Repertoire of the gut microbiota from stomach to colon using culturomics and next-generation sequencing.

BACKGROUND: Most studies on the human microbiota have analyzed stool samples, although a large proportion of the absorption of nutrients takes place in upper gut tract. We collected samples from different locations along the entire gastrointestinal tract from six patients who had simultaneously undergone upper endoscopy and colonoscopy, to perform a comprehensive analysis using culturomics with matrix assisted laser desorption ionisation - time of flight (MALDI-TOF) identification and by metagenomics targeting the 16S ribosomal ribonucleic acid (rRNA) gene. RESULTS: Using culturomics, we isolated 368 different bacterial species, including 37 new species. Fewer species were isolated in the upper gut: 110 in the stomach and 106 in the duodenum, while 235 were isolated from the left colon (p < 0.02). We isolated fewer aero-intolerant species in the upper gut: 37 from the stomach and 150 from the left colon (p < 0.004). Using metagenomics, 1,021 species were identified. The upper gut microbiota was revealed to be less rich than the lower gut microbiota, with 37,622 reads from the stomach, 28,390 from the duodenum, and 79,047 from the left colon (p < 0.009). There were fewer reads for aero-intolerant species in the upper gut (8,656 in the stomach, 5,188 in the duodenum and 72,262 in the left colon, p < 0.02). Patients taking proton pump inhibitors (PPI) were then revealed to have a higher stomach pH and a greater diversity of species in the upper digestive tract than patients not receiving treatment (p < 0.001). CONCLUSION: Significant modifications in bacterial composition and diversity exist throughout the gastrointestinal tract. We suggest that the upper gut may be key to understanding the relationship between the gut microbiota and health.

The nasopharyngeal microbiota in patients with viral respiratory tract infections is enriched in bacterial pathogens.

The nasopharynx is the primary site of colonization by respiratory pathogen that constitutes the port of entrance in the respiratory tract. The role of mucosal respiratory microbiota in infection has been recently emphasized; therefore, we aimed to assess if a specific respiratory microbiota profile was associated with symptomatic infection and/or with presence of respiratory viruses. We performed a case-control study to characterize the healthy respiratory microbiota and its alteration during acute viral infections. Next-generation sequencing of the 16S rRNA gene was applied to 225 nasopharyngeal samples from 177 patients with viral respiratory infection and 48 matched healthy controls. We evidenced an important decrease of bacterial alpha-diversity in patients with symptomatic respiratory infection and a loss of the healthy core microbiota, specifically anaerobes and Prevotella spp. Moreover, eight respiratory pathogens were enriched in these patients, including Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, Dol osigranulum pigrum and Corynebacterium propinquum/pseudodiphtheriticum, whose role in respiratory infection is unclear. The asymptomatic carrier of influenza harbors a microbiota similar to healthy subjects, suggesting a critical role of microbiota in the clinical expression of viruses. These data suggest that the commensal microbiota plays a significant role in susceptibility to viral infection. The frequent co-detection of virus and bacteria raises the question of a strategy to prevent bacterial disease, focusing on the prevention of nasopharyngeal colonization through effective antibiotic treatment. In addition to antibiotics, further studies should test preventive or therapeutic interventions for maintaining or restoring a healthy nasopharyngeal microbiota.

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy.

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR(2), http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.

Midgut microbiota of the malaria mosquito vector Anopheles gambiae and interactions with Plasmodium falciparum infection.

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.

Ultra-deep sequencing of foraminiferal microbarcodes unveils hidden richness of early monothalamous lineages in deep-sea sediments.

