Recent Trends in Research on Intestinal Flora

JBF LECTURE Bifidobacteria Microflora Vol. 1(1), 3-24, 1982

Recent Trends in Research on Intestinal Flora

Tomotari MITSUOKA

The Institute of Physical and Chemical Research, Wako, Saitama 351

(Received 15 October, 1981)

CONTENTS

1. Ecology of the Intestinal Flora

Advances in culture methods

Composition of the intestinal flora of adults

Distribution of the intestinal flora

Succession of the intestinal flora

Composition of the intestinal flora of various animal species

Bacterial species isolated from the intestine

Factors affecting the balance of intestinal flora

2. Role of Intestinal Flora in the Host

Relationships between intestinal flora and host

Nutrition

Defence mechanisms

Autogenous infection

Production of harmful substance

Carcinogenesis and the intestinal flora

Bacterial detoxification in intestine

Drug efficacy and physiological functions

3. Conclusions

Key words: Intestinal flora; intestinal bacteria; host-parasite relationships; de -fence mechanisms; carcinogenesis; drug efficacy; ecology; taxonomy; autogenous

infection

The history of intestinal microecology

began in 1719, when the first microscopic

observations of fecal bacteria were made by Leeuwenhoek. In 1885, Escherich initiated

the study of the intestinal flora with a

description of Bacterium coli communior in

bottle-fed infants. Subsequent studies on

intestinal microecology have mainly been

focussed on the aerobes, such as coliform

bacteria, enterococci, etc.

1. Ecology of the Intestinal Flora

In the past 15 years, however, it has been

recognized that there are enormous quan-

tities of intestinal bacteria which cannot be cultivated in vitro using conventional micro-biological techniques. Such bacteria are extremely sensitive to oxygen, and unless strict anaerobic techniques and suitable media are used, they are liable to be ig-nored.

Advances in culture methods: Hungate (18) has developed an anaerobic roll tube method for the isolation of cellulolytic anaerobic bacteria in the rumen. This culture tech-nique has also been applied to the study of intestinal flora and made possible the

growth of fastidious anaerobes in the intes-tine. Subsequently, the Hungate technique

3

4 T. MITSUOKA

Table 1. Development

of prereduced media

has been modified to make possible the

growth of fastidious anaerobes as surface colonies on agar media (Table 1). One modification is the "Anaerobic glove-box" of Rosebury and Reynold (48), Drasar (9) and Aranki et al. (1), and another is the authors' "Plate-in-bottle" method (28) . The principles of these newly developed methods are the same as those of the Hun-

gate technique, i.e. prereduction and an-aerobic sterilization, with the reliable elim-ination of atmospheric oxygen during hand-

ling of material and cultivation.These technical improvements increase

cultural recoveries from intestinal contents and enable cultivation of over 70% of the microscopic count of bacteria in human feces, and often in excess of 90%. Data from several recent studies show that the cultural counts approach 400 billion per

gram dry weight or about 200 billion per gram wet weight (Table 2) . We have es-tablished a comprehensive system using the "Plate -in-bottle" method, involving the use

of 10 selective and 4 non-selective media, for the study of intestinal frora (Table 3) .

Composition of the intestinal flora of adults: With the use of these strict anaerobic cul-turing techniques, it is now accepted that the most prevalent microorganisms in hu-man adult feces are obligate anaerobes, while easily culturable aerobes are expected to account for less than 10-3 of anaerobe numbers ((28), Fig. 1) . The most prevalent

Table 2. Normal fecal flora of man

RECENT TRENDS IN RESEARCH ON INTESTINAL FLORA 5

Table 3. The media and cultural method for comprehensive investigation of intestinal flora (29)

Fig. 1. Fecal flora of adults (42 specimens) . Where no subjects harbored the bacterial group, numbers in parentheses show inci-dence (%) (Mitsuoka et al. (28)) .

anaerobe is bacteroidaceae and the average count of this organism is 1010.9 per gram of wet feces. The second most prevalent micro-organisms are eubacteria and anaerobic lactobacilli, and are found with average counts of 1010.4 per gram. Third is pep-tococcaceae including Ruminococcus, Coprococ-cus, Peptostreptococcus and Peptococcus. The average count is 1010.2 per gram. Fouth is bifidobacteria with an average number of 1010.0 per gram. Other anaerobes , often found in human feces, include clostridia ,

Fig. 2. Bacterial flora of various sites of alimentary Tract (Mitsuoka (23)) .

megasphaerae, and veillonellae. The aerobic flora is predominantly represented by coli-forms, streptococci and faculative anaerobic lactobacilli, but the counts are less than 108

per gram feces.Distribution of the intestinal flora: There

are now considerable data on normal mi-crobial populations of various parts of the alimentary tract ((23), Fig. 2) .

