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Phylogeny of oral asaccharolytic Eubacterium species determined by 16S ribosomal DNA sequence comparison and proposal of Eubacterium infirmum sp. nov. and Eubacterium tardum sp. nov.

16S rRNA gene sequences of Eubacterium brachy, Eubacterium nodatum, Eubacterium saphenum, Eubacterium timidum, and two previously unnamed taxa were determined. The results of a phylogenetic analysis indicated that all of the strains sequenced belonged to a deep branch of the low-G+C-content gram-positive group. The levels of 16S ribosomal DNA sequence similarity between species were low, suggesting that a number of genera may be represented in this group. The representatives of the two unnamed taxa, which were isolated from patients with periodontitis, were clearly distinct from the previously described species, and, therefore, the following two new species are proposed: Eubacterium infirmum (type strain, NCTC 12940) and Eubacterium tardum (type strain, NCTC 12941).

Base Sequence↗

Characteristics and sites of infection of Eubacterium nodatum, Eubacterium timidum, Eubacterium brachy, and other asaccharolytic eubacteria.

Three new species, Eubacterium nodatum, Eubacterium timidum, and Eubacterium brachy, were described, primarily from subgingival samples taken from patients with moderate and severe adult periodontitis. Except for the isolation of E. brachy from a pleuropulmonary infection, these species have not been reported from other infected body sites. We report on the isolation of these species and an undescribed group (D-6) of asaccharolytic eubacteria also found in periodontal disease from numerous different sites of infection, mostly the head and neck. A similarity in cellular morphological properties of E. nodatum and Actinomyces sp. was noted previously. Additional similarities, particularly to Actinomyces israelii, that we found are the formation of molar tooth colonies and the isolation from cases of lumpy jaw and from the genital tract of women in association with the use of an intrauterine contraceptive device. E. timidum and E. brachy did not occur more often from any particular site outside of the head, neck, and respiratory tract. The group D-6 strains came from a variety of sites in the trunk and pelvis. These species are all obligately anaerobic, asaccharolytic, and generally nonreactive, and they grow poorly and slowly on media commonly used to isolate anaerobic bacteria. L-Lysine (0.5%) markedly stimulated the growth of E. nodatum and, to a lesser extent, another acetate- and butyrate-producing group, Eubacterium sp. group D-6, but we did not find comparable stimulants for the other species. We found the production of phenyl acetate to be a helpful marker in the identification of E. timidum and Eubacterium sp. group D-6. Although the isolation and identification of most of these species remain somewhat difficult, the evidence from dental infections and the present report suggests that these species are potential pathogens that are likely to be overlooked in infected clinical material without special attention to more prolonged incubation and use of enriched isolation media.

Bacterial Infections↗

The family Coriobacteriaceae: reclassification of Eubacterium exiguum (Poco et al. 1996) and Peptostreptococcus heliotrinreducens (Lanigan 1976) as Slackia exigua gen. nov., comb. nov. and Slackia heliotrinireducens gen. nov., comb. nov., and Eubacterium lentum (Prevot 1938) as Eggerthella lenta gen. nov., comb. nov.

16S rRNA gene sequences were determined for Eubacterium exiguum and Peptostreptococcus heliotrinreducens. These species were found to be closely related and, together with Eubacterium lentum, to constitute a branch of the Coriobacteriaceae. Two new genera are proposed on the basis of phenotypic characteristics and 16S rRNA gene sequence comparisons: Slackia to include the bile-sensitive species Eubacterium exiguum and P. heliotrinreducens, and Eggerthella to include the bile-resistant Eubacterium lentum. It is proposed that Eubacterium exiguum and Peptostreptococcus heliotrinreducens are transferred to the genus Slackia gen. nov. as Slackia exigua gen. nov., comb. nov. (type strain ATCC 700122T) and Slackia heliotrinireducens gen. nov., comb. nov. (type strain NTCC 11029T), respectively, and Eubacterium lentum is transferred to the genus Eggerthella gen. nov. as Eggerthella lenta gen. nov., comb. nov. with Eggerthella lenta as the type species.

Base Composition↗

Phylogenetic relationships of the genera Acetobacterium and Eubacterium sensu stricto and reclassification of Eubacterium alactolyticum as Pseudoramibacter alactolyticus gen. nov., comb. nov.