Deep-sea floors represent one of the largest and most complex ecosystems on Earth but remain essentially unexplored. The vastness and remoteness of this ecosystem make deep-sea sampling difficult, hampering traditional taxonomic observations and diversity assessment. This problem is particularly true in the case of the deep-sea meiofauna, which largely comprises small-sized, fragile, and difficult-to-identify metazoans and protists. Here, we introduce an ultra-deep sequencing-based metagenetic approach to examine the richness of benthic foraminifera, a principal component of deep-sea meiofauna. We used Illumina sequencing technology to assess foraminiferal richness in 31 unsieved deep-sea sediment samples from five distinct oceanic regions. We sequenced an extremely short fragment (36 bases) of the small subunit ribosomal DNA hypervariable region 37f, which has been shown to accurately distinguish foraminiferal species. In total, we obtained 495,978 unique sequences that were grouped into 1,643 operational taxonomic units, of which about half (841) could be reliably assigned to foraminifera. The vast majority of the operational taxonomic units (nearly 90%) were either assigned to early (ancient) lineages of soft-walled, single-chambered (monothalamous) foraminifera or remained undetermined and yet possibly belong to unknown early lineages. Contrasting with the classical view of multichambered taxa dominating foraminiferal assemblages, our work reflects an unexpected diversity of monothalamous lineages that are as yet unknown using conventional micropaleontological observations. Although we can only speculate about their morphology, the immense richness of deep-sea phylotypes revealed by this study suggests that ultra-deep sequencing can improve understanding of deep-sea benthic diversity considered until now as unknowable based on a traditional taxonomic approach.

Microbacterium lemovicicum sp. nov., a bacterium isolated from a natural uranium-rich soil.

An actinobacterial strain, designated ViU22(T), was isolated from a natural uranium-rich soil and was studied using a polyphasic approach. Cells formed orange-pigmented colonies, were rod-shaped, Gram-positive (non-staining method), non-motile and non-spore-forming. This organism grew in 0-4.5 % (w/v) NaCl and at 15-37 °C, with optimal growth occurring in 0.5 % (w/v) NaCl and at 30 °C. Comparative 16S rRNA gene sequence analysis revealed that the strain ViU22(T) belonged to the genus Microbacterium. It exhibited highest 16S rRNA gene sequence similarity with the type strains of Microbacterium testaceum (98.14 %) and Microbacterium binotii (98.02 %). The DNA-DNA relatedness of strains ViU22(T) with the most closely related type strains Microbacterium testaceum and Microbacterium binotii DSM 19164(T) was 20.10 % (± 0.70) and 28.05 % (± 0.35), respectively. Strain ViU22(T) possessed a type B2ß peptidoglycan with partial substitution of glutamic acid by 3-hydroxy glutamic acid. The major menaquinones were MK-11 and MK-12. Major polar lipids detected in the strain ViU22(T) were diphosphatidylglycerol, phosphatidylglycerol, an unknown phospholipid and unknown glycolipids. The predominant fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0, a pattern reported for other Microbacterium species. The major cell-wall sugars were galactose, xylose and mannose and the DNA G+C content was 71 mol%. Together, the DNA-DNA hybridization results and the differentiating phenotypic characteristics, showed that strain ViU22(T) should be classified as the type strain of a novel species within the genus Microbacterium, for which the name Microbacterium lemovicicum sp. nov. is proposed. The type strain is ViU22(T) ( = ATCC BAA-2396(T) = CCUG 62198(T) = DSM 25044(T)).

Region-specific microRNA alterations in marmosets carrying SLC6A4 polymorphisms are associated with anxiety-like behavior.

BACKGROUND: Psychiatric diseases such as depression and anxiety are multifactorial conditions, highly prevalent in western societies. Human studies have identified a number of high-risk genetic variants for these diseases. Among them, polymorphisms in the promoter region of the serotonin transporter gene (SLC6A4) have attracted much attention. However, due to the paucity of experimental models, molecular alterations induced by these genetic variants and how they correlate to behavioral deficits have not been examined. In this regard, marmosets have emerged as a powerful model in translational neuroscience to investigate molecular underpinnings of complex behaviors. METHODS: Here, we took advantage of naturally occurring genetic polymorphisms in marmoset SLC6A4 gene that have been linked to anxiety-like behaviors. Using FACS-sorting, we profiled microRNA contents in different brain regions of genotyped and behaviorally-phenotyped marmosets. FINDINGS: We revealed that marmosets bearing different SLC6A4 variants exhibit distinct microRNAs signatures in a region of the prefrontal cortex whose activity has been consistently altered in patients with depression/anxiety. We also identified Deleted in Colorectal Cancer (DCC), a gene previously linked to these diseases, as a downstream target of the differently expressed microRNAs. Significantly, we showed that levels of both microRNAs and DCC in this region were highly correlated to anxiety-like behaviors. INTERPRETATION: Our findings establish links between genetic variants, molecular modifications in specific cortical regions and complex behavioral responses, providing new insights into gene-behavior relationships underlying human psychopathology. FUNDING: This work was supported by France National Agency, NRJ Foundation, Celphedia and Fondation de France as well as the Wellcome Trust.

