Salt in stools is associated with obesity, gut halophilic microbiota and Akkermansia muciniphila depletion in humans.

BACKGROUND/OBJECTIVES: High salt intake has been linked to several diseases including obesity and an increased risk of death; however, fecal salinity and the ability of salt to alter the gut microbiota, which was recently identified as an instrumental factor for health and disease, remains poorly explored. METHODS/SUBJECTS: We analyzed the fecal samples of 1326 human individuals for salinity by refractometry, 572 for gut microbiota by culturomics, and 164 by 16S rRNA-targeted metagenomics. Geographical origin, age, gender, and obesity were tested as predictors of fecal salinity and halophilic diversity. All halophilic isolates were characterized by taxonogenomics and their genome sequenced. RESULTS: Fecal salinity was associated with obesity independently of geographical origin, gender, and age. The first 2 human-associated halophilic archaeal members were isolated along with 64 distinct halophilic species, including 21 new species and 41 known in the environment but not in humans. No halophiles grow in less than 1.5% salinity. Above this threshold, the richness of the halophilic microbiota was correlated with fecal salinity (r = 0.58, p < 0.0001). 16S metagenomics linked high fecal salinity to decreased diversity (linear regression, p < .035) and a depletion in anti-obesity Akkermansia muciniphila and Bifidobacterium, specifically B. longum and B. adolescentis. Genomics analysis suggested that halophilic microbes are not only transient passengers but may be residents of the human gut. CONCLUSIONS: High salt levels are associated with alteration of the gut microbial ecosystem and halophilic microbiota, as discovered during this study. Further studies should clarify if the gut microbiota alterations associated with high salt levels and the human halophilic microbiota could be causally related to human disease, such as obesity.

Aerobic culture of methanogenic archaea without an external source of hydrogen.

Culturing methanogenic archaea is fastidious, expensive, and requires an external source of hydrogen and carbon dioxide. Until now, these microorganisms have only been cultivated under strictly anaerobic conditions. We previously developed a single versatile culture medium containing sugars and anti-oxydants for cultivating all human known methanogens. Performing aerobic cultures in the presence of Bacteroides thetaiotaomicron, which produces hydrogen, allows for cultivation of Methanobrevibacter smithii which itself produces methane. To obtain colonies, we cultivated M. smithii in an agar plate in the upper part of a double chamber flask with a liquid culture of B. thetaiotaomicron in the lower compartment. We subsequently cultured four other methanogenic species for the first time and successfully isolated 13 strains of M. smithii and nine strains of Methanobrevibacter oralis from 100 stools and 45 oral samples. This procedure allows aerobic isolation and antibiotic susceptibility testing. This changes the ability to routinely study methanogens, which have been neglected in clinical microbiology laboratories and may be useful for biogas production.

Aerobic culture of anaerobic bacteria using antioxidants: a preliminary report.

Antioxidants have been shown to help the growth of anaerobic bacteria. We were able to grow six anaerobe species (including Fusobacterium necrophorum and Ruminococcus gravus) and seven aerobic species all aerobically in Schaedler agar tubes and agar plates with high doses of ascorbic acid and/or glutathione. This may deeply change strategies for culturing bacteria.

Bacterial culture through selective and non-selective conditions: the evolution of culture media in clinical microbiology.

Microbiology has been largely developed thanks to the discovery and optimization of culture media. The first liquid artificial culture medium was created by Louis Pasteur in 1860. Previously, bacterial growth on daily materials such as some foods had been observed. These observations highlighted the importance of the bacteria's natural environment and their nutritional needs in the development of culture media for their isolation. A culture medium is essentially composed of basic elements (water, nutrients), to which must be added different growth factors that will be specific to each bacterium and necessary for their growth. The evolution of bacterial culture through the media used for their culture began with the development of the first solid culture medium by Koch, allowing not only the production of bacterial colonies, but also the possibility of purifying a bacterial clone. The main gelling agent used in solid culture media is agar. However, some limits have been observed in the use of agar because of some extremely oxygen-sensitive bacteria that do not grow on agar media, and other alternatives were proposed and tested. Then, the discovery of antimicrobial agents and their specific targets prompted the emergence of selective media. These inhibiting agents make it possible to eliminate undesirable bacteria from the microbiota and select the bacteria desired. Thanks to a better knowledge of the bacterial environment, it will be possible to develop new culture media and new culture conditions, better adapted to certain fastidious bacteria that are difficult to isolate.