In normal persons, the flora of the small intestine is relatively simple and large num.-

6 T.MITSUOKA

bers of organisms are not found. Total counts are generally 104 or less per ml, except for the distal ileum, where counts are usually about 106 per ml. In the duodenum and jejunum, streptococci, lacto-bacilli, and veillonellae are mainly found. Towards the ileum, Escherichia coli and an-aerobic bacteria increase in number.

In the cecum, the composition suddenly changes and is similar to that found in feces, and the concentration may reach 1011

per gram contents. Succession of the intestinal flora: The suc-

cession of the flora in human life has also been studied ((24), Fig. 3) . The fetus exists in a sterile environment until birth. After-birth it is contaminated with various mi-crobes, first from the maternal vagina and the mother's or nurse's fecal and skin mi-croflora and subsequently from the air and from food. A rather haphazard but rapid

colonization of the intestinal tract then follows.

On breast-feeding, fecal flora becomes stabilized and consists mostly of bifidobac-teria and about 1% of E. coli, enterococci, and lactobacilli ((25), Fig. 4) . There also occurs a significant reduction or even an absence of putrefactive bacteria.

In contrast, the fecal flora of bottle-fed infants rather resembles that of adults. In bottle-fed infants, bifidobacteria also con-stitute the major flora, but aerobic bacteria such as E. coli and streptococci and anaero-bic bacteria such as bacteroides, eubacteria and peptococcaceae are present in signifi-

Fig. 3. Succession of fecal flora of neonatals until 7th day (24) .

Fig. 4. Differences of fecal flora between breast-fed infants and bottle-fed infants (25).

Fig. 5. Succession of intestinal flora with age (24).


cantly greater numbers than in breast-fed infants. The microflora of children closely resem-

bles that of adults, where the number of bacteroidaceae, eubacteria and peptococ-caceae may be greater than that of bifido-bacteria, and E. coli and streptococci numbers decrease to less than 108 per gram feces ((24), Fig. 5) .

In older persons bifidobacteria often de-crease or dimnish, lactobacilli and Clostrid-ium perfringens increasing.

Composition of the intestinal flora of various

animal species: The compositions of the fecal flora of healthy adult animals of 12 different species were analysed (Table 4) . In general, animals of the same species had a common pattern of fecal flora but patterns different from those of other species. In almost all animal species, the most pre-dominant fecal bacteria were anaerobes , including bacteroidaceae, bifidobateria , eubacteria, lactobacilli, peptococcaceae , and anaerobic curved rods. Numbers of lacto-bacilli and bifidobacteria varied with the species of animal. In the feces of humans ,

Table 4. Fecal flora of twelve adult animals of different species

a Mean ± SD of log bacterial counts (when present) . b Figures in parentheses refer to the number of subjects that harbor the organism .

Table 4. (Continued)

8 T. MITSUOKA


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monkeys and guinea pigs bifidobacteria out-numbered lactobacilli. Regarding the feces of chickens, pigs, dogs, mice, rats, and hamsters bifidobacteria were present in almost all individuals, but in smaller quan-tity than with lactobacilli. In the feces of rabbits, and horses, bifidobacteria were oc-casionally demonstrated as being present, but not consistently. In the feces of cats and minks they were never demonstrated.

Bacterial species isolated from the intestine: The intestinal bacterial groups are further divided in many bacterial species. Many of these, however, have not been described. The classification and nomenclature of the intestinal anaerobes have progressed remark-ably during the past decade (25, 32, 33) .

Significant progress has been made in the taxonomy of bifidobacteria. For many

years bifidobacteria were included in the genus Lactobacillus as L. bifidus, but they are now classified in a separate genus Bifido-bacterium as suggested by Orla-Jansen on the basis of their characteristics of morphology, biochemical characters, cell wall constituents, and GC contents (8, 39, 47, 51, 52) .

