Metabolic model of necrotizing enterocolitis in the premature newborn gut resulting from enteric dysbiosis.

Necrotizing enterocolitis (NEC) is a leading cause of premature newborn morbidity and mortality. The clinical features of NEC consistently include prematurity, gut dysbiosis and enteral inflammation, yet the pathogenesis remains obscure. Herein we combine metagenomics and targeted metabolomics, with functional in vivo and in vitro assessment, to define a novel molecular mechanism of NEC. One thousand six hundred and forty seven publicly available metagenomics datasets were analyzed (NEC = 245; healthy = 1,402) using artificial intelligence methodologies. Targeted metabolomic profiling was used to quantify the concentration of specified fecal metabolites at NEC onset (n = 8), during recovery (n = 6), and in age matched controls (n = 10). Toxicity assays of discovered metabolites were performed in vivo in mice and in vitro using human intestinal epithelial cells. Metagenomic and targeted metabolomic analyses revealed significant differences in pyruvate fermentation pathways and associated intermediates. Notably, the short chain fatty acid formate was elevated in the stool of NEC patients at disease onset (P = 0.005) dissipated during recovery (P = 0.02) and positively correlated with degree of intestinal injury (r 2 = 0.86). In vitro, formate caused enterocyte cytotoxicity in human cells through necroptosis (P < 0.01). In vivo, luminal formate caused significant dose and development dependent NEC-like injury in newborn mice. Enterobacter cloacae and Klebsiella pneumoniae were the most discriminatory taxa related to NEC dysbiosis and increased formate production. Together, these data suggest a novel biochemical mechanism of NEC through the microbial production of formate. Clinical efforts to prevent NEC should focus on reducing the functional consequences of newborn gut dysbiosis associated metabolic pathways.

Immobilization of a Bifidobacterial Endo-ß-N-Acetylglucosaminidase to Generate Bioactive Compounds for Food Industry.

Conjugated N-glycans are considered next-generation bioactive prebiotic compounds due to their selective stimulation of beneficial microbes. These compounds are glycosidically attached to proteins through N-acetylglucosamines via specific asparagine residue (AsN-X-Ser/Thr). Certain bacteria such as Bifidobacterium longum subspecies infantis (B. infantis) have been shown to be capable of utilizing conjugated N-glycans, owing to their specialized genomic abilities. B. infantis possess a unique enzyme, Endo-ß-N-acetylglucosaminidase (EndoBI-1), which cleaves all types of conjugated N-glycans from glycoproteins. In this study, recombinantly cloned EndoBI-1 enzyme activity was investigated using various immobilization methods: 1) adsorption, 2) entrapment-based alginate immobilization, 3) SulfoLink-, and 4) AminoLink-based covalent bonding immobilization techniques were compared to develop the optimum application of EndoBI-1 to food processes. The yield of enzyme immobilization and the activity of each immobilized enzyme by different approaches were investigated. The N-glycans released from lactoperoxidase (LPO) using different immobilized enzyme forms were characterized using MALDI-TOF mass spectrometry (MS). As expected, regardless of the techniques, the enzyme activity decreased after the immobilization methods. The enzyme activity of adsorption and entrapment-based alginate immobilization was found to be 71.55% ± 0.6 and 20.32% ± 3.18, respectively, whereas the activity of AminoLink- and SulfoLink-based covalent bonding immobilization was found to be 58.05 ± 1.98 and 47.49% ± 0.30 compared to the free form of the enzyme, respectively. However, extended incubation time recovery achieved activity similar to that of the free form. More importantly, each immobilization method resulted in the same glycan profile containing 11 different N-glycan structures from a model glycoprotein LPO based on MALDI-TOF MS analysis. The glycan data analysis suggests that immobilization of EndoBI-1 is not affecting the enzyme specificity, which enables full glycan release without a limitation. Hence, different immobilization methods investigated in this study can be chosen for effective enzyme immobilization to obtain bioactive glycans. These findings highlight that further optimization of these methods can be a promising approach for future processing scale-up and commercialization of EndoBI-1 and similar enzymes.

