Stanislas Mondot

Towards the human intestinal microbiota phylogenetic core.

The paradox of a host specificity of the human faecal microbiota otherwise acknowledged as characterized by global functionalities conserved between humans led us to explore the existence of a phylogenetic core. We investigated the presence of a set of bacterial molecular species that would be altogether dominant and prevalent within the faecal microbiota of healthy humans. A total of 10 456 non-chimeric bacterial 16S rRNA sequences were obtained after cloning of PCR-amplified rDNA from 17 human faecal DNA samples. Using alignment or tetranucleotide frequency-based methods, 3180 operational taxonomic units (OTUs) were detected. The 16S rRNA sequences mainly belonged to the phyla Firmicutes (79.4%), Bacteroidetes (16.9%), Actinobacteria (2.5%), Proteobacteria (1%) and Verrumicrobia (0.1%). Interestingly, while most of OTUs appeared individual-specific, 2.1% were present in more than 50% of the samples and accounted for 35.8% of the total sequences. These 66 dominant and prevalent OTUs included members of the genera Faecalibacterium, Ruminococcus, Eubacterium, Dorea, Bacteroides, Alistipes and Bifidobacterium. Furthermore, 24 OTUs had cultured type strains representatives which should be subjected to genome sequence with a high degree of priority. Strikingly, 52 of these 66 OTUs were detected in at least three out of four recently published human faecal microbiota data sets, obtained with very different experimental procedures. A statistical model confirmed these OTUs prevalence. Despite the species richness and a high individual specificity, a limited number of OTUs is shared among individuals and might represent the phylogenetic core of the human intestinal microbiota. Its role in human health deserves further study.

Comparative assessment of human and farm animal faecal microbiota using real-time quantitative PCR

Pollution of the environment by human and animal faecal pollution affects the safety of shellfish, drinking water and recreational beaches. To pinpoint the origin of contaminations, it is essential to define the differences between human microbiota and that of farm animals. A strategy based on real-time quantitative PCR (qPCR) assays was therefore developed and applied to compare the composition of intestinal microbiota of these two groups. Primers were designed to quantify the 16S rRNA gene from dominant and subdominant bacterial groups. TaqMan probes were defined for the qPCR technique used for dominant microbiota. Human faecal microbiota was compared with that of farm animals using faecal samples collected from rabbits, goats, horses, pigs, sheep and cows. Three dominant bacterial groups (Bacteroides/Prevotella, Clostridium coccoides and Bifidobacterium) of the human microbiota showed differential population levels in animal species. The Clostridium leptum group showed the lowest differences among human and farm animal species. Human subdominant bacterial groups were highly variable in animal species. Partial least squares regression indicated that the human microbiota could be distinguished from all farm animals studied. This culture-independent comparative assessment of the faecal microbiota between humans and farm animals will prove useful in identifying biomarkers of human and animal faecal contaminations that can be applied to microbial source tracking methods.

High-fat diet alters gut microbiota physiology in mice

The intestinal microbiota is known to regulate host energy homeostasis and can be influenced by high-calorie diets. However, changes affecting the ecosystem at the functional level are still not well characterized. We measured shifts in cecal bacterial communities in mice fed a carbohydrate or high-fat (HF) diet for 12 weeks at the level of the following: (i) diversity and taxa distribution by high-throughput 16S ribosomal RNA gene sequencing; (ii) bulk and single-cell chemical composition by Fourier-transform infrared- (FT-IR) and Raman micro-spectroscopy and (iii) metaproteome and metabolome via high-resolution mass spectrometry. High-fat diet caused shifts in the diversity of dominant gut bacteria and altered the proportion of Ruminococcaceae (decrease) and Rikenellaceae (increase). FT-IR spectroscopy revealed that the impact of the diet on cecal chemical fingerprints is greater than the impact of microbiota composition. Diet-driven changes in biochemical fingerprints of members of the Bacteroidales and Lachnospiraceae were also observed at the level of single cells, indicating that there were distinct differences in cellular composition of dominant phylotypes under different diets. Metaproteome and metabolome analyses based on the occurrence of 1760 bacterial proteins and 86 annotated metabolites revealed distinct HF diet-specific profiles. Alteration of hormonal and anti-microbial networks, bile acid and bilirubin metabolism and shifts towards amino acid and simple sugars metabolism were observed. We conclude that a HF diet markedly affects the gut bacterial ecosystem at the functional level.

