A longitudinal study of infant faecal microbiota during weaning

For infants, the introduction of food other than breast milk is a high risk period due to diarrheal diseases, and may be corroborated with a shift in the faecal microbiota. This longitudinal study was the first undertaken to understand the effect of the supplementation on the infant's faecal microbiota and particularly the bifidobacteria. Eleven infants were enrolled. Their faecal microbiota were analysed using temporal temperature gradient gel electrophoresis (TTGE) with bacterial and bifidobacterial primers. In parallel, bifidobacterial counts were followed using competitive PCR. Three periods were distinguished: exclusive breastfeeding (Bf period), weaning (i.e. formula-milk addition, W period) and postweaning (i.e. breastfeeding cessation, Pw period). The bifidobacterial counts were not modified, reaching 10.5 (Log10 cells g(-1) wet weight). In the TTGE profiles, the main identified bands corresponded to Escherichia coli, Ruminococcus sp. and Bifidobacterium sp., more precisely Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium breve. For both TTGE profiles, the analysis of the distance suggested a maturation of the faecal microbiota but no correlation could be established with the diet. Despite a high interindividual variability, composition of the faecal microbiota appeared more homogenous after weaning and this point may be correlated with the cessation of breastfeeding.     

Quantitative assessment of faecal bifidobacterial populations by real-time PCR using lanthanide probes

AIM: To develop real-time quantitative PCR methods, based on the use of probes labelled with a stable fluorescent lanthanide chelate, for the quantification of different human faecal bifidobacterial populations. METHODS AND RESULTS: The designed quantitative PCR assays were found to be specific for the corresponding Bifidobacterium species or groups (Bifidobacterium longum group, Bifidobacterium catenulatum group, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium angulatum, Bifidobacterium bifidum and Bifidobacterium dentium). The detection limits of the methodologies used ranged between 2 x 10(5) and 9 x 10(3) cells g(-1) of faeces. The applicability of the developed assays was tested by analysing 20 human faecal samples. Bif. longum group was found to be the qualitatively and quantitatively predominant bifidobacterial group. CONCLUSIONS: The real-time PCR procedures developed here are specific, accurate, rapid and easy methods for the quantification of Bifidobacterium groups or species in human faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed procedures will facilitate rapid and objective counting of large numbers of samples increasing our knowledge on the role of gut bifidobacterial microbiota in health and disease. This will contribute to the efficient use of intestinal bacterial assays in research, food and pharmaceutical development as well as in the assessment of dietary management of diseases.     

Culture-independent analysis of fecal microbiota in infants, with special reference to Bifidobacterium species

Fecal microbiota of 31 breast-fed, 26 mix-fed, and 11 bottle-fed infants were analyzed by using terminal restriction fragment length polymorphism (T-RFLP), and culture method. We first determined the total and cultivated bacterial counts in infant fecal microbiota. Only approximately 30% of bacteria present in fecal microbiota were cultivable while the remainder was yet-to-be cultured bacteria. Sixty-eight fecal samples were divided into two clusters (I and II) by T-RFLP analysis, and then subdivided into five subclusters (Ia, Ib, IIa, IIb and IIc). There was no clear relationship between clusters and feeding method. A proportion of bifidobacteria was detected in the fecal material by PCR method using species-specific primers. The predominant Bifidobacterium spp. was Bifidobacterium longum longum type (43 samples (63.2%)), followed by B. longum infantis type (23 samples (33.8%)) and B. breve (16 samples (23.5%)). The distribution of Bifidobacterium spp. was similar in the three feeding groups. In contrast, the high incidence of B. breve in cluster I, especially subcluster Ia and B. longum longum type in cluster II, especially subcluster IIa and IIc were characterized by T-RFLP method. Our results showed that the colonization of Bifidobacterium spp. in infant feces correlated with the T-RFLP clusters.     

