Peptoniphilus coli sp. nov. and Peptoniphilus urinae sp. nov., isolated from humans.

Strains Marseille-P3761 and Marseille-P3195 are representatives of two bacterial species isolated from human specimens. Strain Marseille-P3761 was isolated from the stool of a healthy volunteer, while strain Marseille-P3915 was cultivated from the urine of a kidney transplant recipient. Both strains are anaerobic Gram-positive coccoid bacteria. Both are catalase-negative and oxidase-negative and grow optimally at 37 °C in anaerobic conditions. They also metabolize carbohydrates, such as galactose, glucose, fructose, and glycerol. The major fatty acids were hexadecanoic acid for both strains. The highest digital DNA-DNA hybridization (dDDH) values of Marseille-P3761 and Marseille-P3195 strains when compared to their closest phylogenetic relatives were 52.3% and 56.4%, respectively. Strains Marseille-P3761 and Marseille-P3195 shared an OrthoANI value of 83.5% which was the highest value found with Peptoniphilus species studied here. The morphological, biochemical, phenotypic and genomic characteristics strongly support that these strains are new members of the Peptoniphilus genus. Thus, we suggest that Peptoniphilus coli sp. nov., and Peptoniphilus urinae sp. nov., are new species for which strains Marseille-P3761 (CSUR P3761 = CCUG 71,569) and Marseille-P3195 (CSUR P3195 = DSM 103,468) are their type strains, respectively of two new Peptoniphilus species, for which we propose the names Peptoniphilus coli sp. nov. and Peptoniphilus urinae sp. nov., respectively.

Limosilactobacillus caccae sp. nov., a new bacterial species isolated from the human gut microbiota.

Strain Marseille-P3519T isolated from the fecal flora of a 25-year-old healthy French woman was a Gram-positive anaerobic bacterium, non-motile and non-spore forming. The 16S rRNA gene sequence of Marseille-P3519 showed 97.73% of sequence similarity with Limosilactobacillus reuteri DSM 20016, the closest species, phylogenetically. Furthermore, the average nucleotide identity of strain Marseille-3519 with its closest related species was 75.8% that was very below the recommended threshold (>95-96%). Its genome had 2 237 367 bp with 45.42 mol% of G + C content. Major fatty acids were C16:0 (50.8%), C18:1n9 (18.0%), C18:2n6 (9.8%) and C19:1n9 (8.9%). It was catalase negative and fermented glycerol, glucose, fructose, D-maltose, lactose and mannose. These findings support that strain Marseille-P3519 ( = CSURP3519 = CECT 30110) is a new member of the genus Limosilactobacillus for which the name Limosilactobacillus caccae sp. nov., is proposed.

Proof of Concept of Culturomics Use of Time of Care.

Culturomics, a high throughput culture method with rapid identification of the colonies by Matrix Assisted Laser Desorption Ionization/Time Of Flight Mass Spectrometry (MALDI-TOF MS), has demonstrated its contribution to the exploration of the gut microbiota over the past 10 years. However, the cost, work time and workload, considerably limit its use on a large scale or emergency context. Here, by testing two different stool samples, including a stool sample from a patient requiring rapid immunotherapy treatment, we tested a new fast culturomic protocol using two pre-incubation media, blood culture bottle and YCFA modified medium. Both media were supplemented with 2 ml of rumen fluid filtered at 0.2 µm and 2 ml of defibrinated and sterile sheep blood. Unlike the standard culturomics, subculturing of blood culture bottle were performed at reduced incubation time (3 h, 6 h, 9 h, 24 h) and at a longer incubation time (3 days, 7 days, and 10 days) at 37°C. By testing 5,200 colonies per MALDI-TOF MS and obtaining a comparable number of cultured bacterial species (131 to 143) in a stool sample, this new protocol reduced the number of colonies tested by 57%, working time by 78.6% and cost by 72.2%. In addition, we highlighted that the proportion of strict anaerobic species has increased by 24%, known to be the preferential targets for biotherapy, including Faecalibacterium prausnitzii, Akkermansia muciniphila, Christensenella minuta, and Phascolarctobacterium faecium. Finally, this work showed that some bacterial species grew earlier but disappeared with prolonged incubation times.

Mgll Knockout Mouse Resistance to Diet-Induced Dysmetabolism Is Associated with Altered Gut Microbiota.

