and Ecology of Bifidobacteria

Transcription

1 Bifidobacteria Microflora Vol. 3(1), 11-28, 1984 Taxonomy and Ecology of Bifidobacteria Tomotari MITSUOKA Department of Biomedical Science, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, and The Institute of Physical and Chemical Research, Wako, Saitama 351 (Received 28 January, 1984) In recent years there have been some startling advances made in the taxonomy of bifidobacteria. The bifidobacteria are classified as a distinct genus Bifidobacterium, and a number of species and biovars are now recognized. As better techniques for the study of bacteria are devised, more definitive information on importance of bifidobacteria as part of the intestinal flora of humans and animals has been obtained. Some ecological relationships of this organism are reviewed in this paper. Key words: Bifidobacterium; taxonomy; ecology Since the first description of bifidobacteria in 1900 by Tissier, who introduced the name Bacillus b fdus communis or simply B. b /dus, much attention has been directed to the habitat, nutritional and immunological significance, biochemistry and taxonomy of these bacteria. In the past much confusion existed regarding the taxonomy and ecology of the bifidobacteria, partly because of the incomplete methods available for the cultivation of the organisms. During the past two decades, however, significant advances have been made in this field. This paper reviews briefly the information available on the taxonomy and ecology of the bifidobacteria. 1. Taxonomy 1) Classification at genus level: Historically the members of the genus Bifidobacterium were assigned to various genus : Bacillus, Bacteroides, Bacterium, Bifidobacterium, Tissieria, Nocardia, Lactobacillus, Actinomyces, Corynebacterium. For many years bifidobacteria were included in the genus Lactobacillus as Lactobacillus bifidus, but they are now classified in a separate genus Bifidobacterium as already suggested by Orla-Jensen (10), and the genus 11 is included in the family Actinomycetaceae Buchanan. 2) Classification at species level: Prior to 1957 many workers recognized only one species in the genus Bifidobacterium. In 1957, Dehnert first recognized the existence of multiple biotypes of bifidobacteria and proposed a schema for the differentiation of five groups of bifidobacteria based mainly on carbohydrate fermentation (1). Reuter (13) has proposed on the basis of carbohydrate fermentation and serological characteristics seven species of Bifidobacterium, in addition to the known B. bifidum, for strains isolated from human infants and adults. He presented a schema for their identification and recognized the following species and biovars in the genus Bifidobacterium: Bifidobacterium bifidum var. a and b, B. infantis, B. parvulorum var. a and b, B. breve var. a and b, B. lactentis, B. liberorum, B. adolescentis var. a, b, c and d, and B. longum var. a and b. The next major classification schema was presented by Mitsuoka (5). Four hundred eighty-three strains of bifidobacteria, isolated from human as well as a variety of animals, e.g., pig, chicken, calf, sheep, mouse, rat and guinea pig, were studied and classified on the

2 12 T. MITSUOKA basis of physiological and biochemical charac- from human by their carbohydrate fermenteristics, and the results were compared with tation patterns and ability to grow at 46.5 C, those obtained by Reuter. In this study and proposed the creation of the two new he clearly differentiated nonhuman strains species, B. thermophilum var. a, b, c and d, Table 1. Type strains and habitats of species of the genus Bifidobacterium

