Genome sequence and description of Gracilibacillus timonensis sp. nov. strain Marseille-P2481T , a moderate halophilic bacterium isolated from the human gut microflora.

Microbial culturomics represents an ongoing revolution in the characterization of the human gut microbiota. By using three culture media containing high salt concentrations (10, 15, and 20% [w/v] NaCl), we attempted an exhaustive exploration of the halophilic microbial diversity of the human gut and isolated strain Marseille-P2481 (= CSUR P2481 =  DSM 103076), a new moderately halophilic bacterium. This bacterium is a Gram-positive, strictly aerobic, spore-forming rod that is motile by use of a flagellum and exhibits catalase, but not oxidase activity. Strain Marseille-P2481 was cultivated in media containing up to 20% (w/v) NaCl, with optimal growth being obtained at 37°C, pH 7.0-8.0, and 7.5% [w/v] NaCl). The major fatty acids were 12-methyl-tetradecanoic acid and hexadecanoic acid. Its draft genome is 4,548,390 bp long, composed of 11 scaffolds, with a G+C content of 39.8%. It contains 4,335 predicted genes (4,266 protein coding including 89 pseudogenes and 69 RNA genes). Strain Marseille-P2481 showed 96.57% 16S rRNA sequence similarity with Gracilibacillus alcaliphilus strain SG103T , the phylogenetically closest species with standing in nomenclature. On the basis of its specific features, strain Marseille-P2481T was classified as type strain of a new species within the genus Gracilibacillus for which the name Gracilibacillus timonensis sp. nov. is formally proposed.

Halophilic & halotolerant prokaryotes in humans.

Halophilic prokaryotes are described as microorganisms living in hypersaline environments. Here, we list the halotolerant and halophilic bacteria which have been isolated in humans. Of the 52 halophilic prokaryotes, 32 (61.54%) were moderately halophilic, 17 (32.69%) were slightly halophilic and three (5.76%) were extremely halophilic prokaryotes. At the phylum level, 29 (54.72%) belong to Firmicutes, 15 (28.84%) to Proteobacteria, four (7.69%) to Actinobacteria, three (5.78%) to Euryarchaeota and one (1.92%) belongs to Bacteroidetes. Halophilic prokaryotes are rarely pathogenic: of these 52 halophilic prokaryotes only two (3.92%) species were classified in Risk Group 2 (Vibrio cholerae, Vibrio parahaemolyticus) and one (1.96%), species in Risk Group 3 (Bacillus anthracis).

Bacillus kwashiorkori sp. nov., a new bacterial species isolated from a malnourished child using culturomics.

Strain SIT6T was isolated from the fecal flora of a severely malnourished child as part of a broad "culturomics" study aiming to maximize the culture conditions for the in-depth exploration of the human microbiota. An analysis of the 16S rRNA gene sequence showed that strain SIT6T shared 94.1% 16S rRNA gene sequence similarity with Bacillus thermoamylovorans DKPT (NR_029151), the phylogenetically closest type species. Colonies are creamy white, circular, 4-5 mm in diameter after cultivation at 37°C for 24 hr on 5% sheep blood-enriched Colombia agar. Growth occurs at temperatures in the range of 25-56°C (optimally at 37°C). Strain SIT6T is a gram-positive, facultative anaerobic rod and motile by means of peritrichous flagella and sporulating; it is catalase and oxidase positive. The 2,784,637-bp-long genome, composed of 16 contigs, has a G+C content of 35.19%. Of the 2,646 predicted genes, 2,572 were protein-coding genes and 74 were RNAs. The major fatty acids are saturated species (15:0 iso, 16:0 and 17:0 anteiso). Of the 14 detected fatty acids, 11 are saturated, either linear or branched (iso and anteiso). Digital DNA-DNA hybridization (dDDH) estimation and average genomic identity of orthologous gene sequences (AGIOS) of the strain SIT6T against genomes of the type strains of related species ranged between 18.6% and 38.3% and between 54.77% and 65.50%, respectively. According to our taxonogenomics results, we propose the creation of Bacillus kwashiorkori sp. nov. that contains the type strain SIT6T (=CSUR P2452T , =DSM 29059T ).

Culture of previously uncultured members of the human gut microbiota by culturomics.

Metagenomics revolutionized the understanding of the relations among the human microbiome, health and diseases, but generated a countless number of sequences that have not been assigned to a known microorganism1. The pure culture of prokaryotes, neglected in recent decades, remains essential to elucidating the role of these organisms2. We recently introduced microbial culturomics, a culturing approach that uses multiple culture conditions and matrix-assisted laser desorption/ionization-time of flight and 16S rRNA for identification2. Here, we have selected the best culture conditions to increase the number of studied samples and have applied new protocols (fresh-sample inoculation; detection of microcolonies and specific cultures of Proteobacteria and microaerophilic and halophilic prokaryotes) to address the weaknesses of the previous studies3-5. We identified 1,057 prokaryotic species, thereby adding 531 species to the human gut repertoire: 146 bacteria known in humans but not in the gut, 187 bacteria and 1 archaea not previously isolated in humans, and 197 potentially new species. Genome sequencing was performed on the new species. By comparing the results of the metagenomic and culturomic analyses, we show that the use of culturomics allows the culture of organisms corresponding to sequences previously not assigned. Altogether, culturomics doubles the number of species isolated at least once from the human gut.