Impact of smoking cessation, coffee and bread consumption on the intestinal microbial composition among Saudis: A cross-sectional study.

The gut microbiota is often affected by the dietary and lifestyle habits of the host, resulting in a better efficacy that favors energy harvesting from the consumed food. Our objective was to characterize the composition of gut microbiota in adult Saudis and investigate possible association with lifestyle and dietary practices. Feces from 104 Saudi volunteers (48% males) were tested for microbiota by sequencing the V3-V4 region of bacterial 16S ribosomal RNA (rRNA). For all participants, data were collected related to their lifestyle habits and dietary practices. The relative abundance (RA) of Fusobacteria was significantly higher in normal weight Saudis (P = 0.005, false discovery rate-FDR = 0.014). Individuals who consumed more coffee presented marginally significant more RA of Fusobacteria (P = 0.02, FDR = 0.20) in their gut microbiota compared to those reporting low or no coffee intake, but the RA of Fusobacteria was significantly higher in smokers compared to non-smokers (P = 0.009, FDR = 0.027). The RA of Fusobacteria was also significantly higher in those reporting daily consumption of bread (P = 0.005, FDR = 0.015). At the species level, the gut microbiota of people who consumed coffee was dominated by Bacteroides thetaiotaomicron followed by Phascolarctobacterium faecium and Eubacterium rectale. Similarly, the gut microbiota of smokers was also enriched by B. thetaiotaomicron and Lactobacillus amylovorus. Smoking cessation, bread and coffee consumption induce changes in the intestinal microbial composition of Saudis. This indicates the significance of diet and lifestyle practices in the determination of the composition of the gut microbiota, which could possibly lead later to changes in metabolic profile and weight.

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.

Gut Microbiota Alteration is Characterized by a Proteobacteria and Fusobacteria Bloom in Kwashiorkor and a Bacteroidetes Paucity in Marasmus.

Kwashiorkor and marasmus are considered to be two different clinical diseases resulting from severe malnutrition, but this distinction has been questioned. In a previous study comparing children with kwashiorkor and healthy children from Niger and Senegal, we found a dramatic gut microbiota alteration with a predominant depletion of anaerobes and enrichment in Proteobacteria and Fusobacteria in kwashiorkor. However, it remained unknown whether this association was related to malnutrition or was a specific feature of kwashiorkor. In this continuation study, we added 7 new marasmus subjects and 71,162 new colonies from the same countries. Our results showed that, compared to marasmus, the kwashiorkor gut microbiota was characterized by an increased proportion of Proteobacteria (culturomics, Marasmus 5.0%, Kwashiorkor 16.7%, p < 0.0001; metagenomics, Marasmus 14.7%, Kwashiorkor 22.0%, p = 0.001), but there was a decreased proportion of Bacteroidetes in marasmus (culturomics, Marasmus 0.8%, Kwashiorkor 6.5%, p = 0.001; metagenomics, Marasmus 5.4%, Kwashiorkor 7.0%, p = 0.03). Fusobacterium was more frequently cultured from kwashiorkor. All detected potential pathogenic species were enriched in the kwashiorkor gut microbiota. These results provide a biological basis to support the usage of an antibiotic therapy more effective in suppressing the overgrowth of bacterial communities resistant to penicillin, combined with antioxidants and probiotics for nutritional recovery therapies, particularly for kwashiorkor.

Gut Bacteria Missing in Severe Acute Malnutrition, Can We Identify Potential Probiotics by Culturomics?

