Bacteroides fragilis Strain ZY-312 Defense against Cronobacter sakazakii-Induced Necrotizing Enterocolitis In Vitro and in a Neonatal Rat Model.
ABSTRACT: Cronobacter sakazakii is an important pathogen associated with the development of necrotizing enterocolitis (NEC), infant sepsis, and meningitis. Several randomized prospective clinical trials demonstrated that oral probiotics could decrease the incidence of NEC. Previously, we isolated and characterized a novel probiotic, Bacteroides fragilis strain ZY-312. However, it remains unclear how ZY-312 protects the host from the effects of C. sakazakii infection. To understand the underlying mechanisms triggering the probiotic effects, we tested the hypothesis that there was cross talk between probiotics/probiotics-modulated microbiota and the local immune system, governed by the permeability of the intestinal mucosa, using in vitro and in vivo models for the intestinal permeability. The probiotic effects of ZY-312 on intestinal epithelial cells were first examined, and the results revealed that ZY-312 inhibited C. sakazakii invasion, C. sakazakii-induced dual cell death (pyroptosis and apoptosis), and epithelial barrier dysfunction in vitro and in vivo The presence of ZY-312 also resulted in decreased expression of an inflammasome (NOD-like receptor family member pyrin domain-containing protein 3 [NLRP3]), caspase-3, and serine protease caspase-1 in a neonatal rat model. Furthermore, ZY-312 significantly modulated the compositions of the intestinal bacterial communities and decreased the relative abundances of Proteobacteria and Gammaproteobacteria but increased the relative abundances of Bacteroides and Bacillus in neonatal rats. In conclusion, our findings have shown for the first time that the probiotic B. fragilis ZY-312 suppresses C. sakazakii-induced NEC by modulating the proinflammatory response and dual cell death (apoptosis and pyroptosis).IMPORTANCE Cronobacter sakazakii is an opportunistic pathogenic bacterium that can cause necrotizing enterocolitis (NEC). However, the mechanism of pathogenicity of C. sakazakii is largely unknown. Here we have now demonstrated that apoptotic and pyroptotic stimuli are effectors of C. sakazakii-induced NEC. Previously, we isolated a novel probiotic strain candidate from fecal samples from healthy infants and characterized it as Bacteroides fragilis strain ZY-312. Functional characterization reveals that ZY-312 inhibited C. sakazakii invasion, restoring epithelial barrier dysfunction, decreasing the expression of inflammatory cytokines, and reducing dual cell death (pyroptosis and apoptosis). Furthermore, the presence of ZY-132 was sufficient to hinder the adverse reaction seen with C. sakazakii in a C. sakazakii-induced NEC model. Taking the results together, our study demonstrated the utility of ZY-312 as a promising probiotic agent for the prevention of NEC.
Project description:Antibiotic-associated diarrhea (AAD) is iatrogenic diarrhea characterized by disruption of the gut microbiota. Probiotics are routinely used to treat AAD in clinical practice; however, the effectiveness and mechanisms by which probiotics alleviate symptoms remain poorly understood. We previously isolated a non-toxic Bacteroides fragilis strain ZY-312, which has been verified to be beneficial in certain infection disorders. However, the precise role of this commensal bacterium in AAD is unknown. In this study, we successfully established an AAD rat model by exposing rats to appropriate antibiotics. These rats developed diarrhea symptoms and showed alterations in their intestinal microbiota, including overgrowth of some pathogenic bacteria. In addition, gastrointestinal barrier defects, indicated by compromised aquaporin expression, aberrant tight junction proteins, and decreased abundance of mucus-filled goblet cells, were also detected in ADD rats compared with control animals. Of note, oral treatment with B. fragilis strain ZY-312 ameliorated AAD-related diarrhea symptoms by increasing the abundance of specific commensal microbiota. Interestingly, we demonstrated that these changes were coincident with the restoration of intestinal barrier function and enterocyte regeneration in AAD rats. In summary, we identified a potential probiotic therapeutic strategy for AAD and identified the vital roles of B. fragilis strain ZY-312 in modulating the colonic bacterial community and participating in microbiota-mediated epithelial cell proliferation and differentiation.