Members of the genus Bifidobacterium from various animals were classified as shown in Table 5. The great majority of animal strains, with the exception of the strains isolated from monkeys and dogs, have been clearly differentiated from human strains by their growth temperature and carbo-hydrate fermentation patterns, and classified as new species, B. thermophilum, B. pseudo-longum and B. animalis (22) . The isolates from chickens and pigs could be 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, isolates from dogs were identified as B. adolescentis, B. longum, and B. pseudolongum, and the strains from monkeys were identified as B. adolescentis (25,26).

The most numerous species of the fecal flora have been listed in the order of their occurrence in clinically healthy persons by

Table 6. Predominant bacterial species isolated from human feces (34)

a Mean logio count g-1 feces (dry weight) . b Percent of total count g-1 (dry weight) .

Fig. 6. Factors affecting the intestinal flora.

Moore and Holdeman ((34), Table 6) . Statistic analysis by Moore and Holdeman

(34) indicates that there are 400 to 500 dif-ferent bacterial species in the fecal flora of a single person.

Factors affecting the balance of intestinal flora: The remarkable stability or constancy of intestinal flora has frequently been reported

(6) . However, intestinal flora can be alter-ed by many endogenous and exogenous factors (Fig. 6) : animal species, age, sex,


habits, various physiological functions such as adrenal function, peristalsis , secretion of digestive enzymes, bile, mucus etc., immune mechanisms of the host, exogenous micro-organisms, climate, diet and drugs , as well as emotional stress. Disturbances of the intestinal flora have been observed in con-nection with the acidity of gastric juice , disorder of peristalsis, cancer or surgical operations on stomach or small intestine , liver or kidney disease, pernicious anaemia , and blind loop syndrome (10) .

2. Role of Intestinal Flora in the Host

During the past two decades the develop-ment of both broad-spectrum antibiotics and practical methods for maintaining ani-mals in a germ-free environment has stim-ulated renewed interest in the significance to the host of intestinal flora.

Relationships between intestinal flora and host: Intestinal bacteria influence both the health and disease of the host ((21), Fig. 7) . I have postulated three groups of intestinal bacteria : the first group consists of organ-

isms symbiotic to the host and constitutes the predominant flora, the second group consists of ubiquitous organisms such as E . coli or Streptococcus group, but the counts in the normal host not being predominant , and the third group of bacteria consists of true pathogenic bacteria, sometimes produc-ing autogenous infection, and with counts normally low.

Nutrition: Some intestinal microorgan-isms synthesize several vitamins including riboflavin, B12, thiamine, folic acid and

panthotenic acid, and proteins, which are partly absorbed and utilized by the host.

Defence mechanisms: Microbial popula-tions in the gastrointestinal tract are known to form a barrier against the proliferation of exogenous pathogens. Thus , host sus-ceptibility to specific enteric infection is influenced by the nature of the intestinal flora. One of the mechanisms may be related to the colonization of the indigenous flora, which prevents colonization of the invader by competing effectively for essen-tial nutrients or attachment sites on epithe-lium. Freter (13) has analyzed a number

Fig. 7. Interrelationships between the intestinal flora and the host (21) .

12 T. MITSUOKA

of parameters related to mechanisms con-trolling the colonization of the intestinalflora.

Another mechanism to prevent coloniza-tion of exogenous pathogens is production ofbacteriocidal or bacteriostatic agents.

Indigenous flora also stimulates immunemechanisms of the host which resist infectionand carcinogenesis.

Autogenous infection: Many organisms iso-lated from clinical specimens often occur as

part of the normal intestinal flora ofclinically healthy animals and humans.This suggests that some intestinal bacteriaare potential pathogens. They induce auto-

genous infections (Ochi and Miyairi (40))when host resistance mechanisms fail onreceipt of antibiotics, steroids, immunosup-

pressants, radiation therapy, or surgicaloperation, or on aging.

Production of harmful substance: Some or-

ganisms may form substances noxious to thehost (Fig. 8) . Among these are certainvasoconstricting amines such as histamine,tyramine, agmatine and cadaverine, am-monia, phenols, tryptophan metabolites, N-nitrosamines, bacterial toxins, and somesteroid metabolites. These metabolites as-sociate with various diseases of the host.

