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.

, thi-phuong-thao pham, niokhor Dione, issa isaac ngom, camille Valles, Dipankar Bachar, Didier Raoult & Jean christophe Lagier 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. Clostridium difficile is responsible for human diseases ranging from mild diarrhea to pseudomembranous colitis 1. C. difficile was responsible for almost 30,000 deaths in the USA in 2011 2 , illustrating the high morbimortality of the disease and an increase in the number of cases. Gut dysbiosis is the triggering factor of C. difficile infection (CDI) 3,4. One of the current treatments, fecal microbiota transplantation (FMT), is based on the restoration of a healthy microbiota 5. FMT demonstrated its effectiveness in a randomized study 5 with 81% of recovery after treatment. FMT is currently recommended for recurrent CDI 6. FMT has also demonstrated its superiority compared with antibiotics as first-line treatment for severe CDI 7. Nevertheless, FMT using whole stool samples presents some limitations. For instance, despite an important pathogen screening among donors 6 , pathogen transmission through entire stool donations remains possible 8,9. Oral administration by capsules has been proposed 10 but usual methods of administration (nasogastric tube, colonoscopy…) remain invasive 11. Rare but serious adverse events correlated to these routes of administration have been reported: aspirating pneumonia, rectal perforation 11. Although there is no formal evidence, some gut bacteria have been associated to colorectal cancer 12 or obesity 13. An unexplained gain of 8.5 points of BMI following FMT has been reported 14. It is therefore desirable to know exactly which bacteria are transferred to...

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.

Antibiotic resistance genes exist naturally in various environments far from human usage. Here, we investigated multidrug-resistant Klebsiella pneumoniae, a common pathogen of chimpanzees and humans. We screened antibiotic-resistant K. pneumoniae from 48 chimpanzee stools and 38 termite mounds (N=415 samples) collected in protected areas in Senegal. The microsatellite method was used to identify chimpanzee individuals (N=13). Whole genome sequencing was performed on K. pneumoniae complex isolates to identify antibiotic-resistant genes and characterize clones. We found a high prevalence of carbapenem-resistant K. pneumoniae among chimpanzee isolates (18/48 samples from 7/13 individuals) and ceftriaxone resistance among both chimpanzee individuals (19/48) and termite mounds (7/415 termites and 3/38 termite mounds). The bla OXA-48 and the bla KPC-2 genes were carried by international pOXA-48 and pKPC-2 plasmids respectively. The ESBL plasmid carried bla CTX-M-15 , bla TEM-1B and bla OXA-1 genes. Genome sequencing of 56 isolates identified two major clones associated with hospital-acquired infections of K. pneumoniae (ST307 and ST147) in chimpanzees and termites, suggesting circulation of strains between the two species, as chimpanzees feed on termites. The source and selection pressure of these clones in this environment need to be explored.

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Intestinimonas massiliensis sp. nov strain GD2 is a new species of the genus Intestinimonas (the second, following Intestinimonas butyriciproducens gen. nov., sp. nov). First isolated from the gut microbiota of a healthy subject of French origin using a culturomics approach combined with taxono-genomics, it is strictly anaerobic, nonspore-forming, rod-shaped, with catalase- and oxidase-negative reactions. Its growth was observed after preincubation in an anaerobic blood culture enriched with sheep blood (5%) and rumen fluid (5%), incubated at 37°C. Its phenotypic and genotypic descriptions are presented in this paper with a full annotation of its genome sequence. This genome consists of 3,104,261 bp in length and contains 3,074 predicted genes, including 3,012 protein-coding genes and 62 RNA-coding genes. Strain GD2 significantly produces butyrate and is frequently found among available 16S rRNA gene amplicon datasets, which leads consideration of Intestinimonas massiliensis as an important human gut commensal.

Here we report the main features of the proposed new bacterial species “Intestinimonas massiliensis” sp. nov. The type strain GD2T (CSUR = P1930) was isolated from the gut microbiota of a healthy patient using a culturomics approach combined with taxonogenomics.

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