3 TAXONOMY AND ECOLOGY OF BIFIDOBACTERIA 13 and B. pseudolongum a, b, c and d, and of a new variant, B. longum subsp. animalis a and b. In the same year, Scardovi et al. (18) isolated B. ruminale (synonym of B. thermophilum) and B. globosum from the rumen of cattle. Furthermore, Scardovi and Trovatelli found B. asteroides, B. indicum and B. coryneforme in the intestine of the honey-bee, three new species quite different morphologically from the previously described bifidobacteria (15). In 1971, Scardovi and colleagues started to apply DNA homology procedure for assessing the validity at the genetic level of the bifidobacterial species previously described (19), and for recognizing new DNA homology groups among the bifidobacteria isolated from human feces, calf, rabbit, rat, pig and chicken feces, the rumen of cattle, sewage, the human vagina, dental caries, and the honey-bee intestine (Table 1). Some amendments of Reuter and Mitsuoka's species identification were suggested : B. liberorum and B. lactentis were proposed as synonyms of B. infantis, and B. longum subsp. animalis Mitsuoka was elevated to the species rank as B. animalis (16). Four additional species isolated from animal feces have been described and proposed quite recently : B. pseudocatenulatum, B. bourn, B. choerinum and B. cuniculi (17). Actinomyces parabifidus (Weiss and Rettger) Pine and Georg (12) strain ATCC (strain Timberlain) is phenotypically B. longum (4). Actinomyces eriksonii Georg et al. (2) strains ATCC and isolated from human pleural fluid and lung abscess are phenotypically B. adolescentis var. b (8), but are B. dentium at DNA-DNA hybridization (14). The relationship between the genetic groups and phenotypic groups in the genus Bifidobacterium are presented in Table 2. The DNA-DNA hybridization technique is a significant advance in determinative bacteriology and will certainly resolve much of the confusion previously encountered. However, on the basis of their fermentation patterns, Table 2. Relationship between DNA homology groups (genospecies) and phenotypic groups (phenospecies) for example, B. animalis. B. suis, B. magnum, and B. pullorum; B. adolescentis var. b and B. dentium; B. adolescentis var. c and B. catenulaturn; or B. adolescentis var. d and B. angulatum are not differentiated from each other. At the present time, fermentation patterns are still the principle guidelines used for the differentiation of bacterial species, because definitive identification of a bacterial strain in the routine laboratory is difficult to accomplish with procedures such as DNA-DNA hybridization. Therefore, it is especially important to develop additional phenotypic characteristics that correlate best with the genetic groups (genospecies). 3) Identification at genus level: Tentative identification of a bacterial strain as a member of the genus Bifidobacterium can be made on the basis of colony form and micro-

4 14 T. MITSUOKA Table 3. Key for differentiation of Bifidobacterium from related generaa scopy, although Lactobacillus, Propionibacterium, identification of phenotypic species of bifidobacteria Actinomyces and Eubacterium may closely is shown in Table 4. We used this resemble each other in their microscopical schema in the ecological studies. appearances. Reliable differentiation of bifidobacteria from these related genera, 2. Ecology however, can only be achieved by biochemical test as shown in Table 3. The 1) Distribution of bifidobacteria in intestine presence of the fermentation products, among of various animals: Bifidobacteria are which acetic acid generally predominates over lactic acid as the major final product, is considered one of the most important found in the intestine of various animals. Table 5 shows the composition of the fecal flora of adult animals of 12 different species. diagnostic tests for the genus Bifidobacterium. In almost all animal species, the most predominant The most direct, reliable and, most important, fecal bacteria were anaerobes, fruitful assignment of a bacterial including bacteroidaceae, bifidobacteria, strain to the genus Bifidobacterium is the eubacteria, anaerobic lactobacilli, peptococcaceae demonstration in cellular extracts of the and spirillaceae. In the feces presence of fructose-6-phosphate phosphoketolase (F6PPK), the key enzyme of bifidobacterial hexose metabolism. Almost all biochemical tests such as the demonstration of catalase, nitrate reduction, formation of indole, liquefaction of gelatin, gas formation from glucose, and response to rhamnose, sorbose, glycerol, erythritol, adonitol, and dulcitol are negative for the genus Bifidobacterium. 4) Identification at species level: Practical identification of Bifidobacterium species can mainly be achieved with the use of the fermentation tests and morphological tests. The differential schema for a rapid routine of monkeys and guinea pigs the bifidobacteria outnumbered lactobacilli. In the feces of chickens, pigs, dogs, mice, rats and hamsters, bifidobacteria were present in almost all cases, but in smaller numbers than lactobacilli. In the feces of rabbits and horses, they were occasionally demonstrated, but not consistently. In the feces of cats and minks they were never demonstrated. Thus, variations in the Bifidobacterium flora of animals were considered to be associated with diet, age and species of the host animals. 2) Distribution of bifidobacteria in feces of healthy humans: Bifidobacteria constitute

