Microbiol. Immunol., 44(4), 223-227, 2000 Phylogenic and Phenotypic Characterization of Some Eubacterium-Like Isolates from Human Feces: Description of Gen. Nov., Sp. Nov.

Akiko Kageyama*, and Yoshimi Benno

Japan Collection of Microorganisms, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan

Received October 13, 1999; in revised form, December 9, 1999. Accepted January 10, 2000

Abstract: Three isolated strains from human feces were characterized by biochemical tests and 16S rDNA analysis. Phylogenetic analysis revealed that these isolated strains were members of the Clostridium sub- phylum of Gram-positive . The phenotypic characters resembled those of the genus Eubacterium, but these strains were shown to be phylogenetically distant from the type of the genus, Eubacteri- um limosum. The strains showed a specific phylogenetic association with Holdemania filiformis and Erysipelothrix rhusiopathiae. Based on a 16S rDNA sequence divergence of greater than 12% with H. fili- formis and E. rhusiopathiae, a new genus, Solobacterium, is proposed for three strains, with one species, Solobacterium moorei. The type strain of Solobacterium moorei is JCM 106451.

Key words: Solobacterium moorei gen. nov., sp. nov., 16S rDNA

The genus Eubacterium contains all Gram-positive, On the basis of the results presented, we propose that anaerobic, non-spore-forming, rod-shaped bacteria which these strains be classified as Solobacterium moorei gen. do not belong to the genera Propionibacterium, Lacto- nov., sp. nov. bacillus, or Bifzdobacterium. Differentiation among these genera is based on fermentation products from glucose: Materials and Methods propionic acid as a major end product for Propionibac- terium, lactic acid as a major product for Lactobacillus, Bacterial strains studied and cultivation. The bacte- and acetic acid in larger amounts than lactic acid for Bifi- rial strains RCA59-74 JCM 106451, RCA59-75 JCM dobacterium, whereas Eubacterium does not produce 10646, and RCA59-77 JCM 10647 were isolated from any of these acids but produces butyric, acetic or formic humans. All bacterial strains were cultivated for 2 days acid as a major product. Because of its broad definition, at 37 C on EG agar [premixed EG agar (Eiken Chemical this genus, has over the years, acted as a depository for a Co., Ltd.) supplied with 5% horse blood containing 3 g large number of phenotypically diverse species (1). In of beef extract, 5 g of yeast extract, 10 g of peptone, 1.5 addition to this marked phenotypic heterogeneity, it is g of glucose, 0.5 g of L-cysteine HCI, 0.2 g of L-cystine, recognized that eubacteria are not phylogenetically 4 g of Na2HPO,, 0.5 g of soluble starch, 0.5 g of Tween homogeneous, with species dispersed among many of the 80, 0.5 g of silicone, and 15 g of agar in 1,000 ml, pH different groups. In particular, the wide range of genom- 7.7] in an anaerobic jar with 100% CO,. ic DNA G+C contents, 30-55 mol%, indicates that the Phenotypic characteristics. Acid production from genus includes organisms that are not related phyloge- carbohydrates, assimilation of organic acids, nitrate netically. reduction, and hydrolysis of starch and gelatin were In this paper, we report the phenotypic and phyloge- tested (4, 9). netic characterization of three isolates from human feces. DNA studies. DNA was isolated as described by *Address correspondence to Dr. Akiko Kageyama, Japan Col- lection of Microorganisms, The Institute of Physical and Chem- Abbreviations: DDBJ, DNA Data Bank of Japan; G+C, gua- ical Research (RIKEN), Wako, Saitama 351-0198, Japan. Fax: 81- nine plus cytosine; JCM, Japan Collection of Microorganisms, 48-462-4619. E-mail: [email protected] RIKEN, Saitama, Japan.

