Culturomics identified 11 new bacterial species from a single anorexia nervosa stool sample.

The rebirth of bacterial culture has been highlighted successively by environmental microbiologists, the design of axenic culture for intracellular bacteria in clinical microbiology, and, more recently, by human gut microbiota studies. Indeed, microbial culturomics (large scale of culture conditions with the identification of colonies by MALDI-TOF or 16S rRNA) allowed to culture 32 new bacterial species from only four stool samples studied. We performed culturomics in comparison with pyrosequencing 16S rRNA targeting the V6 region on an anorexia nervosa stool sample because this clinical condition has never been explored before by culture, while its composition has been observed to be atypical by metagenomics. We tested 88 culture conditions generating 12,700 different colonies identifying 133 bacterial species, with 19 bacterial species never isolated from the human gut before, including 11 new bacterial species for which the genome has been sequenced. These 11 new bacterial species isolated from a single stool sample allow to extend more significantly the repertoire in comparison to the bacterial species validated by the rest of the world during the last 2 years. Pyrosequencing indicated a dramatic discrepancy with the culturomics results, with only 23 OTUs assigned to the species level overlapping (17 % of the culturomics results). Most of the sequences assigned to bacteria detected only by pyrosequencing belonged to Ruminococcaceae, Lachnospiraceae, and Erysipelotrichaceae constituted by strictly anaerobic species, indicating the future route for culturomics. This study revealed new bacterial species participating significantly to the extension of the gut microbiota repertoire, which is the first step before being able to connect the bacterial composition with the geographic or clinical status.

The gut microbiota of a patient with resistant tuberculosis is more comprehensively studied by culturomics than by metagenomics.

Gut microbiota consists of 10(10) bacteria per gram of stool. Many antibiotic regimens induce a reduction in both the diversity and the abundance of the gut flora. We analyzed one stool sample collected from a patient treated for drug-resistant Mycobacterium tuberculosis and who ultimately died from pneumonia due to a Streptococcus pneumoniae 10 months later. We performed microscopic observation, used 70 culture conditions (microbial culturomics) with identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and 16S rRNA amplification and sequencing, pyrosequencing, and 18S rRNA amplification and clone sequencing. Electron and optical microscopic observations revealed the presence of yeast, but no bacterial species were observed. By culture, only 39 bacterial species were identified, including one new species, as well as three species that have not been previously observed in the human gut. The pyrosequencing showed only 18 phylotypes, detecting a lower number of bacterial species than the culture techniques. Only two phylotypes overlapped with culturomics. In contrast, an amount of chloroplasts was found. Additionally, specific molecular eukaryote detection found three fungal species. We recovered, for the first time, more cultivable than non-cultivable bacterial species in a patient with a low bacterial load in the gut, demonstrating the depth bias of pyrosequencing. We propose that the desertification of gut microbiota in this patient is a reflection of the total body microbiota and may have contributed to the invasive infection of S. pneumoniae. This finding suggests that caution should be applied when treating patients with broad-spectrum antibiotics, and preventive measures should be taken in order to avoid invasive infection.

Microbial culturomics: paradigm shift in the human gut microbiome study.

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.

Obesity-associated gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii.

BACKGROUND: Obesity is associated with increased health risk and has been associated with alterations in bacterial gut microbiota, with mainly a reduction in Bacteroidetes, but few data exist at the genus and species level. It has been reported that the Lactobacillus and Bifidobacterium genus representatives may have a critical role in weight regulation as an anti-obesity effect in experimental models and humans, or as a growth-promoter effect in agriculture depending on the strains. OBJECTIVES AND METHODS: To confirm reported gut alterations and test whether Lactobacillus or Bifidobacterium species found in the human gut are associated with obesity or lean status, we analyzed the stools of 68 obese and 47 controls targeting Firmicutes, Bacteroidetes, Methanobrevibacter smithii, Lactococcus lactis, Bifidobacterium animalis and seven species of Lactobacillus by quantitative PCR (qPCR) and culture on a Lactobacillus-selective medium. FINDINGS: In qPCR, B. animalis (odds ratio (OR)=0.63; 95% confidence interval (CI) 0.39-1.01; P=0.056) and M. smithii (OR=0.76; 95% CI 0.59-0.97; P=0.03) were associated with normal weight whereas Lactobacillus reuteri (OR=1.79; 95% CI 1.03-3.10; P=0.04) was associated with obesity. CONCLUSION: The gut microbiota associated with human obesity is depleted in M. smithii. Some Bifidobacterium or Lactobacillus species were associated with normal weight (B. animalis) while others (L. reuteri) were associated with obesity. Therefore, gut microbiota composition at the species level is related to body weight and obesity, which might be of relevance for further studies and the management of obesity. These results must be considered cautiously because it is the first study to date that links specific species of Lactobacillus with obesity in humans.

Microbial diversity in the sputum of a cystic fibrosis patient studied with 16S rDNA pyrosequencing.

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.