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6 16 T. MITSUOKA

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8 18 T. MITSUOKA Fig. 1. Changes with age of fecal flora of an infant fed maternal milk together with cow's milk. : Enterobacteriaceae, : Bifidobacteria, : Streptococci, œ : Bacteroidaceae, A : Lactobacilli, : C. perfringens. Table 7. Comparison of fecal flora between bottle-fed infants and breast-fed infants a numerically major part of the fecal flora of healthy humans. Table 6 presents the results on the composition of the fecal flora of humans of various age groups (6). Bifidobacteria appeared on the 2nd to 5th day of life and continued to be one of the most numerous bacteria, amounting to about 1010/g of wet feces (Fig. 1). Since the work of Tissier (21), it has been believed that bifidobacteria are found exclusively in feces of breast-fed infants, whereas in bottle-fed infants Lactobacillus acidophilus is the most commonly found organism. According to our recent study (22), however, the differences in the occurrences and numbers of bifidobacteria between breast-fed and bottlefed infants were not significant. Bifidobacteria were present in all of 30 breast-fed and in 29 bottle-fed infants with mean counts (when present) of /g and /g, respectively (Table 7). The principal difference between breast-fed and bottle-fed infants was that in bottle-fed infants the numbers of enterobacteriaceae, streptococci, and anaerobes other than bifidobacteria were significantly greater than in breast-fed infants. 3) Species and biovar distribution in

9 Table 8. Frequency of occurrence of species and biotypes of bifidobacteria in feces of various age groups of humansa a Figures indicate the numbers of specimens. Table 9. Changes with age of fecal bifidobacterial species and biovars of an infant fed maternal milk together with cow's milk humans: Differences in the predominant Bifidobacterium species or biovars among humans although in the senile men the occurrence of tines of children, adults and senile men, of different age groups were observed. The B. adolescentis biovar b was significantly higher results are shown in Table 8 (7). The most than in the other age groups. These species common bifidobacteria isolated from infants and biovars were only occasionally isolated belonged to the species B. bifidum biovar b, from infants. Table 9 shows changes with B. infantis ss. infantis, B. breve ss. breve, B. breve age of bifidobacterial species and biovars in ss. parvulorum, and B. longum biovar b, occurring almost exclusively in infants. In cow's milk. an infant fed maternal milk together with contrast, B. adolescentis, and B. longum biovar The difference in Bifidobacterium species a were found in high numbers in the intes- composition of intestinal flora between bottle-

10 fed and breast-fed infants was not significant, but the frequency of occurrence of B. bifidum in bottle-fed infants were less than that in Table 10. Comparison of Bifidobacterium species between bottle-fed infants and breast-fed infants a Mean }SD of log bacterial counts b Frequency of occurrence (%). Fig. 2. Changes with age of fecal flora of a low birth weight bottle-fed infant (B).. breast-fed infants (Table 10). Such a high number of bifidobacteria found recently in bottle-fed infants may reflect the improvement of formula component, i.e., the enhancement of protein digestibility, decrease in protein and salt contents, and increase in lactose content. 4) Appearance of bifidobacteria in low birth weight bottle-fed infants: The appearance of bifidobacteria in low birth weight bottle-fed infants was more delayed than that in normal bottle-fed infants as shown in Fig. 2, and the species composition of bifidobacteria in low birth bottle-fed infants was simpler than that in normal bottle-fed infants, consisting of only two species (22). Bifidobacterium breve was detected at first, followed by the appearance of either B. bifidum or B. adolescentis (Table 11). The delayed appearance of bifidobacteria might reflect the immaturity of the digestive organs of low birth weight infants. 5) Changes of fecal flora of twins: The fecal flora of twins showed a similar pattern (Figs. 3, 4). The sepcies and biovars of bifidobacteria found in stools of twins were also very similar (Tables 12, 13). 6) Species and biovars distribution in animals: In view of the relationship between Bifidobacterium species or biotypes and ecological distribution in humans and animals, it must be stressed that in general, each animal species harbored definite species or Table 11. Changes of the species and biovars of bifidobacteria with age of a low birth weight bottle-fed infant (B)

11 Fig. 3. Changes with age of fecal flora of a low birth weight bottle-fed infant (twin Ai). : Enterobacteriaceae, : Bifidobacteria, : Streptococci, œ: Bacteroidaceae, A : Lactobacilli, : C. perfringens. Fig. 4. Changes with age of fecal flora of a low birth weight bottle-fed infant (twin A2). biovars of bifidobacteria (Table 14). The great majority of the animal strains, with the exception of the strains isolated from monkeys and dogs could be phenotypically identified as B. thermophilum and B. pseudolongum while the strains from rodents (mice, rats and guinea pigs) and ruminants (cattle and sheep) belonged to B. pseudolongum and/or B. animalis. On the other hand, the bifidobacteria from dogs were identified as B. adolescentis, B. longum and B. pseudolongum, and the strains from monkeys were identified as B. adolescentis. 7) Clinic-dependent bifidobacterial species and biovars in infant feces: Variations were observed in the occurrences of Bifidobacterium species or biovars in infants in different clinics (7). In general, all infants in the same clinic harbored similar predominating Bifidobacterium strains. B. longum biovar b

