A catalog of ethanol-producing microbes in humans.

Aim: Endogenous ethanol production emerges as a mechanism of nonalcoholic steatohepatitis, obesity, diabetes and auto-brewery syndrome. Methods: To identify ethanol-producing microbes in humans, we used the NCBI taxonomy browser and the PubMed database with an automatic query and manual verification. Results: 85 ethanol-producing microbes in human were identified. Saccharomyces cerevisiae, Candida and Pichia were the most represented fungi. Enterobacteriaceae was the most represented bacterial family with mainly Escherichia coli and Klebsiella pneumoniae. Species of the Lachnospiraceae and Clostridiaceae family, of the Lactobacillales order and of the Bifidobacterium genus were also identified. Conclusion: This catalog will help the study of ethanol-producing microbes in human in the pathophysiology, diagnosis, prevention and management of human diseases associated with endogenous ethanol production.

Our bodies are home to a community of tiny living organisms like bacteria, viruses and archaea, collectively known as the microbiota. These microbes are crucial for our well-being and the proper functioning of our bodies. Certain things, like antibiotics or an imbalanced diet, can disturb this microbial community, known as dysbiosis. This can lead to illness. This review focuses on dysbiosis related to the production of ethanol, a type of alcohol, within our bodies. While the disruption of the microbiota has been linked to several health issues, the role of ethanol production in this is not well explored. This review aims to shed light on the microbes involved in this process. We found 85 microbes capable of producing ethanol in the human body, including 61 bacterial and 24 yeast species. This review provides a detailed updated catalog of ethanol-producing microbes in humans. Understanding these microbes and their role in diseases related to ethanol production could pave the way for better diagnostic tools and treatments in the future.

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

Background: Non-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. Aim: To 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. Results: Fecal 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). Conclusion: Elevated 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.

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.

Description of Streptococcus thalassemiae sp. nov., a Bacterium Isolated from Human Blood.

Blood is a precious biological liquid that is normally sterile. Therefore, bacteria in the bloodstream are shown a priori anomaly. A blood culture is systematically performed to diagnose the cause of the bacteremia. Indeed, a patient received in our service had a thalassemia major and underwent a genoidentical transplant. Then, a blood test was performed to diagnose a four-day fever. In this context, we have isolated strain Marseille-Q2617 from the blood sample. It revealed a new bacterial strain that belongs to the genus Streptococcus. It is a Gram-positive coccus, nonmotile, and nonspore forming. The major fatty acid found is hexadecanoic acid, with 49.5%. A taxonomic method was used to characterize the strain by studying their phenotypic, phylogenetic, and genomic characteristics. In addition, sequence analysis of the 16S rRNA gene shows that the strain Marseille-Q2617 has 99.94% sequence similarity to Streptococcus mitis. Average nucleotide identity (ANI) analysis for strain Marseille-Q2617T showed the highest similarity of 92.9% with S. mitis. The DNA-DNA hybridization value obtained (50.2%) between strain Marseille-Q2607 and S. mitis, its closest related species, was below the recommended threshold (<70%). Strain Marseille-Q2617T has a genome size of 2.02 Mbp with 40.5 mol% of G + C content. Based on these results, we propose a new species of the genus Streptococcus, for which the name Streptococcus thalassemiae sp. nov., Marseille-Q2617T (=CSUR Q2617 = CECT 30109) was proposed.

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

Non-alcoholic fatty liver (NAFLD), and its complicated form, non-alcoholic steatohepatitis (NASH), have been associated with gut dysbiosis with specific signatures. Endogenous ethanol production by Klebsiella pneumoniae or yeasts has been identified as a potential physio-pathological mechanism. A species-specific association between Lactobacillus and obesity and metabolic diseases has been reported. In this study, the microbial composition of ten cases of NASH and ten controls was determined using v3v4 16S amplicon sequencing as well as quantitative PCR (qPCR). Using different statistical approaches, we found an association of Lactobacillus and Lactoccocus with NASH, and an association of Methanobrevibacter, Faecalibacterium and Romboutsia with controls. At the species level, Limosilactobacillus fermentum and Lactococcus lactis, two species producing ethanol, and Thomasclavelia ramosa, a species already associated with dysbiosis, were associated with NASH. Using qPCR, we observed a decreased frequency of Methanobrevibacter smithii and confirmed the high prevalence of L. fermentum in NASH samples (5/10), while all control samples were negative (p = 0.02). In contrast, Ligilactobacillus ruminis was associated with controls. This supports the critical importance of taxonomic resolution at the species level, notably with the recent taxonomic reclassification of the Lactobacillus genus. Our results point towards the potential instrumental role of ethanol-producing gut microbes in NASH patients, notably lactic acid bacteria, opening new avenues for prevention and treatment.

