Fabrice Armougom

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.

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

Gut microbiota consists of 1010 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 10xa0months 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.

Non-contiguous finished genome sequence and description of Anaerococcus senegalensis sp. nov.

Anaerococcus senegalensis strain JC48T sp. nov. is the type strain of A. senegalensis sp. nov. a new species within the genus Anaerococcus. This strain whose genome is described here was isolated from the fecal flora of a healthy patient. A. senegalensis is an obligate anaerobic coccus. Here we describe the features of this organism together with the complete genome sequence and annotation. The 1,790,835 bp long genome (1 chromosome but no plasmid) contains 1,721 protein-coding and 53 RNA genes including 5 rRNA genes

High-level colonisation of the human gut by Verrucomicrobia following broad-spectrum antibiotic treatment

The gut microbiota is mainly composed of the phyla Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria; the Verrucomicrobia phylum is occasionally observed. Antibiotics can change the bacterial diversity of the gut, with limited changes in the proportions of phyla. In this study, the gut repertoire of two patients who received a broad-spectrum antibiotic regimen was studied. As part of a large gut microbiota study, two stool samples were analysed: one sample was collected after broad-spectrum antibiotic therapy in a patient with Coxiella burnetii vascular infection (Patient A); and the other sample was collected from a patient admitted to the Intensive Care Unit (Patient B). Samples were subjected to Gram staining, electron microscopy, 16S rRNA V6 amplicon pyrosequencing and fluorescence in situ hybridisation (FISH). In parallel, the antibiotic susceptibility of Akkermansia muciniphila Muc(T) strain was studied and this strain was observed by electron microscopy. Pyrosequencing revealed that a large proportion of the sequences were associated with Verrucomicrobia (proportions of 44.9% and 84.6% for Patients A and B, respectively). All of the phylotypes were represented by a single species (A. muciniphila), and neither patient presented significant gastrointestinal disorders. Electron microscopy and FISH with specific Verrucomicrobia probes confirmed the presence of the bacterium. The Muc(T) strain was susceptible to imipenem and doxycycline but resistant to vancomycin and metronidazole. Dramatic colonisation of the human gut microbiota by the Verrucomicrobia phylum following a broad-spectrum antibiotic regimen occurred without significant gastrointestinal manifestations, suggesting that influenced by external factors such as antibiotics, the gut repertoire remains partially unknown.

Evidence of the megavirome in humans

BACKGROUNDnMegavirales is a proposed new virus order composed of Mimivirus, Marseillevirus and closely related viruses, as well as members of the families Poxviridae, Iridoviridae, Ascoviridae, Phycodnaviridae and Asfarviridae. The Megavirales virome, which we refer to as the megavirome, has been largely neglected until now because of the use of technical procedures that have jeopardized the discovery of giant viruses, particularly the use of filters with pore sizes in the 0.2-0.45-μm range. Concurrently, there has been accumulating evidence supporting the role of Mimivirus, discovered while investigating a pneumonia outbreak using amoebal coculture, as a causative agent in pneumonia.nnnOBJECTIVESnIn this paper, we describe the detection of sequences related to Mimivirus and Marseillevirus in the gut microbiota from a young Senegalese man. We also searched for sequences related to Megavirales in human metagenomes publicly available in sequence databases.nnnRESULTSnWe serendipitously detected Mimivirus- and Marseillevirus-like sequences while using a new metagenomic approach targeting bacterial DNA that subsequently led to the isolation of a new member of the family Marseilleviridae, named Senegalvirus, from human stools. This discovery demonstrates the possibility of the presence of giant viruses of amoebae in humans. In addition, we detected sequences related to Megavirales members in several human metagenomes, which adds to previous findings by several groups.nnnCONCLUSIONSnOverall, we present convergent evidence of the presence of mimiviruses and marseilleviruses in humans. Our findings suggest that we should re-evaluate the human megavirome and investigate the prevalence, diversity and potential pathogenicity of giant viruses in humans.

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 (17xa0% 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.

Culturomics and pyrosequencing evidence of the reduction in gut microbiota diversity in patients with broad-spectrum antibiotics

The human gut flora is currently widely characterised using molecular techniques. Microbial culturomics (large-scale culture conditions with identification of colonies using MALDI-TOF or 16S rRNA) is part of the rebirth of bacterial culture that was initiated by environmental microbiologists for the design of axenic culture for intracellular bacteria in clinical microbiology. Culturomics was performed on four stool samples from patients treated with large-scale antibiotics to assess the diversity of their gut flora in comparison with other culture-dependent studies. Pyrosequencing of the V6 region was also performed and was compared with a control group. Gut richness was also estimated by bacterial counting after microscopic observation. In total, 77 culture conditions were tested and 32,000 different colonies were generated; 190 bacterial species were identified, with 9 species that had not been isolated from the human gut before this study, 7 newly described in humans and 8 completely new species. A dramatic reduction in diversity was observed for two of the four stool samples for which antibiotic treatment was prolonged and uninterrupted. The total number of bacteria was generally preserved, suggesting that the original population was replaced but was sustained in size. Discordances between culture and pyrosequencing biodiversity biomarkers highlight the depth of bias of molecular studies. Stool samples studied showed a dramatic reduction in bacterial diversity. Considering the variable antibiotic concentration in the gut, this reduction in the number of species is possibly linked to the production of bacteriocin in the upper digestive tract by specific bacteria, such as Lactobacillus spp.