The ability of some organisms to increase

plasma cholesterol in germ-free rats mayindicate that the intestinal flora is implicated

in cholesterol metabolism, and, possibly,arteriosclerosis.

Carcinogenesis and the intestinal flora: Re-cently, much attention has been directed tothe production of carcinogens or cocarcino-

gens in the intestine. Epidemiological datahave indicated a positive correlation be-tween dietary components, especially beefor fat, and the incidence of colon cancer

(60) . It is reasonable to postulate that theintestinal flora may produce or potentiatecarcinogen or cocarcinogen. To evaluatethe relationship between cancer and intes-tinal flora, intestinal materials from volun-teers or animals taking test diet were an-alyzed bacteriologically and biochemically.

A correlation between the composition ofthe intestinal flora and the epidemiology ofcolon cancer has been suggested. Some dif-ferences have been noted in the fecal counts ofvarious bacterial groups of individual speciesand/or genera between fecal specimens of

people on a so-called "Western" type ofdiet and specimens from people on high-carbohydrate or non-meat diets (Table 7) .

Studies of human subjects in variouscountries have shown some differences in

groups of fecal bacteria (17) . British andAmerican subjects yield much more Bac-teroides and nuclear dehydrogenating clos-tridia than Ugandans, Indians and Jap-anese. Conversely, Ugandans, Indians and

Fig. 8. Harmful products from intestinal flora.


Table 7. Effect of "Western" vs. high carbohydrate or non -meat diets on human fecal flora

a "Western" diet; b High -Carbohydrate diet; c "Japanese" diet; d "Non -meat" diet;e %;f Mean loglo of counts per gram feces (dry weight) .

Japanese had much more aerobes (strepto-cocci and enterobacteria) . In the work ofPeach et al., the samples of the high-carbo-hydrate diet group contained a larger

proportion of eubacteria and fewer Bac-teroides than did the "Western" samples.Finegold et al. used subjects entirely of

Japanese ancestry; one group took primarilya traditional Japanese diet, the other pri-marily an American diet. The differencesobserved in bacteroides were only of mar-

ginal significance (p<0.08), and differencesin Clostridium paraputrificum were not statis-tically significant. Over 220 distinct typesof bacteria were observed in the "high-carbohydrate" group, as opposed to 160 inthe "Western" diet group. Overall , about300 different types were observed . Extreme-ly oxygen-sensitive anaerobes were recov-ered more often from the Japanese dietthan from the "Western" diet.

Reddy et al. (46) used the same subjectsfor assessing the effects of high-meat andnon-meat diets. Total anaerobic flora wassignificantly higher with the "Western" diet ,as were the counts of bacteroides , bifido-bacteria, peptococci, and anaerobic lacto-bacilli. No differences in total aerobic florawere noted, although streptococci and staph-

ylococci species increase significantly in sub-jects on the non-meat diet.Hentges et al.(16) have also examined the effect of a high-beef diet on the bacterial composition ofhuman feces. Differences in counts be-tween meatless and high-beef diets wereobserved in only a few instances . Countsof bacteroides and clostridia were signifi-cantly higher and counts of Bifidobacteriumadolescentis A were significantly lower duringthe high-beef than during the meatless dietregimen. No other differences were ob-served.

Moore et al. (32) reported that NorthAmericans and polyp patients at high riskmore frequently have Bacteroides vulgatus,Bacteroides distasonis and Peptostreptococcus prod-uctus, and less frequently have Eubacteriumaerofaciens II, Bacteroides fragilis and E. coli intheir feces than Japanese and Africans .

In summary, it seems clear that thedifferences obtained in the earlier studieswere not confirmed in the later studies, andthat studies on dietary changes of intestinalflora in man are largely equivocal. Theseresults seem to show the complexity of theintestinal flora and also of the technical

problems associated with the determinationof intestinal flora composition . More de-

14 T. MITSUOKA

tailed studies of dietary effects on intestinal

flora are certainly needed to elucidate this

important problem. This may indicate that

individual ethnic populations (regardless of

risk) harbor intestinal flora associated with

carcinogenesis or that significant differences

in the flora are too subtle to detect with

present technology.

In addition to the bacteriological ap-

proach, determination of changes in fecal

enzymatic activities (Fig. 9) with dietary

changes provides an excellent and precise

index of intestinal metabolic function which

in turn reflects any changes in intestinal

flora.

Several hypotheses have been proposed

for the high incidence of colon cancer in

beef-eating populations. Bile acids are sus-

pected as being substrates for carcinogen

production .(2). Hill et al. have pointed

out that only four nuclear dehydrogenase

reactions are sufficient to convert bile acids

into carcinogen and that surrogate reactions

thereof can be carried out on related ster-

oids by lecithinase-negative clostridia iso-

lated from feces (14).

Some steroids including deoxycholic acid

estrone, apocholic acid, 3ƒÀ-acetoxy-bis-nor-

•¢5-cholenic acid, and 7-dehydrocholesterol

are carcinogenic in mammals. Deoxycholic

acid is produced from the major bile acid

(cholic acid) by intestinal bacteria; it is

present in relatively large amounts in the

human colon. Fecal deoxycholate excretion

Fig. 9. Fecal enzymatic activities.

among different geographical and ethnic

populations correlates well with the inci-

dence of colorectal cancer (10).

Goldin et al. (15) have measured the

activities of the bacterial enzymes ƒÀ-glucu-

ronidase, ƒÀ-glucosidase, azoreductase, and

nitroreductase in the fecal contents of rats

and humans (Figs. 10-16) . After the rats

were shifted from a grain to a high-beef

diet, a rapid rise in ƒÀ-glucuronidase, azore-

ductase, and nitroreductase activity occurred

within 20 days. Animals over 20 months

of age, consuming a meat diet, showed a

further increase in fecal ƒÀ-glucuronidase

activity, while the levels of the three micro-

bial enzymes increased in old rats fed on a

grain diet. Humans receiving fiber supple-

ments of 30 g of bran or wheat germ added

Fig. 10. Effect of high-beef diet on specific

activity of nitroreductase in fecal flora of

rats. Specific activity is expressed as ƒÊg of

m-aminobenzoic acid formed per hour per

mg of fecal protein. Mean values for 10 an-

imals (Goldin et al. (15)) .

Fig. 11. Effect of high-beef diet on specificactivity of azoreductase in fecal flora of rats.Specific activity is expressed as ,ug of sunsetyellow reduced per hour per mg of fecal pro-tein. Mean values for 10 animals (Goldinet al. (15)).


Fig. 12. Effect of high-beef diet on the specif-

ic activity of ƒÀ-glucuronidase in fecal flora

of rats. Specific activity is expressed as ƒÊg of

phenolphthalein released per minute per mg

fecal protein. Mean values for 8 animals

(Goldin et al. (15)) .

Fig. 13. Effect of high-beef diet on specific

activity of ƒÀ-glucosidase in fecal flora of rats.

Specific activity is expressed as ,ƒÊg of nitro-

phenol released per minute per mg fecal

protein. Mean values for 5 animals (Goldin

et al. (15)).

Fig. 14. Effect of age on specific activity of

ƒÀ-glucuronidase in the fecal flora of rats on

a meat diet (Goldin et al. (15)) .

to their customary diets showed no signifi-

cant changes in fecal enzyme activity.

Although changes in fecal enzymatic ac-

tivities with dietary alterations have not yet

been confirmed, such studies will still help

to elucidate the possible relationships be-

Fig. 15. Effect of age on specific activity of

ƒÀ-glucuronidase in the fecal flora of rats on

a grain diet (Goldin et al. (15)) .

Fig. 16. Effect of age on specific activity ofnitroreductase in the fecal flora of rats on agrain diet (Goldin et al. (15)) .

Fig. 17. Production and circulation of nitro-

socompounds.

tween carcinogenesis and the intestinal flora.A different chemical etiology for colon

cancer is suggested by the isolation fromextracts of human feces of a compound

provisionally identified as a N-nitrosocom-pound.

