Bifidobacterium Species Expressing Phenotypical Similarity to Bifidobacterium adolescentis Isolated from

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1 Full Paper Bifidobacteria Microflora Vol. 11 (1), 25-32, 1992 Bifidobacterium Species Expressing Phenotypical Similarity to Bifidobacterium adolescentis Isolated from the Feces of Human Adults Tomoko YAESHIMA,1 Tomohiko FUJISAWA2 and Tomotari MITSUOKA2,3 1 Nutritional Science Laboratory, Morinaga Milk Industry Co., Ltd., Higashihara, Zama-shi, Kanagawa 228, 2Laboratory for Intestinal Flora, Frontier Research Program, Wako-shi, Saitama and 3Department of Food Hygiene, Nippon Veterinary and Animal Science University, Kyonan-cho, Musashino-shi, Tokyo 180 (Received for publication, August 19, 1991) Abstract Seventy-six strains of Bifidobacterium adolescentis and phenotypically similar Bifidobacterium species, of which 56 strains were isolated from the feces of human adults in our laboratory and 20 strains were obtained from DSM and ATCC as type strains or reference strains, were studied by carbohydrate fermentation patterns, DNA base compositions and DNA/DNA homologies. On the basis of DNA/DNA homology they were divided into six species: B. adolescentis, B. pseudocatenulatum, B. catenulatum, B. angulatum, B. dentium and B. longum. The strains of B. angulatum and B. dentium were not detected in 56 strains isolated from the feces of human adults. B. catenulatum could be distinguished from other four species, except for B. longum, by the inability to ferment glycogen and mannose. The G+C contents of DNAs of B. pseudocatenulatum and B. catenulatum (57.1 }0.7 mol% and 56.5 }1.0 mol%, respectively) were lower than that of B. adolescentis (59.8 }0.9 mol%). It was difficult to differentiate B. adolescentis biovar b and B. dentium, and B. adolescentis biovar c and B. pseudocatenulatum on the basis of carbohydrate fermentation pattern. Key words: Bifidobacterium species; B. adolescentis; feces of human adults; identification Bifidobacteria are part of the predominant species of intestinal microflora in human and animals. Twenty-eight species are now included in genus Bifidobacterium. It was reported that the species isolated from human oral cavity, feces and vagina were B. bifidum, B. infantis, B. breve, B. longum, B. adolescentis, B. catenulatum, B. pseudocatenulatum, B. angulatum, B. dentium (11) and B. gallicum (3). B. infantis, B. breve, B. longum, and B. adolescentis were originally proposed as the species isolated from human infants and adults by Reuter (9). Scardovi et al (12, 13) proposed B. catenulatum, B. dentium, B. angulatum and B. pseudocatenulatum as new species isolated 25

2 26 T. YAESHIMA et al from human feces, human vagina, human dental caries, sewage and calf feces. These species were diverse and distinctive from other bifidobacterial species on the basis of DNA relatedness or on combination of some properties, namely cell wall composition, electrophoretic patterns of enzymes and PAGE patterns of total cellular proteins (11). However, they were found not to differ significantly from B. adolescentis Reuter in their phenotypic characteristics based on sugar fermentation patterns. The similarity in sugar fermentation is considered to be the main cause of incorrect identification and persistent problems with mixed cultures. In the present study, we identified the strains isolated from the feces of human adults by DNA/DNA relatedness which had been attributed to B. adolescentis on the basis of carbohydrate fermentation patterns, and we studied the provisional phenotypic differential characteristics of these species isolated from the feces of human adults for clinical and ecological studies. MATERIALS AND METHODS Bacterial strains. The strains used in the present study are listed in Table 1. Fifty-six strains were isolated from the feces of human adults in our laboratory by the procedure of Mitsuoka et al (8). All of the organisms were grown on glucoseblood-liver (BL) agar (8) in anaerobic steel wool jars in an atmosphere of 100% CO2 and in Briggs liver broth (6) at 37 Ž. Phenotypic characterization. Carbohydrate fermentation pattern was determined according to Mitsuoka (7). Preparation of DNA. Cells were grown in Briggs liver broth for 8 hr, harvested and washed three times with 0.15 M NaCl-0.1 M ethylenediaminetetraacetic acid (ph 8.0). DNA was isolated and purified using a modification of the procedure of Marmur (4). The purity and the amount of DNA were estimated by measuring the hyperchromic shift during thermal denaturation. Tritium-labeled DNA was prepared using the nick translation system (New England Nuclear Corp., Boston, MA, USA) adapted from the procedure of Rigby et al (10). DNA base composition. The guanine-plus-cytosine (G+C) contents of the DNA preparations were determined by the thermal melting point (Tm) method (5), using an automatic recording spectrophotometer (model DU-8B; Beckman Instruments, Inc., Fullerton, CA, USA). Escherichia coli DNA was included in each set as a reference standard. DNA/DNA hybridization. DNA hybridization experiments were performed using the Sl nuclease procedure as described by Johnson et al (2). S1 nuclease digestions were conducted with 0.5 U of Sl nuclease (Tokyo Kasei Ltd., Tokyo Japan). After incubation for 15 min at 37 Ž, the remaining double-stranded DNA segments were precipitated with cold 10% trichloroacetic acid and collected on a membrane filter (type HA : Millipore Corp., Bedford, MA, USA). The membrane filter pads were dried, and the radioactivity was measured in toluene-based scintillation fluid with a liquid scintillation counter (model 3330; Packard Instrument Co., Rockville, MD, USA).,

3 BIFIDOBACTERIUM SPECIES ISOLATED FROM HUMAN FECES 27 Table 1. Strains used a DSM, Deutsche Sammlung von Mikroorganismen, Gottingen, Germany; ATCC, American Type Culture Collection, Rockville, Md.; Reuter, G. Reuter, Institut fur Lebensmittelhygiene, Freie Universitat Berlin, Berlin, Germany. RESULTS AND DISCUSSION Identification of strains of B. adolescentis and phenotypically similar Bifidobacterium. species by DNA relatedness Seventy-six strains of B. adolescentis and phenotypically similar Bifidobacterium

4 28 T. YAESHIMA et al Table 2. Characteristics and identification species, of which 56 strains were isolated from the feces of human adults and 20 strains were obtained from ATCC and DSM as reference strains, were differentiated into six species: B. adolescentis, B. pseudocatenulatum, B. catenulatum, B. angulatum, B. dentium and B. longum. The results are shown in Table 2. The strains considered to belong to B. adolescentis biovar a, which fermented both mannitol and sorbitol, were identified as B. adolescentis, B. pseudocatenulatum, B. angulatum and B. longum by DNA relatedness. None of the strains were assigned to B. adolescentis biovar a except for the type strain of B. adolescentis. Although Scardovi et al (13) reported that most of B. pseudocatenulatum strains failed to ferment mannitol and the strains which fermented both mannitol and sorbitol were

5 BIFIDOBACTERIUM SPECIES ISOLATED FROM HUMAN FECES 29 of the strains employeda,b,c,d lactose, melibiose, raffinose and salicin; all strains negative: rhamnose and inositol. weakly positive; }, very weakly positive or delayed very weakly positive; V, variable reaction. by Mitsuoka (7). B. angulatum, B. dentium and B. longum. exceptionally detected, our twelve isolates of B. pseudocatenulatum did so. One strain identified as B. longum obviously is a new biotype of this species, because it is able to ferment the sugar alcohols. Of twenty-eight strains of B. adolescentis biovar b, which fermented mannitol but not sorbitol, 21 strains showed high levels of DNA relatedness with the type strain of B. adolescentis, which is biovar a, whereas seven additional strains were identified as B. dentium. All isolates of B. adolescentis biovar b isolated from the feces of human adults in the present study were assigned to B. adolescentis. Scardovi reported that the main habitat of their B. dentium strains was dental caries (12). Twenty-six strains, fermenting sorbitol were attributed to B. adolescentis biovar

