Matthieu Million

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.

Comparative meta-analysis of the effect of Lactobacillus species on weight gain in humans and animals

BACKGROUND Obesity is associated with alteration of the gut microbiota. In order to clarify the effect of Lactobacillus-containing probiotics (LCP) on weight we performed a meta-analysis of clinical studies and experimental models. We intended to assess effects by Lactobacillus species. METHODS A broad search with no date or language restriction was performed. We included randomized controlled trials (RCTs) and comparative clinical studies in humans and animals or experimental models assessing the effect of Lactobacillus-containing probiotics on weight. We primarily attempted to extract and use change from baseline values. Data were extracted independently by two authors. Results were pooled by host and by Lactobacillus species and are summarized in a meta-analysis of standardized difference in means (SMDs). RESULTS We identified and included 17 RCTs in humans, 51 studies on farm animals and 14 experimental models. Lactobacillus acidophilus administration resulted in significant weight gain in humans and in animals (SMD 0.15; 95% confidence intervals 0.05-0.25). Results were consistent in humans and animals. Lactobacillus fermentum and Lactobacillus ingluviei were associated with weight gain in animals. Lactobacillus plantarum was associated with weight loss in animals and Lactobacillus gasseri was associated with weight loss both in obese humans and in animals. CONCLUSIONS Different Lactobacillus species are associated different effects on weight change that are host-specific. Further studies are needed to clarify the role of Lactobacillus species in the human energy harvest and weight regulation. Attention should be drawn to the potential effects of commonly marketed lactobacillus-containing probiotics on weight gain.

The relationship between gut microbiota and weight gain in humans

The human gut microbiota is a metabolic organ that is determined by a dynamic process of selection and competition. Age, dietary habits and geographical origin of people have an important impact on the intestinal microbiota. The role of the microbiota is still largely unknown, but the bacteria of the gut flora do contribute enzymes that are absent in humans and play an essential role in the catabolism of dietary fibers. Germ-free mice provide a complementary approach for characterizing the properties of the human gut microbiota. Recently, microbial changes in the human gut were proposed to be one of the possible causes of obesity. This review summarizes the latest research on the association between microbial ecology and host weight.

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.

Human Gut Microbiota: Repertoire and Variations

The composition of human gut microbiota and their relationship with the host and, consequently, with human health and disease, presents several challenges to microbiologists. Originally dominated by culture-dependent methods for exploring this ecosystem, the advent of molecular tools has revolutionized our ability to investigate these relationships. However, many biases that have led to contradictory results have been identified. Microbial culturomics, a recent concept based on a use of several culture conditions with identification by MALDI-TOF followed by the genome sequencing of the new species cultured had allowed a complementarity with metagenomics. Culturomics allowed to isolate 31 new bacterial species, the largest human virus, the largest bacteria, and the largest Archaea from human. Moreover, some members of this ecosystem, such as Eukaryotes, giant viruses, Archaea, and Planctomycetes, have been neglected by the majority of studies. In addition, numerous factors, such as age, geographic provenance, dietary habits, antibiotics, or probiotics, can influence the composition of the microbiota. Finally, in addition to the countless biases associated with the study techniques, a considerable limitation to the interpretation of studies of human gut microbiota is associated with funding sources and transparency disclosures. In the future, studies independent of food industry funding and using complementary methods from a broad range of both culture-based and molecular tools will increase our knowledge of the repertoire of this complex ecosystem and host-microbiota mutualism.

Correlation between body mass index and gut concentrations of Lactobacillus reuteri , Bifidobacterium animalis , Methanobrevibacter smithii and Escherichia coli

Background:Genus and species level analysis is the best way to characterize alterations in the human gut microbiota that are associated with obesity, because the clustering of obese and lean microbiotas increases with the taxonomic depth of the analysis. Bifidobacterium genus members have been associated with a lean status, whereas different Lactobacillus species are associated both with a lean and an obese status.Objectives and methods:We analyzed the fecal concentrations of Bacteroidetes, Firmicutes, Methanobrevibacter smithii, the genus Lactobacillus, five other Lactobacillus species previously linked with lean or obese populations, Escherichia coli and Bifidobacterium animalis in 263 individuals, including 134 obese, 38 overweight, 76 lean and 15 anorexic subjects to test for the correlation between bacterial concentration and body mass index (BMI). Of these subjects, 137 were used in our previous study.Findings:Firmicutes were found in >98.5%, Bacteroidetes in 67%, M. smithii in 64%, E. coli in 51%, Lactobacillus species between 17 and 25% and B. animalis in 11% of individuals. The fecal concentration of Lactobacillus reuteri was positively correlated with BMI (coefficient=0.85; 95% confidence interval (CI) 0.12–0.58; P=0.02) in agreement with what was reported for Lactobacillus sakei. As reported, B. animalis (coefficient=−0.84; 95% CI −1.61 to −0.07; P=0.03) and M. smithii (coefficient=−0.43, 95% CI −0.90 to 0.05; P=0.08) were negatively associated with the BMI. Unexpectedly, E. coli was found here for the first time to negatively correlate with the BMI (coefficient=−1.05; 95% CI −1.60 to −0.50; P<0.001).Conclusion:Our findings confirm the specificity of the obese microbiota and emphasize the correlation between the concentration of certain Lactobacillus species and obesity.

