A significant number of microorganisms from the human oral cavity remain uncultivated. This is a major impediment to the study of human health since some of the uncultivated species may be involved in a variety of systemic diseases. We used a range of innovations previously developed to cultivate microorganisms from the human oral cavity, focusing on anaerobic species. These innovations include (i) in vivo cultivation to specifically enrich for species actively growing in the oral cavity (the "minitrap" method), (ii) single-cell long-term cultivation to minimize the effect of fast-growing microorganisms, and (iii) modifications of conventional enrichment techniques, using media that did not contain sugar, including glucose. To enable cultivation of obligate anaerobes, we maintained strict anaerobic conditions in most of our cultivation experiments. We report that, on a per cell basis, the most successful recovery was achieved using minitrap enrichment (11%), followed by single-cell cultivation (3%) and conventional plating (1%). Taxonomically, the richest collection was obtained using the single-cell cultivation method, followed by minitrap and conventional enrichment, comprising representatives of 13, 9, and 4 genera, respectively. Interestingly, no single species was isolated by all three methods, indicating method complementarity. An important result is the isolation and maintenance in pure culture of 10 strains previously only known by their molecular signatures, as well as representatives of what are likely to be three new microbial genera. We conclude that the ensemble of new methods we introduced will likely help close the gap between cultivated and uncultivated species from the human oral cavity.

The majority of environmental micro-organisms identified with the rRNA approach have never been visualized. Thus, their reliable classification and taxonomic assignment is often difficult or even impossible. In our preliminary 18S rRNA gene sequencing work from the world’s largest anoxic marine environment, the Cariaco Basin (Caribbean Sea, Venezuela), we detected a ciliate clade, designated previously as CAR_H [Stoeck, S., Taylor, G. T. & Epstein, S. S. (2003). Appl Environ Microbiol 63, 5656–5663]. Here, we combine the traditional rRNA detection method of fluorescent in situ hybridization (FISH) with scanning electron microscopy (SEM) and confirm the phylogenetic separation of the CAR_H sequences from all other ciliate classes by showing an outstanding morphological feature of this group: a unique, archway-shaped kinety surrounding the oral apparatus and extending to the posterior body end in CAR_H cells. Based on this specific feature and the molecular phylogenies, we propose a novel ciliate class, Cariacotrichea nov. cl.

Stomatobaculum longum gen. nov., sp. nov., an obligately anaerobic bacterium from the human oral cavity

Three strictly anaerobic, Gram-positive, non-spore-forming, rod-shaped, motile bacteria, designated strains ACB1 T , ACB7 T and ACB8, were isolated from human subgingival dental plaque. All strains required yeast extract for growth. Strains ACB1 T and ACB8 were able to grow on glucose, lactose, maltose, maltodextrin and raffinose; strain ACB7 T grew weakly on sucrose only. The growth temperature range was 30-42 6C with optimum growth at 37 6C. Major metabolic fermentation end products of strain ACB1 T were acetate and lactate; the only product of strains ACB7 T and ACB8 was acetate. Major fatty acids of strain ACB1 T were C 14 : 0 , C 16 : 0 , C 16 : 1 v7c dimethyl aldehyde (DMA) and C 18 : 1 v7c DMA. Major fatty acids of strain ACB7 T were C 12 : 0 , C 14 : 0 , C 16 : 0 , C 16 : 1 v7c and C 16 : 1 v7c DMA. The hydrolysate of the peptidoglycan contained meso-diaminopimelic acid, indicating peptidoglycan type A1c. Genomic DNA G+C content varied from 42 to 43.3 % between strains. According to 16S rRNA gene sequence phylogeny, strains ACB1 T , ACB8 and ACB7 T formed two separate branches within the genus Oribacterium, with 98.1-98.6 % sequence similarity to the type strain of the type species, Oribacterium sinus. Predicted DNA-DNA hybridization values between strains ACB1 T , ACB8, ACB7 T and O. sinus F0268 were ,70 %. Based on distinct genotypic and phenotypic characteristics, strains ACB1 T and ACB8, and strain ACB7 T are considered to represent two distinct species of the genus Oribacterium, for which the names Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov. are proposed. The type strains are ACB1 T (5DSM