Monoglyceride lipase (MGLL) regulates metabolism by catabolizing monoacylglycerols (MAGs), including the endocannabinoid 2-arachidonoyl glycerol (2-AG) and some of its bioactive congeners, to the corresponding free fatty acids. Mgll knockout mice (Mgll-/-) exhibit elevated tissue levels of MAGs in association with resistance to the metabolic and cardiovascular perturbations induced by a high fat diet (HFD). The gut microbiome and its metabolic function are disrupted in obesity in a manner modulated by 2-arachidonoyl glycerol (2-AG's) main receptors, the cannabinoid CB1 receptors. We therefore hypothesized that Mgll-/- mice have an altered microbiome, that responds differently to diet-induced obesity from that of wild-type (WT) mice. We subjected mice to HFD and assessed changes in the microbiomes after 8 and 22 weeks. As expected, Mgll-/- mice showed decreased adiposity, improved insulin sensitivity, and altered circulating incretin/adipokine levels in response to HFD. Mgll-/- mice on a chow diet exhibited significantly higher levels of Hydrogenoanaerobacterium, Roseburia, and Ruminococcus than WT mice. The relative abundance of the Lactobacillaceae and Coriobacteriaceae and of the Lactobacillus, Enterorhabdus, Clostridium_XlVa, and Falsiporphyromonas genera was significantly altered by HFD in WT but not Mgll-/- mice. Differently abundant families were also associated with changes in circulating adipokine and incretin levels in HFD-fed mice. Some gut microbiota family alterations could be reproduced by supplementing 2-AG or MAGs in culturomics experiments carried out with WT mouse fecal samples. We suggest that the altered microbiome of Mgll-/- mice contributes to their obesity resistant phenotype, and results in part from increased levels of 2-AG and MAGs.

Clostridium massiliamazoniense sp. nov., New Bacterial Species Isolated from Stool Sample of a Volunteer Brazilian.

The study of the gut microbiota by the "culturomics concept" permitted us to isolate, from human stool sample, an unknown anaerobic bacterium within the genus Clostridium for which we propose the name Clostridium massiliamazoniense sp. nov. It was isolated from the fecal flora of a healthy 49-year-old Brazilian male. Here, we describe the characteristics of this organism and its complete genome sequencing and annotation. Clostridium massiliamazoniense sp. nov., ND2T (= CSURP1360 = DSMZ 27309) is a Gram-positive, obligate anaerobic member of Firmicutes with a 3,732,600 bp-long genome and a G+C content of 27.6%.

Optimization and standardization of the culturomics technique for human microbiome exploration.

Culturomics is a high-throughput culture approach that has dramatically contributed to the recent renewal of culture. While metagenomics enabled substantial advances in exploring the microbiota, culturomics significantly expanded our knowledge regarding the bacterial gut repertoire through the discovery and the description of hundreds of new taxa. While this approach relies on the variation of culture conditions and media, we have tested so far more than 300 conditions since the beginning of culturomics studies. In this context, we aimed herein to identify the most profitable conditions for optimizing culturomics approach. For this purpose, we have analysed a set of 58 culturomics conditions that were previously applied to 8 faecal specimens, enabling the isolation of 497 bacterial species. As a result, we were able to reduce the number of conditions used to isolate these 497 of more than a half (i.e. to 25 culture conditions). We have also established a list of the 16 conditions that allowed to capture 98% of the total number of species previously isolated. These data constitute a methodological starting point for culture-based microbiota studies by improving the culturomics workflow without any loss of captured bacterial diversity.

Alcohol pretreatment of stools effect on culturomics.

Recent studies have used ethanol stool disinfection as a mean of promoting valuable species' cultivation in bacteriotherapy trials for Clostridium difficile infections (CDI) treatment with a particular focus on sporulating bacteria. Moreover, the culturomic approach has considerably enriched the repertoire of cultivable organisms in the human gut in recent years. This study aimed to apply this culturomic approach on fecal donor samples treated with ethanol disinfection to evidence potential beneficial microbes that could be used in bacteriotherapy trials for the treatment of CDI. Thereby, a total of 254 bacterial species were identified, 9 of which were novel. Of these, 242 have never been included in clinical trials for the treatment of CDIs, representing potential new candidates for bacteriotherapy trials. While non-sporulating species were nevertheless more affected by the ethanol pretreatment than sporulating species, the ethanol disinfection technique did not specifically select bacteria able to sporulate, as suggested by previous studies. Furthermore, some bacteria previously considered as potential candidates for bacteriotherapy have been lost after ethanol treatment. This study, while enriching the bacterial repertoire of the human intestine, would nevertheless require determining the exact contribution of each of species composing the bacterial consortia intended to be administered for CDI treatment.

Extensive culturomics of 8 healthy samples enhances metagenomics efficiency.

