Ecology of Bifidobacterium in the Human Intestinal Flora

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1 Review Bifidobacteria Microflora Vol. 6(2), 33-41, 1987 Ecology of Bifidobacterium in the Human Intestinal Flora Masahiko MUTAI and Ryuichiro TANAKA* Yakult Central Institute, 1796 Yaho, Kunitachi, Tokyo, 186 Japan (Received 17 June, 1987) In this review, we present the evidence for the establishment and the succession of Bifidobacterium flora in the human intestine and suggest that the number of the Bifidobacterium is the potential marker of the stability of human intestinal flora. The significance of Bifidobacterium with regard to colonization resistance is dicussed. Key words: Bifidobacterium; ecology; intestinal flora; colonization resistance; oral administration In 1907, Metchinikoff (15), advanced the theory that intestinal flora exerted important influences on health and longevity. Considerable evidence has since highlighted the importance of intestinal flora to the host. Research done with germfree animals and the introduction of improved anaerobic culture techniques have been particularly useful in clarifying the significance of intestinal flora (5, 8, 17). In the past decade, there has been renewed interest in the ingestion of cultured dairy products containing viable Bifidobacterium to maintain a proper balance of normal intestinal flora and to enhance the beneficial relationship between host and intestinal flora. We have evidence of the feeding effects of Bifidobacterium on healthy humans with or without "Bifidus growth factor TOS" (12, 23, 25), and the therapeutic effects of administration of Bifidobacterium preparation or Bifidus yogurt on human diarrhea (11, 27), constipation (24), and the management of hepatic encephalopathy with hyperammonemia (13). In this paper we review the evidence for the development and succession of Bifidobacterium flora in the human gastrointestinal tract and stress the significance of Bifidobacterium with regard to colonization resistance in health and disease. Establishment of Bifidobacterium during the Early Days in Life From the moment of birth, newborn babies are normally exposed to various microbes from the maternal vagina and other environmental sources. Table 1 shows the composition of oral microbes isolated from newborn babies within 10 min after delivery. In the case of vaginal delivery, many kinds of anaerobes and aerobes were isolated from all subjects studied and Enterococcus, Propionibacterium, Bacteroides, Lactobacillus and Bifidobacterium were the more frequently found species. In contrast, only two kinds of microbes, Enterococcus and Propionibacterium were detected from newborn babies delivered by caesarean section. Evidently, these are initially acquired microbes from the vagina or other environmental sources and they begin to colonize as the candidates of the indigenous intestinal flora through the interactions of host-parasite relationships. It is well known that Lactobacilli are the predominant vaginal flora and are considered to play a protective role in the female genital 33

2 34 M. MUTAI and R. TANAKA Table 1. Composition of oral microflora of newborns at delivery. tract, preventing colonization by virulent were born at a modern university hospital in species (2, 14). However, recent studies on the normal vaginal flora showed that anaerobes Tokyo. All were healthy full-term infants, vaginally delivered, except one by caesarean outnumber aerobes and the most section, after normal pregnancies. Three commonly found isolates are anaerobic cocci infants were breast-fed while seven were and Bacteroidaceae (19, 26). formula-fed. Initially, Enterobacteriaceae As for Bifidobacterium in the vaginal flora, and Enterococcus were predominant, but it is less commonly found in isolates and the Enterobacteriaceae rapidly increased in frequency, as reported by previous workers species reported so far are Bifidobacterium longum, B. breve and B. adolescentis (3, 30) ; (20, 21). these were the same species as our identified Bifidobacterium appeared on the first day of strains isolated from the present newborn babies. The development of the intestinal flora of newborns during the first 5 days of life is shown in Fig. 1. All 10 infants in the study life at levels of per gram of feces in the three babies; then reached levels of per gram in most subjects within the first 5 days of life; however, the number of Bifidobacterium differed by 106 among individuals. In addition, a total of 99 strains consisting of 57 B. breve and 42 B. longum were isolated and identified in this early stage of life. Although the origin of Bifidobacterium is still obscure, the demonstration of B. breve and B. longum may account for the importance of transmission of vaginal Bifidobacterium during delivery. Evidently, the demonstration of Bifidobacterium in newborns delivered with caesarean section may also suggest the importance of environmental factors after delivery. Fig. 1. Establishment of intestinal microflora of newborns during the early days in life. Total bacteria ( œ), Bacteroidaceae ( ), Bifidobacterium ( ), Enterobacteriaceae ( ~), Enterococcus ( ), Staphylococcus ( ). Normal Intestinal Flora and Distribution of Bifidobacterium Table 2 shows the composition of the fecal microflora of healthy humans consisting of 13 breast-fed and 13 formula-fed infants, 11