6 30 T. YAESHIMA et al c. This group of strains could be divided into three species : B. adolescentis, B. catenulatum and B. pseudocatenulatum. The strains of the phenotypic group B. adolescentis biovar d, which failed to ferment mannitol and sorbitol, were attributable either B. adolescentis, B. angulatum or B. longum. The B. longum strains showed a new sugar fermentation pattern of B. longum in the ability to ferment glucosides. No strain belonged to B. adolescentis biovar d except for Reuter's original strain ATCC Consequently, the strains of the phenotypic groups B. adolescentis isolated from the feces of human adults in the present study, could be virtually identified as B. adolescentis, B. catenulatum, B. pseudocatenulatum and B. longum on the basis of DNA/ DNA relatedness. In the present study, B. pseudocatenulatum strains also were isolated from the feces of human adults, and no strain was assigned to B. angulatum and B. dentium. Regarding this point, our results coincided with that of Biavati et al (1). Therefore, it may be assumed that B. angulatum and B. dentium are not indigenous members of the human intestinal microflora. Characteristics and presumptive differentiation of B. adolescentis and phenotypically similar Bifidobacterium species Phenotypic characteristics and G+C contents of the strains of B. adolescentis and phenotypically similar species are summarized in Table 2, and provisionaldiff erential characteristics of these species are shown in Table 3. It is difficult to distinguish B. adolescenti strains which fermented sorbitol and not mannitol, B. catenulatum and B. pseudocatenulatum (mannitol negative, sorbitol positive) on phenotypic characteristics. B. catenulatum strains apparently are different from the other four species by the inability to ferment glycogen and mannose. In our study, the strains B. catenulatum ATCC and 27677, which fermented glycogen, were identified as B. pseudocatenulatum on the basis of DNA/DNA relatedness. Scardovi et al (11, 13) reported that starch and melezitose fermentation properties were useful differential characteristics differentiating between B. catenulatum and B. pseudocatenulatum, and B. catenulatum, and B. adolescentis which fermented sorbitol and not mannitol. Obviously our results do not confirm those. Furthermore, B. catenulatum strains differentiate from B. adolescenti strains in their lower G + C contents. Scardovi described that sorbitol-fermenting strains of B. pseudocatenulatum could be distinguished from B. adolescenti strains by their inability to ferment melezitose. However, in the present study, melezitose fermentation is not a useful differentiating characteristic between these two species. Although these B. adolescentistrains are hardly distinguished from some strains of B. pseudocatenulatum on the basis of carbohydrate fermentation pattern, DNA base composition is a useful differentiating characteristic, because the G+C content of B. pseudocatenulatum is lower than that of B. adolescentis. The strains of B. adolescentis which fermented mannitol and not sorbitol cannot be also differentiated from B. dentium by their sugar fermentation patterns. Though B. angulatum strains show fermentation patterns similar to B. adolescentis (mannitol and sorbitol positive or mannitol and sorbitol negative), this species is distinguishable from other species by

7 BIFIDOBACTERIUM SPECIES ISOLATED FROM HUMAN FECES 31 Table 3. Key for the presumptive identification of B. adolescentis and phenotypically similar Bifidobacterium species its characteristic V or palisade arrangements of cells as Scardovi report ed (12). As a consequence, fermentation properties of glycogen, mannose, mannitol and sorbitol, G+C content and cell morphology are useful characteristics in pr ovisional differentiation of these species (Table 3). Although, in the present study, the strains of B. longum which exhibited the similar fermentation patterns to those of B. adolescentis biovar a or d were isolated, these strains can be distinguished from other five species by the inability to f erment sodium gluconate, starch and glycogen. B. adolescentis and B. pseudocatenulatum are major bifidobacterial species of the intestinal microflora of human adults, and therefore further study on simple phenotypic method differentiating between B. adolescentis and B. pseudocatenulatum is required. Acknowledgments. We are grateful to Noriko Sakurai (Nutritional Science Laboratory ustry Co., Ltd.) for technical assistance, MorinagaMilk Ind. REFERENCES (1) Biavati, B., P. Castagnoli, and L.D. Trovatelli Species of the genus Bifidobacterium in the feces of human adults. Microbiologica 9: (2) Johnson, J.L., C.F. Phelps, C.S. Cummins, J. London, and F. Gasser Taxonomy of the Lactobacillus acidophilus group. Int. J. Syst. Bacteriol. 30: (3) Lauer, E Bifidobacterium gallicum sp. nov. isolated from human feces. Int. J. Syst. Bacteriol. 40: (4) Marmur, J A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3:

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