Ethanol-Producing Enterocloster bolteae is enriched in Hepatitis B Virus-Associated Gut Dysbiosis: A Case-Control Culturomics Study

Abstract

Abstract 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 We analyzed the fecal samples of eight patients with HBV and ten healthy individuals using the high-throughput culturomics approach compared to 16S rRNA sequencing. Quantification of ethanol, known for its damaging effect on the liver, produced from bacterial strains enriched in HBV was carried out by gas chromatography-mass spectrometry. Results By culturomics, 29,120 isolated colonies were analyzed by MALDI-TOF to identify 340 species (240 species in HBV samples, 254 species in control samples) belonging to 169 genera and six phyla. In the HBV group, 48 species were already known in humans but had not been previously found in the gut, 17 known species not previously found in humans, and six new species were isolated. Comparing bacterial species frequency, we serendipitously found three bacterial genera with significantly enriched bacterial diversity in HBV gut dysbiosis: 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, significantly enriched species in HBV almost all belonged to class Clostridia as Clostridium perfringens, Clostridium sporogenes, Enterocloster aldenensis, Enterocloster bolteae, Enterocloster clostridioformis, and Clostridium innocuum. Two E. bolteae strains, isolated from two different HBV patients, showed high ethanol production (27 and 200 mM). Conclusion Culturomics allowed us to identify Enterocloster species, and specifically E. bolteae, that are enriched in the gut microbiota of HBV patients. These species have never been isolated in HBV patients so far. Moreover, ethanol production by E. Boltea strains isolated from HBV patients could play a role in liver disease progression. Additionally, culturomics might be critical for better elucidating the relationship between dysbiosis and HBV infection in the future.