大肠埃希菌—双歧杆菌穿梭表达载体的构建及白介素-10基因的表达

目的构建能够在大肠埃希菌和双歧杆菌中穿梭表达目的基因的载体,并用此载体在大肠埃希菌和双歧杆菌中表达人白介素-10基因（hIL-10）的蛋白产物;为hIL-10基因重组双歧杆菌治疗炎症性肠病做前期准备。方法以质粒pDG7为模板扩增pMB1片段,构建表达质粒pET28B1。用PCR法扩增hIL-10基因,将此目的基因以及pET28B1经酶切后用连接酶连接,形成重组质粒pET28B1-hIL10。pET28B1-IL10转染大肠埃希菌BL21和长双歧杆菌。最后用Western blot检测hIL-10基因在大肠埃希菌和长双歧杆菌中的表达情况。结果pET28B1-hIL10阳性克隆扩增后提取质粒并进行基因测序,结果显示插入片段为hIL-10,序列正确且无突变。hIL-10基因在大肠埃希菌、长双歧杆菌中的诱导表达产物通过Western blot检测验证为IL-10蛋白,显示该hIL-10表达载体在大肠埃希菌阳性克隆中经诱导可高量表达,在长双歧杆菌体中有少量表达。结论成功构建质粒pET28B1,该质粒能够在大肠埃希菌和双歧杆菌中穿梭表达目的基因hIL-10。     

PCR-ELISA II: Analysis of Bifidobacterium populations in human faecal samples from a consumption trial with Bifidobacterium lactis Bb-12 and a galacto-oligosaccharide preparation

A PCR-ELISA method was extended for detection of most common Bifidobacterium species in humans and applied to a feeding trial including administration of Bifidobacterium lactis Bb-12 and galacto-oligosaccharide (GOS)-containing syrup as probiotic and prebiotic preparations, respectively. For PCR-ELISA, oligonucleotide probes based on 16S rDNA sequences were designed and tested for specificity and sensitivity with nine different bifidobacterial species followed by analysis of faecal samples. Bifidobacteria were monitored for their fluctuations during and after the feeding trial. Bifidobacterium longum was the most common species found in the faecal samples, followed by B. adolescentis and B. bifidum. During ingestion of the probiotic B. lactis Bb-12, the strain appeared in the faeces but was absent again one week after finishing of the trial. The species that were observed in the faecal samples taken prior to the feeding experiments persisted also in samples derived from the pre-feeding and feeding periods. The most consistent change observed was the decrease in the relative amount of B. longum in the test group ingesting either B. lactis Bb-12 alone or in combination with GOS-syrup. Since the amounts of B. longum increased again in the post-feeding sample with these subjects, it may suggest that to some extent B. lactis Bb-12 is able to transiently replace B. longum.     

Characterization of human intestinal bifidobacteria using competitive PCR and PCR-TTGE

In this study, a competitive PCR was developed to estimate the quantity of bifidobacteria in human faecal samples using two 16S rRNA gene Bifidobacterium genus-specific primers, Bif164f and Bif662r. A PCR-temporal temperature gradient gel electrophoresis (TTGE) with the same primers also allowed us to describe the Bifidobacterium species present in these faecal samples. The PCR product obtained from the competitor had 467 bp, and was 47 bp shorter than the PCR products obtained from Bifidobacterium strains. The number of bifidobacterial cells was linear from 10 to 10(8) cells per PCR assay. Taking into account the dilutions of the extracted DNA, the linear range was over 8 x 10(5) bifidobacteria g(-1) of faeces. Reproducibility was assessed from 10 independent DNA extractions from the same stool and the coefficient of variation was 0.5%. When the competitive PCR was compared with the culture method, a similar count of seven out of nine Bifidobacterium pure cultures were obtained, or had a difference inferior or equal to 1 log(10). In faecal samples, the enumeration of Bifidobacterium genus in most cases gave higher results with competitive PCR than with culture on selective Columbia-Beerens agar pH 5 (P < 0.05). In conclusion, this competitive PCR allows a rapid, highly specific and reproducible quantification of Bifidobacterium genus in faecal samples. TTGE fragments co-migrating with B. longum CIP64.63 fragment were found in 10 out of 11 faecal samples. Bifidobacterium adolescentis and B. bifidum were detected in five out of 11 subjects. Thus, cPCR and PCR-TTGE can be associated in order to characterize human faecal bifidobacteria.     