223 224 A. KAGEYAMA AND Y. BENNO

Saito and Miura (11). DNA base composition was esti- Table 1. Physiological and biochemical characteristics of Eubac- mated by high-performance liquid chromatography terium-like strains' (HPLC) (13). Levels of DNA-DNA relatedness were determined by the method of Ezaki et al (3) using pho- tobiotin and microplates. One-hundred microliter por- tions of a heat-denatured DNA solution (DNA 10 gg/ml) in phosphate-buffered saline (PBS) containing 0.1 M MgCh_were incubated for 16 hr at 37 C in microdilution plates. Photobiotinylation of DNA was performed as described by Ezaki et al (3). For quantitative detection of biotinylated DNA, 200 ml portions of prehybridization solution containing denatured salmon sperm DNA 100 p.g/ml were added to microdilution wells. The microdi- lution plates were then incubated at 37 C for 30 min. The prehybridization solution was then removed from the wells and replaced with 100 µl portions of hybridization mixture containing 50 ng of biotinylated DNA. The microplates were then covered and hybridized for 3 hr at 40.0 C. A 100 µl portion of a streptavidin-beta-D-galac- tosidase solution was added to the wells, and the prepa- rations were incubated at 37 C for 30 min. A 100 ml portion of 3 X 10-° M 4-methylumbelliferyl-beta-D-galac- topyranoside in PBS was added, and the plates were incubated at 37 C for several periods of time. The fluo- rescence intensity was measured with a MicroFluor reader (Titertek Fluoroskan Il) at a wavelength of 360 nm for excitation and 450 nm for emission. 16S rDNA sequencing. The 16S rRNA gene was amplified using the PCR method and prokaryotic 16S rDNA universal primers 27F (5'-AGAGTTTGATC- CTGGCTCAG-3') and 1492R (5'-GGTTACCTTGT- TACGACTT-3'). PCR was performed with a DNA ther- mal cycler (Perkin-Elmer Cetus, Norwalk, Conn., U.S.A.) using 30 cycles consisting of denaturation at 94 C for 60 sec, primer annealing at 55 C for 150 sec, and primer °' The data are based on the reactions of our three isolated strains . b' + extension at 72 C for 150 sec (with 30 sec per cycle , positive; -, negative; v, variable; I, inhibit. added). Sequencing was performed using the ALFred AutoCycle Sequencing Kit (Pharmacia Biotech) with feces were characterized. The cells of these strains an ALFexpress DNA sequencer (Pharmacia Biotech). were 0.2 by 0.4-0.7 gm in size and occurred singly. These sequences were aligned with previously published All strains produced acid from ribose, glucose, galactose, sequences obtained from the GenBank, including close- fructose, and maltose. None of the strains produced acid ly related microorganisms. Nucleotide substitution rates from arabinose, xylose, rhamnose, mannose, sucrose, (K,,, values) were calculated (10), and phylogenetic cellobiose, lactose, trehalose, raffinose, melezitose, trees were constructed by the neighbor-joining method starch, glycogen, mannitol, sorbitol, inositol, erythritol, (12). The topology of the trees was evaluated by per- esculin, salicin, or amygdalin. Hydrolysis of starch was forming a bootstrap analysis of the sequence data with negative for all, while that of esculin was positive for all. CLUSTAL W software (14). The sequence similarity Gas formation, indole production, nitrate reduction, values were visually determined. gelatin liquefaction, and HZS production were all nega- tive. All strains were non-motile (Table 1). The isolates Results and Discussion produced moderate amounts of acetic, lactic, and butyric acids and some strains also produced a small amount of Three obligately anaerobic, Gram-positive, non- pyruvic acid from PYFG broth. Thus, physiological sporing rod Eubacterium-like strains isolated from human characteristics indicate that the isolates are close to the SOLOBACTERI UM MOOREI GEN . NOV., SP. NOV. 225

Fig. 1. Unrooted phylogenetic tree derived from 16S rDNA sequences. The tree was created using the neighbor joining method and K values. The numbers on the tree indicate bootstrap values for the branch points with greater than 50%.

Table 2. 16S rDNA sequence similarity matrix Table 3. DNA base composition and levels of DNA-DNA relat- edness among the three isolated strains

speciesof the genus Eubacterium. To establish the precise phylogenetic affiliation of RCA59-74, RCA59-75, and RCA59-77 was 37.5 to these three strains, more than 1,400 bases of the 16S 39.1 mol% G+C by the HPLC nucleoside method rDNAsequences (positions 28 to 1510;Escherichia coli (Table 3). Furthermore, to determine the relatedness numberingsystem) were detenninedand these sequences among the three isolated strains, DNA-DNA homology have been depositedin the DDBJdatabase. These acces- was studied. The levels of DNA-DNA relatedness sion numbers are describedin Fig. 1. A database search among the three isolated strains ranged from 97.8 to revealed that these strains belong to the Clostridium 99.6% (Table 3). The results showed that these three iso- subphylumof Gram-positivebacteria, and the 16SrDNA lated strains belong to the same species. sequenceis most similar to that of Clostridium cluster The unknown isolates from human feces clearly XVI (2). A phylogenetictree was preparedincluding all belong to a hitherto unrecognized Gram-positive species taxa from cluster XVI and related clusters (Fig. 1). within the Clostridium subphylum. From a 16S rDNA Table 2 shows that the sequence similarity values gene sequence comparison, it was evident that the bac- between the three isolated strains were 99.3 to 99.5. teria have a close phylogenetic relationship with H. fili- Thesethree isolatedstrains formeda distinctline exhibit- formis and E. rhusiopathiae; however, a sequence diver- ing a specific phylogenetic association (approximately gence of > 1.2% suggested that this relationship is not 87% 16SrDNA sequencerelatedness) with Holdemania sufficient for the same genera. Table 4 shows the dif- filiformis(16), (approximately 86% 16SrDNA sequence ferential characters between Solobacterium and other relatedness)with Erysipelothrixrhusiopathiae. A boot- phenotypically and phylogenetically related genera. strapanalysis (value 100%)showed this associationto be Erysiperothrix is facultatively anaerobic and other gen- statistically significant. era can be differentiated from Solobacterium by fer- The DNA base composition of isolated strains mentation products from glucose. Based on 16S rDNA 226 A. KAGEYAMA AND Y. BENNO

Table 4. Differential characters between Solobacterium and related genera

°' Data from reference 16 . b' Data from reference 6. " Data from reference 15. d1Data from ref- erence 7. e' Data from reference 5."Data from reference 8. sequence considerations and some other characteristics, fructose, and maltose but not from arabinose, xylose, Solobacterium is proposed for the three isolated strains, rhamnose, mannose, sucrose, cellobiose, lactose, tre- with one species, S. moorei. halose, raffinose, melezitose, starch, glycogen, mannitol, sorbitol, inositol, erythritol, esculin, salicin, or amygdalin. Description of Solobacterium Gen. Nov. The hydrolysis of starch is negative and that of esculin is Solobacterium (So.lo. L.a solus, sole Gr. Dim n. bak- positive. Gas formation, indole production, nitrate reduc- terion a small rod; M.L. neut. n. Solusbacterium sole bac- tion, gelatin liquefaction, and HS production are nega- terium). tive. DNA G+C content is 37 to 39 mol%. The type Cells occur singly. The species is Gram-positive and strain of S. moorei is strain JCM 106451. obligately anaerobic. Spores and fragella are absent. Fermentation products of glucose are mainly acetic, lac- References tic, and butyric acids; pyruvic acid is produced in a smaller amount. DNA G+C content is 37 to 39 mol%. 1) Andreesen, J.R. 1992. The genus Eubacterium, p. 1914- The type species is S. moorei. The genus Solusbacteri- 1924. In Balows, A., Truper, H.G., Dworkin, M., Harder, W., and Schleifer, K.-H. (eds), The prokaryotes, 2nd ed, Springer um is a member of the Clostridium subphylum of Gram- Verlag, New York. positive bacteria and exhibits a close phylogenetic asso- 2) Collins, M.D., Lawson, P.A., Willems, A., Cordoba, J.J., ciation with H. filiformis and E. rhusiopathiae. Femandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H., and Farrow, J.A.E. 1994. The phylogeny of the genus Clostridi- Description of Solobacterium moorei Sp. Nov. um: proposal of five new genera and eleven new species Solobacterium moorei (Moo.rei. M.C. gen. n. moorei combinations. Int. J. Syst. Bacteriol. 44: 812-826. of Moore; moorei named in honor of W.E.C. Moore, a 3) Ezaki, T., Hashimoto, Y., and Yabuuchi, E. 1989. Fluoro- contemporary American microbiologist). metric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to Cells are 0.2 by 0.4-0.7 tm in size and occur singly. membrane filter hybridization in which radioisotopes are The species is Gram-positive and obligately anaerobic. used to determine genetic relatedness among bacterial strains. Spores and flagella are absent. It can be cultivated in 2 Int. J. Syst. Bacteriol. 39: 224-229. days at 37 C on EG agar in an anaerobic jar with 100% 4) Holdeman, L.V., Cato, E.P., and Moore, W.E.C. 1977. Anaer- CO,.. Cells produce acid from ribose, glucose, galactose, obic laboratory manual, 4th ed, Anaerobe Laboratory , Vir- SOLOBACTERIUM MOOREI GEN. NOV., SP . NOV. 227

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