Bifidobacterium longum Subspecies infantis Strain EVC001 Decreases Neonatal Murine Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a disease mainly of preterm infants with a 30-50% mortality rate and long-term morbidities for survivors. Treatment strategies are limited and have not improved in decades, prompting research into prevention strategies, particularly with probiotics. Recent work with the probiotic B. infantis EVC001 suggests that this organism may generate a more appropriate microbiome for preterm infants who generally have inappropriate gut colonization and inflammation, both risk factors for NEC. Experimental NEC involving Paneth cell disruption in combination with bacterial dysbiosis or formula feeding was induced in P14-16 C57Bl/6 mice with or without gavaged B. infantis. Following completion of the model, serum, small intestinal tissue, the cecum, and colon were harvested to examine inflammatory cytokines, injury, and the microbiome, respectively. EVC001 treatment significantly decreased NEC in a bacterial dysbiosis dependent model, but this decrease was model-dependent. In the NEC model dependent on formula feeding, no difference in injury was observed, but trending to significant differences was observed in serum cytokines. EVC001 also improved wound closure at six and twelve hours compared to the sham control in intestinal epithelial monolayers. These findings suggest that B. infantis EVC001 can prevent experimental NEC through anti-inflammatory and epithelial barrier restoration properties.

Early probiotic supplementation with B. infantis in breastfed infants leads to persistent colonization at 1 year.

BACKGROUND: Recent studies have reported a dysfunctional gut microbiome in breastfed infants. Probiotics have been used in an attempt to restore the gut microbiome; however, colonization has been transient, inconsistent among individuals, or has not positively impacted the host's gut. METHODS: This is a 2-year follow-up study to a randomized controlled trial wherein 7-day-old infants received 1.8 × 1010 colony-forming unit Bifidobacterium longum subsp. infantis (B. infantis) EVC001 (EVC) daily for 21 days or breast milk alone (unsupplemented (UNS)). In the follow-up study, mothers (n = 48) collected infant stool at 4, 6, 8, 10, and 12 months postnatal and completed the health-diet questionnaires. RESULTS: Fecal B. infantis was 2.5-3.5 log units higher at 6-12 months in the EVC group compared with the UNS group (P < 0.01) and this relationship strengthened with the exclusion of infants who consumed infant formula and antibiotics. Infants in the EVC group had significantly higher Bifidobacteriaceae and lower Bacteroidaceae and Lachnospiraceae (P < 0.05). There were no differences in any health conditions between the two groups. CONCLUSIONS: Probiotic supplementation with B. infantis within the first month postnatal, in combination with breast milk, resulted in stable colonization that persisted until at least 1 year postnatal. IMPACT: A dysfunctional gut microbiome in breastfed infants is common in resource-rich nations and associated with an increased risk of immune diseases. Probiotics only transiently exist in the gut without persistent colonization or altering the gut microbiome. This is the first study to show that early probiotic supplementation with B. infantis with breast milk results in stable colonization of B. infantis and improvements to the gut microbiome 1 year postnatal. This study addresses a key gap in the literature whereby probiotics can restore the gut microbiome if biologically selected microorganisms are matched with their specific food in an open ecological niche.

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum.

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a good source of macro- and micronutrients, bioactive peptides, and growth factors. Bovine colostrum is also a potential source of nutrition and bioactive because of its rich protein content that includes immunoglobulin G (IgG) and lactoferrin. However, the level of lactoferrin and IgG in bovine colostrum changes markedly during the lactation period. Therefore, monitoring the concentration of IgG and lactoferrin for the use of bovine colostrum as a protein source is an important question to study. Methods in this article describe how to determine protein content, as well as specific concentrations of lactoferrin and IgG. These methods include the following steps: Isolation of bovine colostrum proteins, Determination of protein concentration via Bicinchoninic acid assay (BCA), Visualization of proteins via SDS-PAGE, Determination of lactoferrin, and IgG concentration using an ELISA Assay.

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release.

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, including protein folding, stability, enzymatic protection, and biological recognition. N-glycans attached to glycoproteins (such as lactoferrin, lactadherin, and immunoglobulins) cannot be digested by the host and reach the large intestine, where they are consumed by certain beneficial microbes. Therefore, they are considered next-generation prebiotic compounds that can selectively stimulate the gut microbiome's beneficial microorganisms. However, the isolation of these new classes of prebiotics requires novel enzymes. Here, we describe the recombinant production of novel glycosidases from different Bifidobacteria strains (isolated from infants, rabbits, chicken, and bumblebee) for improved N-glycan isolation from glycoproteins. The method presented in this study includes the following steps: molecular cloning of Bifidobacterial genes by an in vivo recombinational cloning strategy, control of transformation success, protein induction, and protein purification.

