A good definition of commensal microflora and an understanding of its relation to health are essential in preventing and combating disease. We hypothesized that the species richness of human oral microflora is underestimated. Saliva and supragingival plaque were sampled from 71 and 98 healthy adults, respectively. Amplicons from the V6 hypervariable region of the small-subunit ribosomal RNA gene were generated by PCR, pooled into saliva and plaque pools, and sequenced by means of the Genome Sequencer 20 system at 454 Life Sciences. Data were evaluated by taxonomic and rarefaction analyses. The 197,600 sequences generated yielded about 29,000 unique sequences, representing 22 taxonomic phyla. Grouping the sequences in operational taxonomic units (6%) yielded 3621 and 6888 species-level phylotypes in saliva and plaque, respectively. This work gives a radically new insight into the diversity of human oral microflora, which, with an estimated number of 19,000 phylotypes, is considerably higher than previously reported.

These findings provide novel insights into microbial succession in the respiratory tract in infancy and link early-life profiles to microbiota stability and respiratory health characteristics. New prospective studies should elucidate potential implications of our findings for early diagnosis and prevention of respiratory infections. Clinical trial registered with www.clinicaltrials.gov (NCT00189020).

The nasopharynx is the ecological niche for many commensal bacteria and for potential respiratory or invasive pathogens like Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. Disturbance of a balanced nasopharyngeal (NP) microbiome might be involved in the onset of symptomatic infections with these pathogens, which occurs primarily in fall and winter. It is unknown whether seasonal infection patterns are associated with concomitant changes in NP microbiota. As young children are generally prone to respiratory and invasive infections, we characterized the NP microbiota of 96 healthy children by barcoded pyrosequencing of the V5–V6 hypervariable region of the 16S-rRNA gene, and compared microbiota composition between children sampled in winter/fall with children sampled in spring. The approximately 1000000 sequences generated represented 13 taxonomic phyla and approximately 250 species-level phyla types (OTUs). The 5 most predominant phyla were Proteobacteria (64%), Firmicutes (21%), Bacteroidetes (11%), Actinobacteria (3%) and Fusobacteria (1,4%) with Moraxella, Haemophilus, Streptococcus, Flavobacteria, Dolosigranulum, Corynebacterium and Neisseria as predominant genera. The inter-individual variability was that high that on OTU level a core microbiome could not be defined. Microbiota profiles varied strongly with season, with in fall/winter a predominance of Proteobacteria (relative abundance (% of all sequences): 75% versus 51% in spring) and Fusobacteria (absolute abundance (% of children): 14% versus 2% in spring), and in spring a predominance of Bacteroidetes (relative abundance: 19% versus 3% in fall/winter, absolute abundance: 91% versus 54% in fall/winter), and Firmicutes. The latter increase is mainly due to (Brevi)bacillus and Lactobacillus species (absolute abundance: 96% versus 10% in fall/winter) which are like Bacteroidetes species generally related to healthy ecosystems. The observed seasonal effects could not be attributed to recent antibiotics or viral co-infection.The NP microbiota of young children is highly diverse and appears different between seasons. These differences seem independent of antibiotic use or viral co-infection.

