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

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: 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.

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

“…The Streptococcus genus was previously associated with NASH (Naka et al, 2014) and includes S. mutans, a known cariogenic species (Alaluusua and Renkonen, 1983). The other two EtOH-producing species, E. bolteae (which ethanol production has been shown in vitro with strains enriched in hepatitis B patients (Magdy Wasfy et al, 2023)) and Mediterraneibacter gnavus (Hynönen et al, 2016), were found to be associated with NASH for the first time in this study. Specifically, E. bolteae was found enriched in NASH patients only using the culturomics approach.…”

Clostridioides difficile infection is associated with differences in transcriptionally active microbial communities

“…Clostridioides difficile is known to form spores during infection (Paredes-Sabja et al, 2014), which fits with the observation of it as a significantly more important contributor to the expression of three differential KOs related to spore formation in CDI+ in this study. Likewise, Enterocloster bolteae can form spores (Magdy Wasfy et al, 2023) and contributed to the differential expression of two of those KOs (Supplementary Table S8). Similarly, though Ruminoccocus gnavus had historically been considered to be non-spore forming, more recent work has demonstrated that it can form spores under certain conditions (Crost et al, 2023).…”