Bangladeshi children with acute diarrhoea show faecal microbiomes with increased Streptococcus abundance, irrespective of diarrhoea aetiology.

We report streptococcal dysbiosis in acute diarrhoea irrespective of aetiology. Compared with 20 healthy local controls, 71 Bangladeshi children hospitalized with acute diarrhoea (AD) of viral, mixed viral/bacterial, bacterial and unknown aetiology showed a significantly decreased bacterial diversity with loss of pathways characteristic for the healthy distal colon microbiome (mannan degradation, methylerythritol phosphate and thiamin biosynthesis), an increased proportion of faecal streptococci belonging to the Streptococcus bovis and Streptococcus salivarius species complexes, and an increased level of E. coli-associated virulence genes. No enteropathogens could be attributed to a subgroup of patients. Elevated lytic coliphage DNA was detected in 2 out of 5 investigated enteroaggregative E. coli (EAEC)-infected patients. Streptococcal outgrowth in AD is discussed as a potential nutrient-driven consequence of glucose provided with oral rehydration solution.

Oral application of Escherichia coli bacteriophage: safety tests in healthy and diarrheal children from Bangladesh.

A T4-like coliphage cocktail was given with different oral doses to healthy Bangladeshi children in a placebo-controlled randomized phase I safety trial. Fecal phage detection was oral dose dependent suggesting passive gut transit of coliphages through the gut. No adverse effects of phage application were seen clinically and by clinical chemistry. Similar results were obtained for a commercial phage preparation (Coliproteus from Microgen/Russia). By 16S rRNA gene sequencing, only a low degree of fecal microbiota conservation was seen in healthy children from Bangladesh who were sampled over a time interval of 7 days suggesting a substantial temporal fluctuation of the fecal microbiota composition. Microbiota variability was not associated with the age of the children or the presence of phage in the stool. Stool microbiota composition of Bangladeshi children resembled that found in children of other regions of the world. Marked variability in fecal microbiota composition was also seen in 71 pediatric diarrhea patients receiving only oral rehydration therapy and in 38 patients receiving coliphage preparations or placebo when sampled 1.2 or 4 days apart respectively. Temporal stability of the gut microbiota should be assessed in case-control studies involving children before associating fecal microbiota composition with health or disease phenotypes.

Genome sequence of the lantibiotic bacteriocin producer Streptococcus salivarius strain K12.

Streptococcus salivarius is a prevalent commensal species of the oropharyngeal tract. S. salivarius strain K12 is an isolate from the saliva of a healthy child, used as an oral probiotic. Here, we report its genome sequence, i.e., the full sequence of the 190-kb megaplasmid pSsal-K12 and a high-quality draft 2.2-Gb chromosomal sequence.

Changing Epidemiology of Acute Respiratory Infections in Under-Two Children in Dhaka, Bangladesh.

Objectives: Risk factors for acute respiratory infections (ARIs) in community settings are not fully understood, especially in low-income countries. We examined the incidence and risk factors associated with ARIs in under-two children from the Microbiota and Health study. Methods: Children from a peri-urban area of Dhaka (Bangladesh) were followed from birth to 2 years of age by both active surveillance of ARIs and regular scheduled visits. Nasopharyngeal samples were collected during scheduled visits for detection of bacterial facultative respiratory pathogens. Information on socioeconomic, environmental, and household conditions, and mother and child characteristics were collected. A hierarchical modeling approach was used to identify proximate determinants of ARIs. Results: Of 267 infants, 87.3% experienced at least one ARI episode during the first 2 years of life. The peak incidence of ARIs was 330 infections per 100 infant-years and occurred between 2 and 4 months of age. Season was the main risk factor (rainy monsoon season, incidence rate ratio [IRR] 2.43 [1.92-3.07]; cool dry winter, IRR 2.10 [1.65-2.67] compared with hot dry summer) in the first 2 years of life. In addition, during the first 6 months of life, young maternal age (<22 years; IRR 1.34 [1.01-1.77]) and low birth weight (<2,500 g; IRR 1.39 [1.03-1.89]) were associated with higher ARI incidence. Conclusions: Reminiscent of industrialized settings, cool rainy season rather than socioeconomic and hygiene conditions was a major risk factor for ARIs in peri-urban Bangladesh. Understanding the causal links between seasonally variable factors such as temperature, humidity, crowding, diet, and ARIs will inform prevention measures.

