Blautia coccoides JCM1395T Achieved Intratumoral Growth with Minimal Inflammation: Evidence for Live Bacterial Therapeutic Potential by an Optimized Sample Preparation and Colony PCR Method.

We demonstrate that Blautia coccoides JCM1395T has the potential to be used for tumor-targeted live bacterial therapeutics. Prior to studying its in vivo biodistribution, a sample preparation method for reliable quantitative analysis of bacteria in biological tissues was required. Gram-positive bacteria have a thick outer layer of peptidoglycans, which hindered the extraction of 16S rRNA genes for colony PCR. We developed the following method to solve the issue; the method we developed is as follows. The homogenates of the isolated tissue were seeded on agar medium, and bacteria were isolated as colonies. Each colony was heat-treated, crushed with glass beads, and further treated with restriction enzymes to cleave DNAs for colony PCR. With this method, Blautia coccoides JCM1395T and Bacteroides vulgatus JCM5826T were individually detected from tumors in mice intravenously receiving their mixture. Since this method is very simple and reproducible, and does not involve any genetic modification, it can be applied to exploring a wide range of bacterial species. We especially demonstrate that Blautia coccoides JCM1395T efficiently proliferate in tumors when intravenously injected into tumor-bearing mice. Furthermore, these bacteria showed minimal innate immunological responses, i.e., elevated serum tumor necrosis factor α and interleukin-6, similar to Bifidobacterium sp., which was previously studied as a therapeutic agent with a small immunostimulating effect.

Method for estimating disease risk from microbiome data using structural equation modeling.

The relationship between the human gut microbiota and disease is of increasing scientific interest. Previous investigations have focused on the differences in intestinal bacterial abundance between control and affected groups to identify disease biomarkers. However, different types of intestinal bacteria may have interacting effects and thus be considered biomarker complexes for disease. To investigate this, we aimed to identify a new kind of biomarker for atopic dermatitis using structural equation modeling (SEM). The biomarkers identified were latent variables, which are complex and derived from the abundance data for bacterial marker candidates. Groups of females and males classified as healthy participants [normal control (NC) (female: 321 participants, male: 99 participants)], and patients afflicted with atopic dermatitis only [AS (female: 45 participants, male: 13 participants)], with atopic dermatitis and other diseases [AM (female: 75 participants, male: 34 participants)], and with other diseases but without atopic dermatitis [OD (female: 1,669 participants, male: 866 participants)] were used in this investigation. The candidate bacterial markers were identified by comparing the intestinal microbial community compositions between the NC and AS groups. In females, two latent variables (lv) were identified; for lv1, the associated components (bacterial genera) were Alistipes, Butyricimonas, and Coprobacter, while for lv2, the associated components were Agathobacter, Fusicatenibacter, and Streptococcus. There was a significant difference in the lv2 scores between the groups with atopic dermatitis (AS, AM) and those without (NC, OD), and the genera identified for lv2 are associated with the suppression of inflammatory responses in the body. A logistic regression model to estimate the probability of atopic dermatitis morbidity with lv2 as an explanatory variable had an area under the curve (AUC) score of 0.66 when assessed using receiver operating characteristic (ROC) analysis, and this was higher than that using other logistic regression models. The results indicate that the latent variables, especially lv2, could represent the effects of atopic dermatitis on the intestinal microbiome in females. The latent variables in the SEM could thus be utilized as a new type of biomarker. The advantages identified for the SEM are as follows: (1) it enables the extraction of more sophisticated information when compared with models focused on individual bacteria and (2) it can improve the accuracy of the latent variables used as biomarkers, as the SEM can be expanded.

Sex Differences in Intestinal Microbiota and Their Association with Some Diseases in a Japanese Population Observed by Analysis Using a Large Dataset.

In recent years, many studies have focused on the relationship between intestinal microbiota and human health, but the impact of sex has not yet been sufficiently investigated. In this study, sex differences in the intestinal microbiota of a Japanese population were investigated by age group, using a large dataset constructed for a cross-sectional study. α-diversity analysis indicated that the impact of sex differences varied among the 20s-50s age groups but tended to be smaller among the 60s-70s age groups. Fusobacterium, Megamonas, Megasphaera, Prevotella, and Sutterella were more common among males, whereas Alistipes, Bacteroides, Bifidobacterium, Odoribacter, and Ruthenibacterium were common among females. Next, intestinal bacteria potentially associated with 12 diseases were investigated for each sex. The results indicate that many of these differ between males and females, and among age groups. Thus, sex and age should be considered for studies on intestinal microbiota and disease association, prevention, and treatment approaches that target them.