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4 36 M. MUTAI and R. TANAKA weaned infants, 19 adults, and 22 Okinawan centenarians. The unique flora of breast-fed infants is well known as "Bifidus flora," predominating only Bifidobacterium. The intestinal flora of formula-fed infants, on the other hand, is more complex and Bacteroidaceae, Peptococcaceae, Enterobacteriaceae and Enterococcus are detected in a proportion similar to Bifidobacterium. The microbial profiles of weaned infants and adults are essentially the same where the most predominant bacteria is only strict anaerobes including Bacteroidaceae, Bifidobacterium, Peptococcaceae, and Eubacterium (7, 18). The pattern of the fecal flora of centenarians in Okinawa the world's most reliable region on the geography of longevity (22) was similar to the healthy senile men; the number of Bifidobacterium significantly decreased while Enterobacteriaceae and Clostridium perfringens showed apparent increase in number (17). Tables 3 and 4 show the composition and distribution of Bifidobacterium in the feces of healthy humans consisting of 23 neonates, 20 infants, 26 adults, and 14 centenarians. A total of 494 strains were identified according to the method of Mitsuoka (16) and the Anaerobic Laboratory Manual (9). The most common Bifidobacterium isolated from both breast-fed and formula-fed infants belonged to the species of B. breve. On the other hand, B. adolescentis and B. longum were the most frequently found species in the intestine of infants and adults. Furthermore, B. adolescentis and B. bifidum were the most commonly found species in the centenarians. The Bifidobacterium flora from different age groups was in general agreement with the findings of other workers (17). In summary, the species of Bifidobacterium can be divided into the infant or adult type due to their capacity for prolonged survival and proliferation in the intestine. However, further studies are needed to determine the influences of the alterations of dietary factors and the development of the infant gut on the succession of Bifidobacterium flora, including the ability to adhere to host tissues. Bifidobacterium and Stability of Intestinal Flora It is well known that the indigenous intestinal flora is stable and prevents the colonization of a number of pathogenic bacteria. The protection against colonization is involved in the natural resistance of humans to intestinal infections. van der Waaij et al (28) propose the concept of colonization resistance and emphasize an important role for anaerobes in the maintenance of colonization resistance in the intestine; however, other studies have suggested that facultative gram negative rods are also important in Table 3. Composition of Bifidobacterium flora in the different age groups.

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6 38 M. MUTAI and R. TANAKA role of Bifidobacterium with regard to colonization resistance. Impact of Administration of Bifidobacterium on the Recovery of Normal Intestinal Flora Fig. 2. Colonization resistance of human intestinal microflora: the ratio of nonanaerobes to anaerobes. this regard (6). In general, the ratio of nonanaerobes to anaerobes seems to be an indicator of the potential stability of intestinal flora. Figure 2 shows the comparative values of the human intestinal flora in the different age groups. The number of Enterobacteriaceae and total bacteria were used as the representatives of nonanaerobes and anaerobes, respectively. In addition, the number of Bifidobacterium was also used instead of total bacteria as the representatives of the most useful bacteria in the human intestine. In the formula-fed infants and centenarians, the ratio of nonanaerobes to anaerobes was much larger than that of breastfed infants, weaned infants, and adults by a factor of 10 to 100. Furthermore, when using the number of Bifidobacterium instead of total bacteria, these differences were more striking, suggesting that Bifidobacterium play an important role in the maintenance of colonization resistance. It is widely recognized that formula-fed infants and senile men are more susceptible to various infections than those of other age, groups. Therefore, further studies are needed to define whether any meaningful associations exist between the susceptibility to the colonization of pathogenic bacteria and the A number of studies demonstrated that the suppression of the normal flora leads to undesirable effects. It is well known, for example, that antibiotic therapy increases the risk of susceptibility to infections (4), bleeding problems (10), and the selection of antibiotic-resistant strains (29). With regard to intestinal infections, the best known is pseudomembranous enterocolitis (1). Recently, we investigated the effects of administration of Bifidobacterium preparations on infantile intractable diarrhea (11). Fifteen patients (11 boys and 4 girls), ranging in age from 1 month to 15 years (mean 2.5 years), were receiving antibiotic therapy for the treatment of such diseases as septicemia and respiratory tract infections. During treatment, watery diarrhea appeared and lasted for 1 to 10 weeks (mean 25 days) with deterioration of the general condition. Conservative therapy such as diet control, infusion and drug therapy could not cure the diarrhea. The antibiotics used included cephems, penicillins, and aminoglycosides. In most cases, abnormal intestinal flora was observed; Candida or Enterococcus often predominated with a marked decrease of anaerobes, especially Bifidobacterium. During the disease, we could not detect any pathogens or toxins responsible for diarrhea such as Clostridium dificile. In all patients, the stool frequency and appearance were dramatically improved within 3 to 7 days after oral administration of Bifidobacterium preparations (Fig. 3). The intestinal flora of all subjects studied also became normal with predominance of resident Bifidobacterium or administered B. breve and the ratio of nonanaerobes to anaerobes remarkably improved to the normal level (Fig. 4). This meant that the metabolic activities of intestinal flora re-