Arabinoxylans and inulin differentially modulate the mucosal and luminal gut microbiota and mucin-degradation in humanized rats

The endogenous gut microbiota affects the host in many ways. Prebiotics should favour beneficial intestinal microbes and thus improve host health. In this study, we investigated how a novel class of potential prebiotic long-chain arabinoxylans (LC-AX) and the well-established prebiotic inulin (IN) modulate the gut microbiota of humanized rats. Six weeks after axenic rats were inoculated with a human faecal microbiota, their colonic microbiota was similar to this inoculum (～ 70%), whereas their caecal microbiota was enriched with Verrucomicrobia and Firmicutes concomitant with lower abundance of Bacteroidetes. Moreover, different Bifidobacterium species colonized the lumen (B. adolescentis) and mucus (B. longum and B. bifidum). Both LC-AX and IN increased SCFA levels and induced a shift from acetate towards health-promoting propionate and butyrate respectively. By applying a high-resolution phylogenetic micro-array (HITChip) at the site of fermentation (caecum), IN and LC-AX were shown to stimulate bacterial groups with known butyrate-producers (Roseburia intestinalis, Eubacterium rectale, Anaerostipes caccae) and bifidobacteria (B. longum) respectively. Prebiotic administration also resulted in lower caecal abundances of the mucin-degrading Akkermansia muciniphila and potentially more mucin production by the host. Both factors might explain the increased caecal mucin levels for LC-AX (threefold) and IN (sixfold). These mucins were degraded along the colon, resulting in high faecal abundances of Akkermansia muciniphila for LC-AX and especially IN-treated rats. Finally, the microbial changes caused an adaptation period for the host with less weight gain, after which the host fine-tuned the interaction with this altered microbiota. Our results demonstrate that next to IN, LC-AX are promising prebiotic compounds by stimulating production of health-promoting metabolites by specific microbes in the proximal regions. Further, prebiotic supplementation shifted mucin degradation to distal regions, where mucin-degraders may produce beneficial metabolites (e.g. propionate by Akkermansia muciniphila), so that prebiotics may potentially improve gut health along the entire length of the intestine.     

In vitro fermentation of rice bran combined with Lactobacillus acidophilus 14 150B or Bifidobacterium longum 05 by the canine faecal microbiota

The fermentability of rice bran (RB), alone or in combination with one of two probiotics, by canine faecal microbiota was evaluated in stirred, pH-controlled, anaerobic batch cultures. RB enhanced the levels of bacteria detected by probes Bif164 (bifidobacteria) and Lab158 (lactic acid bacteria); however, addition of the probiotics did not have a significant effect on the predominant microbial counts compared with RB alone. RB sustained levels of Bifidobacterium longum 05 throughout the fermentation; in contrast, Lactobacillus acidophilus 14 150B levels decreased significantly after 5-h fermentation. RB fermentation induced changes in the short-chain fatty acid (SCFA) profile. However, RB combined with probiotics did not alter the SCFA levels compared with RB alone. Denaturing gradient gel electrophoresis analysis of samples obtained at 24 h showed a treatment effect with RB, which was not observed in the RB plus probiotic systems. Overall, the negative controls displayed lower species richness than the treatment systems and their banding profiles were distinct. This study illustrates the ability of a common ingredient found in pet food to modulate the canine faecal microbiota and highlights that RB may be an economical alternative to prebiotics for use in dog food.     

Quantitative real-time PCR assays to identify and quantify fecal Bifidobacterium species in infants receiving a prebiotic infant formula

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5' nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5' nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% +/- 9.8% to 73.4% +/- 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% +/- 1.92% and 8.11% +/- 4.12%, respectively, versus 0.15% +/- 0.11% and 1.38% +/- 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.     