Vescimonas gen. nov., Vescimonas coprocola sp. nov., Vescimonas fastidiosa sp. nov., Pusillimonas gen. nov. and Pusillimonas faecalis sp. nov. isolated from human faeces.

Six strains of Gram-stain-negative, obligately anaerobic, non-spore-forming, non-motile rods were isolated from human faeces. Based on phylogenetic characteristics, the six isolates were included in the family Ruminococcaceae, and divided into three groups. The six isolates showed 16S rRNA gene sequence similarity values lower than 96.2 % to the closely related species, Oscillibacter ruminantium GH1T, Oscillibacter valericigenes Sjm18-20T and Dysosmobacter welbiomis J115T. Coherently with the 16S rRNA gene sequence results, the in silico DNA-DNA hybridization and average nucleotide identity values clearly indicated that strains MM35T, MM50T and MM59T belong to different species from the closely related three species. Based on phenotypic features and phylogenetic positions, three novel species, Vescimonas coprocola gen. nov., sp. nov., Vescimonas fastidiosa gen. nov., sp. nov. and Pusillimonas faecalis gen. nov., sp. nov. are proposed. The type strain of V. coprocola is strain MM50T (=JCM 34012T=DSM 111893T). The type strain of V. fastidiosa is strain MM35T (=JCM 34016T=DSM 111899T). The type strain of P. faecalis is strain MM59T (=JCM 34011T=DSM 111669T). The DNA G+C contents estimated according to the whole genomes of strains MM35T, MM50T and MM59T were 56.4, 58.2 and 55.2 mol%, respectively.

UVC disinfects SARS-CoV-2 by induction of viral genome damage without apparent effects on viral morphology and proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a pandemic threat worldwide and causes severe health and economic burdens. Contaminated environments, such as personal items and room surfaces, are considered to have virus transmission potential. Ultraviolet C (UVC) light has demonstrated germicidal ability and removes environmental contamination. UVC has inactivated SARS-CoV-2; however, the underlying mechanisms are not clear. It was confirmed here that UVC 253.7 nm, with a dose of 500 µW/cm2, completely inactivated SARS-CoV-2 in a time-dependent manner and reduced virus infectivity by 10-4.9-fold within 30 s. Immunoblotting analysis for viral spike and nucleocapsid proteins showed that UVC treatment did not damage viral proteins. The viral particle morphology remained intact even when the virus completely lost infectivity after UVC irradiation, as observed by transmission electronic microscopy. In contrast, UVC irradiation-induced genome damage was identified using the newly developed long reverse-transcription quantitative-polymerase chain reaction (RT-qPCR) assay, but not conventional RT-qPCR. The six developed long RT-PCR assays that covered the full-length viral genome clearly indicated a negative correlation between virus infectivity and UVC irradiation-induced genome damage (R2 ranging from 0.75 to 0.96). Altogether, these results provide evidence that UVC inactivates SARS-CoV-2 through the induction of viral genome damage.

SARS-CoV-2 Disinfection of Air and Surface Contamination by TiO2 Photocatalyst-Mediated Damage to Viral Morphology, RNA, and Protein.

SARS-CoV-2 is the causative agent of COVID-19, which is a global pandemic. SARS-CoV-2 is transmitted rapidly via contaminated surfaces and aerosols, emphasizing the importance of environmental disinfection to block the spread of virus. Ultraviolet C radiation and chemical compounds are effective for SARS-CoV-2 disinfection, but can only be applied in the absence of humans due to their toxicities. Therefore, development of disinfectants that can be applied in working spaces without evacuating people is needed. Here we showed that TiO2-mediated photocatalytic reaction inactivates SARS-CoV-2 in a time-dependent manner and decreases its infectivity by 99.9% after 20 min and 120 min of treatment in aerosol and liquid, respectively. The mechanistic effects of TiO2 photocatalyst on SARS-CoV-2 virion included decreased total observed virion count, increased virion size, and reduced particle surface spike structure, as determined by transmission electron microscopy. Damage to viral proteins and genome was further confirmed by western blotting and RT-qPCR, respectively. The multi-antiviral effects of TiO2-mediated photocatalytic reaction implies universal disinfection potential for different infectious agents. Notably, TiO2 has no adverse effects on human health, and therefore, TiO2-induced photocatalytic reaction is suitable for disinfection of SARS-CoV-2 and other emerging infectious disease-causing agents in human habitation.

