Increased fecal ethanol and enriched ethanol-producing gut bacteria Limosilactobacillus fermentum, Enterocloster bolteae, Mediterraneibacter gnavus and Streptococcus mutans in nonalcoholic steatohepatitis

BackgroundNon-alcoholic steatohepatitis (NASH) has become a major public health issue as one of the leading causes of liver disease and transplantation worldwide. The instrumental role of the gut microbiota is emerging but still under investigation. Endogenous ethanol (EtOH) production by gut bacteria and yeasts is an emerging putative mechanism. Microbial metagenomics and culture studies targeting enterobacteria or yeasts have been reported, but no culturomics studies have been conducted so far.AimTo assess fecal EtOH and other biochemical parameters, characterize NASH-associated dysbiosis and identify EtOH-producing gut microbes associated with the disease, fecal samples from 41 NASH patients and 24 controls were analyzed. High-performance liquid chromatography (HPLC) was used for EtOH, glucose, total proteins, triglyceride and total cholesterol. Viable bacteria were assessed with microbial culturomics. Microbial genetic material was assessed using 16S metagenomics targeting the hypervariable V3V4 region.ResultsFecal EtOH and glucose was elevated in the stools of NASH patients (p < 0.05) but not triglyceride, total cholesterol or proteins. In culturomics, EtOH-producing Enterocloster bolteae and Limosilactobacillus fermentum were enriched in NASH. V3V4 16S rRNA amplicon sequencing confirmed the enrichment in EtOH-producing bacteria including L. fermentum, Mediterraneibacter gnavus and Streptococcus mutans, species previously associated with NASH and other dysbiosis-associated diseases. Strikingly, E. bolteae was identified only by culturomics. The well-known Lacticaseibacillus casei was identified in controls but never isolated in patients with NASH (p < 0.05).ConclusionElevated fecal EtOH and glucose is a feature of NASH. Several different EtOH-producing gut bacteria may play an instrumental role in the disease. Culturomics and metagenomics, two complementary methods, will be critical to identify EtOH-producing bacteria for future diagnostic markers and therapeutic targets for NASH. Suppression of EtOH-producing gut microbes and L. casei administration are options to be tested in NASH treatment

Identification and Characterization of an HtrA Sheddase Produced by Coxiella burnetii

International audienceHaving previously shown that soluble E-cadherin (sE-cad) is found in sera of Q fever patients and that infection of BeWo cells by C. burnetii leads to modulation of the E-cad/β-cat pathway, our purpose was to identify which sheddase(s) might catalyze the cleavage of E-cad. Here, we searched for a direct mechanism of cleavage initiated by the bacterium itself, assuming the possible synthesis of a sheddase encoded in the genome of C. burnetii or an indirect mechanism based on the activation of a human sheddase. Using a straightforward bioinformatics approach to scan the complete genomes of four laboratory strains of C. burnetii, we demonstrate that C. burnetii encodes a 451 amino acid sheddase (CbHtrA) belonging to the HtrA family that is differently expressed according to the bacterial virulence. An artificial CbHtrA gene (CoxbHtrA) was expressed, and the CoxbHtrA recombinant protein was found to have sheddase activity. We also found evidence that the C. burnetii infection triggers an over-induction of the human HuHtrA gene expression. Finally, we demonstrate that cleavage of E-cad by CoxbHtrA on macrophages-THP-1 cells leads to an M2 polarization of the target cells and the induction of their secretion of IL-10, which “disarms” the target cells and improves C. burnetii replication. Taken together, these results demonstrate that the genome of C. burnetii encodes a functional HtrA sheddase and establishes a link between the HtrA sheddase-induced cleavage of E-cad, the M2 polarization of the target cells and their secretion of IL-10, and the intracellular replication of C. burnetii

Microvirga massiliensis sp nov., the human commensal with the largest genome

International audienceMicrovirga massiliensis sp. nov. strain JC119(T) is a bacteria isolated in Marseille from a stool sample collected in Senegal. The 16S rRNA (JF824802) of M.massiliensis JC119(T) revealed 95% sequence identity with Microvirga lotononidis WSM3557(T) (HM362432). This bacterium is aerobic, gram negative, catalase positive, and oxidase negative. The draft genome of M.massiliensis JC119(T) comprises a 9,207,211-bp-long genome that is the largest bacterial genome of an isolate in humans. The genome exhibits a G+C content of 63.28% and contains 8685 protein-coding genes and 77 RNA genes, including 21 rRNA genes. Here, we describe the features of M.massiliensis JC119(T), together with the genome sequence information and its annotation

Identification of Algerian Field-Caught Phlebotomine Sand Fly Vectors by MALDI-TOF MS