BackgroundAn understanding of the relation of commensal microbiota to health is essential in preventing disease. Here we studied the oral microbial composition of children (N = 74, aged 3 - 18 years) in natural transition from their deciduous to a permanent dentition and related the microbial profiles to their oral health status. The microbial composition of saliva was assessed by barcoded pyrosequencing of the V5-V6 hypervariable regions of the 16 S rRNA, as well as by using phylogenetic microarrays.ResultsPyrosequencing reads (126174 reads, 1045 unique sequences) represented 8 phyla and 113 higher taxa in saliva samples. Four phyla - Firmicutes, Bacteriodetes, Proteobacteria and Actinobacteria - predominated in all groups. The deciduous dentition harboured a higher proportion of Proteobacteria (Gammaproteobacteria, Moraxellaceae) than Bacteroidetes, while in all other groups Bacteroidetes were at least as abundant as Proteobacteria. Bacteroidetes (mainly genus Prevotella), Veillonellaceae family, Spirochaetes and candidate division TM7 increased with increasing age, reflecting maturation of the microbiome driven by biological changes with age.Microarray analysis enabled further analysis of the individual salivary microbiota. Of 350 microarray probes, 156 gave a positive signal with, on average, 77 (range 48-93) probes per individual sample.A caries-free oral status significantly associated with the higher signal of the probes targeting Porphyromonas catoniae and Neisseria flavescens.ConclusionsThe potential role of P. catoniae and N. flavescens as oral health markers should be assessed in large-scale clinical studies. The combination of both, open-ended and targeted molecular approaches provides us with information that will increase our understanding of the interplay between the human host and its microbiome.

The gut microbiota is essential for numerous aspects of human health. However, the underlying mechanisms of many host-microbiota interactions remain unclear. The aim of this study was to characterize effects of the microbiota on host epithelium using a novel ex vivo model based on mouse ileal organoids. We have explored the transcriptional response of organoids upon exposure to short-chain fatty acids (SCFAs) and products generated by two abundant microbiota constituents, Akkermansia muciniphila and Faecalibacterium prausnitzii. We observed that A. muciniphila metabolites affect various transcription factors and genes involved in cellular lipid metabolism and growth, supporting previous in vivo findings. Contrastingly, F. prausnitzii products exerted only weak effects on host transcription. Additionally, A. muciniphila and its metabolite propionate modulated expression of Fiaf, Gpr43, histone deacetylases (HDACs), and peroxisome proliferator-activated receptor gamma (Pparγ), important regulators of transcription factor regulation, cell cycle control, lipolysis, and satiety. This work illustrates that specific bacteria and their metabolites differentially modulate epithelial transcription in mouse organoids. We demonstrate that intestinal organoids provide a novel and powerful ex vivo model for host-microbiome interaction studies.

Yeast cell wall proteins, including Cwp1p and alpha-agglutinin, could be released by treating the cell wall with either beta-1,3-or beta-1,6-glucanases, indicating that both polymers are involved in anchoring cell wall proteins. It was shown immunologically that both beta-1,3- and beta-1,6-glucan were linked to yeast cell wall proteins, including Cwp1p and alpha-agglutinin. It was further shown that beta-1,3-glucan was linked to the wall protein through a beta-1,6-glucan moiety. The beta-1,6-glucan moiety could be removed from Cwp1p and other cell wall proteins by cleaving phosphodiester bridges either enzymatically using phosphodiesterases or chemically using ice-cold aqueous hydrofluoric acid. These observations are consistent with the notion that cell wall proteins in Saccharomyces cerevisiae are linked to a beta-1,3-/beta-1,6-glucan heteropolymer through a phosphodiester linkage and that this polymer is responsible for anchoring cell wall proteins. It is proposed that this polymer is identical to the alkali-soluble beta-1,3-/beta-1,6-glucan heteropolymer characterized by Fleet and Manners (1976, 1977).