Phascolarctobacterium wakonense sp. nov., isolated from common marmoset (Callithrix jacchus) faeces.

Two strictly anaerobic strains (MB11T and MB56) were isolated from common marmoset (Callithrixjacchus) faeces. Cells of the two strains were Gram-stain-negative, pleomorphic short (strain MB11T) or long (strain MB56) rods. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both isolates were related to the genus Phascolarctobacterium. They had 16S rRNA gene sequences similarities lower than 93 % to previously described species, Phascolarctobacterium faecium ACM 3679T and Phascolarctobacterium succinatutens YIT 12067T, and 98.7 % between themselves. DNA-DNA hybridization values showed that strains MB11T and MB56 were the same species. The genomic DNA G+C content of strains MB11T and MB56 were 47.3-47.4 mol% and 47.7-48.0 mol%. The isolates had different enzymatic activities compared with P. succinatutens JCM 16074T and different major cellular fatty acids compared with P. faecium ACM 3679T. Substrate availability revealed that they utilized not only succinate, but also pyruvate. With pyruvate supplementation, they produced both propionate and acetate, while only propionate production occured with succinate. As suggested by the phylogenic and physiological properties of strains MB11T and MB56, we propose the name Phascolarctobacteriumwakonense sp. nov. with the type strain MB11T (=JCM 32899T=DSM 107697T).

Gut microbiota development in mice is affected by hydrogen peroxide produced from amino acid metabolism during lactation.

The development of gut microbiota during infancy is an important event that affects the health status of the host; however, the mechanism governing it is not fully understood. l-Amino acid oxidase 1 (LAO1) is a flavoprotein that catalyzes the oxidative deamination of particular l-amino acids and converts them into keto acids, ammonia, and H2O2. Our previous study showed that LAO1 is present in mouse milk and exerts protection against bacteria by its production of H2O2. The data led us to consider whether LAO1, H2O2, or both could impact infant gut microbiota development via mother's milk consumption in mice. Different gut microbiota profiles were observed in the wild-type (WT) and LAO1-knockout mouse pups. The WT pups' microbiota was relatively simple and composed of only a few dominant bacteria, such as Lactobacillus, whereas the lactating knockout pups had high microbiota diversity. Cross-fostering experiments indicated that WT milk (containing LAO1) has the ability to suppress the diversity of microbiota in pups. We observed that the stomach content of pups fed WT milk had LAO1 proteins and the ability to produce H2O2. Moreover, culture experiments showed that Lactobacillus was abundant in the feces of pups fed WT milk and that Lactobacillus was more resistant to H2O2 than Bifidobacterium and Escherichia. Human breast milk produces very little H2O2, which could be the reason for Lactobacillus not being dominant in the feces of breast-fed human infants. In mouse mother's milk, H2O2 is generated from the process of free amino acid metabolism, and H2O2 may be a key player in regulating the initial acquisition and development of gut microbiota, especially growth of Lactobacillus, during infancy.-Shigeno, Y., Zhang, H., Banno, T., Usuda, K., Nochi, T., Inoue, R., Watanabe, G., Jin, W., Benno, Y., Nagaoka, K. Gut microbiota development in mice is affected by hydrogen peroxide produced from amino acid metabolism during lactation.

Comparison of gut microbiota composition between laboratory-bred marmosets (Callithrix jacchus) with chronic diarrhea and healthy animals using terminal restriction fragment length polymorphism analysis.

Chronic diarrhea in laboratory-bred marmosets poses a serious health problem during experiments. Despite a growing demand for laboratory-bred experimental marmosets, the mechanisms underlying the development of diarrhea and measures for its treatment and prevention remain unclear. To explore the factors affecting development of chronic diarrhea in laboratory-bred marmosets, the gut microbiota composition (GMC) of 58 laboratory-bred marmosets, including 19 animals with chronic diarrhea, was analyzed using terminal restriction fragment length polymorphism. We found that the GMCs in these animals cluster into two groups that differ significantly in rate of chronic diarrhea (56.5% in one group, Cluster 1, and 17.1% in Cluster 2). Additionally, a higher α-diversity and a lower proportion of Bifidobacterium spp. according to quantitative PCR was found the animals in the Cluster 1 than in those in Cluster 2. Taken together, our findings indicate that there is a relationship between GMC and development of chronic diarrhea in laboratory-bred marmosets. This is the first study to highlight the potential of assessing GMC in relation to development of chronic diarrhea in laboratory-bred marmosets.

Bioactive polyamine production by a novel hybrid system comprising multiple indigenous gut bacterial strategies.

Metabolites of the intestinal microbiota are thought to be generated through metabolic pathways spanning multiple taxa of intestinal bacteria. We have previously shown that the level of putrescine, a polyamine found abundantly in the human intestinal lumen, is increased in the colonic lumen following administration of arginine and the probiotic Bifidobacterium sp.; however, the underlying mechanism remained poorly understood. We report a novel pathway for putrescine production from arginine through agmatine involving the collaboration of two bacterial groups, and triggered by environmental acidification (drop in pH to below 6.5 from neutral). This pathway comprises the acid tolerance system of Escherichia coli, representing bacteria that have an arginine-dependent acid resistance system; the energy production system of Enterococcus faecalis, representing bacteria that have an agmatine deiminase system; and the acid production system of the acid-producing bacteria, represented by Bifidobacterium spp. This pathway is unique in that it represents a relationship between the independent survival strategies of multiple bacteria.

Colonic Absorption of Low-Molecular-Weight Metabolites Influenced by the Intestinal Microbiome: A Pilot Study.

Low-molecular-weight metabolites produced by the intestinal microbiome play a direct role in health and disease. However, little is known about the ability of the colon to absorb these metabolites. It is also unclear whether these metabolites are bioavailable. Here, metabolomics techniques (capillary electrophoresis with time-of-flight mass spectrometry, CE-TOFMS), germ-free (GF) mice, and colonized (Ex-GF) mice were used to identify the colonic luminal metabolites transported to colonic tissue and/or blood. We focused on the differences in each metabolite between GF and Ex-GF mice to determine the identities of metabolites that are transported to the colon and/or blood. CE-TOFMS identified 170, 246, 166, and 193 metabolites in the colonic feces, colonic tissue, portal plasma, and cardiac plasma, respectively. We classified the metabolites according to the following influencing factors: (i) the membrane transport system of the colonocytes, (ii) metabolism during transcellular transport, and (iii) hepatic metabolism based on the similarity in the ratio of each metabolite between GF and Ex-GF mice and found 62 and 22 metabolites that appeared to be absorbed from the colonic lumen to colonocytes and blood, respectively. For example, 11 basic amino acids were transported to the systemic circulation from the colonic lumen. Furthermore, many low-molecular-weight metabolites influenced by the intestinal microbiome are bioavailable. The present study is the first to report the transportation of metabolites from the colonic lumen to colonocytes and somatic blood in vivo, and the present findings are critical for clarifying host-intestinal bacterial interactions.

Comprehensive analysis of the fecal microbiota of healthy Japanese adults reveals a new bacterial lineage associated with a phenotype characterized by a high frequency of bowel movements and a lean body type.