Molecular approaches have long led to the assumption that the human gut microbiota is dominated by uncultivable bacteria. The recent advent of large-scale culturing methods, and in particular that of culturomics have demonstrated that these prokaryotes can in fact be cultured. This is increasing in a dramatic manner the repertoire of commensal microbes inhabiting the human gut. Following eight years of culturomics approach applied on more than 900 samples, we propose herein a remake of the pioneering study applying a dual approach including culturomics and metagenomics on a cohort of 8 healthy specimen. Here we show that culturomics enable a 20% higher richness when compared to molecular approaches by culturing 1 archaeal species and 494 bacterial species of which 19 were new taxa. Species discovered as a part of previous culturomics studies represent 30% of the cultivated isolates, while sequences derived from these new taxa enabled to increase by 22% the bacterial richness retrieved by metagenomics. Overall, 67% of the total reads generated were covered by cultured isolates, significantly reducing the hidden content of sequencing methods compared to the pioneering study. By redefining culture conditions to recover microbes previously considered fastidious, there are greater opportunities than ever to eradicate metagenomics dark matter.

Metagenomic and culturomic analysis of gut microbiota dysbiosis during Clostridium difficile infection.

Recently, cocktail of bacteria were proposed in order to treat Clostridium difficile infection (CDI), but these bacteriotherapies were selected more by chance than experimentation. We propose to comprehensively explore the gut microbiota of patients with CDI compared to healthy donors in order to propose a consortium of bacteria for treating C. difficile. We compared stool samples composition from 11 CDI patients and 8 healthy donors using two techniques: metagenomics, 16S V3-V4 region amplification and sequencing and culturomics, high throughout culture using six culture conditions and MALDI-TOF identification. By culturomics, we detected 170 different species in the CDI group and 275 in the control group. Bacteroidetes were significantly underrepresented in the CDI group (p = 0.007). By metagenomics, 452 different operational taxonomic units assigned to the species level were detected in the CDI group compared to 522 in the control group. By these two techniques, we selected 37 bacteria only found in control group in more than 75% of the samples and/or with high relative abundance, 10 of which have already been tested in published bacteriotherapies against CDI, and 3 of which (Bifidobacterium adolescentis, Bifidobacterium longum and Bacteroides ovatus) have been detected by these two techniques. This controlled number of bacteria could be administrated orally in a non-invasive way in order to treat CDI.

Description and genomic characterization of Massiliimalia massiliensis gen. nov., sp. nov., and Massiliimalia timonensis gen. nov., sp. nov., two new members of the family Ruminococcaceae isolated from the human gut.

Using the culturomics approach, we isolated two strains, Marseille-P2963 and Marseille-P3753, from the intestinal microbiota of a 19-year-old healthy Saudi Arabian Bedouin male and from a 32-year-old healthy Senegalese male faecal transplant donor. Here, we studied their phenotypic, phylogenetic and genomic characteristics. Both strains were phylogenetically related, but different from Ruminococcus species. Bacterial cells were anaerobic, rod-shaped, non-spore-forming and not motile, with neither catalase nor oxidase activities. Their growth temperatures ranged from 28 to 45 °C, with an optimal growth at 37 °C. The genomes are 2,842,720 bp- and 2,707,061 bp-long respectively. The G + C contents are 47.18% and 46.90%, respectively. Based on these characteristics, we propose the creation of a new genus within the family Ruminococcaceae named Massiliimalia gen. nov., that contains the new species Massiliimalia massiliensis gen. nov., sp. nov., and Massiliimalia timonensis gen. nov., sp. nov. Strains Marseille-P2963T (= CSUR P2963 = DSM 106837) and Marseille-P3753T (= CSUR P3753 = CCUG 71632) are their type strains, respectively.

Anaerococcus jeddahensis sp. nov., a New Bacterial Species Isolated From Healthy Nomadic Bedouin Woman From Saudi Arabia.

An understanding of the microbial diversity of the human body has generated significant interest in recent years. With the advent of MALDI-TOF mass spectrometry, high-speed sequencing, and the rebirth of microbial culture, knowledge of human microbiota is growing. Using culturomics, a strategy to explore the microbial diversity of samples, coupled with a taxono-genomic strategy, we isolated a new bacterium named Anaerococcus jeddahensis sp. nov. strain SB3T. This strain was isolated from the stool sample of a healthy nomadic Bedouin woman from Saudi Arabia. Here, we describe the characteristics of this organism, and the complete genome sequence and annotation. Strain SB3T is a Gram-positive obligate anaerobic coccus which is non-motile and non-spore forming. Fatty acid analysis shows that the major fatty acid is by far hexadecanoic acid (C16:0; 52%). Its genome is 1,903,534 bp long and has 29.70 mol% of G+C content. It contains 1756 protein-coding genes and 53 RNA genes. These results show that strategy provides a better understanding of the microorganism and that is a good methodology for microbial identification and characterization.

