de Doença , Método Duplo-Cego , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Recidiva , Indução de Remissão , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Galactooligosaccharides (GOS) are mixed oligosaccharides that are mainly composed of galactosyllactoses (GLs), which include 3'-GL, 4'-GL, and 6'-GL. Data from numerous in vitro and in vivo studies have shown that GOS selectively stimulate the growth of bifidobacteria. Previously, we identified the gene locus responsible for 4'-GL utilization, but the selective routes of uptake and catabolism of 3'- and 6'-GL remain to be elucidated. In this study, we used differential transcriptomics to identify the utilization pathways of these GLs within the Bifidobacterium breve YIT 4014T strain. We found that the BBBR_RS 2305-2320 gene locus, which includes a solute-binding protein (SBP) of an ATP-binding cassette (ABC) transporter and ß-galactosidase, were up-regulated during 3'- and 6'-GL utilization. The substrate specificities of these proteins were further investigated, revealing that ß-galactosidase hydrolyzed both 3'-GL and 6'-GL efficiently. Our surface plasmon resonance results indicated that the SBP bound strongly to 6'-GL, but bound less tightly to 3'-GL. Therefore, we looked for the other SBPs for 3'-GL and found that the BBBR_RS08090 SBP may participate in 3'-GL transportation. We also investigated the distribution of these genes in 17 bifidobacterial strains, including 9 B. breve strains, and found that the ß-galactosidase genes were present in most bifidobacteria. Homologues of two ABC transporter SBP genes were found in all B. breve strains and in some bifidobacteria that are commonly present in the human gut microbiota. These results provide insights into the ability of human-resident bifidobacteria to utilize the main component of GOS in the gastrointestinal tract.
RESUMO
Bifidobacteria exert beneficial effects on hosts and are extensively used as probiotics. However, due to the genetic inaccessibility of these bacteria, little is known about their mechanisms of carbohydrate utilization and regulation. Bifidobacterium breve strain JCM1192 can grow on water-insoluble yeast (Saccharomyces cerevisiae) cell wall glucans (YCWG), which were recently considered as potential prebiotics. According to the results of 1H nuclear magnetic resonance (NMR) spectrometry, the YCWG were composed of highly branched (1â3,1â6)-ß-glucans and (1â4,1â6)-α-glucans. Although the YCWG were composed of 78.3% ß-glucans and 21.7% α-glucans, only α-glucans were consumed by the B. breve strain. The ABC transporter (malEFG1) and pullulanase (aapA) genes were transcriptionally upregulated in the metabolism of insoluble yeast glucans, suggesting their potential involvement in the process. A nonsense mutation identified in the gene encoding an ABC transporter ATP-binding protein (MalK) led to growth failure of an ethyl methanesulfonate-generated mutant with yeast glucans. Coculture of the wild-type strain and the mutant showed that this protein was responsible for the import of yeast glucans or their breakdown products, rather than the export of α-glucan-catabolizing enzymes. Further characterization of the carbohydrate utilization of the mutant and three of its revertants indicated that this mutation was pleiotropic: the mutant could not grow with maltose, glycogen, dextrin, raffinose, cellobiose, melibiose, or turanose. We propose that insoluble yeast α-glucans are hydrolyzed by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics.IMPORTANCE In general, Bifidobacterium strains are genetically intractable. Coupling classic forward genetics with next-generation sequencing, here we identified an ABC transporter ATP-binding protein (MalK) responsible for the import of insoluble yeast glucan breakdown products by B. breve JCM1192. We demonstrated the pleiotropic effects of the ABC transporter ATP-binding protein in maltose/maltooligosaccharide, raffinose, cellobiose, melibiose, and turanose transport. With the addition of transcriptional analysis, we propose that insoluble yeast glucans are broken down by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics.