N-nitrosocompounds are formed in areaction between nitrite and either a sec-ondary or a tertiary amine (Fig. 17) . Thereaction can take place in an acid environ-ment such as that in the stomach or can be

16 T. MITSUOKA

mediated by intestinal bacteria. The ni-

trosable amines required for the formation

of N-nitrosocompounds are produced by

intestinal bacteria in the lower intestinal

tract. However, Tannenbaum and his

coworkers (56) recently demonstrated that

much nitrite is formed in the small intestine

from ammonia through heterotrophic ni-

trification by intestinal flora and that a part

of the nitrite formed reacts further with

amines in the lower intestinal tract. Thus,

N-nitrosamine is formed in the presence of

the intestinal flora. The amount of N-

nitrosocompounds formed by these processes

may be substantially greater than previously

recognized. Tannenbaum (55) estimated

the N-dimethylnitrosamine formed in human

body per day to be 1 to 10 ƒÊg/kg. How-

ever, what kinds of bacteria relate to this

process is not known. Recently, the ni-

trosamine concentration of the feces was

analysed in our laboratory. Nitrosamines

were remarkably increased on changing

from normal Japanese diet to "Western"

diet (Fig. 18) .

Bruce and his coworkers (7) showed thatthe feces of normal humans taking Westerndiet contained a mutagen, which is anendogenously produced nitrosocompound.In our laboratory (54), the mutagenicity ofcell extracts or cell-free extracts of culturesof 45 bacterial strains predominant inhuman feces was examined. Thirty-six

percent of the bacteria showed mutagenicactivity which could be classified into two

groups, base-pair substitutions and frame-shifts (Table 8).

Gnotobiotic experimentation has beenused to assess the role of the intestinal florain the carcinogenesis of the host.

Intrarectal injection of direct-acting car-cinogens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) have been shown toinduce adenomas and adenocarcinomas ofthe colon in germ-free and conventional ratsand in conventional mice (35-37, 45, 57) .Carcinogenic compounds that require meta-bolic activation for tumor induction, suchas 1,2-dim.ethylhydrazine or cycasin, the

Fig. 18. Changes of volatile nitrosamines in human feces on adhering to Japanese- and Western-

style diets. , nitrosodimethylamine; •¡, nitrosodiethylamine; •¢, nitrosodibutylamine; •›, nitroso-

piperidine; •£, nitrosopyrrolidine, TJ, typical Japanese diet; MJ, mixed Japanese diet; BW, bal-

anced Western-style diet. The values represent the mean+s.e. of 3 or 4 subjects.


Table 8. Lethal activities of the extracts of intestinal microbes in Bacillus subtilis

Activities were expressed as follows :—-, no difference of inhibition length; •}, less

than 5 mm difference of inhibition length; +, 5-10 mm difference of inhibition

length; œO, more than 10 mm difference of inhibition length; ™¿, more than 20 mm

of difference of inhibition length; it, more than 30 mm of difference of inhibition

length. (Suzuki & Mitsuoka (54))

18 T. MITSUOKA

ƒÀ-glucoside of methylazoxymethanol, have

also been shown to induce a high incidence

of colon tumors in germ-free and conven-

tional rats (45).

Reddy et al. (43, 44) have tested both

primary and secondary bile acid as well as

cholesterol metabolites for their carcinogenic

activity in germ-free and conventional rats.

They have indicated that cholic acid and

chenodeoxycholic acid act as colon tumor

promotors in conventional rats treated with

MNNG whereas deoxycholic acid and litho-

cholic acid act as colon tumor promotors

in both germ-free and conventional rats,

but not as complete carcinogens (Tables

9-11).

Sumi and Miyakawa (53) compared tumor

production between male germ-free and

conventional Wistar rats given 100 ƒÊg

MNNG/ml in drinking water and found

that 91% of conventional MNNG-treated

rats developed tumors in the gastrointestinal

tract by day 314 of the experiment, whereas

only 17% of germ-free MNNG-treated rats

developed such tumors (Table 12) . In ad-

dition, large tumors, some 5 cm or more in

diameter, were frequently observed in the

conventional rats, whereas only small

tumors 0.4 to 1.2 cm in diameter were

present in the germ-free rats. The results

suggested that gut flora might exert a

promoting influence on tumorigenesis caused

by MNNG in the gastrointestinal tract.