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.

Increased Gut Redox and Depletion of Anaerobic and Methanogenic Prokaryotes in Severe Acute Malnutrition.

Severe acute malnutrition (SAM) is associated with inadequate diet, low levels of plasma antioxidants and gut microbiota alterations. The link between gut redox and microbial alterations, however, remains unexplored. By sequencing the gut microbiomes of 79 children of varying nutritional status from three centers in Senegal and Niger, we found a dramatic depletion of obligate anaerobes in malnutrition. This was confirmed in an individual patient data meta-analysis including 107 cases and 77 controls from 5 different African and Asian countries. Specifically, several species of the Bacteroidaceae, Eubacteriaceae, Lachnospiraceae and Ruminococceae families were consistently depleted while Enterococcus faecalis, Escherichia coli and Staphylococcus aureus were consistently enriched. Further analyses on our samples revealed increased fecal redox potential, decreased total bacterial number and dramatic Methanobrevibacter smithii depletion. Indeed, M. smithii was detected in more than half of the controls but in none of the cases. No causality was demonstrated but, based on our results, we propose a unifying theory linking microbiota specificity, lacking anaerobes and archaea, to low antioxidant nutrients, and lower food conversion.

The aerobic activity of metronidazole against anaerobic bacteria.

Recently, the aerobic growth of strictly anaerobic bacteria was demonstrated using antioxidants. Metronidazole is frequently used to treat infections caused by anaerobic bacteria; however, to date its antibacterial activity was only tested in anaerobic conditions. Here we aerobically tested using antioxidants the in vitro activities of metronidazole, gentamicin, doxycycline and imipenem against 10 common anaerobic and aerobic bacteria. In vitro susceptibility testing was performed by the disk diffusion method, and minimum inhibitory concentrations (MICs) were determined by Etest. Aerobic culture of the bacteria was performed at 37°C using Schaedler agar medium supplemented with 1mg/mL ascorbic acid and 0.1mg/mL glutathione; the pH was adjusted to 7.2 by 10M KOH. Growth of anaerobic bacteria cultured aerobically using antioxidants was inhibited by metronidazole after 72h of incubation at 37°C, with a mean inhibition diameter of 37.76mm and an MIC of 1µg/mL; however, strains remained non-sensitive to gentamicin. No growth inhibition of aerobic bacteria was observed after 24h of incubation at 37°C with metronidazole; however, inhibition was observed with doxycycline and imipenem used as controls. These results indicate that bacterial sensitivity to metronidazole is not related to the oxygen tension but is a result of the sensitivity of the micro-organism. In future, both culture and antibiotic susceptibility testing of strictly anaerobic bacteria will be performed in an aerobic atmosphere using antioxidants in clinical microbiology laboratories.

Culturomics and pyrosequencing evidence of the reduction in gut microbiota diversity in patients with broad-spectrum antibiotics.

The human gut flora is currently widely characterised using molecular techniques. Microbial culturomics (large-scale culture conditions with identification of colonies using MALDI-TOF or 16S rRNA) is part of the rebirth of bacterial culture that was initiated by environmental microbiologists for the design of axenic culture for intracellular bacteria in clinical microbiology. Culturomics was performed on four stool samples from patients treated with large-scale antibiotics to assess the diversity of their gut flora in comparison with other culture-dependent studies. Pyrosequencing of the V6 region was also performed and was compared with a control group. Gut richness was also estimated by bacterial counting after microscopic observation. In total, 77 culture conditions were tested and 32,000 different colonies were generated; 190 bacterial species were identified, with 9 species that had not been isolated from the human gut before this study, 7 newly described in humans and 8 completely new species. A dramatic reduction in diversity was observed for two of the four stool samples for which antibiotic treatment was prolonged and uninterrupted. The total number of bacteria was generally preserved, suggesting that the original population was replaced but was sustained in size. Discordances between culture and pyrosequencing biodiversity biomarkers highlight the depth of bias of molecular studies. Stool samples studied showed a dramatic reduction in bacterial diversity. Considering the variable antibiotic concentration in the gut, this reduction in the number of species is possibly linked to the production of bacteriocin in the upper digestive tract by specific bacteria, such as Lactobacillus spp.