Assuntos
Bifidobacterium breve/metabolismo , Glucanos/metabolismo , Saccharomyces cerevisiae/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bifidobacterium breve/efeitos dos fármacos , Bifidobacterium breve/genética , Bifidobacterium breve/crescimento & desenvolvimento , Parede Celular/química , Parede Celular/metabolismo , Dextrinas/farmacologia , Glicogênio/farmacologia , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hidrólise , Maltose/metabolismo , Maltose/farmacologia , Mutação , Solubilidade , Simbióticos , Água , beta-Glucanas/metabolismoRESUMO
PURPOSE: Rotavirus (RV) is the leading cause of severe diarrhoea among infants and young children, and although more standardized studies are needed, there is evidence that probiotics can help to fight against RV and other infectious and intestinal pathologies. On the other hand, the effects of prebiotics have not been properly addressed in the context of an RV infection. The aim of this study was to demonstrate a protective role for a specific scGOS/lcFOS 9:1 prebiotic mixture (PRE) separately, the probiotic Bifidobacterium breve M-16V (PRO) separately and the combination of the prebiotic mixture and the probiotic (synbiotic, SYN) in a suckling rat RV infection model. METHODS: The animals received the intervention from the 3rd to the 21st day of life by oral gavage. On day 7, RV was orally administered. Clinical parameters and immune response were evaluated. RESULTS: The intervention with the PRO reduced the incidence, severity and duration of the diarrhoea (p < 0.05). The PRE and SYN products improved clinical parameters as well, but a change in stool consistency induced by the PRE intervention hindered the observation of this effect. Both the PRE and the SYN, but not the PRO, significantly reduced viral shedding. All interventions modulated the specific antibody response in serum and intestinal washes at day 14 and 21 of life. CONCLUSIONS: A daily supplement of a scGOS/lcFOS 9:1 prebiotic mixture, Bifidobacterium breve M-16V or a combination of both is highly effective in modulating RV-induced diarrhoea in this preclinical model.
Assuntos
Bifidobacterium breve , Gastroenterite/terapia , Gastroenterite/virologia , Infecções por Rotavirus/terapia , Animais , Animais Recém-Nascidos , Anticorpos Antivirais/sangue , Peso Corporal , Diarreia/terapia , Diarreia/virologia , Modelos Animais de Doenças , Ácidos Graxos Voláteis/metabolismo , Fezes/microbiologia , Fezes/virologia , Gastroenterite/microbiologia , Imunoglobulina A/sangue , Imunoglobulina M/sangue , Prebióticos/administração & dosagem , Probióticos/administração & dosagem , Ratos , Ratos Endogâmicos Lew , Rotavirus , Infecções por Rotavirus/microbiologia , Manejo de Espécimes , SimbióticosRESUMO
AIM: To characterise the secreted and inducible antimicrobial protein and peptide (APP) levels in a prospective cohort of preterm infants (<30 weeks gestational age) with or without Bifidobacterium breve M16V supplementation during the first month of life. METHODS: We analysed serial biosamples of infants who did (n = 13) or did not receive (n = 62) B. breve (3 × 109 cfu/day). Peripheral blood was obtained on days 1, 14 and 28, and infant stool prior to commencement of probiotic supplementation and on day 21. Levels of APP (bactericidal/permeability inducing protein (BPI), beta defensins 1 and 2, lactoferrin, human cathelicidin, secretory phospholipase A2) in plasma and stool were determined. Further, we characterised induced APP levels in whole blood cultured with live S. epidermidis or with agonists of Toll-like receptors 2/6 and 4. RESULTS: Stool, plasma and stimulated blood APP levels changed significantly during the first month of life. Supplementation with B. breve did not affect basal or stimulated APP levels except for a transient increase in inducible BPI. CONCLUSION: Supplementation with B. breve does not appear to act via modulation of systemic or enteric APP expression in preterm infants although small effects cannot be excluded. Further work with other probiotic preparations is warranted.
Assuntos
Peptídeos Catiônicos Antimicrobianos/sangue , Bifidobacterium breve , Recém-Nascido Prematuro/sangue , Probióticos , Fezes/química , Feminino , Humanos , Recém-Nascido , Lactoferrina/sangue , Masculino , Fosfolipases A2 Secretórias/sangue , Estudos ProspectivosRESUMO
BACKGROUND: Since 1982, viable cells of Bifidobacterium breve (BBG-01) have been used in pediatric patients for clinical conditions such as intractable infantile diarrhea, preterm status, and pediatric surgery. Although the efficacy of BBG-01 has been widely reported, adverse events related to the use of BBG-01 have been reported in very few cases. METHODS: In order to trace adverse events seen by 109 doctors in 88 medical institutions where BBG-01 was used, a questionnaire survey of the number of occurrences and details of each case was conducted. RESULTS: Eighty-six clinicians (70 institutions) responded to the questionnaire (response rate, 78.9%). Number of respondents according to department of diagnosis (no. BBG-01-treated infants) was as follows: pediatrics, 29 respondents (10 938 patients); premature and newborn medicine, 26 (10 677 patients); obstetrics and gynecology, 1 (1212 patients) and pediatric surgery, 22 (169 patients). More than 90% of the total BBG-01-treated patients (23 092 patients) were in the departments of premature and newborn medicine and pediatrics, and BBG-01 had been used mainly in preterm infants and children with intractable diarrhea. Adverse events occurred in two extremely premature infants with functional ileus due to starch aggregates as vehicle, and in two surgical neonates with bacteremia caused by B. breve genetically identical to BBG-01, and no serious adverse events with poor outcome were reported. CONCLUSION: Adverse events related to the use of BBG-01 have an extremely low incidence and are mild in severity, thus ensuring the superior safety of this preparation.