Project description:Commensal non-toxigenic Bacteroides fragilis confers powerful health benefits to the host, and has recently been identified as a promising probiotic candidate. We previously isolated B. fragilis strain ZY-312 and identified it as a novel strain based on 16S rRNA sequencing and morphological analyses. We also determined that ZY-312 displayed desirable probiotic properties, including tolerance to simulated digestive fluid, adherence, and in vitro safety. In this study, we aim to investigate whether ZY-312 meets the safety criteria required for probiotic bacteria through comprehensive and systematic evaluation. Consequently, the fatty acid profile, metabolite production, and biochemical activity of strain ZY-312 were found to closely resemble descriptions of B. fragilis in Bergey's manual. Taxonomic identification of strain ZY-312 based on whole genome sequencing indicated that ZY-312 and ATCC 25285 showed 99.99% similarity. The 33 putative virulence-associated factors identified in ZY-312 mainly encoded structural proteins and proteins with physiological activity, while the lack of bft indicated that ZY-312 was non-toxigenic. In vivo safety was proven in both normal and immune-deficient mice. The 11 identified antibiotic resistance genes were located on the chromosome rather than on a plasmid, ruling out the risk of plasmid-mediated transfer of antibiotic resistance. In vitro, ZY-312 showed resistance to cefepime, kanamycin, and streptomycin. Finally, and notably, ZY-312 exhibited high genetic stability after 100 passages in vitro. This study supplements the foundation work on the safety evaluation of ZY-312, and contributes to the development of the first probiotic representative from the dominant Bacteroidetes phylum.
Project description:Colonizing bacteria interacting with the immature, unlike the mature, human intestine favors inflammation over immune homeostasis. As a result, ten percent of premature infants under 1500 grams weight develop an inflammatory necrosis of the intestine after birth, e.g., necrotizing enterocolitis (NEC). NEC is a major health problem in this population causing extensive morbidity and mortality and an enormous expenditure of health care dollars. NEC can be prevented by giving preterm infants their mother's expressed breast milk or ingesting selective probiotic organisms. Vaginally delivered, breast fed newborns develop health promoting bacteria ("pioneer" bacteria) which preferentially stimulate intestinal host defense and anti-inflammation. One such "pioneer" organism is Bacteroides fragilis with a polysaccharide (PSA) on its capsule. B. fragilis has been shown developmentally in intestinal lymphocytes and dendritic cells to produce a balanced T-helper cell (TH1/TH2) response and to reduce intestinal inflammation by activity through the TLR2 receptor stimulating IL-10 which inhibits IL-17 causing inflammation. No studies have been done on the role of B. fragilis PSA on fetal enterocytes and its increased inflammation. Accordingly, using human and mouse fetal intestinal models, we have shown that B. fragilis with PSA and PSA alone inhibits IL-1?-induced IL-8 inflammation in fetal and NEC intestine. We have also begun to define the mechanism for this unique inflammation noted in fetal intestine. We have shown that B. fragilis PSA anti-inflammation requires both the TLR2 and TLR4 receptor and is in part mediated by the AP1 transcription factor (TLR2) which is developmentally regulated. These observations may help to devise future preventative treatments of premature infants against NEC.
Project description:BACKGROUND:Probiotic therapy can reduce the incidence of NEC. Therapeutic use of probiotics after NEC diagnosis reduces the severity of NEC in preterm infants or full-term infants is unclear. To evaluate the effect of probiotics on preventing the deterioration of necrotizing enterocolitis (NEC) from stage I to II/III. METHODS:A retrospective matched cohort study was performed. Included patients were ultimately divided into two groups: the probiotic treatment group (probiotics were used ?4?days) and the no probiotic treatment group. The differences in deterioration trends between the two groups were compared. Additionally, the risk factors associated with the deterioration of NEC were further analyzed with a case-control study. RESULTS:A total of 231 infants met the inclusion criteria. Eighty-one pairs were matched according to similar gestational age and birth weight. Before matching, we found that the rate of deterioration of NEC from stage I to II/III in the group with probiotic treatment was similar to that in the group without probiotic treatment (23.1% [25/108] vs 26.0% [32/123], P?=?0.614). After matching, the rate of deterioration of NEC between the two groups still had no significant difference (21.0% [17/81] vs 27.2% [22/81], P?=?0.358). Logistic regression analysis showed that sepsis after NEC was an independent risk factor for NEC deteriorating from stage I to II/III (OR 2.378, 95% CI 1.005-5.628, P?=?0.049). CONCLUSION:Probiotics may not prevent the deterioration of NEC from stage I to II/III in infants, but this conclusion should be treated with caution.
