Hauke Smidt

Comparative analysis of pyrosequencing and a phylogenetic microarray for exploring microbial community structures in the human distal intestine

Background Variations in the composition of the human intestinal microbiota are linked to diverse health conditions. High-throughput molecular technologies have recently elucidated microbial community structure at much higher resolution than was previously possible. Here we compare two such methods, pyrosequencing and a phylogenetic array, and evaluate classifications based on two variable 16S rRNA gene regions. Methods and Findings Over 1.75 million amplicon sequences were generated from the V4 and V6 regions of 16S rRNA genes in bacterial DNA extracted from four fecal samples of elderly individuals. The phylotype richness, for individual samples, was 1,400–1,800 for V4 reads and 12,500 for V6 reads, and 5,200 unique phylotypes when combining V4 reads from all samples. The RDP-classifier was more efficient for the V4 than for the far less conserved and shorter V6 region, but differences in community structure also affected efficiency. Even when analyzing only 20% of the reads, the majority of the microbial diversity was captured in two samples tested. DNA from the four samples was hybridized against the Human Intestinal Tract (HIT) Chip, a phylogenetic microarray for community profiling. Comparison of clustering of genus counts from pyrosequencing and HITChip data revealed highly similar profiles. Furthermore, correlations of sequence abundance and hybridization signal intensities were very high for lower-order ranks, but lower at family-level, which was probably due to ambiguous taxonomic groupings. Conclusions The RDP-classifier consistently assigned most V4 sequences from human intestinal samples down to genus-level with good accuracy and speed. This is the deepest sequencing of single gastrointestinal samples reported to date, but microbial richness levels have still not leveled out. A majority of these diversities can also be captured with five times lower sampling-depth. HITChip hybridizations and resulting community profiles correlate well with pyrosequencing-based compositions, especially for lower-order ranks, indicating high robustness of both approaches. However, incompatible grouping schemes make exact comparison difficult.

The gut microbiota and host health: a new clinical frontier

Over the last 10–15 years, our understanding of the composition and functions of the human gut microbiota has increased exponentially. To a large extent, this has been due to new ‘omic’ technologies that have facilitated large-scale analysis of the genetic and metabolic profile of this microbial community, revealing it to be comparable in influence to a new organ in the body and offering the possibility of a new route for therapeutic intervention. Moreover, it might be more accurate to think of it like an immune system: a collection of cells that work in unison with the host and that can promote health but sometimes initiate disease. This review gives an update on the current knowledge in the area of gut disorders, in particular metabolic syndrome and obesity-related disease, liver disease, IBD and colorectal cancer. The potential of manipulating the gut microbiota in these disorders is assessed, with an examination of the latest and most relevant evidence relating to antibiotics, probiotics, prebiotics, polyphenols and faecal microbiota transplantation.

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

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 Pearsons 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.

S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions

Dendritic cells (DCs) are antigen-presenting cells that play an essential role in mucosal tolerance. They regularly encounter beneficial intestinal bacteria, but the nature of these cellular contacts and the immune responses elicited by the bacteria are not entirely elucidated. Here, we examined the interactions of Lactobacillus acidophilus NCFM and its cell surface compounds with DCs. L. acidophilus NCFM attached to DCs and induced a concentration-dependent production of IL-10, and low IL-12p70. We further demonstrated that the bacterium binds to DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a DC- specific receptor. To identify the DC-SIGN ligand present on the bacterium, we took advantage of a generated array of L. acidophilus NCFM mutants. A knockout mutant of L. acidophilus NCFM lacking the surface (S) layer A protein (SlpA) was significantly reduced in binding to DC-SIGN. This mutant incurred a chromosomal inversion leading to dominant expression of a second S layer protein, SlpB. In the SlpB-dominant strain, the nature of the interaction of this bacterium with DCs changed dramatically. Higher concentrations of proinflammatory cytokines such as IL-12p70, TNFα, and IL-1β were produced by DCs interacting with the SlpB-dominant strain compared with the parent NCFM strain. Unlike the SlpA-knockout mutant, T cells primed with L. acidophilus NCFM stimulated DCs produced more IL-4. The SlpA–DC-SIGN interaction was further confirmed as purified SlpA protein ligated directly to the DC-SIGN. In conclusion, the major S layer protein, SlpA, of L. acidophilus NCFM is the first probiotic bacterial DC-SIGN ligand identified that is functionally involved in the modulation of DCs and T cells functions.

Intestinal microbiota in human health and disease: the impact of probiotics.

The complex communities of microorganisms that colonise the human gastrointestinal tract play an important role in human health. The development of culture-independent molecular techniques has provided new insights in the composition and diversity of the intestinal microbiota. Here, we summarise the present state of the art on the intestinal microbiota with specific attention for the application of high-throughput functional microbiomic approaches to determine the contribution of the intestinal microbiota to human health. Moreover, we review the association between dysbiosis of the microbiota and both intestinal and extra-intestinal diseases. Finally, we discuss the potential of probiotic microorganism to modulate the intestinal microbiota and thereby contribute to health and well-being. The effects of probiotic consumption on the intestinal microbiota are addressed, as well as the development of tailor-made probiotics designed for specific aberrations that are associated with microbial dysbiosis.