A metagenomic insight into our gut's microbiome

Advances in sequencing technology and the development of metagenomic and bioinformatics methods have opened up new ways to investigate the 1014 microorganisms inhabiting the human gut. The gene composition of human gut microbiome in a large and deeply sequenced cohort highlighted an overall non-redundant genome size 150 times larger than the human genome. The in silico predictions based on metagenomic sequencing are now actively followed, compared and challenged using additional ‘omics’ technologies. Interactions between the microbiota and its host are of key interest in several pathologies and applying meta-omics to describe the human gut microbiome will give a better understanding of this crucial crosstalk at mucosal interfaces. Adding to the growing appreciation of the importance of the microbiome is the discovery that numerous phages, that is, viruses of prokaryotes infecting bacteria (bacteriophages) or archaea with a high host specificity, inhabit the human gut and impact microbial activity. In addition, gene exchanges within the gut microbiota have proved to be more frequent than anticipated. Taken together, these innovative exploratory technologies are expected to unravel new information networks critical for gut homeostasis and human health. Among the challenges faced, the in vivo validation of these networks, together with their integration into the prediction and prognosis of disease, may require further working hypothesis and collaborative efforts.

Complete genome sequence of the fish pathogen Flavobacterium psychrophilum

We report here the complete genome sequence of the virulent strain JIP02/86 (ATCC 49511) of Flavobacterium psychrophilum, a widely distributed pathogen of wild and cultured salmonid fish. The genome consists of a 2,861,988–base pair (bp) circular chromosome with 2,432 predicted protein-coding genes. Among these predicted proteins, stress response mediators, gliding motility proteins, adhesins and many putative secreted proteases are probably involved in colonization, invasion and destruction of the host tissues. The genome sequence provides the basis for explaining the relationships of the pathogen to the host and opens new perspectives for the development of more efficient disease control strategies. It also allows for a better understanding of the physiology and evolution of a significant representative of the family Flavobacteriaceae, whose members are associated with an interesting diversity of lifestyles and habitats.

Highlighting New Phylogenetic Specificities of Crohn's Disease Microbiota

Background: Recent studies suggest that gastrointestinal (GI) microbes play a part in the pathogenesis of Crohns disease (CD). Methods: Fecal samples were collected from 16 healthy individuals and 16 CD patients (age‐ and sex‐matched). The DNA extracted from these samples were subjected to two different methods of microbiome analysis. Specific bacterial groups were quantified by real‐time polymerase chain reaction (PCR) methods using primers designed using a high‐throughput in‐house bioinformatics pipeline. The same DNA extracts were also used to produce fluorescently labeled cRNA amplicons to interrogate a custom‐designed phylogenetic microarray for intestinal bacteria. Results: Even though the intersubject variability was high, differences in the fecal microbiomes of healthy and CD patients were detected. Faecalibacterium prausnitzii and Escherichia coli were more represented in healthy and ileal CD patients, respectively. Additionally, probes specific for Ruminococcus bromii, Oscillibacter valericigenes, Bifidobacterium bifidum, and Eubacterium rectale produced stronger hybridization signals with the DNA samples from healthy subjects. Conversely, species overrepresented in CD patients were E. coli, Enterococcus faecium, and species from the Proteobacteria not normally found in the healthy human GI tract. Furthermore, we detected “healthy specific” molecular species or operational taxonomic units (OTUs) that are not closely related to any known species (Faecalibacterium, Subdoligranulum, and Oscillospora species), indicating that the phylogenetic dysbiosis is broader than at strain or species level. Conclusions: These two techniques of microbiome analysis provided a statistically robust new picture of the dysbiosis in fecal microbiota from ileal CD patients. Specifically, we identified a set of six species discriminant for CD, which provides a preliminary diagnostic tool. (Inflamm Bowel Dis 2011;)