Endogenous Ethanol and Triglyceride Production by Gut Pichia kudriavzevii, Candida albicans and Candida glabrata Yeasts in Non-Alcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) increases with fructose consumption and metabolic syndrome and has been recently linked with endogenous ethanol production, notably by high alcohol-producing Klebsiella pneumoniae (HiAlc Kpn). Candida yeasts are the main causes of auto-brewery syndromes but have been neglected in NASH. Here, the fecal ethanol and microbial content of 10 cases and 10 controls were compared. Ethanol was measured by gas chromatography-mass spectrometry. Species identification was performed by MALDI-TOF MS, and triglyceride production was assessed by a colorimetric enzymatic assay. The fecal ethanol concentration was four times higher in patients with NASH (median [interquartile range]: 0.13 [0.05-1.43] vs. 0.034 [0.008-0.57], p = 0.037). Yeasts were isolated from almost all cases but not from controls (9/10 vs. 0/10, p = 0.0001). Pichia kudriavzevii was the most frequent (four patients), while Candida glabrata, Candida albicans, and Galactomyces geotrichum were identified in two cases each. The concentration of ethanol produced by yeasts was 10 times higher than that produced by bacteria (median, 3.36 [0.49-5.60] vs. 0.32 [0.009-0.43], p = 0.0029). Using a 10% D-fructose restricted medium, we showed that NASH-associated yeasts transformed fructose in ethanol. Unexpectedly, yeasts isolated from NASH patients produced a substantial amount of triglycerides. Pichia kudriavzevii strains produced the maximal ethanol and triglyceride levels in vitro. Our preliminary human descriptive and in vitro experimental results suggest that yeasts have been neglected. In addition to K. pneumoniae, gut Pichia and Candida yeasts could be linked with NASH pathophysiology in a species- and strain-specific manner through fructose-dependent endogenous alcohol and triglyceride production.

Risk factors for symptoms of infection and the acquisition of pathogens among pilgrims at the Grand Magal of Touba, 2017-2021.

BACKGROUND: Respiratory and gastrointestinal symptoms are frequent in pilgrims at the Grand Magal of Touba (GMT). METHODS: Pilgrims were prospectively investigated in 2017-2021 for demographics, chronic conditions, preventive measures, respiratory and gastrointestinal symptoms, and pathogen carriage using PCR assays. RESULTS: 535 pilgrims were included. 54.8% and 13.3% reported respiratory and gastrointestinal symptoms, respectively. 18.4% acquired respiratory viruses, notably rhinovirus (10.1%) and coronaviruses (5.6%) and 39.9% bacteria, notably Haemophilus influenzae (18.9%) and Streptococcus pneumoniae (14.1%). The acquisition of gastrointestinal pathogens was lower, with enteroaggregative Escherichia coli (18.9%) and enteropathogenic Escherichia coli (10.5%) being the most frequent. A decrease was observed in the acquisition rates of pathogens in 2020-2021 GMT. Female pilgrims were more at risk of respiratory and gastrointestinal symptoms. Respiratory symptoms were associated with virus acquisition (aRR: 2.20, 95%CI [1.38-3.50]) and S. pneumoniae acquisition (aRR: 2.76, 95%CI = [1.64-4.62]). Using hand soap was associated with a decrease in the acquisition of rhinovirus (aRR: 0.42, 95%CI [0.22-0.80]) and coronavirus (aRR: 0.42, 95%CI [0.22-0.81]). Using face masks was associated with a decrease in reporting of respiratory symptoms (aRR: 0.54, 95% [0.35-0.86]). CONCLUSION: Hand washing with soap and wearing face masks should be recommended to GMT pilgrims.

Peptoniphilus coli sp. nov. and Peptoniphilus urinae sp. nov., isolated from humans.

Strains Marseille-P3761 and Marseille-P3195 are representatives of two bacterial species isolated from human specimens. Strain Marseille-P3761 was isolated from the stool of a healthy volunteer, while strain Marseille-P3915 was cultivated from the urine of a kidney transplant recipient. Both strains are anaerobic Gram-positive coccoid bacteria. Both are catalase-negative and oxidase-negative and grow optimally at 37 °C in anaerobic conditions. They also metabolize carbohydrates, such as galactose, glucose, fructose, and glycerol. The major fatty acids were hexadecanoic acid for both strains. The highest digital DNA-DNA hybridization (dDDH) values of Marseille-P3761 and Marseille-P3195 strains when compared to their closest phylogenetic relatives were 52.3% and 56.4%, respectively. Strains Marseille-P3761 and Marseille-P3195 shared an OrthoANI value of 83.5% which was the highest value found with Peptoniphilus species studied here. The morphological, biochemical, phenotypic and genomic characteristics strongly support that these strains are new members of the Peptoniphilus genus. Thus, we suggest that Peptoniphilus coli sp. nov., and Peptoniphilus urinae sp. nov., are new species for which strains Marseille-P3761 (CSUR P3761 = CCUG 71,569) and Marseille-P3195 (CSUR P3195 = DSM 103,468) are their type strains, respectively of two new Peptoniphilus species, for which we propose the names Peptoniphilus coli sp. nov. and Peptoniphilus urinae sp. nov., respectively.