Abnormal weight gain and gut microbiota modifications are side effects of long-term doxycycline and hydroxychloroquine treatment

ABSTRACT Doxycycline has been proposed for the treatment of malnourished children in developing countries, and its use has been associated with weight gain in healthy volunteers. No previous studies have assessed abnormal weight gain as a putative side effect of long-term doxycycline treatment; thus, the objective of the present study was to characterize this phenomenon. We also analyzed the role of the gut microbiota in this effect. We assessed changes in the body mass index in Q fever endocarditis patients treated with doxycycline and hydroxychloroquine and healthy individuals with no antibiotic treatment. Abnormal weight gain was defined as a gain in weight above that of the controls. The fecal samples were examined using molecular assays for Methanobrevibacter smithii, Bacteroidetes, Firmicutes, Escherichia coli, Lactobacillus, Lactobacillus reuteri, and total bacterial concentrations. We examined 82 patients, including 48 patients with Q fever endocarditis and 34 controls. Approximately 23% of the treated patients showed abnormal weight gain (P = 0.001). Patients treated with doxycycline and hydroxychloroquine presented significantly lower concentrations of Bacteroidetes (P = 0.002), Firmicutes (P = 0.01), and Lactobacillus (P = 0.02). The linear regression analysis revealed that the duration of treatment was significantly associated with a decrease in Bacteroidetes (P = 0.0001), Firmicutes (P = 0.002), and total bacteria (P < 0.00001). Abnormal weight gain is a side effect of long-term doxycycline and hydroxychloroquine treatment. Gut microbiota modifications at the phylum level could play an instrumental role in this effect. We highlight the need for specific nutritional care in patients undergoing long-term antibiotic treatment, particularly treatment involving the use of doxycycline.

A Metagenomic Investigation of the Duodenal Microbiota Reveals Links with Obesity.

Background Few studies have tested the small intestine microbiota in humans, where most nutrient digestion and absorption occur. Here, our objective was to examine the duodenal microbiota between obese and normal volunteers using metagenomic techniques. Methodology/Principal Findings We tested duodenal samples from five obese and five normal volunteers using 16S rDNA V6 pyrosequencing and Illumina MiSeq deep sequencing. The predominant phyla of the duodenal microbiota were Firmicutes and Actinobacteria, whereas Bacteroidetes were absent. Obese individuals had a significant increase in anaerobic genera (p < 0.001) and a higher abundance of genes encoding Acyl-CoA dehydrogenase (p = 0.0018) compared to the control group. Obese individuals also had a reduced abundance of genes encoding sucrose phosphorylase (p = 0.015) and 1,4-alpha-glucan branching enzyme (p = 0.05). Normal weight people had significantly increased FabK (p = 0.027), and the glycerophospholipid metabolism pathway revealed the presence of phospholipase A1 only in the control group (p = 0.05). Conclusions/Significance The duodenal microbiota of obese individuals exhibit alterations in the fatty acid and sucrose breakdown pathways, probably induced by diet imbalance.

Identification of Bacterial Strains Isolated from the Mediterranean Sea Exhibiting Different Abilities of Biofilm Formation

The Mediterranean Sea has rarely been investigated for the characterization of marine bacteria as compared to other marine environments such as the Atlantic or Pacific Ocean. Bacteria recovered from inert surfaces are poorly studied in these environments, when it has been shown that the community structure of attached bacteria can be dissimilar from that of planktonic bacteria present in the water column. The objectives of this study were to identify and characterize marine bacteria isolated from biofilms developed on inert surfaces immersed in the Mediterranean Sea and to evaluate their capacity to form a biofilm in vitro. Here, 13 marine bacterial strains have been isolated from different supports immersed in seawater in the Bay of Toulon (France). Phylogenetic analysis and different biological and physico-chemical properties have been investigated. Among the 13 strains recovered, 8 different genera and 12 different species were identified including 2 isolates of a novel bacterial species that we named Persicivirga mediterranea and whose genus had never been isolated from the Mediterranean Sea. Shewanella sp. and Pseudoalteromonas sp. were the most preponderant genera recovered in our conditions. The phenotypical characterization revealed that one isolate belonging to the Polaribacter genus differed from all the other ones by its hydrophobic properties and poor ability to form biofilms in vitro. Identifying and characterizing species isolated from seawater including from Mediterranean ecosystems could be helpful for example, to understand some aspects of bacterial biodiversity and to further study the mechanisms of biofilm (and biofouling) development in conditions approaching those of the marine environment.