16S rRNA gene sequences of the type strains of the seven previously described Acetobacterium species were determined. The Acetobacterium species were found to form a tight phylogenetic cluster within the Clostridium subphylum of the gram-positive bacteria. Within this subphylum these organisms belong to cluster XV as defined by Collins et al. (M.D. Collins, P.A. Lawson, A. Willems, J.J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994) together with Eubacterium alactolyticum barkeri, Eubacterium callanderi, and Eubacterium limosum. Our data indicate that Clostridium cluster XV consists of at least the following three genera: the genus Acetobacterium, the genus Eubacterium sensu stricto (comprising E. limosum, E. barkeri, and E. callanderi), and the genus Pseudoramibacter gen. nov., which is created for E. alactolyticum, which we reclassify as Pseudoramibacter alactolyticus comb. nov.

Base Sequence↗

Eubacterium minutum is an earlier synonym of Eubacterium tardum and has priority.

The recently proposed species Eubacterium minutum and Eubacterium tardum appeared to be similar from their published descriptions. The aim of this study was to perform phenotypic and genetic analyses of strains of both species to clarify their taxonomic position. The type strains of E. minutum and E. tardum exhibited identical biochemical and protein profiles and their 16S rRNA gene sequences displayed 99.9% similarity. The G + C content of the DNA of both strains was estimated at 45 mol%. It is concluded that E. minutum and E. tardum are synonyms; E. minutum has priority. An emended description of E. minutum is given.

Bacterial Typing Techniques↗

Description of Mogibacterium pumilum gen. nov., sp. nov. and Mogibacterium vescum gen. nov., sp. nov., and reclassification of Eubacterium timidum (Holdeman et al. 1980) as Mogibacterium timidum gen. nov., comb. nov.

A new genus, Mogibacterium, is proposed for anaerobic, non-spore-forming, Gram-positive, rod-shaped bacteria which have been isolated from the periodontal pockets of adult human patients with periodontal disease and infected root canals. The novel isolates, strains D2-18T, BA11a-f and D5-2T, were inert in most of the conventional biochemical tests and phenotypically resemble asaccharolytic Eubacterium species. The protein profiles of whole cells on SDS-PAGE gels and Western immunoblotting reaction analysis distinguished these organisms from type strains belonging to the previously described Eubacterium species. The G + C content of the DNA is 45-46 mol% for Mogibacterium pumilum and 46 mol% for Mogibacterium vescum. The levels of DNA-DNA relatedness of these new species to other Eubacterium species, including Eubacterium limosum, Eubacterium brachy, Eubacterium lentum, Eubacterium nodatum, Eubacterium saphenum, and the more recently proposed Eubacterium minutum and Eubacterium exiguum (reclassified as Slackia exigua), are less than 2%. The DNA-DNA hybridization value between M. pumilum and M. vescum was 30%. Eubacterium timidum exhibited DNA homologies with Mogibacterium species which were low (17 and 18%) but clearly higher than with all the other Eubacterium species. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the closest phylogenetic neighbour of Mogibacterium species was E. timidum, and that these three species represent a novel lineage distinct from the previously described genera of Gram-positive, rod-shaped bacteria. On the basis of phenotypic characteristics and 16S rRNA gene sequence comparisons, it is also proposed that E. timidum is transferred to the genus Mogibacterium gen. nov. as Mogibacterium timidum gen. nov., comb. nov. (type strain ATCC 33093T).

Adult↗

Phylogenetic evidence for the transfer of Eubacterium lentum to the genus Eggerthella as Eggerthella lenta gen. nov., comb. nov.

Eubacterium lentum has unique phenotypic characters within the genus Eubacterium. The 16S rRNA sequence of Eubacterium lentum was determined and its phylogenetic position was defined. This micro-organism is a member of the genus Eubacterium but it is not closely related to Eubacterium limosum, the type species of the genus Eubacterium, and is nearer to Collinsella aerofaciens and Coriobacterium glomerans. A PCR-based identification system using species-specific primers designed on the basis of DNA sequences encoding the 16S rRNA of strains of Eubacterium lentum, Collinsella aerofaciens and Coriobacterium glomerans is described. A species-specific primer set can distinguish Eubacterium lentum from Eubacterium limosum or closely related species including Collinsella aerofaciens, Coriobacterium glomerans and Atopobium species. This species-specific PCR method can be used to identify Eubacterium lentum-like species isolated from human faeces. On the basis of the 16S rRNA sequence divergence from Collinsella aerofaciens and Coriobacterium glomerans and the presence of unique phenotypic characters, a new genus, Eggerthella gen. nov., is proposed for Eubacterium lentum, with one species, Eggerthella lenta comb. nov. The type strain of Eggerthella lenta is JCM 9979T.