The Influence of FUT2 and FUT3 Polymorphisms and Nasopharyngeal Microbiome on Respiratory Infections in Breastfed Bangladeshi Infants from the Microbiota and Health Study.

Acute respiratory infections (ARIs) are one of the most common causes of morbidity and mortality in young children. The aim of our study was to examine whether variation in maternal FUT2 (α1,2-fucosyltransferase 2) and FUT3 (α1,3/4-fucosyltransferase 3) genes, which shape fucosylated human milk oligosaccharides (HMOs) in breast milk, are associated with the occurrence of ARIs in breastfed infants as well as the influence of the nasopharyngeal microbiome on ARI risk. Occurrences of ARIs were prospectively recorded in a cohort of 240 breastfed Bangladeshi infants from birth to 2 years. Secretor and Lewis status was established by sequencing of FUT2/3 genes. The nasopharyngeal microbiome was characterized by shotgun metagenomics, complemented by specific detection of respiratory pathogens; 88.6% of mothers and 91% of infants were identified as secretors. Maternal secretor status was associated with reduced ARI incidence among these infants in the period from birth to 6 months (incidence rate ratio [IRR], 0.66; 95% confidence interval [CI], 0.47 to 0.94; P = 0.020), but not at later time periods. The nasopharyngeal microbiome, despite precise characterization to the species level, was not predictive of subsequent ARIs. The observed risk reduction of ARIs among infants of secretor mothers during the predominant breastfeeding period is consistent with the hypothesis that fucosylated oligosaccharides in human milk contribute to protection against respiratory infections. However, we found no evidence that modulation of the nasopharyngeal microbiome influenced ARI risk. IMPORTANCE The observed risk reduction of acute respiratory infections (ARIs) among infants of secretor mothers during the predominant breastfeeding period is consistent with the hypothesis that fucosylated oligosaccharides in human milk contribute to protection against respiratory infections. Respiratory pathogens were only weak modulators of risk, and the nasopharyngeal microbiome did not influence ARI risk, suggesting that the associated protective effects of human milk oligosaccharides (HMOs) are not conveyed via changes in the nasopharyngeal microbiome. Our observations add to the evidence for a role of fucosylated HMOs in protection against respiratory infections in exclusively or predominantly breastfed infants in low-resource settings. There is no indication that the nasopharyngeal microbiome substantially modulates the risk of subsequent mild ARIs. Larger studies are needed to provide mechanistic insights on links between secretor status, HMOs, and risk of respiratory infections.

Activation of the G-protein coupled receptor GPR35 by human milk oligosaccharides through different pathways.

Numerous benefits of breastfeeding over infant formula are fully established. The superiority of human milk over bovine milk-based formula is partly due to human milk oligosaccharides (HMOs), a family of over 100 molecules present specifically and substantially in human milk that resemble mucosal glycans. To uncover novel physiological functions and pathways of HMOs, we screened a panel of 165 G-protein coupled receptors (GPCRs) using a blend of 6 HMOs (3'-O-sialyllactose (3'SL), 6'-O-sialyllactose (6'SL), lacto-N-tetraose (LNT), lacto-N-neo-tetraose (LNnT), 2-O-fucosyllactose (2'FL), and difucosyllactose (diFL)), and followed up positive hits with standard receptor assays. The HMO blend specifically activated GPR35. LNT and 6'SL individually activated GPR35, and they showed synergy when used together. In addition, in vitro fermentation of infant stool samples showed that 2'FL upregulates the production of the GPR35 agonist kynurenic acid (KYNA) by the microbiota. LNT + 6'SL and KYNA showed additive activation of GPR35. Activation by 6'SL and LNT of GPR35, a receptor mediating attenuation of pain and colitis, is to our knowledge the first demonstration of GPCR activation by any HMO. In addition, we demonstrated a remarkable cooperation between nutrition and microbiota towards activation of a host receptor highlighting the close interplay between environment and host-microbe interactions.