Bovine Colostrum and Its Potential for Human Health and Nutrition.

Colostrum is the first milk produced post-partum by mammals and is compositionally distinct from mature milk. Bovine colostrum has a long history of consumption by humans, and there have been a number of studies investigating its potential for applications in human nutrition and health. Extensive characterization of the constituent fractions has identified a wealth of potentially bioactive molecules, their potential for shaping neonatal development, and the potential for their application beyond the neonatal period. Proteins, fats, glycans, minerals, and vitamins are abundant in colostrum, and advances in dairy processing technologies have enabled the advancement of bovine colostrum from relative limitations of a fresh and unprocessed food to a variety of potential applications. In these forms, clinical studies have examined bovine colostrum as having the substantial potential to improve human health. This review discusses the macro-and micronutrient composition of colostrum as well as describing well-characterized bioactives found in bovine colostrum and their potential for human health. Current gaps in knowledge are also identified and future directions are considered in order to elevate the potential for bovine colostrum as a component of a healthy diet for a variety of relevant human populations.

Bifidobacteria-mediated immune system imprinting early in life.

Immune-microbe interactions early in life influence the risk of allergies, asthma, and other inflammatory diseases. Breastfeeding guides healthier immune-microbe relationships by providing nutrients to specialized microbes that in turn benefit the host's immune system. Such bacteria have co-evolved with humans but are now increasingly rare in modern societies. Here we show that a lack of bifidobacteria, and in particular depletion of genes required for human milk oligosaccharide (HMO) utilization from the metagenome, is associated with systemic inflammation and immune dysregulation early in life. In breastfed infants given Bifidobacterium infantis EVC001, which expresses all HMO-utilization genes, intestinal T helper 2 (Th2) and Th17 cytokines were silenced and interferon ß (IFNß) was induced. Fecal water from EVC001-supplemented infants contains abundant indolelactate and B. infantis-derived indole-3-lactic acid (ILA) upregulated immunoregulatory galectin-1 in Th2 and Th17 cells during polarization, providing a functional link between beneficial microbes and immunoregulation during the first months of life.

Impact of Probiotic B. infantis EVC001 Feeding in Premature Infants on the Gut Microbiome, Nosocomially Acquired Antibiotic Resistance, and Enteric Inflammation.

Background: Preterm birth is a major determinant of neonatal survival and morbidity, but the gut microbiome and associated enteric inflammation are also key factors in neonatal development and the risk of associated morbidities. We prospectively and longitudinally followed two cohorts of preterm infants, one of which was fed activated Bifidobacterium longum subsp. infantis (B. infantis) EVC001 8 × 109 CFU daily, and the other was not fed a probiotic. Hospital feeding protocol assigned all infants born at <1500 g and/or < 32 weeks corrected gestational age to the probiotic feeding protocol, whereas infants born at >1500 g and/or >32 weeks corrected gestational age were not fed a probiotic. Fecal samples were opportunistically collected from 77 infants throughout the hospital stay, and subjected to shotgun metagenomic sequencing and quantification of enteric inflammation. De-identified metadata was collected from patient medical records. Results: The gut microbiome of preterm infants was typified by a high abundance of Enterobacteriaceae and/or Staphylococcaceae, and multivariate modeling identified the probiotic intervention, rather than degree of prematurity, day of life, or other clinical interventions, as the primary source of change in the gut microbiome. Among infants fed B. infantis EVC001, a high abundance of total Bifidobacteriaceae developed rapidly, the majority of which was B. infantis confirmed via subspecies-specific qPCR. Associated with this higher abundance of Bifidobacteriaceae, we found increased functional capacity for utilization of human milk oligosaccharides (HMOs), as well as reduced abundance of antibiotic resistance genes (ARGs) and the taxa that harbored them. Importantly, we found that infants fed B. infantis EVC001 exhibited diminished enteric inflammation, even when other clinical variables were accounted for using multivariate modeling. Conclusion: These results provide an important observational background for probiotic use in a NICU setting, and describe the clinical, physiological, and microbiome-associated improvements in preterm infants associated with B. infantis EVC001 feeding.

Metagenomic insights of the infant microbiome community structure and function across multiple sites in the United States.

The gut microbiome plays an important role in early life, protecting newborns from enteric pathogens, promoting immune system development and providing key functions to the infant host. Currently, there are limited data to broadly assess the status of the US healthy infant gut microbiome. To address this gap, we performed a multi-state metagenomic survey and found high levels of bacteria associated with enteric inflammation (e.g. Escherichia, Klebsiella), antibiotic resistance genes, and signatures of dysbiosis, independent of location, age, and diet. Bifidobacterium were less abundant than generally expected and the species identified, including B. breve, B. longum and B. bifidum, had limited genetic capacity to metabolize human milk oligosaccharides (HMOs), while B. infantis strains with a complete capacity for HMOs utilization were found to be exceptionally rare. Considering microbiome composition and functional capacity, this survey revealed a previously unappreciated dysbiosis that is widespread in the contemporary US infant gut microbiome.

Production of Bovine Colostrum for Human Consumption to Improve Health.

Colostrum contains all essential nutrients for the neonate during the first days of life, with impacts that continue far beyond these first days. Bovine colostrum has been used for human consumption due to the high concentrations of bioactive proteins, vitamins, minerals, growth factors, as well as free and conjugated oligosaccharides. Processes involved in the preparation of bovine colostrum for human consumption play a pivotal role in preserving and maintaining the activity of the bioactive molecules. As bovine colostrum is a multifunctional food that offers a myriad of benefits for human health, assessing the main processes used in preparing it with both advantages and disadvantages is a crucial point to discuss. We discuss major processes effects for colostrum production on the nutritional value, some advanced technologies to preserve processed bovine colostrum and the end-product forms consumed by humans whether as dairy products or dietary supplements.

Indole-3-lactic acid associated with Bifidobacterium-dominated microbiota significantly decreases inflammation in intestinal epithelial cells.

BACKGROUND: Bifidobacterium longum subsp. infantis (B. infantis) is a commensal bacterium that colonizes the gastrointestinal tract of breast-fed infants. B. infantis can efficiently utilize the abundant supply of oligosaccharides found in human milk (HMO) to help establish residence. We hypothesized that metabolites from B. infantis grown on HMO produce a beneficial effect on the host. RESULTS: In a previous study, we demonstrated that B. infantis routinely dominated the fecal microbiota of a breast fed Bangladeshi infant cohort (1). Characterization of the fecal metabolome of binned samples representing high and low B. infantis populations from this cohort revealed higher amounts of the tryptophan metabolite indole-3-lactic acid (ILA) in feces with high levels of B. infantis. Further in vitro analysis confirmed that B. infantis produced significantly greater quantities of the ILA when grown on HMO versus lactose, suggesting a growth substrate relationship to ILA production. The direct effects of ILA were assessed in a macrophage cell line and intestinal epithelial cell lines. ILA (1-10 mM) significantly attenuated lipopolysaccharide (LPS)-induced activation of NF-kB in macrophages. ILA significantly attenuated TNF-α- and LPS-induced increase in the pro-inflammatory cytokine IL-8 in intestinal epithelial cells. ILA increased mRNA expression of the aryl hydrogen receptor (AhR)-target gene CYP1A1 and nuclear factor erythroid 2-related factor 2 (Nrf2)-targeted genes glutathione reductase 2 (GPX2), superoxide dismutase 2 (SOD2), and NAD(P) H dehydrogenase (NQO1). Pretreatment with either the AhR antagonist or Nrf-2 antagonist inhibited the response of ILA on downstream effectors. CONCLUSIONS: These findings suggest that ILA, a predominant metabolite from B. infantis grown on HMO and elevated in infant stool high in B. infantis, and protects gut epithelial cells in culture via activation of the AhR and Nrf2 pathway.

Comparative Genome Analysis of Bifidobacterium longum subsp. infantis Strains Reveals Variation in Human Milk Oligosaccharide Utilization Genes among Commercial Probiotics.

Dysbiosis is associated with acute and long-term consequences for neonates. Probiotics can be effective in limiting the growth of bacteria associated with dysbiosis and promoting the healthy development of the infant microbiome. Given its adaptation to the infant gut, and promising data from animal and in vitro models, Bifidobacterium longum subsp. infantis is an attractive candidate for use in infant probiotics. However, strain-level differences in the ability of commercialized strains to utilize human milk oligosaccharides (HMOs) may have implications in the performance of strains in the infant gut. In this study, we characterized twelve B. infantis probiotic strains and identified two main variants in one of the HMO utilization gene clusters. Some strains possessed the full repertoire of HMO utilization genes (H5-positive strains), while H5-negative strains lack an ABC-type transporter known to bind core HMO structures. H5-positive strains achieved significantly superior growth on lacto-N-tetraose and lacto-N-neotetraose. In vitro, H5-positive strains had a significant fitness advantage over H5-negative strains, which was also observed in vivo in breastfed infants. This work provides evidence of the functional implications of genetic differences among B. infantis strains and highlights that genotype and HMO utilization phenotype should be considered when selecting a strain for probiotic use in infants.

Expression profiling of human milk derived exosomal microRNAs and their targets in HIV-1 infected mothers.

Despite the use of antiretroviral therapy (ART) in HIV-1 infected mothers approximately 5% of new HIV-1 infections still occur in breastfed infants annually, which warrants for the development of novel strategies to prevent new HIV-1 infections in infants. Human milk (HM) exosomes are highly enriched in microRNAs (miRNAs), which play an important role in neonatal immunity. Furthermore, HM exosomes from healthy donors are known to inhibit HIV-1 infection and transmission; however, the effect of HIV-1 on HM exosomal miRNA signatures remains unknown. In this study, we used nCounter NanoString technology and investigated miRNAs expression profiles in first week postpartum HM exosomes from HIV-1 infected and uninfected control mothers (n = 36). Our results indicated that HIV-1 perturbed the differential expression patterns of 19 miRNAs (13 upregulated and 6 downregulated) in HIV-1 infected women compared to healthy controls. DIANA-miR functional pathway analyses revealed that multiple biological pathways are involved including cell cycle, pathways in cancer, TGF-ß signaling, FoxO signaling, fatty acid biosynthesis, p53 signaling and apoptosis. Moreover, the receiver operating characteristics (ROC) curve analyses of miR-630 and miR-378g yielded areas under the ROC curves of 0.82 (95% CI 0.67 to 0.82) and 0.83 (95% CI 0.67 to 0.83), respectively highlighting their potential to serve as biomarkers to identify HIV-1 infection in women. These data may contribute to the development of new therapeutic strategies in prevention of mother-to-child transmission (MTCT) of HIV-1.

Integrating the Ecosystem Services Framework to Define Dysbiosis of the Breastfed Infant Gut: The Role of B. infantis and Human Milk Oligosaccharides.

Mounting evidence supports a connection between the composition of the infant gut microbiome and long-term health. In fact, aberrant microbiome compositions during key developmental windows in early life are associated with increased disease risk; therefore, making pertinent modifications to the microbiome during infancy offers significant promise to improve human health. There is growing support for integrating the concept of ecosystem services (the provision of benefits from ecosystems to humans) in linking specific microbiome functions to human well-being. This framework is widely applied in conservation efforts of macro-ecosystems and offers a systematic approach to guide restoration actions aimed to recover critical ecological functions. The aim of this work is to apply the ecosystem services framework to integrate recent studies demonstrating stable alteration of the gut microbiome of breastfed infants when Bifidobacterium longum subsp. infantis EVC001, a gut symbiont capable of efficiently utilizing human milk oligosaccharides into organic acids that are beneficial for the infant and lower intestinal pH, is reintroduced. Additionally, using examples from the literature we illustrate how the absence of B. infantis results in diminished ecosystem services, which may be associated with health consequences related to immune and metabolic disorders. Finally, we propose a model by which infant gut dysbiosis can be defined as a reduction in ecosystem services supplied to the host by the gut microbiome rather than merely changes in diversity or taxonomic composition. Given the increased interest in targeted microbiome modification therapies to decrease acute and chronic disease risk, the model presented here provides a framework to assess the effectiveness of such strategies from a host-centered perspective.