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.

Konateibacter massiliensis gen. nov. sp. nov. and Paenibacillus faecalis sp. nov., Two New Species Isolated from the Stool Samples of Infants Suffering from Marasmus.

Two bacterial strains were isolated and identified using microbial culturomics and characterised according to the taxono-genomics strategy. The strictly anaerobic strain, Marseille-P3773T, forms smooth and translucent colonies consisting of Gram-stain negative, non-motile and non-spore-forming rod-shaped cells. Strain Marseille-P3787T consists of Gram-stain positive, motile and spore-forming cells resulting in grey and translucent colonies. The phylogenetic analysis of the 16S rRNA gene of strains Marseille-P3773T and Marseille-P3787T revealed a 96.9% similarity level with Lachnotalea glycerini strain DLD10 and 97% identity with Paenibacillus uliginis strain N3/975, respectively. The genome of strain Marseille-P3773 is 4,260,534 bp long with a 40.3 mol% G + C content and includes 3879 predicted genes of which 3769 are protein-coding genes, 76 RNAs and 34 are pseudo-genes. Strain Marseille-P3787 had a genome size of 4,833,032 bp with a 47.9 mol% G + C and has 4481 predicted genes of which 4265 are protein-coding genes, 101 RNAs and 115 are pseudo-genes. According to the data collected on these strains and, more specifically to the genomic comparison, we suggest the creation of a new genus and species, Konateibacter massiliensis gen. nov., sp. nov. with strain Marseille-P3773T (=CSURP3773 and CCUG71331) as its type strain within the Lachnospiraceae family, as well as a new species, Paenibacillus faecalis sp. nov. with strain Marseille-P3787T (=CSURP3787 and CCUG71650) as its type strain within the Paenibacillus genus.

A Listeria monocytogenes clone in human breast milk associated with severe acute malnutrition in West Africa: A multicentric case-controlled study.

BACKGROUND: Severe acute malnutrition (SAM) is a major public health problem affecting children under the age of five in many low- and middle-income countries, and its resolution would contribute towards achieving the several sustainable development goals. The etiology of SAM is pluri-factorial, including delayed maturation of the gut microbiota, suboptimal feeding practices and dysfunctional breastfeeding. The recent serendipitous detection of Listeria monocytogenes in the breast milk of Malian women, in contrast to French women, suggests a possible association with SAM. METHODOLOGY/ PRINCIPAL FINDINGS: To investigate the possible association of L. monocytogenes carriage in breast milk and SAM, a case-control study was performed in Senegal, with subjects recruited from two areas. Using 16S amplicon sequencing, a culture independent method, 100% (152/152) of the mothers were positive for L. monocytogenes in their breast milk while qPCR analysis gave lower recovery rates. Interestingly, after enrichment in Fraser broth and seeding on PALCALM agar, all 10 isolated strains were isolated from the milk of 10 mothers who had SAM children which also had a significantly increased relative abundance of L. monocytogenes (0.34 (SD 0.35) vs 0.05 (SD 0.07) in controls, p<0.0001). The high genomic similarity between these strains and Malian breast milk strains from a previous study supports the hypothesis of endemic clone carriage in West Africa. Moreover, the in vitro growth inhibition of L. monocytogenes using breast milk samples was obtained from only 50% of the milk of mothers who had SAM children, in contrast to control samples which systematically inhibited the growth of L. monocytogenes with a higher inhibition diameter (15.7 mm (SD 2.3) in controls versus 3.5 mm (SD 4.6) in SAM, p = 0.0001). Lactobacillus and Streptococcus isolated from the breast milk of controls inhibit L. monocytogenes in a species-dependent manner. CONCLUSIONS/SIGNIFICANCE: Our study reveals a previously unsuspected carriage of L. monocytogenes in the breast milk of West African women, which is associated with SAM. The inhibitory effect of human selected lactic acid bacterial species against L. monocytogenes might provide new therapeutic and inexpensive options to prevent and treat this neglected public health issue.