Ethanol-Producing Enterocloster bolteae Is Enriched in Chronic Hepatitis B-Associated Gut Dysbiosis: A Case-Control Culturomics Study.

BACKGROUND: Hepatitis B virus (HBV) infection is a global health epidemic that causes fatal complications, leading to liver cirrhosis and hepatocellular carcinoma. The link between HBV-related dysbiosis and specific bacterial taxa is still under investigation. Enterocloster is emerging as a new genus (formerly Clostridium), including Enterocloster bolteae, a gut pathogen previously associated with dysbiosis and human diseases such as autism, multiple sclerosis, and inflammatory bowel diseases. Its role in liver diseases, especially HBV infection, is not reported. METHODS: The fecal samples of eight patients with chronic HBV infection and ten healthy individuals were analyzed using the high-throughput culturomics approach and compared to 16S rRNA sequencing. Quantification of ethanol, known for its damaging effect on the liver, produced from bacterial strains enriched in chronic HBV was carried out by gas chromatography-mass spectrometry. RESULTS: Using culturomics, 29,120 isolated colonies were analyzed by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-TOF); 340 species were identified (240 species in chronic HBV samples, 254 species in control samples) belonging to 169 genera and 6 phyla. In the chronic HBV group, 65 species were already known in the literature; 48 were associated with humans but had not been previously found in the gut, and 17 had never been associated with humans previously. Six species were newly isolated in our study. By comparing bacterial species frequency, three bacterial genera were serendipitously found with significantly enriched bacterial diversity in patients with chronic HBV: Enterocloster, Clostridium, and Streptococcus (p = 0.0016, p = 0.041, p = 0.053, respectively). However, metagenomics could not identify this enrichment, possibly concerning its insufficient taxonomical resolution (equivocal assignment of operational taxonomic units). At the species level, the significantly enriched species in the chronic HBV group almost all belonged to class Clostridia, such as Clostridium perfringens, Clostridium sporogenes, Enterocloster aldenensis, Enterocloster bolteae, Enterocloster clostridioformis, and Clostridium innocuum. Two E. bolteae strains, isolated from two patients with chronic HBV infection, showed high ethanol production (27 and 200 mM). CONCLUSIONS: Culturomics allowed us to identify Enterocloster species, specifically, E. bolteae, enriched in the gut microbiota of patients with chronic HBV. These species had never been isolated in chronic HBV infection before. Moreover, ethanol production by E. bolteae strains isolated from the chronic HBV group could contribute to liver disease progression. Additionally, culturomics might be critical for better elucidating the relationship between dysbiosis and chronic HBV infection in the future.

Identification and Characterization of an HtrA Sheddase Produced by Coxiella burnetii.

Having 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/ß-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.

Buttiauxella massiliensis sp. nov., Isolated from a Human Bone Infection.

Strain Marseille-P9829 was isolated from a bone sample collected from an open right fibula fracture from a 46-years old patient. Strain Marseille-P9829 (= CSUR P9829 = DSM 110695) was a Gram-negative, non-spore-forming and non-motile bacterium. This strain had a positive catalase activity but was oxidase-negative. The major fatty acids methyl esters were hexadecanoic acid (45.6%) and 9-hexadecenoic acid (28.4%). Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry analysis suggested that this strain belongs to the species Buttiauxella gaviniae. Since there were few reports of clinical infections with this species in humans, whole genome sequencing was performed and a polyphasic taxono-genomic approach was followed in order to verify the classification of strain Marseille-P9829. The 16S rRNA gene sequence BLAST against the NCBI database yielded the highest similarity of 99.8% with Buttiauxella agrestis, suggesting that strain Marseille-P9829 belongs to this species. However, genomic comparison by digital DNA-DNA hybridization showed that values between strain Marseille-P9829 and other validly published Buttiauxella species were all lower than 70%. Furthermore, all average nucleotide identities were lower than 95-96%. Therefore, these results confirmed that strain Marseille-P9829 belonged to a new Buttiauxella species for which we propose the name Buttiauxella massiliensis sp. nov., with strain Marseille-P9829 as type strain.

New human-associated species of the family Atopobiaceae and proposal to reclassify members of the genus Olsenella.

Five novel bacterial strains, Marseille-P1476T (=CSURP1476T=DSM 100642T), Marseille-P3256T (=CSURP3256T=CECT 9977T), Marseille-P2936T (=CSURP2936T=DSM 103159T), Marseille-P2912T (=CSURP2912T=DSM 103345T) and Marseille-P3197T (=CSURP3197T=CCUG 71847T), were isolated from various human specimens. These five strains were not identified at the species level by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Following 16S rRNA gene sequence comparisons with the GenBank database, the highest nucleotide sequence similarities of all studied strains were obtained to members of the paraphyletic genus Olsenella. A polyphasic taxono-genomic strategy (16S rRNA gene-based and core genome-based phylogeny, genomic comparison, phenotypic and biochemical characteristics) enabled us to better classify these strains and reclassify Olsenella species. Among the studied strains, Marseille-P1476T, Marseille-P2936T and Marseille-P3197T belonged to new species of the genus Olsenella for which we propose the names Olsenella massiliensis sp. nov., Olsenella phocaeensis sp. nov. and Olsenella urininfantis sp. nov., respectively. Strains Marseille-P2912T and Marseille-P3256T belonged to a new genus for which the names Thermophilibacter provencensis gen. nov., sp. nov. and Thermophilibacter mediterraneus gen. nov., sp. nov. are proposed, respectively. We also propose the creation of the genera Parafannyhessea gen. nov., Tractidigestivibacter gen. nov. and Paratractidigestivibacter gen. nov. and the reclassification of Olsenella umbonata as Parafannyhessea umbonata comb. nov., Olsenella scatoligenes as Tractidigestivibacter scatoligenes comb. nov., and Olsenella faecalis as Paratractidigestivibacter faecalis comb. nov.