Project description:In this review, we summarize existing knowledge regarding the effects of probiotics on necrotizing enterocolitis (NEC). We review the role of the microbiome in NEC and pre-clinical data on mechanisms of probiotic action. Next, we summarize existing randomized controlled trials and observational studies of probiotics to prevent NEC. We also summarize findings from several recent meta-analyses and report a new cumulative meta-analysis of probiotic trials. Finally, we review data from cohorts routinely using commercially available probiotics. Our goal is to inform clinicians about the risks and benefits of probiotics, which may be helpful for those considering use in preterm infants to prevent NEC, death, or sepsis.
Project description:Necrotizing enterocolitis is the most common gastrointestinal disorder in premature neonates. This disease is characterized by massive epithelial necrosis, gut barrier dysfunction and improper mucosal defense development. Studies have shown that probiotic administration can decrease NEC incidence and mortality. The proposed mechanisms of probiotics for the prevention of NEC are: promotion of intestinal development; improved barrier function through decreased apoptosis and improved mucin production; decreased expression of proinflammatory cytokines IL6, IL8, and TNF?, and modulation of microbiota dysbiosis in preterm infants. However, reported sepsis in the immunocompromised preterm host has deterred routine prophylactic administration of probiotics in the neonatal intensive care unit. We hypothesize that maternal administration of probiotics to pregnant mouse dams can recapitulate the beneficial effects observed in neonates fed with probiotics directly. We exposed pregnant mice to the probiotics and monitored the changes in the developing intestines of the offspring. Pregnant mice were fed daily with the probiotics Lactobacillus acidophilus and Bifidobacterium infantis (LB) from embryonic day15 to 2-week-old postnatally. Intraperitoneal administration of IL-1? in the pups was used to model proinflammatory insults. Sera were collected at 2 weeks of age and evaluated for inflammatory cytokines by enzyme-linked-immunosorbent-assay and gut permeability by Fluorescein isothiocyanate-dextran tracer assay. Ileal tissues were collected for the evaluation of apoptosis and proliferation of the intestinal epithelium; as well as mucin and tight junction integrity at mucosal surface by immunofluorescent staining. We find that maternal LB exposure facilitated intestinal epithelial cell differentiation, prevented loss of mucin and preserved the intestinal integrity and barrier function and decreased serum levels of IL-1?, TNF-? and IL-6 in the preweaned offsprings. in LB exposed pups. We demonstrate that maternal probiotic supplementation promotes gut maturation in developing offspring. This is potentially a safe alternative therapy to induce intestinal maturation and prevent prematurity-associated neonatal disorders.
Project description:Clostridium difficile is currently the leading cause of nosocomial infection. Antibiotics remain the first-line therapy for C. difficile-associated diseases (CDAD), despite the risks of resistance promotion and further gut microbiota perturbation. Notably, the abundance of Bacteroides fragilis was reported to be significantly decreased in CDAD patients. This study aimed to clarify the prophylactic effects of B. fragilis strain ZY-312 in a mouse model of C. difficile infection (CDI). The CDI mouse model was successfully created using C. difficile strain VPI 10463 spores, as confirmed by lethal diarrhea (12.5% survival rate), serious gut barrier disruption, and microbiota disruption. CDI model mice prophylactically treated with B. fragilis exhibited significantly higher survival rates (100% in low dosage group, 87.5% in high dosage group) and improved clinical manifestations. Histopathological analysis of colon and cecum tissue samples revealed an intact gut barrier with strong ZO-1 and Muc-2 expression. The bacterial diversity and relative abundance of gut microbiota were significantly improved. Interestingly, the relative abundance of Akkermansia muciniphila was positively correlated with B. fragilis treatment. In vitro experiments showed that B. fragilis inhibited C. difficile adherence, and attenuated the decrease in CDI-induced transepithelial electrical resistance, ZO-1 and MUC-2 loss, and apoptosis, suggesting that B. fragilis protected against CDI possibly by resisting pathogen colonization and improving gut barrier integrity and functions. In summary, B. fragilis exerted protective effects on a CDI mouse model by modulating gut microbiota and alleviating barrier destruction, thereby relieving epithelial stress and pathogenic colitis triggered by C. difficile. This study provides an alternative preventative measure for CDI and lays the foundations for further investigations of the relationships among opportunistic pathogens, commensal microbiota, and the gut barrier.