Long-term outcome of Q fever endocarditis: a 26-year personal survey

BACKGROUND Q fever endocarditis caused by Coxiella burnetii is a potentially fatal disease characterised by a chronic evolution. To assess the long-term outcome and identify prognostic factors for mortality, surgical treatment, and serological changes in Q fever endocarditis, we did a retrospective study in the French National Referral Centre. METHODS Patients included were diagnosed with Q fever endocarditis at our centre from May, 1983, to June, 2006, and followed up for a minimum of 3 years for each patient, history and clinical characteristics were recorded with a standardised questionnaire. Prognostic factors associated with death, surgery, serological cure, and serological relapse were assessed by Cox regression analysis. Excised heart valve analysis was assessed according to duration of treatment. FINDINGS 104 patients were identified for inclusion in the study, although one was lost to follow-up; median follow-up was 100 months (range 37-310 months). 18 months of treatment was sufficient to sterilise the valves of all the patients except three, and 2 years of treatment sterilised all valves except one. In a multivariate Cox regression analysis, the major determinants associated with mortality were age (hazard ratio 1.11, 95% CI 1.05-1.18, p=0.003), stroke at diagnosis (7.09, 2.00-25.10, p=0.001), endocarditis on a prosthetic valve (6.04, 1.47-24.80, p=0.044), an absence of a four-times decrease of phase I IgG and IgA at 1 year (5.69, 1.00-32.22, p=0.049), or the presence of phase II IgM at 1 year (12.08, 3.11-46.85, p=0.005). Surgery was associated with heart failure (2.68, 1.21-5.94, p=0.015) or a cardiac abscess (4.71, 1.64-13.50, p=0.004). The determinants of poor serological outcome were male sex (0.47, 0.26-0.86, p=0.014), a high level of phase I IgG (0.65, 0.45-0.95, p=0.027), and a delay in the start of treatment with hydroxychloroquine (0.20, 0.04-0.91, p=0.037). Factors associated with relapse were endocarditis on a prosthetic valve (21.3, 2.05-221.86, p=0.01) or treatment duration less than 18 months (9.69, 1.08-86.72, p=0.042). INTERPRETATION The optimum duration of treatment with doxycycline and hydroxychloroquine in Q fever endocarditis is 18 months for native valves and 24 months for prosthetic valves. This duration should be extended only in the absence of favourable serological outcomes. Patients should be serologically monitored for at least 5 years because of the risk of relapse. FUNDING French National Referral Centre for Q Fever.

Gut bacterial microbiota and obesity

Although probiotics and antibiotics have been used for decades as growth promoters in animals, attention has only recently been drawn to the association between the gut microbiota composition, its manipulation, and obesity. Studies in mice have associated the phylum Firmicutes with obesity and the phylum Bacteroidetes with weight loss. Proposed mechanisms linking the microbiota to fat content and weight include differential effects of bacteria on the efficiency of energy extraction from the diet, and changes in host metabolism of absorbed calories. The independent effect of the microbiota on fat accumulation has been demonstrated in mice, where transplantation of microbiota from obese mice or mice fed western diets to lean or germ-free mice produced fat accumulation among recipients. The microbiota can be manipulated by prebiotics, probiotics, and antibiotics. Probiotics affect the microbiota directly by modulating its bacterial content, and indirectly through bacteriocins produced by the probiotic bacteria. Interestingly, certain probiotics are associated with weight gain both in animals and in humans. The effects are dependent on the probiotic strain, the host, and specific host characteristics, such as age and baseline nutritional status. Attention has recently been drawn to the association between antibiotic use and weight gain in children and adults. We herein review the studies describing the associations between the microbiota composition, its manipulation, and obesity.

From Q Fever to Coxiella burnetii Infection: a Paradigm Change

SUMMARY Coxiella burnetii is the agent of Q fever, or “query fever,” a zoonosis first described in Australia in 1937. Since this first description, knowledge about this pathogen and its associated infections has increased dramatically. We review here all the progress made over the last 20 years on this topic. C. burnetii is classically a strict intracellular, Gram-negative bacterium. However, a major step in the characterization of this pathogen was achieved by the establishment of its axenic culture. C. burnetii infects a wide range of animals, from arthropods to humans. The genetic determinants of virulence are now better known, thanks to the achievement of determining the genome sequences of several strains of this species and comparative genomic analyses. Q fever can be found worldwide, but the epidemiological features of this disease vary according to the geographic area considered, including situations where it is endemic or hyperendemic, and the occurrence of large epidemic outbreaks. In recent years, a major breakthrough in the understanding of the natural history of human infection with C. burnetii was the breaking of the old dichotomy between “acute” and “chronic” Q fever. The clinical presentation of C. burnetii infection depends on both the virulence of the infecting C. burnetii strain and specific risks factors in the infected patient. Moreover, no persistent infection can exist without a focus of infection. This paradigm change should allow better diagnosis and management of primary infection and long-term complications in patients with C. burnetii infection.