Bifidobacterium population analysis in the infant gut by direct mapping of genomic hybridization patterns: potential for monitoring temporal development and effects of dietary regimens.

A bifidobacterial mixed-species microarray platform was used in a proof-of-principle study to address the composition and development of bifidobacteria in DNA extracted from faecal samples. These were collected in a time-course of 2 years since birth and derived from human infants that were breastfed, standard formula-fed or received a prebiotic formula during their weaning period. A set of over 50 samples was analysed, testifying for the throughput of the designed platform for multiple genome hybridizations. The generated data revealed that faecal samples of breastfed infants contained a high abundance of genomic DNA homologous to Bifidobacterium breve. In contrast, faecal samples from standard formula-fed infants lacked detectable amounts of this B. breve DNA but contained genes with high similarity to B. longum. Remarkably, infants that received breastmilk and later a prebiotic formula consisting of a standard formula milk containing a mixture of specific galacto- and fructo-oligosaccharides, continued to harbour a B. breve-dominant faecal population. One infant that received standard formula in combination with the additional B. lactis Bb12 culture, contained significant amounts of faecal DNA belonging to Bb12 but only during the period of ingestion. The microarray platform showed sufficient sensitivity to analyse the B. breve group at the strain level. Overall, the B. breve populations observed in the faecal samples of the studied infants showed a stable composition over time and were unique per infant. In conclusion, our results show the applicability of comparative genome hybridization to study bifidobacterial populations in infant faecal samples without the use of any amplification step.

Mixed-species genomic microarray analysis of fecal samples reveals differential transcriptional responses of bifidobacteria in breast- and formula-fed infants.

Although their exact function remains enigmatic, bifidobacteria are among the first colonizers of the newborn infant gut and further develop into abundant communities, notably in response to diet. Therefore, the transcriptional responses of bifidobacteria in rapidly processed fecal samples from young infants that were fed either breast milk or a formula containing a mixture of galacto- and fructo-oligosaccharides were studied. The presence and diversity of the bifidobacterial fecal communities were determined using PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR for specific species. Changes in the total number of bifidobacteria as well as in species diversity were observed, indicating the metabolic activities of the bifidobacteria within the infant gut. In addition, total RNAs isolated from infant feces were labeled and hybridized to a bifidobacterium-specific microarray comprising approximately 6,000 clones of the major bifidobacterial species of the human gut. Approximately 270 clones that showed the most prominent hybridization with the samples were sequenced. Fewer than 10% of the hybridizing clones contained rRNA genes, whereas the vast majority of the inserts showed matches with protein-encoding genes predicted to originate from bifidobacteria. Although a wide range of functional groups was covered by the obtained sequences, the largest fraction (14%) of the transcribed genes assigned to a functional category were predicted to be involved in carbohydrate metabolism, while some were also implicated in exopolysaccharide production or folate production. A total of three of the above-described protein-encoding genes were selected for quantitative PCR and sequence analyses, which confirmed the expression of the corresponding genes and the expected nucleotide sequences. In conclusion, the results of this study show the feasibility of obtaining insight into the transcriptional responses of intestinal bifidobacteria by analyzing fecal RNA and highlight the in vivo expression of bifidobacterial genes implicated in host-related functions.

Early impairment of gut function and gut flora supporting a role for alteration of gastrointestinal mucosa in human immunodeficiency virus pathogenesis.

Our results show that impairment of the gastrointestinal tracts in human immunodeficiency virus (HIV)-positive patients is present in the early phases of HIV disease. This impairment is associated with alterations in gut microbiota and intestinal inflammatory parameters. These findings support the hypothesis that alterations at the gastrointestinal-tract level are a key factor in HIV pathogenesis.

Enhanced severity of virus associated lower respiratory tract disease in asthma patients may not be associated with delayed viral clearance and increased viral load in the upper respiratory tract.

BACKGROUND: Viral respiratory infections, particularly human rhinovirus (HRV) infections, are the most common cause of asthma exacerbation. HRV infections usually lead to more severe and longer duration of lower respiratory tract (LRT) symptoms in asthmatics than in otherwise healthy individuals. However, the exact mechanism by which viruses contribute to exacerbation of asthma is unknown. OBJECTIVES: The main objective of our study was to investigate the relationship of the enhanced severity of LRT symptoms to viral dynamics or cytokine responses in the upper respiratory tract (URT). STUDY DESIGN: Therefore, we conducted a longitudinal study in which asthmatics and healthy controls were followed during natural viral respiratory tract infections. RESULTS: Our study confirmed that viral respiratory tract infections caused more severe problems of the LRT in asthma patients as compared to healthy controls. However, for all subjects, the severity of LRT symptoms were not related to viral load or prolonged viral shedding in the URT. In addition, we did not detect differences in proinflammatory cytokines in the URT between asthmatics and controls. CONCLUSION: Persistence of the virus, as well as viral load in the URT, may not be associated with the induction and/or persistence of asthmatic symptoms.

Effects of galactooligosaccharide and long-chain fructooligosaccharide supplementation during pregnancy on maternal and neonatal microbiota and immunity--a randomized, double-blind, placebo-controlled study.

BACKGROUND: Galactooligosaccharides (GOS) and long-chain fructooligosaccharides (lcFOS) proliferate bifidobacteria in infant gut microbiota. However, it is not known how GOS and FOS influence the microbiota of pregnant women and whether a potential prebiotic effect is transferred to the offspring. OBJECTIVES: We aimed to test how supplementation with GOS and lcFOS (GOS/lcFOS) in the last trimester of pregnancy affects maternal and neonatal gut microbiota. Variables of fetal immunity were assessed as a secondary outcome. DESIGN: In a randomized, double-blind, placebo-controlled pilot study, 48 pregnant women were supplemented 3 times/d with 3 g GOS/lcFOS (at a ratio of 9:1) or maltodextrin (placebo) from week 25 of gestation until delivery. Percentages of bifidobacteria and lactobacilli within total bacterial counts were detected by fluorescent in situ hybridization and quantitative polymerase chain reaction in maternal and neonatal (days 5, 20, and approximately 182) stool samples. Variables of fetal immunity were assessed in cord blood by using flow cytometry and cytokine multiplex-array analysis. RESULTS: The proportions of bifidobacteria in the maternal gut were significantly higher in the supplemented group than in the placebo group (21.0% and 12.4%, respectively; P = 0.026); the proportion of lactobacilli did not differ between the groups. In neonates, bifidobacteria and lactobacilli percentages, diversity and similarity indexes, and fetal immune parameters did not differ significantly between the 2 groups. Mother-neonate similarity indexes of bifidobacteria decreased over time. CONCLUSIONS: GOS/lcFOS supplementation has a bifidogenic effect on maternal gut microbiota that is not transferred to neonates. The increased maternal bifidobacteria did not affect fetal immunity as measured by a comprehensive examination of cord blood immunity variables.

Dietary supplementation of neutral and acidic oligosaccharides enhances Th1-dependent vaccination responses in mice.

Immunomodulatory effects of oligosaccharide preparations that resemble chemical and functional aspects of human milk oligosaccharides (HMOS) were studied for the development of new concepts in infant nutrition. A dose range of 1-5% (w/w) dietary pectin-derived acidic oligosaccharides (AOS) was tested in a murine influenza vaccination model. In addition, combinations of AOS and a 9:1 mixture of galacto-oligosaccharides and long-chain fructo-oligosaccharides (GOS/FOS) were tested at a fixed total dietary dose of 2% (w/w). It was found that AOS significantly enhanced vaccine-specific delayed-type hypersensitivity (DTH) responses in a dose-dependent manner. This was accompanied by a reduction in T-helper2 (Th2) cytokine production by splenocytes in vitro. Overall, this indicates that the systemic immune response to the vaccine was Th1-skewed by the dietary intervention. Combinations of GOS/FOS and AOS were more effective in enhancing DTH responses than either of the oligosaccharides alone, suggesting interaction effects between these agents. Similar to effects in infants, supplementation of the murine diets with GOS/FOS and combinations of GOS/FOS and AOS for 6-wk enhanced the proportion of fecal bifidobacteria and lactobacilli, but AOS alone did not. In conclusion, these data indicate that GOS/FOS and AOS enhance systemic Th1-dependent immune responses in a murine vaccination model. As Th1-responses are weak in early life in humans, this might suggest that application of these oligosaccharides in infant formulas will be beneficial for the development of the infant's immune system.

A specific prebiotic oligosaccharide mixture stimulates delayed-type hypersensitivity in a murine influenza vaccination model.

Analogous to reported immunomodulatory effects of probiotics, this study was performed to analyse the immunomodulatory properties of prebiotic oligosaccharides that share chemical characteristics with human milk oligosaccharides. A mixture containing galacto- and fructo-oligosaccharides (GOS/FOS; ratio 9:1) was tested at dietary doses between 1% and 10% (w/w of total diet) in an influenza vaccination model, using 10 C56BL/6JolaHsd mice per group. The modulation of vaccine specific delayed-type hypersensitivity (DTH) responses was studied as a marker of T-helper 1 (Th1) immunity, as well as other immune parameters. GOS/FOS enhanced DTH responses dose-dependently (optimum at 5% w/w of total diet; 41.4+/-14.1% increased compared to controls, p<0.05). No significant changes were detected on splenocyte proliferation or vaccine-specific antibody concentrations. Simultaneously, GOS/FOS dose-dependently increased the proportion of faecal bifidobacteria and lactobacilli (maximal effect at 10% w/w of total diet; 16.8+/-2.4% and 5.8+/-1.3% increased compared to controls respectively, p<0.01 for both parameters). In a comparative experiment, GOS/FOS and FOS/inulin (both at 2% w/w of total diet) induced similar significant effects on the gut microbiota. In contrast to GOS/FOS, FOS/inulin did not enhance DTH responses, indicating that an increase in the proportions of bifidobacteria and lactobacilli is not sufficient for an immunomodulatory effect in this model. The use of GOS/FOS in dietary products might provide an opportunity to stimulate the adaptive immune response in a Th1-direction and subsequently inhibit infections and Th2-related immune disorders in humans, for instance allergies. Clinical studies are being performed to confirm this.

Quantitative real-time PCR analysis of fecal Lactobacillus species in infants receiving a prebiotic infant formula.

The developing intestinal microbiota of breast-fed infants is considered to play an important role in the priming of the infants' mucosal and systemic immunity. Generally, Bifidobacterium and Lactobacillus predominate the microbiota of breast-fed infants. In intervention trials it has been shown that lactobacilli can exert beneficial effects on, for example, diarrhea and atopy. However, the Lactobacillus species distribution in breast-fed or formula-fed infants has not yet been determined in great detail. For accurate enumeration of different lactobacilli, duplex 5' nuclease assays, targeted on rRNA intergenic spacer regions, were developed for Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus rhamnosus. The designed and validated assays were used to determine the amounts of different Lactobacillus species in fecal samples of infants receiving a standard formula (SF) or a standard formula supplemented with galacto- and fructo-oligosaccharides in a 9:1 ratio (OSF). A breast-fed group (BF) was studied in parallel as a reference. During the 6-week intervention period a significant increase was shown in total percentage of fecal lactobacilli in the BF group (0.8% +/- 0.3% versus 4.1% +/- 1.5%) and the OSF group (0.8% +/- 0.3% versus 4.4% +/- 1.4%). The Lactobacillus species distribution in the OSF group was comparable to breast-fed infants, with relatively high levels of L. acidophilus, L. paracasei, and L. casei. The SF-fed infants, on the other hand, contained more L. delbrueckii and less L. paracasei compared to breast-fed infants and OSF-fed infants. An infant milk formula containing a specific mixture of prebiotics is able to induce a microbiota that closely resembles the microbiota of BF infants.

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.