The yeast cell wall contains ␤1,3-glucanase-extractable and ␤1,3-glucanase-resistant mannoproteins. The ␤1,3-glucanase-extractable proteins are retained in the cell wall by attachment to a ␤1,6-glucan moiety, which in its turn is linked to ␤1,3-glucan (J. C. Kapteyn, R. C. Montijn, E. Vink, J. De La Cruz, A. Llobell, J. E. Douwes, H. Shimoi, P. N. Lipke, and F. M. Klis, Glycobiology 6:337-345, 1996). The ␤1,3-glucanase-resistant protein fraction could be largely released by exochitinase treatment and contained the same set of ␤1,6-glucosylated proteins, including Cwp1p, as the ␤1,3-glucanase-extractable fraction. Chitin was linked to the proteins in the ␤1,3-glucanase-resistant fraction through a ␤1,6-glucan moiety. In wild-type cell walls, the ␤1,3-glucanase-resistant protein fraction represented only 1 to 2% of the covalently linked cell wall proteins, whereas in cell walls of fks1 and gas1 deletion strains, which contain much less ␤1,3-glucan but more chitin, ␤1,3-glucanase-resistant proteins represented about 40% of the total. We propose that the increased crosslinking of cell wall proteins via ␤1,6-glucan to chitin represents a cell wall repair mechanism in yeast, which is activated in response to cell wall weakening.The cell wall is crucial for the integrity of Saccharomyces cerevisiae. Its rigid structure maintains the shape of the cell and offers protection against harmful environmental conditions (6, 19). The wall is mainly composed of ␤-glucans and mannoproteins, in addition to smaller amounts of chitin and lipids (6). The glucans, which are interwoven with the chitin fibrils, form the inner skeletal layer of the cell wall, whereas the outer layer consists of mannoproteins. The majority of the cell wall mannoproteins are anchored into the wall through covalent linkages to heteropolymers of ␤1,6-and ␤1,3-glucan (18, 26, 41, 43) (Fig. 1). The ␤1,6-glucosyl moiety of these polymers is phosphodiester-linked to protein as shown by its sensitivity to treatment with ice-cold aqueous hydrofluoric acid (HF) and phosphodiesterases (18). This observation, together with data from other studies, pointed to a glycosylphosphatidylinositol (GPI)-derived structure as the attachment site for ␤1, 6-glucan (3, 23, 40, 42, 45). The ␤1,3/␤1,6-glucan heteropolymer was proposed to be identical to the alkali-soluble ␤1,3/␤1,6-glucan studied by Fleet and Manners (7,8). In S. cerevisiae, this alkali-soluble glucan becomes alkali insoluble through a linkage with chitin (11, 24) via a ␤1,4 bond from the terminal reducing residue of chitin to the nonreducing end of ␤1,3-glucan (20). Furthermore, by digesting cell walls with ␤1,3-glucanase, followed by incubation with exochitinase, a heterogeneous high-molecular-weight complex was isolated (21). Structural studies indicated that in this complex the terminal reducing residue of chitin is linked to ␤1,6-glucan (21). The nonreducing end of the ␤1,6-glucan polymer is bound to the GPI-derived glycan part of a cell wall protein, whereas its reducing terminus is linked to ␤1,3-glucan (...

Abstract. The cell adhesion protein t~-agglutinin is bound to the outer surface of the Saccharomyces cerevisiae cell wall and mediates cell-cell contact in mating, a-Agglutinin is modified by addition of a glycosyl phosphatidylinositol (GPI) anchor as it traverses the secretory pathway. The presence of a GPI anchor is essential for cross-linking into the wall, but the fatty acid and inositol components of the anchor are lost before cell wall association (Lu, C.-E, J. Kurjan, and P. N. . A pathway for cell wall anchorage of Saccharomyces cerevisiae a-agglutinin. Mol. . Cell wall association of ot-agglutinin was accompanied by an increase in size and a gain in reactivity to antibodies directed against ~l,6-glucan. Several kre mutants, which have defects in synthesis of cell wall/~l,6-glucan, had reduced molecular size of cell wall ot-agglutinin. These findings demonstrate that the cell wall form of oz-agglutinin is covalently associated with/31,6-glucan. The ot-agglutinin biosynthetic precursors did not react with antibody to/~l,6-glucan, and the sizes of these forms were unaffected in kre mutants. A COOHterminal truncated form of a-agglutinin, which is not GPI anchored and is secreted into the medium, did not react with the anti-/31,6-glucan. We propose that extracellular cross-linkage to/31,6-glucan mediates covalent association of a-agglutinin with the cell wall in a manner that is dependent on prior addition of a GPI anchor to ot-agglutinin.