In a previous investigation of bifidobacteria isolated from human dental caries (V. Scardovi and F. Crociani, Int. J. Syst. Bacteriol. 24:6-20, 1974), 40 strains were assigned to the new species Bifidobacterium dentiurn. In this study we examined 70 new strains of bifidobacteria isolated from dental caries. The morphological characteristics, biochemical reactions, fermentation patterns, end products from glucose metabolism, protein electrophoretic patterns, levels of DNA hybridization, and DNA G+C contents of these organisms revealed that they belong to three different taxa. One of these taxa was identified as B. dentium. The other two are described as the following new Bifidobacterium species in this paper: Bijidobacterium inopinaturn (type strain, DSM 10107) and Bijidobacterium denticolens (type strain, DSM 10105). The two new species differ from other Bijidobacterium species in their morphological characteristics (especially B. inopinatum, with its very small coccoid cells), in their carbohydrate fermentation patterns (most strains ferment dextran, and B. inopinatum does not ferment galactose), and in their DNA base compositions (especially B. inopinatum).

Genetic relatedness among 179 strains representing 13 named species and several unnamed taxa of the genus Bifidobacterium from three main habitats, i.e., (i) feces of man, (ii) feces of various other animals, and (iii) the bovine rumen, was assessed by means of deoxyribonucleic acid (DNA)-DNA hybridization by using a filter-paper technique in competition experiments. Assignment of these strains to the genus Bifidobacterium was based on the production of lactic and acetic acids as chief products from glucose, lack of gas production, fructose-6-phosphate phosphoketolase activity, and on morphology. About 180 DNA competitors were tested with 23 reference systems. Several genetically distinct groups were recognized. Little or no DNA homology was demonstrated between some of the groups, suggesting large evolutionary divergence in this genus. B. infantis, B. liberorum, and B. lactentis form one of these groups; the ecological significance of this relatedness was discussed. The DNA of the following pairs are homologous: (i) B. breve and B. parvulorum, (ii) B. thermophilum and B. ruminale, and (iii) B. pseudolongum and B. globosum. Within a number of strains assigned to B. adolescentis, many of which were isolated from waste waters, a large genetic heterogeneity was demonstrated: in addition t o B. adolescentis, at least three unrelated groups were recognized and are provisionally referred to as "dentium, " "catenulatum, " and "angulatum. " Since these groups are not related genetically to any species of the genus and are phenotypically distinct, they may represent new species. The validity of the species B. bifidum, B. longum, and B. suis was confirmed at the genetic level.

Among the several thousand bifidobacteria of our collection, 244 strains from the feces of rabbits and suckling pigs, the rumen of cattle, the feces of breast-and bottle-fed infants, the feces of calves, and from sewage are recognized by means of deoxyribonucleic acid (DNA)-DNA hybridization (competition filter method) as belonging to four new, distinct DNA homology groups. Twenty reference DNA preparations from the type strains of the currently known species or from reference strains of the homology groups of Bifidobacterium were used for comparison. The phenotypic traits which distinguish these four groups from previously described species of the genus Bifidobacterium include gross morphology, fermentation characteristics, guanine plus cytosine content of the DNA, the interpeptide bridge of the cell-wall peptidoglycan, and the transaldolase and 6-phosphogluconate dehydrogenase isozyme pattern (by starch gel electrophoresis). The four groups are named and described as new species of the genus Bifido bacterium: B. cuniculi, B. choerinum, B. boum, and B. pseudocatenulatum. The type strains of these species are RA93 (=ATCC 27916), SU806 (=ATCC 27686), RU917 (=ATCC 27917), and B1279 (=ATCC 27919), respectively.Several species and provisional groups of bifidobacteria isolated from the feces of adult rabbits have been named and described (17,20,24). One of the provisional groups was referred to by Trovatelli et al. (24) as deoxyribonucleic acid (DNA) homology group 11. In the present study, this group of bifidobacteria, here referred to as the cuniculi DNA homology group, is described in detail and is recognized as a new species.Bifidobacteria from the feces of piglets were isolated and studied by Zani et al. (26). Most of the strains were identified on the basis of DNA homology relationships as members of Bifidobacterium suis, B. thermophilum, B. globosum, or B. pseudolongum. These authors also reported that 13 of 95 strains examined were distinct from previously described species. This group was designated homology group coirinense. A few fermentation characteristics and the DNA homology relationships to other organisms from this habitat were reported (26), but the taxonomic status of this group was not established. In the present work, this group, referred to here as the choerinum DNA homology group, is also described and recognized as a new species.The rumen is an important habitat of bifidobacteria, especially when the animals are fed carbohydrate-rich diets (25). Indeed, isolates from sheep rumen were used for our fmst studies on the fructose 6-phosphate shunt mechanism of glucose degradation by bifidobacteria (15). Species reported from this habitat include B. globosum and B. ruminale. B. ruminale was found to be identical with B. thermophilum, a species isolated by Mitsuoka (6) from the feces of swine. Other bifidobacteria from the rumen (strains RU276, RU348, and RU354) at first appeared to be variants of B. ruminale (ref. 18, p. 290 Table 10, p. 292). In recent investigations on the effect of diet on the ...