BACKGROUND: In Japan, a variety of traditional dietary habits and daily routines have developed in many regions. The effects of these behaviors, and the regional differences in the composition of the gut microbiota, are yet to be sufficiently studied. To characterize the Japanese gut microbiota and identify the factors shaping its composition, we conducted 16S metagenomics analysis of fecal samples collected from healthy Japanese adults residing in various regions of Japan. Each participant also completed a 94-question lifestyle questionnaire. RESULTS: We collected fecal samples from 516 healthy Japanese adults (325 females, 191 males; age, 21-88). Heatmap and biplot analyses based on the bacterial family composition of the fecal microbiota showed that subjects' region of residence or gender were not strongly correlated with the general composition of the fecal microbiota. Although clustering analysis for the whole cohort did not reveal any distinct clusters, two enterotype-like clusters were observed in the male, but not the female, subjects. In the whole subject population, the scores for bowel movement frequency were significantly correlated with the abundances of Christensenellaceae, Mogibacteriaceae, and Rikenellaceae in the fecal microbiota (P < 0.001). These three bacterial families were also significantly more abundant (P < 0.05 or 0.01) in lean subjects (body mass index (BMI) < 25) than in obese subjects (BMI > 30), which is consistent with previously published results. However, a previously reported correlation between BMI and bowel movement frequency was not observed. In addition, the abundances of these three families were positively correlated with each other and comprised a correlative network with 14 other bacterial families. CONCLUSIONS: The present study showed that the composition of the fecal microbiota of healthy Japanese adults at the national level was not strongly correlated with subjects' area of residence or gender. In addition, enterotype partitioning was ambiguous in this cohort of healthy Japanese adults. Finally, the results implied that the abundances of Christensenellaceae, Mogibacteriaceae, and Rikenellaceae, along with several other bacterial components that together comprised a correlative network, contributed to a phenotype characterized by a high frequency of bowel movements and a lean body type.

Membrane filter method to study the effects of Lactobacillus acidophilus and Bifidobacterium longum on fecal microbiota.

A large number of commensal bacteria inhabit the intestinal tract, and interbacterial communication among gut microbiota is thought to occur. In order to analyze symbiotic relationships between probiotic strains and the gut microbiota, a ring with a membrane filter fitted to the bottom was used for in vitro investigations. Test strains comprising probiotic nitto strains (Lactobacillus acidophilus NT and Bifidobacterium longum NT) and type strains (L. acidophilus JCM1132(T) and B. longum JCM1217(T) ) were obtained from diluted fecal samples using the membrane filter to simulate interbacterial communication. Bifidobacterium spp., Streptococcus pasteurianus, Collinsella aerofaciens, and Clostridium spp. were the most abundant gut bacteria detected before coculture with the test strains. Results of the coculture experiments indicated that the test strains significantly promote the growth of Ruminococcus gnavus, Ruminococcus torques, and Veillonella spp. and inhibit the growth of Sutterella wadsworthensis. Differences in the relative abundances of gut bacterial strains were furthermore observed after coculture of the fecal samples with each test strain. Bifidobacterium spp., which was detected as the dominant strain in the fecal samples, was found to be unaffected by coculture with the test strains. In the present study, interbacterial communication using bacterial metabolites between the test strains and the gut microbiota was demonstrated by the coculture technique. The detailed mechanisms and effects of the complex interbacterial communications that occur among the gut microbiota are, however, still unclear. Further investigation of these relationships by coculture of several fecal samples with probiotic strains is urgently required.

Parabacteroides faecis sp. nov., isolated from human faeces.

A bacterial strain, designated 157(T), isolated from human faeces was characterized by using a polyphasic taxonomic approach, which included analysis of physiological and biochemical features, cellular fatty acid profiles, menaquinone profiles and its phylogenetic position, based on 16S rRNA gene sequence analysis. The strain was obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-stain-negative rods. The isolate was able to grown on medium containing 20% (w/v) bile. 16S rRNA gene sequence analysis showed that the strain was a member of the genus Parabacteroides . Strain 157(T) was closely related to Parabacteroides gordonii JCM 15724(T) (96% sequence similarity). The results of hsp60 gene sequence analysis indicated that strain 157(T) was different from P. gordonii JCM 15724(T), with a hsp60 gene sequence similarity of 96.1%. The major cellular fatty acids of strain 157(T) were anteiso-C(15 : 0), iso-C(17 : 0) 3-OH, C(18 : 1)ω9c and anteiso-C(17 : 0) 3-OH. The major menaquinone of the isolate was MK-9. The DNA G+C content of strain 157(T) was 41.8 mol%. On the basis of these data, strain 157(T) represents a novel species of the genus Parabacteroides , for which the name Parabacteroides faecis sp. nov. is proposed; the type strain is 157(T) ( = JCM 18682(T) = CCUG 66681(T)).

Application of a single-colony coculture technique to the isolation of hitherto unculturable gut bacteria.