Colonization by B. infantis EVC001 modulates enteric inflammation in exclusively breastfed infants.

BACKGROUND: Infant gut dysbiosis, often associated with low abundance of bifidobacteria, is linked to impaired immune development and inflammation-a risk factor for increased incidence of several childhood diseases. We investigated the impact of B. infantis EVC001 colonization on enteric inflammation in a subset of exclusively breastfed term infants from a larger clinical study. METHODS: Stool samples (n = 120) were collected from infants randomly selected to receive either 1.8 × 1010 CFU B. infantis EVC001 daily for 21 days (EVC001) or breast milk alone (controls), starting at day 7 postnatal. The fecal microbiome was analyzed using 16S ribosomal RNA, proinflammatory cytokines using multiplexed immunoassay, and fecal calprotectin using ELISA at three time points: days 6 (Baseline), 40, and 60 postnatal. RESULTS: Fecal calprotectin concentration negatively correlated with Bifidobacterium abundance (P < 0.0001; ρ = -0.72), and proinflammatory cytokines correlated with Clostridiaceae and Enterobacteriaceae, yet negatively correlated with Bifidobacteriaceae abundance. Proinflammatory cytokines were significantly lower in EVC001-fed infants on days 40 and 60 postnatally compared to baseline and compared to control infants. CONCLUSION: Our findings indicate that gut dysbiosis (absence of B. infantis) is associated with increased intestinal inflammation. Early addition of EVC001 to diet represents a novel strategy to prevent enteric inflammation during a critical developmental phase.

TLR10 Senses HIV-1 Proteins and Significantly Enhances HIV-1 Infection.

Toll-like receptors (TLRs) play a crucial role in innate immunity and provide a first line of host defense against invading pathogens. Of the identified human TLRs, TLR10 remains an orphan receptor whose ligands and functions are poorly understood. In the present study, we sought to evaluate the level of TLR10 expression in breast milk (BM) and explore its potential function in the context of HIV-1 infection. We evaluated HIV-1-infected (Nigerian: n = 40) and uninfected (Nigerian: n = 27; Canadian: n = 15) BM samples for TLR expression (i.e., TLR10, TLR2, and TLR1) and report here that HIV-1-infected BM from Nigerian women showed significantly higher levels of TLR10, TLR1, and TLR2 expression. Moreover, the level of TLR10 expression in HIV-1-infected BM was upregulated by over 100-fold compared to that from uninfected control women. In vitro studies using TZMbl cells demonstrated that TLR10 overexpression contributes to higher HIV-1 infection and proviral DNA integration. Conversely, TLR10 inhibition significantly decreased HIV-1 infection. Notably, HIV-1 gp41 was recognized as a TLR10 ligand, leading to the induction of IL-8 and NF-κBα activation. The identification of a TLR10 ligand and its involvement in HIV-1 infection enhances our current understanding of HIV-1 replication and may assist in the development of improved future therapeutic strategies.

Corrigendum: Use of UV Treated Milk Powder to Increase Vaccine Efficacy in the Elderly.

[This corrects the article DOI: 10.3389/fimmu.2018.02254.].

Use of UV Treated Milk Powder to Increase Vaccine Efficacy in the Elderly.

Aging populations experience a decline in adaptive immune system function also known as immunosenesence. Protein nutrition has been shown to stimulate and strengthen the immune system, and such approaches are needed for this growing segment of the population. A controlled, randomized, double blind pilot study was conducted to compare two different protein sources (soy and dairy) as nutritional supplementation to enhance vaccine response. Our objective was to examine the immune stimulating effects of dairy protein subjected to ultraviolet radiation (UV-C) radiation treatment process instead of pasteurization. Participants were 21 healthy individuals over 60 years of age who consumed 6 g of the dairy protein or a comparison, soy isoflavone protein, twice a day for 8 weeks. DTaP vaccine administered at week 4. Non-parametric t-tests revealed a significant increase in Tetanus antibodies in the dairy group compared to the soy group at week 8. These findings suggest additional benefits of UV-C treated unheated dairy protein as a solution to counteract immunosenescence, but warrant further study in elderly and other populations that might benefit from immune system stimulation.