Adherence and cytokine induction in Caco-2 cells by bacterial populations from a three-stage continuous-culture model of the large intestine

Adherence of bacteria to epithelial cells is an important step in colonization and immune modulation in the large bowel. The aims of this study were to use a three-stage continuous-culture system (CCS) to investigate how environmental factors affect bacterial attachment to Caco-2 cells and modulation of cytokine expression by gut microorganisms, including a probiotic Bifidobacterium longum strain, DD2004. The CCS simulated environmental conditions in the proximal large intestine (vessel 1 [V1]) and distal colon (V2 and V3) at two different system retention times (R) within the range of normal colonic transits (20 and 60 h). The model was inoculated with human fecal material, and fluorescence in situ hybridization (FISH) was used to characterize microbial populations and to assess bacterial attachment to Caco-2 cells. Real-time quantitative PCR (qPCR) was employed to measure cytokine gene expression following challenge with bacteria from different components of the CCS in the presence and absence of B. longum. At an R of 60 h, bacterial adherence increased from V1 to V3, but this trend was reversed at an R of 20 h. Atopobia were the predominant adherent organisms detected at both system retention times in each culture vessel. Modulation of transforming growth factor β1 (TGF-β1), interleukin 6 (IL-6), and IL-18 gene expression by CCS bacteria was marked at an R of 60 h, while at an R of 20 h, IL-4, IL-10, TGF-β2, IL-1α, and tumor necrosis factor alpha (TNF-α) were significantly affected. The addition of B. longum affected cytokine expression significantly at both retention times. This study demonstrates that environmental determinants regulate the adherence properties of intestinal bacteria and their abilities to regulate cytokine synthesis.     

Quantitative detection of probiotic Bifidobacterium strains in bacterial mixtures by using real-time PCR

Strain-specific rRNA-targeted primers were designed for the quantitative detection of Bifidobacterium infantis Y1, B. breve Y8 and B. longum Y10 used in a pharmaceutical probiotic product (VSL-3). PCR and real-time PCR techniques with the selected primers were employed for the direct enumeration of the bifidobacteria in the probiotic preparation and for studying their kinetic characteristics in batch cultures. These analysis revealed that B. infantis Y1 was the predominant strain in the probiotic product and that its growth rate was the highest. Since B. infantis Y1, B. breve Y8 and B. longum Y10 are co-cultured during the industrial production of VSL-3, the kinetic characteristics of these strains can explain their different concentrations in the probiotic preparation. A validation of the PCR quantification method was performed by identifying a representative number of isolates from the bacterial mixtures with automated ribotyping. The methodology described represents a useful tool for the specific quantitative detection of bacterial strains and species in complex mixtures such as pharmaceutical preparations, dairy starter cultures, faecal samples and biopsies.     

Multiplex PCR using 16S rRNA gene-targeted primers for the identification of bifidobacteria from human origin

Three multiplex polymerase chain reactions (PCRs) targeted on Bifidobacterium and related species were designed to identify human species. The selected primers yielded amplified products of various sizes, each specific for a species. Three to four pairs were gathered in one PCR reaction and their specificity under multiplex conditions was confirmed using DNA from 26 reference strains. Using this technique on unidentified faecal strains, B. bifidum, B. longum and B. breve species were commonly recovered in infants while B. adolescentis, B. catenulatum/B. pseudocatenulatum continuum and B. longum species were predominant in adults. Thus, a single PCR can provide the assignment of a strain to one these species, reducing the number of PCR reactions and hands-on time for the identification of human isolates of bifidobacteria. Moreover, this technique is also applicable for the in situ detection of bifidobacteria in DNA extracts from human stools.     

Endo-β-N-acetylglucosaminidases from Infant Gut-associated Bifidobacteria Release Complex N-glycans from Human Milk Glycoproteins

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-β-N-acetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man(3)GlcNAc(2)(α1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies.     

