Faecal microbiota and short-chain fatty acid levels in faeces from infants with cow's milk protein allergy.

BACKGROUND: The present study was designed to compare the faecal microbiota and concentrations of faecal short-chain fatty acid and ammonia between healthy and cow's milk protein allergic (CMPA) infants. METHODS: The population comprised 92 infants aged 2-12 months who were nonallergic (n = 46) or diagnosed as having CMPA (n = 46). Faecal samples were analyzed by fluorescent in situ hybridization and flow cytometry, using a panel of 10 rRNA targeted group- and species-specific oligonucleotide probes. Acetic, propionic, butyric, isocaproic and branched-chain short fatty acids (BCSFA) were measured by gas-liquid chromatography, lactate by enzymatic reaction, and pH and ammonia levels were determined. RESULTS: CMPA infant faeces had significantly higher proportions of the Clostridium coccoides group and Atopobium cluster and a higher sum of the proportions of the different bacterial groups in comparison to healthy infant faeces. Faecal pH and ammonia did not significantly differ between CMPA and healthy infants. Faeces concentrations and percentages of butyric acid and BCSFA were higher in CMPA infants than in healthy infants. CONCLUSIONS: The findings clearly set a link between a dysbiosis in gut microbiota composition and the pathogenesis of CMPA. No single species or genus appeared to play an essential role, but dysbiosis led to biomarkers of CMPA among bacterial fermentation products.

Determinants of the human infant intestinal microbiota after the introduction of first complementary foods in infant samples from five European centres.

Although it is well established that early infant feeding has a major influence on the establishment of the gut microbiota, very little is understood about how the introduction of first solid food influences the colonization process. This study aimed to determine the impact of weaning on the faecal microbiota composition of infants from five European countries (Sweden, Scotland, Germany, Italy and Spain) which have different lifestyle characteristics and infant feeding practices. Faecal samples were collected from 605 infants approximately 4 weeks after the introduction of first solid foods and the results were compared with the same infants before weaning (6 weeks of age) to investigate the association with determining factors such as geographical origin, mode of delivery, previous feeding method and age of weaning. Samples were analysed by fluorescence in situ hybridization and flow cytometry using a panel of 10 rRNA targeted group- and species-specific oligonucleotide probes. The genus Bifidobacterium (36.5 % average proportion of total detectable bacteria), Clostridium coccoides group (14 %) and Bacteroides (13.6 %) were predominant after weaning. Similar to pre-weaning, northern European countries were associated with a higher proportion of bifidobacteria in the infant gut microbiota while higher levels of Bacteroides and lactobacilli characterized southern European countries. As before weaning, the initial feeding method influenced the Clostridium leptum group and Clostridium difficile+Clostridium perfringens species, and bifidobacteria still dominated the faeces of initially breast-fed infants. Formula-fed babies presented significantly higher proportions of Bacteroides and the C. coccoides group. The mode of birth influenced changes in the proportions of bacteroides and atopobium. Although there were significant differences in the mean weaning age between countries, this was not related to the populations of bifidobacteria or bacteroides. Thus, although the faecal microbiota of infants after first complementary foods was different to that before weaning commenced, many of the initial influences on microbiota composition were still evident.

Molecular methods to study complex microbial communities.

Microbes, which constitute a major fraction of the total biomass, are the main source of biodiversity on our Planet and play an essential role in maintaining global processes, which ultimately regulate the functioning of the Biosphere. Recent emergence of "metagenomics" allows for the analysis of microbial communities without tedious cultivation efforts. Metagenomics approach is analogous to the genomics with the difference that it does not deal with the single genome from a clone or microbe cultured or characterized in laboratory, but rather with that from the entire microbial community present in an environmental sample; it is the community genome. Global understanding by metagenomics depends essentially on the possibility of isolating the entire bulk DNA and identifying the genomes, genes, and proteins more relevant to each of the environmental sample under investigation. Following on this, in this chapter, we provide an analysis of methods available to isolate environmental DNA and to establish metagenomic libraries that can further be used for extensive activity screens.

Changes in faecal microbiota of infants with cow's milk protein allergy--a Spanish prospective case-control 6-month follow-up study.

Major differences have been found in gut microbiota between healthy and allergic children, and a possible association between allergy and altered microbiota patterns have been postulated. The main object of the study was to compare the faecal microbiota between healthy and cow's milk protein allergy (CMPA) infants at the baseline immediately after the diagnosis, and to evaluate the changes in the faecal microbiota after 6 months of treatment of CMPA infants fed on extensively cow's milk protein hydrolyzed formulae without pre- or probiotics, compared with healthy children fed on standard milk formulae. The population comprised 92 infants aged 2-12 months who were non-allergic (n=46) or diagnosed with IgE-mediated CMPA (n=46). At baseline and at 6 months, faecal samples were collected into sterile plastic tubes, immediately placed into anaerobic jars and processed within 2 h of their collection. Weighed faeces samples were diluted from 10(-1) to 10(-7) and cultured in selective media for total count of aerobes, anaerobes, enterobacteria, bifidobacteria, lactobacilli, clostridia and yeasts. Samples from non-allergic and allergic infants were compared at baseline and at 6 months using appropriate statistical tests, considering p<0.05 to be significant. In comparison with healthy infants, CMPA infants had higher total bacteria and anaerobic counts and a lower yeast count at baseline, finding no difference in the proportions of each bacterial group between groups. After 6 months, CMPA infants showed higher anaerobic and lactobacilli counts, a higher proportion of lactobacilli, a lower count and proportion of bifidobacteria, and lower proportions of enterobacteria and yeasts. Comparison of faecal samples from CMPA infants between baseline and at 6 months showed an increase in count and proportion of lactobacilli and a decrease in counts and proportions of enterobacteria and bifidobacteria. Differences in the composition of gut microbiota between CMPA and healthy infants may influence in the development of or protection from this allergy.

Reactome array: forging a link between metabolome and genome.

We describe a sensitive metabolite array for genome sequence-independent functional analysis of metabolic phenotypes and networks, the reactomes, of cell populations and communities. The array includes 1676 dye-linked substrate compounds collectively representing central metabolic pathways of all forms of life. Application of cell extracts to the array leads to specific binding of enzymes to cognate substrates, transformation to products, and concomitant activation of the dye signals. Proof of principle was shown by reconstruction of the metabolic maps of model bacteria. Utility of the array for unsequenced organisms was demonstrated by reconstruction of the global metabolisms of three microbial communities derived from acidic volcanic pool, deep-sea brine lake, and hydrocarbon-polluted seawater. Enzymes of interest are captured on nanoparticles coated with cognate metabolites, sequenced, and their functions unequivocally established.

Metagenomics approaches in systems microbiology.

The world of microorganisms comprises a vast diversity of live organisms, each with its individual set of genes, cellular components and metabolic reactions that interact within the cell and communicate with the environment in many different ways. There is a strong imperative to gain a broader view of the wired and interconnected cellular and environmental processes as a whole via the systems microbiology approach in order to understand and predict ecosystem functioning. On the other hand, currently we experience a rise of metagenomics as an emerging tool to study communities of uncultured microorganisms. In this review, we conducted a survey of important methodologies in metagenomics and describe systems microbiology-like approaches for gaining a mechanistic understanding of complex microbial systems to interrogate compositional, evolutionary and metabolic properties. The review also discusses how metagenomics can be used as a holistic indicator for ecosystem response in terms of matter, nutrient and energy sources and functional networking.