A total of 1461 bacterial isolates, representing 24 different species of the genus Bifidobacterium, were examined for the presence of plasmid DNA. Approximately 20% of the isolates contained detectable plasmids, but only four species were presented: B. longum, the predominant bifid species in the human intestine; B. globosum, the most common in animals; and B. asteroides and B. indicum, species found exclusively in the intestines of western and asiatic honey bees, respectively. Multiple plasmids were common among isolates of B. longum and B. asteroides, while all plasmid-bearing isolates of B. globosum and 60% of B. indicum isolates contained only one plasmid each. Certain multiple plasma profiles were predominant among the B. longum and B. asteroides isolates.

The polyacrylamide gel electrophoretic patterns of soluble cellular proteins from 1,094 strains of bifidobacteria were compared with available deoxyribonucleic acid (DNA)-DNA homology data and with the phenotypic and biochemical reactions of these strains. There was excellent correlation between the 25 distinct protein patterns and 24 DNA-DNA homology groups in the genus. Differentiation among species on the basis of common phenotypic properties often was unreliable. Our results demonstrate that the species previously known as "Bifidobacterium eriksonii" is a synonym of Bifidobacterirrm dentium Scardovi and Crociani; that "Actinomyces parabijdus" is a synonym of Among the 20 species of the genus Bifidobacterium cited on the Approved Lists of Bacterial Names ( 3 3 ) , several genetically distinct species from humans and other animals cannot be differentiated reliably by commonly used enzymatic or fermentation reactions (5) or by zymograms of transaldolases and 6-phosphogluconate dehydrogenases (23). Several authors have reported the use of polyacrylamide gel electrophoresis (PAGE) of soluble cellular proteins to distinguish species or serovars (serotypes) of bacteria (1, 4, 8, 9, 11, 32, 35) or to determine the similarity of unidentified isolates (15). The present study was designed to determine the reliability and sensitivity of a PAGE procedure (15) to distinguish genetically related and unrelated species of bifidobacteria. For this purpose we compared the electrophoretograms of BiJidobacterium type strains and many other strains that were previously identified according to their deoxyribonucleic acid (DNA)-DNA homology characteristics or phenotypic characteristics or both. MATERIALS AND METHODSBacterial strains. The strains examined are listed in Table 1.Methods. The biochemical properties of the strains were determined by methods previously described by Holdeman et al. ( 5 ) . The DNA-DNA homology values used were from previously published data (10. 23, 24, 26, 27, 29-31, 37, 38) and were obtained by the methods described by Scardovi et al. (24. 29).For electrophoretic analyses, cells were inoculated into 5 ml of supplemented brain heart infusion broth ( 5 ) containing 0.1% calcium carbonate and 0.025% (final concentration) Tween 80 and then incubated at 37°C for 24 h. Growth was harve3ted by centrifugation at 8,000 X g for 10 min. Electrophoretic analysis of cellular soluble proteins was performed by using the procedures of Moore et al. (15). The similarity of patterns on different gels was determined by placing cut photographs adjacent to each other. Strains with similar protein patterns were reanalyzed on single gels for direct visual comparisons. RESULTS AND DISCUSSIONThe DNA-DNA homology relationships among species of the genus Bifdobacterium have been studied extensively. Many of the species show close genetic relationships, and several species have similar phenotypic characteristics. Therefore, the species in this genus provide an ideal test of the sensitivity and reliability of the application of PAG...