Description of Prevotella rectalis sp. nov., a new bacterium isolated from human rectum.

Using a taxonogenomics method, we describe here a Gram-negative bacterium named Prevotella rectalis sp. nov., strain Marseille-P4334T (= CSUR P4334) isolated from the rectum. Strain Marseille-P4334T has a genome that measure 3.03 Mbp with 43.3 mol% G + C content.

Massilistercora timonensis gen. nov., sp. nov., a new bacterium isolated from the human microbiota.

Massilistercora timonensis gen. nov., sp. nov. strain Marseille-P3756T is a new species of the phylum Firmicutes; it was isolated from the human gut microbiota and has a genome of 2 769 591 bp (51.2% G + C). The closest species based on 16S rRNA sequence was Merdimonas faecis strain BR31 with 95.2 % sequence similarity. Considering phenotypic features and comparative genome studies, we proposed the strain Marseille-P3756T as the type strain of Massilistercora timonensis sp. nov., a new species within the genus Massilistercora.

Corrigendum to "'Bacteroides mediterraneensis' sp. nov., a new human-associated bacterium isolated from ileum specimen".

[This corrects the article DOI: 10.1016/j.nmni.2016.06.003.].

Isolation and culture of Methanobrevibacter smithii by co-culture with hydrogen-producing bacteria on agar plates.

OBJECTIVES: Methanogenic Archaea are considered as extremely oxygen-sensitive organisms, and their culture is fastidious, requiring specific equipment. We report here conditions allowing the cultivation of Methanobrevibacter smithii in an anaerobic chamber without the addition of hydrogen. METHODS: We first enriched the stool sample in an anaerobic liquid medium. To cultivate M. smithii with Bacteroides thetaiotaomicron and other hydrogen-producing bacteria on solid medium in an anaerobic chamber, we divided the agar plates into two compartments and seeded each strain on each compartment. Methane production was assessed by gas chromatography, and the growing colonies were authenticated by MALDI-TOF MS. RESULTS: We successfully cultured M. smithii from a liquid culture medium inoculated with stool collected from a healthy donor in an anaerobic chamber. The isolation in pure culture permitted successful culture on agar medium by our performing a co-culture with B. thetaiotaomicron. We also successfully tested the co-cultivation of M. smithii with other known hydrogen-producing bacteria. Gas chromatographic tests showed that these strains produced hydrogen in different amounts. Agar colonies of methanogens were obtained by co-culture with these bacteria, and methane production was detected. CONCLUSIONS: We propose a new approach to isolate and cultivate new strains of M. smithii by using a co-culture-based technique that can facilitate and make available the isolation of new methanogenic Archaea strains in clinical microbiology laboratories.

Neoactinobaculum massilliense gen. nov., a new genesis and Pseudopropionibacterium massiliense sp. nov., a new bacterium isolated from the human oral microbiota.

Neoactinobaculum massilliense gen. nov., strain Marseille-P6182T (= CSUR P6182) and Pseudopropionibacterium massiliense sp. nov., strain Marseille-P6184T (= CSUR P6184) are a new bacterial genus and new bacterial species belonging to the Actinobacteria phylum that have been isolated from the human oral microbiota.

Corrigendum to '"Mediterranea massiliensis" gen. nov., sp. nov., a new human-associated bacteria isolated from the right and left colon lavage of a 58-year-old patient'.

[This corrects the article DOI: 10.1016/j.nmni.2016.06.002.].

Massilimicrobiota timonensis gen. nov., sp. nov., a new bacterium isolated from the human gut microbiota.

Massilimicrobiota timonensis gen. nov., sp. nov. strain Marseille-P2264 is a new species from Firmicutes phylum isolated from the human gut. Its genome was 2,849,574 bp-long with a 31.8% G+C content. The closest species based on 16S rRNA sequence was Longibaculum muris with 95.6% sequence similarity. Considering phenotypic features, 16S rRNA sequence and comparative genome studies, we proposed Marseille-P2264 as the type strain of Massilimicrobiota timonensis gen. nov., sp. nov.

Genome sequence and description of Bacteroides bouchesdurhonensis sp. nov., a new anaerobic bacterium isolated from the human gut.