Although many studies have indicated

Table 9. Colon tumor incidence in germ-free rats treated i.r. with MNNG and/orbile acids

a CA, cholic acid; CDA, chenodeoxycholic acid.

b Of 24 germ-free rats, 2 rats were lost due to technical problems during the 4th week

of the experiment.

c Mean•}S.E.

d Not significantly different from group given MNNG alone by X2 test; p <0.05.

e Significantly different from group given MNNG alone by t test; p<0.05. (Reddy

et al. (44))

Table 10. Colon tumor cincidence in conventional rats treated i.r. with MNNGand/or bile acids

a CA, cholic acid; CDA, chenodeoxycholic acid.

b Mean•}S .E.

c Significantly different from group given MNNG alone by X2 test; p <0.025.d Significantly different from group given MNNG alone by t test; p <0

.05. (Reddy

et al. (44))


Table 11. Colon tumor incidence in germ-free and conventional rats treated i .r. with MNNGand/or lithocholic acid

a LC, lithocholic acid.b Significantly different from the MNNG group by x2 test (p<0 .05 or better). (Reddy et al. (43))

Table 12. Incidence, and structural classification of gastrointestinal

tumors in germ-free and conventional rats given MNNG (100

ƒÊg/ml) in drinking water until Day 314

a Number of rats with tumors/number of rats examined .b Including rats with a double tumor . (Surni & Miyakawa (53))

that the microflora may be producingcarcinogens in vivo and play a role in theetiology of cancer, the influence of themicroflora on temporal tumor induction bydirect-acting carcinogens such as MNU orMNNG has also been assessed. Ratherthan forming carcinogens or cocarcinogens ,the metabolic activity of the microorganismsin the gut could also interfere with theaction of carcinogens or cocarcinogens bydegrading them or providing a reservoir ofnonhost macromolecules for alkylation reac-tions.

Balish et al. (4) intrarectally injectedMNU or MNNG in low doses over a pro-longed time period into germ-free and con-ventional rats to assess the effect of intestinalbacteria on temporal effects of direct-acting

colon carcinogens. They observed that the

germ-free colon was more sensitive to thecarcinogenic actions of MNNG or MNUthan were the colons of rats with complexmicroflora (Fig. 19) . Tumors developedsooner and morbidity was earlier in the

germ-free rats. Both conventional andgerm-free female rats were more resistant toboth carcinogens than were the male rats .Exposure of younger germ-free animals (30days old vs. 60 days old at the start of theexperiment) resulted in a more rapid induc-tion of colon tumors than when injectionswere started in older (60-day-old) germ-freeanimals. The experiments indicated that,at least with direct-acting carcinogens, themicrobial flora may interfere with theirtumor-inducing activity. This interference

20 T. MITSUOKA

Fig. 19. Comparison of the tumor-inducingcapacity of MNNG or MNU, by i.r. injec-tion of 1 mg per week, in germ-free and con-ventional Sprague-Dawley rats; MNNG—GF30=MNNG injected into 8 30-day-old maleand female germ-free rats; MNNG—GF30=MNNG injected into 60 60-day-old germ-free rats; MNU—GF60=MNU injected into60 60-day-old germ-free rats; MNNGCONV60=MNNG injected into 60 60-day-old conventional rats; MNU—CONV60=MNU injected into 60 60-day-old conven-tional rats (Balish et al. (4)).

could be brought about by microbial en-zymes that are capable of degrading N-nitrosamines (49) or possibly by the alkyl-ation of microbial nucleic acids by thecarcinogens and the resulting reduction ofoverall exposure of the colon epithelium tothe direct-acting carcinogens.

Recently, we have investigated the effectof intestinal bacteria on liver tumorigenesis

in gnotobiotic C3H/He male mice monoas-sociated, diassociated, or polyassociated withvarious intestinal bacteria (30, 31) . Asshown in Table 13-15, the incidence of livertumors was higher in most of the gnotobiotes

(42-100%) and conventionalized mice(75%) derived from the germ-free micethan in germ-free mice (30%). The aver-age number of tumor nodules in the con-ventionalized mice (1.3) was also higherthan that in the germ-free mice (0.4) .Liver tumorigenesis in the gnototiotes wasmarkedly promoted by association with abacterial combination of E. coli, Streptococcus

faecalis, and Clostridium paraputrificum (100%in incidence and 2.8 in average number oftumor nodules) . This promoting effect wassuppressed by addition of certain intestinalbacteria such as Bifidobacterium longum, Lac-tobacillus acidophilus, and Eubacterium rectale.These results suggested that intestinal bac-teria are related to both promotion andinhibition of liver tumorigenesis in mice,depending upon the kind of bacteria.