Linking Human Milk Oligosaccharides, Infant Fecal Community Types, and Later Risk To Require Antibiotics.

Human milk oligosaccharides (HMOs) may provide health benefits to infants partly by shaping the development of the early-life intestinal microbiota. In a randomized double-blinded controlled multicentric clinical trial, healthy term infants received either infant formula (control) or the same formula with two HMOs (2'-fucosyllactose and lacto-N-neotetraose; test) from enrollment (0 to 14 days) to 6 months. Then, all infants received the same follow-up formula without HMOs until 12 months of age. Breastfed infants (BF) served as a reference group. Stool microbiota at 3 and 12 months, analyzed by 16S rRNA gene sequencing, clustered into seven fecal community types (FCTs) with marked differences in total microbial abundances. Three of the four 12-month FCTs were likely precursors of the adult enterotypes. At 3 months, microbiota composition in the test group (n = 58) appeared closer to that of BF (n = 35) than control (n = 63) by microbiota alpha (within group) and beta (between groups) diversity analyses and distribution of FCTs. While bifidobacteriaceae dominated two FCTs, its abundance was significantly higher in one (FCT BiH for Bifidobacteriaceae at high abundance) than in the other (FCT Bi for Bifidobacteriaceae). HMO supplementation increased the number of infants with FCT BiH (predominant in BF) at the expense of FCT Bi (predominant in control). We explored the association of the FCTs with reported morbidities and medication use up to 12 months. Formula-fed infants with FCT BiH at 3 months were significantly less likely to require antibiotics during the first year than those with FCT Bi. Previously reported lower rates of infection-related medication use with HMOs may therefore be linked to gut microbiota community types. (This study has been registered at ClinicalTrials.gov under registration number NCT01715246.)IMPORTANCE Human milk is the sole and recommended nutrition for the newborn infant and contains one of the largest constituents of diverse oligosaccharides, dubbed human milk oligosaccharides (HMOs). Preclinical and clinical association studies indicate that HMOs have multiple physiological functions largely mediated through the establishment of the gut microbiome. Until recently, HMOs were not available to investigate their role in randomized controlled intervention trials. To our knowledge, this is the first report on the effects of 2 HMOs on establishing microbiota in newborn infants. We provide a detailed description of the microbiota changes observed upon feeding a formula with 2 HMOs in comparison to breastfed reference infants' microbiota. Then, we associate the microbiota to long-term health as assessed by prescribed antibiotic use.

Transcriptomic Analysis Links Eosinophilic Esophagitis and Atopic Dermatitis.

Background: Eosinophilic esophagitis (EoE) is commonly associated with concomitant atopic diseases including atopic dermatitis (AD) and allergic airway (AA) diseases including asthma. Despite this link and the shared pathologic features across these three disorders, detailed analyses of the unifying molecular pathways are lacking. Objectives: We sought to investigate the mRNA expression profile overlap between EoE, AA, and AD and to identify the involvement of interleukin 13 (IL-13) in modulating gene expression. Methods: Whole-genome mRNA expression analyses were performed on tissue specimens (biopsies or nasal brushes) from patients with EoE, AD, and AA, and IL-13-stimulated primary human epithelial cells from the esophagus, the skin, and the airways. Results: By human disease expression profiles, EoE evidenced a significantly higher overlap (p = 0.0006) with AD (181 transcripts; 10%) than with AA (124 transcripts, 7%). Only 18 genes were found to be commonly dysregulated among the three diseases; these included filaggrin, histamine receptor H1, claudin 1, cathepsin C, plasminogen activator urokinase receptor, and suppressor of cytokine signaling 3. Ontogenetic analysis revealed a common immune/inflammatory response among the three diseases and a different epithelial response (epidermal cell differentiation) between EoE and AA. The overlap between the IL-13-stimulated epithelial cell transcriptome and the respective disease transcriptome was 22, 9, and 5% in EoE, AD, and AA, respectively, indicating a greater involvement of the IL-13 pathway in EoE than AA (p = 0.0007) or AD (p = 0.02). Conclusion: EoE, AD, and AA share a common set of disease-specific transcripts, highlighting common molecular etiology. Their comparative analysis indicates relatively closer relationships between EoE and AD, particularly centered around IL-13-driven pathways. Therefore, these findings provide an increased rationale for shared therapeutic strategies.

