Comprehensive determination of the microbial composition of the gut microbiota and the relationships with health and disease are major challenges in the 21st century. Metagenomic analysis of the human gut microbiota detects mostly uncultured bacteria. We studied stools from two lean Africans and one obese European, using 212 different culture conditions (microbial culturomics), and tested the colonies by using mass spectrometry and 16S rRNA amplification and sequencing. In parallel, we analysed the same three samples by pyrosequencing 16S rRNA amplicons targeting the V6 region. The 32 500 colonies obtained by culturomics have yielded 340 species of bacteria from seven phyla and 117 genera, including two species from rare phyla (Deinococcus-Thermus and Synergistetes, five fungi, and a giant virus (Senegalvirus). The microbiome identified by culturomics included 174 species never described previously in the human gut, including 31 new species and genera for which the genomes were sequenced, generating c. 10 000 new unknown genes (ORFans), which will help in future molecular studies. Among these, the new species Microvirga massiliensis has the largest bacterial genome so far obtained from a human, and Senegalvirus is the largest virus reported in the human gut. Concurrent metagenomic analysis of the same samples produced 698 phylotypes, including 282 known species, 51 of which overlapped with the microbiome identified by culturomics. Thus, culturomics complements metagenomics by overcoming the depth bias inherent in metagenomic approaches.

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 microorganism 1 . The pure culture of prokaryotes, neglected in recent decades, remains essential to elucidating the role of these organisms 2 . 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 identification 2 . 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 studies 3-5 . 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.

BackgroundThere is strong evidence that culture-based methods detect only a small proportion of bacteria present in the respiratory tracts of cystic fibrosis (CF) patients.Methodology/Principal FindingsStandard microbiological culture and phenotypic identification of bacteria in sputa from CF patients have been compared to molecular methods by the use of 16S rDNA amplification, cloning and sequencing. Twenty-five sputa from CF patients were cultured that yield 33 isolates (13 species) known to be pathogens during CF. For molecular cloning, 760 clones were sequenced (7.2±3.9 species/sputum), and 53 different bacterial species were identified including 16 species of anaerobes (30%). Discrepancies between culture and molecular data were numerous and demonstrate that accurate identification remains challenging. New or emerging bacteria not or rarely reported in CF patients were detected including Dolosigranulum pigrum, Dialister pneumosintes, and Inquilinus limosus.Conclusions/SignificanceOur results demonstrate the complex microbial community in sputa from CF patients, especially anaerobic bacteria that are probably an underestimated cause of CF lung pathology. Metagenomic analysis is urgently needed to better understand those complex communities in CF pulmonary infections.

BackgroundMicrobial eukaryotes represent an important component of the human gut microbiome, with different beneficial or harmful roles; some species are commensal or mutualistic, whereas others are opportunistic or parasitic. The diversity of eukaryotes inhabiting humans remains relatively unexplored because of either the low abundance of these organisms in human gut or because they have received limited attention from a whole-community perspective.Methodology/Principal FindingIn this study, a single fecal sample from a healthy African male was studied using both culture-dependent methods and extended molecular methods targeting the 18S rRNA and ITS sequences. Our results revealed that very few fungi, including Candida spp., Galactomyces spp., and Trichosporon asahii, could be isolated using culture-based methods. In contrast, a relatively a high number of eukaryotic species could be identified in this fecal sample when culture-independent methods based on various primer sets were used. A total of 27 species from one sample were found among the 977 analyzed clones. The clone libraries were dominated by fungi (716 clones/977, 73.3%), corresponding to 16 different species. In addition, 187 sequences out of 977 (19.2%) corresponded to 9 different species of plants; 59 sequences (6%) belonged to other micro-eukaryotes in the gut, including Entamoeba hartmanni and Blastocystis sp; and only 15 clones/977 (1.5%) were related to human 18S rRNA sequences.ConclusionOur results revealed a complex eukaryotic community in the volunteer’s gut, with fungi being the most abundant species in the stool sample. Larger investigations are needed to assess the generality of these results and to understand their roles in human health and disease.