International audienceBackground Phlebotomine sand flies are known to transmit Leishmania parasites, bacteria and viruses that affect humans and animals in many countries worldwide. Precise sand fly identification is essential to prevent phlebotomine-borne diseases. Over the past two decades, progress in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an accurate tool for arthropod identification. The objective of the present study was to investigate the usefulness of MALDI-TOF MS as a tool for identifying field-caught phlebotomine.Methodology/Principal Findings Sand flies were captured in four sites in north Algeria. A subset was morphologically and genetically identified. Six species were found in these areas and a total of 28 stored frozen specimens were used for the creation of the reference spectrum database. The relevance of this original method for sand fly identification was validated by two successive blind tests including the morphological identification of 80 new specimens which were stored at -80 degrees C, and 292 unknown specimens, including engorged specimens, which were preserved under different conditions. Intra-species reproducibility and inter-species specificity of the protein profiles were obtained, allowing us to distinguish specimens at the gender level. Querying of the sand fly database using the MS spectra from the blind test groups revealed concordant results between morphological and MALDI-TOF MS identification. However, MS identification results were less efficient for specimens which were engorged or stored in alcohol. Identification of 362 phlebotomine sand flies, captured at four Algerian sites, by MALDI-TOF MS, revealed that the subgenus Larroussius was predominant at all the study sites, except for in M'sila where P.(Phlebotomus) papatasi was the only sand fly species detected.Conclusion The present study highlights the application of MALDI-TOF MS for monitoring sand fly fauna captured in the field. The low cost, reliability and rapidity of MALDI-TOF MS analyses opens up new ways in the management of phlebotomine sand fly-borne diseases

Limosilactobacillus fermentum, Lactococcus lactis and Thomasclavelia ramosa are enriched and Methanobrevibacter smithii is depleted in patients with non-alcoholic steatohepatitis

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Multidisciplinary evaluation of Clostridium butyricum clonality isolated from preterm neonates with necrotizing enterocolitis in South France between 2009 and 2017

Abstract The association between Clostridium species identification from stool samples in preterm neonates and the occurrence of necrotizing enterocolitis has been increasingly reported. To confirm the specific impact of Clostridium butyricum in this pathology, selective culture procedure was used for Clostridia isolation. Whole-genome analysis was employed to investigate genomic relationships between isolates. Stool samples from present study, as well as from previously investigated cases, were implicated including 88 from preterm neonates with necrotizing enterocolitis and 71 from matched controls. Quantitative real-time polymerase chain reaction was performed to evaluate the presence of C. butyricum from stools of new cases. Clostridium species prevalence isolated by culture was compared between patients with necrotizing enterocolitis and controls. By combining results of both culture and quantitative polymerase chain reaction methods, C. butyricum was significantly more frequent in stool samples from preterm neonates with necrotizing enterocolitis than in controls. Whole-genome analysis of 81 genomes including 58 neonates’ isolates revealed that cases were clustered depending on geographical origin of isolation. Controls isolates presented genomic relations with that of patients suggesting a mechanism of asymptomatic carriage. Overall, this suggests an epidemiology comparable to that observed in Clostridium difficile colitis in adults

Genome sequence and description of Haloferax massiliense sp nov., a new halophilic archaeon isolated from the human gut

International audienceBy applying the culturomics concept and using culture conditions containing a high salt concentration, we herein isolated the first known halophilic archaeon colonizing the human gut. Here we described its phenotypic and biochemical characterization as well as its genome annotation. Strain Arc-Hr(T) (= CSUR P0974 = CECT 9307) was mesophile and grew optimally at 37 A degrees C and pH 7. Strain Arc-Hr(T) was also extremely halophilic with an optimal growth observed at 15% NaCl. It showed gram-negative cocci, was strictly aerobic, non-motile and non-spore-forming, and exhibited catalase and oxidase activities. The 4,015,175 bp long genome exhibits a G + C% content of 65.36% and contains 3911 protein-coding and 64 predicted RNA genes. PCR-amplified 16S rRNA gene of strain Arc-Hr(T) yielded a 99.2% sequence similarity with Haloferax prahovense, the phylogenetically closest validly published species in the Haloferax genus. The DDH was of 50.70 +/- 5.2% with H. prahovense, 53.70 +/- 2.69% with H. volcanii, 50.90 +/- 2.64% with H. alexandrinus, 52.90 +/- 2.67% with H. gibbonsii and 54.30 +/- 2.70% with H. lucentense. The data herein represented confirm strain Arc-Hr(T) as a unique species and consequently we propose its classification as representative of a novel species belonging to the genus Haloferax, as Haloferax massiliense sp. nov

Passive Filtration, Rapid Scanning Electron Microscopy, and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Treponema Culture and Identification from the Oral Cavity

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