The Mouse Microbiome Is Required for Sex-Specific Diurnal Rhythms of Gene Expression and Metabolism.

The circadian clock and associated feeding rhythms have a profound impact on metabolism and the gut microbiome. To what extent microbiota reciprocally affect daily rhythms of physiology in the host remains elusive. Here, we analyzed transcriptome and metabolome profiles of male and female germ-free mice. While mRNA expression of circadian clock genes revealed subtle changes in liver, intestine, and white adipose tissue, germ-free mice showed considerably altered expression of genes associated with rhythmic physiology. Strikingly, the absence of the microbiome attenuated liver sexual dimorphism and sex-specific rhythmicity. The resulting feminization of male and masculinization of female germ-free animals is likely caused by altered sexual development and growth hormone secretion, associated with differential activation of xenobiotic receptors. This defines a novel mechanism by which the microbiome regulates host metabolism.

Interaction of bifidobacteria with Caco-2 cells-adhesion and impact on expression profiles.

The aim of the present study was to study different strains of bifidobacteria for adhesion to Caco-2 intestinal epithelial cells (IECs) and to test for the mRNA response of these cells following interaction with bifidobacteria. Adhesion was tested at different pH conditions using model epithelia consisting of transwell cultures of fully differentiated Caco-2 cells. Microarrays were used to characterize changes in global expression profiles of Caco-2 cells co-cultured with peripheral blood mononuclear cells (PBMCs) and challenged with non-pathogenic Escherichia coli D2241 or four different strains of bifidobacteria. Furthermore, cytokine mRNA of IECs in responses to challenge with Bifidobacterium bifidum S17 or E. coli D2241 was tested in PBMC-sensitised Caco-2 cells using RT-PCR. Bifidobacteria showed strain-specific adhesion to Caco-2. Shift of apical pH from 7 to 4.5 resulted in strain-specific changes of adhesion. Global expression profiles of PBMC-sensitised Caco-2 cells revealed differential expression of a significant number of genes only after challenge with E. coli D2241 while cells were essentially unresponsive to challenge with four strains of bifidobacteria showing different adhesion properties. Using a RT-PCR approach, in the same system a similar differential expression after challenge with E. coli D2241 or B. bifidum S17 was observed for various immune markers. The presented results suggest that Caco-2 cells might be specifically unresponsive to challenge with bifidobacteria irrespective of the level of adhesion.

Anti-inflammatory effects of bifidobacteria by inhibition of LPS-induced NF-kappaB activation.

AIM: Different strains of bifidobacteria were analysed for their effects on HT-29 intestinal epithelial cells (IECs) in in vitro models both of the non-inflamed and inflamed intestinal epithelium. METHODS: A reporter gene system in HT-29 cells was used to measure levels of NF-kappaB activation after challenge with bifidobacteria or after bacterial pre-treatment following LPS challenge. IL-8 protein and pro-inflammatory gene expression was investigated using normal HT-29 cells. RESULTS: None of the bifidobacteria tested induced activation of nuclear factor kappaB (NF-kappaB) indicating that bifidobacteria themselves do not induce inflammatory events in IECs. However, six out of eight bifidobacteria tested inhibited lipopolysaccharide- (LPS-) induced NF-kappaB activation in a dose- and strain-dependent manner. In contrast, NF-kappaB activation in response to challenge with tumor necrosis factor-alpha (TNF-alpha) was affected by none of the tested bifidobacteria, indicating that the inhibitory effect of bifidobacteria is specific for LPS-induced inflammation in IECs. As shown with two of the six inhibition-positive bifidobacteria, LPS-induced inhibition of NF-kappaB activation was accompanied by a dose-dependent decrease of interleukin 8 (IL-8) secretion and by lower mRNA levels for IL-8, TNF-alpha, cyclooxygenase 2 (Cox-2), and intercellular adhesion molecule 1 (ICAM-1). CONCLUSION: Some strains of bifidobacteria are effective in inhibiting LPS-induced inflammation and thus might be appropriate candidates for probiotic intervention in chronic intestinal inflammation.

Anti-inflammatory effects of Lacto-Wolfberry in a mouse model of experimental colitis.

AIM: To investigate the anti-inflammatory properties of Lacto-Wolfberry (LWB), both in vitro and using a mouse model of experimental colitis. METHODS: The effects of LWB on lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) and interleukin (IL)-6 secretion were assessed in a murine macrophage cell line. in vitro assessment also included characterizing the effects of LWB on the activation of NF-E2 related 2 pathway and inhibition of tumor necrosis factor-α (TNF-α)-induced nuclear factor-κB (NF-κB) activation, utilizing reporter cell lines. Following the in vitro assessment, the anti-inflammatory efficacy of an oral intervention with LWB was tested in vivo using a preclinical model of intestinal inflammation. Multiple outcomes including body weight, intestinal histology, colonic cytokine levels and anti-oxidative measures were investigated. RESULTS: LWB reduced the LPS-mediated induction of ROS production [+LPS vs 1% LWB + LPS, 1590 ± 188.5 relative luminescence units (RLU) vs 389 ± 5.9 RLU, P < 0.001]. LWB was more effective than wolfberry alone in reducing LPS-induced IL-6 secretion in vitro (wolfberry vs 0.5% LWB, 15% ± 7.8% vs 64% ± 5%, P < 0.001). In addition, LWB increased reporter gene expression via the anti-oxidant response element activation (wolfberry vs LWB, 73% ± 6.9% vs 148% ± 28.3%, P < 0.001) and inhibited the TNF-α-induced activation of the NF-κB pathway (milk vs LWB, 10% ± 6.7% vs 35% ± 3.3%, P < 0.05). Furthermore, oral supplementation with LWB resulted in a reduction of macroscopic (-LWB vs +LWB, 5.39 ± 0.61 vs 3.66 ± 0.59, P = 0.0445) and histological scores (-LWB vs +LWB, 5.44 ± 0.32 vs 3.66 ± 0.59, P = 0.0087) in colitic mice. These effects were associated with a significant decrease in levels of inflammatory cytokines such as IL-1ß (-LWB vs +LWB, 570 ± 245 µg/L vs 89 ± 38 µg/L, P = 0.0106), keratinocyte-derived chemokine/growth regulated protein-α (-LWB vs +LWB, 184 ± 49 µg/L vs 75 ± 20 µg/L, P = 0.0244), IL-6 (-LWB vs +LWB, 318 ± 99 µg/L vs 117 ± 18 µg/L, P = 0.0315) and other pro-inflammatory proteins such as cyclooxygenase-2 (-LWB vs +LWB, 0.95 ± 0.12 AU vs 0.36 ± 0.11 AU, P = 0.0036) and phosphorylated signal transducer and activator of transcription-3 (-LWB vs +LWB, 0.51 ± 0.15 AU vs 0.1 ± 0.04 AU, P = 0.057). Moreover, antioxidant biomarkers, including expression of gene encoding for the glutathione peroxidase, in the colon and the plasma anti-oxidant capacity were significantly increased by supplementation with LWB (-LWB vs +LWB, 1.2 ± 0.21 mmol/L vs 2.1 ± 0.19 mmol/L, P = 0.0095). CONCLUSION: These results demonstrate the anti-inflammatory properties of LWB and suggest that the underlying mechanism is at least in part due to NF-κB inhibition and improved anti-oxidative capacity.

Bifidobacterium lactis attenuates onset of inflammation in a murine model of colitis.

AIM: To assess the anti-inflammatory effect of the probiotic Bifidobacterium lactis (B. lactis) in an adoptive transfer model of colitis. METHODS: Donor and recipient mice received either B. lactis or bacterial culture medium as control (deMan Rogosa Sharpe) in drinking water for one week prior to transfer of a mix of naive and regulatory T cells until sacrifice. RESULTS: All recipient mice developed signs of colonic inflammation, but a significant reduction of weight loss was observed in B. lactis-fed recipient mice compared to control mice. Moreover, a trend toward a diminution of mucosal thickness and attenuated epithelial damage was revealed. Colonic expression of pro-inflammatory and T cell markers was significantly reduced in B. lactis-fed recipient mice compared to controls. Concomitantly, forkhead box protein 3, a marker of regulatory T cells, was significantly up-regulated by B. lactis. CONCLUSION: Daily oral administration of B. lactis was able to reduce inflammatory and T cells mediators and to promote regulatory T cells specific markers in a mouse model of colitis.

Intake of a milk-based wolfberry formulation enhances the immune response of young-adult and aged mice.

Aging is associated with alterations of immune responses. Wolfberry, a popular Chinese functional ingredient, is prized for its anti-aging properties; however, little is known about the immunological effect of wolfberry intake. The purpose of this study was to examine the effect of dietary intake of a milk-based formulation of wolfberry, named Lacto-Wolfberry, on in vivo and ex vivo parameters of adaptive immunity in young-adult and aged mice. Over 44 days, young-adult (2 months) and aged (21 months) C57BL/6J mice were fed ad libitum with a controlled diet and received drinking water supplemented or not with 0.5% (wt/vol) Lacto-Wolfberry. All mice were immunized on day 15 and challenged on day 22 with a T cell- dependent antigen, keyhole limpet hemocyanin (KLH). Lacto-Wolfberry supplementation significantly increased in vivo systemic immune markers that are known to decline with aging. Indeed, both antigen-(KLH) specific humoral response and cell-mediated immune responses in young-adult and aged mice were enhanced when compared to their respective controls. No significant effect of Lacto-Wolfberry supplementation was observed on ex vivo spleen cells proliferative response to mitogens and on splenocyte T cell subsets. In conclusion, dietary intake of Lacto-Wolfberry may favorably modulate the poor responsiveness to antigenic challenge observed with aging.

N(epsilon)-carboxymethyllysine-modified proteins are unable to bind to RAGE and activate an inflammatory response.

Advanced glycation endproducts (AGEs) containing carboxymethyllysine (CML) modifications are generally thought to be ligands of the receptor for AGEs, RAGEs. It has been argued that this results in the activation of pro-inflammatory pathways and diseases. However, it has not been shown conclusively that a CML-modified protein can interact directly with RAGE. Here, we have analyzed whether beta-lactoglobulin (bLG) or human serum albumin (HSA) modified chemically to contain only CML (10-40% lysine modification) can (i) interact with RAGE in vitro and (ii) interact with and activate RAGE in lung epithelial cells. Our results show that CML-modified bLG or HSA are unable to bind to RAGE in a cell-free assay system (Biacore). Furthermore, they are unable to activate pro-inflammatory signaling in the cellular system. Thus, CML probably does not form the necessary structure(s) to interact with RAGE and activate an inflammatory signaling cascade in RAGE-expressing cells.

A serpin from the gut bacterium Bifidobacterium longum inhibits eukaryotic elastase-like serine proteases.

Serpins form a large class of protease inhibitors involved in regulation of a wide spectrum of physiological processes. Recently identified prokaryotic members of this protein family may provide a key to the evolutionary origins of the unique serpin fold and the associated inhibitory mechanism. We performed a biochemical characterization of a serpin from Bifidobacterium longum, an anaerobic Gram-positive bacterium that naturally colonizes human gastrointestinal tract. The B. longum serpin was shown to efficiently inhibit eukaryotic elastase-like proteases with a stoichiometry of inhibition close to 1. Porcine pancreatic elastase and human neutrophil elastase were inhibited with the second order association constants of 4.7 x 10(4) m(-1) s(-1) and 2.1 x 10(4) m(-1) s(-1), respectively. The B. longum serpin is expected to be active in the gastrointestinal tract, because incubation of the purified recombinant serpin with mouse feces produces a stable covalent serpin-protease adduct readily detectable by SDS-PAGE. Bifidobacteria may encounter both pancreatic elastase and neutrophil elastase in their natural habitat and protection against exogenous proteolysis may play an important role in the interaction between these commensal bacteria and their host.