12 Table 12. Changes of the species and biovars of bifidobacteria with age of a twin (A1) Table 13. Changes of the species and biovars of bifidobacteria with age of a low birth weight bottle-fed infant (twin A2) was a relatively common species isolated from the infants of Clinic 1, but was only occasionally isolated from the infants in Clinics 2 and 3. B. infantis ss. infantis biovar a was the most common bifidobacteria isolated from infants in Clinic 3 but was not found in other clinics (Table 15). This finding suggested that one Bifidobacterium strain may be transmitted from infant to infant through contact with nurses. 8) The number of species or biovars of bifidobacteria harbored in single infants, child, adult and senile male adult individuals: One or two of the most commonly found species or biovars were mostly demonstrated in infants. On the other hand, more than two species or biovars were demonstrated in children, adults and senile men at given time (Table 16). 9) Succession of species and biovars found in infants: In 1900, Tissier discovered B. bifidum in the feces of infants. In 1950, Norris et al. isolated Lactobacillus parabifidus strain Timberlain (ATCC 17930) from infant feces (9). This strain is considered as synonymous with B. infantis ss. liberosum Gyorgy (3) originally designated the organism L. bifidus var. penn (subsequently changed to L. bifidus var. pennsylvanicus) isolated from infants. This strain belongs to B. bifidum. Dehnert (1) reported his group IV (B. infantis ss. lactentis) as the predominant bifidobacterial group in the stools of breastfed infants. Reuter (13) and Mitsuoka (5) reported that B. infantis ss. infantis predom-

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16 Table 16. Numbers of Bifidobacterium species or biovars found in feces of respective individuals from various age groups Table 17. Succession of species and biovars of bifidobacteria occurring in feces of infants a Br: Breast-fed infant, b Bo: bottle-fed infant, c number of specimens, d frequency of isolation, e number of specimens harbored. inated in the feces of breast-fed infants. Seeliger and Werner (20) reported that Dehnert's group III (B. infantis) was predominant in Bonn (West Germany), although group IV (B. infantis ss. lactentis) was also present. Petuely and Lindner (11) noted the predominance of Dehnert's group IV (B. infantis ss. lactentis) in the stools of breastfed infants. Werner (24) also reported the prevalence of Dehnert's group IV in breast-fed infants and stated that group IV had been found only in the stools of breastfed infants or of infants fed maternal milk together with cow's milk. It has recently

