Development and application of the human intestinal tract chip, a phylogenetic microarray: analysis of universally conserved phylotypes in the abundant microbiota of young and elderly adults

Abstract

In this paper we present the in silico assessment of the diversity of variable regions of the small subunit ribosomal RNA (SSU rRNA) gene based on an ecosystem-specific curated database, describe a probe design procedure based on two hypervariable regions with minimal redundancy and test the potential of such probe design strategy for the design of a flexible microarray platform. This resulted in the development and application of a phylogenetic microarray for studying the human gastrointestinal microbiota – referred as the human intestinal tract chip (HITChip). Over 4800 dedicated tiling oligonucleotide probes were designed based on two hypervariable regions of the SSU rRNA gene of 1140 unique microbial phylotypes (< 98% identity) following analysis of over 16 000 human intestinal SSU rRNA sequences. These HITChip probes were hybridized to a diverse set of human intestinal samples and SSU rRNA clones to validate its fingerprinting and quantification potential. Excellent reproducibility (median Pearson's correlation of 0.99) was obtained following hybridization with T7 polymerase transcripts generated in vitro from SSU rRNA gene amplicons. A linear dose–response was observed with artificial mixtures of 40 different representative amplicons with relative abundances as low as 0.1% of total microbiota. Analysis of three consecutively collected faecal samples from ten individuals (five young and five elderly adults) revealed temporal dynamics and confirmed that the adult intestinal microbiota is an individual-specific and relatively stable ecosystem. Further analysis of the stable part allowed for the identification of a universal microbiota core at the approximate genus level (90% sequence similarity). This core consists of members of Actinobacteria, Bacteroidetes and Firmicutes. Used as a phylogenetic fingerprinting tool with the possibility for relative quantification, the HITChip has the potential to bridge the gaps in our knowledge in the quantitative and qualitative description of the human gastrointestinal microbiota composition.

Modulation of the gut microbiota by prebiotic fibres and bacteriocins

“…Subsequently, the microbiota slowly develops into an adult-like, more diverse and stable state at around 3 years of age [21,22]. In the adulthood of healthy humans, the gut ecosystem is in a homeostatic equilibrium with temporal balance between different microbial groups, epithelial tissue of the intestine and the immune system of the host [21,23]. However, after approximately 65 years of age, the composition of the gut microbiota alters, with high interindividual variability, which is probably due to the physiological changes in the intestines that affect food digestion and absorption, and immune function [24].…”

Human gut microbiota: does diet matter?

“…Interest, as well as the number of studies, in the GI microbiota of elderly people has grown as life expectancy in the Western world has rapidly increased. The elderly have been reported to have relatively stable microbiota (93)(94)(95) . However, the microbiota of the elderly has been reported to be more diverse and to contain partly different core microbiota as compared with younger adults (93,94,96,97) .…”

“…The elderly have been reported to have relatively stable microbiota (93)(94)(95) . However, the microbiota of the elderly has been reported to be more diverse and to contain partly different core microbiota as compared with younger adults (93,94,96,97) . Moreover, inter-individual variation is greater in elderly people as compared with younger adults (2,94,98) .…”

Ageing, immunity and influenza: a role for probiotics?

“…A reduction in total gene counts in faecal samples from 178 elderly subjects in long-term care compared with those in the community illustrates the reduction in gut microbiota biodiversity, which is associated with frailty, morbidity and poor nutritional status in elderly subjects (1) . Also commonly reported is an age-related increase in facultative anaerobes, including streptococci, staphylococci, enterococci and enterobacteria (4)(5)(6)(7) . These are sometimes referred to as 'pathobionts'; bacteria, which are present at low concentrations in the healthy gut microbiota, but thrive in inflamed conditions and actively promote the inflammation by producing inflammatory stimuli (7) .…”