Project description:Background: Bowel preparation is necessary for successful colonoscopy, while it can seriously affect intestinal microbial composition and damage the intestinal mucosal barriers in humans. Methods: To figure out whether probiotics can sustain intestinal homeostasis and guard people's health, the probiotic drug of Bifidobacterium Tetragenous viable Bacteria Tablets (P group, n = 16) or placebo (C group, n = 16) was used for volunteers receiving bowel preparation, and high-throughput sequencing method was applied to monitor their intestinal microbial changes. Results: The present results suggested that bowel preparation obviously reduced the intestinal microbial diversity, while taking probiotics significantly restored it to normal level. In addition, probiotics sharply reduced the abundance of pathogenic Proteobacteria, and obviously lowered the ratio of Firmicutes/Bacteroidetes compared with control group at phylum level (P < 0.05). And probiotics markedly decreased the abundance of pathogenic Acinetobacter and Streptococcus, while greatly enriched the relative abundance of beneficial bacteria Bacteroides, Roseburia, Faecalibacterium, and Parabacteroides at genus level (P < 0.05). Conclusion: Probiotic drugs, e.g., Bifidobacterium Tetragenous viable Bacteria Tablets, can be used to restore intestinal dysbacteriosis caused by bowel preparation, and reduce side effects during colonoscopy.
Project description:BACKGROUND:Some oral probiotics have been shown to prevent necrotizing enterocolitis (NEC) and decrease mortality effectively in preterm very low birth weight (PVLBW) infants. However, it is unclear whether a single probiotic or a mixture of probiotics is most effective for the prevention of NEC. OBJECTIVE:A meta-analysis was conducted by reviewing the most up to date literature to investigate whether multiple strains probiotics are more effective than a single strain in reducing NEC and death in PVLBW infants. DATA SOURCES:Relevant studies were identified by searches of the MEDLINE, EMBASE, and Cochrane CENTRAL databases, from 2001 to 2016. DATA EXTRACTION AND SYNTHESIS:The inclusion criteria were randomized controlled trials of any enteral probiotic supplementation that was initiated within the first 7 days and continued for at least 14 days in preterm infants (? 34 weeks' gestation) and/or those of a birth weight ?1500 g. RESULTS:A total of 25 trials (n = 7345 infants) were eligible for inclusion in the meta-analysis using a fixed-effects model. Multiple strains probiotics were associated with a marked reduction in the incidence of NEC, with a pooled OR of 0.36 (95% CI, 0.24-0.53; P < .00001). Single strain probiotic using Lactobacillus species had a borderline effect in reducing NEC (OR of 0.60; 95% CI 0.36-1.0; P = .05), but not mortality. Multiple strains probiotics had a greater effectiveness in reducing mortality and were associated with a pooled OR of 0.58 (95% CI, 0.43-0.79; P = .0006). Trials using single strain of Bifidobacterium species and Saccharomyces boulardii did not reveal any beneficial effects in terms of reducing NEC or mortality. CONCLUSION:This updated report found that multiple strains probiotics appear to be the most feasible and effective strategy for the prevention of NEC and reduction of mortality in PVLBW neonates. Further clinical trials should focus on which probiotic combinations are most effective.
Project description:This was a pilot study aiming to evaluate the effects of probiotics as adjunctive treatment for ulcerative colitis (UC). Twenty-five active patients with UC were assigned to the probiotic (n = 12) and placebo (n = 13) groups. The probiotic group received mesalazine (60 mg kg-1 day-1 ) and oral probiotics (containing Lactobacillus casei Zhang, Lactobacillus plantarum P-8 and Bifidobacterium animalis subsp. lactis V9) twice daily for 12 weeks, while the placebo group received the same amounts of mesalazine and placebo. The clinical outcomes were assessed. The gut mucosal microbiota was profiled by PacBio single-molecule, real-time (SMRT) sequencing of the full-length 16S rRNA of biopsy samples obtained by colonoscopy. A significantly greater magnitude of reduction was observed in the UC disease activity index (UCDAI) in the probiotic group compared with the placebo group (P = 0.043), accompanying by a higher remission rate (91.67% for probiotic-receivers versus 69.23% for placebo-receivers, P = 0.034). The probiotics could protect from diminishing of the microbiota diversity and richness. Moreover, the gut mucosal microbiota of the probiotic-receivers had significantly more beneficial bacteria like Eubacterium ramulus (P < 0.05), Pediococcus pentosaceus (P < 0.05), Bacteroides fragilis (P = 0.02) and Weissella cibaria (P = 0.04). Additionally, the relative abundances of the beneficial bacteria correlated significantly but negatively with the UCDAI score, suggesting that the probiotics might alleviate UC symptoms by modulating the gut mucosal microbiota. Our research has provided new insights into the mechanism of symptom alleviation in UC by applying probiotic-based adjunctive treatment.