Molecular studies have led to postulation of a relationship between gut microbiota and certain diseases. However, because studies of hitherto uncultured species in vivo are essential for characterizing the biology and pathogenic properties of gut bacteria, techniques for culturing and isolating such bacteria must be developed. Here, a technique is described that partially overcomes the obstacles that prevent detection of interbacterial communication in vitro and are thus responsible for the failure to culture certain bacterial species. For this purpose, a ring with a membrane filter at the bottom was designed and a relatively simple nutrient medium was used instead of conventional media. Gut bacteria were cocultivated in soft agar separated by the membrane filter to simulate interbacterial communication in vitro. Use of this soft agar coculture technique led to the successful isolation of hitherto uncultured bacteria and the demonstration of multistage interbacterial communication among gut bacteria in vitro. Cultivation and isolation of single colonies of bacteria that require other bacteria for growth will enhance efforts to better understand the physiological and pathogenic roles of gut microbiota.

Alteration of a human intestinal microbiota under extreme life environment in the Antarctica.

The human intestinal microbiota (HIM) settles from birth and continues to change phenotype by some factors (e.g. host's diet) throughout life. However, the effect of extreme life environment on human HIM composition is not well known. To understand HIM fluctuation under extreme life environment in humans, fecal samples were collected from six Japanese men on a long Antarctic expedition. They explored Antarctica for 3 months and collected their fecal samples at once-monthly intervals. Using terminal restriction fragment length polymorphism (T-RFLP) and real time polymerase chain reaction (PCR) analysis, the composition of HIM in six subjects was investigated. Three subjects presented restoration of HIM after the expedition compared versus before and during the expedition. Two thirds samples collected during the expedition belonged to the same cluster in dendrogram. However, all through the expedition, T-RFLP patterns showed interindividual variability. Especially, Bifidobacterium spp. showed a tendency to decrease during and restore after the expedition. A reduction of Bifidobacterium spp. was observed in five subjects the first 1 month of the expedition. Bacteroides thetaiotaomicron, which is thought to proliferate during emotional stress, significantly decreased in one subject, indicating that other factors in addition to emotional stress may affect the composition of HIM in this study. These findings could be helpful to understand the effect of extreme life environment on HIM.

Innate lymphoid cells regulate intestinal epithelial cell glycosylation.

Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host-microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria-dependent and -independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation.

Butyricimonas faecihominis sp. nov. and Butyricimonas paravirosa sp. nov., isolated from human faeces, and emended description of the genus Butyricimonas.

Two bacterial strains, designated 180-3(T) and 214-4(T), isolated from human faeces were characterized by using a polyphasic taxonomic approach that included analysis of their phenotypic and biochemical features, cellular fatty acid profiles, menaquinone profiles and phylogenetic positions based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that these strains represented members of the genus Butyricimonas. These strains shared 97.9 % 16S rRNA gene sequence similarity with each other and were related to Butyricimonas virosa JCM 15149(T) (97 % sequence similarity) and Butyricimonas synergistica JCM 15148(T) (94-95 %). Although strain 180-3(T) was related to (but distinct from) B. virosa JCM 15149(T) and B. synergistica JCM 15148(T), with hsp60 gene sequence similarities of 89.4 and 84.6 %, respectively, strain 214-4(T) exhibited high hsp60 gene sequence similarity (100 %) with B. virosa JCM 15149(T) and was different from B. synergistica JCM 15148(T) (83.5 %). DNA-DNA hybridization experiments demonstrated a genomic distinction of strains 180-3(T) and 214-4(T) from B. virosa JCM 15149(T) and B. synergistica JCM 15148(T). The strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-stain-negative rods. Growth of the strains was inhibited on medium containing 20 % bile. The two strains produced butyric and isobutyric acids as the end products from glucose, as has been observed in the other two species of the genus Butyricimonas. The major cellular fatty acid of strains 180-3(T) and 214-4(T) was iso-C15 : 0. The major menaquinone of the isolates was MK-10 (>50 %). Strains 180-3(T) and 214-4(T) have DNA G+C contents of 45 mol%. On the basis of these data, strains 180-3(T) and 214-4(T) represent two novel species of the genus Butyricimonas, for which the names Butyricimonas faecihominis sp. nov. and Butyricimonas paravirosa sp. nov., respectively, are proposed. The type strains of B. faecihominis and B. paravirosa are 180-3(T) ( = JCM 18676(T) = CCUG 65562(T)) and 214-4(T) ( = JCM 18677(T) = CCUG 65563(T)), respectively.