Genus- and species-specific PCR primers for the detection and identification of bifidobacteria

16SrDNA-targeted genus- and species-specific PCR primers have been developed and used for the identification and detection of bifidobacteria. These primers cover all of the described species that inhabit the human gut, or occur in dairy products. Identification of cultured bifidobacteria using PCR primer pairs is rapid and accurate, being based on nucleic acid sequences. Detection of bifidobacteria can be achieved using DNA extracted from human faeces as template in PCR reactions. We have found that, in adult faeces, the Bifidobacterium catenulatum group was the most commonly detected species, followed by Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum. In breastfed infants, Bifidobacterium breve was the most frequently detected species, followed by Bifidobacterium infantis, B. longum and B. bifidum. It was notable that the B. catenulatum group was detected with the highest frequency in adults, although it has often been reported that B. adolescentis is the most common species. Real-time, quantitative PCR using primers targeting 16S rDNA shows promise in the enumeration of bifidobacteria in faecal samples. The approach to detect the target bacteria with quantitative PCR described in this review will contribute to future studies of the composition and dynamics of the intestinal microflora.     

Bifidobacterium longum CECT 7347 modulates immune responses in a gliadin-induced enteropathy animal model

Coeliac disease (CD) is an autoimmune disorder triggered by gluten proteins (gliadin) that involves innate and adaptive immunity. In this study, we hypothesise that the administration of Bifidobacterium longum CECT 7347, previously selected for reducing gliadin immunotoxic effects in vitro, could exert protective effects in an animal model of gliadin-induced enteropathy. The effects of this bacterium were evaluated in newborn rats fed gliadin alone or sensitised with interferon (IFN)-γ and fed gliadin. Jejunal tissue sections were collected for histological, NFκB mRNA expression and cytokine production analyses. Leukocyte populations and T-cell subsets were analysed in peripheral blood samples. The possible translocation of the bacterium to different organs was determined by plate counting and the composition of the colonic microbiota was quantified by real-time PCR. Feeding gliadin alone reduced enterocyte height and peripheral CD4+ cells, but increased CD4+/Foxp3+ T and CD8+ cells, while the simultaneous administration of B. longum CECT 7347 exerted opposite effects. Animals sensitised with IFN-γ and fed gliadin showed high cellular infiltration, reduced villi width and enterocyte height. Sensitised animals also exhibited increased NFκB mRNA expression and TNF-α production in tissue sections. B. longum CECT 7347 administration increased NFκB expression and IL-10, but reduced TNF-α, production in the enteropathy model. In sensitised gliadin-fed animals, CD4+, CD4+/Foxp3+ and CD8+ T cells increased, whereas the administration of B. longum CECT 7347 reduced CD4+ and CD4+/Foxp3+ cell populations and increased CD8+ T cell populations. The bifidobacterial strain administered represented between 75-95% of the total bifidobacteria isolated from all treated groups, and translocation to organs was not detected. These findings indicate that B. longum attenuates the production of inflammatory cytokines and the CD4+ T-cell mediated immune response in an animal model of gliadin-induced enteropathy.     

Complete genome sequence of Bifidobacterium longum subsp. longum KACC 91563

Bifidobacterium longum strains predominate in the colonic microbiota of breast-fed infants. Here we report the complete genome sequence of B. longum subsp. longum KACC 91563, isolated from feces of neonates. A single circular chromosome of 2,385,301 bp contains 1,980 protein-coding genes, 56 tRNA genes, and 3 rRNA operons.     

Diversity of gut Bifidobacterium species is not altered between allergic and non-allergic French infants

Some clinical studies have suggested a relationship between allergic diseases and gut microbiota. We aimed to study bifidobacterial colonization at species and strain levels in ten allergic French infants included at their first clinical consultation and 20 controls matching for age at sampling, mode of delivery, per partum antibiotics, type of feeding and antibiotics in the first weeks of life. The faecal microbiota was analyzed by culture methods and TTGE. Bifidobacterial species and strains were identified using multiplex PCR and Box-PCR fingerprinting. No differences were observed between groups in the number of colonized infants or in the levels of colonization by the main aerobic and anaerobic genera. All infants were colonized with high levels of Bifidobacterium except for one in each group. One to 5 Bifidobacterium species and 1 to 7 strains were observed per subject independently of allergic status and age at sampling. Our study showed the infants to be colonized by several species and strains, including several strains from the same species. This diversity in Bifidobacterium colonization was not related with the allergic status and showed that the link between Bifidobacterium colonization and allergic diseases is complex and cannot be restricted to the role attributed to Bifidobacterium species.     