Bacterial detoxification in intestine: Whilethe intestinal bacteria are possibly relatedto the production of carcinogens, they canconversely participate in detoxification ofsome toxic substances.

The degradation of N-nitrosamines has

Table 13. Incidence of liver tumor in germ-free, conventionalized, and gnotobiotic C3H/He malemice associated with 1 to 4 strains of intestinal bacteria

a Mice were killed under ether anesthesia when they were 12 months old .


Table 14. Incidence of liver tumor in gnotobiotic C3H/He male mice associated with 2 to 5 strainsof intestinal bacteria

a Mice were killed under ether anesthesia when they were 12 months old.

Table 15. Incidence of liver tumor in gnotobiotic C3H/He male mice associated with 5 or 6 strainsof intestinal bacteria

a Mice were killed under ether anesthesia when they were 12 months old.

recently been reported by Rowland andGrasso (49). A large portion of intestinalbacterial groups (Escherichia coli, Bacteroides,Bifidobacterium, Lactobacillus, and Streptococcus

faecalis) degrade diphenylnitrosamine anddimethylnitrosamine in vitro.

The degradation of benzo [a] pyrene hasbeen detected in some bacteria includingPseudomonas sp. E. coli, and Bacillus sp. (5,20).

Escherichia coli has been shown to carryout N-dehydroxylation of N-hydroxyacetyl-

22 T. MITSUOKA

aminofluorene (59). More recently, the

reduction of N-hydroxy-4-acetylaminobi-

phenyl to 4-acetylaminobiphenyl has been

demonstrated in several intestinal isolates,

e.g., Peptostreptococcus productus I and Bac-

teroides thetaiotaomicron and Bacteroides vulgatus

(58). Thus, the flora, by hydrolyzing glu-

curonides and reducing N-hydroxyaryl

amines and amides, may exert a significant

influence on the concentration of proximate

carcinogens in the bowel.

The intestinal flora also participates in

some of the interconversion of inorganic and

organic mercury that may occur in the

host. Mercury is eliminated in the inor-

ganic form in the feces even when adminis-

tered to rats in the form of methylmercury

(38).

Drug efficacy and physiological functions: The

metabolic transformations of exogenous

compounds by the intestinal flora have

implications either for drug action or toxi-

city, and for physiological functions. It is

well known that the intestinal flora played

a critical role in the activation of the early

antibacterial agents prontosil and neopron-

tosil.

Sandler et al. (50) have reported that the

trace metabolites of L-dopa are of possible

clinical importance, and that the intestinal

flora seems to have an obligate role in the

formation of these metabolites such as m-

tyramine and m-hydroxyphenylacetic acid.

Purgative activities of "Rhei rhizoma" and

Senna have been suggested to be related to in-

testinal bacterial action. Kobashi et al. (19)

have demonstrated that the active principle

is probably rheinanthrone, which is formed

by ƒÀ-glucosidase-possessing bacteria such as

Clostridium sphenoides, C. butyricum and Bifido-

bacterium adolescentis through the reduction

and subsequent hydrolysis of sennosides.

3. Conclusions

In recent years, the introduction of im-

proved anaerobic culture technique and the

application of modern biochemical methods,

as well as the utilization of gnotobiotic

animal technique have greatly promoted studies related to microecological research of the intestine.

The purposes of intestinal microecology are to clarify the interrelationships of the intestinal flora, environmental factors and the host, and to define the optimum pattern of the intestinal flora in order to improve human health and well-being, and finally to develop the method to do so by means of diet, bacterial culture, etc.

Intestinal flora interacts profoundly with the host both beneficially and harmfully and may manifest pathophysiological effects. These include nutritional status, effects of drugs, physiological functions, aging, cancer incidence, immune response, as well as resistance to infection (Fig. 20). However, little is known on which bacterial species are implicated in these phenomena. Like-wise, little is known about the mechanisms of the microbe-host interactions involved. A solution to these problems will only be achieved by the cooperation of the workers in different research areas such as microbi-ology, immunology, physiology, biochemistry,

pathology, pharmacology, toxicology, mo-lecular biology, nutrition, and clinical medicine.

Fig. 20. Influences of intestinal flora on the

host.


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