Background & aims: Ageing is associated with an increased risk of frailty, intestinal microbiota perturbations, immunosenescence and oxidative stress. Prebiotics such as galacto-oligosaccharides (GOS) may ameliorate these ageing-related alterations. We aimed to compare the faecal microbiota composition, metabolite production, immune and oxidative stress markers in prefrail elderly and younger adults, and investigate the effects of GOS supplementation in both groups. Methods: In a randomised controlled cross-over study, 20 prefrail elderly and 24 healthy adults received 21.6 g/day Biotis™ GOS (containing 15.0 g/day GOS) or placebo. Faecal 16S rRNA gene-based microbiota and short-chain fatty acids were analysed at 0, 1 and 4 weeks of intervention.Volatile organic compounds were analysed in breath, and stimulated cytokine production, CRP, malondialdehyde, trolox equivalent antioxidant capacity (TEAC) and uric acid (UA) in blood at 0 and 4 weeks. Results: Principle coordinate analysis showed differences in microbial composition between elderly and adults (P 0.05), with elderly having lower bifidobacteria (P 0.033) at baseline. In both groups, GOS affected microbiota composition (P 0.05), accompanied by increases in bifidobacteria (P<0.001) and decreased microbial diversity (P 0.023). Faecal and breath metabolites, immune and oxidative stress markers neither differed between groups (P ! 0.125) nor were affected by GOS (P ! 0.236). TEAC values corrected for UA were higher in elderly versus adults (P<0.001), but not different between interventions (P ! 0.455). Conclusions: Elderly showed lower faecal bifidobacterial (relative) abundance than adults, which increased after GOS intake in both groups. Faecal and breath metabolites, parameters of immune function and oxidative stress were not different at baseline, and not impacted by GOS supplementation. Clinicaltrials.gov with study id number: NCT03077529.

The first year of life is a crucial period during which the composition and functionality of the gut microbiota develop to stabilize and resemble that of adults. Throughout this process, the gut microbiota has been found to contribute to the maturation of the immune system, in gastrointestinal physiology, in cognitive advancement and in metabolic regulation. Breastfeeding, the “golden standard of infant nutrition,” is a cornerstone during this period, not only for its direct effect but also due to its indirect effect through the modulation of gut microbiota. Human milk is known to contain indigestible carbohydrates, termed human milk oligosaccharides (HMOs), that are utilized by intestinal microorganisms. Bacteria that degrade HMOs like Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, and Bifidobacterium breve dominate the infant gut microbiota during breastfeeding. A number of carbohydrate active enzymes have been found and identified in the infant gut, thus supporting the hypothesis that these bacteria are able to degrade HMOs. It is suggested that via resource-sharing and cross-feeding, the initial utilization of HMOs drives the interplay within the intestinal microbial communities. This is of pronounced importance since these communities promote healthy development and some of their species also persist in the adult microbiome. The emerging production and accessibility to metagenomic data make it increasingly possible to unravel the metabolic capacity of entire ecosystems. Such insights can increase understanding of how the gut microbiota in infants is assembled and makes it a possible target to support healthy growth. In this manuscript, we discuss the co-occurrence and function of carbohydrate active enzymes relevant to HMO utilization in the first year of life, based on publicly available metagenomic data. We compare the enzyme profiles of breastfed children throughout the first year of life to those of formula-fed infants.

Background Formalin-fixed paraffin embedded (FFPE) tissues may provide an exciting resource to study microbial associations in human disease, but the use of these low biomass specimens remains challenging. We aimed to reduce unintentional bacterial interference in molecular analysis of FFPE tissues and investigated the feasibility of conducting quantitative polymerase chain reaction (qPCR) and 16S rRNA amplicon sequencing using 14 colorectal cancer, 14 normal adjacent and 13 healthy control tissues. Results Bacterial contaminants from the laboratory environment and the co-extraction of human DNA can affect bacterial analysis. The application of undiluted template improves bacterial DNA amplification, allowing the detection of specific bacterial markers (Escherichia coli and Faecalibacterium prausnitzii) by qPCR. Nested and non-nested PCR-based 16S rRNA amplicon sequencing approaches were employed, showing that bacterial communities of tissues and paired paraffin controls cluster separately at genus level on weighted Unifrac in both non-nested (R2 = 0.045; Pr(> F) = 0.053) and nested (R2 = 0.299; Pr(> F) = 0.001) PCR datasets. Nevertheless, considerable overlap of bacterial genera within tissues was seen with paraffin, DNA extraction negatives (non-nested PCR) or PCR negatives (nested PCR). Following mathematical decontamination, no differences in α- and β diversity were found between tumor, normal adjacent and control tissues. Conclusions Bacterial marker analysis by qPCR seems feasible using non-normalized template, but 16S rRNA amplicon sequencing remains challenging. Critical evaluation of laboratory procedures and incorporation of positive and negative controls for bacterial analysis of FFPE tissues are essential for quality control and to account for bacterial contaminants.

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The Amadori product fructoselysine is formed upon heating of food products and is abundantly present in infant formula while being almost absent in breast milk. The human gut microbiota can degrade fructoselysine for which interindividual differences have been described for adults. The aim of this study is to compare functional differences in microbial fructoselysine degradation between breast-fed and formula-fed infants, in view of their different diets and resulting different fructoselysine exposures. First, a publicly available metagenomic dataset with metagenome assembled genomes (MAGs) from infant fecal samples was analyzed and showed that query genes involved in fructoselysine degradation (frlD/yhfQ) were abundantly present in multiple bacterial taxa in the fecal samples, with a higher prevalence in the formula-fed infants. Next, fecal samples collected from exclusively breast-fed and formula-fed infants were anaerobically incubated with fructoselysine. Both groups degraded fructoselysine, however the fructoselysine degradation activity was significantly higher by fecal samples from formula-fed infants. Overall this study provides evidence that infant formula feeding, leading to increased dietary fructoselysine exposure, seems to result in an increased fructoselysine degradation activity in the gut microbiota of infants. This indicates that the infant gut microbiota adapts towards dietary fructoselysine exposure.