Cytokine deviation induced by intrathymic injection of staphylococcal enterotoxin B (SEB)

We have previously shown that intrathymic (i.t.) injection of staphylococcal enterotoxin B (SEB) to mice induces both T cell clonal deletion and IL-2-dependent anergy. In the present study, we have used a quantitative RT-PCR to demonstrate that i.t. administration of SEB induced a significant decrease in the levels of the IL-2 and IFN-gamma mRNAs in total splenocytes, from day 7 to day 28 post-injection. I.t. SEB injection also induced a significant increase in the levels both of IL-10 and TGF-beta mRNAs on day 7, leading to a significant enhance in the IL-10 + TGF-beta/IL-2 + IFN-gamma mRNA ratio on days 7 and 28. By contrast, IL-10 and TGF-beta mRNAs were unchanged after intraperitoneal (i.p.) or subcutaneous (s.c.) SEB injections, although both IL-2 and IFN-gamma mRNA levels were decreased. The cytokine mRNA ratio was enhanced on days 7 and 28 after i.p. injection. Interestingly, a cytokine mRNA ratio of a least 10 in favour of IL-10 plus TGF-beta mRNAs was correlated with the hyporesponsive state observed in vitro after i.t. and i.p. injections. Our results clearly demonstrate that i.t. SEB administration induces a switch from Th1-type to Th2-type cytokine expression in the spleen. The deviation from IL-2 plus IFN-gamma towards IL-10 plus TGF-beta expression could be responsible for the immunoregulatory effect exerted upon SEB-reactive T cells, which is characterized by an IL-2-dependent, specific anergy in vitro. Moreover, it highlights the crucial role of the route of SEB injection in the pattern of cytokine expression.     

Influence of milk-feeding type and genetic risk of developing coeliac disease on intestinal microbiota of infants: the PROFICEL study

Interactions between environmental factors and predisposing genes could be involved in the development of coeliac disease (CD). This study has assessed whether milk-feeding type and HLA-genotype influence the intestinal microbiota composition of infants with a family history of CD. The study included 164 healthy newborns, with at least one first-degree relative with CD, classified according to their HLA-DQ genotype by PCR-SSP DQB1 and DQA1 typing. Faecal microbiota was analysed by quantitative PCR at 7 days, and at 1 and 4 months of age. Significant interactions between milk-feeding type and HLA-DQ genotype on bacterial numbers were not detected by applying a linear mixed-model analysis for repeated measures. In the whole population, breast-feeding promoted colonization of C. leptum group, B. longum and B. breve, while formula-feeding promoted that of Bacteroides fragilis group, C. coccoides-E. rectale group, E. coli and B. lactis. Moreover, increased numbers of B. fragilis group and Staphylococcus spp., and reduced numbers of Bifidobacterium spp. and B. longum were detected in infants with increased genetic risk of developing CD. Analyses within subgroups of either breast-fed or formula-fed infants indicated that in both cases increased risk of CD was associated with lower numbers of B. longum and/or Bifidobacterium spp. In addition, in breast-fed infants the increased genetic risk of developing CD was associated with increased C. leptum group numbers, while in formula-fed infants it was associated with increased Staphylococcus and B. fragilis group numbers. Overall, milk-feeding type in conjunction with HLA-DQ genotype play a role in establishing infants' gut microbiota; moreover, breast-feeding reduced the genotype-related differences in microbiota composition, which could partly explain the protective role attributed to breast milk in this disorder.     

Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria

The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono- and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.