Nutritional management of gut health in pigs around weaning

Early weaning of piglets is often accompanied by a severe growth check and diarrhoea. It is well established that this process is multi-factorial and that post-weaning anorexia and undernutrition are major aetiological factors. Gastrointestinal disturbances include alterations in small intestine architecture and enzyme activities. Recent data indicate transiently-increased mucosal permeability, disturbed absorptive-secretory electrolyte balance and altered local inflammatory cytokine patterns after weaning. These responses appear to operate according to two distinct temporal patterns, an acute response followed by a long-lasting adaptation response. Pigs coexist with a diverse and dense commensal microbiota in their gastrointestinal tract. Most of these microbes are beneficial, providing necessary nutrients or protection against harmful pathogens for the host. The microbial colonisation of the porcine intestine begins at birth and follows a rapid succession during the neonatal and weaning period. Following the withdrawal of sows milk the young piglets are highly susceptible to enteric diseases partly as a result of the altered balance between developing beneficial microbiota and the establishment of intestinal bacterial pathogens. The intestinal immune system of the newborn piglet is poorly developed at birth and undergoes a rapid period of expansion and specialisation that is not achieved before early (commercial) weaning. Here, new insights on the interactions between feed components, the commensal microbiota and the physiology and immunology of the host gastrointestinal tract are highlighted, and some novel dietary strategies are outlined that are focused on improving gut health. Prebiotics and probiotics are clear nutritional options, while convincing evidence is still lacking for other bioactive substances of vegetable origin.

Specific Response of a Novel and Abundant Lactobacillus amylovorus-Like Phylotype to Dietary Prebiotics in the Guts of Weaning Piglets

ABSTRACT Using 16S rRNA gene-based approaches, we analyzed the responses of ileal and colonic bacterial communities of weaning piglets to dietary addition of four fermentable carbohydrates (inulin, lactulose, wheat starch, and sugar beet pulp). An enriched diet and a control diet lacking these fermentable carbohydrates were fed to piglets for 4 days (n = 48), and 10 days (n = 48), and the lumen-associated microbiota were compared using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes. Bacterial diversities in the ileal and colonic samples were measured by assessing the number of DGGE bands and the Shannon index of diversity. A higher number of DGGE bands in the colon (24.2 ± 5.5) than in the ileum (9.7 ± 4.2) was observed in all samples. In addition, significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the colonic microbial community of weaning piglets fed the fermentable-carbohydrate-enriched diet for 10 days than in the control. Selected samples from the ileal and colonic lumens were also investigated using fluorescent in situ hybridization (FISH) and cloning and sequencing of the 16S rRNA gene. This revealed a prevalence of Lactobacillus reuteri in the ileum and Lactobacillus amylovorus-like populations in the ileum and the colon in the piglets fed with fermentable carbohydrates. Newly developed oligonucleotide probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH. The results indicate that addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ilea and colons of weaning piglets.

The effects of selected probiotic strains on the development of eczema (the PandA study)

Background:  Modification of the intestinal microbiota by administration of probiotic bacteria may be a potential approach to prevent allergic disease. We aimed to study primary prevention of allergic disease in high‐risk children by pre‐ and postnatal supplementation of selected probiotic bacteria.

High temporal and inter‐individual variation detected in the human ileal microbiota

The diversity and temporal stability of the predominant bacteria in the human ileum was studied with the use of ileal effluent samples of seven individuals with Brooke ileostomies. The total number of bacteria within the ileal effluent was in the range of 10⁷ -10⁸ bacteria per gram (wet weight). The diversity of the bacteria in the ileal effluent showed marked differences compared with that in faecal samples from age-matched healthy adults. The ileal effluent had a higher relative abundance of species within the orders Lactobacillales and Clostridiales, mainly Streptococcus bovis-related species, and the Veillonella group, and a lower proportion of species related to Ruminococcus gnavus, R. obeum and Bacteroides plebeius. In addition, inter-individual differences were found, indicative of a highly personal ileal microbiota profile. Furthermore, temporal profiles showed large fluctuations per individual over a period of 9-28 days (average similarity over a period of 9 days was as low as 44%), and differences between morning and afternoon profiles were observed. Parallel cloning and sequencing efforts revealed several phylotypes that were not identified in previous studies (12 out of 65 phylotypes showed less than 97% sequence similarity with previously reported sequences). Achaea were found to be below detection limit by quantitative PCR. Overall, the results indicate that the microbiota of the human ileum is relatively unstable, less complex and consisting of different dominating phylotypes when compared with the colonic microbiota.

Isolation of DNA from bacterial samples of the human gastrointestinal tract.

The human gastrointestinal (GI) tract contains a complex microbial community that develops in time and space. The most widely used approaches to study microbial diversity and activity are all based on the analysis of nucleic acids, DNA, rRNA and mRNA. Here, we present a DNA isolation protocol that is suitable for a wide variety of GI tract samples, including biopsies with minute amounts of material. The protocol is set up in such a way that sampling can be performed outside the laboratory, which offers possibilities for implementation in large intervention studies. The DNA isolation is based on mechanical disruption, followed by isolation of nucleic acids using phenol:chloroform:isoamylalcohol extraction. In addition, it includes an alternative DNA isolation protocol that is based on a commercial kit. These protocols have all been successfully used in our laboratory, resulting in isolation of DNA of sufficient quality for microbial diversity studies. Depending on the number of samples and sample type, the whole procedure will take approximately 2.5–4 hours.