Bacterial Typing Techniques↗

Coaggregation studies of the Eubacterium species.

Eubacterium species are gram-positive anaerobic rods that are frequently isolated from subgingival plaque of periodontal pockets. Five Eubacterium species were tested for their ability to coaggregate with 33 oral bacterial strains. Using visual and turbidimetric assays, coaggregation was observed among Eubacterium brachy, Eubacterium nodatum, Eubacterium alactolyticum and Eubacterium limosum strains only when tested with Fusobacterium nucleatum strains; Eubacterium saburreum displayed only weak coaggregation ability. Coaggregation between F. nucleatum and the Eubacterium species was observed over a wide range of concentrations of each organism. The F. nucleatum strains contained a heat labile and the Eubacterium species a heat stabile coaggregation receptor. Arginine, histidine, lysine and glycine inhibited the coaggregation between F. nucleatum and the Eubacterium species. Sugars and other amino acids tested did not inhibit the observed coaggregation. Rabbit anti-F. nucleatum serum completely inhibited coaggregation, but anti-E. brachy serum and normal rabbit serum did not. As these anaerobic microorganisms are frequently isolated from the same oral lesions, the surface interactions observed may be important in the pathogenesis of these polymicrobic infections.

Arginine↗

Phylogenetic and phenotypic evidence for the transfer of Eubacterium aerofaciens to the genus Collinsella as Collinsella aerofaciens gen. nov., comb. nov.

Three strains of Eubacterium aerofacien, JCM 10188T, JCM 7790 and JCM 7791, and 178 freshly isolated strains of the Eubacterium aerofaciens group from human faeces were characterized by biochemical tests, cell wall peptidoglycan type and 16S rRNA analysis. The Eubacterium aerofaciens group was divided into four groups by fermentation patterns of sucrose and cellobiose, and were further divided into 16 sub-groups by fermentation patterns of aesculin, salicin and amygdalin. All of the strains of the Eubacterium aerofaciens group were shown to be phylogenetically distantly related to Eubacterium limosum, which is the type species of genus Eubacterium. Eubacterium aerofaciens was shown to have a specific phylogenetic association with Coriobacterium glomerans. All the strains belonging to Eubacterium aerofaciens resembled Coriobacterium glomerans in possessing a high G + C content (60 mol%). Cell wall analysis, however, revealed the presence of different A4 beta (L-Ala)-D-Glu-L-Orn-L-Asp peptidoglycan types. Based on a 16S rRNA sequence divergence of greater than 9% with Coriobacterium glomerans and the presence of a unique peptidoglycan type, a new genus, Collinsella, is proposed for Eubacterium aerofaciens, with one species, Collinsella aerofaciens. The type strain of Collinsella aerofaciens is JCM 10188T.

Bacteria, Anaerobic↗

Restriction fragment-length polymorphism analysis of 16S rDNA from oral asaccharolytic Eubacterium species amplified by polymerase chain reaction.

Restriction fragment-length polymorphism (RFLP) analysis of 16S rDNA amplified by polymerase chain reaction was used to generate restriction profiles of the type strains of oral asaccharolytic Eubacterium species, that is, Eubacterium brachy, Eubacterium exiguum, Eubacterium lentum, Eubacterium minutum, Eubacterium nodatum, Eubacterium saphenum, Eubacterium timidum and 33 asaccharolytic Eubacterium strains isolated from oral sites. The 16S rRNA gene sequences from isolated genomic DNA samples were amplified by polymerase chain reaction (PCR). PCR products were purified and characterized by single digestions with 7 restriction endonucleases. Among the 7 endonucleases, HpaII was found to discriminate the respective reference strains. Twenty-three isolates, out of 33, were assigned to one of the reference species, on the basis of their restriction profiles by digestion with HpaII. The remaining 10 isolates could not be assigned to any of the established species and constituted 4 distinct groups, each of which may be a new species.

DNA, Bacterial↗

Tissue distribution and persistence of arthritogenic and non-arthritogenic Eubacterium cell walls.