Altered gut microbiota composition in immune-impaired Nod2−/− mice

We read with great interest the recently published study by Rehman and colleagues.1 Investigating microbial diversity on faecal and ileal samples from 48 mice using Sanger and 454 techniques, Rehman et al detected increased percentages of Bacteroidetes in the faeces of adult Nod2 −/− mice, whereas Firmicutes levels were significantly higher only in the ileum of Nod2 −/− mice. Since we demonstrated that antibiotic …

ZAP-70 Genotype Disrupts the Relationship Between Microbiota and Host, Leading to Spondyloarthritis and Ileitis in SKG Mice

The spondyloarthritides share genetic susceptibility, interleukin‐23 (IL‐23) dependence, and the involvement of microbiota. The aim of the current study was to elucidate how host genetics influence gut microbiota and the relationship between microbiota and organ inflammation in spondyloarthritides.

Population structure of the fish-pathogenic bacterium Flavobacterium psychrophilum.

ABSTRACT Flavobacterium psychrophilum is currently one of the main bacterial pathogens hampering the productivity of salmonid farming worldwide, and its control mainly relies on antibiotic treatments. To better understand the population structure of this bacterium and its mode of evolution, we have examined the nucleotide polymorphisms at 11 protein-coding loci of the core genome in a set of 50 isolates. These isolates were selected to represent the broadest possible diversity, originating from 10 different host fish species and four continents. The nucleotide diversity between pairs of sequences amounted to fewer than four differences per kilobase on average, corresponding to a particularly low level of diversity, possibly indicative of a small effective-population size. The recombination rate, however, seemed remarkably high, and as a consequence, most of the isolates harbored unique combinations of alleles (33 distinct sequence types were resolved). The analysis also showed the existence of several clonal complexes with worldwide geographic distribution but marked association with particular fish species. Such an association could reflect preferential routes of transmission and/or adaptive niche specialization. The analysis provided no clues that the initial range of the bacterium was originally limited to North America. Instead, the historical record of the expansion of the pathogen may reflect the spread of a few clonal complexes. As a resource for future epidemiological surveys, a multilocus sequence typing website based on seven highly informative loci is available.

Bacterial protein signals are associated with Crohn’s disease

Objective No Crohn’s disease (CD) molecular maker has advanced to clinical use, and independent lines of evidence support a central role of the gut microbial community in CD. Here we explore the feasibility of extracting bacterial protein signals relevant to CD, by interrogating myriads of intestinal bacterial proteomes from a small number of patients and healthy controls. Design We first developed and validated a workflow—including extraction of microbial communities, two-dimensional difference gel electrophoresis (2D-DIGE), and LC-MS/MS—to discover protein signals from CD-associated gut microbial communities. Then we used selected reaction monitoring (SRM) to confirm a set of candidates. In parallel, we used 16S rRNA gene sequencing for an integrated analysis of gut ecosystem structure and functions. Results Our 2D-DIGE-based discovery approach revealed an imbalance of intestinal bacterial functions in CD. Many proteins, largely derived from Bacteroides species, were over-represented, while under-represented proteins were mostly from Firmicutes and some Prevotella members. Most overabundant proteins could be confirmed using SRM. They correspond to functions allowing opportunistic pathogens to colonise the mucus layers, breach the host barriers and invade the mucosae, which could still be aggravated by decreased host-derived pancreatic zymogen granule membrane protein GP2 in CD patients. Moreover, although the abundance of most protein groups reflected that of related bacterial populations, we found a specific independent regulation of bacteria-derived cell envelope proteins. Conclusions This study provides the first evidence that quantifiable bacterial protein signals are associated with CD, which can have a profound impact on future molecular diagnosis.