J. Delerce

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.

Loss of TSS1 in hypervirulent Coxiella burnetii 175, the causative agent of Q fever in French Guiana

In French Guiana, the unique Coxiella burnetii circulating genotype 17 causes 24% of community-acquired pneumonia, the highest prevalence ever described. To explain this unusual virulence, we performed a comparative genomic analysis of strain Cb175, which was isolated from a patient from French Guiana. Cb175 has a greater number of mutations in genes involved in metabolism compared with the Nine Mile I strain. We found a 6105bp fragment missing in Cb175, which corresponds to the Type 1 secretion systems (T1SS) hlyCABD operon region. This deletion was detected by a specific qPCR in the 8 other strains available from this territory an in none of 298C.burnetii strains from other areas and other genotypes (8/8 vs 0/298, Fishers exact test, p<0.0000001). Loss of genes implicated in secretion systems has been observed in other epidemic bacterial strains. Thus, the virulence of Cb175 may be linked to this genome reduction.

Comparison of a Modern and Fossil Pithovirus Reveals Its Genetic Conservation and Evolution

Most theories on viral evolution are speculative and lack fossil comparison. Here, we isolated a modern Pithovirus-like virus from sewage samples. This giant virus, named Pithovirus massiliensis, was compared with its prehistoric counterpart, Pithovirus sibericum, found in Siberian permafrost. Our analysis revealed near-complete gene repertoire conservation, including horizontal gene transfer and ORFans. Furthermore, all orthologous genes evolved under strong purifying selection with a non-synonymous and synonymous ratio in the same range as the ratio found in the prokaryotic world. The comparison between fossil and modern Pithovirus species provided an estimation of the cadence of the molecular clock, reaching up to 3 × 10−6 mutations/site/year. In addition, the strict conservation of HGTs and ORFans in P. massiliensis revealed the stable genetic mosaicism in giant viruses and excludes the concept of a bag of genes. The genetic stability for 30,000 years of P. massiliensis demonstrates that giant viruses evolve similarly to prokaryotes by classical mechanisms of evolution, including selection and fixation of genes, followed by selective constraints.

Draft genome sequence of Coxiella burnetii Dog Utad, a strain isolated from a dog-related outbreak of Q fever

Coxiella burnetii Dog Utad, with a 2 008 938 bp genome is a strain isolated from a parturient dog responsible for a human familial outbreak of acute Q fever in Nova Scotia, Canada. Its genotype, determined by multispacer typing, is 21; the only one found in Canada that includes Q212, which causes endocarditis. Only 107 single nucleotide polymorphisms and 16 INDELs differed from Q212, suggesting a recent clonal radiation.

Fournierella massiliensis, gen. nov., sp. nov., a new human-associated member of the family Ruminococcaceae.

An anaerobic bacterium, strain AT2T, was isolated from the fresh stool sample of a healthy French man using the culturomics approach. The 16S rRNA gene sequence analysis showed that strain AT2T had 95.2 % nucleotide sequence similarity with Gemmiger formicilisATCC 27749T, the phylogenetically closest species with standing in nomenclature. Cells are Gram-stain-negative, catalase- and oxidase-negative, obligately anaerobic, non-motile, non-spore-forming, rod-shaped, and the bacilli were mesothermophilic. The major fatty acids were C16 : 0 (43.8 %) and C18 : 1n9 (20 %). The DNA G+C content of the strain based on its genome sequence was 56.8 mol%. Based on the phenotypic, biochemical and phylogenetic analysis, we propose the creation of the genus Fournierella gen. nov., which contains strain AT2T (=CSUR P2014T=DSM 100451T) as the type strain of the type species Fournierella massiliensis gen. nov., sp. nov.

“Anaerotruncus massiliensis,” a new species identified from human stool from an obese patient after bariatric surgery

We report the main characteristics of a new bacterial species strain AT3T (CSUR = P2007, DSM = 100567) that was isolated from the stool sample collected from a 47-year-old obese French man after he underwent bariatric surgery.

‘Urmitella timonensis’ gen. nov., sp. nov., ‘Blautia marasmi’ sp. nov., ‘Lachnoclostridium pacaense’ sp. nov., ‘Bacillus marasmi’ sp. nov. and ‘Anaerotruncus rubiinfantis’ sp. nov., isolated from stool samples of undernourished African children

We report here the main characteristics of five new species ‘Urmitella timonensis’ strain Marseille-P2918T (CSUR P2918), ‘Blautia marasmi’ strain Marseille-P2377T (CSUR P2377), ‘Lachnoclostridium pacaense’ strain Marseille-P3100T (CSUR P3100), ‘Bacillus marasmi’ strain Marseille-P3556T (CSUR P3556) and ‘Anaerotruncus rubiinfantis’ strain MT15T (CSUR P2276), which were isolated recently from stool samples taken from undernourished children in Niger and Senegal using microbial culturomics.

‘Collinsella phocaeensis’ sp. nov., ‘Clostridium merdae’ sp. nov., ‘Sutterella massiliensis’ sp. nov., ‘Sutturella timonensis’ sp. nov., ‘Enorma phocaeensis’ sp. nov., ‘Mailhella massiliensis’ gen. nov., sp. nov., ‘Mordavella massiliensis’ gen. nov., sp. nov. and ‘Massiliprevotella massiliensis’ gen. nov., sp. nov., 9 new species isolated from fresh stool samples of healthy French patients

Here we report of summary of the characteristics of ‘Collinsella phocaeensis’ strain Marseille-P3245T sp. nov., ‘Clostridium merdae’ strain Marseille-P2953T, ‘Sutterella massiliensis’ strain Marseille-P2435T sp. nov., ‘Sutturella timonensis’ strain Marseille-P3282T sp. nov., ‘Enorma phocaeensis’ Marseille-P3242T sp. nov., ‘Mailhella massiliensis’ strain Marseille-P3199T gen. nov., sp. nov., ‘Mordavella massiliensis’ strain Marseille-P3246T sp. nov. and ‘Massiliprevotella massiliensis’ strain Marseille-P2439T sp. nov. isolated from fresh stool samples of healthy French patients.

Description of ‘Arabia massiliensis’ gen. nov., sp. nov., ‘Gordonibacter massiliensis’ sp. nov., and ‘Bacilliculturomica massiliensis’ gen. nov., sp. nov., isolated from a faecal specimen of a 50-year-old Saudi Bedouin woman

We report here the main characteristics of ‘Arabia massiliensis’ strain Marseille-P3078T gen. nov., sp. nov., ‘Gordonibacter massiliensis’ Marseille-P2775T sp. nov. and ‘Bacilliculturomica massiliensis’ strain Marseille-P3303 gen. nov., sp. nov. The culturomics approach combined with taxonogenomics was used to characterize these strains, which were all isolated from a faecal specimen of a 50-year-old Saudi Bedouin woman.

Genome sequence and description of Anaeromassilibacillus senegalensis gen. nov., sp. nov., isolated from the gut of patient with kwashiorkor

Using the culturomics strategy, a wide range of anaerobic bacteria was discovered including Anaeromassilibacillus senegalensis strain mt9T (= CSUR P1511 = DSM 102954), isolated from the gut microbiota of a 1-year-old Senegalese patient with kwashiorkor. This Gram-negative strain is a strictly anaerobic, spore-forming rod motile by a polar flagellum. The 3 511 289 bp long genome of this strain contains 3046 protein-coding and 49 RNA genes, including 45 tRNA and four rRNA genes, and exhibits a G+C content of 52.94%. Here we describe the features of this organism, together with the complete genome sequence and annotation.