Severe acute malnutrition is the world-leading cause of children under-five's death. Recent metagenomics studies have established a link between gut microbiota and severe acute malnutrition, describing an immaturity with a striking depletion in oxygen-sensitive prokaryotes. Amoxicillin and therapeutic diet cure most of the children with severe acute malnutrition but an irreversible disruption of the gut microbiota is suspected in the refractory and most severe cases. In these cases, therapeutic diet may be unable to reverse the microbiota alteration leading to persistent impaired development or death. In addition, as enteric sepsis is a major cause of death in this context, identification of missing gut microbes to be tested as probiotics (live bacteria that confer a benefit to the host) to restore rapidly the healthy gut microbiota and prevent the gut pathogenic invasion is of foremost importance. In this study, stool samples of malnourished patients with kwashiorkor and healthy children were collected from Niger and Senegal and analyzed by culturomics and metagenomics. We found a globally decreased diversity, a decrease in the hitherto unknown diversity (new species isolation), a depletion in oxygen-sensitive prokaryotes including Methanobrevibacter smithii and an enrichment in potentially pathogenic Proteobacteria, Fusobacteria and Streptococcus gallolyticus. A complex of 12 species identified only in healthy children using culturomics and metagenomics were identified as probiotics candidates, providing a possible, defined, reproducible, safe, and convenient alternative to fecal transplantation to restore a healthy gut microbiota in malnourished children. Microbiotherapy based on selected strains has the potential to improve the current treatment of severe acute malnutrition and prevent relapse and death by reestablishing a healthy gut microbiota.

Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies.

Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum level, species richness (Chao index, 227 to 2,714) and diversity (non parametric Shannon, 1.37 to 4.4). Moreover DNA was extracted from stools obtained from 83 different individuals by the fastest extraction assay and by an extraction assay that degradated exopolysaccharides. The fastest extraction method was able to detect 68% to 100% genera and 42% to 95% species whereas the glycan degradation extraction method was able to detect 56% to 93% genera and 25% to 87% species. To allow a good liberation of DNA from exopolysaccharides commonly presented in stools, we recommend the mechanical lysis of stools plus glycan degradation, used here for the first time. Caution must be taken in the interpretation of current metagenomic studies, as the efficiency of DNA extraction varies widely among stool samples.

Gut microbiome and dietary patterns in different Saudi populations and monkeys.

Host genetics, environment, lifestyle and proximity between hosts strongly influence the composition of the gut microbiome. To investigate the association of dietary variables with the gut microbiota, we used 16S rDNA sequencing to test the fecal microbiome of Bedouins and urban Saudis and we compared it to the gut microbiome of baboons living in close contact with Bedouins and eating their leftovers. We also analyzed fermented dairy products commonly consumed by Bedouins in order to investigate their impact on the gut microbiome of this population. We found that the gut microbiomes of westernized urban Saudis had significantly lower richness and biodiversity than the traditional Bedouin population. The gut microbiomes of baboons were more similar to that of Bedouins compared to urban Saudis, probably due the dietary overlap between baboons and Bedouins. Moreover, we found clusters that were compositionally similar to clusters identified in humans and baboons, characterized by differences in Acinetobacter, Turicibacter and Collinsella. The fermented food presented significantly more bacteria genera common to the gut microbiome of Bedouins compared to urban Saudis. These results support the hypothesis that dietary habits influence the composition of the gut microbiome.

Gut microbiota associated with HIV infection is significantly enriched in bacteria tolerant to oxygen.

OBJECTIVES: Gut microbiota modifications occurring during HIV infection have recently been associated with inflammation and microbial translocation. However, discrepancies between studies justified a comprehensive analysis performed on a large sample size. DESIGN AND METHODS: In a case-control study, next-generation sequencing of the 16S rRNA gene was applied to the faecal microbiota of 31 HIV-infected patients, of whom 18 were treated with antiretroviral treatment (ART), compared with 27 healthy controls. 21 sera samples from HIV-infected patients and 7 sera samples from control participants were used to test the presence of 25 markers of inflammation and/or immune activation. RESULTS: Diversity was significantly reduced in HIV individuals when compared with controls and was not restored in the ART group. The relative abundance of several members of Ruminococcaceae such as Faecalibacterium prausnitzii was critically less abundant in the HIV-infected group and inversely correlated with inflammation/immune activation markers. Members of Enterobacteriaceae and Enterococcaceae were found to be enriched and positively correlated with these markers. There were significantly more aerotolerant species enriched in HIV samples (42/52 species, 80.8%) when compared with the control group (14/87 species, 16.1%; χ(2) test, p<10(-5), conditional maximum-likelihood estimate (CMLE) OR=21.9). CONCLUSIONS: Imbalance between aerobic and anaerobic flora observed in HIV faecal microbiota could be a consequence of the gut impairment classically observed in HIV infection via the production of oxygen. Overgrowth of proinflammatory aerobic species during HIV infection raises the question of antioxidant supplementation, such as vitamin C, E or N-acetylcysteine.

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.

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.