Bacteroides bouchesdurhonensis sp. nov., strain Marseille-P2653T (= CSUR; P2653=DSM103120) is a new bacterial species belonging to the Firmicutes phylum in the family Bacteroidaceae that was isolated from the human gut microbiota.

Genome sequence and description of Coprococcus phoceensis gen. nov., sp. nov., a new bacterial genus isolated from the human left colon.

We report here the main characteristics of Coprococcus phoceensis strain Marseille-P3062T (CSUR P3062). The 16S rDNA sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectrum analysis were used to identify and characterize this new anaerobic bacterial species, which was isolated from the left colon cleansing of a 25-year-old French man with Crohn's disease.

Study of Human Gastrointestinal Microbiota by Culturomics in Africa.

The interest in studying gut microbiota has been rekindled with the advent of molecular techniques, in particular, metagenomics. Culturomics (high throughput microbial culture with identification of the colonies by Maldi-TOF) has demonstrated its complementarity with metagenomics for comprehensive study of the microbiota. The main metagenomic studies have revealed an increase in biodiversity, with in particular an increase of Spirochaetes and Prevotella in subjects of African origin compared with Western subjects. Studies on malnutrition have shown a reduction of all bacteria and in particular of anaerobic bacteria and methanogenic archaea. Of the 1,162 bacteria isolated by culturomics studies, 476 were isolated only from non-African samples, 445 were isolated in African and non-African groups, and 241 bacteria were isolated from samples of African origin including 68 new species. Further studies of African microbiota by culturomics and metagenomics will make it possible to assess whether some bacteria have particular specificities and if these might play a role in certain pathologies such as malnutrition.

Olsenella timonensis sp. nov., a new bacteria species isolated from the human gut microbiota.

Olsenella timonensis sp. nov., strain Marseille-P2300T (= CSUR P2300; =DSM102072), is a new bacterial species from the phylum Firmicutes in the family Atopobiaceae.This bacteria species was isolated from the human gut microbiota.

Massilicoli timonensis sp. nov., a new bacterium isolated from the human microbiota.

Massilicoli timonensis sp. nov., strain Marseille-P3755T (= CSUR P3755 = DSM 103513) is a new bacterial species from the phylum Firmicutes and the family Clostridiales which was isolated from the human gut microbiota.

Genome sequence and description of Traorella massiliensis gen. nov., sp. nov., a new bacterial genus isolated from human left colon.

A strictly anaerobic, motile, non-spore-forming, Gram-negative, rod-shaped bacterium designated Marseille-P3110T was isolated from the left colon cleansing of a 76-year-old Frenchwoman. Its 16S ribosomal RNA (rRNA) gene showed a 93.2% similarity level with the 16S rRNA of Dielma fastidiosa strain JC13, the closest species with a validly published name. The genome of Marseille-P3110T is 2 607 061 bp long with 35.99% G+C content. Of the 2642 predicted genes, 2582 were protein-coding genes and 60 were RNAs, including five 16S rRNA genes.

Anaerotruncus massiliensis sp. nov., a succinate-producing bacterium isolated from human stool from an obese patient after bariatric surgery.

A new bacterium, strain AT3T, was isolated by microbial culturomics from a faecal sample from a Frenchman after bariatric surgery. The isolate exhibited 96.6% 16S ribosomal RNA gene nucleotide sequence similarity with Anaerotruncus colihominis strain WAL 14565T = CCUG 45055T = CIP 107754T. Phenotypic and genomic characteristics showed that the new strain represents a novel species, for which the name Anaerotruncus massiliensis sp. nov. is proposed. The type strain is strain AT3T = CSUR P2007T = DSM 100567T.

Non-contiguous finished genome sequencing and description of Enterococcus timonensis sp. nov. isolated from human sputum.

Enterococcus timonensis sp. nov., strain Marseille-P2817T, is a facultatively anaerobic, motile and non-spore-forming Gram-positive coccus which was isolated from the sputum of a healthy adult man in Marseilles. We present herein its phenotypic description together with MALDI-TOF (matrix-assisted laser-desorption/ionization time-of-flight) mass spectrometry analysis and genome sequencing and comparison. The genome of Enterococcus timonensis is 2 123 933 bp long with 38.46 mol% of G+C content, and it contains 1983 protein-coding genes and 65 RNA genes (including nine rRNA genes).