Assuntos
Bifidobacterium breve , Diarreia Infantil/terapia , Doenças do Prematuro/terapia , Probióticos/efeitos adversos , Feminino , Pesquisas sobre Atenção à Saúde , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Japão , Masculino , Padrões de Prática Médica/estatística & dados numéricos , Probióticos/uso terapêutico , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: An altered compositional signature and reduced diversity of early gut microbiota are linked to development of allergic disease. We investigated the relationship between dominant Bifidobacterium species during the early post-natal period and subsequent development of allergic disease in the first year of life. METHODS: Faecal samples were collected at age 1 week, 1 month and 3 months from 117 infants at high risk of allergic disease. Bifidobacterium species were analysed by quantitative PCR and terminal restriction fragment length polymorphism. Infants were examined at 3, 6 and 12 months, and skin prick test was performed at 12 months. Eczema was diagnosed according to the UK Working Party criteria. RESULTS: The presence of B. catenulatum at 3 months was associated with a higher risk of developing eczema (ORadj = 4.5; 95% CI: 1.56-13.05, padj = 0.005). Infants colonized with B. breve at 1 week (ORadj = 0.29; 95% CI: 0.09-0.95, padj = 0.04) and 3 months (ORadj = 0.15; 95% CI: 0.05-0.44, padj = 0.00001) had a reduced risk of developing eczema. Furthermore, the presence of B. breve at 3 months was associated with a lower risk of atopic sensitization at 12 months (ORadj = 0.38; 95% CI: 0.15-0.98, padj = 0.05). B. breve colonization patterns were influenced by maternal allergic status, household pets and number of siblings. CONCLUSIONS: Temporal variations in Bifidobacterium colonization patterns early in life are associated with later development of eczema and/or atopic sensitization in infants at high risk of allergic disease. Modulation of the early microbiota may provide a means to prevent eczema in high-risk infants.
Assuntos
Infecções Bacterianas/epidemiologia , Bifidobacterium breve/imunologia , Eczema/epidemiologia , Hipersensibilidade/epidemiologia , Bifidobacterium breve/genética , DNA Bacteriano/análise , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/imunologia , Humanos , Lactente , Recém-Nascido , Masculino , Probióticos , Risco , Testes CutâneosRESUMO
PURPOSE: The incidence and severity of allergic asthma is rising, and novel strategies to prevent or treat this disease are needed. This study investigated the effects of different mixtures of non-digestible oligosaccharides combined with Bifidobacterium breve M-16V (BB) on the development of allergic airway inflammation in an animal model for house dust mite (HDM)-induced allergic asthma. METHODS: BALB/c mice were sensitized intranasally (i.n.) with HDM and subsequently challenged (i.n.) with PBS or HDM while being fed diets containing different oligosaccharide mixtures in combination with BB or an isocaloric identical control diet. Bronchoalveolar lavage fluid (BALF) inflammatory cell influx, chemokine and cytokine concentrations in lung homogenates and supernatants of ex vivo HDM-restimulated lung cells were analyzed. RESULTS: The HDM-induced influx of eosinophils and lymphocytes was reduced by the diet containing the short-chain and long-chain fructo-oligosaccharides and BB (FFBB). In addition to the HDM-induced cell influx, concentrations of IL-33, CCL17, CCL22, IL-6, IL-13 and IL-5 were increased in supernatants of lung homogenates or BALF and IL-4, IFN-γ and IL-10 were increased in restimulated lung cell suspensions of HDM-allergic mice. The diet containing FFBB reduced IL-6, IFN-γ, IL-4 and IL-10 concentrations, whereas the combination of galacto-oligosaccharides and long-chain fructo-oligosaccharides with BB was less potent in this model. CONCLUSION: These findings show that synbiotic dietary supplementation can affect respiratory allergic inflammation induced by HDM. The combination of FFBB was most effective in the prevention of HDM-induced airway inflammation in mice.