Herein, the mycobiota was characterized in fecal samples from sick patients and healthy subjects, collected from different geographical locations and using both culturomics and amplicon-based metagenomics approaches. Using the culturomics approach, a total of 17,800 fungal colonies were isolated from 14 fecal samples, and resulted in the isolation of 41 fungal species, of which 10 species had not been previously reported in the human gut. Deep sequencing of fungal-directed ITS1 and ITS2 amplicons led to the detection of a total of 142 OTUs and 173 OTUs from the ITS1 and ITS2 regions, respectively. Ascomycota composed the largest fraction of the total OTUs analyzed (78.9% and 68.2% of the OTUs from the ITS1 and ITS2 regions, respectively), followed by Basidiomycota (16.9% and 30.1% of the OTUs from the ITS1 and ITS2 regions, respectively). Interestingly, the results demonstrate that the ITS1/ITS2 amplicon sequencing provides different information about gut fungal communities compared to culturomics, though both approaches complete each other in assessing fungal diversity in fecal samples. We also report higher fungal diversity and abundance in patients compared to healthy subjects. In conclusion, combining both culturomic and amplicon-based metagenomic approaches may be a novel strategy towards analyzing fungal compositions in the human gut.

Recent studies using 16S rRNA gene amplification followed by clonal Sanger sequencing in cystic fibrosis demonstrated that cultured microorganisms are only part of the infecting flora. The purpose of this paper was to compare pyrosequencing and clonal Sanger sequencing on sputum. The sputum of a patient with cystic fibrosis was analysed by culture, Sanger clone sequencing and pyrosequencing after 16S rRNA gene amplification. A total of 4,499 sequencing reads were obtained, which could be attributed to six consensus sequences, but the length of reads leads to fastidious data analysis. Compared to clonal Sanger sequencing and to cultivation results, pyrosequencing recovers greater species richness and gives a more reliable estimate of the relative abundance of bacterial species. The 16S pyrosequencing approach expands our knowledge of the microbial diversity of cystic fibrosis sputum. The current lack of phylogenetic resolution at the species level for the GS 20 sequencing reads will be overcome with the next generation of pyrosequencing apparatus.

Respiratory infections remain a major threat to cystic fibrosis (CF) patients. The detection and correct identification of the bacteria implicated in these infections is critical for the therapeutic management of patients. The traditional methods of culture and phenotypic identification of bacteria lack both sensitivity and specificity because many bacteria can be missed and/or misidentified. Molecular analyses have recently emerged as useful means to resolve these problems, including molecular methods for accurate identification or detection of bacteria and molecular methods for evaluation of microbial diversity. These recent molecular technologies have increased the list of new and/or emerging pathogens and epidemic strains associated with CF patients. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of intact cells has also emerged recently as a powerful and rapid method for the routine identification of bacteria in clinical microbiology laboratories and will certainly represent the method of choice also for the routine identification of bacteria in the context of CF. Finally, recent data derived from molecular culture-independent analyses indicate the presence of a previously underestimated, complex microbial community in sputa from CF patients. Interestingly, full genome sequencing of some bacteria frequently recovered from CF patients has highlighted the fact that the lungs of CF patients are hotspots for lateral gene transfer and the adaptation of these ecosystems to a specific chronic condition.

Background: Staphylococcus aureus is a major human pathogen responsible for a variety of nosocomial and community-acquired infections. Recent reports show that the prevalence of Methicillin-Resistant S. aureus (MRSA) infections in cystic fibrosis (CF) patients is increasing. In 2006 in Marseille, France, we have detected an atypical MRSA strain with a specific antibiotic susceptibility profile and a unique growth phenotype. Because of the clinical importance of the spread of such strain among CF patients we decided to sequence the genome of one representative isolate (strain CF-Marseille) to compare this to the published genome sequences. We also conducted a retrospective epidemiological analysis on all S. aureus isolated from 2002 to 2007 in CF patients from our institution.