7 ECOLOGY OF BIFIDOBACTERIUM 39 Fig. 3. Effects of the administration of Bifidobacterium breve preparation intractable diarrhea. (Hotta, M. et al.ti) 1987) (BBG -01) on infantile Fig. 4. Effects of the administration of Bifidobacterium breve preparation (BBG -01) on the recovery of colonization resistance in the children with diarrhea.

8 40 M. MUTAI and R. TANAKA turned to physiological conditions and the balance between the host and its intestinal physiology also returned to normal. In summary, as described in more detail elsewhere (11), malabsorption of water and electrolytes, probably induced by antibiotic therapy, may be corrected through normal floral metabolic activities such as organic acid fermentation, bile acid metabolism, and utilization of mucin, resulting in a cure of diarrhea. Concluding Remarks Much has been written on the significance of human intestinal flora in health and disease. It is now considered that Bifidobacterium in the human intestine plays an important role in the maintenance of normal intestinal flora. Our findings also suggest that the number of Bifidobacterium is the potential marker of the stability of human intestinal flora. Further studies are needed to determine the precise role of the intestinal Bifidobacterium in the colonization resistance, which can serve to develop feasible and practical measures to enhance the host's defense. References (1) Bartlett, J.G Antibiotic associated pseudomenbranous colitis. Rev. Infect. Dis. 1: (2) Bartlett, J.G., and B.F. Polk Bacterial flora of the vagina: quantitative study. Rev. Infec. Dis. 6: (3) Crociani, F., D. Matteuzzi, and H. Ghazvinizadeh Species of the genus Bifidobacterium found in human vagina. Zbl. Bakt. Hyg., I. Abt. Orig. A223: (4) Dineen, P The effect of alterations in intestinal flora on host resistance to systemic bacterial infection. J. Infec. Dis. 109: (5) Finegold, S.M., V.L. Sutter, and G.E. Mathisen Normal Indigenous Intestinal Flora, p In D.J. Hentges (ed.), Human intestinal microflora in health and disease, Academic Press, New York. (6) Freter, R., and G.D. Abrams Function of various intestinal bacteria in converting germfree mice to the normal state. Infec. Immun. 6: (7) Gorbach, S.L., L. Nahas, P.I. Lerner, and L. Weinstein Studies of intestinal microflora. I. Effects of diet, age, and periodic sampling on numbers of fecal microorganisms in man. Gastroenterology 53: (8) Hentges, D.J Role of the Intestinal Microflora in Host Defense against Infection, p In D.J. Hentges (ed.), Human intestinal microflora in health and disease, Academic Press, New York. (9) Holdeman, L.V., and W.E.C. Moore. (ed.) Anaerobe laboratory manual, 2nd ed. Virginia Polytechnic Institute and State University, Balcksburg. (10) Hooper, C.A., B.B. Haney, and H.H. Stone Gastrointestinal bleeding due to vitamin K deficiency in patients on parenteral cefamandole. Lancet 1: (11) Hotta, M., Y. Sato, S. Iwata, N. Yamashita, K. Sunakawa, T. Oikawa, R. Tanaka, K. Watanabe, H. Takayama, M. Yajima, S. Sekiguchi, S. Arai, T. Sakurai, and M. Mutai Clinical effects of Bifidobacterium preparations on pediatric intractable diarrhea. Keio J. Med. 36: (in press). (12) Kan, T., S. Suzuki, M. Harada, T. Terashima, M. Mutai, S. Kataoka, and T. Futaki Effects of administration of Bifidobacterium bifidum 4002 on the intestinal flora of formula-fed infants. Jpn. J. Pediatr. 30: (13) Koizumi, T., N. Mitsuya, T. Mukuta, S. Fujita, H. Ishizu, and H. Yoshioka Effects of administration of Bifidobacterium preparation (BBG- 02) on hepatic coma. Sogo Rinsho 29: (in Japanese). (14) Larsen, B., and R.P. Galask Vaginal microflora: composition and influences on host physiology. Ann. Intern. Med. 96: (15) Metchinikoff, E The Prolongation of Life, Heinemann, London. (16) Mitsuoka, T., and C. Kaneuchi Ecology of the bifidobacteria. Am. J. Clin. Nutr. 30: (17) Mitsuoka, T Recent trends in research on intestinal flora. Bifidobacteria Microflora 1: (18) Moore, W.E.C., and L.V. Holdeman Human fecal flora: the normal flora of 20 Japanese- Hawaiians. Appl. Microbiol. 27: (19) Ohm, M.J., and R.P. Galask Bacterial flora of the cervix from 100 prehysterectomy patients. Am. J. Obstet. Gynecol. 122: (20) Rotimi, V.O., and B.I. Duerden The development of the bacterial flora in normal neonates. J. Med. Microbiol. 14: (21) Stark, P.L., and A. Lee The microbial ecology of the large bowel of breast-fed and formula-fed infants during the first year of life. J. Med. Microbiol. 15: (22) Suzuki, M., H. Mori, T. Asato, H. Sakugawa, T. Ishii, and Y. Hosoda Medical researches upon centenarians on Okinawa-case controlled study of family history as hereditary influence on