OBJECTIVE: To study the tissue distribution and persistence of arthritogenic and non-arthritogenic Eubacterium cell walls (CWs), using arthritogenic Eubacterium aerofaciens and non-arthritogenic Eubacterium limosum. METHODS: Eubacterium aerofaciens or Eubacterium limosum CW was injected into Lewis rats intraperitoneally. Inflammatory changes in the synovium and periarticular tissues were graded histologically. On days 14, 28 and 56 after the injection, the presence of CW in the liver, spleen, mesenteric lymph nodes and synovium was studied by indirect immunofluorescence. In parallel, CW-derived muramic acid in the liver and spleen was measured by gas chromatography-mass spectrometry. In addition, serum TNF-alpha, IL-1 beta and IL-10 concentrations were determined by ELISA. RESULTS: Systemic injection of Eubacterium aerofaciens CW, but not of Eubacterium limosum CW, resulted in chronic arthritis. Both E. aerofaciens and E. limosum CWs were observed in the liver and spleen at all of the time points studied. In addition, Eubacterium limosum CW was present in non-arthritic synovium on day 14. It was not, however, detected in the synovium or lymph nodes on days 28 and 56, in clear contrast to the rats injected with E. aerofaciens CW. According to the analysis by gas chromatography-mass spectrometry, non-arthritogenic E. limosum CW had accumulated in the liver cells on days 14 and 28 after the injection to a greater extent than arthritogenic E. aerofaciens CW, leading to a lesser distribution in the other organs. A weak trend was observed suggesting that the production of TNF-alpha and IL-1 beta, but not of IL-10, is stimulated better by arthritogenic CW than by non-arthritogenic CW. CONCLUSION: Our results indicate that non-arthritogenic CWs are handled by the rat's defence mechanisms in a different way than arthritogenic CWs. The tissue distribution and persistence of CWs play a role in arthritogenicity, but additional factors must exist to determine why the CWs of certain bacteria are arthritogenic and those of others are not.

Animals↗

Immunological specificity of oral Eubacterium species.

Antigens of Eubacterium species including E. alactolyticum, E. brachy, E. nodatum, E. saburreum, E. timidum, E. yurii subsp. yurii and E. yurii subsp. margaretiae, which have been isolated frequently from periodontal pockets and associated with periodontal diseases, were extracted by ultrasonication from whole bacterial cells. Antigens were also prepared from E. aerofaciens, E. lentum and E. rectale, which have been found in intestinal tracts and infected abscesses in human oral cavities. The antigens of the oral Eubacterium species were compared with antigens from E. limosum, the type species of the genus Eubacterium, by using SDS-PAGE and Western immunoblot assays. SDS-PAGE gels stained with Coomassie brilliant blue indicated that no major peptide bands were common among the Eubacterium species examined. The protein profile patterns were distinctly different from each other. Western immunoblotting reactions with rabbit antisera showed that the Eubacterium species could be clearly distinguished serologically, and that the species-specific antigens were peptide components of ultrasonic extracts from the whole bacterial cells. The present study demonstrates that these Eubacterium species show great heterogeneity in their peptide components and immunological reactions, which may be useful for identification of the Eubacterium species from human oral specimens.

Antigens, Bacterial↗

Expression of the bile acid-inducible NADH:flavin oxidoreductase gene of Eubacterium sp. VPI 12708 in Escherichia coli.

The intestinal microorganism Eubacterium sp. VPI 12708 synthesizes a bile acid-inducible NADH:flavin oxidoreductase (NADH:FOR) which presumably functions in the 7 alpha-dehydroxylation of cholic acid to deoxycholic acid. The baiH gene encoding NADH:FOR was subcloned into an IPTG-inducible expression vector, pBaiH2.2. Escherichia coli DH5 alpha cells transformed with pBaiH2.2 expressed 10-fold higher levels of NADH:FOR upon induction with IPTG than did Eubacterium sp. VPI 12708 cells induced with cholic acid. The NADH:FOR produced by E. coli DH5 alpha(pBaiH2.2) was purified to > 95% electrophoretic homogeneity in three steps. The purified NADH:FOR was similar to that of Eubacterium sp. VPI 12708 in subunit and native M(r) (ca. 72,000 and 210,000, respectively), pH optimum, sensitivity to inhibitors, and electron acceptor specificity. It contained 1 mol of FAD, up to 2 mol of iron, and 1 mol of copper per mol of subunit. The enzyme reduced synthetic quinones, dyes, flavins, and O2 with NADH as the electron donor, but did not reduce disulfide compounds, various unsaturated bile acids, cytochrome c, physiological quinones, or cell fractions from Eubacterium sp. VPI 12708. Addition of purified NADH:FOR to Eubacterium sp. VPI 12708 cell extracts altered the balance of oxidized and reduced bile acid intermediates produced during cholic acid 7 alpha-dehydroxylation, suggesting that the enzyme may regulate the cellular ratio of NAD to NADH.

Amino Acid Sequence↗

Diversity of oral asaccharolytic Eubacterium species in periodontitis--identification of novel phylotypes representing uncultivated taxa.

Oral asaccharolytic Eubacterium species are associated with periodontal disease and other oral infections. The aim of this study was to use a culture-independent molecular technique to determine the diversity of asaccharolytic Eubacterium species in subgingival plaque in periodontitis. An oligonucleotide PCR primer designed to amplify 16S rRNA genes of the oral asaccharolytic Eubacterium branch of the phylogenetic tree was constructed. This primer was used together with a universal primer in PCRs to amplify gene sequences directly from a single subgingival plaque sample. Fifty PCR products were purified by cloning, fully sequenced and subjected to molecular phylogenetic analysis. The sequences were assigned to four groups within a single lineage of the low G + C gram-positive bacteria. Group I (58% of the cloned sequences) was assigned to a branch that included Eubacterium nodatum, and Group II (22%) to a branch including Eubacterium infirmum. Group III (8%) was distinct from but related to E. infirmum at the species level, and Group IV (12%) was another novel taxon more distantly related to E. infirmum and E. nodatum.

Adult↗

Rapid detection of human fecal Eubacterium species and related genera by nested PCR method.

PCR procedures based on 16S rDNA gene sequence specific for seven Eubacterium spp. and Eggerthella lenta that predominate in the human intestinal tract were developed, and used for direct detection of these species in seven human feces samples. Three species of Eggerthella lenta, Eubacterium rectale, and Eubacterium eligens were detected from seven fecal samples. Eubacterium biforme was detected from six samples. It was reported that E. rectale, E. eligens, and E. biforme were difficult to detect by traditional culture method, but the nested PCR method is available for the detection of these species. This result shows that the nested PCR method utilizing a universal primer pair, followed by amplification with species-specific primers, would allow rapid detection of Eubacterium species in human feces.

Bacteriological Techniques↗

Influence of resistant starch on the SCFA production and cell counts of butyrate-producing Eubacterium spp. in the human intestine.

AIMS: The genus Eubacterium, which is the second most common genus in the human intestine, includes several known butyrate producers. We hypothesized that Eubacterium species play a role in the intestinal butyrate production and are inducible by resistant starch. METHODS AND RESULTS: In a human pilot study species-specific and group-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labelled oligonucleotide probes were used to quantify butyrogenic species of the genera Eubacterium, Clostridium and Ruminococcus. Following the intake of RS type III a significant increase in faecal butyrate but not in total SCFA was observed. However, increase in butyrate was not accompanied by a proliferation in the targeted bacteria. CONCLUSIONS: The tested Eubacterium species have the capacity to produce butyrate but do not appear to play a major role for butyric acid production in the human intestine. SIGNIFICANCE AND IMPACT OF THE STUDY: In view of the fact that the bacteria responsible for butyrate production are largely unknown, it is still difficult to devise a dietary intervention to stimulate butyrogenic bacteria in a targeted way.

Acetates↗

Reclassification of Eubacterium formicigenerans Holdeman and Moore 1974 as Dorea formicigenerans gen. nov., comb. nov., and description of Dorea longicatena sp. nov., isolated from human faeces.

Two strains of a gram-positively staining, obligately anaerobic, non-spore-forming, rod-shaped bacterium, designated strains 111-13A and 111-35T, were isolated from human faeces. Analysis of the 16S rRNA gene sequences indicated that these strains were members of the Clostridium coccoides rRNA group of organisms. The nearest relatives of the unknown bacterium were Eubacterium formicigenerans (having a sequence similarity of 94%) and an uncultured bacterium (similarity > 99%). Characterization studies indicated that the unidentified faecal bacterium was biochemically distinct from Eubacterium formicigenerans, members of the Clostridium coccoides group and all other described Eubacterium species. On the basis of the data from these studies, it is proposed that the hitherto unknown rod-shaped bacterium be designated a species of a novel genus, namely Dorea longicatena gen. nov., sp. nov., and that Eubacterium formicigenerans be transferred to this genus as Dorea formicigenerans gen. nov., comb. nov.

Eubacterium↗