17 been found that all of the subspecies of B. infantis are very difficult to isolate from infant feces, and that B. breve ss. breve predominates in the feces of both breast-fed and bottle-fed infants (Table 17). The factors affecting such succession of the predominant bifidobacterial species and biovars is still unknown. 10) Abnormal intestinal flora: Disturbances of intestinal flora have been observed in connection with gastric juice acidity, peristalsis disorders, cancer or surgical operations of stomach or small intestine, liver or kidney disease, pernicious anaemia, blind loop syndrome, radiation effects, impairment of gastrointestinal function in old age, administration of antibiotics, etc. Abnormal intestinal flora are usually characterized by an increase of aerobes, mostly enterobacteriaceae and streptococci, and by the reduction or disappearance of bifidobacteria. (1) Dehnert, J Untersuchung uber die grampositive Stuhlflora des Brustmilchkindes. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. I. Abt. Orig. 169: (2) Georg, L. K., G. W. Robertstad, S. A. Brinkman, and M. D. Hicklin A new pathogenic anaerobic Actinomyces species. J. Infect. Dis. 115: (3) Gyorgy, P A hitherto unrecognized biochemical difference between human milk and cow's milk. Pediatrics 11: (4) Kandler, O., and E. Lauer Neuere Vorstellungen zur Taxonomie der Bifidobakterien. Zentralbl. Bakteriol. Mikrobiol. Hyg. I. Abt. Orig. A288: (5) Mitsuoka, T Vergleichende Untersuchungen uber die Bifidobakterien aus dem Verdauungstrakt von menschen and Tieren. Zentralbl. Bakteriol. Parsitenk. Infektionskr. Hyg. I. Abt. Orig. 210: (6) Mitsuoka, T., and K. Hayakawa Die Faekalflora bei Menschen. I. Mitteilung: Die Zusammensetzung der Faekalflora der verschiedenen Altersgruppen. Zentralbl. Bakteriol. Hyg. I. Abt. Orig. A223: (7) Mitsuoka, T., K. Hayakawa, and N. Kimura Die Faekalflora bei Menschen. II. Mitteilung: Die Zusammensetzung der Bifidobakterienflora der verschiedenen Altersgruppen. Zentralbl. Bakteriol. Hyg. I. Abt. Orig. A226: (8) Mitsuoka, T., Y. Morishita, A. Terada, and K. Watanabe Actinomyces eriksonii Georg, Robertstad, Brinkman and Hicklin 1965 identisch mit Bifidobacterium adolescentis Reuter Zentralbl. Bakt. Hyg. I. Abt. A226: (9) Norris, R.F., T. Flanders, R.M. Tomarelli, and P. Gyorgy The isolation and cultivation of Lactobacillus bifidus. A comparison of branched and unbranched strains. J. Bacteriol. 60: (10) Orla-Jensen, S La classification des bacteries lactiques. Lait 4: (11) Petuely, F., and G. Linder Kritische Untersuchung uber die Darmflora. III. Mitteilung: Bedeutung quantitativer Zuchtungsmethoden. Die Darmflora des Brustkindes. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. I. Abt. Orig. 195: (12) Pine, L., and L. Georg The classification and phylogenetic relationships of the Actinomycetales. Int. Bull. Bact. Nomenclature Taxonomy 15: (13) Reuter, G Vergleichende Untersuchungen uber die Bifidus-Flora im Sauglingsund Erwachsenenstuhl. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. I. Abt. Orig. 169: (14) Scardovi, V The genus Bifidobacterium, p In M.P. Starr, H. Stolp, H.G. Truper, A. Balows and H.G. Schlegel (eds.), The Prokaryotes, Springer-Verlag, Berlin, Heidelberg, New York. (15) Scardovi, V., and L.D. Trovatelli New species of bifid bacteria from Apis mellifica L. and Apis indica F. A contribution to the taxonomy and biochemistry of the genus Bifidobacterium. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. I. Abt. Orig. 123: (16) Scardovi, V., and L.D. Trovatelli Bifidobacterium animalis (Mitsuoka) comb. nov. and the "minimum" and "subtile" groups of new bifidobacteria found in sewage. Int. J. Syst. Bacteriol. 24: (17) Scardovi, V., L.D. Trovatelli, B. Biavati, and G. Zani Bifidobacterium cuniculi, Bifidobacterium choerinum, Bifidobacterium boum, and Bifidobacterium pseudocatenulatum: Four new species and their deoxyribonucleic acid homology relationships. Int. J. Syst. Bacteriol. 29: (18) Scardovi, V., L.D. Trovatelli, F. Crociani, and B. Sgorbati Bifid bacteria in bovine rumen. Arch. Mikrobiol. 68: (19) Scardovi, V., L.D. Trovatelli, G. Zani, F. Crociani, and D. Matteuzzi Deoxyribonucleic acid homology relationships among species of the genus Bifidobacterium. Int. J. Syst. Bacteriol. 21:

18 (20) Seeliger, H.P.R., and H. Werner Recherches qualitatives et quantitatives sur la flora intestinale de l'homme. Ann. Inst. Pasteur 105: (21) Tissier, H Recherches sur la flora intestinal des nourrissons. Theses Paris, pp (22) Yuhara, T., S. Isojima, F. Tsuchiya, and T. Mitsuoka On the intestinal flora of bottlefed infant. Bifidobacteria Microflora 2: (23) Weiss, J.E., and L.F. Rettger, Taxonomic relationship of Lactobacillus bifidus (Bacillus bifidus (Tissier)) and Bacteroides bifidus (Eggerth). J. Bacteriol. 35: (24) Werner, H The gram positive nonsporing anaerobic bacteria of the human intestine with particular reference to the Corynebacteria and Bifidobacteria. J. Appl. Bacteriol. 29: