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Bacterial surface (S) layers are the outermost proteinaceous cell envelope structures found on members of nearly all taxonomic groups of bacteria and Archaea . They are composed of numerous identical subunits forming a symmetric, porous, lattice-like layer that completely covers the cell surface. The subunits are held together and attached to cell wall carbohydrates by non-covalent interactions, and they spontaneously reassemble in vitro by an entropy-driven process. Due to the low amino acid sequence similarity among S-layer proteins in general, verification of the presence of an S-layer on the bacterial cell surface usually requires electron microscopy. In lactobacilli, S-layer proteins have been detected on many but not all species. Lactobacillus S-layer proteins differ from those of other bacteria in their smaller size and high predicted p I . The positive charge in Lactobacillus S-layer proteins is concentrated in the more conserved cell wall binding domain, which can be either N- or C-terminal depending on the species. The more variable domain is responsible for the self-assembly of the monomers to a periodic structure. The biological functions of Lactobacillus S-layer proteins are poorly understood, but in some species S-layer proteins mediate bacterial adherence to host cells or extracellular matrix proteins or have protective or enzymatic functions. Lactobacillus S-layer proteins show potential for use as antigen carriers in live oral vaccine design because of their adhesive and immunomodulatory properties and the general non-pathogenicity of the species.

Gut barrier function is key in maintaining a balanced response between the host and its microbiome. The microbiota can modulate changes in gut barrier as well as metabolic and inflammatory responses. This highly complex system involves numerous microbiota-derived factors. The gut symbiont Akkermansia muciniphila is positively correlated with a lean phenotype, reduced body weight gain, amelioration of metabolic responses and restoration of gut barrier function by modulation of mucus layer thickness. However, the molecular mechanisms behind its metabolic and immunological regulatory properties are unexplored. Herein, we identify a highly abundant outer membrane pili-like protein of A. muciniphila MucT that is directly involved in immune regulation and enhancement of trans-epithelial resistance. The purified Amuc_1100 protein and enrichments containing all its associated proteins induced production of specific cytokines through activation of Toll-like receptor (TLR) 2 and TLR4. This mainly leads to high levels of IL-10 similar to those induced by the other beneficial immune suppressive microorganisms such as Faecalibacterium prausnitzii A2-165 and Lactobacillus plantarum WCFS1. Together these results indicate that outer membrane protein composition and particularly the newly identified highly abundant pili-like protein Amuc_1100 of A. muciniphila are involved in host immunological homeostasis at the gut mucosa, and improvement of gut barrier function.

The human gastrointestinal tract contains a complex community of microbes, fulfilling important health-promoting functions. However, this vast complexity of species hampers the assignment of responsible organisms to these functions. Recently, Akkermansia muciniphila, a new species from the deeply branched phylum Verrucomicrobia, was isolated from the human intestinal tract based on its capacity to efficiently use mucus as a carbon and nitrogen source. This anaerobic resident is associated with the protective mucus lining of the intestines. In order to uncover the functional potential of A. muciniphila, its genome was sequenced and annotated. It was found to contain numerous candidate mucinase-encoding genes, but lacking genes encoding canonical mucus-binding domains. Numerous phage-associated sequences found throughout the genome indicate that viruses have played an important part in the evolution of this species. Furthermore, we mined 37 GI tract metagenomes for the presence, and genetic diversity of Akkermansia sequences. Out of 37, eleven contained 16S ribosomal RNA gene sequences that are >95% identical to that of A. muciniphila. In addition, these libraries were found to contain large amounts of Akkermansia DNA based on average nucleotide identity scores, which indicated in one subject co-colonization by different Akkermansia phylotypes. An additional 12 libraries also contained Akkermansia sequences, making a total of ∼16 Mbp of new Akkermansia pangenomic DNA. The relative abundance of Akkermansia DNA varied between <0.01% to nearly 4% of the assembled metagenomic reads. Finally, by testing a large collection of full length 16S sequences, we find at least eight different representative species in the genus Akkermansia. These large repositories allow us to further mine for genetic heterogeneity and species diversity in the genus Akkermansia, providing novel insight towards the functionality of this abundant inhabitant of the human intestinal tract.

ObjectiveAn adequate bowel cleansing is essential for a successful colonoscopy. Although purgative consumption is safe for the patient, there is little consensus on how the intestinal microbiota is affected by the procedure, especially regarding the potential long-term consequences.Design23 healthy subjects were randomised into two study groups consuming a bowel preparation (Moviprep), either in two separate doses of 1 L or as a single 2-L dose. Participants donated faecal samples at the baseline, after bowel cleansing, 14 and 28 days after the treatment. The intestinal microbiota composition was determined with phylogenetic microarray as well as quantitative PCR analysis and correlated with the previously quantified faecal serine proteases.ResultsThe lavage introduced an instant and substantial change to the intestinal microbiota. The total microbial load was decreased by 31-fold and 22% of the participants lost the subject-specificity of their microbiota. While the bacterial levels and community composition were essentially restored within 14 days, the rate of recovery was dose dependent: consumption of the purgative in a single dose had a more severe effect on the microbiota composition than that of a double dose, and notably increased the levels of Proteobacteria, Fusobacteria and bacteria related to Dorea formicigenerans. The abundance of the latter also correlated with the amount of faecal serine proteases that were increased after purging.ConclusionsOur results suggest that the bowel cleansing using two separate dosages introduces fewer alterations to the intestinal microbiota than a single dose and hence may be preferred in clinical practice.

It is generally believed that the infant's microbiota is established during the first 1-2 years of life. However, there is scarce data on its characterization and its comparison to the adult-like microbiota in consecutive years. To characterize and compare the intestinal microbiota in healthy young children (1-4 years) and healthy adults from the North Carolina region in the U.S. using high-throughput bacterial phylogenetic microarray analysis. Detailed characterization and comparison of the intestinal microbiota of healthy children aged 1-4 years old (n = 28) and healthy adults of 21-60 years (n = 23) was carried out using the Human Intestinal Tract Chip (HITChip) phylogenetic microarray targeting the V1 and V6 regions of 16S rRNA and quantitative PCR. The HITChip microarray data indicate that Actinobacteria, Bacilli, Clostridium cluster IV and Bacteroidetes are the predominant phylum-like groups that exhibit differences between young children and adults. The phylum-like group Clostridium cluster XIVa was equally predominant in young children and adults and is thus considered to be established at an early age. The genus-like level show significant 3.6 fold (higher or lower) differences in the abundance of 26 genera between young children and adults. Young U.S. children have a significantly 3.5-fold higher abundance of Bifidobacterium species than the adults from the same location. However, the microbiota of young children is less diverse than that of adults. We show that the establishment of an adult-like intestinal microbiota occurs at a later age than previously reported. Characterizing the microbiota and its development in the early years of life may help identify 'windows of opportunity' for interventional strategies that may promote health and prevent or mitigate disease processes.

While our knowledge of the intestinal microbiota during disease is accumulating, basic information of the microbiota in healthy subjects is still scarce. The aim of this study was to characterize the intestinal microbiota of healthy adults and specifically address its temporal stability, core microbiota and relation with intestinal symptoms. We carried out a longitudinal study by following a set of 15 healthy Finnish subjects for seven weeks and regularly assessed their intestinal bacteria and archaea with the Human Intestinal Tract (HIT) Chip, a phylogenetic microarray, in conjunction with qPCR analyses. The health perception and occurrence of intestinal symptoms was recorded by questionnaire at each sampling point. A high overall temporal stability of the microbiota was observed. Five subjects showed transient microbiota destabilization, which correlated not only with the intake of antibiotics but also with overseas travelling and temporary illness, expanding the hitherto known factors affecting the intestinal microbiota. We identified significant correlations between the microbiota and common intestinal symptoms, including abdominal pain and bloating. The most striking finding was the inverse correlation between Bifidobacteria and abdominal pain: subjects who experienced pain had over five-fold less Bifidobacteria compared to those without pain. Finally, a novel computational approach was used to define the common core microbiota, highlighting the role of the analysis depth in finding the phylogenetic core and estimating its size. The in-depth analysis suggested that we share a substantial number of our intestinal phylotypes but as they represent highly variable proportions of the total community, many of them often remain undetected. A global and high-resolution microbiota analysis was carried out to determine the temporal stability, the associations with intestinal symptoms, and the individual and common core microbiota in healthy adults. The findings provide new approaches to define intestinal health and to further characterize the microbial communities inhabiting the human gut.

As an ecological niche, the mammalian intestine provides the ideal habitat for a variety of bacterial microorganisms. Purportedly, some commensal genera and species offer a beneficial mix of metabolic, protective, and structural processes that help sustain the natural digestive health of the host. Among these sort of gut inhabitants is the Gram-positive lactic acid bacterium Lactobacillus ruminis, a strict anaerobe with both pili and flagella on its cell surface, but also known for being autochthonous (indigenous) to the intestinal environment. Given that the molecular basis of gut autochthony for this species is largely unexplored and unknown, we undertook a study at the genome level to pinpoint some of the adaptive traits behind its colonization behavior. In our pan-genomic probe of L. ruminis, the genomes of nine different strains isolated from human, bovine, porcine, and equine host guts were compiled and compared for in silico analysis. For this, we conducted a geno-phenotypic assessment of protein-coding genes, with an emphasis on those products involved with cell-surface morphology and anaerobic fermentation and respiration. We also categorized and examined the core and accessory genes that define the L. ruminis species and its strains. Here, we made an attempt to identify those genes having ecologically relevant phenotypes that might support or bring about intestinal indigenousness.

Development of the human gut microbiota commences at birth, with bifidobacteria being among the first colonizers of the sterile newborn gastrointestinal tract. To date, the genetic basis of Bifidobacterium colonization and persistence remains poorly understood. Transcriptome analysis of the Bifidobacterium breve UCC2003 2.42-Mb genome in a murine colonization model revealed differential expression of a type IVb tight adherence (Tad) pilus-encoding gene cluster designated \"tad₂₀₀₃.\" Mutational analysis demonstrated that the tad₂₀₀₃ gene cluster is essential for efficient in vivo murine gut colonization, and immunogold transmission electron microscopy confirmed the presence of Tad pili at the poles of B. breve UCC2003 cells. Conservation of the Tad pilus-encoding locus among other B. breve strains and among sequenced Bifidobacterium genomes supports the notion of a ubiquitous pili-mediated host colonization and persistence mechanism for bifidobacteria.

Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects.

Non-aureus staphylococci (NAS) are most commonly isolated from subclinical mastitis. Different NAS species may, however, have diverse effects on the inflammatory response in the udder. We determined the genome sequences of 20 staphylococcal isolates from clinical or subclinical bovine mastitis, belonging to the NAS species Staphylococcus agnetis, S. chromogenes, and S. simulans , and focused on the putative virulence factor genes present in the genomes. For comparison we used our previously published genome sequences of four S. aureus isolates from bovine mastitis. The pan-genome and core genomes of the non-aureus isolates were characterized. After that, putative virulence factor orthologues were searched in silico . We compared the presence of putative virulence factors in the NAS species and S. aureus and evaluated the potential association between bacterial genotype and type of mastitis (clinical vs. subclinical). The NAS isolates had much less virulence gene orthologues than the S. aureus isolates. One third of the virulence genes were detected only in S. aureus . About 100 virulence genes were present in all S. aureus isolates, compared to about 40 to 50 in each NAS isolate. S. simulans differed the most. Several of the virulence genes detected among NAS were harbored only by S. simulans , but it also lacked a number of genes present both in S. agnetis and S. chromogenes . The type of mastitis was not associated with any specific virulence gene profile. It seems that the virulence gene profiles or cumulative number of different virulence genes are not directly associated with the type of mastitis (clinical or subclinical), indicating that host derived factors such as the immune status play a pivotal role in the manifestation of mastitis.