9 ECOLOGY OF BIFIDOBACTERIUM 41 longevity. Jpn. J. Geriat. 22: (23) Tanaka, R., T. Kan, H. Teshima, T. Kuroshima, S. Kodaira, S. Suzuki, T. Terashima, and M. Mutai Effects of administration of Bifidobacterium bifidum 4007 and B. breve 4006 on the intestinal flora of infants and adults. Jpn. J. Pediatr. 33: (in Japanese). (24) Tanaka, R., and K. Shimosaka Investigation of the stool frequency in elderly who were bedridden and its improvements by ingesting of bifidus yogurt. Jpn. J. Geriat. 19: (in Japanese with English summary). (25) Tanaka, R., H. Takayama, M. Morotomi, T. Kuroshima, S. Ueyama, M. Matsumoto, A. Kuroda, and M. Mutai Effects of administration of TOS and Bifidobacterium breve 4006 on the human fecal flora. Bifidobacteria Microflora 2: (26) Taylor, E., A.L. Blackwell, D. Barlow, and I. Phillips Gardnerella vaginalis, anaerobes, and vaginal discharge. Lancet 1: (27) Tojo, M., T. Oikawa, Y. Morikawa, N. Yamashita, S. Iwata, Y. Sato, J. Hanada, and R. Tanaka The effects of Bifidobacterium breve administration on Campylobacter entertis. Acta Paediatr. Jpn. 29: (28) van der Waaij, D., J.M. Berghuis, and J.E.C. Lekkerkerk Colonization resistance of the digestive tract of mice during systemic antibiotic treatment. J. Hyg. 70: (29) Vergnes, D., N. Moatti, X. Monrozies, F. Lazorthes, and L. Enjalbert Pre-operative colonic preparation using kanamycin and metronidazole : Qualitative and quantitative effects on the bacterial flora of the intestine. J. Antimicrob. Chemother. 6: (30) Werner, H., and Seeliger, H.P.R Cultural studies of the vaginal flora with special reference to L. bifidus. Pathol. Microbiol. 26: