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Role of gut bacterial flora in nutrition and health:

A review of recent advances in bacteriological
techniques, metabolism, and factors affecting flora
composition
a b
Joseph P. Brown & Carl Lamanna
a
Senior Research Biologist, Department of Biological Sciences, Dynapol, Palo Alto,
California
b
Associate Director for Pharmaceutical Research and Testing, Food and Drug Administration,
Washington, D.C.
Published online: 29 Sep 2009.

To cite this article: Joseph P. Brown & Carl Lamanna (1977) Role of gut bacterial flora in nutrition and health: A review of
recent advances in bacteriological techniques, metabolism, and factors affecting flora composition, C R C Critical Reviews in
Food Science and Nutrition, 8:3, 229-336

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 ROLE OF GUT BACTERIAL FLORA IN NUTRITION AND HEALTH: A REVIEW
OF RECENT ADVANCES IN BACTERIOLOGICAL TECHNIQUES, METABOLISM,
AND FACTORS AFFECTING FLORA COMPOSITION

Author: Joseph P. Brown

Department of Biological Sciences
Dynapol
Palo Alto, California
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Referee: Carl Lamanna

Food and Drug Administration
Washington, D.C.

I. INTRODUCTION both the composite flora of a given specimen and

The study of man's intestinal microbiota dates
back to 1681, when the first observations of fecal
bacteria and protozoa were made by Leeuwen-
hoek. 129 Subsequent developments in medical
microbiology include the isolation and identifica-
tion of various enteric pathogens. Only in the past
10 to 15 years have advances in culturing tech-
niques allowed recovery of significant fractions
(1/3 to 2/3) of the numerous microbes observed
by direct microscopic examination of fecal
specimens. Data from several recent sources put
the viable count in normal human feces at 2 to 4 X
10 1 ' per gram dry weight. In normal Western
European or North American dietaries, the vast
majority of fecal bacteria are obligate nonsporing
anaerobes. They may outnumber aerobes and
facultative microbes by as much as 5000:1. A
fraction (5 to 20%) of the fecal anaerobes are
extremely sensitive to molecular oxygen, and
unavoidable exposure of them is probably a major
cause of the variation in recovery figures reported
by different workers.
Rigorous anaerobic culturing techniques have
revealed a disconcerting degree of complexity in
the flora variations within and between hosts.
Moore and Holdeman 369 have estimated that the
20 or so species comprising 55 random isolates
yield a statistical coverage of 70 to 80% of all
types present in a fecal specimen. However, the
remaining 20 to 30% of the population may
include over 400 additional species. In what may
be the most detailed study 3 6 8 available of indi-
vidual fecal specimens (20 hosts), the same authors
randomly isolated 113 species and types repre-
senting a 94% coverage of the fecal flora of these
20 individuals as a population. The species most
frequently observed and their number in propor-
tion to the total flora were the Gram-negative rod
Bacteroides fragilis ss. vulgatus (12.1%) and Fuso-
bacterium prausnitzii (7.15%), the Gram-positive
rods Bifidobacterium adolescentis (6.45%) and
Eubacterium aerofaciens (6.02%), and Pepto-
streptococcus productus II (5.58%). The dominant
genera were Eubacterium, 26 types (25.5%);
Bacteroides, 20 types (22.6%); Bifidobacterium, 8
types (11.5%); Peptostreptococcus productus I
and II (8.9%); Fusobacterium, 5 types (7.7%);
Ruminococcus, 11 types (4.5%); and Lacto-
bacillus, 7 types (2.4%). Microorganisms belonging

January 1977 229

 to more familiar groups comprised a comparatively
small part of the flora, e.g., streptococci (1.56%),
clostridia (0.6%), and enterobacteria (0.5%).
While the microbial composition of human
feces is generally accepted as being fairly represen-
tative of the luminal flora of the lower large bowel
or rectosigmoid, the luminal flora of the ileocecal
region is significantly different. 246 It is not
known how closely luminal flora throughout the
intestinal tract reflects indigenous flora in close
association with the various habits3 2 s afforded by
the villi, crypts, and enterocyte surfaces. A variety
of mammalian studies, 221 particularly with
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rodents, indicate that significant differences exist.
In all organisms studied (i.e., mice, rats, dogs, pigs,
monkeys, and humans), anaerobic spirochetes
apparently occur in close association with the
mucosal epithelia of the large bowels. While these
spiral-shaped microbes are largely absent from the
lumen of the cecum and colon, studies with
colonization of germfree and gnotobiotic rodents
show them to be part of the autochthonous or
symbiotic flora of the large bowel. 121 > 3 0 4 ' 3 7 S In
addition to spirochetes, certain autochthonous
fusiform bacteria are also found in the cecum and
colon. In the ileum, coccobacilli are found
attached to the mucosal villa, 458 whereas lacto-
bacilli predominate in the stomach and upper
small intestine. 146
Since ancient times, the influence of Man's
intestinal contents upon his health and well being
has been a matter of personal concern and
associated superstitions.298 Early in this century,
these unpleasant suspicions were explained
biochemically by the postulation that ptomaines
or toxic amines are produced by intestinal
bacteria, to the disadvantage of the host. S31 The
concept that a given dietary might have a major
influence on the health and survival of the host
(through "favorable" alternatives of the gut micro-
biota) was first popularized through the philosoph-
ical writings of Metchnikoff. 144>3S3 He believed
that the harmful effects of intestinal putrefying
organisms could be minimized or prevented by
establishing the proper lactobacilli flora in the gut.
This led to a popular interest in yogurt and other
cultured milk products and to a rather colorful
period of applied bacteriological research on
Lactobacillus bulgaricus and L. acidophilus.421
While interest in acidified milk and its alleged
benefits eventually waned (see Section II. D),
there has been some renewal of scientific interest
230 Critical Reviews in Food Science and Nutrition
in the basic idea of intestinal microbial toxigenesis
and in the influences of various nutritional
regimens. During the past few years, this interest
has been stimulated by the ideas of workers at the
Wright-Fleming Institute, who first suggested the
association between colorectal cancer, gut micro-
flora, and d i e t . 2 3 9 ' 2 4 2 ' 2 4 5 ' 2 4 7
Current studies of intestinal microecology are
aimed at better defining the interrelationships of
normal and autochthonous components of the
indigenous flora to the physiology, diet, and
nutrition of the host. The task is enormously
difficult, because of the complexity of human
fecal flora. While man lacks such specialized
gastrointestinal organs as the rumen or cecum of
the agricultural and laboratory animals studied so
often, his diet is undoubtedly more complex. In
addition to being omnivorous in the strict sense,
he ingests hundreds of synthetic and naturally
occurring food additives, both intentionally and
incidentally.324 Expected future trends in food
production involve novel sources of proteins, s4 °
vitamin supplements, etc. which will make man's
diet even more heterogenous.
Despite the inherent stability of the intestinal
microflora, 1 3 9 ' 1 9 2 ' 3 7 0 it can be altered by
changes in diet. Significant changes have been
noted in the fecal concentrations of various groups
of microbes (e.g., coliforms, other facultative
anaerobes, clostridia)1 >33S of individual species
and/or genera (e.g., Eubacterium contortum,
Bifidohacterium infantis, Peptostreptococcus
sp., 1 6 3 Bacteroides sp., 3 3 5 Fusobacterium sp., s 2 8
Lactobacillus*23) or even in the metabolic activity
of individual species.24 At present, it is impossible
to assess the short- or long-term effects which such
changes in flora will have upon host physiology. In
numerous studies of high meat content, (so-called
"Western") diets and nonmeat or vegetarian diets,
reductions in anaerobic flora caused by vegetarian
diets are associated with reduced fecal bile acids
and neutral sterols. These findings appear to be
consistent with the hypothesis that extensive
microbial metabolism of these steroid metabolites
leads to carcinogenic or cocarcinogenic inter-
mediates and with the epidemiological data
implicating diets high in meat and animal fat as
predisposing factors in the incidence of colorectal
cancer. However, the situation is undoubtedly
more complex. Wilkins and Hackmans s 1 have
studied neutral steroid conversion in 31 North
American subjects and found about 25% to be low
 converters, irrespective of dietary habits (i.e., the
total quantity of plant and animal steroids
excreted in both groups was equivalent). These
differences in steroid conversion could be due to
factors other than differences in the indigenous
microbiota, e.g., the physical state of cholesterol
in the lumen or fecal transit times. It is not known
whether the low-conversion subjects represent a
population at lower risk (consistent with the
above-mentioned hypothesis) or at greater risk,
because they are not able to detoxify carcinogenic
steroids. 110
The role of anaerobic bacteria in disease
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processes has been discussed in recent reviews66'
192
and in a symposium monograph.39 Clinical
syndromes (such as gas gangrene, tetanus, and
botulism) caused by the anaerobic spore-forming
clostridia have long been recognized. However, the
prevalence of infections due to nonspore-forming
Gram-negative bacteria (the bacteroides and the
fusobacteria) has only recently been realized and
given proper attention. This is partially due to
advances in isolation, culturing, and associated
follow-up techniques. Other anaerobic Gram-
positive nonspore-formers (propionibacteria,
eubacteria, actinomyces, and bifidobacteria)
together with the Gram-negative veillonella are
recovered from clinical specimens less often. As
these pathogenic anaerobes do not produce
distinct clinical pictures (e.g., bacteremia,
peritonitis, abscesses), their isolation and identifi-
cation have frequently been confounded by the
presence of indigenous flora contaminants. Fre-
quently, it has been difficult to determine whether
a member of the indigenous flora associated with a
given clinical state caused the state or was merely a
side effect. Also, the distinction between
indigenous and pathogenic flora is arbitrary. Under
certain conditions, some members of the normal
flora may manifest pathophysiological effects,
whereas other species long recognized as pathogens
may persist in the host with no ill effects.
Numerous agents and actions can disrupt the
ecological equilibrium of the intestinal milieu
predisposing the host to pathological processes,
viz., nutritional deficiencies, stress, and toxic
substances, including antibiotics and immuno-
suppressants.
The major emphasis of this review is on the role
of the intestinal microbiota in the nutrition and
physiology of normal human hosts. The following
discussion is roughly divided into subsections on
"digestion," "absorption and malabsorption,"
"host defenses," "aging and survival," and
toxigenic capabilities of the flora. Separate
sections follow, covering more specialized, if not
recondite, literature — "methods" of isolation and
cultivation, speciation, etc. of normal intestinal
flora; factors influencing the composition and
distribution of the gut flora; the normal human
microflora with descriptions of some important
species; and genera and selected aspects of gut
flora metabolism.
II. T H E GUT F L O R A IN
NUTRITION AND HEALTH
A. Digestion
Although bacterial products are absorbed from
the human colon, it has not been demonstrated
conclusively that the indigenous flora augments
the nutrient content of man's diet. Experience
with germfree animals clearly demonstrates that
the intestinal flora is not essential for
l i f e . 1 9 7 ' 3 2 2 ' 3 2 3 Animals raised in clean sur-
roundings have a relatively simple gut flora and
actually utilize their nutrients more efficiently.144
Paradoxically, the notion that our intestinal flora
contributes certain nutrients has come from
studies with germfree animals. Germfree rats
maintained on a folate-deficient diet develop
symptoms of folate deficiency, while the same diet
has no effect on conventional rats.1 i 7 Deficiency
symptoms could apparently be prevented by
monoassociation of germfree animals with various
species of enterobacteria (Aerobacter sp., Alcali-
genes sp., Proteus sp., or Escherichia coli).
Escherichia, Lactobacillus, Clostridium, and other
intestinal microbes are capable of synthesizing a
complex variety of folate coenyzmes which are in
many respects similar to dietary folates from
animal and plant sources. 7 1 ' 1 1 5 ' 3 9 6 Similarly,
conventional rats fed a diet deficient in panto-
thenic acid were able to utilize microbial panto-
thenate only via coprophagy.
Vitamin K deficiency has also been observed in
germfree rats fed a K-deficient diet. 2 ! ' The
symptoms can be relieved by conventionalization
or dietary supplementation; vitamin K is most
effective. 212 ' 567 It was found that E. coli or an
unidentified micrococcus was able to reverse the
deficiency symptoms.
While thiamine was once thought to be a useful
product of intestinal microbial action, Wostmann
January 1977 231
 et al., 5 6 6 using radiolabeled precursors of thia-
mine, showed that very little microbial thiamine
was available for absorption by the host.
It has also been suggested that the host utilizes
other gut-synthesized vitamins (biotin, riboflavin,
nicotinic acid, B i 2 . Bi, B 2 , B6) and essential
amino acids. In this connection, the oral biota may
also play a contributory role. 13 9
Although germfree animals lack microbial
protein and other microbial products in their
intestines, they do contain increased amounts of a
variety of substances, including free amino acids,
urea, hexosamines, glycoproteins, and glycos-
aminoglycans. 1 9 7 ' 3 1 4 ' 3 1 6 ' 3 1 7 Quantities of
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trypsin and chymotrypsin are also elevated in the
germfree gut, 317 ' 4 * 9 suggesting that the gut flora
plays a role in deactivating these enzymes. The
production of ammonia as an end product of
protein dissimulation appears to be wholly due to
the intestinal biota. 197 Cecal contents of germfree
rats produce only very small quantities of
ammonia. 1 0 2 ' 1 0 3 When 14C-labeled urea was
administered parenterally to germfree and
conventional control rats, only the latter gave off
14
CO 2 in exhaled air. 308 Also, monoassociation
of germfree rats with genera such as Lactobacillus,
Actinobacillus, and Staphylococcus induced the
formation of ammonia from urea in the ceca of
these animals. 147
The role of gut flora in carbohydrate catabo-
lism has not been studied in great detail.
Experiments with germfree and conventional rats
suggest that intestinal glucosidases and disac-
charidases are partially inactivated by the gut
flora.1 l&'Als On the other hand, the degradation
of gastrointestinal mucins and blood group
substances seems to be entirely due to the action
of bacterial enzymes. 262 " 264 It is conceivable that
other dietary polysaccharides and glycoproteins
from plant and animal sources, while only poorly
digested, may serve as substrates for microbial
dissimilation and as possible sources of additional
nutrients to the host. 3 5 2 This is supported by a
study in which a high percentage of streptococci
isolated from sheep rumen exhibited pectinolytic
activity. The extracellular pectin lyase elaborated
by these streptococci is a constitutive enzyme
which produces oligogalacturonides. Neither the
oligomers nor galacturonic acid is utilized by the
streptococci. 579 Numerous plant glycosides
present in man's diet (e.g., the flavonoids, cyano-
genic glucosides, amygdalin, cycasin, esculin, etc.)
232 Critical Reviews in Food Science and Nutrition
are hydrolysed by gut bacteria releasing various
aglycones. The latter may exert important
physiological or toxic effects. While several groups
of bacteria (viz., E. coli, enterococci, lactobacilli,
clostridia, bacteroides, and bifidobacteria) possess
/3-glucosidase, the dominant position of lacto-
bacilli, bacteroides, and bifidobacteria among the
bowel flora indicates their dominant role in the
lower intestinal metabolism of /3-glucosides. Most
of the hydrolysis of 0-glycosides and a- and
/3-galactosides is also carried out by lactobacilli and
other nonsporing anaerobes.226 Intestinal bacteria
(e.g., E. coli, Aerobacter aerogenes, and Bacter-
oides sp.) also exhibit 0-glucuronidase activity. 500
Although 0-glucuronides are primarily endogenous,
the aglycones may have synthetic, plant, or
endogenous origins. Bacterial action on endo-
genous 0-glucuronides frequently results in an
enterohepatic circulation of the aglycones and
related derivatives.
The influence of the gut flora and diet on fecal
steroid metabolism in man has been mentioned
above. The effect on fecal fatty acids of rats seems
to be an increase in saturated acids and in the
cyclic and branched-chain acids which are
synthesized wholly by bacteria. 1 5 3 ' 1 5 4 Germfree
rats contain only cholesterol and phytosterols in
their feces whereas conventional controls also
contain a mixture of coprostanol derivatives and
other s t e r o l s . 1 5 4 ' 2 8 0 ' 2 8 1 These microbial
transformations involve oxidation and reduction
of the steroid nucleus and reduction of un-
saturated fatty acids. Strains of Eubacterium sp.
which have been isolated from rat cecum are
probably involved in both reductive processes in
vivo. 156 >39 7 One isolate converted cholesterol and
A 5 -3j3-hydroxy plant sterols into the cor-
responding 5 /3-H-saturated derivatives. Other
organisms (such as Bacteroides sp., Bifido-
bacterium and Clostridium sp.) are also cholesterol
reducers. While there is probably more than one
microbe involved in the reduction of unsaturated
fatty acids in vivo, 362 a Eubacterium sp. under
strict anaerobic conditions reduces octadecadi-
enoic (18:2) acids (mainly linoleic) to octa-
decenoic (18:1) (probably frans-vaccenic) acid.
Although a suspension which combines various
types of fecal bacteria found in rats reduces both
oleic (18:1) and linoleic acids to stearic acid
(18:0), it has not been possible to isolate a single
organism that would carry out this reduction.
Furthermore, although fecal cultures from
 cecectomized rats reduce linoleic acid, they do not
produce stearic acid. It has not yet been
determined whether the cecum is the site of
production of essential growth factors or whether
(as is more likely) it permits the growth of
fastidious oleic acid-reducing anaerobes.
The oxidation of the steroid nucleus of the bile
acids has been studied in detail by Drasar and
Hill 139 and Hill. 241 Gut bacteria have been found
to introduce double bonds into the nucleus by
four mechanisms: (a) a double bond conjugated to
an oxo group, (b) a double bond via dehydration,
(c) removal of C-10 methyl group allowing
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ketoenolization of l,4-dien-3-oxosteroids to
phenolic forms, and (d) a double bond conjugated
with other double bonds. Three of the above
reaction types are carried out in vitro by certain
lecithinase-negative clostridia, chiefly Qostridium
paraputrificum. It is not known whether such
aromatization reactions occur in vivo or what the
physiological oxidants might be. Conceivably, such
anaerobic oxidations could be coupled with a
variety of reductive processes, some of which have
been mentioned above.
The role of bile acid degradation in the ecolog-
ical equilibrium of intestinal microbiota has re-
ceived considerable attention during the past few
years. Normal human bile contains cholic acid
(3a,7a,12a-trihydroxy-50-cholanic acid) and
chenodeoxycholic acid (3a,7a-dihydroxy-50-
cholanic acid), which are both synthesized by the
liver and secreted as taurine or glycine conjugates
and as the conjugates of a so-called secondary bile
acid (deoxycholic acid) which results from
microbial 7a-dehydroxylation of cholic acid.
These bile.salts assist in the absorption of dietary
lipid and are reabsorbed in the ileum. The fecal
bile acids, normally representing about 5% .of the
entrohepatically circulating pool, are entirely
deconjugated and consist of a variety of secondary
bile acids in addition to small quantities of the
primary bile acids. The former are produced by
three principal types of reactions carried out by
the gut flora: (a) hydrolysis of the amide bond
releasing free bile acids (i.e., deconjugation); (b)
oxidoreduction of hydroxyl groups at C-3, C-7 and
C-12 (yielding ketobile acids and those with
0-hydroxyl groups) via ketoenolization followed
by reduction; and (c) dehydroxylation at C-7 and
to a much lesser extent at C-3 and
£ _ | 2 13 9,3 1 1 ,3 5 7
Under normal circumstances, bile salt decon-
jugation occurs in the large bowel. Anaerobic
microbes like Bacteroides, Bifldobacterium,
lactobacilli, and clostridia appear to be mainly
responsible.4 8 3 Curiously, deconjugation is largely
absent in the "viridans" streptococci and the
enterobacteria; only two strains of the latter (e.g.,
Aerobacter aerogenes and Proteus mirabilis, among
several hundred strains tested) exhibited acti-
vity. 3 5 7 ' 3 5 8 In general, intestinal deconjugating
bacteria act upon both conjugates, although
several species have been found to be specific for
only one of the conjugates.3 5 7
A wide range of bacterial genera is involved in
the oxidoreduction of hydroxyl groups. Of 65
strains tested, 22 types belonging to six genera
were found capable of hydroxyl group dehydro-
genation (e.g., E. coli, Pseudomonas aeruginosa,
Bacteroides sp., Eubacterium sp., Bacillus cereus,
and Clostridium sp.). The formation of 3-/J-0H
derivatives via reduction of the 3-keto group is
carried out primarily by anaerobic bacteria, e.g.,
Eubacterium cadaverus, E. parvum, E. lentum, and
Clostridium perfringens.3 5 8 Intestinal bacteria
capable of forming 12/3-OH or 7/3-OH derivatives
have not yet been described in detail.3 S 7 The
latter compound is known to be formed in the
liver.
The dehydroxylation of the 7a-0H group of
cholic acid and chenodeoxycholic acid, leading to
the production of deoxycholic and lithocholic
acids, respectively, has been demonstrated in
comparatively few species of intestinal
bacteria. 5 3 ' 2 1 3 ' 2 2 4 ' 2 2 8 In all strains studied,
dehydroxylation was found to be specific for the
free bile acids in vitro and after association of
germ free animals. 214 However, in man, dehy-
droxylation of glycocholic but not taurocholic
acid has been demonstrated. 231 ' 232 Both
deconjugating 2 4 3 and nondeconjugating
strains3 S 9 may be capable of dehydroxylation.
Deconjugated or free bile acids, particularly
deoxycholate and chenodeoxycholic acid, inhibit
the growth of a variety of intestinal bacteria (e.g.,
Bacteroides sp., Lactobacillus sp., Clostridium sp.,
and enterococci) in vitro. 4 8 ' 1 6 8 The finding that
the extent of in vitro antibacterial action was
strongly pH dependent 402 led to the suggestion
that bile acids may participate in a self-regulatory
system preventing microbial colonization of the
upper bowel. To date, studies on the small
intestinal flora of man 3 3 7 have provided no
evidence of bile acid inhibition in vivo.
January 1977 233
 Presumably, the total concentration of free bile
acids, which is 1 mM or less 383 throughout the
ileum and lower jejunum, was insufficient to cause
bacteriostasis. Alternatively, other factors besides
bile acids are significant in determining the
microbial population in the upper intestine.
B. Absorption and Malabsorption
The role of intestinal flora in absorption has
recently been reviewed by Gordon. 196 Studies
with germfree and conventional animals indicate
that the flora affects the intestinal function by
causing a mild or "physiological" inflam-
mation 145 of the mucosa and by neutralizing
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physiologically active metabolites in the large
bowel. Upon accumulation, the metabolites appear
to depress water absorption, intestinal muscle tone
(in rodents only), and blood circulation. In the
large intestine below the ileocecal valve, the
intestinal microflora could be considered
indispensable to normal bowel function, despite
experimental evidence (see II.D) apparently
indicating greater longevity of germfree animals.
An essential biochemical action of the flora seems
to be the degradation of the so-called "colloid
pool," 1 9 6 resulting from accumulation in the
lumen of partially degraded polymeric and oligo-
meric components of intestinal secretions and
from mucosal desquamation. The accumulation of
nonabsorbable acidic colloids causes water
retention in the lumen and may also trap cations
required for solute-coupled water absorption. Lack
of adequate muscle tone and peristalsis in germfree
rodents is thought to be due to the accumulation
of depressant compounds in the bowel lumen.
Free amino acids and hypotensive peptides may be
involved.196 Other depressant substances, which
are normally deactivated by the gut flora upon
absorption by the host, cause contractile re-
fractoriness of vascular smooth muscle and
probably reduce cardiac output and metabolic
rate. Several antagonists are involved (e.g., those
affecting the agonists epinephrine, vasopressin, and
angiotensin), and ferritin components arising from
epithelial desquamation seem to be involved in the
epinephrine inhibitory effects. It has been
postulated that, by neutralizing such antiepineph-
rine agents, the intestinal flora indirectly raises the
metabolic rate by supporting the action of
endogenous epinephrine. However, Levenson and
Kan 3 0 7 and Levenson et al. 3 0 9 speculate that the
higher metabolic rate of conventional and
234 Critical Reviews in Food Science and Nutrition
conventionalized rats (compared with germfree
controls) results from either the formation by
some gut bacteria, (e.g., E. coli) of certain amines
(e.g., tyramine) or possibly from an effect of
specific bacteria on mammalian catecholamine
metabolism or other (nonthyroid) hormonal
agents. Such amine agents have been shown to
increase oxygen consumption in chicks.s
Although water absorption from the small
intestine seems to be identical in conventional and
germfree animals, a few studies have shown
increased absorption of some monosaccharides
(viz., 3-methylglucose, glucose, xylose) and B
vitamins in the latter group. 170 j l 9 6 >2 2 9 However,
germfree rats contain higher concentrations of free
amino acids 102 in cecal contents than do con-
ventional controls. This finding has been difficult
to interpret and has been ascribed to higher food
intake in germfree animals, 102 to increases in
digestive enzymes, or to higher levels of fecal
nitrogen excretion observed in germfree
animals.153 ' 3 ° 6 No marked difference in nitrogen
balance has been observed between conventional
and germfree groups fed complete diets. 406
The upper bowel flora of man is normally quite
sparse and consists largely of Gram-positive
facultative bacteria. A variety of conditions may
lead to overgrowth of fecal-type microorganisms in
the small bowel and may result in further
metabolic disturbances (notably malabsorption of
certain nutrients). The predisposing conditions
include abnormalities of gastric function, such as
those resulting from gastric surgery or gastric
achlorhydria (frequently associated with perni-
cious anemia); conditions resulting in stasis, such
as surgical blind loops, enteroanastomosis,
strictures or adhesions, diverticulosis, or abnormal
motllity; and free communication between the
large and small bowel, resulting from fistulas or
intestinal resection.s 19
Colonization of the small intestine with ab-
normal flora frequently results in steatorrhea or
impaired absorption of dietary lipid. In the case of
stagnant or blind loop syndrome, this is believed
to be the result of altered bile salt metabol-
ism5 ' 6 " 5 1 8 which may cause abnormally high
concentrations of enterococci, bacteroides,
bifid obacterium, clostridia and other fecal
organisms to deconjugate and dehydroxylate bile
salts to the extent where lipid micelle formation is
impaired. While this notion has received some
experimental support from both direct measure-
 ments of luminal bile salt concentrations in
patients with bacterial overgrowth and steatorrhea
and from the demonstration of impaired intra-
luminal micelle formation in animals with steator-
rhea, other factors may be involved. Some studies
have suggested that mucosal cells may have a
direct effect upon fat absorption. 8 ' 93
Diarrhea can also result from microbial over-
growth, though the mechanism of action is not
clear. Studies in man show that increased quanti-
ties of free bile acids in the colon cause colonic se-
cretion of water and electrolytes, which may lead to
a condition called choleric enteropathy; 3sl
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similar results have been obtained in the small
intestine of rats. 220 An alternate explanation
involves the microbial production of hydroxy
fatty acids. Such acids are common constituents of
purgative oils, e.g., ricinoleic acid (12-hydroxy-CH1-
9-octadecenoic acid), of castor oil, 1 8 2 ' 5 0 9 and
they are also found in the fecal fat of steatorrheic
patients. 400 One third of the 22 anaerobic species
tested by Thomas 524 were able to convert oleic
acid to 10-hydroxy-stearic acid; these included
various clostridia and Bifidobacterium bifidum.
Among four facultative species tested, only
Streptococcus faecalis exhibited significant
hydroxy-stearate formation. Similarly, Pearson et
al. 4 0 0 a tested 207 strains of human fecal bacteria
and found all but one of the enterococci able to
convert oleic to 10-hydroxy-stearic acid. Other
groups showing activity include bacteroides (18%),
bifidobacteria (32%), clostridia (50%) and
enterobacteria (21%). Microbial synthesis of
unsaturated hydroxy acids has also been
reported. 476
Malabsorption of D-xylose has been observed in
patients with bacterial overgrowth. It is not known
how much of this results from bacterial utilization
of xylose, 188 as opposed to true malabsorption.
Inhibition of glucose transport by deconjugated
bile acids has been observed in jejunal mucosa in
vitro 517 and in perfused rat jejunum. 408 More
recent studies by Gracey et a l . 2 0 2 ' 2 0 3 indicate
that deconjugated bile acid inhibition of active
monosaccharide transport is reversible, which may
explain temporary malabsorption.
Proliferation of enteric bacteria in the small
intestine has frequently caused vitamin B J 2
malabsorption, which may lead to vitamin B 1 2
deficiency and megaloblastic anemia. 109 It is not
known how the bacteria interfere with vitamin
B 12 absorption. Three possibilities have been
studied, viz., competitive uptake by gut bacteria,
destruction of the carrier glycoprotein "intrinsic
factor," and the production of enterocyte toxins.
For some time, it has been known that bacteria
can take up vitamin B 1 2 in vitro. 4 7 0 ' 4 7 3 Certain
bacteria have an avidity for vitamin B 1 2 ap-
proaching that of the human intrinsic factor (IF).
IF-bound B 1 2 may exchange with adsorbed
extracellular vitamin B i 2 , but not with intra-
cellular vitamin B i 2 . 1 8 3 ' 1 8 4 In vivo studies with
human subjects support the bacterial uptake
hypothesis. Intubated subjects were fed a test meal
containing S7Co-labeled vitamin Bi 2 and,
subsequently, ileal aspirates were collected and
centrifuged. In the case of subjects with both
jejunal diverticulosis and Bj 2 malabsorption, the
sediments from ileal aspirates were found to
contain a significant amount (46 to 74% of dose)
of S 7 Co-Bi 2 ) whereas normal control subjects and
those who had jejunal diverticulosis but normal
B i 2 absorption exhibited little (<10%)
57
Co-B 1 2 . 5 ' 9 Antibiotic treatment of one subject
led to a reduction in labeled sediment in ileal
aspirates and in anaerobic microflora colonizing
the small intestine, as well as to a return to normal
vitamin Bi 2 absorption.4 7 4 >s 16
In contrast to the bacterial B 1 2 uptake
mechanism, relatively little experimental data
supports the theory that either the destruction of
intrinsic factors 4 7 1 ' 4 7 2 or a direct toxicity to
enterocytes 2 7 9 ' 4 7 1 plays a significant role in
vitamin B 1 2 malabsorption associated with
bacterial overgrowth.
Folate deficiency rarely occurs in subjects with
blind loop syndrome. Serum folate levels may be
higher than normal and independent of vitamin
Bj 2 status per se, and urinary excretion of folate
may also increase. As mentioned earlier, bacteria
in the upper gut lumen are probably capable of
synthesizing significant quantities of folates, which
could subsequently be absorbed by the host. In
subjects with excess bacterial growth in the ileum,
this absorption is less likely, 251 probably because
of the relatively poor absorption of folate in this
region of the intestinal tract4 7 or perhaps because
of the inhibition or degradation of "conjugase"
enzymes (pteroyl-L-glutamate-poly-7-L-glutamate
carboxypeptidases) required for folate absorption.
By contrast, folate deficiency frequently occurs
in tropical sprue; this condition may result from
an abnormal flora in the small bowel. 3 7 ' 1 0 8 ' 1 9 4
Folate conjugase impairment has been implicated
January 1977 235
 in the development of folate deficiency in tropical
sprue; the finding that products of bacterial bile
acid degradation inhibit intestinal conjugase in
vitro seems to support this notion. 46 These
products of bacterial colonization of the ileum
could undergo enterohepatic circulation and
interfere with folate polyglutamate hydrolysis in
the upper small intestine. However, normal
enzymatic activity has been observed in both the
succus entericus 287 and in jejunal biopsies of
subjects with the syndrome. 250 While the etiology
of tropical sprue is by no means established, a
majority of the evidence indicates an infectious
agent. Bacteriological studies of upper bowel
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flora 194 show contamination by coliforms.
Recently Klebsiella pneumoniae and Enterobacter
cloacae have been identified as dominant
organisms in the jejunal flora of affected
individuals. 288 It is not known whether the
disease symptoms result from bacterial overgrowth
or whether the latter results via the damaging
action of enterotoxins in the intestinal mucosa.
C. Host Defenses
Animals reared under germfree conditions
usually exhibit marked susceptibility to various
forms of stress, as compared to conventional
controls. 145 For instance, they show much less
resistance when first challenged by experimental
or accidental infections. This deficiency of
response has been interpreted as evidence that
indigenous flora play a role in the development of
normal host defenses. Resistance to infection can
be increased by association of germfree animals
with the proper microflora. In some cases, even
monoassociation is a sufficient stimulus for
immunological development. Although germfree
mice contain only about one third of the potential
antibody-forming cells found in conventional
controls, association with indigenous flora results
in an increase in the amount of mucosal lymphoid
tissue. 4 5 ' 9 0 ' 1 9 7 A similar development is seen in
the normal association of conventional neonatal
mice and their indigenous flora.
Specific pathogen-free (SPF) mice form
antibodies which react with indigenous coliform,
(Bacteroides or Lactobacillus) antigens following
vaccination with homologous strains. Adult
control animals (unvaccinated) contain natural
antibody-forming spleen cells reactive to indi-
genous bacterial antigens, but only in lower
numbers. SPF mice show a stronger immune
236 Critical Reviews in Food Science and Nutrition
response after vaccination with nonindigenous
bacteria. Germfree mice exhibit only a moderate
immunological response after monoassociation
with an indigenous Bacteroides sp. Thus, it seems
that the mouse is less responsive to members of its
indigenous flora than to transient or pathogenic
species.9 °
Examination of the intestinal tract of germfree
animals has revealed a relative lack of plasma cells.
The normal intestine is replete with these cells
which produce IgA, the major coproimmuno-
globulin.112 Also, germfree rats lack the salivary
lysozyme and associated leukocytes present in
normal animals.
The gut flora may also be instrumental in the
elaboration of human anti-A and anti-B isoanti-
bodies. s06 An Escherichia coli strain with high
human blood group B activity was fed to healthy
human subjects and to persons with intestinal
disorders. Eighty percent of the diseased subjects
of blood groups O and A responded with increases
in anti-B antibodies (often de novo in the case of
infants), while more than one third of the healthy
subjects exhibited isoantibody increases.
Numerous enterobacteria are known to possess
blood group activity, although this has not been
readily demonstrable in fecal specimens.
An intriguing and poorly understood problem is
the degree of influence the host's immunological
system exerts on the colonization of the intestinal
tract by the indigenous flora. The immunological
milieu of the host may protect it from hostile
pathogenic microbes as well as exert selective
pressure for the development of the indigenous
flora.45
In addition to their indirect role in host
defense, indigenous microbiota constitute a major
defense against enteric infection. The effects of
antibiotic therapy in man and animals and
experimental work with gnotobiotic animals have
clearly revealed the protective function of the
normal intestinal flora.145
Most of the earlier studies have been reviewed
by Freter. 174 Attempts to induce intestinal
infection in experimental animals by using Vibrio
or Shigella were uniformly unsuccessful when
there was no pretreatment with oral doses of
streptomycin. A number of streptomycin-resistant
pathogens could be successfully implanted by this
approach. When administered simultaneously with
certain challenging pathogens (e.g., V. cholerae, S.
flexneri, Pseudomonas aeruginosa, and Staphylo-
 coccus aureus); streptomycin-resistant strains of E.
coli completely inhibited the implantation of the
pathogens. Other pathogens (viz., Candida albicans
and Salmonella enteritidis) were unaffected by E.
coll In some cases implantation of E. coli
provided greater prophylaxis than that induced by
immunological methods. Further studies with
implantation of P. aeruginosa following X-
irradiation of mice indicated that E. coli could
provide complete immunity to Pseudomonas
infection under conditions where normal immune
response (even after artificial vaccination) was
wholly ineffective. Although E. coli has been used
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in many of these experiments, there is no direct
evidence indicating that it is responsible for any
significant part of these antagonistic activities in
the normal intestinal flora. The mechanisms
involved in the normal microbiota's reaction
against pathogenic invaders are not known with
certainty. Apan from direct competition for
available nutrients, antagonistic actions probably
result in part from the production of inhibitory
fatty acids by the normal anaerobic flora.
Implantation of gnotobiotic mice containing E.
coli with cecal homogenates from a normal mouse
resulted in a large reduction (~1000:1) in the
cecal E. coli population concomitant with the
appearance of short-chain fatty acids.
The host's local immune system also seems to
play a role in the control of enteric pathogens. In
studies of experimental cholera, Freter 1 7 3 ' 1 7 6 >
177
and Fubara and Freter 178 have identified two
separate mechanisms. One is involved in the
inhibition of adsorption or adhesion of bacteria to
the mucosa, mediated by local antibodies in-
cluding secretory IgA. 179 The other mechanism is
antibacterial in that it exerts bacteriostatic or even
bacteriocidal action upon bacteria on the mucosal
surfaces.17S This mechanism requires antibacterial
antibodies and viable mucosal cells; it is
independent of complement. 179 Studies of
experimental cholera in baby mice 443 have also
provided evidence for an antibody-mediated
vibrio-killing mechanism. Starvation had a strong .
inhibitory effect on the rate of killing, but this
could be reversed by the administration of
histamine or prostigmine to the starved mice.
Presumably, secretions of the epithelial mucosa
milieu are associated with the antivibrio action.
While such local immune mechanisms un-
doubtedly play an important role in host defenses,
it is still poorly understood which parameters
govern the relative effectiveness of microflora
antagonisms and local immunity throughout the
GI tract. As suggested by Freter 1 7 4 ' 1 7 7 and
Shedlofsky and Freter, 482 local immunity may
primarily affect mucosa-associated, nonluminal
types, while bacterial antagonisms act mainly on
luminal species, resulting in their elimination via
the feces. In combination, these two mechanisms
might act synergistically, providing an efficient
control of bacterial growth in the intestine. Thus,
gnotobiotic mice monoassociated with V. cholerae
contained 109 to 10 1 0 cells per cecum, and this
number could be reduced to about one third by
vaccination. However, in germfree mice implanted
with antagonistic bacteria prior to infection with
V. cholerae only 10 s to 108 vibrios were found
per cecum. In this case, immunization led to a
significantly greater decrease in the vibrio count
(1/5 to 1/60) as compared with nonimmunized
controls.4 8 2 These findings appear to support the
synergy hypothesis of Freter, in that local
antibacterial immunity was considerably enhanced
by the operation of bacterial antagonism. Other
findings consistent with the hypothesis were
obtained by Kenny et al.,2 8 3 who found that local
immunity has no effect on the implantation of
streptomycin-resistant E. coli 0127 in mice. The
animals were treated with streptomycin prior to
challenge, thus abolishing the antagonism of the
normal flora for the invader.
Studies of the succession of vibrio serotypes in
monoassociated m i c e 4 4 8 ' 4 8 2 suggest another role
of local immune response. It enables antigenic
mutants to outgrow the parent strain of the
predominant gut flora when the host becomes
immune to the latter. Recent findings by Fubara
and Freter 178 indicate that the intestinal flora can
affect the availability of serum derived antibodies
in the gut lumen. Antibodies in the lumen of
germfree mice immunized locally or parenterally
were exclusively secretory IgA, whereas similarly
immunized conventional animals had appreciable
titers of IgM and IgG (as well as IgA) in their
intestines. Thus, the intestinal flora, while possibly
controlled by local immune mechanisms and
acting synergistically with them, paradoxically
may also to some extent control local immunity.
D. Aging and Survival
The idea that gut flora may play a role in
limiting the human life span was popularized by
Metchnikoff354 and his "orthobiosis" theory. In
January 1977 237
 recent years, several findings have initiated a
reexamination of this concept.
Two studies of longevity in the germfree state
have been conducted. The first 198 was carried out
with 300 germfree mice and an equal number of
conventional control mice. Germfree males and
females survived an average of 723 and 681 days,
respectively. For conventional mice, the survival
figures were 580 and 516 days, respectively. These
survival rates were relatively constant throughout
the course of the experiment.
In the second study,5 3 3 50 germfree mice and
conventional controls were tested. Survival rates
were 556 and 535 days for male and female
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germfree mice, respectively, and 536 and 547 days
for control animals. While both groups in this
latter study showed equal survival in the first
trimester, germfree mice exhibited increased
survival in the second trimester and increased
mortality in the final trimester; thus, the mean
figures appear more similar than observed in the
previous study. This latter finding cannot be
explained easily; it may have resulted from the
fact that a different mouse strain was used and a
smaller number of animals were involved. Also,
differences in the diet may have had an effect. The
autoclaved diet employed in both studies was
considered inadequate for aging studies. Therefore,
while there are indications that a germfree state
prolongs life span, the data are inconclusive. More
significant in the studies cited was the different
patterns of lesions found at death in the two
groups. The prevalent lesions in germfree mice of
both studies were related to distension of the
cecum with a lack of muscle tone and bowel
propulsive movements. Conventional control
animals had primarily inflammatory lesions.
With regard to the early theories of
Metchnikoff concerning the effect of intestinal
lactobacilli on longevity, it is of some interest that
dairy products, primarily of the yogurt type,
containing Lactobacillus acidophilus or other
implantable intestinal lactobacilli are still being
marketed in several countries.
In the U.S. an unfermented, so-called "Sweet
Acidophilus®"milk has recently been developed
by Prof. M. L. Speck and colleagues at North
Carolina State University. The Sweet Acidophilus
product results from adding a concentrated culture
of a bile resistant L. acidophilus strain to cold
pasteurized milk. Viable concentrations of several
hundred million cells per ml are present in the
238 Critical Reviews in Food Science and Nutrition
marketed product and human feeding trials have
demonstrated definite increases in fecal lactobacilli
following consumption.5 ° 2 a
While many investigators feel that the presence
of adequate numbers of L. acidophilus or other
intestinal lactobacilli in the intestinal tract are a
requisite of healthy gut function, it has been
difficult to fully elucidate the specific actions of
these flora components.5 0 2 a > b It is known that
some lactic acid bacteria produce antibiotic
substances (e.g., nisin, diplococcin, and lactolin).
Lactobacillus acidophilus probably owes its main
antimicrobial activity to end products of
metabolism such as lactic and other organic acids
and hydrogen peroxide. However, it also produces
small amounts of other antibiotic substances
including acidophilin, lactocidin, and acido-
lin. 3 5 9 a Antagonisms of indigenous lactobacilli
including strains of L. acidophilus for entero-
pathogenic E. coli and pathogenic salmonellae and
staphylococci have been well documented and are
discussed in a recent review by Sandine et a i 4 5 S a
Various lactobacillus preparations have been
employed in a number of intestinal disease
therapies including infant diarrhea due to anti-
biotic resistant E. coli, reestablishment of normal
intestinal flora following antibiotic therapy, and
treatment of portal-systemic encephalopathy.
However, such Lactobacillus therapies have
frequently been unsuccessful1048 and much
further research will doubtless be required to
identify and select particular strains or biotypes of
L. acidophilus for use in dietary or therapeutic
preparations. Factors such as bile resistance and
host specificity (i.e. compatibility) may be of
major importance in achieving the desired
results. 1843
In view of recent advances in the cultivation,
enumeration, and identification of many human
gut microflora components and an increasing
appreciation of the complexity of their inter-
relationships and interactions with host
physiology, it is perhaps an opportune time for a
renaissance of research on man's indigenous
lactobacilli.
E. Toxic Metabolites
The possibility that the indigenous flora may
influence man's survival through the production of
toxic metabolites has yet to receive conclusive
support. Yet a number of findings point to such a
role in several disease processes. The numerous
 microbial products of potential toxicity include
ammonia, physiologically active amines, phenols,
phenolic acids, and ethanol. These metabolites,
which result from the digestion of meat, are
absorbed and subjected to hepatic detoxification
before excretion in the urine. The concentrations
of the substances are usually well below toxic
levels in the systemic circulation. Conditions
resulting in impaired liver function or in circula-
tory by-pass can lead to acute toxicity, as seen in
the hepatic coma of cirrhotic patients.
1. Ammonia and Amines
As mentioned earlier, intestinal ammonia is
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thought to be primarily a product of microbial
urea hydrolysis. Both urea and ammonia undergo
enterohepatic circulation, with urea being syn-
thesized in the liver. Several fecal microorganisms
have urease activity (e.g., Klebsiella, Proteus, and
Bacteroides), but species in close association with
the mucosa may play a dominant role. Wolpert et
al. s 6 3 found that urea provided via mesenteric
circulation was more efficiently hydrolysed than
that given intraluminally. Portal-systemic
encephalopathy is thought to result mainly from
bacterial degradation of nitrogenous compounds in
the large bowel. Hepatic coma in hyperam-
monemia results from the direct inhibition of
brain oxidative metabolism caused by
ammonia. 469 A reduction in dietary protein
provides less urea for microbial urease action.
Different sources of protein also influence am-
monia production, with milk and cheese protein
producing lower levels than meat. 139 It is not
known whether this is a direct effect of an altered
substrate protein or an indirect result of the gut
flora's "change to less ureolytic types (e.g.,
lactobacilli and bifidobacteria). However, direct
attempts to alter the flora by feeding powdered
cells of nonureolytic types have had little success,
thus somewhat discounting the latter notion.
Production of ammonia in the bowel may be
decreased by shortening the fecal transit period:
the rationale of various purgative palliatives. The
use of lactulose therapy has been more promising.
Lactulose ((3-1,4-galactosylfructose) is not hydro-
lysed by the mucosal /3-galactosidase, but is
fermented by a variety of microbes in the large
bowel (enterococci, bifidobacteria, and lacto-
bacilli). The resultant acidic end products lower
colonic pH (fecal pH values of 4 to 5) and .retard
or even reverse portal ammonia absorption by
intraluminal production of ammonium ion.
Decarboxylation of amino acids by gut micro-
flora leads to the production of a variety of
amines, some still unidentified (e.g., tryptamine,
histamine, cadavarine, agmatine, octopamine, and
piperidine). Tyramine may have an enteric
origin12S in addition to arising endogenously.26
Failure of the liver to detoxify such amines may
also contribute significantly to hepatic coma
associated with cirrhosis. The administration of
the antibiotic oxytetracycline abolishes these
amines from the blood. Furthermore, while certain
amines arising in the gut are excreted in the urine
(viz., histamine, tyramine, octopamine, and
piperidine), treatment of cirrhotic patients with
monoamine oxidase inhibitors results in excretion
of at least four additional amines. 403 The urinary
excretion of the normal amines was also greatly
increased in cirrhotic subjects. Thus, it seems
reasonable to suppose that the inactivation or
by-pass of hepatic monoamine oxidase plays a key
role in amine toxicity. One may wonder if the
action of amines (or possibly their secondary
metabolites) on detoxifying enzymes might be
analogous to the 'vicious circle' proposed by
Iieber et al.3 * 3 for ethanol hepatotoxicity. Thus,
increasing toxic exposure leads to decreasing
ability to detoxify, etc.
The pharmacologic actions of various amines
are well known. Tyramine (like cadaverine and
putrescine) is a depressor, while histamine and
several other monoamines are pressors. The fact
that histamine can stimulate gastric acid secretion,
as well as possessing general inflammatory activity
in increasing vascular permeability, has led to the
suggestion that intestinal histamine is involved in
the etiology of peptic ulcer associated with cir-
rhosis.
2. Phenols/Tyrosine
The production of a variety of phenolic
compounds by the enteric microflora has also been
demonstrated in vitro and in vivo. Many of the
studies have concerned the fate of dietary tyro-
sine. An increase in dietary tyrosine leads to
greater excretion of urinary phenols.3 The anaero-
bic metabolism of tyrosine by rat cecal microflora
proceeds via p-hydroxyphenylpyruvic and p-
hydroxyphenylacetic acids and yields p-cresol
and phenol. 32 " 34 While the formation of phloretic
January 1977 239
 acid from tyrosine has been observed, it is unlikely
that this is an intermediate of phenol formation
(as was previously thought), since no phenols are
formed from it by rat cecal extracts,3 5 and it is
readily dehydroxylated to 3-phenylpropionic acid
by sheep rumen microorganisms.478 It is not
known which components of the gut flora are
involved in tyrosine degradation, although
formation of phenol from tyrosine has been
demonstrated in Escherichia coli110 and Clost-
ridium tetanomorphwn.6B
Apart from tyrosine metabolism, gut microflora
are known to effect a variety of metabolic
conversions of phenolic acids (e.g., phenylacetic,
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phenylpropionic, and cinnamic acid derivatives),
many of which are plant metabolites and dietary
c o n s t i t u e n t s . 4 6 4 ' 4 6 6 Decarboxylation of
p-hydroxyphenylacetic acid yielded p-cresol, while
p-hydroxycinnamic acid (p-coumaric acid) gave
p-ethyl- and p-vinyl- phenols. p-Hydroxyphenyl-
propionic acid was not decarboxylated. Other
substrates and phenolic products of anaerobic
microbial metabolism include: protocatechuic acid
-> catechol; gallic acid -*• pyrogallol -*• resorcinol;
homoprotocatechuic acid -> p-methylcatechol;
caffeic acid -»• p-vinylcatechol, p-ethylcatechol;
and ferulic acid -*• p-ethylcatechol, p-vinylguaiacol.
The intestinal bacteria associated with these
decarboxylating activities include two Bacillus
s p p . , 2 7 1 ' 5 0 0 A erobacter aerogenes,'6 6 and
Streptococcus faeciumf" ° l
The role of enteric phenols in human disease is
uncertain. Some of the phenols reaching the liver
via the portal vein are conjugated prior to systemic
circulation and urinary excretion. Circulating
phenols may play a role in human carcinogenesis.
Small quantities of phenol, 2-ethylphenol, and
p-cresol promote development of benign and
malignant tumors when applied to mouse skin
pretreated with suitable carcinogens.3 6 >5 9 >5 6 9
Furthermore, Roe and Grant 2 0 4 ' 4 3 2 found
that germfree status protected male C3H mice
from development of hepatic tumors in response
to dimethylbenzanthracene administered neon-
atally. Germfree mice of both sexes were found to
have a lower incidence of malignant lymphoma,
mammary, ovarian, and uterine tumors. This is
consistent with the postulated role of the gut flora
in producing carcinogenic or cocarcinogenic
substances. Taken together with other findings on
protein intake and carcinogenic incidence, 139 the
concept is at least plausible.
240 Critical Reviews in Food Science and Nutrition
Since certain phenols have convulsant acti-
vity 13 (e.g., phenol and catechol), they may also
play a role in hepatic coma attendant with
cirrhosis.
In addition to simple phenols a number of
other enteric microbial metabolites (Table 1),
although produced only in minute quantities, are
thought to play a role in the etiology of can-
cer. 138 The large majority of cancers in man are
of environmental origin and affect the epithelium
and related tissues (intestine, lung, and
breast). 85 ' 86 The environmental carcinogens or
precarcinogens may be of plant, fungal, bacterial,
synthetic, or even of endogenous origin and may
be potentiated via the gut flora and/or the hosts'
detoxifying enzymes.3 3 4 ' 3 6 4
3. Colorectal Cancer
Several authors have recently reviewed the
numerous factors involved in gastrointestinal 43 '
321
and particularly colorectal cancer. 4 4 ' 8 2 " 8 4 '
54 0,568,570 y ^ ^ ^ tQ ^ j ^ ^ p r e d js.
posing factors include a "westernized" urban life
style, high fat and protein diet, and high
anaerobe-aerobe fecal flora. Primarily a disease of
later life, the age dependence of colorectal cancer
correlates well with diverticular disease of the
colon, cholesterol gallstones, ischemic heart
disease, and hiatus hernia. Protective factors may
include dietary fiber or an unavailable carbo-
hydrate (which is thought to suppress bile acid
degradation, shorten intestinal transit times, and
result in large soft stools) and a vegetarian or low
meat diet. 83 Recent highly detailed bacteriological
studies carried out in the U.S. have failed to find
significant differences in the composition of the
colonic microflora among different risk groups of
human subjects. 1 6 5 ' 3 7 0 This may indicate that
each population (regardless of risk) harbors in-
testinal flora associated with carcinogenesis or that
significant differences in the flora are too subtle
to detect with present technology.
4. Secondary Bile Acids/Steroids
The most studied etiological agents are the
microbially produced biliary steroid derivatives,
nitrosamines and amino acid metabolites-. Some-
what less studied, but perhaps of equal signifi-
cance, are conjugated carcinogens or precarcino-
gens hydrolysed by the gut flora.
Some steroids (including deoxycholic acid, 105
estrone, 116 apocholic acid, 295 3/3-acetoxy-bis-

Possible Sources of Dietary Procarcinogens or Promutagens That Might Be
Potentiated by Gut Microflora
Source
Protein related
Tryptophan
Tyrosine
Lysine and Arginine
Methionine
Lipid related
Lecithin
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Bile acids and sterols
Plant glycones
Cycasin
Hydroxylated
anthraquinones
nor-As-cholenic acid, and 7-dehydrochoIester-
ol 2 9 6 ) are carcinogenic in mammals. Some car-
cinogenic activity has even been noted for
cholesterol itself238 and its a-oxide. 77a
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 among differ-
ent geographical and ethnic populations correlates
well with the incidence of colorectal cancer. 139
Drasar and Hill estimate United Kingdom lifetime
excretion of deoxycholate to average about 1.5
kg/50 years per individual; thus, with an incidence
of 18 cases per 100,000 individuals, deoxycholate
need only be a weak carcinogen to play a
significant role in colon carcinogenesis. Deoxy-
cholate may undergo only slight conversion to a
I on
more potent carcinogen.
It is uncertain if acetoxy-bis-nor-A5-cholenic
acid is produced by action of the gut flora,
although its 3/3-hydroxy derivative has been re-
ported to be a product of microbial cholesterol
degradation.478 Similarly, it is not known if
apocholic acid (3a, 12a dihydroxy-A8 -5/3-
chqlanoic acid) is produced by gut bacteria from
cholic acid. However, in view of the types of
microbial bile acid transformations discussed
earlier,3 5 7 both of these reactions appear likely.
Numerous minor bile acids in the feces have not
yet been described.
The carcinogenicity of estrone has been demon-
TABLE 1
Intermediates and products
Carcinogenic metabolites
Cocarcinogenic phenols
Cyclic secondary amines, pynolidine, piperi-
dine, A^-nitrosamines
Ethionine
Dimethylamine, Mnitrosamines, methylamine,
dimethylhydrazine
Carcinogenic or cocarcinogenic metabolites
Methylazoxymethanol
Emodin, alizarin, purpurin, anthragallol
strated in rats. 1 1 6 Breast tumors induced in these
animals were of epithelial origin and were com-
pletely dependent upon the external source of
estrogen. Hypophysectomy conferred complete
immunity to mammary tumor induction and
resulted in total regression of established tumors,
indicating that pituitary hormones play a role in
oncogenesis. Estrone formation from A l > 4 -
androstadien-3,17-dione via oxidative C-10
demethylation has been observed in vitro in
Bacillus cyclooxidans, Pseudomonas testosteronii,
and Nocardia restrictus, as well as in mammalian
systems. A strain of Escherichia coli has been
shown to produce estradiol from A4-androsten-
3,17-dione. A strain of Clostridium paraputrifi-
cum, isolated from the same substrate from the
human intestine, produced 17-methoxy-A1'3 >s
(10)
-estratrien-3-ol. 186 ' 187 These conversions
have not been demonstrated in vivo. If these
conversions occurred in vivo, methoxy "estradiol"
produced in the colon would be absorbed, con-
jugated by the liver to the 3-sulfate and/or the
17-glucuronide with attendant 17-O-demethyla-
tion, and excreted in the urine. Such a microbial
metabolite would not be readily detectable in
urine specimens.
The role of the gut flora of humans in
metabolizing steroids remains relatively unclear.
The end products of endogenous metabolism of
corticoids in man are excreted mainly via the
urine; fecal excretion is of "quantitatively" minor
January 1977 241
 importance. Biliary corticosteroids are reabsorbed,
but undergo microbial metabolism in the intestine.
Studies with mixed human fecal flora in vitro 54
indicate that 21-dehydroxylation is a main ele-
ment of this metabolism in man. The model
substrate 11-deoxycorticosterone was converted to
3a,21-dihydroxy-5/3-pregnan-20-one, 3a-hydroxy-
5j3-pregnan-20-one (pregnanolone), and two un-
identified compounds. Conceivably, gut bacteria
might produce polycyclic aromatic hydrocarbons
from these and other steroids. The types of
reactions which could cause this to occur have
been mentioned above. Thus the ultimate forma-
tion of substituted cyclopenta [a] phenanthrenes
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(cyclopentadienophenanthrenes) from the bile
acid in other steroid nuclei is a possibility.
However, this has not yet been demonstrated.
Both deoxycholic and cholic acids may be
converted into 20-methyl-cholanthrene (a pofent
carcinogen), by using drastic chemical proce-
dures 49 (including pyrolysis at 330°). The rela-
tionship between structure and carcinogenicity in
more than 20 cyclopenta [a] phenanthrenes and
related derivatives has been investigated by
Croft 106 and Coombs et al. 1 0 7 Approximately
half of the substances exhibited carcinogenic
activity when painted on the skin of mice. Among
the more active cyclopenta [a] phenanthrenes were
1 l-methyl-17-ol, 7-methyl-ll-methoxy-17-one,
15,16-dihydro-ll-methyl-17-one, and a chrysene
analogue (1,2,3,4-tetrahydro-l 1-methylchrysene-
1-one). The presence of small electron-donating
methyl or methoxy groups at C-ll appears to
promote carcinogenicity in this class of polycyclic
aromatics. While no naturally occurring cyclo-
penta [a] phenanthrenes have been found, it would
seem reasonable to search for them or their
metabolic precursors, particularly in subjects with
high risk of colorectal cancer.
5. Tryptophan
A number of authors have suggested a relation-
ship between the increased excretion of trypto-
phan metabolites and the incidence of bladder
cancer. 73 >S7S In studies by Dunning and
Curtis 148 and Dunning et al., 149 the addition of
DL-tryptophan to a synthetic diet containing
2-acetylaminofluorene increased the incidence of
bladder tumors 100% in rats. Similar results were
obtained with indole. Marked focal hyperplasia of
the transitional epithelium of the bladder was
produced in dogs fed a diet supplemented with
242 Critical Reviews in Food Science and Nutrition
DL-tryptophan.415 Various tryptophan metab-
olites have.a carcinogenic effect when implanted
directly in the bladder.73 These metabolites in-
clude L-kynurenine, acetyl kynurenine, 3-hy-
droxyanthranilic acid, xanthurenic acid, 8-methyl
ether of xanthurenic acid, 8-hydroxyquinaldic
acid, and quinaldic acid. Subsequent tests with
indole administered orally and subcutaneously
revealed malignant tumors of the lymphoreticular
system or leukemia.151 ' 4 * 7 More recently, Oyasu
et al. 3 9 4 reported that indole administered with
2-acetylaminofluorene enhanced the induction of
bladder tumors in hamsters.
It is not known how tryptophan metabolites and
other compounds participate in the carcinogenic
process in the bladder. Both 3-hydroxykynurenine
and 3-hydroxyanthranilic acid are mutagenic in
cultured mammalian tissue cells. There is a
strong relationship between mutagenesis and car-
cinogenesis. Tryptophan or its metabolites may act
as cocarcinogens or promoters of bladder carcino-
genesis. 414 ' 415 The findings of Bryan and Spring-
berg 74 suggest that subcutaneous administration
of xanthurenic acid-8-methyl ether induces blad-
der tumors in mice only if the animals have
cholesterol pellets in the bladder; this demon-
strates the limitations of the implantation tech-
nique and the complexity of the bladder carcino-
genic process. Thus, xanthurenic acid-8-methyl
ether and possibly other tryptophan metabolites
are "incomplete" carcinogens requiring the in-
fluence of other factors.
The chemical instability of tryptophan metab-
olites (particularly the aminophenols) has been
noted by Bryan.73 Aminophenols are generally
highly autooxidizable and yield a variety of
products. Pipkin et al. 4 0 5 suggested that such
oxidative phenomena might play a critical role in
carcinogenesis. Three metabolites (viz.,
3-hydroxykynurenine, 3-hydroxyanthranilic acid,
and 3-hydroxy-2-aminoacetophenone) definitely
undergo oxidative dimerization to substituted
phenoxazinones. The oxidation of 3-hydroxy-
anthranilic acid is dependent upon oxygen partial
pressure (pO 2 ), pH, and the presence of certain
metal ions (such as copper); it could certainly
degrade substantially under conditions existing
in the bladder. Oxidative dimerization of 3-
hydroxyanthranilic acid could produce two
phenoxazinones (viz., cis or trans dicarboxylic
phenoxazinones). Nishimura et al.3 8 2 found that
overnight incubation of 3-hydroxyanthranilic in
 urine at 37° in the presence of air leads to the
formation of cinnabarinic acid (2-amino-3-oxo-
3H-phenoxazine-l,9-dicarboxylic acid), the cis
dicarboxylic phenoxazinone. Furthermore, greater
quantities of cinnabarinic acid were found in the
urine of bladder cancer patients than in normal
subjects; oral administration of ascorbic acid
prevented the formation of cinnabarinic acid in
both groups. It is not known how much cinnabar-
inic acid is formed in vivo, because the pO2 values
for the normal bladder rarely exceed 36 mm Hg.
Also, it is not known whether cinnabarinic acid is
formed by enzymatic or nonenzymatic means.
Enzyme systems of animal and microbial origin
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catalyze the dimerization of aminophenols to
phenoxazinones. Cinnabarinic acid and cinnibarin
occur in the fungus Polystictus sanguineus. The
enzyme "phenoxazinone synthase" (which
dimerizes substrates such as 3-hydroxyanthranilic
acid, 4-methyl-3-hydroxyanthranilic acid, and
3-hydroxykynurenine) is widely distributed and
occurs in Streptomyces antibioticus, the source of
actinomycin. In vitro the enzyme catalyses the
dimerization of 4-methyl-3-hydroxyanthranilic
acid to actinocin, the chromophore of actinomy-
cin. Phenoxazinones are also formed from o-
aminophenols by 'L-DOPA-oxidase' of fungal or
mammalian origin. Because of the wide occurrence
of enzymes capable of catalysing the formation of
phenoxazinones, it is conceivable that enzyme
activity in the urine or bladder mucosa may play a
significant role in carcinogenesis.
Cinnabarinic acid has carcinogenic effects when
the bladder implantation technique is used; other-
wise, little is known of its carcinogenic potential.
However, the carcinogenicity of actinomycin has
been convincingly demonstrated.19 Many tricyclic
heteroaromatic compounds react with DNA in
vitro, 378 and some phenazines70 and at least one
phenoxazine 68a have exhibited mutagenicity in
Salmonella typhimurium.
Of the tryptophan metabolites known to occur
in the urine, indole, quinaldic, and 8-hydroxyquin-
aldic acids are solely the result of intestinal
bacteria. The flora may also contribute to the
development of other metabolites found in urine.
Indole formation is widely distributed among
bacterial species(notably Escherichia, coli, Paraco-
lobactrum coliforme, Proteus vulgaris, Bacteroides
sp., and Sphaerophorus varius)124 and may be
accomplished either by degradation of indole
glycerol phosphate or by the hydrolysis of trypto-
phan by the enzyme tryptophanase. A recent
study of 23 intestinal anaerobes98 reported that
only four types are capable of producing indole
from tryptophan (viz., three strains of Bacteroides
fragilis ss. thetaiotaomicron and a Citrobacter sp.).
The tryptophanase of these indole-positive anaer-
obes was induced by tryptophan and repressed by
glucose. Rats fed an all meat diet contained higher
tryptophan content and greater tryptophanase
activity in their feces than did animals on a normal
diet. These data further suggest that the increased
enzyme activity results from the enzyme's in-
ducible nature and not from removal of an enzyme
inhibitor present in the normal diet. 98 In view of
indole's rapid absorption via large bowel epithelial
cells and its carcinogenic or cocarcinogenic poten-
tial, indole may also play a role in colorectal
cancer. 171
6. N-Nitrosamines
A^-Nitrosamines exhibit potent carcinogenesis
when administered to laboratory animals. 3 3 3 ' 3 3 4
They may be produced by the acid-catalyzed
reaction of secondary amines and nitrous acid;
most studies have suggested that the stomach may
be the site of synthesis, 455 > 4 8 0 as the optimal pH
value is 3.4. 3 6 3 Several types of enteric bacteria
Af-nitrosate diphenylamine at neutral pH in the
presence of nitrate. 3 1 > 2 2 7 - 2 4 4 > 4 5 4 >523 These in-
clude enterobacteria, enterococci, clostridia,
bacteroides, and bifidobacteria. The efficiency of
nitrosation varies from 68% for the weak base
diphenylamine to less than 0.01% for dimethylam-
ine. It is still uncertain whether this N-nitrosa-
tion is an enzymatic or nonenzymatic reaction.
Strains of Escherichia coli may form nitrosamines
from nitrate or nitrite and secondary amines;
several other bacteria carry out the reaction with
nitrite, suggesting that nitrate reduction is a
preliminary step in nitrosation. The occurrence of
nitrosation at pH 6.5 only in the presence of
bacteria and the demonstration of strain
specificity suggest that an enzymatic reaction is
involved. As yet, no cell-free enzymatic activity
has been demonstrated.
In a normal diet, nitrate is found in vegetables,
cured meats, and some cheese products; it may
also be present in relatively high concentrations in
drinking water. 161 Three secondary amines are
produced in the large intestine, viz., dimethylam-
ine (from lecithin or choline), piperidine (from
lysine), and pyrrolidine (from arginine). All are
January 1977 243
 potentially nitrosatable by gut microflora. How-
ever, studies in rats show that nitrate is rapidly
excreted in the urine and presumably is absorbed
in the upper small intestine before reaching the
large bowel microflora. Therefore, little if any
nitrosation would occur enterically. The urinary
bladder of individuals with urinary tract infections
is probably the most likely site of microbial
nitrosation. In a recent study, 223 a high correla-
tion was found between the ability of a variety of
bacteria to nitrosate dimethylamine in vitro and
their human pathogenicity. Pathogenic E. coli
(H-140, H-146, and H-158), Pseudomonas aeru-
ginosa, Klebsiella pneumoniae, Proteus mirabilis,
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Proteus vulgaris, and Proteus morganii are nitro-
saters; indigenous strains of E. coli and Proteus, as
well as a number of other indigenous enteric
species (e.g., Lactobacillus, Bacteroides, Propioni-
bacterium, Ruminococcus bromii, Eubacterium
aerofaciens, Peptostreptococcus productus, Bifldo-
bacterium sp., and Actinomyces parabifidus), did
not nitrosate dimethylamine.
Since E. coli is frequently a cause of urinary
tract infections and an efficient nitrosater of
dimethylamine, this hypothesis warrants serious
investigation, particularly in view of the potency
of the carcinogens found. Drasar and Hill 139 and
Hill and Hawksworth 244 have demonstrated in
vivo the production of nitrosamines in rats with
experimental E. coli bladder infection. The rats
were fed nitrate and piperidine or pyrrolidine;
N-nitrosamines were examined in urine collected
during a 48-hr period. No nitrosamine was
detected in the urine of rats fed only nitrate or
secondary amine. The production of urinary nitro-
samines in man has not yet been demonstrated.
Other studies by Drasar and Hill 139 have
shown that radioactively labeled nitrosamines
(dimethylnitrosamine and nitrosopiperidine) intro-
duced into the bladder were quickly absorbed into
the bloodstream. Using the label to determine
tissue distribution, they found the liver and
kidneys to be the major target organs of dimethyl-
nitrosamine in rats.
Subjects with gastric achlorhydria or anacidity
may provide a site where nitrosating bacteria,
nitrate, and secondary amines can exist simul-
taneously (although it is somewhat less likely than
in persons with urinary infection). In the former
case the stomach would contain both lower
numbers of bacteria (10 s to 107 per milliliter) and
a lower secondary amine concentration (dietary
244 Critical Reviews in Food Science and Nutrition
amines only). In addition, due to gastric emptying,
the incubation period would also be abbreviated in
comparison to bladder infection. Production of
nitrosopiperidine2 by bacteria has been demon-
strated in the isolated rat stomach. The normal
gastric pH in a rat is similar to that of an
achlorhydric human. Some epidemiologic data
indicates a relationship between gastric cancer and
drinking water with a high nitrate content. 111 >139
Pernicious anemia, which is frequently associated
with achlorhydria, may also predispose patients to
gastric cancer. Much of the evidence relating to
human risk is circumstantial. However, ill-defined
areas of environmental carcinogenesis will cer-
tainly be investigated in the near future, because
of recent increases in cancer incidence in the U.S.
7. Azo Compounds
Azo compounds are derivatives of aromatic
amines and are commonly used as synthetic
colorings in foods, Pharmaceuticals, and cosmetics.
Although they have been used for many years,
knowledge of their toxicity and possible genetic
hazards has developed slowly.4 8 s During the past
20 years, 12 FD&C dyes have been prohibited for
use in food and another three have been subjected
to limitations in their usage.14 The toxicology of
food colors in general has recently been reviewed
by Radomski; 413 the metabolism of azo com-
pounds in particular has been reviewed by
Walker.S36 The most important metabolic reac-
tion of azo compounds is reduction to component
primary amines. Azo reductase activity has been
demonstrated in the mammalian liver; it may
be the result (at least in part) of microsomal
NADPH-cytochrome C reductase. 2 3 5 ' 2 3 6 Sur-
prisingly, the dominant role of the intestinal
microflora in azo reduction has been discovered
only relatively recently. Radomski and
Mellinger4-16 were the first to realize the quan-
titative significance of microbial azo reduction of
water-soluble azo food dyes (Red No. 2 and 4 and
Yellow No. 6). Childs et al. 96 demonstrated azo
reduction in vitro with intestinal contents (minced
intestinal tissue or cultures of intestinal bacteria),
and Fore et al. 1 7 2 showed that intestinal azo
reduction was largely confined to the terminal
ileum and cecum of the rat and similar portions of
the pig intestinal tract.
The microbial reduction of azo dyes was first
demonstrated in vitro with lactic acid bacteria
 nearly 40 years ago. 65 A subsequent survey of 21
species and 14 azo compounds by Dieckhues128
showed that azo reduction was a fairly general
property of the species tested. Cell-free studies
with bacteria (e.g., Streptococcus faecalis) have
failed to yield a specific enzyme associated with
azo reductase activity (see further discussion
below, Section VI.C.l).
The subsequent metabolism of primary aro-
matic amines arising via enteric microbial azo
reduction has not yet been fully documented.
Certainly, some naphthylamines could be poten-
tiated to more serious toxicants or carcinogens by
hepatic microsomal enzymes. 1 9 ' 3 3 4 ' 3 6 1 The re-
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duction of four benzidine (di)azo dyes to free
benzidine (a known human bladder carcinogen)
has recently been demonstrated in the rhesus
monkey. Benzidine dyes are widely used in indus-
try and may present a health hazard via accidental
ingestion.430 Data on the carcinogenic risk of a
variety of azo compounds may be found in a
recent I.A.R.C. monograph.564
8. Glycosides/Cycasin
Apart from therapeutic glycosides, conjugated
metabolites reaching the large bowel originate
from dietary constituents and/or biliary secretions.
Numerous glycosides occur widely in plant mater-
ials, where they may serve a defensive function
(the glycosides or aglycones being toxic or anti-
microbial). A number of polyhydroxy-
anthraquinone purgatives occur as glycosides
(senna, rhubarb, and cascara sagrada), and their
pharmacological action on the large bowel depends
on release of the active aglycones by gut bacterial
action. Several hydroxylated anthraquinones
recently have been found to be mutagenic for
Salmonella typhimurium; these compounds could
participate in host carcinogenesis.70 Glucuronides
primarily arise from the normal detoxification
processes of the liver and are secreted in the bile.
The liver does not produce significant quantities of
other glycosides. The major glycosidases produced
by the gut microflora are /3-galactosidase, /3-gIu-
cosidase and j3-glucuronidase.
The /3-glucuronidase activity of Escherichia coli
has been recognized for some years.79 In a
comparative study, 226 50 strains of intestinal
bacteria from six groups (viz., E. coli, enterococci,
lactobacilli, clostridia, Bacteroides sp., and bifido-
bacteria) were tested for /3-glucuronidase, a- and
/3-glucosidases and a- and /3-galactosidases. Entero-
cocci produced large quantities of /3-glucosidase
and /3-galactosidase, but only small amounts of
/3-glucuronidase. E. coli produced large amounts of
|3-glucuronidase and /3-galactosidase, but little
|3-glucosidase. Nonsporing anaerobes (enterococci,
bifidobacteria, and lactobacilli) produced inter-
mediate levels of all enzymes except /3-glucuroni-
dase. The latter enzyme was produced in greatest
quantity by E. coli and clostridia; Bacteroides sp.
yielded an intermediate amount, while other non-
sporing anaerobes yielded very little. The lacto-
bacilli and clostridia exhibited the greatest
a-galactosidase activity, whereas clostridia, lacto-
bacilli, and bifidobacteria showed larger amounts
of a-glucosidase than the other groups. 226 The
same authors have estimated that the major action
of rat intestinal /3-glucuronidase occurs in bifido-
bacteria, followed by bacteroides and lactobacilli,
respectively. The total amount of /3-glucuronidase
in the small intestine of the rat is much greater
than in humans, rabbits, or guinea pigs; its content
is greatest in the colon and cecum of all animals.
In addition to harmless dietary glycosides and
the cathartic anthraquinone glycosides of uncer-
tain hazard, there are a few types of glycosides
which can be quite toxic if taken orally. 104
Amygdalin, present in bitter almonds, is extremely
toxic when ingested. The glucoside is probably
hydrolysed in vivo by plant and/or microbial
/3-glucosidases into mandelo-nitrile, which decom-
poses and produces toxic hydrogen cyanide.
Amygdalin is hydrolysed in vitro by several bacte-
rial genera {Proteus, Escherichia, Klebsiella,
Streptococcus, Lactobacillus, and Bifidobacte-
rium). Other cyanogenic glucosides such as dhurrin
(p -h y d r o xybenzaldehydecyanohydrin-glucoside),
lotaustralin (2-butanonecyanohydrin-glucoside),
and linamarin (acetonecyanohydrin-glucoside)
may be found in various foodstuffs (e.g., maize,
chick peas, sweet potatoes, yams, lima beans,
sorghum, and cassava. Two enzymes usually found
in cyanogenic plants (viz., /3-glucosidase and hy-
droxynitrile lyase) are also involved in HCN
production in vivo. Other important factors
include the speed of ingestion, the type of food
ingested along with the cyanogen, and the sub-
ject's ability to detoxify HCN.
The best documented example of intestinal
microbial potentiation of a potent carcinogen
involves cycasin. This toxic glucoside occurs in the
pulp and husk of the cycad nut and in other parts
of cycad trees (Cycadaceae), which are found in
January 1977 245
 tropical and subtropical regions. 346 The cycads
are a staple food for natives of these regions.
Investigations of the toxic principals in cycads
resulted in isolation of a series of glycosides with a
common aglycone, the principal component being
cycasin (methylazoxymethanol-/3-D -glucose).13 9 '
360
Laqueur et al. 3 0 1 found that unextracted
cycad nuts are highly carcinogenic when fed to
rats; the liver and kidneys are the major sites of
action. Tumor induction by cycasin has also been
noted in mice, guinea pigs, hamsters, and fish.302
Studies with gnotobiotic and conventional animals
revealed that cycasin was nontoxic when ingested
by germfree animals, and it can be recovered
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quantitatively and unchanged from the urine and
feces of these animals. However, in conventional
animals as much as 82% of the administered dose
was nonrecoverable. 300 ' 502 These results indicate
that the aglycone is the proximate carcinogen and
primary toxicant, and the intestinal microflora is
the source of deconjugating activity. Differences in
cycasin toxicity found among animals exposed to
comparable doses of cycasin also might be ex-
plained by individual variations in the intestinal
flora. Germfree rats monoassociated with bacteria
of known /3-glucosidase activity (viz., Strepto-
coccus faecalis and salicin positive and negative
varieties of Lactobacillus salivarius) showed a
strong correlation between cycasin toxicity and
microbial /3-glucosidase. Thus, S. faecalis with high
activity caused severe liver damage and death in all
animals while, the salicin negative L. salivarius was
indistinguishable from germfree control animals
fed cycasin. Salicin-positive L. salivarius which had
less /3-glucosidase than S. faecalis caused mild
toxicity . s 0 2
Target organs in cycasin carcinogenesis vary
with the species, age, and sex of experimental
animals; dosage and administration period of
cycasin; and routes of cycasin administration.142
When adult rats are subjected to gastric administra-
tions of cycasin or cycad extract (which contains
cycasin, neocycasins, and macrozamin), tumors
develop mainly in the intestinal mucosa. Animals
receiving rectal infusions of cycasin exhibited
tumors primarily in the lower large intestine, while
another group of animals with external colostomy
exhibited tumors in the small intestine and
proximal large intestine (which were not directly
exposed to cycad extract infusate). 541 These
studies (which are inconclusive) seem to indicate
that systemic and/or enterohepatic circulation of
246 Critical Reviews in Food Science and Nutrition
the aglycone proximate carcinogen may be in-
volved in cycasin carcinogenesis.
9. 1,2-Dimethylhydrazine
The only other documented case indicating gut
microbial potentiation of a known (pre)carcinogen
involves 1,2-dimethylhydrazine (DMH). Reddy et
a j 424,42s j n j ec ted DMH subcutaneously into
germfree and conventional rats and observed an
induced colonic tumor rate of 93% (14/15) for the
conventional rats and only 21% (5/24) for the
germfree animals. The results suggest that the gut
flora played a direct or indirect role in promoting
or accelerating colon tumor production by DMH.
In experiments with conventional rats, the same
authors 420 noted that injection of DMH resulted
in excretion of higher concentrations (milligram/
kilogram body weight/day) of acid and neutral
sterols when compared to control animals. The
mechanism of this effect is unclear but it did not
seem to influence colon carcinogenesis by DMH.
Animals fed a Purina Lab Chow® diet excreted the
largest quantities of sterols and bile acids, but
showed the lowest incidence of DMH-induced
colon tumors. The highest incidence of tumors was
noted in rats receiving a high-fat diet.
10. Ethionine
In 1938, Dyer first synthesized ethionine, the
S-ethyl analog of methionine, as an antagonist of
methionine. It is toxic in several rodent species,
causing morphological changes in various tissues
including the liver and pancreas. Chronic feeding
of 0.25% ethionine in the diet of rats leads to a
high incidence of hepatocellular carcinomas.508
Ethionine also inhibits the growth of various
microorganisms {Escherichia coli, Staphylococcus
aureus, and lactobacilli) and poliomyelitis virus.
The natural occurrence of ethionine in bacteria
was first reported by Fisher and Mallette. 167
Ethionine was found in cell extracts and in spent
growth media of E. coli, Bacillus megaterium,
Pseudomonas aeruginosa, and Aerobacter aero-
genes, but not in the alga Scenedesmus, the yeast
Saccharomyces cerevisiae, or bovine lympho-
sarcoma cells. Resting cell suspensions of E. coli
formed ethionine from 3 5 S0 4 = and [3 s S] -
methionine. Growing cells of E. coli labeled
ethionine rapidly with 3 S SO 4 = , but no incorpora-
tion of this amino acid into cellular proteins was
found.
In the rat, ethionine is incorporated into
 protein in several tissues. Alkylation of nucleic
acids by the S-ethyl group, particularly tRNAs,
occurs only in the liver of rats. The formation of
S-adenosylethionine in the liver of rats has also
been noted, although more recent evidence sug-
gests that this may not be the main ethyl donor
for tRNAs.39 J It is not known to what extent (if
any) mammals are exposed to ethionine produced
by the gut microflora.
11. Polycyclic Aromatic Hydrocarbons
The production of certain polycyclic aromatic
hydrocarbons (notably benzo(a)pyrene) by
Clostridium putride, Escherichia coli, and Bacillus
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badius in vitro has been reported. 3 3 6 ' 3 8 1 Simi-
larly, the significance of these findings vis-a-vis the
intestinal flora and mammalian exposure is un-
known.
In view of the rather speculative nature of
information regarding intestinal microbial potenti-
ation of procarcinogens, there seems to be a
pressing need for the development of new or more
sensitive techniques to investigate the problem.
During the past few years there has been a rapid
development of short-term microbial tests for
detecting chemical carcinogens as mutagens. These
tests (particularly those advocated by Ames and
co-workers employing specialized mutants of
Salmonella typhimurium) are relatively specific
and extremely sensitive (compared with other in
vitro methods) for detecting a broad range of
chemicals which are carcinogenic in man.9"11 The
tests usually employ mammalian microsomal prep-
arations in vitro to simulate the hepatic potentia-
tion of procarcinogens that is often required for
carcinogenesis.3 6 1 By employing a wide variety of
mutagenicity tests with appropriate tissue or
bacterial enzyme activation, it should be possible
to detect and identify mutagens and promutagens
formed by bacterial action in the gut.
12. Microbial Detoxification
While much of the above discussion has em-
phasized .the possibilities for microbial potentia-
tion of toxicants, mutagens, and carcinogens, it
should be realized that (like mammalian tissue) 544
gut bacteria can detoxify some hazardous chemi-
cals.
The degradation of A^-nitrosamines has recently
been reported by Rowland and Grasso.442 A large
portion of intestinal bacterial types (Escherichia
coli, Bacteroides, Bifidobacterium, Lactobacillus,
and Streptococcus faecalis) degraded diphenyl-
nitrosamine and dimethylnitrosamine in vitro. At
low concentrations (<50 JJM), about 55% of
diphenylnitrosamine, 30% of nitrosopyrrolidine,
and 4% of dimethylnitrosamine were degraded, the
products were the parent secondary amines, nitrite
ions, and unidentified volatile metabolites. The
rates of nitrosamine degradation varied consider-
ably among species tested, with strains of lacto-
bacilli and E. coli being the most active. Some
strains degraded diphenylnitrosamine but were
unable to act on dimethylnitrosamine, suggesting
that two separate mechanisms are operative. These
mechanisms are apparently distinct from those
observed in mammalian tissues in that neither
[14C]-formaldehyde nor 14 CO 2 are found after
I1 4 C] -dimethylnitrosamine degradation. The
activity of E. coli and other bacteria associated
with urinary tract infection (viz., Klebsiella aero-
genes, Pseudomonas aeruginosa, and Proteus sp.)
suggests that under conditions where bacterial
synthesis of nitrosamines is possible, the net
synthesis could be reduced in the presence of
degrading species.
The degradation of a variety of polycyclic
aromatic hydrocarbons, notable benzo[a]pyrene,
has been detected in a number of bacteria (in-
cluding Pseudomonas sp., E. coli, and Bacillus
s p . ) . 4 2 ' 3 2 0 ' 4 0 7 like the mammalian microsomal
benzpyrene hydroxylase system of the liver or
intestinal mucosa, 545 the microbial benzpyrene
degradation requires molecular oxygen and is also
stimulated by cyanide. It is not known whether
benzpyrene degrading strains could survive in the
mammalian gut or what role they might play in
the detoxification of dietary carcinogens.
Components of the intestinal flora (e.g.,.£*. coli)
have been shown to carry out N-dehydroxylation
of A^-hydroxyacetylaminofluorene.555 More re-
cently, the reduction of A^-hydroxy-4-acetylamino-
biphenyl to 4-acetylaminobiphenyl has been
demonstrated in several anaerobic intestinal iso-
lates, e.g., Peptostreptococcus productus I and B.
fragilis ss. thetaiotaomicron and ss. vulgatus.54"7
Thus, the flora, by hydrolyzing glucuronides and
reducing iV-hydroxy aryl amines and amides, may
exert a significant influence on the concentration
of proximate carcinogens in the bowel.
The degradation of afiatoxin Bi by Flavo-
bacterium aurantiacum, Corynebacteriwn rubrum,
and Mycobacterium phlei in vitro has been ob-
served by Mann and Rehm. 338 A number of fungi
January 1977 247
 and the yeast Candida sp. were also active in
reducing the cyclopentadiene ring of aflatoxin Bi.
III. METHODOLOGY OF
INTESTINAL MICROECOLOGY
A. Theoretical Basis of Obligate Anaerobiosis
According to currently popular views of chemi-
cal and biological evolution, the world was once
anaerobic. 9 9 ' 3 3 9 ' 3 4 7 The first living forms
probably arose about 3 billion (10 9 ) years ago in
the virtual absence of molecular oxygen,40 and
the earth seems to have remained anaerobic for
another billion years. The evolution of anaerobic
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microbes probably proceeded during this per-
iod.2 ' 6 >261 Slow accumulation of oxygen (and
ozone) resulting from ultraviolet photolysis of
water eventually enabled the evolution of photo-
synthetic plants and algae. Associated with the rise
of oxygen-evolving photosynthesis was the evolu-
tion of aerobic nonphotosynthetic protists, organ-
isms that adapted to using oxygen as a terminal
oxidant of energy-yielding metabolism. The pro-
tective mechanisms that were involved in this
evolutionary adaptation to O2 were not shared by
their anaerobic counterparts.
The strict requirements of anaerobic microbial
life have been a source of puzzlement to bacteri-
ologists for more than a generation. One theory
emphasizes the notion that the toxicity of O 2
itself is the paramount f a c t o r . 8 7 ' 1 9 5 ' 2 3 0 ' 2 9 0 '
319,345,376 ^ n alternative hypothesis is that the
redox potential of the culture medium is the
dominant factor, with oxygen serving to increase
the potential and indirectly inhibiting anaerobic
growth.218'291'377'499'532
In studies in which the effects of O2 and redox
potentials were measured independently, oxygen
appeared as the significant 131 ' 386 factor; how-
ever, only clostridia were studied. More recently,
Walden and Hentges 534 were able to separately
assess the effects of oxygen and redox potential by
employing K3 Fe(CN)6 and dithiothreitol to poise
the potential of the culture medium at positive
(E'h ~ +500 mV) or negative (E(, 50 mV)
values, respectively. While only three organisms
were studied (Clostridium perfringens, Bacil-
lus fragilis, and Peptococcus magnus), all
three were growth-inhibited in the presence of
oxygen, irrespective of the Ej, value. In the
absence of O 2 , anaerobic growth was observed at
an average E^ value of +325 mV. These authors
248 Critical Reviews in Food Science and Nutrition
also observed differential oxygen sensitivity in the
species studied, in agreement with the earlier
findings of Loesche. 319 The latter found two
groups of anaerobes. The first group was com-
posed of "strict anaerobes," which had an upper
oxygen tolerance limit of <0.5% (PO2 < 0.8 mm
Hg) and included such species as Clostridium
haemolyticum, Butyrivibrio fibriosolvens, Trepo-
nema macrodentium, and Selenomonas rumin-
antium. The second group, "moderate anaerobes,"
tolerated oxygen tensions up to 3% (5 mm Hg)
and included B. fragilis, Fusobacterium nucleatum,
Clostridium novyi, and Peptostreptococcus
elsdenii.
Several explanations have been suggested to
account for the apparent toxicity of molecular
oxygen. Perhaps the earliest postulated the in-
volvement of bacterial catalase,87 an enzyme
frequently present in aerobic microbes but usually
absent in anaerobic species. Presumably, anaer-
obes, when exposed to O2, formed lethal or
bacteriostatic quantities of hydrogen peroxide,
probably via autooxidation of reduced flavopro-
teins. This theory is not generally acceptable since
not all anaerobes form detectable amounts of
H2O2 upon O2 exposure. Also, while some anaer-
obes do synthesize catalases or other H 2 O 2 -
utilizing peroxidases, they do not tolerate O 2
exposure. Exogenous catalase has little growth-
promoting action for anaerobes in the presence of
oxygen.
A more recent t h e o r y 2 0 5 ' 2 0 7 ' 3 3 0 involves the
postulated physiological function of the enzyme
superoxide dismutase. This enzyme brings about
disproportionation of the superoxide anion radi-
cal, O2" + O2" + 2H+ -> O2 + H 2 O 2 . The toxic
superoxide anion radical is produced as an inter-
mediate in the reduction of O2 by certain metallo-
flavoproteins (e.g., xanthine oxidase). The enzyme
is widely distributed and occurs in many forms;
enzymes containing copper and zinc are found in
eukaryotes such as fungi, mammalian tissues, and
higher plants, while separate manganese- and/or
iron-containing enzymes are found mainly in
prokaryotes (e.g., E. coli,206 the blue-green alga
Plectonema boryanum,25 and Streptococcus
mutans).
A comparative study of microorganisms330
revealed the presence of both superoxide dis-
mutase and catalase in all eight aerobic and
facultative species tested, whereas the former
enzyme was absent in ten strictly anaerobic species
 (of Clostridium, Veillonella, and Butyrivibrio).
Another group of moderate anaerobes all con-
tained (with the exception of Lactobacillus
plantaruni) superoxide dismutase but lacked
catalase activity. Oxygen induces superoxide dis-
mutase synthesis in E. coli and Saccharomyces
cerevisiae, and higher levels of the enzyme pro-
vided greater protection from the lethal effects of
hyperbaric oxygen, thus supporting the dismu-
tase/oxygen toxicity hypothesis.2 ° 5 ' 2 ° 7
Although McCord et al. 3 3 0 were unable to
detect either superoxide dismutase or respiratory
activity in L. plantarum, other strains of this
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organism are known to possess oxygen-
metabolizing enzymes and to respire under certain
conditions. 7 6 ' 5 1 0 > 5 1 1 > 5 3 S In addition, some
strains of L. plantarum contain nonheme cata-
lase; 2 7 S - 2 7 7 ' s s 0 > S 7 6 Youstenetal. 5 7 6 reportlow
levels of superoxide dismutase in both a catalase-
positive and catalase-negative strain.
While the presence of superoxide dismutase
may be a significant factor in aerotolerance of
many moderate anaerobes, it probably is not the
dominant factor for all of them. Some strict
anaerobes (e.g., C. haemolyticium) appear to be
more sensitive to oxidized constituents of the
culture medium than to molecular oxygen or even
H 2 O 2 itself.496 The influence of dissolved O2 on
the redox potential of the medium cannot be
ignored. All anaerobes probably have a limiting
redox potential above which they cannot grow.
The strain involved, pH of the medium, and the
size and physiological state of the inoculum are all
important factors. Futter and Richardson 180 ' 181
found that the gaseous atmosphere is an important
factor in the viability of C. perfringens spores.
Although medium prepared with N2 had a higher
redox potential (+150 mV) than that with H2
atmosphere (-325 mV), recoveries of spores were
as good or better with the N2 atmosphere. The
difference was even greater with spores damaged
by gamma irradiation. Such results may be indi-
cative of hydrogen toxicity, since spores treated in
argon were undistinguishable from those exposed
to nitrogen. Similar results were obtained with
other clostridial spores (C. septicum, C. histoly-
ticium, and C. sporogenes) and vegetative cells (C
cellobioparum)?7 Thus, while hydrogen is gener-
ally considered innocuous and nontoxic to even the
most fastidious anaerobes, one can appreciate the
difficulties involved in making generalities about
so diverse a group of organisms.
B. Anaerobic Media and Isolation Techniques
The evolution of anaerobic methodology has
been reviewed by Sonnenwirth, 50! and techniques
have been developed that insure the growth of all
anaerobes for which the media are nutritionally
adequate. However, the collection of nonfecal
specimens from the human gastrointestinal tract
has seen fewer advances. The collection of such
specimens relies upon surgical techniques involving
abnormal or deceased subjects, intubation tech-
niques (which have been used extensively), or the
use of remote control self-opening capsules. The
advantages and limitations of these methods have
been discussed by Drasar and Hill. 139 The tech-
niques for the collection of fecal specimens have
been critically evaluated by Attebery et al. 29
In view of the previous discussion, methods for
the isolation of indigenous intestinal microflora
should ideally provide for "complete" exclusion of
molecular oxygen from the culture media as well
as from the microbiological specimens. Three
methods are currently in use for the isolation and
surface cultivation of anaerobic bacteria, viz., the
anaerobic jar, roll tube, and glove box, but only
the latter two are considered adequate for the
isolation of normally dominant intestinal microbes
that are extremely sensitive to O2 and/or oxidized
medium constituents.
The use of anaerobic jars dates from the early
part of this century, but they were not widely
used until the disposable hydrogen generator was
developed. 60 ' 61 This generator (later marketed as
the now familiar Gas Pak®) eliminated the need
for compressed gas cyclinders and pumps and in its
simplest form could be used with a cold palladium
catalyst (palladium on alumina pellets) which
required no external heating to affect oxygen
removal as water.
The Gas Pak system has been evaluated and
compared with other methods by several re-
s e a r c h e r s . 1 0 1 ' 1 3 2 ' 2 8 4 ' 3 4 0 ' 4 4 0 When appropriate
precautions are used for media preparation and
specimen transport, the method appears to be
adequate for the isolation of clinically significant
anaerobes. Recent evaluation of reducible solid
media (containing PdCl2) with the Gas Pak 1 5 2 a
demonstrates the effectiveness of this combination
in high-volume clinical laboratories. Incorporation
of PdCl2 as a reducing agent should be evaluated
for the particular medium employed since it does
not yield significantly increased recoveries in all
393
cases:
January 1977 249
 The first effective method for isolating strictly
anaerobic microbes was developed by
Hungate2 6 6"2 6 8 for the study of ruminant micro-
flora. The procedure combines certain successful
features of earlier methods in providing a low
redox potential, an oxygen-free atmosphere, and
media preparation under anaerobic conditions.
Media are autoclaved, inoculated, and incubated in
the absence of oxygen, preventing the formation
of toxic oxidation or peroxidation products. Agar
medium is distributed in a thin layer over the inner
surface of tubes filled with an anaerobic atmos-
phere (preferably carbon dioxide). The roll tube
technique has been adapted by several workers 75 '
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120,252,494 an( j n a s given rise to the commercial
availability of prereduced anaerobically sterilized
media (PRAS). While the technique and modified
procedures are rather complicated and time con-
suming, they remain effective and reliable ways to
isolate fastidious O2-sensitive anaerobes from a
wide variety of sources. Detailed descriptions of
preferred techniques and commercially available
supplies and equipment are provided by Bryant75
and Holdeman and Moore.2 5 3
Another reliable technique for isolating strict
anaerobes on surface media employs an anaerobic
chamber or glove box. The first boxes used for this
purpose 1 3 5 > 4 3 9 > 4 9 8 were of rigid construction
and successfully isolated spirochetes and other
fastidious intestinal bacteria. This approach was
somewhat simplified by the introduction of flex-
ible vinyl plastic film (20 mil). 1 7 ' 1 8 The box is
fitted with neoprene or Mylar® gloves and usually
has a vacuum-type entry lock for introducing and
removing test materials. The flexibility of vinyl
allows easy fabrication into chambers of any size
or shape; the chamber walls move to accommodate
volume changes caused by internal extension of
the gloves. Initial setup and filling of the chamber
with oxygen-free gas is considerably simplified by
the chamber's ability to collapse. Rigid boxes must
either be flushed for lengthy periods of time or
filled by internal displacement of a large balloon.
While any gas or mixture may be employed in an
anaerobic chamber, the common use of neoprene
gloves (which are permeable to O 2 ) necessitates
the continual removal of O2 from the chamber.
This may be accomplished either by catalytic
removal or continuous replacement. The use of a
palladium catalyst in conjunction with 3 to 10%
H2 in the glove box atmosphere will continuously
remove oxygen from a static atmosphere. The
250 Critical Reviews in Food Science and Nutrition
chamber may be thermostatted and used as an
incubator; one manufacturer has marketed a ther-
mostatted fan heater designed to hold a tray of
catalyst pellets and continuously circulate the
chamber gas over the catalyst (Coy Laboratory
Products, Inc., Ann Arbor, Mich.). Other devices
are available for flameless sterilization of loops,
needles, etc. inside the chamber. The performance
of an anaerobic chamber may be measured with an
oxygen analyzer 237 ' 512 or with the less costly
redox indicator. The most useful of the latter type
is probably phenosafranine (E^ ~ -250 mV).
Some reduction of the color of this indicator
should be noted in media with the reducing agent
PdCl2 1 to 2 days after introduction into the glove
box.
The principal advantages of glove box methods
include the use of standard disposable petri dishes,
the possibility of using opaque media, sampling of
specimens with less risk of oxygen exposure, and
greater flexibility afforded by the relatively large
anaerobic work area. Agar plates containing PdCl2
as a reducing agent may be poured on the bench;
according to Aranki and Freter, 17 these may be
stored for considerable periods of time before
being introduced into the glove box. Plated media
are usually introduced into the box as soon as
possible following preparation; one may also pour
plates of PRAS media in the box to insure
minimum exposure to oxygen. Disadvantages of
anaerobic chambers include larger space require-
ments and the cost in comparison with other
methods.
Variations on this basic approach include the
use of a continuous flow of CO2 through the
chamber to displace oxygen 303 and the use of
combusted natural gas in the chamber atmos-
phere. 292 If the former technique is used with a
rigid cabinet, it dispenses with the need for the
usual double entry air lock, catalysts, vacuum
pumps, etc.; it relies solely on a continuous flow
(ca. 3 to 6 1 min ~l) of oxygen-free CO 2 . Anaer-
obic conditions can be attained in as little as 3 hr;
thus it is unnecessary to keep the chamber
anaerobic while not actually processing specimens.
The system described by Koopman et al. 2 9 2
attempts to combine some of the advantages of
the continuous and discontinuous gas flow anaer-
obic chambers by continuously purging a chamber
with an extremely low-cost O 2 free gas, viz.,
combusted natural gas. The composition of this
inert gas is largely N2 with 12% CO 2 ,0.3% CO and
 H 2 , 30 ppm O2, and traces of nitrogen oxides. The
H2 and CO content is utilized to remove the
remaining O 2 ; this is carried out via an in-line
heated copper catalyst. Media introduced into an
anaerobic chamber and purged with the oxygen-
free gas had potentials (E^) of-250 to -300 mV
after 24 hr; this is similar to values obtained by
Aranki et al. 18 It is uncertain what adverse effects
CO, NO, and NO2 might exert on the isolation of
anaerobic bacteria. Many strains of Bacteroides
ruminicola, B. fragilis, B. oralis, and B. melanino-
genicus synthesize b, o, or c type cytochromes.
The latter pigment is known to bind CO. 431
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A variety of nonselective media have been
employed by various workers in isolating and
enumerating human fecal microflora. The most
successful media appear to be those composed of
Trypticase® soy agar (TSA), veal infusion agar
(VIA), brain-heart infusion agar (BHI), and parti-
cularly media originally designed for isolation of
ruminant bacteria such as rumen fiuid-glucose-
cellobiose agar (RGCA). 1 8 ' 7 7 ' 2 5 2 One of the
latter media (M10) was systematically evaluated
by Eller et al. 1 5 2 The medium was initially
composed of relatively small quantities of glucose,
cellobiose, soluble starch, Trypticase, yeast ex-
tract, hemin, volatile fatty acids, minerals,
cysteine-HCl, sulfide and carbonate buffer. The
efficacy of the various constituents could be
evaluated using the roll tube technique. Recoveries
of viable fecal bacteria with M10 averaged 1.15 X
10 11 g"1 wet weight. Recovery was not reduced
by the deletion of volatile fatty acids, Trypticase,
yeast extract, or sulfide. Deletion of hemin or
both Trypticase and yeast extract significantly
reduced observed counts. Addition of a fecal
extract, rumen fluid, 1% dehydrated BHI, or up
to 6% liver infusion had no effect, whereas 1%
dehydrated bile or 3.7% BHI depressed viable
numbers. Decreasing atmospheric CO2 from 100
to 5% with N2 had little effect during this study,
although other workers have found reduced re-
coveries under these conditions. 369 The counts
obtained with dehydrated commercial media (viz.,
Brewer thioglycollate, BHI, and TSA) supple-
mented with hemin and cysteine HC1 were equiva-
lent to M10 or lower. The study indicates that
simpler media might satisfactorily recover signifi-
cant numbers of human fecal flora which seem to
be somewhat less fastidious than the majority of
rumen microbes. While the work of Eller et al. 1 5 2
is flawed by its small sample size (1 g), lack of
microscopic counts, and dry weight measurements,
it amply demonstrates the growth-promoting ef-
fects of hemin in an isolation medium. Further
modification and simplification of an MIO-like
medium has recently been reported by Grubb and
Dehority. 210
How truly nonselective any of these media are
is open to question. Addition of Tween® 80 is
known to stimulate the growth of several species
while inhibiting others. Rumen fluid can also
stimulate certain fecal organisms. Bile salts and
blood may inhibit a variety of intestinal isolates.
Only hemin and vitamin K (not menadione) may
be added with virtual impunity to nonselective
media. 369 It would seem prudent to employ a
variety of nonselective media in isolating fecal
anaerobes.
In addition to the nonselective media, various
selective media can be useful in recovering specific
bacteria or groups of bacteria from complex
mixtures such as those found in feces or other
intestinal specimens. For studies of "total flora" it
is essential to use such media; otherwise, import-
ant groups of organisms might be overlooked due
to their relatively sparse numbers. The degree of
selectivity varies considerably, and very few media
are highly selective. Furthermore, selective media
frequently inhibit the very organisms for which
they are designed. Many substances have been
employed as selective agents, viz., brilliant green,
crystal violet, ethyl violet, bile, sodium azide,
chloral hydrate, phenylethyl alcohol, and various
antibiotics. The efficacy of antibiotics has been
stressed by Finegold et a l . 1 6 2 ' 1 6 4 Table 2 lists
several selective media employed in the isolation
of human intestinal microorganisms. The use of
such selective media does not preclude use of
adequate nonselective media which provide a
useful control. The identity of organisms obtained
with selective media must be verified by using
suitable tests. For instance, recovery of a Gram-
positive spore-forming bacillus does not necessarily
indicate the presence of a Clostridium sp. Many
Bacillus spp. will grow anaerobically, producing a
colonial morphology resembling clostridia.
C. Identification of Intestinal Anaerobes
The majority of anaerobes isolated from the
human gastrointestinal tract may be assigned to
the correct genus on the basis of five character-
istics, e.g., spore formation, cellular morphology,
Gram-staining reaction, fermentation end
January 1977 251
 TABLE 2

Selective Media for the Isolation of Intestinal Bacteria

Medium Major uses Comments

Rogosa'sagarV 4 3 3 - 4 3 8 Veillonella
Willis and Hobbs' agar2 * ' >55 7 Clostridia Neomycin 40 jug/ml
(lactose-egg yolk-milk)
Neomycin blood agai Anaerobic cocci and clostridia Inhibits facultative Gram-negative
bacilli
Egg yolk-neomycin agar Clostridium perfringens and Inhibits facultative Gram-negative
Nagler plates other clostridia bacilli
Azide agai4 ' ' Enterococci
KF streptococcus agar Enterococci
MacConkey's agar Enterobacteria With crystal violet
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Enterococci Without crystal violet

Mannitol salt agar Staphylococcus aereus and Incubate 30°
Bacillus spp.
Rogosa's agar (L)4 3 ' Lactobacilli 90% CO2
Neomycin-vancomycin Fusobacterium and Inhibits most facultative bacteria
blood agar Veillonella
Modified FM medium Fusobacterium Inhibits Bacteroides and Gram-
positive organisms
Rifampin blood agar Fusobacterium varium, Inhibits B. fragilis and most
F. mutiferum, some facultative bacteria
Eubacterium spp., and clostridia
Phenethyl alcohol Anaerobic cocci and nonspore-forming Inhibits Proteus, facultative Gram-
blood agar anaerobes negative bacilli
Biildobacterium medium Bifidobacterium spp. Inhibits Gram-negative bacteria
Sabouraud's dextrose agar Yeasts and filamentous fungi 40 Mg/ml of chloramphenicol at
37 and 22°C
S, agar 5 5 s a Streptococcus salivarens
Nutrient agar Bacillus spp. Inoculin heated at 70° for 10 min
Enterococcosel (BBL) S ' s Streptococcus bovis and other
group D streptococci
Clostrisel agar (BBL) Clostridia
Kanamycin-vancomycin blood agar Bacteroides fragilis
Kanamycin-vancomycin laked blood agar Bacteroides melaninogenicus

Note: Data from Drasar and Crowther,13 7 Feingoldet a l . , 1 6 2 ' 1 6 4 Sutter et al., 5 ' 4 and Dowell.13 3 >' 3 4 Neomycin, The
Upjohn Company, Kalamazoo, Mich.;kanamycin, Bristol Laboratories, Syracuse, N.Y.; vancomycin, Eli Lilly & Co.,
Indianapolis, Ind.; rifampin, Ciba Pharmaceutical Co., Summit, N.J.;Chloromycetin® (chloramphenicol), Parke-
Davis, Detroit, Mich.

products, and arrangement of flagella for motile
cells (Table 3). Kopeloffs modification of Gram's
staining procedure and Leifson's flagella staining
technique are generally preferred. No one medium
is satisfactory for the production of spores by all
spore-forming microbes. In addition, vacuoles may
frequently be mistaken for spores; thus, question-
able spore-forming ability should be further
assessed by heating older cultures (80°, 10 min) to
test for survival.366
The identification of several anaerobes is
assisted by gas-liquid chromatography (GLC) of
fermentative end products. 63 This is particularly
true of Gram-positive cocci, certain species of
Clostridium, and certain genera of Gram-positive
nonspore-forming rods (e.g., Lactobacillus,
Bifidobacterium, Propionibacterium, Arachnid,
Actinomyces, and Eubacterium).
The analysis of free fatty acids (C2 to C 6 ) has
recently been reviewed by Cochrane. 100 A variety
of methods have been developed for analysis of
volatile and nonvolatile organic acids. 6 7 ' 8 9 ' 2 2 S >
297,312,392 ^ relatively simple instrument
equipped with a thermal conductivity detector
(TC) will identify volatile fatty acids and alcohols
and methyl esters of nonvolatile acids (pyruvic,
lactic, fumaric, and succinic acids). In both
instances, alcohols and acids or their derivatives

252 Critical Reviews in Food Science and Nutrition

 TABLE 3

Properties of Anaerobic Generaa

Morphology and Gram stain Fermentation products Genusb

Rods (+ or -) with spores Varied - acetic, formic, butyric, Qostridium

lactic, and valeric acids
Rods (+) without spores Propionic and acetic acids Propionibacterium0
Acetic:lactic acids = 1 to 1.5 Bifido bacterium ^
Lactic acid Lactobacillus
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Acetic, formic, succinic, and Actinomyces

lactic acids
Butyric and acetic, formic and Eubacteriume
lactic acids; none
Rods (-), nonmotile or motile Butyric, acetic, and lactic acids Fusobacterium*
with peritrichous flagella
Lactic acid Leptotrichia buccalis
No butyric or butyric, isovaleric, Bacteroides^
and isobutyric acids; succinic,
acetic and formic; proprionic
and acetic acids;none
Rods (-), polar flagella Butyric and lactic acids Butyrivibrio
Succinic acid Succinivibrio
(curved cells)
Rods (-), flagella in tufts on Succinic and acetic acids Succinimonas
concave side of cells (ovoid cells)
Cocci (+), growth in peptone Varied - acetic, propionic, Pcptococcus and
butyric, formic, succinic, lactic, Pcptostreptococcus
isobutyric, valeric, isovaleric,
caproic and isocaproic acids
Cocci (+), require carbohydrate Acetic and formic acids or Ruminococcus
ethanol and/or succinic acid
Cocci (-) Butyric acid Acidaminococcus
Acetic and propionic acids Veillonella
Complex - acetic, propionic, Megasphaera ehderni
butyric, isobutyric and valeric
acids

a
Adapted from Moore and Holdeman. 3 "
''Designations according to Bergey's Manual of Determinative Bacteriology, 8th edition. 1 '
c
Includes organisms formerly in Corynebacterium.
d
Includes Lactobacillus catenaforme (formerly in Catenabacterium), L. disciformans and
L. crispatus (formerly in Eubacterium).
e
Includes Butyribacterium and some species formerly in Ramibacterium, Catenabacterium,
and Cellobacterium.
includes some species formerly in Sphaerophorus and Bacteroides.
^Includes the genus Dialister and some species formerly in Sphaerophorus and Fusobacterium.

are extracted from the spent medium (peptone
yeast extract ± glucose) into an organic solvent
(usually chloroform or diethyl ether) prior to GLC
separation. A disadvantage of this procedure is the
possible differential degree of extraction of the
various components of the experimental aqueous
mixture. Thermal conductivity makes it possible
to detect formic acid and gases, particularly
hydrogen. 1 3 4 ' 2 S 3 Somewhat more costly instru-
ments, equipped with flame ionization detectors
(FI), have certain advantages over TC; they have
far greater sensitivity, enabling the direct analysis
of small volumes (~1 pi) of culture supernatant for
volatile metabolites. 62 ' 314 While it is possible to
identify nonvolatile acids in an aqueous solution,
"quantitative" analysis necessitates their derivati-
zation.
A recent rnethod described by Salanitro and
Muirhead 450 employs butylation of the sodium
salts of both volatile and nonvolatile acids from

January 1977 253

 freeze-dried culture supernatants. Recoveries
averaged 85% with this technique, ranging from
68% for fumaric acid to 95% for valeric acid. The
chief advantage of this method is that all acidic
metabolites are analyzed simultaneously on a
single column (Chromosorb® W/Dexsil® 300GC)
in as little as 24 min. In the direct FI analysis of
volatile acids, formic acid is not detected; this
requires separate analysis either by TC or derivati-
zation. Because of FI's complete lack of response
to formic acid Cochrane 100 has included formic
acid in the carrier gas to prevent ghosting and
tailing of free fatty acids. Isolating the acids as
alkali salts and solution using formic acid/acetone,
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followed by separation on Chromosorb W/NPGA,
gives nearly quantitative (95 to 101%) recovery of
several organic acids (acetic, propionic, butyric,
isovaleric and caproic) with good precision. More
detailed descriptions of equipment, materials, and
operating conditions for GLC analysis may be
found in recent editions of the Anaerobe Labora-
tory Manual,253 Wadsworth Anaerobic Bacteri-
ology Manual,s'4 and Laboratory Methods in
Anaerobic Bacteriology.J 3 4
While the relation of microbial types based on
metabolic patterns and end products has long been
accepted by the large majority of microbiologists
and is employed extensively in the current 8th
edition of Bergey's Manual,1* an alternative classi-
fication of the anaerobic genera based almost
solely on morphological characteristics has been
advanced by Prevot. 411 Prevot's scheme divides
the anaerobes into approximately twice as many
genera (Table 4) as Bergey's system; it does not
yet enjoy widespread acceptance. 366
In lieu of a complete genotypic characterization
of microorganisms, it would seem useful to
employ as many stable phenotypic characters as is
feasible in assessing taxonomic relatedness. In
most cases, the investigator is uncertain that his
"key" characters (e.g., terminal vs. subterminal
spore location) represent major genotypic varia-
tions. In a few instances where more detailed
chemical and genetic data have been obtained
(e.g., DNA base composition, % GC, DNA and/or
ribosomal RNA homologies, and cell wall composi-
tion) the classification of test organisms within
phenotypically defined groups has corresponded
remarkably well with previous classifications.114'
123,273,274 Once the larger groups (genera and
subgenera) are well established by nucleic acid
homologies, etc., it will be necessary to select
254 Critical Reviews in Food Science and Nutrition
those phenotypic tests that correlate best with
such groupings. This will permit classification
without further resort to lengthy homology
tests. 272
Present methods of characterization and identi-
fication of intestinal isolates at the species level
usually requires about 20 biochemical tests. For
example, 12 biochemical media usually suffice to
differentiate cocci, whereas clostridia require 17
media; Gram-negative nonspore-forming rods, 26
media; and Gram-positive nonspore formers, 32
media.3 6 6 These numbers can be reduced in some
cases if the genus of the isolate is determined
initially. A number of the tests commonly
employed are presented in Table 5, and further
details concerning their preparation and use can be
found in the laboratory manuals cited above.
Generally speaking, the precautions taken
during subculturing and testing of anaerobic
isolates need not be as rigorous as for initial
isolation. Transfers and inoculation of biochemi-
cals may be accomplished on the bench by use of
gas cannulae and Pasteur pipettes (as per Anaerobe
Laboratory Manual)253 or with an inoculating
syringe and PRAS media contained in Hungate
tubes. 332 The biochemical tests may be read in 7
to 10 days or considerably less time if growth is-
rapid. Detailed keys for the identification of many
anaerobic species can be found in the Anaerobe
Laboratory Manual, although the emphasis here
and in the other manuals is on clinically relevant
anaerobes and not normal microflora. As mention-
ed earlier, the normal flora are not well character-
ized at present.
In the past few years there have been several
reports of methods designed for the rapid identifi-
cation of anaerobic bacteria. These are generally
extensions of earlier commercial systems
(Pathotec® and API® Analytab) for rapid testing
of the Enterobacteriaceae.4 7 5 ' 5 ° 7
A recent micromethod for carbohydrate
fermentation tests uses microtiter plates and a
replicator device. It enables simultaneous inocula-
tion with 48 separate cultures and has been
designed for use in a glove b o x . 2 5 2 ' 2 5 3 The
accuracy claimed for this system is 97%, based on
conventional control tests; this is somewhat higher
than in other tests.
D. In Vivo Cultivation of Gut Microflora
Studies on the cultivation and interactions of
specific gut anaerobes in vivo have been confined
 TABLE 4

The Genera of Anaerobes According to Prevota-4 " • " '

Morphology and Gram reaction Genus5

Eubacteria

Spore-forming rods
Central or subterminal spores
Gram negative
Motile Endosporus Pre'vot
Nonmotile Paraplectrum Fischer
Gram positive
Nonmotile, nonencapsulated Inflabilis Pre'vot
Nonmotile, encapsulated Welchia Pribram
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Motile Clostridium Prazmowski

Terminal spores
Gram negative
Motile Terminosporus*'b Pre'vot
Nonmotile Caduceus Pre'vot
Gram positive
Motile Plectridium Fischer
Nonmotile Acuformis Pre'vot
Spore-forming vibrios, Gram negative Sporovibrio Starkey
Rods, Gram negative
Small with bipolar staining Pasteurella^ Trevisan
Small without bipolar staining Dialister Bergey
Nonencapsulated Ristellab Pre'vot
Encapsulated Capsularis PreVot
Motile Zuberella Pre'vot
Highly pleomorphic/bipolar staining Sphaerophorus Pre'vot
Pointed ends/long spindles Fusiformis0 Hoefling
Very long, filamentous rods, ovoid, Leptotrichia Trevisan .
spherical, or fusiform swellings; stain-
ing Gram positive in young cultures
Short, rigid, comma-shaped bacteria Vibrio* <A Muller
occurring singly or in spirals; motile
via peritrichous or polar flagella
Rods, Gram positive
Singles, pairs, very short chains Eubacterium* Janke
Long chains and filaments Catenabacterium PreVot
Singles, short chains with pseudo- Ramibacterium Pre'vot
branching
Motile Gllobacterium § Pre'vot
Salmom pink colonies, catalase Corynebacterium Lehmann and Neumann
positive produce acetic acid
Microaerophilic produce propionic Proprionibacterium Orla-Jensen
acid
White colonies, catalase negative, Actinobacterium Haass
branching and club-shaped in
cultures; acetic acid
Bifid or doubly bifid termini; acetic Bifidobacterium Orla-Jensen
and lactic acids produced
Filamentous, ramified, conidia at Micromonospora Orskov
end of short conidiophores
Cocci, Gram negative
Pairs with adjacent sides flattened Neisseria Trevisan
Very small cells in irregular masses Veillonella Pre'vot
Cocci, Gram positive
Usually in pairs or short chains Diplococcus® Weichselbaum

January 1977 255

 TABLE 4 (continued)

The Genera of Anaerobes According to PreVota'4 " ' * "

Morphology and Gram reaction Genusb

Short chains (seldom in pairs) Streptococcus® Billroth

Tetrads seen after division Gaffkya Trevisan
Cells grouped in single-plane Staphylococcus Rosenbach
irregular masses
Cubical packets of eight Sarcina Goodsir
Irregular masses of more than one Micrococcus* Cohn
plane

Protozoobacteria

Helecoidal cells 4-16 /im,

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Terminal filament, vertebrate Treponema Schaudin

parasites poorly stained by aniline
dyes
Well stained by usual dyes Borrelia Swellengrebel
a
Excluded from this table are the sulfur bacteria and nonsulfur purple and brown bacteria.
b
Genera of uncertain affiliation, symbols indicate tentative or subgeneric relationship:
*Methanobacterium Omelianski
^Methanobacterium Kluyver and van Neil
* Ruminobacter Kaars Sypersteyn
Selenomonas (Prowazek) Boskamp
Butyrivibrio Bryant and Small
Succinovibrio Bryant and Small
^Butyribacterium Barker and Haas
§Lachnospira Bryant and Small
^Ruminococcus Kaars Sypersteyn
*Methanococcus (Groeneweg) Kluyver and van Neil
c
Fusiformis probably a sub genus of Sphaerophorus.
"Contains both aerobic and anaerobic species.

largely to germfree animals. Early work demon-
strated that germfree rats were readily convention-
alized by oral inoculation with normal cecal
contents or even by physical contact with conven-
tional animals. Subsequent studies attempted to
elucidate host-normal flora interactions by
comparing the effects of inoculation with known,
artificial normal flora with those of undefined
mixed cecal flora.197 Oral administration of
Lactobacillus sp., Streptococcus sp., and
Bacteroides sp. from NCS mice containing a
simplified flora, to germfree mice produced
ex-germfree animals with numbers and distribu-
tions of microbes similar to those found in the
NCS mice. 462 Loesche 318 studied the effects of
various bacterial associations of germfree rats and
mice upon cecal size (a measure of conventional-
ization). Bacteroides oralis and Fusobacterium
nucleatum were unable to develop in germfree
mice. Streptococcus mutans, Clostridium difficile,
and a Neisseria sp. were able to establish them-
selves but had no effect on cecal size. However, a
group K Streptococcus sp. and B. fragilis effected
an enlargement of the cecum. Both Peptococcus
sp. and a mixture of two Clostridium sp. were able
to reduce cecal size somewhat, and the combina-
tion of these strains gave still further reduction
(ca. 65% overall). More recent results by Sacquet
et al. 4 4 9 show a similar pattern. Of 15 strains
{Streptococcus, Sphaerophorus, Butyribacterium,
Catenabacterium, Ramibacterium, Fusiformis,
Endosporus, and Clostridium) used to mono-
contaminate germfree mice, only two (viz., an
anaerobic Streptococcus and a Butyribacterium)
decreased cecal enlargement appreciably. These
authors found that association with six strains
(Streptococcus, Sphaerophorus, Butyribacterium,
Catenabacterium, Clostridium bifermentans, and
Plectridium) produced very marked reduction in
cecal weights of ex-germfree rats and mice on a
semisynthetic diet.
Sasaki et al. 4 5 6 demonstrated the persistence

256 Critical Reviews in Food Science and Nutrition

 TABLE 5
Tests Employed in the Identification of Anaerobic Bacteria
Medium Test/observation
Peptone yeast extract (PY) Spore stain and heat test
on sample of PY before
pH, pH, chromatography
Peptone yeast extract pH, GLC of PYG (or other
glucose (PYG), carbohydrate containing
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sp.; combined oral administration resulted in
multiple association (gnotoxenic) with no
apparent signs of antagonism.
Aside from studies to elucidate the role of
normal flora components in maintaining the
normal physiology of the intestinal tract,
monoxenic and oligoxenic animals have been
employed to study enteric pathogen-normal flora
interactions, 387 * 390 the development of humoral
and cellular immunity in neonates,3 8 • ' ' and the

cellobiose, m edia if pH is signif ican tly

levulose, lower) etiology of intestinal strangulation. 573 These
maltose, studies and others have shown that an undefined
Threonine, pH, GLC (propionate indigenous microflora and many of its known
lactate formation)
components can be established in germfree
Adonitol, pH
amygdalin, animals. The animal, in a sense, acts as a selective,
arabinose, although far more complicated, medium than
erythritol those mentioned above, since growth is continuous
Esculin pH, hydrolysis or discontinuous and ample selective pressure can
Glycogen, pH be exerted to favor more competitive biotypes. A
inositol,
lactose, good example is the finding of Schaedler et al. 4 6 2
mannitol, that axenic mice monoassociated with a slow
mannose, lactose-fermenting (SLF) coliform bacterium
melezitose, subsequently harbored mainly a rapid lactose-
melibiose, fermenting (RLF) E. coli type. If the SLF strain
raffinose,
rhamnose, was administered to conventional NCS mice, it
ribose, could only establish itself at very low levels (ca.
salicin, 103 cells g" 1 ), and it retained its SLF activity.
sorbitol Similarly, when ex-germfree mice, associated with
Starch pH, hydrolysis Lactobacillus sp., Streptococcus sp., and
Sucrose, pH
Bacteroides sp. were given the SLF strain, it was
trehalose,
xylose, able to establish itself throughout the intestinal
Gelatin Liquefaction tract but retained its SLF character. Administra-
Milk pH, curd, digestion tion of feces from conventional mice to these
Indole-ni trite Indole, nitrate reduction "tetraxenic" mice rapidly reduced coliform levels
Chopped meat slant Spore stain, heat test in to those observed in conventional animals (10 3
starch
Chopped meat Digestion, indole, motility g~'). Subsequent studies by Franzese and Wilkins
Urea broth Urease employing an SLF Paracolobactrum coliforme
PYG-bile Growth comparison with supported these findings. They observed that RLF
PYG-Tween® 80 PYG after 1-2 days mutants had a faster growth rate in all areas of the
PYG-Fildes intestinal tract except the ileum and proposed that
GMB
their dominance in the cecum was probably due to
Egg yolk agar Lecithinase
BHIA Antibiotic susceptibilities utilization of carbohydrate produced by the
host. 560 This shift from SLF to RLF coliforms
Note: Abbreviations — GLC, gas liquid chromatography; has also been demonstrated with antibiotic-
BHIA, brain-heart infusion agar; GMB, glucose- resistant coliforms (Escherichia and Aerobacter
minimal salts-biotin. types). 560

of several bacterial species normally found in the
flora as monoassociates in the intestines of
ex-germfree mice. The normal flora components
included Escherichia coli, Streptococcus sp.,
Lactobacillus sp., Clostridium sp., and Bacteroides
E. Continuous Cultivation of Gut Microflora
Several workers have noted the similarities
between various open microbial ecosystems (e.g.,
the rumen, large intestine, and cecum) and labora-
tory continuous culture (chemostat) systems.3 2 5 •

January 1977 257

 562
Such ecosystems contain diverse populations
of microorganisms and more or less continuously
receive nutrients and water. The catabolism of
exogenous and endogenous organic substrates
provides energy for the growth and maintenance
of these populations. The dilution rates are
variable and depend upon such factors as the body
weight, structure of the intestinal tract, feeding
habits, and composition of the diet. S62
While such natural ecosystems cannot be
duplicated in vitro, laboratory continuous cultures
of formidable complexity may be maintained
temporarily. Several simplifications assist in the
interpretation of results obtained with mixed
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chemostat cultures. For example, a number of
environmental factors may be kept constant:
temperature, pH, Eh, PO2, dilution rate, and
medium composition. The inoculum may be
restricted to known organisms or a more complex
undefined flora may be restricted by the natural
selection operating in a chemostat or by use of
certain antibiotics or other selective agents.
Experimental work involving mixed cultures of
bacteria has been reviewed in detail. 8 0 ' 2 4 9 ' 3 4 9
Although microbial interactions in natural
ecosystems may be very complex, some of the
simpler types of interaction discussed in these
reviews are listed in Table 6. A variety of terms
have been used by different investigators to
describe these interactions, and no single classifica-
tion of terms is accepted by all. While intestinal
microbial interactions in vivo are greatly compli-
Terms for Simple Microbial Interactions
Term
Neutralism
Competition
Commensalism
Mutualism
Symbiosis
Synergism
Amensalism
Predation
Parasitism
258 Critical Reviews in Food Science and Nutrition
cated by host immunity and localized adhesion of
various species to intestinal cell surfaces, 325 it is
likely that many of the simple types of interaction
are operative as well. This is particularly true of
competition, amensalism, commensalism, and
mutualism. Competition and amensalism have
been observed to operate simultaneously in
continuous cultures of normal intestinal flora
found in mice. 177
Considering the potential value of continuous
culture techniques for controlled studies of
intestinal microecology, metabolism of drugs, food
additives, and environmental toxicants and
protoxicants, it is somewhat surprising that so few
attempts have been made to model such systems
on the human large bowel. Ozawa and Freter have
employed continuous cultures based on mouse
intestinal bacteria, 395 and Wang s39 has continu-
ously cultured mixed human fecal flora. The large
majority of studies employing these techniques are
based on rumen models. A number of devices and
modifications have been described, notably those
of Kafekewitz et al., 2 7 8 Rufener et a l . 4 4 6
Slyter, 491 Slyter et al., 4 9 3 and Hobson. 2483
Recent studies of the nutrition and physiology of
some important human gut bacteria (Bacteroides
fragilis, Ruminococcus bromii, and Peptostrepto-
coccus productus) have demonstrated their
marked similarity to dominant rumen species.76
Thus, R. bromii requires ammonia and branched-
chain volatile organic acids (e.g., isobutyric acid),
as do ruminant inhabitants like Ruminococcus
TABLE 6
Definition
Lack of interaction
Population of two or more species are
mutually limiting due to common dependence
on particular nutrient(s)
One species stimulated in the presence
of another, nonreciprocal
Growth of each species promoted by the
other
Two species with mutual obligate dependence
Metabolic production of specific products
is greater in mixed than in pure culture
One species produces substances antagonistic
to a second species
One organism engulfs and digests another
One organism feeds on tissues or body
fluids of another
 albus and Bacteroides succinogenes. As in the
rumen, this organism probably relies on other
intestinal cohabitants to produce these nutrients.
Because of the many similarities between
fermentations carried out in the rumen and other
intestinal tracts, interactions between ruminant
microbes may, to some extent, serve as a model
for the cecum and large bowel. 562 Wolin 463 and
co-workers have studied some key rumen
fermentations in both pure and mixed cultures of
rumen bacteria. The catabolism of cellulose to
acetate, propionate, methane, and other products
is mediated by three groups of bacteria. Cellulo-
lytic species (such as B. succinogenes) produce
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succinate, acetate, formate, and CO2 from
cellulose, cellobiose, or glucose. Propionate-
producing species such as Selenomonas
ruminantium utilize carbohydrate and succinate
but do not ferment cellulose. Finally, the
"methanogens," such as Methanobacterium
ruminantium, produce methane from hydrogen
and CO2 or formate. Mixed batch cultures of B.
succinogenes and S. ruminantium have demon-
strated complete utilization of succinate from B.
succinogenes by S. ruminantium; the quantities of
propionate formed by the latter species were
greater than would be expected in a pure
culture.4 6 3 Although S. ruminantium is unable to
use cellulose, it will grow in mixed culture with
cellulose as the sole energy source; the cellulolytic
B. succinogenes provides the necessary soluble
carbohydrates. A similar interaction in mixed
culture was noted between the cellulolytic species
Ruminococcus flavefaciens (which produces small
quantities of H 2 , formate, and CO 2 , in addition to
succinate and acetate) and the methanogen M.
ruminantium (which utilizes the former metabo-
lites for CH4 production). Transfer of H2 gas has
also been shown to occur between R. albus and
Vibrio succinogenes in continuous mixed
cultures 269 and between Clostridium cellobio-
parum and M. ruminantium.9'1 Kistner and
Kotze 285 and Kistner and van Zyl 286 have used a
"chemostat" device to study the enzymes of
intermediary carbohydrate metabolism in glucose
limited pure cultures of R. albus and Butyrivibrio
fibrisolvens.
The use of continuous culture devices to
simulate the complexities of mixed rumen flora
was developed largely by Slyter et al. 4 9 1 ~ 4 9 3 A
direct comparison of the distributions of species in
vivo (steer rumen) and in vitro, with equivalent
nutrient inputs, showed that the same species and
groups of bacteria predominated in both cases
(viz., Bacteroides ruminicola, B. amylophilus,
Butyrivibrio fibrisolvens, and Butyrivibrio sp.). 492
Furthermore, while less rumen protozoa existed in
vitro, more viable bacteria were found (6 to 7 X
109 cells ml" 1 ). Important physiological proper-
ties of bacterial isolates were quite similar in both
cases (viz., cellulolytic, amylolytic, and acid-
producing activities). Butyric, propionic and acetic
acids were formed by 72, 10, and 16% of the
isolates from the steer rumen, while these acids
averaged 66, 9, and 18%, respectively, in two
chemostat vessels. In an earlier study, 4 9 3 a the
quantities of volatile organic acids and methane
produced in continuous culture decreased
markedly at pH values below 6.0, while viable cells
appeared to increase. In these studies an "artificial
saliva" was continuously infused into the ferment-
er vessels while solid nutrients were added at 12-hr
intervals; therefore, the devices used were not,
strictly speaking, "chemostats." Recently
Slyter4 9 ' has improved this system by developing
an automated input for solid substrates in pellet
form. Feeding intervals may be varied from 2 min
to 60 hr.
The utilization of NH3 as a major nitrogen
source in most rumen bacteria and their reduced
capacity to use exogenous amino acids suggests
that these microbes depend on the de novo
synthesis of amino acids. Sauer et al.,4 s 7 using a
continuous culture device similar to that of Slyter,
were able to follow the biosynthesis of amino
acids from radioactively labeled precursors in
mixed rumen cultures. They demonstrated the
reductive carboxylation of a variety of precursors:
viz., acetate -*• pyruvate -> oxaloacetate; proprio-
nate -»• 2-oxobutyrate; isovalerate -*• 4-methyI-2-
oxo-pentanoate; phenylacetate and hydroxy-
phenylacetate to corresponding phenyl and
hydroxyphenylpyruvic acids; and succinate -»•
2-oxoglutarate. Only 3-hydroxypyruvate
(precursor of serine) was formed via an oxidative
path, viz., 3 phosphoglyceric acid -*• 3-phospho-
hydroxypyruvic acid. These results show that
acetate and CO2 comprise a precursor C-pool for
de novo amino acid synthesis by rumen bacteria.
On the other hand, propionate carbon was poorly
utilized, participating only in isoleucine biosyn-
thesis. The data are consistent with a ferredoxin-
dependent carboxylation of 2-keto acids first
described in a photosynthetic bacterium.
January 1977 259
 The continuous cultivation of microorganisms
has perhaps received more than its share of
theoretical interpretation (see annual reviews of
Ricica) 4 2 ":" 2 9 The best known treatment of the
behavior of pure cultures is probably that of
Herbert et a!.,234 based on the earlier work of
Monod. 365 The fundamental balance equations
describing the net rates of increase in concentra-
tion of organisms and limiting substrate concentra-
tion are as follows:
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increase in = growth - output
organisms
dx
^ = nx-Dx
dt
1 dx _ S*m - D (la)
x dt " S + K
increase in = input - output - consumption
substrate growth
concentration =
yield constant
d S _= D S ° -
dt
dS = D(so _ S) _ " (2a)
dt
where
S substrate concentration in chemostat
(gr1);
S° input substrate concentration (con-
stant, g I" 1 );
concentration of microorganisms
(gr1);
K saturation constant equal to S where /i
= Mm/2;
Mm maximum specific growth rate (hr ' ) ,
i.e., maximum value of n at saturation
levels of substrate;
Y cell growth yield (dimensionless);
D dilution rate of the chemostat vessel
(hr" 1 ), i.e., the number of volume
changes per hour.
The theory for competition between a
continuously cultured microbe and a mutant or
exogenous contaminating organism has been
developed by Powell.4' ° Contaminant organisms
or mutants can grow successfully only if their
maximum growth rate (jim) a n d saturation
constant "exceed" those for the native organism.
If they are successful in establishing themselves,
they will displace the native organism entirely. The
first few generations are critical for an invader
organism. Even if it fulfills the above conditions, it
260 Critical Reviews in Food Science and Nutrition
still suffers a certain probability of being washed
out.
These basic ideas have been extended by Taylor
and Williamss 2 ' to describe the coexistence of
competing species under continuousflow condi-
tions. The fundamental equations (la and 2a) may
be extended for mixed populations, concen-
trations Xj with a single limiting substrate:
i dx _ S M i
- D ; i = 1,2, . . . , n (3)
x dt S + K;
(1) )- 2 (4)
where
M;= Mm» Yi and Kj are the growth yield, and
saturation constants for each of the n-
competing organisms.
(2) With a single limiting nutrient, it is unlikely that
more than one species would survive long, even pro-
viding oscillations in D (or in S°). Further extension
to coyer the case where there are m possible limiting
substrates and n species or types gives the fol-
lowing equations:
(5)
(S.o -
.
- ^ + xiD]Yij-j = l,2,...m.(6)
where Mj, Yy, and Ky are constants for species i
and substrate j (note M is independent of sub-
strate).
The authors note that it was impossible to find
equilibrium values for Sj unless M > k; thus, to
sustain a mixed population of a given number of
species, at least as many growth-limiting nutrients
are required. Of course, this treatment does not
take into account such factors as amensalism,
commensalism, or population density, 3 4 8 ' 3 5 0 but
it does provide a framework for more detailed
studies of mixed continuous cultures.
The population densities achieved in anaerobic
continuous cultures, while similar to those occur-
ring in the rumen, are at best only about 1% of
those found in the cecum or colon. An alternative
approach could employ continuous filtration or
dialysis of the culture, thereby removing low
molecular weight end products and allowing higher
 cell concentrations. A fully continuous dialysis
culture system, in which both fermenter and
dialysis reservoir are fed continuously with sterile
medium, has received some mathematical treat-
ment from Schultz and Gerhardt. 477 The most
striking feature of this system (as opposed to
conventional nondialysis continuous cultures) is
the higher cell densities (MO 1 ° cells ml" 1 ) attain-
able especially at low dilution rates (D < 0.1
hr" 1 ). While the maximum production rate is
achieved at a lower dilution rate with the continu-
ous dialysis system, the efficiency of cell produc-
tion is apparently lower than in the nondialysis
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continuous system (efficiency = actual production
rate/production rate equivalent to complete
nutrient utilization). The theory of continuous
dialysis culture has not been extended to treat-
ment mixed populations.
IV. COMPOSITION AND DISTRIBUTION
OF THE INTESTINAL MICROFLORA
A. Small Intestine
Despite a wide diversity in specimen collection
and culturing techniques, most investigators agree
that the upper and mid regions of the human small
intestine contain only a small number of micro-
organisms, usually less than 104 per milliliter.
These are predominantly Gram-positive facultative
organisms (streptococci, lactobacilli, diphtheroids,
staphylococci, and fungi), although anaerobic
types (similar in some respects to those found in
the large intestine) may also be encountered. 130 '
1 3 6 , 1 3 9 , 1 4 0 , 1 9 0 , 2 1 7 , 3 3 7,5 16
The survival of bacteria in the stomach depends
largely upon the pH level. Gastric samples with pH
values below 3 usually contain few viable bacteria.
Food and saliva entering the stomach tend to
neutralize gastric acidity, allowing viable bacteria
from these sources to pass into the small intestine
during and following meals. These bacteria con-
stitute a transient flora and are subsequently
removed by peristalsis, intestinal secretions, or
other factors. Stasis at the ileocecal valve probably
allows some concentration of bacteria in the lower
small bowel which explains the consistently richer
flora found there. Many organisms found in the
colon are also found in the terminal ileum,
although in smaller numbers, and may originate in
the large bowel.
Therefore, the distribution of normal luminal
microflora in the upper small intestine can be
expected to vary somewhat during the day. The
flora in the upper small intestines of infants and
children has recently been reviewed by Anderson
et al.; 12 in general it appears to be similar to flora
in adults.
Histological and bacteriological analyses of
biopsy specimens from the human gastrointestinal
mucosa reveal a lack of organisms in the normal
stomach and duodenum. 380 In samples of jejunal
mucosa, as in lower portions of the tract (colon
and appendix), anaerobic streptococci seemed to
predominate over Gram-positive and Gram-
negative bacilli. The organisms were observed in
the mucus of the mucosae; the average numbers
found were quite low, viz., 104 g"1 tissue in the
jejunum; 106 g" 1 in the colon; and 107 g"1 in the
appendix.
B. Large Intestine
There have been numerous surveys of human
fecal flora. Data from some recent studies are
summarized in Tables 7 and 8. 2 9 > 1 4 0 > 1 6 3 > 1 9 1 >
199,200,344,368,423 g ^ j ^ ^^ . ^ ^
Q f
not strictly comparable, e.g., the first three studies
(Table 7) report no microscopic counts or fecal
dry weights. The latter is particularly important
for comparisons since the moisture content can
vary considerably. The work of Drasar et al. 1 4 0
has been criticized by Gorbach, 190 particularly
with respect to their methodology for determining
the upper bowel flora. In the work reported here,
greater than 99% of the "cultivable" flora were
comprised of bacteroides and bifidobacteria. The
description of the methods used for handling fecal
specimens is not very detailed. Presumably, the
specimens were initially diluted 1:10 in an in-
fusion broth with 10% glycerol. Subsequently,
serial decimal dilutions were performed and 0.1-ml
aliquots of the dilution series were plated. There-
fore, the lowest dilution plated was 10 ~3 and lack
of growth on a single selective plate would indicate
a count of <10 3 g"1 or, on 10 plates, <10 2 g ~ \
etc. While several of the ranges listed by Drasar et
al. 1 4 0 indicate a lower limit of log 10 = 0 or 1 g" 1 ,
these probably indicate "undetectable at the level
tested." Determination of a concentration of one
organism g"1 would require 1000 plates per
specimen with the methods described. Similarly, in
the study of Mallory et al., 3 3 7 logio values of
zero are stated by the authors as undetectable at
10~3 dilution or <10 3 g" 1 . Instead of mean values
of logio g" 1 , the medians are provided by these
January 1977 261
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TABLE 7

Normal Fecal Flora of Man (Mixed Western Diet)

Gossling and Moore and
Experimental parameter Drasar et al. 1 ' ° Mata et a l . " ' Mallory et al. 3 ' 1 Attebery et al." Finegold et al.1 h J Slack1"" Holdeman" 1 Reddy et al. 4 : s

Number of hosts 25 12 12 7 18 10 20 8
2. Number of specimens 25 12 12 7 20 47 20 8
Percent solids - mean (range) 23.9(11-41) 22(13-34) 29.5 21.5 (12-35)
1S' Total microscopic count 11.88" 11.92b 11.66 ±0.23*' 11.70b
Q1 Total anaerobic count (10-ll)a 10.5 ± 0.7 (9-11)" 10(9-ll) c 11.46*= 11.52b 11.39 ±0.31 b 1I.681' 11.54 ± 0.101'
o Total aerobic count 8.8 ± 0.6(7-9) 7(4-8) 9.14 9.59 8.38 ± 0.49
<*> Anaerobic Gram-negative rods 10.47 ±0.27 (10-11) 11.15
2. Bactcroides spp. 10.5(10-11) 10.3 ± 0.6(9-11) 9(8-11) 10.5 (6-11) 11.06(6-11) 11.03 10.98 ±0.11
Fusobacterium spp. 3.5(1.5-5) 9(0 d -10)8 6.0 f 5.53(5-10)8 10.56
Anaerobic Gram-positive rods 10.26 ±0.40(9-11) 11.22
Eubactcrium spp. 9(5-11) 10.10(4-11)8 10.40 11.08 10.84 ± 0.40
0. Bifidobacterium spp. 10.5(9-11) 9.4 ± 0.9(<8-10)8 9(0-10)8 (6-11)8 9.52(6-11)8 10.03 10.73 10.90 ± 0.08
Propionibactcrium spp. 7.3' 8.99
5^ Lactobacilli 9.0 ± 0.9 (8-10)8 (6-10)8 10.14 ± 0.10
5* Anaerobic Gram-negative cocci 0(0-9)8 9.60± 0.49(9-11)8 8.78
Veillonella spp. 3(0-6)8 9.2 ± 0.8(<8-10)8 (4-7)8 8.85 (3-10)8
Acidaminoeoccus spp. (6-8)8 9.57(3-10)8 8.78
Mcgasphocfa spp. 10.86
10.1 ± 0 . 6 « 8 - l l ) 8 9(0-10)8 10.04 ± 0.50(9-11)
Anaerobic Gram-positive cocci
Ruminococcits spp. 10f 10.30(9-11)8 10.23 10.32
Peptococcus spp. 8 (4-10)8 9.95 15-10)8 9.10 10.61 ± 0.09
Peptostrcptococcus spp. 10(7-10)8 10.89(7-11)8 9.93 10.62 10.54 ± 0.26
Clostridium spp. 2.5 (0-5)8 9.3±0.9«8-10)8 0(0-9)8 6 (4-10) 9.53(3-11) 9.48
Enterobacteria/if. coli 6 (4-8) 8.7 ±0.7 (6-9) 7(4-8) 10(8-10) 9.50(7-10)8 9.42 8.37 ± 0.36
Lactobacilli 4(2-7) 8.6 ± 0.5 « 7 - 9 ) 8 0(0-5)8 (5-10)8 (6-10)8 10.04 5.41 ± 0.63
Total streptococci 4(2-7) 8.7 ± 0.8«7-10)8 (4-11)8 11.21 (4-11)8 6.95 ±0.21
Enterococci 3.6(2-6) 7.9 ± 1.0 (< 6-9)8 4 (0-7)8 8.48 8.5 (3-11)8 9.07
Staphylococci 1.3 (0-4)8 5.0 ± I.I «4-7)S 0 (0-4)8 (8-10)8 9(8-10)8 8.78 4.85 ± 0.46
Yeasts/fungi 1.0(0-4)8 4.0 ± 1.0(<4-5)S 0 (0-3)8 (3-9)8 (3-9)8 5.03 ± 0.45
PH 7.24 6.8(5.7-7.7)
Percent recovery 35 40 88

a
"Average" log,0 of counts g ' ' feces fresh weight ± standard deviation (range of counts rounded to nearest log, „ ).
b
Average log,0 of counts g' 1 feces dry weight ± standard deviation.
c
Medianlog,o of count g' 1 feces fresh weight.
d
Log, 0 count = 0 indicates "undetectable at level tested" usually < 103 g"1 (see text for explanation.)
e
Median log,0 count g "' feces dry weight.
''Single observation.
SNot observed in all specimens.
 workers, thus several medians were <10 3 g"1 feces
(fresh weight). Likewise, in the work of Attebery
et al., 29 median logjo values are given, but only in
some cases; in others, only ranges are given
because the organisms were detected in only one
or two specimens. These authors also present
figures similar to those of Drasar et al., with ranges
extending to logio values of zero (for bifido-
bacteria and anaerobic cocci), which represent
counts g"1 dry weight of feces. In this study, 19
diluents are compared for recovery of anaerobic
bacteria. Five were found superior; they ranged in
pH from 7.0 to 7.7 and in Eh from -220 to -330
mV. In addition, the homogeneity of fecal speci-
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mean counts ranging from 2.01 to 3.83 X 101 *
g"1 (dry weight), whereas homogenized whole
specimens yielded a mean of 2.83 X 10 11 g" 1 (N
= 6, in both cases). The authors caution against
sampling stool specimens at random.
Gossling and Slack 200 restricted their investiga-
tion to predominant Gram-positive anaerobic
bacteria and used immunological and other tech-
niques to characterize the persistence of species in
given hosts over extended periods (281 to 672
days).
The most extensive and quantitative published
study of human fecal flora is that of Moore and
Holdeman;368 they worked with a group of

mens was examined. Samples from different areas elderly Japanese-Hawaiians. The recoveries claimed
(e.g., surface, interior, terminal, etc.) gave total by these authors averaged nearly 90% or roughly

TABLE 8

Predominant Bacterial Species Isolated from Human Feces

Rank Organism Counta

Finegoldetal.' 63

1 Bacteroidesfragilisss. vulgatus 10.91 9.8

2 Bacteroidesfragilisss. other 10.84 8.3
3 Bacteroidesfragilisss. fragilis 10.72 6.3
4 Bacteroidesfragilisss. thetaiotaomicron 10.56 4.4
5 Peptostreptococcus micros 10.56 4.4
6 Bacillus spp. 10.51 3.9
7 Bifidobacterium adolescentis D 10.35 2.7
8 Eubacterium aerofaciens 10.33 2.6
9 Bifidobacterium infantis 10.29 2.3
10 Ruminococcus albus 10.28 2.3
11 B. fragilis ss. distasonis 10.20 1.9
12 Peptostreptococcus intermedius 10.17 1.7
13 Peptostreptococcus sp. Z 10.16 1.7
14 Peptostreptococcus productus 10.11 1.6
15 Eubacterium lentum 10.07 1.4
16 Streptococci, facultative 10.00 1.2
17 Fusobacterium russii 9.99 1.2
18 Bifidobacterium adolescentis A 9.97 1.1
19 Bifidobacterium adolescentis C 9.95 1.1
20 Bacteroides clostridiiformis ss. clostridiiformis 9.92 1.0
21 Peptococcus prevotii 9.82 0.80
22 Bifidobacterium infantis ss. liberorum 9.80 0.76
23 Clostridium indolis 9.79 0.74
24 Streptococcus faecium 9.78 0.73
25 Bifidobacterium longum ss. longum 9.77 0.71

Total 65
a 1
Mean log, 0 count g" feces (dry weight).
b
Percent of total microscopic count g"' (dry weight).
c
Gram-positive organisms only, these represented 47% of isolates and ranged 16
to 84% in individual specimens.
d
Percent of 865 isolates, 10 hosts.
e
Percent of 1442 isolates, 3 hosts.

January 1977 263

 TABLE 8 (continued)

Predominant Bacterial Species Isolated from Human Feces

Organism0 Count a

Gossling and Slack'°"

Eubacterium aerofaciens 10.23 18

Ruminococcus bromii 10.23 13
Bifidobacterium adolescentis 10.01 10
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Eubacterium rectale 9.93 9

Peptostreptococcus productus 9.93 9
Eubacterium cylindroides - 4
Eubacterium contortum _ 2
Eubacterium ventriosum _ 1
Eubacterium tortuosum _ 1
Propionibacterium acnes - 1
Ruminococcus albus _ 1
Sarcina ventriculi 1

Total 71

Organism Count a

Moore and Holdeman 3

Bacteroides fragilis ss. vulgatus 10.76 12.1

Fusobacterium prausnitzii 10.53 7.2
Bifidobacterium adolescentis 10.49 6.4
Eubacterium aerofaciens 10.46 6.0
Peptostreptococcus productus - II 10.42 5.6
Bacteroides fragilis ss. thetaiotaomicron 10.32 4.4
Eubacterium eligens 10.24 3.7
Peptostreptococcus productus - I 10.20 3.3
Eubacterium biforme 10.18 3.2
E. aerofaciens — III 10.07 2.4
Eubacterium rectale — I 10.05 2.4
B. fragilis ss. distasonis 10.05 2.4
E. rectale — II 10.03 2.3
B. fragilis ss. a 9.99 2.1
E. rectale - IV 9.96 .9
Bifidobacterium longum 9.94 .8
Gemmiger formicilis nov. gen. nov. sp.2 °' 9.92 .8
Bifidobacterium infantis 9.85 .5
Ruminococcus bromii 9.82 .4
Lactobacillus acidophilus 9.80 L.3
Bifidobacterium breve 9.74 .1
Ruminococcus albus 9.70 1.1
B. fragilis ss. b 9.58 . 0.8
E. rectale - III-F 9.58 0.8
Eubacterium ventriosum 9.53 0.7

Total .78

264 Critical Reviews in Food Science and Nutrition

 TABLE 8 (continued)

Piedominant Bacterial Species Isolated from Human Feces

Organism Count3 %e

Holdeman et al.2 57

B. fragilis ss. vulgatus 10.48 11.8

E. aerofaciens 10.41 9.9
B. fragilis ss. thetaiotaomicron 10.36 8.9
P. productus — II 10.23 6.6
B. fragilis ss. distasonis 10.18 6.0
F. prausnitzii 10.05 4.4
Coprococcus eutactus 9.95 3.5
E. aerofaciens III 9.89 3.0
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P. productus I 9.86 2.8

R. bromii 9.84 2.7
B. adolescentis 9.83 2.6
Gemmiger formicilis 9.76 2.2
B. longum 9.76 2.2
Eubacterium siraeum 9.73 2.1
Ruminococcus torques 9.67 1.8
E. rectale III-H 9.63 1.7
E. rectale IV 9.62 1.6
E. eligens 9.62 1.6
Bacteroides eggerthii 9.58 1.5
Clostridium leptum 9.55 1.4
B. fragilis ss. a 9.57 1.3
Eubacterium biforme 9.49 1.2
B. infantis 9.37 0.91
E. rectale III-F 9.34 0.84
Coprococcus comes 9.16 0.57

Total 82

twice those of other studies. The increase is
presumably due to both higher cultural counts and
lower microscopic counts. As they point out, 368
microscopic counts (particularly those using the
Petroff-Hausser counting chamber) are prone to
serious errors and are generally much less repro-
ducible than the cultural counts. For this reason,
they have counted clumps in Gram-stained smears
(10~6 dilution cm' 2 ). The loss of cells during the
staining procedure and the counting of clumps
instead of single cells probably resulted in lower
microscopic counts. In a subsequent study of 25
specimens, (unpublished study of Moore and
Holdeman cited in 368) a mean recovery of only
64% was obtained.
While the moisture contents of the fecal speci-
mens varied considerably (60 to 90%) when they
were measured, it is somewhat surprising that in
three of the studies cited the mean moisture
contents were within a few percent of each other.
Perhaps the most striking feature of the data in
Table 7 is the extreme variability of the so-called
"normal" flora. Very few typical genera were
observed in all specimens, viz., Bacteroids and
probably Escherichia coli (19/20 subjects in
Finegold et al. 1 6 3 ). In the study of Moore and
Holdeman, 368 Bacteroides, Peptococcus and
Eubacterium species were observed in all speci-
mens. The profusion of species is even more
staggering; the predominant species from the
major studies cited are ranked in Table 8. As can
be seen, the top 25 species or types comprise
roughly 65 to 85% of the total composite flora.
There are numerous other species present in
quantities considerably less than 1%. In the work
of Finegold et al., 1 6 3 over 160 distinguishable
types were isolated from 18 subjects, while Moore
and Holdeman 368 detected 113 different kinds
among 1147 isolates from 20 subjects. Statistical
treatment of the latter data reveals that the total
number of "different" bacteria in the gut at any
given time is approximately 400 to 500. However,

January 1977 265

 most of these types are present at less than 108
cells per g"1 feces (i.e., <0.1% of the total). Many
of the fecal isolates obtained in these studies have
not been fully characterized; therefore, descrip-
tions and keys for their identification are not
available in the various laboratory manuals. Since
most of the isolates are related to clinically
significant species, the methods advocated for
their characterization (particularly by the Anaer-
obe Laboratory Manual)2 s3 are still applicable.
Several new species and two new genera (viz.,
Coprococcus and Gemmiger) have been proposed
based on these studies.2 0 1 ' " 4
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the populations sampled) indicate a significant
dietary effect. 247 The biggest differences were
between fecal specimens of people in the U.K. and
U.S. on a so-called "mixed Western" type of diet
(high in meat, fat, etc.) and specimens from people
in Uganda, Japan, or India (where the diet is high
in carbohydrates). Some recent studies of high
meat and "meatless" dietary effects in somewhat
more homologous populations are summarized in
Table 10. In the work of Peach et al., 3 9 9 the high
carbohydrate diet group consisted of Indian (17
samples), Ugandan (9 samples), and Japanese (31
samples) villagers. They concluded that these
samples contained a larger proportion of

C. Dietary Factors eubacteria and fewerBacteroides spp. than did the

As mentioned previously, the nature of the diet
(Table 9) affects the rate of gastric emptying and
can indirectly influence the flora of the upper
small intestine. The diet has frequently been
considered a major factor in determining the
composition of the fecal flora. While such a
dietary influence has been demonstrated in ani-
mals,1 4 4 >4 9 5 >s 4 6 studies in man have been largely
equivocal.
Studies of human subjects in various countries
have shown some differences in groups of fecal
bacteria which (despite other differences between
"mixed Western" samples. Finegold et al. 1 6 3 used
subjects entirely of Japanese ancestry; one group
ate primarily a traditional Japanese diet, the other
primarily an American diet. The differences ob-
served in Bacteroides spp. were only of marginal
significance (P = 0.08), and differences in Clostrid-
ium paraputrificum were not statistically signifi-
cant. Although Streptococcus faecalis ss. faecalis
was significantly different in the two groups,
comparisons of all enterococci and Group D and
non-Group D streptococci showed no differences.
Over 220 distinct types of bacteria were observed

TABLE 9

Sources of Nutrients in the Large Intestine of Man

Souice(s) Nutrient(s) Quantity

Intestinal residue Pancreatic enzymes Autodigestion

Bile salts 300-500 mg/day
Neutral steroids 500-1000 mg/day
Intestinal mucus
Secretory IgA
Exfoliated cells 50-200 g/day shed mucosa
Dietary residue Fiber (unavailable 4 - 8 g/day enters colon
carbohydrate)
Natural nutrients
(e.g., plant steroids,
glycones)
Food additives <4 g/day intake
Biliary excretions Endogenous
(e.g., bile pigments,
retinoic acid, thyroxine, and
estriol)
Exogenous
(e.g. coumarin,
stilbestrol, amaranth, and
sunset yellow)
Microbial Dead cells
Extracellular products, etc.

Adapted from Drasar.1 3* With permission.

266 Critical Reviews in Food Science and Nutrition
 TABLE 10

Effects of "Mixed Western" vs. High Carbohydrate or Nonmeat Diets on Human Fecal Flora
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Finegoldetal." 3
3 99 • 1 j
Peach et al Reddy et al.

"Mixed Western" "High CHO"a "Mixed Western" "HighCHO"b "Mixed Western" "High CHO"C

Number of subjects 55 57 18 15 8h 8h
Total anaerobes 3.3 ( l . l - 6 . 8 ) k 2.5 ( 0 . 1 - l l ) k 11.54 ± 0.10 11.12 ± 0.11
Anaerobic Gram-negative rods
Bacteroides spp. 61.6 d 37.7e 11.06f 10.06J 10.98 ± 0.1 l j 10.52 ±0.10
Fusobacterium spp. 0.9 1-4
Anaerobic Gram-positive rods
Eubacterium spp.
con torturn 6.7 23.2 0 9.58
lentum 10.07 10.20
Bifidobacterium spp. 25.0 27.0 10.29 0 10.90 ± 0.08 10.42 ± 0.10
Propionibacterium spp. 3.1 5.1
Anaerobic Gram-negative cocci
Anaerobic Gram-positive cocci
Peptococcus spp. 10.61 ±0.09 10.20 ± 0.08
Peptostreptococcus spp. . 4.64 8.29
sp. 0 10.53
Enterobacteria
Enterococci 8.46' 9.83 6.95 ± 0.21 h 8.20 ± 0.53
Lactobacilli 0 1.4 10.14 t 0.108 9.42 ± 0.09 s
Closiridium
Staphylococci 4.85 t 0.46 5.92 ± 0.20
Other facultative 4.75 7.20

a
Native diet of Indian, Japanese, and Ugandan villagers.
'Predominantly traditional Japanese diet.
'Nonmeat diet.
d
Percent of 224 isolates.
e
Percent of 215 isolates
f
l'=0.080, marginally significant.
8
Anaerobic lactobacilli only.
Same subjects.
'Streptococcus faccalis ss. faccalis only.
J
Mean log, 0 count g"' feces dry weight ± standard error of the mean.
k
Anaerobic count (X 1 0 " g' 1 dry weight).
 in the "high carbohydrate" group, as opposed to
160 in the "mixed Western" diet group. Overall
about 300 different types were observed. Ex-
tremely oxygen-sensitive anaerobes were recovered
more often from the Japanese diet than from the
"mixed Western." Similar numbers of fusobacteria
were observed in both groups, despite earlier
findings that this group of organisms was more
plentiful in the Japanese than in Americans. 388 '
s28
This fact may be due to the greater degree of
ethnic homogeneity of.the test subjects.
Reddy et al. 4 2 3 used the same subjects for
assessing the effects of high meat and nonmeat
diets. Bacterial isolates in this study were not
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characterized to the species level as in the study
first discussed,399 and only six subjects were
tested in obtaining the mean counts for the
respective diets. Total anaerobic flora was signifi-
cantly higher in the "mixed Western" diet, as were
the counts of Bacteroides, Bifidobacterium, Pepto-
coccus, and anaerobic Lactobacillus species. No
differences in the total aerobic microflora were
noted, although Streptococcus and Staphylo-
coccus species increased significantly in subjects
on the nonmeat diet. A decline in the number of
Bacillus species and the total disappearance of
diphtheroids were also observed in the nonmeat
group, while enterobacteria were not detected in
the high meat group. The fecal excretion of total
neutral sterols, coprostanol, and coprostanone was
significantly elevated in subjects receiving the high
meat diet. The elevated ratio of coprostanol:
cholesterol in these specimens suggests increased
microbial degradation of cholesterol, although
cholesterol excretion was similar in subjects on
both diets. While no differences were noted in
total bile acids, there were significant differences
between the two groups in several individual bile
acids. The concentrations of primary bile acids
were lower in fecal samples from the "mixed
Western" diet, whereas secondary bile acids (e.g.,
deoxycholic acid; 3,12-diketo-5j3-cholanic acid;
12-keto-lithocholic acid, and 3-keto-5/3-cholanic
acid), arising from microbial action, were more
numerous. In general, these results of Reddy et
al. 42 3 agree with the earlier work of Aries et al., 23
Hill, 240 and Hill et al. 2 4 7 It is interesting that the
differences described above occurred after 4 weeks
on the respective diets, whereas in an earlier study
by Moore et al., 3 7 2 12 subjects fed a strict
vegetarian diet for 10 weeks elicited no substantial
differences from their "normal" nonvegetarian
fecal flora.
268 Critical Reviews in Food Science and Nutrition
Several groups have looked into the effects that
chemically defined diets have upon fecal flora.2 8 '
S 8,1 1 3 , 5 0 3 , 5 5 9 ^ 559
W m U z aJ
normal subjects receiving a low residue synthetic
diet (Vivonex Corporation, Mountain View, Calif.)
showed rapid reductions in the variety and concen-
trations of fecal flora (to 103 to 10 s g" 1 feces,
wet weight) in less than 2 weeks. The rates of
reduction in the flora could be increased further
by administration of an enema 1 day after
initiation of the dietary regimen. Fecal bulk was
also reduced considerably from a mean value of
160 g day" 1 (range 100 to 210) to 50 gd" 1 (range
30 to 98) after several days on the diet. The usual
glucose-based diet resulted in simplification of the
flora to essentially three types: bacteroides, coli-
forms, and enterococci (i.e., no Gram-positive
rods). When the synthetic diet was based on
sucrose, the reduction in the number and diversity
of the gut flora was less rapid and complete, with
bacteroides as the predominant type in all sub-
jects.
Subsequent studies with essentially the same
glucose-based diet (Vivonex CDD6) have failed to
confirm some of the findings of Winitz et al. Both
Attebery et al. 28 arid Crowther et al.1 a 3 found no
profound differences in the overall fecal flora of
subjects on the defined diet for 10 days or more.
Bornside and Cohns 8 used a similar diet (W-T Low
Residue Food®) and produced results similar to
those obtained with Vivonex CDD6. The most
obvious effects seen were diminished stool mass
and prolonged transit times.
Attebery et al.2 8 noted some marked decreases
in numbers of Clostridium perfringens, while other
Clostridium species were abundant with the de-
fined diet. Bifidobacterium species were undetect-
able in two subjects who had the genus in their
control feces. Although 19% of the prediet micro-
flora consisted of "extremely oxygen-sensitive"
anaerobes, these anaerobes were absent from ,the
diet feces. Among the disappearing species were
Eubacterium rectale, Peptococcus anaerobius,
Peptococcus intermedius (sic), and Butyrivibrio
fibrisolvens. Average aerobic plate counts increase
in subjects on the diet, largely due to E. coli.
In both studies, reductions in enterococci and
"viridans" streptococci were seen in some of the
subjects. The concentrations of acid and neutral
steroids (cholesterol metabolites) decreased during
the administration of the defined diet. The latter
decrease correlated well with the number of
enterococci per gram feces. 113 No significant
 alterations in fecal flora components were noted
by Bornside and Cohn. s8
In summary, it seems clear that the nature of
the diet can affect the composition of the in-
testinal microflora and that substantial changes
may have beneficial or detrimental effects. How-
ever, it is also apparent that variations within
population groups and even within given subjects
sampled at different times2 0 0 ' 2 S 7 point to con-
siderably more complex relationships. The indivi-
dual physiology of the host is probably a
dominant factor controlling the composition of
the flora. Alterations in the flora are probably not
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seen until the host's physiology has adapted to the
dietary change. Factors such as intestinal motility;
enzyme, bile acid, or mucin secretion; and rate of
turnover of intestinal epithelium may be in-
volved.370
In addition, a recent report by Holdeman et
al. 2S7 suggests that emotional stress in the host
can effect changes in fecal flora components. Their
study involved detailed bacteriological analyses of
fecal specimens from three Skylab astronauts over
a 5-month period (25 specimens, 1442 isolates).
The subjects ate their normal diet under normal
living conditions and a Skylab diet under both
normal conditions and simulated Skylab isolation.
While increases in two species of Eubacterium
were noted in response to the diet, a surprising
finding was the simultaneous increase in B. fragilis
ss. thetaiotaomicron and the decrease in P. pro-
ductus II which occurred in all subjects during the
simulated isolation. The authors ascribe these
marked changes in microflora components to an
anger stress situation in the subjects; the micro-
floras are presumably affected by hormonal altera-
tions in intestinal physiology (motility, blood
flow, secretions, absorption, etc.).
While more detailed longer term studies of
dietary effects on gut flora are certainly needed,
the complexity of the normal flora necessarily
limits the usefulness of bacteriological techniques
alone. Hill and Drasar246 have advocated the use
of more extensive microbial enzyme analyses to
aid in distinguishing subtle but significant altera-
tions in gut microflora physiology. Reddy et
al. 4 2 1 noted lower levels of fecal 0-glucuronidase
activity when subjects had been on the nonmeat
diet for 4 weeks. Other fecal enzymes need to be
correlated with dietary microflora shifts to further
assess the usefulness of this approach.
V. CHARACTERISTICS OF
IMPORTANT INTESTINAL BACTERIA
A. Spore-forming Rods: Qostridium
The members of this genus are generally Gram-
positive, motile, and strictly anaerobic. They differ
considerably in other characteristics; some species
are saccharolytic, others proteolytic, still others
may be both or neither. Fermentation occurs in
sugars, poly alcohols, amino acids, organic acids,
purines, and other organic materials. The %GC
(guanine and cytosine) content of DNA in
Qostridium spp. ranges from 23 to 45 mol%. A
recent study of ribosomal ribonucleic acid (rRNA)
homologies among 56 species of Qostridium has
been published by Johnson and Francis. 274 Four
rRNA homology groups were defined. The major-
ity of species were included in groups 1(30) and
11(11); these species had low %GC values (27 to
28%). A third group (6) with low %GC had very
low homologies with groups I and II, while a
fourth had high %GC values (41 to 45%) and
appeared as a distinct rRNA homology group. The
correlation of rRNA homologies with various
phenotypic characteristics was somewhat mixed.
Good correlations were found in cellular mor-
phology, motility, nutritional requirements
(saccharolytic vs. proteolytic), and patterns of
fermentation products, (i.e., species with high
homology showed similar patterns). On the other
hand, there was little or no correlation with
characteristics traditionally used to subdivide the
genus, viz., spore position (terminal vs. subter-
minal) and gelatin liquefaction. Also, a poor
correlation was seen between homology and cell
wall chemical composition, although the latter
correlated well with DNA homologies in a previous
study. 114 The subdivision of genus given in the
8th edition of Bergey'sManual** is based on spore
position and gelatin hydrolysis (four groups); one
group includes species with special growth require-
ments. The various biochemical and carbohydrate
reactions of some of the Qostridium species
isolated from human feces are presented in Table
11. Since some species are aerotolerant, it is
occasionally necessary to distinguish these from
facultative Bacillus species. The latter rarely
produce spores anaerobically and are generally
catalase positive, whereas the aerotolerant
clostridia rarely form spores aerobically and are
usually catalase negative. 496 Further details con-
cerning many individual species may be found in
January 1977 269
 the monograph of Smith 496 and the papers of
Moore et al.3 7 ' and Holdeman et al.2 5 5
B. Gram-positive Nonspore-forming Rods
1. Propionibacterium
This genus includes nonmotile propionic- and
acetic acid-producing species. They are anaerobic
or aerotolerant 126 and are usually pleomorphic,
diphtheroid, or club shaped in appearance. Metab-
olism' of carbohydrates, peptone, pyruvate, or
lactate yields a variety of fermentation products,
including combinations of acetic and propionic
acids, with lesser quantities of isovaleric, formic,
succinic, or lactic acids and carbon dioxide. All
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formed with little or no acetate, propionate, and
succinate. The propionic bacteria appear to be
capable of oxidative phosphorylation.12 6 Cell wall
composition and DNA similarities among the
propionibacteria have been studied by Johnson
and Cummins. 273 Eighty strains of anaerobic
coryneforms were compared with 29 strains of
classical propionibacteria. The former had DNA
base compositions of 58 to 64% GC and exhibited
at least three homology groups, typified by P.
acnes, P. avidum, and P. granulosum. The classical
propionibacteria had DNA compositions in the
range of 65 to 68% GC and exhibited four
homology groups which corresponded closely to

species form acid from glucose. Under aerobic earlier classification (i.e., 7th edition of Bergey's
conditions, large amounts of pyruvate may be Manual). These groups have been provisionally

TABLE 11

Biochemical and Carbohydrate Reactions of Some Clostridium Species of the Normal Human Fecal Flora

ft>
e o2 w'"
a
3 &
— -5
a .s -a
Species g < < o u o
a
oroticum + + +* - + + +
paraperfringens -* + + + +
fallax - +

ghoni +* + +* +> _ _ _ _ w
bifermentans +* d +* d _ +* V +"' - - - - - d - +» d
sordellii +* d +" + d + +* V + + d - _ _ d - +* d
botulinum B, E, F +• - ±" - — + v" + -<* + d d - - - + d +' d
perfringens +* d d d + + • d -' - - d - + + +" d
felsineum +* - + _ ' + _<* - d + + + d + + +* -
chauvoei +* - + w _ - + - d d - + + -
septicum +* d + _ > + + - _ _ d + d +• _
difficile +s -* > - w - ' - d - - - + - -
sphenoides _* + -" d _ - - + - - - d + - d + d d
indolis + -* + + _ - > _ - d d + _ _ + _ + + _
malenominalum •

sartagoformum
%

pseudotetanicum *
paraputrifwum _* * - d _ _ _ _ d d d d
1 _
aminovalericum _* _
- d - - w +' - w _ _ - - -
glycolicum .^
sporosphaeroides
cochlearium
ramosum _• d _ _ _ - + d + - d + _ + +
innocuum
barkeri
tetani _ _ +
putrefaciens

Adapted from Buchanan and Gibbons.7 *

Note: +, >90% of strains positive; -, >90% of strains negative; d, 11-89% of strains positive; w, weak reaction; s,
very slow; v, strain instability; V, variable; *, distinguishing characteristics of species within main subgeneric
groups; L, lactic acid; P, propionic acid; Pr, propanol; Bu, butanol; Am, amyl alcohol; A, acetic acid; B, butyric
acid; F, formic acid; V, valeric acid.

270 Critical Reviews in Food Science and Nutrition

 TABLE 11 (continued)

Biochemical and Carbohydrate Reactions of Some Clostridium Species of the Normal Human Fecal Flora

s s Major
a .s 2 I 2 •g 2 fermentation
Species 5 S S -a S •§ -a S S 2 H X products

oroticum

+
• + '

i
paraperfringens • +* - +' • - d* + -* - +* + d ABL

ii

ii
ii
fallax - ' +' " - d* - d* d +• - - +
ghoni
K
hifermentans d* -* d — AFPV
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sordellii K A* - d* - AFV

1 1 73 73 1
1

73 1 1 1
botulinum B, E. F d' - '* d - d d d
73 + 1 1

• - d*

1
1 73 73 1

1 73 73 1

1 + + +i

perfringens * +* ' - d* d * - - + + d ABL

' d< 1 11 • + -* +
felsineum
chauvoei ' +< • - +* - AB
septicum AB
difficile - d - - • d - - - d d d - - - d AFB
sphenoides d d - d• + » d - + + d * + - d - + d* + AFV
indolis * + d" d« * +
malenominalum APBV
73 1 1

sartagoformum • + +" 73 + 73
+ +1

+ + +

+ ++

+ + +

+ + 73
• +
1
1 + 1

1
pseudotetanicum * + -* * +
1 73

1 73

1 73
paraputrificum + - d +
aminovalericum
glycolicum • + APVPr, Bu, Am
sporosphaeroides
cochlearium
ramosum +< » + d' + - + + d d * + - - _ + +« d AFLV
- - - "
innocuum _ + _ _ d * + - d - d + ' d ABL
barken * - +* +* - + * +
tetani APB, Bu
putrefaciens

named P. freudenreichii, P. thoeni, P. jensenii, and the feces of man and other animals and from the
P. acidi-propionici. The characteristics. of the rumen of cows. Several distinct homology groups
Propionibacterium species are given in Table 12. were recognized. In some cases, little or no DNA
homology was observed, possibly indicating wide
2. Bifidobacterium evolutionary diversity in the genus. The DNA of
The bifidobacteria form diphtheroid- or bifid the following pairs were found to be homologous:
shaped cells and are usually nonfilamentous. Car- B. breve and B. parvulorum, B. thermophulum and
bohydrates are fermented by bifidobacteria and B. rumunale, and B. pseudolongum and B.
products from glucose are mainly acetic and lactic globosum. A large genetic heterogeneity was ob-
acids (ca 1.5:1.0); gas is not produced. Their cell served among strains assigned to B. adolescentis. In
wall composition lacks diaminopimelic acid and addition to the latter, three unrelated homology
arabinose, and the cells are catalase negative. In groups, (provisionally named "dentium" "caten-
much of the early literature, many species of ulatum," and "angulatum") were observed. The
Bifidobacterium were designated Lactobacillus validity of the species B. bifidum, B. longum, and
bifidus, as was Lactobacillus acidophilus. The B. suis was confirmed in this study. Surprisingly,
morphology, physiology, and classification of the DNA base composition of 28 strains of
bifidobacteria have been reviewed in detail by Bifidobacterium appears remarkably consistent at
Poupard et al. 4 0 9 The genetic relatedness of 179 60.1 ± 0.33% GC (range 57.2 to 64.5% GC for 211
strains (including 13 named species and several strains) and probably constitutes a valid dis-
unnamed taxa) was assessed by a DNA hybridiza- tinguishing characteristic with respect to other
tion technique. 460 The strains were obtained from genera, particularly Lactobacillus (%GC < 50).

January 1977 271

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TABLE 12

Biochemical and Carbohydrate Reactions of the Genus Propionibacterium

aj „ .£ *O .S 41

Galac ose
u
4)

ol
Cellobio
% 1 Si 2 §

Dulcitol
^JjcH o * I •§ S Fermentation
1 a I1 I1 -3
1 ts o
1& »I 2
8 s ~ 3 S . S l - S - S s S - g ^ products
Species < <: < 's S s
~ s s s s a W-" CO-3 OT
-? - 5 H X fromPYC
o S £ z o 3 £ 5 J
freudenreichii ss. w 2* - vc _w +w -w PASfl
freudenreichii6
w
freudenreichii ss. - PAsl
globosum
•f freudenreichii ss. _b +w . w" d w" PAS(fl)
S3 shermanifi
I thoeni
acidi-propionici c"
_c
-
+
v +1 _w 4- _ _w
b
w*
+W
w" +w —
+w
w" + + + V +
d +w - + V
+w H +" +w +w +

I jensenii
avidum
acnes*
c
d -
dc"
- - - 4* - b" V V
d
_w +w
h + + V + -• + +" - Hh « V -+
-w
w
+ + w
-
w
PA(sfiv)
PA(sfiv)
PA(LslflV)
I lymphophilum
granulosum*1
d v
+
+"
V b w w v d _w _w
_
+w w" -
PASU
PA(s/v)
7
Adapted from Buchanan and Gibbons.

Note: Fermentation products from peptone yeast extract glucose broth: Upper case letters indicate >1 meq acid/100 ml broth; lower case letters indicate <1 meq/100 ml broth. A,
acetic; F, formic, iv, isovaleric; L, lactic; P, propionic; S, succinic; ( ), products of occasional strains. Biochemical reactions: +, positive reaction in >90% of strains; -, negative
reaction in >90% of strains; v, variable within strains. Hemolysis: b, beta; w, weak. Gas: relative amounts indicated by *, 2, 3,4. Milk: c, curd; d, digested. Gelatin: w, liquefac-
tion if chilled cultures in less than half the time of controls. Superscript symbols represent reactions of 10-40% of strains. Carbohydrate reactions: +, strong acid (i.e., pH <5.5
in >90% of strains); w, weak acid (i.e., pH 5.5—6.0 in >90% of strains, except xylose and arabinose which may have relatively low initial pH); -, acid not produced (i.e., pH
>6.0 in >90% of strains); d, positive reaction in 40-60% of strains; v, variable within strain. Superscript symbols represent 10—40% of strains.
a
Distinguishing characteristics.
b
Formerly P. freudenreichii.
c
FormerlyP. shermanii.
Formerly Corynebacterium acnes.
e
Formerly Corynebacterium granulosum.
 TABLE 13

Carbohydrate Reactions of the Genus Bifidobacterium

Ara binose
ygdalii

Glu conate

h alose
Maiinitol

Maiinose
O o
"3

Ino sitol

Lac tose
Esc ulin

ose
Sali cin
Inu lin
| CD o

Rib

Stai
Mel
E •>,
Species CO H X
bifidum
adolescentis + + + + + d + V _ V + + d V V +
infantis - - V - - d d + - - — + V _ V V _
breve + - + + - . - - + + d V + V d V V
longum - + - - - - - + - d + + - _ V V +
pseudolongum + V - - - V - - V + V - + _ +
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thermophilum d - V d - - - V - - V - V - + V -
mis + - - - - + - d - - - _ - _ +
asteroides + + + _ - _ - _ - + + _ _ +
indicum + + - - - - - — + + _ _ _ _
coryneforme + + + - _ - - - — + + _ _ _ +

Note: +, positive; - , negative; v, variable, slight, or delayed reactions by the same or related strains; d,
reaction differs with strain.

The morphology of the bifidobacteria may be end product (as Propionibacteriwri); lactic as the
highly variable, including uniform or branched major product (as Lactobacillus); succinic (in
rods, bifurcated Y and V forms, and club or presence of CO2) and lactic, with traces of acetic
spatulate forms. Their development may be in- or formic (as Actinomyces); and acetic and lactic
fluenced by nutritional conditions. In the stools of (A:L > 1), with or without formic (as Bifido-
infants, the bifidobacteria are curved, Gram- bacterium). Eubacteria species are usually catalase
positive rods which are ocasionally bifid. These negative and do not hydrolyze hippurate. Bio-
could be cultured on the medium of Norris 409 for chemical and carbohydrate reactions of selected
as long as 22 years without alteration of morphol- species are given in Table 14. Further details on
ogy. In other media, such as tomato agar and individual species have been published by
thioglycollate, branched and other bizarre forms Holdeman et al., 2S6 Moore and Holdeman, 367
are frequently seen. While Bifidobacterium species and Moore et al. 3 7 3
are generally anaerobic, some aerotolerance may
be seen in the presence of C 0 2 . The distinguishing
carbohydrate reactions of the bifidobacteria are 4. Lactobacillus
given in Table 13. All members of the genus The lactobacilli vary in morphology from long
ferment glucose, galactose, and fructose; nearly all slender rods to short coccobacilli; they frequently
ferment maltose, melibiose, raffinose, and sucrose. form chains. Metabolism is fermentative, some
Bifidobacteria do not ferment adonitol, dulcitol, species are aerotolerant and may utilize 0 2 via
erythritol, glycerol, rhamnose, or a-methyl-D- flavoprotein oxidases, while others are strictly
mannoside. anaerobic (e.g., L. acidophilus and L. leichmannii
from human intestine). Glucose fermentation
3. Eubacterium produces at least 50% of end product carbon as
The eubacteria are obligately anaerobic rods, lactic acid. Other products include acetate, for-
uniform or pleomorphic and motile or nonmotile. mate, succinate, CO2, or ethanol. Volatile organic
They may or may not exhibit saccharoclastic acids with a chain length greater than two carbons
activity. Generally, mixtures of organic acids are not formed. The pH of the growth medium
(often including butyric, acetic, or formic acids) may be reduced considerably, since the lactobacilli
are produced from carbohydrates or peptones. are aciduric, growing optimally at pH 5.5 to 5.8
However, they are characterized more by the acids and only somewhat slower at pH 5.0 or lower.
they do not produce, viz., propionic as a major Nitrate reduction is rare. Gelatin is not lique-

January 1977 273

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TABLE 14

Biochemical and Carbohydrate Reactions of the Genus Eubacterium

c
I s 00
evie

Neu1:ral red
u a 8 Fermentation

sis
^J
PS 8

Fruc tose
jnito

ygda

Celllobio
Ara bino
products from

Lecil hin
s >
% •>. § a *o .S
Gelai

carbi

Grov
Nitra
Indo

Acet

Moti
Hem
20%
68
Milk

a'

Gas
»o
Species Z < < PYG Pyruvate
3"
ft.
Q

Q
rcctale _w c~ _ + +3 _w + +3 _ d _ d d +w _w + LB(sfa) AB"
r?
| Hmnosum -* - - - + +* +" + • 4* _w + - _b - _ w" +w + LAb(sp) ABiV-
ruminantium w a - d - +- - w" +7 - w - w LFb(ap) -
§ nitrogenes _w - - + d 4' d t - +- _b - +w LBAs(fp) AB F
ventriosum - - - ' + 2" _w d V +
-+ - - - +w Fbla(s) AF"
cylindroides _w - - - + w" w 4" d ++ - _w - +w Lb(asf) AB F "
moniliforme - - - d + 4 + +
4* - +" _w + + LBa(fspe) AB K "
15' tortuosum - a" - d + _2
w" _w -4 -
-
- _a - Lbs(af) ABFL"
tenue + cd + - • + > +w d +2 - -* + w" d _w Af(lsp ibiv) AF"
+
contortium - c - - + 2" - d 2" - d w - +w Af(sle) AF e
aerofaciens _w ac~ - - + 4" + • 4" +- - - + LAF(se) AFL-
ten turn _w _ _ + • + + _ +' d +- _ _ _ _ (afsl) _AF

Adapted from Buchanan and Gibbons.'

 TABLE 14 (continued)

Biochemical and Caibohydrate Reactions of the Genus Eubacterium

Fermentation
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%
oo « I 9,
o B products from
1 I

cin
Ribos
el o .

XyU
u
3
re
Species u PYG Pyruvate
3 O 5 £
rectale + + +w _* d —
+ LB(sfa) AB"
limnosum + + +w _vv _w —w d LAb(sp) ABiV "
ruminantium - + d d w _ - + d d LFb(ap) -
nitrogenes d + +w - + w" _w _w - LBAs(fp) AB F
ventriosum + + +" + + - w+ d Fbla(s) AF"
cylindroides - + - - + d Lb(asf) ABF"
moniliforme d +w - d + - LBa(fspe) ABF"
tortuosum + d w* - _ Lbs(af) ABFL"
tenue - w _ •
_w _ - Af(lspi6;V) AF"
contortium +w + d + _ d d Af(sle) AF e
aerofaciens + +- +w - + - LAF(se) AfL"
lentum _ (afsl) _AF

Adapted from Buchanan and Gibbons."

Note: Biochemical reactions: +, >90% of strains; - , >90% of strains; d, + reaction for 40-60% of strains. Hemolysis: a, alpha; b, beta; w, weak reaction. Gas/growth relative quanti-
ties: +, 2, 3, 4. Milk: c, curd; d, digestive. Gelatin: w, liquefaction of chilled cultures in less than half the time of controls. Products from peptone yeast extract glucose (PYG)
broth or pyruvate: Upper case letters indicate >1 meq/100 ml broth; lower case letters indicate <1 meq/100 ml broth. A, acetic; B, butyric; F, formic; ib, isobutyric; iv,
isovaleric; L, lactic; P, propionic; S, sucdnic; e, ethanol; ( ), product of occasional strains. Carbohydrate reactions: +, strong acid (pH < 5.5 for >90% of strains); -, no acid,
pH > 6.0 for >90% of strains); w, weak acid (pH 5.5-6.0 for >90% of strains) except xylose and arabinose, where + is < pH 5.4 and w ispll 5.4-5.7); di, positive reaction in
40-60% of strains. Use of any of the above as superscripts indicates 10-40% of strains.

in
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TABLE 15

Selected Carbohydrate and Biochemical Reactions of the Lactobacillus Species of Human Intestinal Origin

•1
a o Z * > - l Z o o £ "o <u b

rowl
S I I tJ 8 88 82 2 | g Sfi | § S I s 2 1 _

ome
Species u o 3 3 s s s l s ^ S s ^ c g ^ ^ ^ S O

^ leichmannii + - + + ~ w + - - + + - + - - - - - + - + + - + D • + +
2. acidophilus + + + + + - - + + - + - d d - - + - + + _ + DL - +
* salivarius - - - + + + - - + + + + - + + * _ * + + +'_ * DL - +
n casei + + - + + + + - + * ( d ) + + + _ _ * + + + (d) + _ + LDL _ d
+
a. plantarum + d + ++ + - ++ + + + d + + - + + + + +d+ + DL _ +
j§ fermentum - d - ++ ++ ++ + - w - + + - + - - + dd DL + +
3. brevis - + - + # + + +# + ( # ) _ _ + # - + - - d - d d DL +
I
Adapted from Buchanan and Gibbons."
Note: +, >90% of strains positive; d, 11-89% of strains positive; -, negative reaction in >90% of strains; ( ), delayed reaction; w, weak reaction; #, weak, slow, or negative;
*, distinguishing characteristics for L. salivarius ss. salivarius and salicinius, and L. casei ss. casei, alactosus, and rhamnosus; +, strains of/., plantarum fermenting arabinose
and xylose, formerly known as L. arabinosus and L. pentosus, respectively. Negative reactions from all strains with 1-erythritol, D-erythrose, D-fucose, D-glucoheptose,
a-methyl-D-xyloside, perseitol, and a-methyl-D-mannoside. L. brevis variable for a-methyl-Z>-glucoside. Some strains of/,, casei ferment adonitol and sorbose, other species
do not. Glycerol, inositol, inulin, starch, dextrin, and dulcitol are only rarely fermented. Tagatose, turanose, and D-xylose are fermented by L. casei and turanose by L.
plantarum. The pentitols L-arabitol and xylitol are fermented only by L. salivarius ss. salicinius and L. salivarius ss. salivarius, respectively.
 fied, and indole or H 2 S are not produced. Casein,
while not digested, may be slightly degraded to
soluble nitrogen. Porphyrins are absent from the
lactobacilli but, as mentioned earlier, some strains
possess nonheme or "pseudo" catalase. The lacto-
bacilli have complex nutritional requirements for
amino acids, peptides, nucleotide bases, vitamins,
minerals, fatty acids, and carbohydrates. The
growth of lactobacilli on solid media is often
stimulated by anaerobiosis with a 5 to 10% CO2
atmosphere. The %GC of Lactobacillns species
ranges from about 35 to 53 mol%. The genus is
subdivided into three groups based on fermenta-
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tion patterns. The first group is homofermentative,
producing lactic acid from glucose fermentation in
excess of 85%. A second group comprises hetero-
feimentative species producing only about 50% of
their end products as lactic acid, with considerable
quantities of acetic acid, ethanol, and CO2. The
third group comprises less well known hetero-
fermentative species producing DL-lactic acid,
CO 2 , and acetic acid. These species generally
ferment carbohydrates less efficiently than mem-
bers of the previous groups. The carbohydrate
reactions of the lactobacilli are presented in Table
15.
C. Gram-negative Nonspore-forming Rods
1. Bacteroides
The members of this genus consist of uniform,
slightly curved, or, occasionally, pleomorphic rods.
They metabolize carbohydrates or peptone.
Saccharolytic species produce combinations of
succinic, lactic, acetic, formic, and propionic acids.
Occasionally, short-chain alcohols are also formed.
Butyric acid is usually not a major product and
when formed is accompanied by isobutyric and
isovaleric acids. Nonsaccharoclastic species degrade
peptone to small to moderate amounts of succinic,
formic, acetic, and lactic acids or to major
quantities of acetic, butyric, and (occasionally)
succinic acids, with lesser amounts of alcohols and
isovaleric, propionic, and isobutyric acids.
Carbon dioxide is required by many intestinal
strains and may be incorporated into succinic acid.
Bacteroides species are obligately anaerobic and
usually catalase negative. Hippurate, lecithin, and
lipids are usually not hydrolyzed, nor is meat
digested. The %GC of the DNA in the various
species ranges from about 40 to 55 mol%. The
subdivision of the genus is based upon pigment
production, type of nutrient catabolism (saccharo-
clastic or nonsaccharoclastic), and fermentation
end products. More detailed characteristics of
selected intestinal species and subspecies are
presented in Table 16.
2. Fusobacterium
These spindle-shaped rods metabolize carbo-
hydrates or peptone, frequently producing
butyric, acetic, and lactic acids with lesser
amounts of propionic, succinic, and formic acids,
butanol, and ethanol. Some strains convert threo-
nine to propionic acid. Adonitol, dulcitol, erythri-
tol, glycerol, inositol, melezitose, rhamnose,
ribose, sorbitol, and sorbose are generally not
fermented. The fusobacteria are obligately anaer-
obic and usually lack catalase. They have a %GC
DNA content ranging from 26 to 34 mol%.
Biochemical, carbohydrate, and fermentation
reactions of some relevant species of this genus are
provided in Table 17.
D. Gram-positive Cocci
l.Peptococcus
These spherical cells occur singly, in pairs,
tetrads, irregular masses, or, occasionally, in short
chains. They utilize peptones and amino acids as
sole sources of energy and have very limited
saccharoclastic activity. 436 Fermentation of nitro-
genous compounds, amino acids, purines, and
pyrimidines has been observed. Fermentation
products (which vary with substrate) include
acetic, propionic, butyric, formic, succinic, and
lactic acids, CO 2 , ammonia, and (sometimes)
hydrogen. Lactate and malate are not fermented.
The peptococci are anaerobic but may exhibit
weak or variable catalase activity. They are rarely
hemolytic or coagulase positive. The %GC content
of DNA in Peptococcus species ranges from 35.7
to 36.7 mol%.
2. Peptostreptococcus
The members of this strictly anaerobic genus
occur in pairs or in short or long chains. Carbo-
hydrates are usually fermented, with formation of
acid and/or gas. Pyruvate is fermented in most
cases, but malate, citrate, and tartrate are not.
Fermentation products include one or more of the
following; acetic, formic, propionic, butyric, iso-
butyric, valeric, isovaleric, isocaproic, caproic, and
succinic acids. Ammonia, amines, and various
alcohols may also be formed. Lactic acid is not
January 1977 277
 uotiujntf pun aouapg pooj ut SMarnsy jootjto SLZ
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Black pigment °

MilV r^
iviUJv U
Indole §!

U i+ i " + «+ ' i ' ' * '«. i + i t <+ H2S 3

i i i i i a. I I I i + i i i i i Nitrate 3

i i i ii i + £ i + t+ I i ' ' ' < Acetyl methyl carbinol g

g.
N u
'+ t + t * " t '. '. ' H i " » " " " 20% bile §
25
% 25
'o- «S ' ' ' ' 'cr ' * 'a- ' • 'a- 'a-l* 'a- ' Hemolysis | g2
3- ffff-" I IIB-B-S-B-3-B-3-3- Growth enhancement I £
3" « ~ -> ©
I I i I i i i ^ i^ • i i^ • i
Adonitol
< +
%** *+ Amygdalin
1
^ I I I I Z + I + < + % + + ' Arabinose

i i i^ i i i P. + + + < + ^ < ^ ^ Cellobiose

1
l? i i I I i < i + i^ l l i i l i Dulcitol

1
i i i i i I i i^ i + i^ i i i^ i I Erythritol
1
i i i i i i^ " i + < o. < z ^ i^ i^ Esculin

I < I £ I I Fructose
&
' ^ ' ' %* W^+WW** Galactose
1 1 + +
' ^ * ^ ? " ? ^ ^ ^ " s " ^ ^ ^ Glucose
>. CO CO t—< P 63 >.*0COOOOOCOCOCOCOCO

S O
•a S ^??S?^ 9
O O ^ ^ ^- ~_/ ._ o o
 TABLE 16 (continued)

Biochemical, Caibohydiate and Feimentation Reactions of Selected Species of the Genus Bacteroides

— _ 2a o a} 23 <D

S S | | 2 2 g g S s | | | j | | | 2 | l Productsfrom
Species ^ 8 | S i ! I H 3 § S i ^ l I I ^ PYG
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fragilis ss. fragilis - w+ - v w+ +w - +w - w+ +w - -w - - - +w +w - +w SAp(lifcivf)

fragilis ss. vulgatus - +" - v +w +w - +w - +" +w +w d - - - +w +w - +w SA(LfpiMv)
fragilis ss. distasonis - -+ - v +w +w - +w v +w +w v v -+ - -w w+ +w - +w SAp(fltoiV)
fragilis ss. ovatus - v - w+ +w +w w +w -w w+ +" v v. v -w -w w+ +w v +w SA(Lfp ib iv)
fragilis ss. thetaiotomicron - +w -' +" +w +w - +w v +w +w v v d - - +w +w +" +w SA(Lpftfn>)
ruminicola ss. ruminicola -w +" - w -+ +w -+ -w +w - -+ -+ -w -w -+ -+ -w d +" -w -+ SA(flp ib b iV)
ruminicola ss. brevis -+ +" - w +" +w +w - + -w +" +w -+ v v -w -w +w + -+ +" SA(lfp ib iv v)
oralis -w +w - w d +w +" - +w -w v +w -+ v -+ -w -w + +w -w -w SA(lfftii>)
hypermegas -+ - +" +" + + + + -w + + +" + w +" + -w -w + +w + PAl(fs)
clostridiiformis ss. - -w - - w+ v - v - -+ v d d v - -w -w +w -w d Af(sle)
clostridiiformis
putredinis _ _ _ _ _ _ _ _w _ _ _ _ _ _ _ _ _ _ _ _ asp/vb ib(lt)
coagulans _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ aisf
fitrcosus _ _ _ w _ _ _ _ _ _ _ _ _ _ _ w _ _ _ v _ _ Lsae(f)
capillosus - - - - _w _w _ _w _ _ _ _ _w _ _ _ _w _w _w _w Sa(1Q
melaninogenicus ss. _ + +- _w _ w _+ v _ d _ _ _+ _ _w _ _ _ +w v _w _w $Aibiv
intermedius
melaninogenicus ss. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ AB ib iv (splf)
asaccharoly ticus

Adapted from Buchanan and Gibbons.7 *

Note: Biochemical reactions: +, positive reaction in >90% of strains; - , negative reaction in >90% of strains. Hemolysis: a, alpha; b, beta; w, weak. Gas
and growth, relative amounts: +, 2, 3, 4; s, stimulated. Growth enhancement by bile (b), hemin (h), rumen fluid (r), serum (s), Tween® 80 (t).
Milk: c, curd; d, digested. Gelation: weak (w) liquefaction in less than half the time of controls after chilling.
Carbohydrate reactions: +, strong acid (i.e., pH < 5.5 in >90% of strains); w, weak acid (pH 5.5-6.0 in >90% of strains); - , no acid produced
(i.e., pH > 6.0 in >90% of strains, except xylose and arabinose); d, positive reaction in 40-60% of strains; v, reaction variable within given strains.
Any of the above symbols used as superscripts, reaction in 10-40% of strains.
Fermentation products from peptone yeast extract glucose (PYG) broth: Upper case letters indicate >1 meq acid/100 ml broth; lower case letters
indicate <1 meq acid or alcohol/100 ml broth; A, acetic; B, butyric; F, formic; ib, isobutyric; iv, isovaleric; L, lactic; P, propionic; S, succinic; v,
valeric; e, ethanol; ( ) products of occasional strains.
 TABLE 17

Biochemical, Carbohydrate and Fermentation Reactions of Selected Species of the Genus Fusobacterium

1 carbino

n cement
)lysis

drol.

Thr
lysis
;>» cd
•a o >, Si 4.
>> •o _: C V _c

jllobios
smolysi

rowth e
arch h>

mygdal
iculin h

rabinos
opiona
:etylm
u

ippurat
otility
;latin

itrate

pase
dole
ccj Products from
Species O is 2 w Si O a S a O CM < < u PYG

gonidiaformans w~ - + _ - +- 42 _ _ a" - V - + - - - Bap(lfs)

A a
varium _w - BLA(p)
necrophorum V cd" + - - - 4' - ba" - - - + - - - Bap(Lsf)
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aquatile - c + - + + 41 + - - + d b d w w+ + Ba(ls)
mortiferum - - - - + • _+ 4 - - - - - b + - w~ BAp(LF/i'sbu)
symbosium _w _c - - - - 42 V - _b d - - - - - + —* BA(Lebuf)
necrogenes - - - - + - 4 - - _b - - + - _w w" Ba(lfpsvbu)
prausnitzii _w - - - + - -2 _w - _a - - rh - - - _w B(AFlspe)
plauti - - - + - + - _+ - _ - +- - - _ w" _w LBas
bullosum - - - - - + 2 - - - + + - - - - - Abpbu
russi _w — +
— — 2' — — — — — — — — Ba(lfsebu)

eha lose
tose

elibiiose
yco gen

_c S* VI
_> 0 0
'3c

arc!
Q

licii
cs
UCO

ulin

icro
iffir
tj o o

Species Q
><
3
*-•
ca
o O O c
1as S H
Products from
PYG

gonidiaformans _ _ _ w~ Bap(lfs)
varium - W _w w+ - - -
- - w - - - - - - - BLA(p)
necrophorum w w Bap(Lsf)
+ w w _w w +w w+
aquatile d W + +w w + +w - + w + - Ba(ls)
mortiferum —w w+ V +w - - w+ w" - w* V V w" V _w + BAp (LF/vsbu)
symbosium _ + w +- +w - - _w - w" + - - - - - - _w - BA(Lebuf)
necrogenes _w w+ w+ +w - - - - - w+ _w _w _w - w" w " - Ba(lfpsvbu)
prausnitzii _w _w w" V _w - w" - _w - - _w - _w _w - B(AFlspe)
plauti +- _w _ w* w+ _w - _ _ _w _ _ - + _ - - LBas
bullosum w - - - Abpbu
russi w - Ba(lfsebu)
Adapted from Buchanan and Gibbons.7 8
Note: Biochemical reactions: +, positive reaction in >90% of strains; - , negative reaction in >90% of strains. Hemolysis:
a, alpha; b, beta; w, weak. Gas and growth, relative amounts: +, 2, 3, 4. Growth enhanced by bile (b), hemin (h),
rumen fluid (r). Milk: c, curd, d, digested. Gelatin: w, weak (i.e., liquefaction in less than half the time as controls).
Carbohydrate reactions: +, strong acid (i.e., pH < 5.5 in >90% of strains); w, weak acid (i.e., pH 5.5—6.0 in>90%
of strains); - , acid not formed (pH >6.0 for >90% of strains). Superscripts denote reaction of 10—40% of strains in
any of the above. Fermentation products from peptose yeast extract glucose (PYG) broth: Upper case, >1 meq
product/100 ml broth; lower case, <1 meq product/100 ml broth; A, acetic; B, butyric; F, formic; IV, isovaleric;
L, lactic; P, propionic; S, succinic; V, valeric; e, ethanol; bu, butanol; ( ) , products of occasional strains.

formed. Catalase, nitrate reduction, and indole 3. Ruminococcus

formation are also negative. Gelatin is usually not The Ruminococcus species have spherical to
liquified, and denatured proteins are not degraded. elongated coccoid morphology. The latter have
Peptostreptococcus is rarely hemolytic. The DNA rounded or flattened termini rather than pointed.
of Peptostreptococcus species contains 33.5 mol% The sides of cells may be flattened where they
GC. come in contact with other cells. They may occur

280 Critical Reviews in Food Science and Nutrition

 as singles, pairs, short chains of 3 to 4 (e.g., R.
albus), or long chains of 8 to 50 (e.g., R.
flavefaciens). The cells stain nearly Gram positive.
Cellulose is usually dissimilated, and cellobiose is
always fermented. Xylan is fermented by ca. 85%
of strains. Fermentation of certain other sugars
varies among strains with glucose, D-xylose,
esculin, fructose, sucrose, lactose, and L-arabinose
as likely substrates. A wide variety of other
carbohydrates and polyols are not fermented.
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Fermentation of cellobiose yields either acetic and
formic acids or ethanol and/or succinic acid as
dominant products. When ethanol is a major
product, more hydrogen and little succinic acid are
observed, as in the case of/?, albus. When succinic
acid is a major product, small quantities of
hydrogen and ethanol are observed (e.g., R.
flavefaciens)?1* Nutrient requirements are com-
plex, with some strains requiring rumen or fecal
extracts. However, many other strains (particularly
of R. bromii) may be grown in a chemically
defined medium containing minerals; NH4* as a
nitrogen source; sulfide or sulfate as a sulfur
source; fructose as an energy and carbon source;
isobutyrate or 2-methyl butyrate and H 2 CO 3 /
HCO3 " as additional C-sources; and the vitamins
biotin, riboflavin, pyridoxine, vitamin B l 2 (re-
placed by L-methionine), pantethine, and tetra-
hydrofolate. 233 As mentioned previously, am-
monia is the primary N-source for all ruminococci;
exogenous amino acid nitrogen is poorly utilized.
Catalase,indole,and H 2 Sare not formed. Starch
hydrolysis and nitrate reduction are also negative.
Ammonia is not formed from amino acids or
peptides. The DNA of Ruminococcus species
contains 39.8 to 45.4 mol% GC.
4. Sarcina
This genus is characterized by rather large
nearly spherical cells (2 to 3 jum) occurring in
packets of eight or more cells as a result of division
in three perpendicular planes. Spore formation has
been reported. The sarcina are strictly anaerobic
and ferment carbohydrates; the main products are
CO 2 , H 2 , acetic acid, and ethanol or butyric acid.
Minimal nutritional requirements include various
amino acids (arginine, glutamate, histidine, iso-
leucine, leucine, methionine, phenylalanine, serine,
tryptophan, tyrosine, and valine), a few vitamins
(biotin and nicotinic acid), glucose or another
fermentable carbohydrate, and inorganic salts. The
DNA composition of Sarcina ranges from 28 to 31
mol% GC. Two species are described in Bergey's
Manual, viz., S. ventriculi and S. maxima. Other
species previously classified as Sarcina are now
found in the genera Methanosarcina, Sporosarcina,
Micrococcus, and Halococcus. Additional charac-
teristics of Gram-positive anaerobic cocci may be
found in Table 18.
E. Gram-negative Cocci
1. Veillonella
Veillonella are small spherical cocci occurring as
pairs, short chains, and masses. Carbohydrates are
not fermented, but several organic acids are
attacked. Lactate is converted to acetate, pro-
pinate, CO 2 , and H 2 , whereas succinate yields
propionate and CO 2 . Pyruvate, oxaloacetate,
malate, and fumarate are also fermented.
Hydrogen sulfide is produced from a variety of
substrates, viz., reduced glutathione, cysteine,
cystine thiosulfate, thiocyanate, and thioglycolate.
The veillonella do not liquify gelatin, reduce
nitrate, produce indole, or exhibit hemolytic
activity. Nonheme catalase (pseudocatalase)
activity may be present. Veillonella species have
complex nutritional requirements including amino
acids, vitamins, CO 2 , and pyruvate (or lactate,
malate, fumarate, and oxaloacetate). They are
strictly anaerobic, and the DNA composition of
species ranges from 40 to 44 mol% GC. Two
species are described in the 8th edition of Bergey's
Manual1* viz., V. parvula and V. alcalescens.
2. Acidaminococcus
This genus was first proposed by Rogosa4 3 4 for
anaerobic Gram-negative diplococci that used
amino acids as their sole energy source. The
diplococci may be oval or kidney shaped. Acid-
aminococcus is weakly saccharoclastic, with only a
minority of strains fermenting glucose weakly.
Lactate, fumarate, malate, succinate, citrate, and
pyruvate are not used as energy sources. Amino
acids, particularly glutamic acid, serve as sole
energy sources. Fermentation products include
acetic and butyric acids (2:1 molar ratio) and
CO 2 . Nutritional requirements are complex; they
include the amino acids tryptophan, glutamic acid,
valine, and arginine (and, occasionally, cysteine,
histidine, tyrosine, phenylalanine, and serine) and
vitamins such as vitamin B I 2 , pyridoxal, panto-
thenate, biotin, and p-aminobenzoic acid. Acid-
aminococcus fails to grow in lactate or pyruvate
media which support growth of Veillonella.
Furthermore, there are no serological cross
reactions between strains of Acidaminococcus and
Veillonella or Peptococcus serotypes.
Acidaminococcus produces ammonia, and
January 1977 281
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TABLE 18

Biochemical, Fermentation, and Carbohydrate Reactions of Selected Peptococcus, Peptostreptococcus, and Ruminococcus Species

abinoss

ilactose
tidine

ose

ycoge
c
•s
;latin

itrate
.5

dole
o S
I I 3 u
Fermentation
ilk
o 3
Species 6 S 2 c S. 3 3 .12 2 S
w «
O a — &
O O 111 products

P. asaccharolyticus _ - + +
+ + + + _w Ab(slf)
I" P. prevottii + _c _+ - _w Ab(Lspf)
8-
Pstc. productus ac ASlffp)
Pstc. micros - AL(sQ
Pstc. parvulus - ac AL(psb)
R. flavefaciens - - - w ASFdj
R. albus +
- - w A(fls)e
R. bromiP _ _ _ _ a(flpb)e
R. callidusb c" SA(Fl,pyj
R. torques^ c w - - - LAe(f;
Sarcina ventriculi _ a" - + +w + w* - EA1

Adapted from Buchanan and Gibbons.7"

Note: +, positive reaction in >90% of strains; -, negative reaction in >90% of strains; w, weak reaction; d, variable reaction. Gas, relative amounts: w, +, 2, 3, 4. Milk: a, acid; c,
curd. Fermentation products: uppercase, major products; lower case, minor products. A, acetic; B, butyric; S, succinic; F, formic; E, ethanol; P, propionic; L, lactic; Py,
pyruvate. Superscripts indicate reaction of minority of strains; ( ) indicates variable production.
a
Sp.nov. (Moore etal., 1972). 374 '
b
Sp. nov. (Holdeman and Moore, 1974). 254
 gelatin liquefaction is weak or negative. Nitrate
reduction, H2 S or indole production, and catalase
and cytochrome oxidase tests are negative. The
DNA composition of 15 strains averaged 56 ±0.9
mol% GC. Only one species has been described in
the 8th edition of Bergey's Manual,1* viz., A.
fermentans.
3. Megasphaera
These are relatively large cocci (2 /im in
diameter), occurring in pairs or occasionally in
chains of pairs. Lactate and glucose are fermented,
producing short-chain organic acids (acetate, pro-
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pionate, butyrate, isobutyrate, and valerate), CO2,
and some H 2 . Succinate, fumarate, and malate are
not fermented. Megasphaera has complex
nutritional requirements and will grow well in a
medium containing yeast extracts, salts, thio-
glycolic acid, soluble starch, and sodium lactate.
Bergey's Manual describes a single species, viz., M
elsdenii.7 8 In addition to glucose and lactate, M.
elsdenii ferments fructose well; glycerol, maltose,
mannitol, sorbitol, and sucrose are fermented
weakly. Although H 2 S is produced, gelatin lique-
faction, nitrate reduction, catalase, and indole are
negative for this species. The DNA composition of
M. elsdenii and two similar strains averaged 53.6 +
0.5 mol% GC.
Acidaminococcus fermentans and M. elsdenii
have been found in 25% and 10%, respectively, of
normal human fecal specimens, 5 ' 3 suggesting that
they are normal flora components.
F. Miscellaneous Genera
1. Butyrivibrio
This genus includes motile, strictly anaerobic,
Gram-negative, curved rods with monotrichous
polar or subpolar flagella. Carbohydrates are fer-
mented, producing butyric or lactic acids in major
proportions. Only one species, B. fibrisolvens, is
described in Bergey's Manual.1* It ferments
glucose to butyric and formic acids with lesser
amounts of lactic acid, H 2 , and CO 2 . Acetic and
propionic acids and ethanol may also be produced.
B. fibrisolvens also ferments D -xylose, fructose,
glucose, cellobiose, lactose, maltose, sucrose,
esculin, salicin, and inulin. Trehalose, dextrin,
pectin (polygalacturonic acid), starch, and xylan
are frequently attacked. Cellulose is fermented by
some strains. Indole and catalase production and
nitrate reduction are negative. Hydrogen sulfide
and acetyl methyl carbinol production and gelatin
liquefaction are variable.
Nutritional requirements of B. fibrisolvens are
variable, but most strains will grow in chemically
defined media containing glucose, minerals, B-
vitamins, cysteine or sulfide, and ammonia.
2. Coprococcus gen. nov.
This genus was first proposed by Holdeman and
Moore, 254 to describe many strains of anaerobic
Gram-positive cocci isolated from human feces
which could not be easily included in described
genera. Unlike the ruminococci, they produce
butyric acid; they do not occur in packets as do
the Sarcina. They are distinguished from the
peptococci and peptostreptococci because carbo-
hydrates are their main source of energy. Thus,
Coprococcus includes those anaerobic Gram-
positive cocci that actively ferment carbohydrates,
forming butyric and acetic acids along with formic
or propionic and/or lactic acids. The DNA compo-
sition of six strains ranged from 39 to 42 mol%
GC. Three species have been described, viz., C.
eutactus, C. catus, and C. comes.2 s4
3. Gemmiger gen. nov.
Gossling and Moore2 ° * have suggested that this
genus describes Gram-negative to Gram-variable
bacteria that appear to reproduce by a budding
process. They grow anaerobically, using carbo-
hydrates as their sole or major energy source. One
species has been described, G. formicilis, which
requires glucose, fructose, maltose, or some other
fermentable carbohydrate. Fermentation products
include formic and n-butyric acids with small
quantities of acetic, lactic, succinic, malonic, and
pyruvic acids. G. formicilis does not ferment
lactate or amino acids, hydrolyze proteins or
lipids, reduce nitrate, or produce catalase. Pairs of
cells are attached by the smaller (daughter) ends
and chains are commonly seen. The DNA composi-
tion is 59 mol% GC. The species is common in the
feces of humans and the cecum of chickens.
4. Streptococcus
Rogosa 435 and Holdeman and Moore 254 have
suggested including in the genus Streptococcus
obligately anaerobic and fermentative species of
Gram-positive cocci which occur in chains and
produce lactic acid as their primary product. Thus,
species known as Peptostreptococcus intermedius,
Peptostreptococcus morbillorum, or Peptococcus
constellatus would be placed in the genus Strepto-
coccus. Holdeman and Moore 254 have also
described a new species of anaerobic Strepto-
January 1977 283
 coccus isolated from human feces, viz., S. hansenii.
This species grew well in PYG broth at 37 to 45°;
growth was not enhanced by supplementation
with Tween 80, rumen fluid, or bile. Fermentation
of glucose, lactose, maltose, raffinose, galactose,
and inulin was observed, as was H 2 S production.
Products formed in PYG were D-lactic acid, acetic
acid, and succinic acid (20:2.5:1). Lactate and
gluconate were not utilized, and no propionate
was produced from threonine. The DNA composi-
tion was 37 to 38 mol% GC. Unlike S. hansenii, S.
constellatus and S. morbillorum do not ferment
lactose. S. intermedius may be distinguished from
S. hansenii in that it ferments cellobiose.
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5. Methanobacterium
The members of this genus are quite variable in
morphology (filamentous to coccoid rods) and
Gram reaction. They may be nonmotile or motile,
with monotrichous polar flagella. Methano-
bacterium is chemolithotrophic, obtaining energy
via the oxidation of hydrogen to methane (CO2 as
terminal oxidant). The species are very strict
anaerobes and range from autotrophic to hetero-
trophic in nutritional requirements. The only
member of the genus to be isolated from the
human gastrointestinal tract is M. ruminan-
tium.3 8 3 a This species is Gram positive and forms
nonmotile coccoid rods. Formate may replace H2
as the electron donor for energy-yielding CO2
reduction. Formate may also replace CO2 as the
source of carbon for methane production
(probably via a formate hydrogenlyase). Carbo-
hydrates, amino acids, fatty acids, and alcohols are
not utilized as electron donors. Acetate is essential
as the major source of carbon, ammonia as the
N-source, and sulfide as the sulfur source. Amino
acids, peptides, and other organic nutrients in
yeast extract are not effectively utilized as C-, N-,
or S- sources. Some strains require 2-methyl-
butyric acid, a few amino acids, and other uniden-
tified growth factors present in rumen fluid.
VI. SELECTED ASPECTS O F
GUT FLORA METABOLISM
In considering the mass and variety of viable
bacteria contained in the human gastrointestinal
tract at any given time (roughly 10 14 cells or 1 kg
wet cell weight), it has been suggested that the gut
flora might be regarded as an "organ" of the body.
This "organ" is subject to alteration by various
284 Critical Reviews in Food Science and Nutrition
selective pressures and possesses great potential for
metabolic diversity. 139 Studies in laboratory
animals5 s indicate that the maintenance of the
intestinal flora requires about 10% of the animal's
dietary caloric intake, a relatively expensive
"organ" in the host's overall economy. This cost
may be somewhat discounted by animals prac-
ticing coprophagy.
Due to the breadth of the subject of gut
bacterial metabolism, a comprehensive treatment
is beyond the scope of this review. Many aspects
of the subject have been dealt with in depth in
recent reviews and monographs. 1 3 9 ' 4 6 5 ' 4 6 7 '
497,556
A. Carbohydrates
The possibility that the nature and quantity of
the dietary residue may be a predisposing factor in
colorectal cancer83 has recently caused attention
to be focused on this ill-defined part of the diet.
Dietary fiber has been roughly defined as un-
available carbohydrate or all plant polysaccharides
not dissimilated by intestinal secretion, plus lignin.
Unfortunately, the plant polysaccharides and
lignin are themselves poorly defined and vary
chemically in different plant substances. It has
been estimated that more than half of the dietary
fiber in Western diets is degraded by microbial
action in the colon to various short-chained
organic acids, water, carbon dioxide, hydrogen,
and, in some cases, methane. A fiber content high
in lignin is much less susceptible to microbial
attack. The production of short-chain acids may be
related to the frequent observation that subjects
ingesting larger quantities of dietary fiber produce
larger, more hydrated stools.4 S 9 At colonic pH
these acids would be largely ionized and poorly
lipid soluble; they could probably exert an
osmotic effect on the colon. 352 The role of
dietary fiber in human nutrition has recently been
reviewed in depth in this journal.5 o s
B. Nitrogen Compounds
Some aspects of enteric metabolism of TV-
compounds have been discussed earlier, e.g., the
decarboxylation or deamination of amino acids
(such as tryptophan and tyrosine), /V-nitrosation
and denitrosation of dietary secondary amines,
and the hydrolysis of urea. Some other topics are
discussed in a recent symposium monograph.64
 1. Primary Amines: Deamination
The ability to deaminate amino acids is
common among gut bacteria, although the
ammonia released makes only a minor contribu-
tion to the total intestinal ammonia. Five
mechanisms of deamination are recognized: (a)
oxidative, with production of an aldehyde; (b)
reductive, with production of a saturated organic
acid; (c) hydrolytic, producing an alcohol or
a-hydroxy acid from amine or amino acid, respec-
tively; (d) desaturative, producing an unsaturat'ed
compound by removal of ammonia; and (e) the
Stickland reaction in which pairs of amino acids
are deaminated, one serving as oxidant, the other
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deamination of amino acids. The deamination of
putrescine and cadaverine by this mechanism has
been observed in many strains of Escherichia coli,
bacteroides, bifidobacteria, and clostridia. These
amines may be subsequently converted into the
cyclic secondary amines piperidine and pyrro-
lidine. 139
The production of "saturated" organic acids
and alcohols via reductive deamination of amino
acids is quite common among gut bacteria,
tyramine, 3,4-dihydroxyphenylalanine (L-DOPA),
and ethanolamine, which is reductively
deaminated by gut bacteria.
Hydrolytic deamination is probably carried out

as reductant (Figure 1). Oxidative deamination is in the formation of p-hydroxyphenyl lactate from
of relatively minor importance in microbial tyrosine. Desaturative deamination appears to be

(a) CO2H CO2H

CH-NH, C=O NH,

a-Keto Acid

(b) CO2H 2H CO2H

CH-NH, CH2 NH3

(c) CO2H H2O CO2H

CH-NH, CHOH + NH3

(d) CO2H -2H CO2H

CH-NH, CH + NH,

CH,
I
CH

R
(e) CO2H CO2H CO2H CO2H

CH-NH2 + 2 CH-NH, H2O R + 2 CH2 + 3NH 3 + C 0 2

R R
FIGURE 1. Types of microbial deamination reactions.

January 1977 285

 the mechanism of aspartate deamination to
fumarate (and subsequently to succinate) in
enterobacteria and other facultative anaerobes.
Fermentations of pairs of amino acids, the
so-called Stickland reaction, are carried out by
clostridia. The initial evidence for this mechanism
was obtained by observing the interaction of
amino acids and suitable redox dyes with cell
suspensions of Clostridium sporogenes.41 While
alanine, leucine, isoleucine, and some other amino
acids were found to serve as reductants for
methylene blue, others (such as glycine, proline,
and hydroxyproline) were ineffective as reduc-
tants. The latter amino acids, on the other hand,
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were able to oxidize the reduced form of benzyl
viologen (a lower potential redox dye). When 1
mol of the "reducing" alanine was mixed with 2
mol of the "oxidizing" glycine, 3 mol of ammonia
were formed, showing that both acids of the redox
couple were deaminated. The mechanism of the
reaction seems to involve a two-step NAD-linked
oxidation of the reducing amino acid via a 2-oxo
acid intermediate:
RCH(NH2 )CO2 H + NAD+ + H2 O
= RCO CO2 H + NH3 + NADH + H*
The subsequent oxidative decarboxylation of the
2-oxo acid is probably catalyzed by a 2-oxo acid
dehydrogenase and a phosphotransacylase:
RCOCO 2 H + NAD* + CoASH
= RCOSCoA + CO 2 + NADH + H*
RCOSCoA + HPO4= = RCO, P 0 3 = + HSCoA
RCO 2 PO 3 = + ADP=
= " + ATP" 3
The reoxidation of NADH is presumably carried
out by the appropriate amino acid oxidoreductase
for the "oxidizing" amino acid:
2 R'CH(NH 2 )CO 2 H +
= 2R'CH 2 CO 2 H + 2NAD* + 2NH3
When proline is the oxidizing species, it is reduced
by ring cleavage without formation of N H 3 . 2 9 9
2H2O
H 3 CC-N- C-N-CH 2 C0 2 H
FIGURE 2. Hydrolysis of amide linkages of p-acetylaminohippuric acid.
286 Critical Reviews in Food Science and Nutrition
2. Primary Amines:N-Esterification and Hydrolysis
iV-Esterification by gut bacteria is instrumental
in the detoxification of antimicrobials, such as the
sulfonamides and aminoglycosides (e.g., strepto-
mycin). The reverse reaction, amide hydrolysis,
serves a similar function in the metabolism of
chloramphenicol by enterobacteria.
Enterobacteria are also largely responsible for
the iV-acetyl histamine excreted in human
urine 1 3 9 following oral administration of the
amine. The reverse reaction is also carried out by
the gut bacteria in vivo and in v i t r o . 4 8 9 ' 4 9 0 A
variety of iV-acetylated amino acids are also
hydrolyzed by gut microfiora. The deconjugation
of bile acids and glycocholic and taurocholic acids
and the hydrolysis of both hippuric acid and
p-acetylaminohippuric acid have been demon-
strated in the human gut. 2 6 S Hydrolysis of
hippurate by enterococci and of p-aminohippurate
and p-acetyl-aminohippurate (Figure 2) by rat
cecal flora and pure cultures of Streptococcus
faecalis and Aerobacter aerogenes has been
reported. 4 6 7 ' 5 0 0
Degradation of pteroyl-L-glutamic acid (folic
acid) and the antimetabolite methotrexate (4-
amino-4-deoxy-10-methyl-pteroyl-L-glutamate)
has been observed in various pseudomonads
(Figure 3 ) . 3 1 0 While similar amidase activity has
been demonstrated with methotrexate and mouse
cecal microfiora, the nature of the organisms
responsible is unknown. 529 It is not known to
what extent amidase acts upon dietary folates and
folic acid in the human intestinal tract. Such
derivatives would be expected to have little or no
biological activity for higher organisms. Aryl
amidase activity for a variety of amino acid-/3-
naphthyl-amides (e.g., alanyl-, lysyl-, and leucyl-)
has been observed in some enterobacteria (e.g.,
Escherichia coli, Proteus vulgarus, Salmonella spp.,
and Pseudomonas aeruginosa).481 The aryl
amidase/esterase of the aerobic Pseudomonas acid-
ovorans has been investigated in some detail by Alt
et al.6 and Alt and Krisch.7 The enzyme differs
from the P. aeruginosa amidase in that it does not
transfer acyl groups to hydroxylamine; however, it
does exhibit esterase activity for several substrates,
e.g., phenyl acetate, ethyl acetate, methyl
butyrate, and ethyl propionate.
¥ H 2 N- - C 0 2 H + H 2 NCH 2 C0 2 H + CH3CO2H
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R R'
OH H pteroyl-L-glutamic acid — • pteroic acid + glu
NH 2 CH3 methotrexate — • 4-amino-4-deoxy-10-methyl pteroic acid + glu
FIGURE 3. Folic acid and methotrexate degradation by inicrobial amidase
action.
The deamidation of nicotinamide has been
observed in the stomachs of normal rats, but not
in axenic rats. 484 Several organisms have been
associated with this deamidase activity: Bacillus
sp., Flavobacterium peregrinum, E. coli, Strepto-
coccus faecalis, and Lactobacillus acidophilus.
3. N-Dealkylation
The dealkylation of choline by various gut
bacteria has been recognized for some time. The
metabolic sequence via trimethyl-, dimethyl-, and
methylamine to ammonia is carried out by
clostridia, bacteroides, and bifidobacteria. Choline
itself is produced from lecithin by bacterial phos-
pholipase and phosphatase actions. Studies with
antibiotics in humans and axenic animals indicate
that approximately 50% of urinary dimethylamine
is of intestinal bacterial origin.2 7 Other examples
of JV-dealkylation by gut bacteria include the
herbicide trifluralin, drugs such as imipramine and
methamphetamine (Figure 4), and various quarter-
nary ammonium compounds (Figure 5). 4 6 7
«.
4. N-Nitrosation
The chemical formation of nitrosamines may be
significant and widespread, with respect to
environmental carcinogenesis.561 The possibilities
for gastrointestinal and urogenital microbial for-
mation of nitrosamines from secondary amine
precursors have been discussed above (Section
II.B.6) (Figure 6). While several nitrosamines are
carcinogenic for rodents, none has been linked to
human carcinogenesis as yet.
5. Reduction of Nitrate
Many microorganisms may use nitrate as their
sole nitrogen source, indicating that nitrate is
reduced to ammonia and incorporated into amino
acids, etc. Thus, a distinction must be made
between such "assimilatory" nitrate reduction and
bulk "dissimilatory" reduction of nitrate to
nitrite, nitrogen, or ammonia, etc. (Figure 7). The
latter is quite common to a variety of intestinal
microorganisms, e.g., enterobacteria, particularly
Escherichia coli; bifidobacteria; bacteroides; clos-
January 1977 287
 2 X
N ^ CH2CH2CH3
N
N

Trifluralin

CH,

Imipramine
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CH,

NH

CH3

Methamphetamine

FIGURE 4. Examples of N-dealkylation by gut microflora: trifluralin degraded

by rumen bacteria; imipramine metabolism by human fecal flora; methampheta-
mine demethylation by enterococci, Iactobacilli, and clostridia.

CH, CH,

N CH,

CH, CH,

Trimethylphenylammonium iodide Dibenzyldimethylammonium iodide

CH, |-
CH,
. C H 2 — N — C H 2 —/( ^ I Br-

CH,

CH 3

Tribenzylinethylammonium iodide Acetyltrimethylammonium bromide

. - FIGURE 5. Some quarternary ammonium compounds degraded by gut microflora.

288 Critical Reviews in Food Science and Nutrition

 R-
NH + NO,
R'-

N N
oo N —CH,

II
o
FIGURE 6. Formation of N-nitrosamines by gut microflora, particularly E. coli
and S. faecalis.

(a) N03" — • N02" — • ? —• NH

NH220H
0 — • NH3 — • amino acids
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(b) N03" — • N0 2 "

(c) NO3- — * N O / — • NO—>• N2O - • N2

FIGURE 7. Types of microbial nitrate reduction: (a) assimilatory reduction as carried

out by Clostridium, Enterobacter, Escherichia, Pseudomonas, Vibrio, etc.; (b) nitrate
respiration, e.g., Corynebacterium, Escherichia, Eubacterium, Propionibacterium,
Proteus, and Staphylococcus; (c) denitriiication, e.g., Alcaligenes, Corynebacterium,
Micrococcus, Propionibacterium, and Pseudomonas.

tridia; streptococci and lactobacilli. In dis- solely by an A-type membrane-bound enzyme

similatory nitrate reduction or so-called "nitrate
respiration," it is thought that NQ3 ~ serves as an
alternative general electron acceptor in the micro-
bial electron transport chains. It has been
suggested that nitrate respiration may have pre-
ceded oxygen respiration in evolution. 389
Pichinoty et al. 4 0 4 have described two separate
types of nitrate reductase associated with these
two physiologically distinct processes. A particle-
bound "A" enzyme has a respiratory function in
those bacteria producing it. When cells of Micro-
coccus denitrificans are grown anaerobically in the
presence of nitrate, they produce the "A" nitrate
reductase, which has the ability to reduce chlorate
as well as nitrate. In the presence of hydrogenase,
cell-free extracts may use H 2 as a nitrate reduc-
tant, with flavin mononucleotide (FMN) or redox
dyes as electron mediators. A second soluble "B"
enzyme found in cell extracts is characterized by
its lack of chlorate reductase activity. This soluble
"B" reductase appears to serve a nutritive or
"assimilatory" role in many, if not all, organisms
in which it is found. In general, assimilative nitrate
reduction is much less common among bacterial
species than is nitrate respiration.398 Further-
more, there appears to be little taxonomic cor-
relation between nitrate-reducing microorganisms.
In Escherichia coli and several other species of
enterobacteria, nitrate reduction is carried out
complex. Formation of the complex is induced by
nitrate and repressed by molecular oxygen. Cells
grown anaerobically carry out nitrate respiration,
whereas those grown aerobically assimilate nitrate.
The complex has been studied in a number of
nitrate reductase-deficient mutants 447 of E. coli
and found to be comprised of formate dehydro-
genase, cytochrome b , , and a molybdenum con-
taining nitrate reductase. The latter component
was recently solubilized and purified by MacGregor
et al. 3 2 9 It has a very high molecular weight of
nearly 800,000 daltons and is itself composed of
four large and four small polypeptide subunits and
four Mo atoms per molecule of enzyme. The data
of Ruiz-Herrera et al. 4 4 7 suggested that only
formate could donate electrons for nitrate reduc-
tion in vivo, a finding at odds with the postulated
role of nitrate as a general electron acceptor. The
product of the complex (e.g., nitrite) can serve as
an electron acceptor for reduced pyridine nucleo-
tides, but under anaerobic conditions it is not
removed at a rate commensurate with that
expected of a general electron acceptor. 558 A
generalized electron transport scheme for dis-
similatory nitrate reduction is shown in Figure 8.
Many recent studies of the structure and
function of the nitrate reductase complex in E.
coli have employed chlorate-resistant mutants (chl
A-G). Such mutants are viable anaerobically

January 1977 289


RH 2 — NAD Flavin
Formate •
-•
substrate
t
Substrate
FIGURE 8. Composite scheme of electron flow in dissimilatory nitrate reduction in several species.
(Taken from Payne, W. J., Bacteriol. Rev., 37,409, 1973. With permission.)
because they lack the ability to reduce chlorate to
the toxic chlorite. The loci probably represent
many of the structural and regulatory genes of the
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complex. 3 2 6 " 3 2 8 ' 5 0 4 The effects of the chl genes
studied so far are quite varied. The gene products
of chl A and chl B appear to be proteins which
comprise part of the complex. Cell-free extracts of
chl A and chl B mutants are complementary; they
yield a soluble enzyme capable of reducing nitrate
and chlorate, with reduced viologen • dyes or
reduced flavins as reductants. The chl D gene is
involved in molybdenum activation of the nitrate
reductase. S04 The nitrate reductase complex of E.
coli appears to be the most complicated of those
studied, although few others have been studied in
great detail. For instance, in Staphylococcus
aureus the nitrate reductase is not linked to
cytochrome bj and formate dehydrogenase,
although the oxidation of L-lactate probably
provides electrons for reduction. 8 ' The nitrate
reductases of certain lactobacilli (L. plantarum and
L. fermenti) do not have any requirements for
anaerobiosis; they are somewhat low in fer-
mentable carbohydrates. Formation of the enzyme
is pH dependent and occurs only above pH 5.5.
6. Nitrite Reduction
In Escherichia coli, nitrite reduction is also a
relatively complicated process. 282 This organism
may utilize nitrate, nitrite, hydroxylamine, or
nitric oxide as its sole source of nitrogen and may
employ at least three distinct nitrite-reducing
enzyme systems. A soluble NADPH-linked enzyme
reduces nitrite, although its main function appears
to be sulfite reduction. An FMNH2-linked or
reduced viologen-linked enzyme may also reduce
nitrite. Anaerobic growth in the third enzyme is
induced by nitrate or nitrite. The enzyme is
NADH-linked and presumably has a function in
the reduction of nitrite and hydroxylamine to
ammonia. A low-potential cytochrome (C 5 s 2 )
290 Critical Reviews in Food Science and Nutrition
other c-type
b cytochrome cytochrome
Quinone
t
Cytochrome b. [Mo v FV»*
1
NO,
may be involved in the latter mechanism although
the evidence is somewhat equivocal.39 8
The intermediates of nitrite reduction are un-
known. However, enzyme systems have been
reported which will reduce either nitric oxide
(NO) or nitrous oxide (N 2 O), which both have
intermediate oxidation states with respect to
nitrogen for the NO2 ~/N2 couple. The enzymes
catalyzing nitrite reduction in the several
organisms studied (Micrococcus denitroficans,
Pseudomonas aeruginosa, Alcaligenes faecalis, and
Achromobacter fischeri) do not contain Mo (as do
those catalyzing nitrate reduction), but rather iron
and copper associated with c-d and c-type cyto-
chromes. A more detailed discussion of the micro-
bial reduction of nitrogenous oxides can be found
in a recent review.3 9 8
7. Nitro Groups
The reduction of aromatic nitro groups by the
intestinal micro flora is probably of considerable
significance with respect to the toxicology of the
parent compounds and their metabolites. While
Scheline466 found a lack of reduction of p-nitro-
catechol or its sulfate by rat cecal flora in vitro, a
subsequent study s ° ° revealed a wide variety of rat
cecal microbes able to reduce 4-nitrobenzoic acid.
Most of the bacteria studied (viz., Streptococcus
faecalis, Lactobacillus sp., Bacillus spp., Proteus
vulgams, Aerobacter aerogenes, Clostridium sp.,
and Bacteriodes sp.) produced 4-aminobenzoic
acid. One species of Lactobacillus, however,
yielded 4-Af-hydroxylaminobenzoic acid and only
small amounts of 4-aminobenzoic acid (PABA).
Significantly, two strains of Pseudomonas
aeruginosa possessed no nitro reductase activity. It
would seem that the reduction mechanism involves
4-hydroxylamino- rather than 4-nitroso-benzoic
acid. Zachariah and Juchau5 7 7 first demonstrated
the quantitative significance of the gut microflora
in reducing 4-nitrobenzoic acid (PNBA). The rate
 of anaerobic reduction (jumol PABA formed/g
protein/hr) in gut contents was more than twice
that of the gut wall arid was nearly four times the
rate observed with liver homogenates. A carbon
monoxide atmosphere inhibited the latter
activities much more than those, of the gut
microflora. An oxygen atmosphere inhibited nitro
reduction in all cases by 90% or more. After
intraperitoneal or oral administration of 50 mg
PNBA, 1 and 2% of the dose, respectively, was
recovered during a 48-hr period as PABA in urine.
Treatment with antibiotics (bacitracin, tetra-
cyclines, and neomycin) caused an 80 to 90%
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decrease in PABA excretion over the same period,
indicating a dominant role of the intestinal micro-
flora in the host metabolism of 4-nitrobenzoic
acid. The antibiotic treatment had no significant
effect on hepatic nitro reduction, but it virtually
eliminated the nitro reduction capacity of gut
contents within 12 hr. Assays of five intestinal
isolates showed that Escherichia coli had the
highest specific activity for nitro group reduction
(200 /imol/g/hr) and A. aerogenes the lowest (87
jtzmol/g/hr). P. vulgaris, Salmonella typhimurium,
and S. faecalis had equivalent activities (140 to
150/umol/g/hr).
A more recent study by Wheeler et al. 5 4 8 used
a 25-mg dose of PNBA in conventional and
germfree rats. The former converted 25% of the
dose to PABA, while the germfree animals meta-
bolized only 1%. A number of normal flora
components were selectively associated with the
germfree animals to assess their contribution to
nitro group reduction. Gnotobiotic rats associated
with Lactobacillus plantarum converted 3.9% of
the PNBA dose. Further colonization with Clos-
tridium sp. and S. faecalis increased the amount of
PNBA converted to about 12%. Studies of micro-
bial nitro reduction in vitro seem to correlate well
with the studies in vivo. The most active reducers
of PNBA were Clostridium perfringens (88%),
Gostridium sp. (84%), Peptostreptococcus pro-
ductus I (82%), and S. faecalis (76%). Among the
moderately active species were Bacillus fragilis ss.
vulgatus (68%), E. coli (67%), and Bacteroides
fragilis ss. thetaiotaomicron (54%). The least active
species tested were Proteus mirabilis (21%), L.
plantarum (9%), and Propionibacterium acnes
(2%).
The microsomal enzymes in the liver of rats
may be induced by administration of DDT or
phenobarbital to exhibit increased reduction of
PNBA in vitro, but induced animals do not
demonstrate an increased capacity for PNBA
reduction in vivo.88 Thus, overall it appears that
mammalian enzymes play a small role in the
reduction of PNBA.
An analogous reaction (the reduction of
•/V-hydroxy-4-acetylaminobiphenyl to 4-acetyl-
aminobiphenyl) has been demonstrated in cultures
of C. perfringens, P. productus I, Bacteroides
fragilis ss. vulgatus, and thetaiotaomicron, but
not in E. coli or L plantarum.547 Previously, the
carcinogen ./v*-hydroxy-iv*-2-fluorenylacetamide was
shown to be similarly reduced by intestinal bac-
teria. 555
The reduction of an aromatic nitro group
appears to be the obligatory first step in the
activation of several procarcinogens and pro-
mutagens. 4 4 1 ' 5 7 1 ' 5 7 2 Therefore, it is interesting
that Wheeler et al. 5 4 9 observed the mutagenicity
of 2-nitrofluorene and 4-nitrobiphenyl for the
Salmonella typhimurium tester strain TA1538 in
monoxenic rats associated with this organism.
Their negative results with 4-nitrobenzoic acid
(PNBA) are probably due to strain specificity, as
this compound is mutagenic for strains TA1535
and TA100. 6 8 a There is some evidence that many
aromatic nitro drugs in general and 4-nitrobenzoic
acid in particular may be converted under
anaerobic conditions to free radical anions
(ArNO2 ") as a first step in their reduction. 341 >342
Such free radicals may play an important role in
the mutagenicity which many of these compounds
exhibit irt microbial test systems.
C. Food Additives And Other Xenobiotics
/. Azo Food Colors
The association of hepatic azo reductase
activities with microsomal NADPH-cytochrome c
reductase, 2 3 S ' 2 3 6 soluble NAD(P)H-dehydro-
genase (DT-diaphorase),3 ° and possibly other
enzymes has stimulated the search for microbial
equivalents. Roxon et al. 4 4 4 first demonstrated
the association of flavins (riboflavin, FAD, and
FMN) with azo reduction in whole cells and crude
cell-free preparations of Proteus vulgaris. They
showed that:
1. Whole-cell suspensions did not reduce
tartrazine (FD & C Yellow No. 5) unless they were
preincubated for 24 hr and that azo reductase
activity increased thereafter to ca. 50 hr starva-
tion.
January 1977 291
 2. Cell-free extracts gave the same level of
activity, regardless of starvation time.
3. No activity was found in the supernatant
from 50-hr starved cells, but the supernatant was
necessary for optimum activity, which it restored
even after boiling.
4. A heat-stable cofactor was found to
increase linearity in concentration in the super-
natant upon starvation and was identified as
riboflavin.
With cell-free extracts, they investigated the
specificity of the azo reduction process for FAD,
FMN, and riboflavin and found that FMN was by
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far the most active. They postulated a soluble
flavokinase affecting conversion of riboflavin to
FMN. The enzyme(s) in P. vulgaris which reduces
FMN is apparently NADPH specific. Subsequent
studies with cell-free extracts of Streptococcus
faecalis and the azo dyes Acid Yellow 468 and Red-
2 G i 8 5,5 3 8 c o n f i r m e ( i a n d extended the findings
of Roxon et al. Gingell and Walker 185 found that:
1. Reduced flavins acting as two electron
donors could rapidly reduce Red 2G nonenzy-
matically and thus could act as an "electron
shuttle" from NAD(P)H-linked flavoproteins to
the acceptor azo dye.
2. The effects of inhibitors were consistent
with direct participation of soluble flavins and the
noninvolvement of cytochromes.
3. Enzyme fractions possessing azo reduc-
tase activity also showed cytochrome c reductase,
diaphorase activities, and respired 0 2 .
Thus, azo reduction under anaerobic conditions
probably represents a nonenzymatic reduction by
enzymatically generated reduced flavins; oxygen
inhibition results from autooxidation of the
reduced flavin.
While the role of soluble flavins in the essen-
tially nonenzymatic reduction of azo dyes seems
reasonably well established, the cellular site of azo
reduction is still not certain. The work of Roxon
et al. 4 4 4 implicated cellular permeability as a
prime factor either with respect to the efflux of
reduced flavin and/or the influx of the charged
aromatic azo dye. Studies carried out in the
author's laboratory 69 with whole cell suspensions
of Proteus vulgaris, Streptococcus faecium, and
Salmonella typhimurium have confirmed the
marked stimulation in the rates of reduction of FD
292 Critical Reviews in Food Science and Nutrition
& C Yellow No. 5 and 6 (tartrazine and sunset
yellow) obtained with exogenous flavins. Experi-
ments carried out with the same azo chromo-
phores coupled with linear high polymers (peak
molecular weights of ca. 200,000 daltons) 122
indicate that penetration of the chromophore
through the plasma membrane is not a rate-
limiting factor in microbial azo reduction. The
rates of reduction seen with the polymeric azo
dyes were very similar to their monomeric equiva-
lents and showed the same stimulation with
exogenous FMN. Furthermore, it was found that
exogenous flavins could be replaced by a variety of
low redox potential (E'o < -200 mV) dyes in
stimulating azo reduction by whole cells of P.
vulgaris and S. typhimurium. Methyl viologen,
benzyl viologen, phenosafranin, and neutral red
were the most potent electron mediators and, in
many instances, exceeded FMN at equivalent
concentrations. A deep rough (rfa) mutant of S.
typhimurium exhibited particularly high azo
reductase activity with benzyl viologen alone,
perhaps indicating the importance of the chemical
nature of the cell envelope in azo reduction.69
The molecular parameters of the dye influencing
the rate of azo reduction have been investigated by
Walker and Ryan,s 3 7 in addition to the nature of
reducing agents. They found that the predominant
factor was the electron density in the region of the
azo group, although hydrogen bond stabilization
had to be considered in some cases. The rates of
reduction (zero order with respect to dye) in cell
suspensions of P. vulgaris correlate reasonably well
with the redox potential of the azo dye (i.e., lower
potential corresponds to slower reduction). 143
All available experimental evidence supports a
general model based on an "electron shuttle" in
which the rate determining factors include:
1. The redox potential of the mediator
vis-a-vis that of the azo dye substrate.
2. The permeability of the mediator to the
"cellular" site of the reduction which may be
soluble intracellular, plasma membrane associated,
periplasmic (i.e., external to plasma membrane but
not to cell wall), or extracellular and could involve
molecular size and charge.
3. Specificity of flavoprotein-reducing
enzymes with respect to the mediator.
4. Steric and electrostatic factors influenc-
ing the reduction of the azo dye by the mediator
in its reduced form.
 5. The permeability of the azo dye substrate
to the cellular site of reduction, probably a
significant factor for some low molecular weight
or low charge azo dyes.
Some of these ideas are summarized in Figure 9.
Association of the flavoprotein diaphorase with
the plasma membrane is likely but not certain.
The similarity of this general model and the
mode of azo reduction in intestinal microorgan-
isms in vivo is only implied. However, the findings
of Williams et al. 5 5 4 may support his theory; they
found that riboflavin stimulates azo reduction by
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rat cecal bacteria. Also, Allan and Roxon4 found
that exogenous bile salts can increase fivefold the
rate of tartrazine reduction by cell suspensions of
log phase P. vulgaris; this may be due to an
alteration in the cell envelope's permeability to
tartrazine. A similar explanation might be
advanced for the specificity shown by S. faecalis
for azo reduction of the polar azo dye Acid
Yellow. Bacillus spp. and Pseudomonas aeruginosa
were unable to reduce this dye, while activities
seen with P. vulgaris, Aerobacter aerogenes,
Clostridium sp., Bacteroides sp., and Lactobacillus
sp. were weak to moderate. 500
The intestinal reduction of azo" compounds
proceeds via the hydrazo intermediate
(R-NH-NH-R') to the component primary amines.
While the reduction of certain azo dyes (e.g.,
amaranth) 343 by mammalian microsomal prepara-
tions may also involve preliminary generation of
the azo anion free radical (R-N-N~-R') such
radicals have not yet been observed during
microbial azo reduction.
A recent study of rats involving absorption,
metabolism, and excretion of the major food
colorants used in the U.S. (viz., amaranth, FD & C
Red No. 2; sunset yellow, FD & C Yellow No. 6;
and tartrazine, FD & C Yellow No. 5, employed
radiolabeled dyes and metabolic products (Figure
10). 258 The results, in most instances, confirmed
the findings of earlier studies which used less
effective techniques. All three dyes and their
metabolites are absorbed from the intestinal tract
to some extent (2 to 25 mg dose range). Very
small quantities of the intact dyes were excreted in
the feces, indicating very effective microbial reduc-
tion in the lower intestine.
The urinary and biliary excretion of amaranth
and its reduction products naphthionic acid
(l-naphthylamine-4-sulfonic acid) and amino R
acid (l-amino-2-naphthol-3,6-disulfonic acid) was
consistent with earlier findings331'412'416;
excretion amounted to 9.8 mol% and 0.9 mol%,
respectively, during the 72-hr period following an
oral dose. Rats fed sunset yellow excreted 0.3 and
1.5 mol% of the oral dose in urine and bile,
respectively. Since little intact dye was detected,
the main labeled metabolite is thought to be the
l-amino-2-naphthol-6-sulfonic[8-14C] acid. Uri-
nary sulfanilic acid from sunset yellow amounted
to 37 mol% of the dose.
After tartrazine administration, about 4% of
the urinary sulfanilic acid is derived from the
further degradation of the 1-p-sulfophenylamino-
pyrazolone moiety of the azo dye, as opposed to
about 40% from the 4-p-sulfophenylazo portion.
These findings do not agree with those of Roxon
et a l . 4 4 5 who used tartrazine labeled with 3 5 S in
the 1-p-sulfophenylaminopyrazolone and esti-
mated a value of 25% from aminopyrazolone.
These authors also observed a total urinary excre-
tion of 20% of the tartrazine dose, whereas only
4% was found by Honohan et al. 2 S 8 Oral
administration of [14C]-aminopyrazolone in the
latter study resulted in about 9% urinary excre-
tion, with little or no radioactivity retained in
internal organs. The urinary metabolites were
roughly equal amounts of [ 14 C] -aminopyrazolone
(i.e., nonmetabolized) and [14C]-sulfanilic acid
(free and N-acetylated). Only traces of [ 14 C]-p-
sulfophenylhydrazine were observed in this study,
as opposed to 5 to 10% by Roxon et al. 44 s
In vitro studies on the degradation of [ 3 S S]-
tartrazine by rat cecal microflora indicated that
after 72 hr of incubation about 7 mol% of the
starting material was present as sulfanilic acid and
1% as p-sulfophenylhydrazine. A subsequent
experiment with Proteus sp. yielded 12 mol%
sulfanilic acid and no p-sulfophenyl hydrazine.
While the quantitative differences in these two
studies may be related to different strains of
animal, dietary regimen, and possibly composition
of the gut microflora, both studies support the
path of degradation of tartrazine carried out by
the microflora of the large bowel, which is
illustrated in Figure 11.
The degradation of azo food dyes, particularly
tartrazine, is thought to be related to a variety of
clinical maladies such as aspirin intolerance, 451 '
4S2
asthma,92 purpura, 356 and urticaria. 3s5 At
the present time the mechanisms behind these
hypersensitivity conditions are unknown.31 s It
January 1977 293
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Intracellular Extracellular

Periplasmic space

9. Gram positive
Cytoplasm Plasma membrane Medium
Cell wall
Peptidoglycan

Gram negative
3"
Cell wall
Substrate
LPS, PL, P
Co
RH, FMNH 2 ; FADH 2 ; RFH 2 -NHSO2-R'-N=N-R'

5.
I
FMN, FAD, RF -NHSO2-R'-NH-NH-R'

Oxidized
substrate Flavoprotein Kinase E'Q, stericand
"diaphorase" electrostatic factors
specificity for for mediator and poly
RF
Cellular electron mediator azo dye
oxido/reductases (X,Y)
may be NAD* or Permeability Permeability
NADP + specific barrier for barrier for poly
electron azo dyes and probably
mediator (X,Y) some mono azo dyes

FIGURE 9. Proposed model for poly and mono azo dye reduction by bacterial cells. (X,Y) Di- and monovalent electron mediators; (FP) flavoprotein; (PL) phospholipid;
(LPS) lipopolysaccharide; (P) protein; (FMN) flavin mononucleotide; (FAD) flavin adenine dinucleotide; (RF) riboflavin.

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Amaranth Sunset Yellow
(FD & C Red No. 2) (FO & C Yellow No. 6)
Acid Yellow
FIGURE 10. Structures of azo dyes mentioned in the text. Asterisk indicates position of ' 4 C radiolabeling.
has been suggested 5458 that the aminopyrazolone
reduction product of tartrazine could undergo
subsequent oxidation in the liver to form imino-
quinone- or 4,5-dihydroxy-derivatives. These py-
razolone derivatives would be highly reactive
towards nucleophiles including NH2 or SH groups
of proteins. In the latter case, the pyrazolones
could act as haptens producing immunogenic pro-
teins. As mentioned previously, naphthylamine
sulfonate reduction products (see Azo Compounds,
II.E.7) may be involved in human carcinogene-
sis. 564 General concern over this possibility and
the difficulty of unequivocally establishing the
safety of certain colors has led to the recent ban-
ning of FD & C Reds Nos. 2 and 4 from use in
foods. A related food color, FD & C Red No. 40, is
also being scrutinized for possible carcinogenic
activity. 51 ' 52
NHCOCH3
Red2G
SO 3 Na
Tartrazine
(FD & C Yellow No. 5)
2. Flavonoids
Flavonoids represent the largest group of
naturally occurring phenols. 486 Citrus fruits
contain a large number of flavonoid glycosides,
methoxylated flavonoid aglycones, and other
simpler phenols. These compounds occur in great-
est quantity in the peel, particularly the white
portion, but are also found in the edible portions
of the fruit. 260 A number of flavonoid derivatives
elicit a sweet taste in man, e.g., hesperetin,
hesperetin dihydrochalcone, and the chalcone and
dihydrochalcone derivatives of naringin and
neohesperetin. Neohesperetin dihydrochalcone has
recently been marketed as an artificial nonnutri-
tive sweetening agent (FLAV-O-LAST®, Nutrilite
Products, Inc., Buena Park, Calif.). Other bioflavo-
noids (such as rutin, quercetin, hesperidin, and
naringin) have been evaluated as antioxidants in
January 1977 295
 Hydrolytic NH2 Reductive
• —f— *
ring NH fission
cleavage ?
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SO,Na SO,Na
SO,Na

Tartrazine 1-lo-Sulfophenyl)- p-Sulfo- Sulfanilic

3-carboxy-4- phenylhydrazine acid
'aminopyrazolone'

FIGURE 11. Ultimate intestinal metabolic fate of tartrazine (FD & C Yellow No. 5).

Chalcone Dthydrochalcone

FIGURE 12. Structural formulas of parent flavonoid compounds.

foods. The basic structural types of flavonoids
discussed are shown in Figure 12.
The importance of the gut microflora in the
metabolism of these compounds was first suggest-
ed by the studies of Booth and Williams.57 They
observed the hydrolysis of rutin (quercetin-3-|3-
rutinoside) to 3-hydroxyphenylpropionic acid
(3-[3-hydroxy phenyl] propanoic acid) by fecal
and cecal extracts from rats. In an earlier study, s6
it was observed that hesperidin (2S-hesperetin-7-/3-
rutinoside) was similarly degraded when adminis-
tered orally to rats, being excreted in urine as the
aglycone hesperetin, its glucuronide and at least
seven ring fission products including 3,4-dihy-
droxyphenylpropionic acid, 3-methoxy-4-hydroxy-
phenylpropionic acid, 3-hydroxycinnamic acid
(m-coumaric acid), 3-hydroxyphenylpropionic
acid, 3-hydroxyhippuric acid, 3-hydroxybenzoic
acid, and 3-methoxy-4-hydroxybenzoic acid.
Scheline 467 found that 3-hydroxyphenylpro-
pionic acid could be generated by incubating rat
cecal contents anaerobically with hesperetin.
Similar incubation of hesperidin yielded hesperetin
and hydroxyphenylpropionic acid. Rutin was
degraded to is aglycone quercetin and 3-hydroxy-

296 Critical Reviews in Food Science and Nutrition

 phenyl-acetic and -propionic acids. Griffiths and
Smith 208 >209 studied the metabolic fate of a wide
variety of flavonoids: (1) a group of polyphenolic
compounds, many with 3',4',5'-Jrihydroxylation
of the B ring as in myricetin (3,5,7,3',4',5'-hexa-
hydroxyflavone), and (2) a group of compounds
related in structure to apigenin (4',5,7-trihydroxy-
flavone). In the case of the polyhydroxy
compounds, those with free 5,7,4'-hydroxyl
groups were susceptible to ring fission after oral
administration to rats. Similar products were
formed from compounds with 3',4',5'-hydroxyla-
tion during anaerobic incubation with rat
intestinal micro flora in vitro. The importance of
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the microflora in vivo was indicated by the
suppression of the products 3,5-dihydroxyphenyl-
acetic acid and 3-hydroxyphenylacetic acid follow-
ing oral administration of the antibiotic neomycin.
For the group of flavonoids related to apigenin,
the presence of free 5-,7- and 4'-hydroxyl groups
was also an essential structural requirement for
ring fission in vitro or in vivo.
The metabolic fate of (+>[ I4 C]-catechin
labeled both uniformly and in the A ring only has
been evaluated in rats and guinea pigs by Das and
Griffiths.119 Catechin-[U-14C] gave rise to
labeled phenolic acids (3-hydroxyphenylpropionic
and 3-hydroxyhippuric acids), phenyl-7-valerolac-
tones, and CO 2 . Catechin labeled in the A ring did
not yield labeled phenolic acids, but labeled
phenyl-7-valerolactones and greater quantities of
14
CO 2 were observed. This indicates a selective
catabolism of ring A to CO2 and accounts for the
absence of metabolites containing intact ring A in
the urine.
A recent study2 s 9 on the metabolic fate of
hesperetin-[3-14C] (which is the aglycone of
hesperidin, the predominant flavonoid in lemons
and sweet oranges [Citrus sinensis]) has demon-
strated the cooperative nature of microbial and
mammalian metabolism. The primary product of
hesperetin-[3-14C] found in the urine of rats
following oral administration and after anaerobic
incubation with rat cecal contents was 3-hydroxy-
4-methoxy-phenylpropionic acid. The labeled
flavanone was completely degraded to phenylpro-
pionic acids, benzoic acids, their conjugated
derivatives, and CO 2 ; the latter accounted for 40%
of the dose. However, very little 1 4 C 0 2 was
produced when hesperetin-[3-14C] was incubated
with rat cecal flora. These data suggest that the
catabolism of the propyl chain of the B ring
metabolites is mediated by mammalian and not
bacterial enzymes. This is in agreement with the
earlier findings of Griffiths and Smith 2 0 8 that
4-hydroxybenzoic acid could not be formed by
gut microbial action in vitro. Intraperitoneal injec-
tion of hesperetin-p- 14 C] to a bile duct cannulat-
ed rat resulted in biliary excretion of the entire
dose, showing that the animal is unable to affect
the initial ring fission. Several of the above
mentioned studies are summarized in Table 19 and
Figure 13.
The specific microflora components responsible
for the intestinal degradation of various dietary
flavonoids are not known. Cheng et al. 94 isolated
a number of strains of Butyrivibrio sp. from
bovine rumen capable of degrading rutin anaerobi-
cally. In addition they observed that Butyrivibrio
fibrisolvens Dl and Selenomonas ruminantium
GA192 hydrolyzed the glycosidic bond of rutin
and further dissimulated the sugar without affect-
ing ring fission, yielding the insoluble aglycone.
However, Peptostreptococcus sp. B178 degraded
rutin to soluble products and Butyrivibrio sp. C3
similarly catabolized rutin, quercitrin, and naringin
to water-soluble products, indicating ring fission.
The latter organism fermented the sugar moiety of
hesperidin, but did not cleave the heterocyclic
ring. Also, it could not catabolize quercetin,
toxifolin, protocatechuic acid, or phloroglucinol.
It is of particular significance with regard to the
Butyrivibrio C3 that flavonoid glycosides undergo
ring fission whereas the aglycones are inert;
suggesting a role for the glycosidic bond in enzyme
induction, specificity, or perhaps membrane trans-
port. Unfortunately, the phenolic acids resulting
from ring fission in these pure culture studies were
not characterized. In addition to the species
mentioned above, about 40 additional strains of
rumen bacteria were screened for ability to
degrade rutin, and all proved negative; these
included Bacteroides spp., Borrelia sp.,
Eubacterium spp., Lachnospira multiparus, Lacto-
bacillus spp., Peptostreptococcus spp., Rumino-
coccus spp., Succinimonas amylolytica, and
Succinivibrio spp.
In a related study with mixed bovine rumen
microflora,488 rutin, quercitrin, naringin, and
hesperidin were found to be rapidly degraded
under anaerobic conditions. Phloroglucinol was
detected as a transitory intermediate in the
fermentation of rutin, quercitrin, and naringin, but
not of hesperidin. Phenolic acids found were
probably 3,4-dihydroxyphenyIacetic and
3,4-dihy droxyphenylpropionic.
Tsai et al. 5 2 6 and Tsai and Jones 527 recently
January 1977 297
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to TABLE 19
3
Metabolism of Flavonoids by Intestinal Bacteria: Summary of Relevant Studies
Compound Studied Experiment
Hesperidin (2S-hesperetin-7-0-rutinoside) Metabolic fate in
rats, 1 g intragastric
dose
ence i
Rat cecal microflora
incubated anaerobically
I 22 hr
3. Hesperetin (3',5,7-trihydroxy-4'- Rat cecal microflora
5' methoxyflavanone) incubated anaerobically
a 22 hr
Hesperetin [3-14C] Metabolic fate in rat,
0.3-mg oral dose
Rat cecal microflora
incubated anaerobically
24 hr; 5.0, 0.5, and 0.05
mg/ml
Rutin (quercetin-3-)3-rutinoside) Rat fecal and cecal
microflora in vitro
Rat cecal microflora
in vitro
Rutin[4-14C] Butyrivibrio C3
Quercitrin (quercetin-3-0-rhamnoside) Butyrivibrio C3
Products observed Reference
Urine: traces of hesperetin 56
and its glucuronide;
3,4-diOH phenylpropionic acid;
3-OH-4-CH3O-phenylpropionic acid;
3-OH-cinnamic acid; 3-OH phenyl-
propionic acid; 3-OH hippuric
acid; 3-OH benzoic acid;
3-OH-4-CH3 O benzoic acid
Hesperetin; 466
3-OH phenylpropionic acid
3-OH phenylpropionic acid 466
Urine: 3-OH4-CH3O phenyl [ 1 4 C]- 259
propionic acid (8 mol%); 3,4-diOH-
phenyl[14C] propionic acid (3.3 mol%);
3-OH-phenyl [' 4 C ] propionic acid
(4.6 mol%) and their conjugates
(10mol%)
Expired air: 40 mol% of dose as
14
CO 2
3-OH4-CH3O phenyl[l4C]propionic acid 259
(55-85 mol%);3-OH-phenyl[14C]propionic
acid (16-44 mol%); 3,4-diOH phenyl
[' 4C]propionic acid (7 mol%) at 5 mg/ml
only;' "CO, 0.4 mol% at 0.5 mg/ml
3OH-phenylpropionic acid 57
Quercetin; 3-OH-phenylacetic acid; 466
3-OH-phenylpropionic acid
Phloroglucinol; 293
3,4-diOH benzaldehyde;
3,4-diOH "phenylacetic acid;
14
C0,
Phloroglucinol; 293
3,4-diOH phenylacetic acid
 TABLE 19 (continued)
Metabolism of Flavonoids by Intestinal Bactetia: Summary of Relevant Studies
Compound studied Experiment
Taxifolin Butyrivibrio C3
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Catechin(3,5,7,3',4'-pentahydroxyflavan) Metabolic fate in rat and
(+)-[U-14C] catechin guinea pig; 50 mg intra-
(+)-[RingA-14C] catechin gastric doses
Myricetin (3,5,7,3',4',5'-hexahydroxy- Metabolic fate in rats,
flavone) oral and intragastric
(I.G.)
Rat cecal microflora
in vitro
Myricitrin (myricetin-3-rhamnoside) Rat cecal microflora
in vitro
Delphlnldin (3,5,7,3',4',5'- Rat, 100 mg I.G.
hcxahydroxy flavylium chloride) i
Cecal microflora
in vitro
Robinetin (3,7,3',4',5'-pentahydroxy- As above, but 200 mg
flavone) oral dose
Tricetin(5,7,3',4',5'-pentahydroxy- Rat,
flavone) 100 mg oral
Rat cecal microflora
in vitro
Products observed Reference
No metabolites detected 293
Urine: 50-63 mol% of dose 119
S-(3,4-diOH phenyl)-7-vaIerolactone;
6-(4-OH-3CH3 O phenylH-valero-
lactone; 5-(3-OH phenyl)-r
valerolactone; 3-hydroxy-hippuric
acid; 3-hydroxy-phenylpropionic
acid (rat only); 3-hydroxy-
benzoic acid (guinea pig only)
Feces: 1.3-1.5 mol%
5-(3-OH phenyl)->-valerolactone (rats
only); 6 -(3,4-diOH phenyl)-?-
valerolactone (rats only)
Expired Air: 35-41 mol% of
[ring A-14C] catechin as ' 4 CO,
Urine: myricetin 209
3,5-diOH phenylacetic acid,
both unconjugated
3,5-diOH phenylacetic acid;
3-OH-phenylacetic acid (trace);
3,4,5-triOH phenylacetic acid;
unaltered myricetin
As above, but none of the 209
aglycone myricetin
Unidentified urinary metabolite
Two unidentified metabolites
Unaltered robinetin; 209
no metabolites found in vivo or
in vitro
Urine: unaltered tricetin, 209
3,5-diOH phenylpropionic acid (trace)
3,5-diOH phenylpropionic acid;
3-OH DhenvIcroDionic acid
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TABLE 19 (continued)
s
Metabolism of Flavonoids by Intestinal Bactetia: Summaiy of Relevant Studies

Compound studied Experiment Products observed Reference

Tricin (5,7,4'-trihydroxy- 100 mg oral Urine: 3,5-diOH phenylpropionic 209

3',5'-dimethoxyflavone)
Rat cecal microflora
in vitro
acid (3.1% of dose);
tricin (1.6% of dose),
no conjugates detected
3,5-diOH phenylpropionic acid (trace)

I
3
Malvin(3,5,7,4'-tetrahydroxy-3',5'-
dimethoxy flavylium chloride 3,5-
diglucoside)
Rat lOOmgl.G.

Rat cecal microflora

Three unidentified urinary metabolites

No metabolites detected
209

a.
in vitro
3. 5,7-Dihydroxy-3',4',5'-trimethoxy- Rat lOOmgl.G. Urine: unaltered dose,
I flavone)
Rat cecal microflora
3,5-diOH phenylpropionic acid (trace)
No metabolites detected
Apigenin (4',5,7-trihydroxyflavone) Rat 200 mg oral Urine: 4-OH phenypropionic acid; 208
4-OH cinnamic acid;
4-OH benzoic acid;
apigenin;
apigenin-0-glucuronide?
Rat cecal microflora 4-OH phenylpropionic acid
in vitro
Apiin (apigenin-7-apioglucoside) Rat 200 mg oral Urine: 4-OH phenylpropionic acid 208
4-OH cinnamic acid;
4-OH benzoic acid;
apigenin and conjugates
Rat cecal microflora 4-OH phenylpropionic acid;
in vitro apigenin and unaltered apiin
Naringin (4',5,7-trihydroxyflavone- Rat 200 mg oral Urine: 4-OH phenylpropionic acid; 208
7-rhamnoglucoside) 4-OH cinnamic acid;
4-OH benzoic acid;
naringenin (aglycone)
Rat cecal microflora 4-OH phenylpropionic acid
in vitro naringenin (aglycone)
 TABLE 19 (continued)

Metabolism of Flavonoids by Intestinal Bacteria: Summaiy of Relevant Studies

Compound studied Experiment Products observed Reference

Butyrivibrio C3 Naringenin 95
4-OH phenylpropionic acid
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Phlorizin(2',4,4',6'-tetrahydroxy- Rat 200 mg oral
dihydrochalcone-2'-|3-glucoside)
Rat cecal microflora
in vitro
Acacetin (4'-methylapigenin) Rat 200 mg oral
Rat cecal microflora
in vitro
Kaempferol (3,4',5,7-tetrahydroxy- Rat 100 mg oral
flavone)
Rat cecal microflora
in vitro
Robinin (kaempferol-7-rhamnosido-3- Rat 100 mg oral
galactorhamnoside)
Rat cecal microflora
in vitro
Chrysin (5,7-dihydroxyflavone) Rat 200 mg I.G.
Rat cecal microflora
in vitro
Tectochrysin (5-hydroxy-7-mcthoxy- Rat 100 mg oral
flavone)
Rat cecal microflora
in vitro
4 '-7-Dihydroxy flavone Rat 100 mg oral
Rat cecal microflora
in vitro
phloroglucinol
Urine: 4-OH phenylpropionic acid; 208
phloretin (aglycone);
4-OH cinnamic acid (trace);
4-OH-benzoic (trace)
4-OH phenylpropionic acid;
phloretin;
phloroglucinol'
Urine: 4-OH phenylpropionic acid
(trace); apigenin (trace)
4-OH phenylpropionic acid; apigenin
(in demethylated fractions only)
Urine: 4-OH phenylacetic acid 208
kaempferol
As above
Urine: kaempferol;
4-OH phenylacetic acid
As above plus unaltered robinin and 208
kaempferol-7-rhamnoside
Urine: apigenin and unaltered
chrysin only
No metabolites detected
Urine: apigenin; genkwanin (apigenin- 208
7-methyI ester); tectochrysin
No metabolites detected
Urine: 4'-7-diOH flavone and
unidentified metabolite
No metabolites detected

o
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TABLE 19 (continued)
s
Metabolism of Flavonoids by Intestinal Bacteria: Summary of Relevant Studies

Compound studied Experiment Products observed Reference

55
(-)-Epiafzelechin (4',5,7-trihydroxy- As above 4-OH phenylpropionic acid and 208
flavan-3-ol) two unidentified products
Pelargonin (3,4',5,7-tetrahydroxy- As above 4-OH phenyllactic acid? 208
flavylium-3,5-di-glucoside)
Genistein (4',5,7-trihydroxyisoflavone) As above 4-Ethyl phenol 208
Biochanin A (5,7-dihydroxy-4'-methoxy- As above Only traces of unidentified 208
isoflavone) metabolites, no 4-ethyl-anisole
detected
g. Daidzein (4',7-dihydroxyi'soflavone) As above Equol (4',7-dihydroxyisoflavan) 208
Formononetin (7-hydroxy-4'-methoxy- As above Trace of equol? 208
a. isoflavone)
I /3-Neohesperidin dihydrochalcone
(hesperetin-7-/J-D-glucoside)
As above 3OH4CH3O phenylpropionic acid
(18 mol%);
68a

30H-phenylpropionic acid (6.9 mol%)

Hesperetin dihydrochalcone As above 3OH-4CH3 O phenylpropionic acid
(0.7 mol%);
30H-phenylpropionic acid (0.3 mol%)
 OH
Hesperidin
R •= 0-rutinoside
^t Microbial 0-glycosidase
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OH O
Hesperetin
Conjugate
HO \V_y/ ""? HO
Oxidation
3OH-Benzoic acid 3-OH Cinnamlc
• acid
Acetate • CO,
FIGURE 13. Microbial and mammalian metabolism of hesperidin.
isolated eight strains of rumen bacteria capable of
anaerobically degrading phloroglucinol in pure
culture. Five strains were facultatively anaerobic
cocci identified as Streptococcus bovis, while the
remaining three anaerobic strains were assigned to
the genus Coprococcus. The intermediates of
phloroglucinol catabolism by anaerobic bacteria
are still unknown. The anaerobic degradation of
the benzene nucleus by a facultatively anaerobic
Pseudomonas sp. has been reported by Taylor et
al. 520 This organism grows rapidly on4-hydroxy-
benzoate under strictly anaerobic conditions,
provided nitrate is present in the medium. Cell
suspensions obtained under these conditions
oxidized benzoic acid with nitrate producing 4 to
5 mol CO2/mol benzoic acid. No benzoic acid
oxidation was noted aerobically. Thus, the
anaerobic catabolism of the benzene ring appears
to be quite distinct from the pathway seen in the
aerobically grown organism. In the latter case,
protocatechuic acid is the key intermediate leading
to a-hydroxy-7-carboxymuconic semialdehyde.
Ring fission followed
by gtucosidase action
Phenylpropionic acids
O
Microbial
O demethylation
dehvdroxylation
H
Anaerobic degradation
oxidativs
Anzymei
-2H
I-2H)
Vinyl and ethyl phenols?
Phenyl acetic acids
The hypothetical scheme proposed by these
authors (Figure 14) for the degradation of benzoic
acid may also apply to phloroglucinol. Guyer and
Hegeman2! 5 and Dutton and Evans' s ° proposed
that a monohydroxy reductive path via cyclohex-
1-ene-l-carboxylate, 2-oxocyclohexane carboxylic
acid, and pimelic acid operates in the anaerobic
photometabolism of Rhodopseudomonas palustris.
The production of methane from benzoic acid by
a strictly anaerobic digest of sewage bacteria has
been observed by Nottingham and Hungate. 384
Thompson et al. 5 2 5 studied a strain of rumen
Coprococcus sp. (Pe 5) which anaerobically
degrades 1 mol of phloroglucinol to 2 mol of
acetate and 2 mol of CO 2 . They attempted to
obtain mutants blocked in phloroglucinol degrada-
tion which may have permitted the isolation of
intermediates. While mutants were obtained that
were unable to use phloroglucinol as an energy
source, they retained the ability to dissimilate
phloroglucinol. Hence, the mutations affected
either a step in energy transfer peculiar to
January 1977 303

HO'NH
Benzoic Trihydroxy-
acid cyclohexane-
carboxy tic-
acid
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CO2 + H2O
FIGURE 14. Anaerobic degradation of the benzene nucleus. (Taken from Taylor, B. F., Campbell,
W. I., and Chinoy, I., /. Bacterial., 102,430, 1970. With permission.)
phloroglucinol or, perhaps, a step in phloroglu-
cinol assimilation. Indahl and Scheline2 7 ' isolated
two Bacillus strains from the intestines of rats
which decarboxylate 4-hydroxycinnamic acid.
These strains were also found to decarboxylate
3,4-dihydroxycinnamic (caffeic) acid and
4-hydroxy-3-methoxycinnamic (ferulic) acid. The
primary metabolites formed were 4-vinylphenol,
4-vinylcatechol, and 4-vinylguaiacol, respectively.
Similar decarboxylations were previously observed
in strains of Aerobacter166 and in Streptococcus
faecium.401
3. Cyclamate (Cyclohexyhulphamic Acid)
Although the use of cyclamate as a sweetener in
foods has been prohibited since 1969, there is a
continuing debate over its safety and possible
future acceptance as an approved additive.1 s>16 >
3 8s
The metabolic fate of cyclamate and the role
of the gut microflora have been investigated by
Drasar et al. 1 4 1 and Renwick and Williams.426 In
these studies, [U-14C]-cyclamic acid was adminis-
tered to guinea pigs, rabbits, rats, and humans.
When naive animals received cyclamate orally, the
compound was excreted unchanged; urinary and
fecal excretion in man amounts to 30% and 50%
304 Critical Reviews in Food Science and Nutrition
CO2H
Dihydroxy
cyclohexan-2-one
carboxylic acid
Dihydroxypimelic
acid
of the dose, respectively. Animals receiving a diet
containing cyclamate developed the ability to
convert oral cyclamate into cyclohexylamine and
possibly metabolites of the latter compound. This
adaptation to cyclamate metabolism was quite
variable among the animals studied; it occurred
more readily in rats than in rabbits or guinea pigs.
Of three human subjects studied, only one
developed the ability to metabolize cyclamate in
10 days, while the other subjects showed no
activity during 30 days of cyclamate pretreatment.
Removal of dietary cyclamate resulted in a reduc-
tion of metabolizing ability. In cyclamate-
pretreated rats, the conversion of cyclamate into
cyclohexylamine occurred only when the dose was
administered orally. Intraperitoneal administration
of [14C]-cyclamate and simultaneous oral
administration of unlabeled cyclamate followed by
analysis of urinary metabolites showed that
practically all the labeled cyclamate was excreted
unchanged in the urine. This finding clearly
implicated the gastrointestinal tract as the site of
cyclamate metabolism. [14C]-Cyclamate was
converted into cyclohexylamine when incubated
anaerobically in vitro with the contents of the
cecum, colon, or rectum or with feces of
 cyclamate-pretreated rats or rabbits. The feces of
the human cyclamate converter were also active in
effecting the reaction.
The ability to metabolize cyclamate was largely
lost when mixed flora from the colon or rectum
were subcultured three times at 1:10 dilutions.
This loss was somewhat diminished if cyclamate
was added to the subculturing medium (thiogly-
collate broth). The effect of dietary cyclamate on
various components of the fecal flora of the rats
and the human subject was also studied. little
change was noted in the human fecal flora, while
the only significant change observed in the rats
was an apparent increase in the number of fecal
clostridia (from <10 3 to 8 X 107 to 9 X 108 g' 1
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wet wt) in rats fed cyclamate for 3 months. Pure
culture studies of rat fecal and cecal isolates
confirmed the specificity of clostridia for
cyclohexylamine formation. Only one of ten
isolates of enterobacteria converted cyclamate; all
the enterococci, bacteroides, and bifidobacteria
(ten isolates each) were negative in this respect. A
similar analysis of fecal isolates from the human
cyclamate converter revealed that enterococci
were the only organisms capable of cyclohexyl-
amine formation.
Cyclamate metabolism is highly variable among
host species and individuals; however, it also
appears to be carried out by different flora
components in the different animal species.
The studies carried out so far do not describe
the mechanism of anaerobic cyclamate conversion.
Drasar et al. suggest two possibilities viz hydrolytic
or reductive fission (Figure 15). Sulfur metabolism
in the large intestine also appears to play a role in
cyclohexylamine formation. Studies with
[3 s S] -cyclamate and washed suspensions of fecal
bacteria from cyclamate-converting rats showed
that cyclamate-derived sulfur was incorporated
into proteins and possibly other macromolecules
FIGURE 15. Possible mechanisms for anaerobic bacterial conversion of cycla-
mate to cyclohexylamine. (Adapted from Drasar et al.) 1 4 '
of some intestinal bacteria. The sulfur incorpora-
tion probably follows an assimilative reductive
pathway and is inhibited by the presence of
cysteine. Prolonged cyclamate feeding caused
increases in H2 S-producing anaerobic species in
fecal specimens.522
In general, sulfate esters are not readily hydro-
lyzed by the intestinal microflora (the case of
p-nitrocatechol sulfate has already been men-
tioned). Drasar and Hill 139 mention three C-
sulfonates thought to be hydrolyzed in the gut,
viz., nitrophenylsulfate, 3,5,3'-tri-iodo-L-thyronine
sulfate, and taurine.
D. Cholesterol Metabolism: 5/3H Reduction
During their movement through the large
bowel, cholesterol and plant sterols (such as
campesterol, 0-sitosterol, and stigmasterol) are
converted into their respective 5/3-cholestan- and
5/3-stigmastan derivatives by microbial 5/J-H reduc-
tion of the 5,6 double bond of the steroid nucleus.
Usually in excess of 50% of human fecal
cholesterol is recovered as the reduced metabolite
coprostanol (5/?-cholestan-3j3-ol), while another 5
to 10% is present as coprostanone. Unmetabolized
cholesterol accounts for 25 to 45% of the fecal
neutral steroids. 139
Two separate pathways have been advanced to
describe the reductive process. In 1964, Rosenfeld
and Gallagher 4403 incubated [3a-3H] cholesterol
with human feces and found that the coprostanol
produced had retained most of the label at the C-3
position. They concluded that the biohydro-
genation proceeded via direct reduction of the
5,6-double bond. A subsequent study by
Bjorkhem and Gustafsson50 employed deuterated
water, [4j3-3H, 4- I 4 C]-, and [3a-3H, 4- 1 4 C]-
cholesterol, and rat cecal microflora incubated
anaerobically. On the basis of their data, they
concluded that the conversion of cholesterol to
January 1977 305
 coprostanol proceeded to at least 50% via the
A4-cholestenone intermediate (i.e., the indirect
pathway, Figure 16). Although these experiments
provided no direct evidence for the participation
of the direct reduction pathway, this possibility
could not be discounted. Since a complex mixture
of intestinal microorganisms was used, it is con-
ceivable that the two pathways might be employed
by different microorganisms.
In more recent work by Eyssen et al., 1 5 5 > 1 S 7
the reduction of sterols was studied with a
Eubacterium sp. (21,408) isolated from rat cecum.
This species was tentatively assigned to the genus
Eubacterium and differs from the described
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species in its need for a As-3|3-hydroxysteroid.
Growth of the organism was proportional to
As-sterol concentration in the medium (0.2 to 2.0
mg cholesterol per milliliter). Quantitation of the
reduction in cholesterol suggested that the Eubac-
terium uses it as an oxidant for anaerobic oxida-
tive reactions. A variety of cholesterol derivatives,
plant sterols, and hormones underwent 5j3-reduc-
tion when incubated with pure cultures of Eubac-
terium 21,408 (Table 20). The structure of the
side chain was not a dominant factor in reduction
of the 5,6-double bond in the steroid nucleus.
The mechanism of biohydrogenation was
Cholesterol
Indirect
mechanism
A 4 -Cholesten-3-one
FIGURE 16. Proposed pathways for microbial cholesterol reduction.
306 Critical Reviews in Food Science and Nutrition
studied with [3a-3 H,4- 14 C] cholesterol or [40-3H,
4- 14 C] cholesterol in brain thioglycollate medium.
The reduction of [3a-3 H, 4- 1 4 C]-cholesterol to
coprostanol by the Eubacterium sp. resulted in the
loss of 65% of the tritium label. Much of this loss
of label apparently occurred after the reduction,
since incubation of Eubacterium 21,408 with
presynthesized [3a- 3 H, 4- 14 C] -coprostanol
resulted in a 40% loss of tritium. The authors
concluded that the organism carries out reversible
redox reactions of the C-3 hydroxyl, and neither
loss nor retention of the radiolabel occurring
during reduction can be used in assessing the
mechanism of reduction. The conversion of
[4j3-3H, 4 - 1 4 C ] cholesterol to coprostanol
occurred with a retention of 81% of the original
3
H label; however, >90% of the tritium had been
transferred to the C-6 position in coprostanol,
indicating isomerization of the 5,6-double bond to
a 4,5-double bond and an intramolecular shift of H
atoms. These data suggest that the major pathway
for cholesterol reduction in Eubacterium 21,408
involves the A4-cholesten-3-one. Other findings
(such as the ability of the Eubacterium to reduce
A4-cholesten-3-one to coprostanol; the formation
of small quantities of A4-cholesten-3-one during
large scale preparation of coprostanol; and the
inability of Eubacterium to reduce A s -cholesten,
50-Cholestanol
(coprostanol)
Coprostanone
 TABLE 20

Substrate Specificity and Metabolism of Steroids by Eubacterium 2 1 , 408

Supports
Substrate growth Metabolism

Cholesterol and related compounds

Cholesterol Yes >95% 50H reduction
A4 -Cholesten-3-one Yes 40% coprostanol
Allocholesterol (A4 -cholesten-3/3-ol) Coprostanol
7-Dehydrocholesterol (A5 >'-cholestadien-3/3-ol); 5£-Cholest-7en-30-ol'
lathosterol (5a-cholest-7en-3j3-ol)
Cholesteryl acetate No No reduction
Cholesteryl stearate No No reduction
Cholesteryl heptadecanoate No No reduction
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Cholesteryl chloride No No reduction

Cholesteryl bromide No No reduction
A5-Cholesten No No reduction
Epicholesterol (A5 -cholester-3a-ol) No Cholestanol
Cholestanone (5a-cholestan-3-one) Cholestanol
Coprostanone Coprostanol
(5(3-cholestan-3-one)
Plant steroids
0-Sitosterol, Yes >95% 50-H reduction
(24R)-24-ethyl-As-cholesten-3/3-ol
Campesterol, Yes >95% 5/3-H reduction
(24R)-24-methyl-A5-cholesten-30-ol
Stigmasterol, Yes >95% 50-H reduction
(24R)-24-ethyl-A5 >* 2-cholestadiene-30-ol
Hormones
A5 -Androsten-3j3-ol-l 7-one Yes >80% SP-H reduction
5/3-Androstan-3(3-ol-17-one No No reduction
5a-Androstan-3/3-ol-17-one No No reduction
A5-Pregnen-3,20/3-diol Yes >80% 5fi-H reduction

Adapted from Eyssen and Parmentier.' 5 6 > 1 5

epicholesterol or, cholesteryl esters) also point to a
3-oxo intermediate.
The functioning of the indirect cholesterol
reduction pathway implies the production of two
enzyme systems for (a) oxidation and isomeriza-
tion of cholesterol into A4-cholesten-3-one and (b)
reduction of the cholestenone to coprostanol.
While both reactions have been reported to occur
separately in a wide variety of microorganisms,
occurrence of both systems appears to be less
common. It is found in 67% of Bacteroides spp.,
75% of Bifidobacterium spp., 45% of Gostridium
spp., and is absent in Escherichia coli and Strepto-
coccus faecalis.139
Since coprostanol is generally less well absorbed
from the gastrointestinal tract than choles-
terol, 565 microbial reduction of the latter com-
pound may influence serum and liver cholesterol
levels. This possibility was investigated by Eyssen
and Parmentier 156 with conventional and
germfree rats and gnotobiotic rats associated with
Gostridium sp. and Eubacterium 21,408. The data
showed that the cholesterol -> coprostanol conver-
sion was not the cause of the higher fecal
excretion of C27 sterols or the lower absorption of
cholesterol seen in conventional rats. Other micro-
flora components or mechanisms must be respon-
sible. The flora may promote sterol excretion via
increased exfoliation of intestinal epithelium,2 8 *
or it may inhibit cholesterol absorption by bile
acid degradation.530
In view of the cecum's role as the site of
microbial cholesterol reduction in the rat, it is
significant that Eubacterium 21,408 was unable to
maintain itself in the intestine of cecectomized
rats. 1 5 8 Changes in pH or Eh of intestinal con-
tents were apparently unrelated to the dis-
appearance of the Eubacterium, although more
efficient peristaltic action of the bowel could not
be ruled out as an important factor.

January 1977 307

 A study of serum cholesterol and bowel flora in
bottle-fed human neonates showed that lacto-
bacilli predominated in stools of infants when
serum cholesterol levels were low suggesting that
these microorganisms play a major role in intes-
tinal cholesterol metabolism in early life. 222
Other aspects of cholesterol metabolism, e.g., side
chain cleavage and demethylation, are discussed by
Drasar and Hill. 139
E. Bile Acid Degradation
The microbial degradation of bile acids in the
intestinal tract includes deconjugation, oxidation,
dehydroxylation, isomerization, and possibly
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reactions leading to aromatization of the steroid
nucleus (Figure 17). The bile acids in man are
primarily conjugates of taurine and glycine; the
amide bonds of the respective conjugates are
resistant to hydrolysis by the hosts' intestinal
enzymes.
1. Deconjugation
The hydrolysis of the amide bond of con-
jugated bile acids by a large number of pre-
HO'
Glycocholic acid
Taurocholic acid
Lithocholic acid
Cholic acid
Deoxycholic acid
Chenodeoxycholic acid
FIGURE 17. Type reactions of bile acid degradation by anaerobic intestinal bacteria.
308 . Critical Reviews in Food Science and Nutrition
dominantly anaerobic bacteria has been reviewed
by Shimada et al. 4 8 3 The large majorities of
strains of Bacteroides fragilis (17/22), Sphaero-
phorus necrophorus (10/11), Eubacterium sp.
(6/11), and Bifidobacterium spp. (15/16) were
able to hydrolyze the bile acid conjugates, as were
some other Gram-positive anaerobes and faculta-
tive organisms (Table 21). Aries and Hill 20 investi-
gated the properties of cholanylglycine (glyco-
cholic acid) hydrolase obtained from cell-free
extracts of Streptococcus faecalis, Bifidobacterium
spp., Oostridium welchii, and Bacteroides spp.
The hydrolase activities in all of the cell-free
preparations required either the presence of a
reducing agent (thioglycollic acid, cysteine, /3-
mercaptoethanol, or glutathione) or the complete
absence of O2. The rates of hydrolysis of glyco-
cholate, glycodeoxycholate, taurocholate, and
taurodeoxycholate were compared for the various
preparations; enzymatic activity on the glycine
and trihydroxy conjugates appeared greater than
on the taurine and dihydroxy conjugates (i.e.,
taurodeoxycholate was the least readily hydro-
lyzed of the substrates). The bifidobacteria
HO
-OH -OH -HNCHaCO2H
-OH -OH -HNCH2CH2SO3H
-H -H -OH
-OH -OH -OH
-H -OH -OH
-OH -H -OH
 TABLE 21

Bile Acid Deconjugation by Intestinal Bacteria In Vitro

Conjugate hydrolyzed

Glycocholic Taurocholic Glycodeoxycholic Taurodeoxycholic

Test organism acid acid acid acid

Bacteroides oralis
Bacteroides fragilis
Sphaerophorus necrophorus
Bacteroides melaninogenicus
Bifidobacterium adolescentis
Bifidobacterium longum
Bifidobacterium breve
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_ _ _ _
+ + + +
- + - +
± ± + ±
+ + + +
+ + + +
+ + + +

Bifidobacterium bifidum + + +
Bifidobacterium liberorum + + + +
Bifidobacterium parvulorum + + + +
Catenabacterium catenaforme + + + +
Gostridium perfringens + + - -
Oostridium paraputrificum + + + +
Streptococcus faecalis - + - +
Streptococcus lactis + + + +
Lactobacillus buchneri + + + +
Corynebacterium acnes - - _
Veillonella sp. + + + ±
Escherichia coli - - - _
Proteus mirabilis ± _ _
Pseudomonas aeruginosa - - - -
Eubacterium sp. + +
Butyribacterium rettgeri + +
Ramibacterium sp. + +

Adapted from Hill and Drasar,243 Midtvedt and Norman,3 5 I and Shimada et al.4

preparations and one of the Streptococcus faecalis
enzymes exhibited no activity on the taurine
conjugates. All enzyme activities were sensitive to
sulfhydryl reagents and had pH optima between 5
and 6.
The findings that the synthesis of cholanyl-
glycine hydrolase by clostridia and bacteroides
require strictly anaerobic conditions, and that, once
synthesized, the enzymes are much more stable
under reducing conditions, suggest that anaerobic
conditions are required in vivo for effective bile
acid deconjugation. In this connection, it is
interesting to note that deconjugating enzymes
elaborated by aerobic bacteria derived from
human feces appear to be quite different than the
enzymes from anaerobic bacteria described above;
they have pH optima of about 7.0 and relatively
high stability.
The metabolic fate of orally administered
cholyl-[2,4-3H]-glycine [1-14C] and cholyl-[2,4-
3
H]-taurine-[35S] in human subjects has been
investigated by Hepner and co-workers. 231 ' 232
The major portion of these conjugated trihydroxy
bile acids is absorbed without deconjugation, with
glycocholate being hydrolyzed at more than twice
the rate of taurocholate. More than half of the
cholic acid produced is conserved and recon-
jugated with glycine or taurine (i.e., enterohepatic
circulation). The glycine or taurine liberated by
deconjugation is partly absorbed, and the
remainder is further dissimilated by bacterial
action. Little fecal excretion of radiolabel from
glycine-[l- l 4 C] or taurine-[ 3s S] occurred.
Hepner et a l . 2 3 1 ' 2 3 2 found good correlations
between the degrees of deconjugation of the
conjugates and either expired I 4 CO 2 or urinary
3S
SO 4 = excretion.
2. Redox Reactions of the Hydroxyl Groups
Hydroxycholanyl dehydrogenases which act on
the 3a-, la-, and 12a-hydroxyl groups have been
studied mainly with cholic, chenodeoxycholic, and
lithocholic acids. The enzymes are produced by a
wide variety of bacteria, viz., 22/65 strains in six

January 1977 309

 genera.3 5 7 ) 3 S 8 Nineteen of the 22 strains formed
monoketo derivatives from cholic acid, while 3
strains formed a diketo derivative and only one
formed a triketo derivative. Chenodeoxycholic
acid was oxidized mainly at C-7 (19 strains) and
C-3 (6 strains). Those strains oxidizing the C-3
hydroxyl of chenodeoxycholic acid were also
active on the monohydroxylated bile and litho-
cholic acid (Table 22).
The 7a-dehydrogenases from five strains of
enteric bacteria were studied in cell-free extracts
by Aries and Hill.2 ' Two strains of Clostridium
perfringens exhibited NADPH-linked activity,
whereas the enzymes from Escherichia coli and
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The significance of hydroxycholanyl dehydro-
genase activity in vivo is still uncertain. Dickinson
et al. 1 2 7 did not observe such activity in the
intestinal tracts of ex-germfree rats associated with
clostridal strains capable of oxidizing cholic acid in
vitro.
The oxidation of hydroxyl groups followed by
reduction may lead to epimerization (as described
above in the case of cholesterol). The formation of
3/3-OH derivatives has been observed in several
bacteria in vitro (Table 22). Microbial formation
of 7|3-OH derivatives has not been described;
however, 12/5-OH derivatives were reported by
Aries and Hill. 2 '

two strains of Bacteroides sp. were NADH linked.

The pH optima were at values of 8.8 or above.
Oxidation activities for 3a-0H and 12a-0H groups
in cholic acid were also observed and were found
to be of the same order as 7a-0H dehydrogenase
activity. Rates of oxidation were substantially
reduced by the presence of glycine or taurine
moieties in the conjugates; hence, the C-24 car-
boxyl function may play a role in the mechanism
of the dehydrogenase action.
3. Dehydroxylation
A comparison of human fecal bile acids with
those undergoing biliary excretion indicates that
more than 80% have been dehydroxylated in the
C-7 position. Several authors have isolated
organisms synthesizing 7a-hydroxycholanyl
dehydroxylases from rat, rabbit, or human feces,
although this capability appears to be relatively
rare among intestinal flora components. 127 Hill

TABLE 22

Products of Microbial Hydroxycholanyl Dehydrogenase In Vitro

Bile acid substrate

Lithocholic Chenodeoxycholic Cholic

Test organism (3a-OH) (3,7-diaOH) (3,7,12-triaOH)

Escherichia coli (2) a None 3a-OH, 7-keto Monoketo

Pseudomonas aeruginosa (1) None 3a-0H, 7-keto Monoketo
Bacteroides spp. (8) None 3a-OH, 7-keto Monoketo
Eubacterium spp. (3) None 3a-OH, 7-keto Monoketo
Eubacterium minutum (1) None 3<*-OH, 7-keto Mono-, diketo
Eubacterium cadaveris (1) 3/3-OH 3/3OH, 7a-0H Monoketo
3-Keto 3-Keto, 7<x-OH
Eubacterium parvum (1) 3j3-OH 3/3OH, 7a-0H Monoketo
3-Keto 3-Keto, 7a-0H Diketo
3aOH, 7-keto
3,7-Diketo
Eubacterium lentum (1) 3/3-OH 3-Keto, 7a-OH Monoketo
3-Keto 3a-OH, 7-keto Diketo
3,7-Diketo Triketo
Bacillus cereus (1) 3/3-OH 3-Keto, 7a-OH Monoketo
3-Keto 3a-OH, 7-keto
Clostridium difficile (1) None 3a-OH, 7-keto Monoketo
Clostridium perfringens (2) 3/3-OH 3/3-OH, 7a-0H Monoketo
3-Keto 3-Keto, 7a-OH

Adapted from Midtvedt. 3 5 7 With permission.

a
Number of strains showing the reactions.

310 Critical Reviews in Food Science and Nutrition

 and Drasar 243 reported the enzyme in a small
number of strains of Bacteroides spp., Bifido-
bacterium spp., Gostridium spp., and Strepto-
coccus faecalis. Subsequent studies by the same
authors revealed that larger numbers of these
strains (30 to 50%) and some Veillonella spp.
could be induced to form the 7-dehydroxylase, if
grown in a buffered medium (pH >6.5) with
0.01% cholic acid. 139 Bokkenheuser et al. 53
isolated a bacteroides type organism from rabbit
feces capable of dehydroxylating both cholic and
allocholic acids in vitro. The former substrate was
only partially converted to deoxycholic acid (ca.
40 mol%), after 7 days incubation. Other products
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observed were the 7-ketocholic acid, 3a-hydroxy-
12-keto-5j3-cholanoic acid and 12a-hydroxy-3-
keto-5j3-cholanoic acid. Over a similar incubation
period, less than 10% of allocholic acid was
converted to allodeoxycholic acid. A similar
Bacteroides (Zuberella) sp. capable of 7-
dehydroxylating cholic acid has been isolated by
Hattori and Hayakawa.224 These authors also
observed the same reaction as that catalyzed by
enzymes from typed strains of Clostridium bifer-
mentans and Clostridium sordellii.22S The strains
produced both deoxycholic acid and 7-keto cholic
acid.
The dehydroxylation reaction appears to
require anaerobic conditions and is irreversible.
Studies using cell-free extracts 2 0 ' 1 3 9 have shown
that the enzyme is very labile and not subject to
reactivation by a variety of reducing agents or
cofactors (e.g., cysteine, thioglycollate, gluta-
thione, NAD+, NADP+, NADH, NADPH, FAD, and
FMN). The enzyme had a pH optimum of 7 to 8
and exhibited specificity for the free bile acids
cholic and chenodeoxycholic acids (i.e., conjugates
or methyl esters were not dehydroxylated). Sub-
strate inhibition was seen at concentrations above
6 mM. Enzyme activity was completely inhibited
by 30 mM Cu2+ or 3 mAf periodate.
It is curious that in several studies in vitro
microbial dehydroxylation was specific for the
free bile acids, since the work of Hepner et
3! 231,232 s h o w e d t h a t cholyl-[24-14C]-glycine-
[1- 14 C] could undergo 7-dehydroxylation both in
vivo and in vitro with human fecal homogenates.
There appears to be no correlation between
deconjugation and 7-dehydroxylation, as the.latter
activity has been observed in strains with and
without deconjugating activity. 357 In a rather
early study, Samuelsson453 uncovered the
mechanism of 7-dehydroxylation of cholic acid in
the rabbit in vivo. An earlier study employing
cholic acid [7j3-3H, 24- 14 C] had eliminated the
possibility that the 7-keto derivative (3a,12a
dehydroxy-7-keto-cholanoic acid) was a key inter-
mediate. Synthesis of cholic acid [6a,6|3,8j3-3 H,
24- 14 C] and cholic acid [6a-3 H] enabled
Samuelsson to propose that a mechanism involving
a diaxial trans elimination of water (60-H, 7/J-OH)
followed by reduction of a A6 intermediate is
consistent with the labeling pattern of the fecal
metabolites (Figure 18). The A6 intermediate has
never been isolated and presumably is quite
unstable. The 7/3-OH epimer of cholic acid is also
converted to deoxycholate in the rat cecum in vivo
but, as yet, little is known of the organisms
effecting this conversion.
4. Aromatization and Related Reactions
The postulate that intestinal bacteria play an
essential role in the mechanism of colorectal
cancer by producing carcinogenic or co-
carcinogenic metabolites has been advanced by
Drasar and Hill,1 2 8 • ' 3 9 Goddard and Hill, 1 8 6 '' 8 7
Goddard et al., 1 8 8 Hill, 241 and Hill and
Drasar. 245 Dehydrogenation of the steroid
nucleus of bile acids could theoretically lead to the
development of cyclopenta[a]phenanthrenes,
some of which are known to cause squamous
carcinoma. 106 ' 107 The production of such poly-
cyclic aromatic metabolites requires four types of
nuclear dehydrogenation reactions (referred to
above, Sections II.A and II.E.4) which have been
demonstrated with human gut bacteria in vitro.
The C6-7 dehydration reaction, which is the first
step in 7a-dehydroxylation, is carried out by
strictly anaerobic nonsporeformers. The other
three reactions have been demonstrated only with
clostridia, primarily Clostridium paraputrificum,
C. indolis, C. tertium, and a few strains of C.
welchii.18 8
Dehydrogenation in conjugation with a 3-keto
group (Figure 19a) was demonstrated with the
conversion of 5/5-androstan-3,17-dione to A4-
androsten-3,17-dione presumably by a A4-steroid
dehydrogenase. Nearly 1100 strains were tested
for their ability to produce the A4-dehydrogenase,
including 100 Gram-negative nonsporing anaerobes
(Bacteroides fragilis), 100 Gram-positive non-
sporing anaerobes (Bifidobacterium spp. and
Eubacterium spp.), 200 facultative anaerobes
(Escherichia coli and Streptococcus faecalis), 200
January 1977 311
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CH CH CH

3"

Cholic acid "A 6 Intermediate" Deoxycholic acid

[6a, 6/3, 8/3-3H] [60,8j3-3Hl
FIGURE 18. Distribution of the tritium label in cholic acid [6a,6/3,80-'H,24-I4C] at successive stages in the 7-dehydroxylation mechanism as proposed by Samuelsson.4 5 3
 lecinthinase-positive clostridia (C. welchii and C.
bifermentans) and 500 lecithinase-negative
clostridia (C. paraputrificum, C. indolis, C. ter-
tium, and others). About half of the latter group
and a few strains of C. welchii exhibited activity.
Demethylation at the C-10 position was
accompanied by Cl-2 dehydrogenation and
aromatization of the A ring (Figure 19b). Thus,
estradiol is the product expected from A4-
androsten-3,17-dione. The product normally
found is the 17-methoxyestradiol (17-methoxy-
A 1 ' 3 ' 5(10) -estratrien-3-ol), indicating methyl
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transfer to the 17 position.1 8 7 a About 850 strains
(as above, but with about 250 fewer lecithinase-
negative clostridia) were screened for aromati-
zation of the A ring and, as in the case of the
A4-dehydrogenase, the only positive strains were
lecithinase-negative clostridia or C. welchii. In an
earlier report, 186 the same group described a
strain of E. coli capable of producing estradiol
from A4-androsten-3,17-dione. The mechanisms of
demethylation may differ markedly in E. coli and
the clostridia. The former presumably follows an
oxidative demethylation via 10-hydroxy-methyl

FIGURE 19. Four types of nuclear dehydrogenation reactions which could lead to the development of
polycyclic aromatic hydrocarbons similar to cyclopenta[a]phenanthrene. R = C 4 H,CO 2 H side chain of
bile acids. (Taken from Goddard, P. and Hill, M. J., Biochem. Soc. Trans., 1,1113,1973. With permission.)

January 1977 313

 and 10-carboxylic acid, as seen in Bacillus cycla-
oxidans and Pseudomonas testosteronii. In the
clostridia, an anaerobic mechanism, probably
involving transmethylation, is operative.
Both the A4 dehydrogenase and the aromati-
zation reactions have been studied in cell-free
extracts of C. paraputrificum.22'186 The dehydro-
genase had a pH optimum between 7 and 8 and
was inactivated by 10 mM Cu2+, 10 mM Ca2+, 30
roM HCHO and 1.0 mM merthiolate. A variety of
substrates as oxidized although 3,7-diketo-5j3
and 5a steroids were inactive as substrates. In
cell-free extracts, menaphthone and phenazine
methosulfate may serve as artificial oxidants, while
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a variety of physiological oxidants had no effect
(e.g. NAD(P)*, FMN, FAD, cytochrome c).
Washed cell suspensions had the same properties as
the extracts, except that O2 could also be used as
a terminal oxidant. The aromatization reaction
occurred optimally at pH 8.5 and was inhibited by
0.25 M Cu2+ , 0.1 M Ca2+ , 0.1 M Mg2+ , 0.1 M F",
0.05 M N3~, 0.05Miodoacetate,6 mMperiodate,
and 0.3 M HCHO. The enzyme activity was wholly
inducible, and the reaction with cell-free extracts
in vitro required NAD(P)+ and phenazine metho-
sulfate.
A A8-steroid dehydrogenase (Figure 19d) has
been demonstrated in the conversion of equilin
(3 -hydroxy-A 1 >3>5 ( l o ) ' 6 -estratetren-17-one) to
equilenin (3-hydroxy-A l i 3 ' 5 ( l 0 ) ' 6 ' 8 ' ( 9 ) -estra-
penten-17-one). Equilin is formed by the action of
the ring A aromatization enzyme(s) on A4 ' 6 andro-
stadien-3,17-dione. All strains which have been
found to carry out this further ring B aromati-
zation also produce enzymes capable of A 4 -
dehydrogenation and ring A aromatization (Figure
20). Of 20 strains of C. paraputrificum tested,
only 5 were positive for ring B aromatization. 188
However, the methods employed for the deter-
mination of equilin and equilenin 187 ' 479 were
Androstadienone
FIGURE 20. Aromatization of A and B rings of the steroid nucleus in the formation of equilenin by
lecithinase negative clostiidia. (Taken from Drasar, B. S. and Hill, M. J., Human Intestinal Flora, Academic
Press, San Francisco, 1974. With permission.)
314 Critical Reviews in Food Science and Nutrition
considered somewhat less than adequate by the
authors. As yet, the dehydrogenation or aromati-
zation of ring C or D has not been observed, nor
have any of the above-mentioned reactions been
shown to occur in vivo. Since the anaerobic
nonspore-forming bacteria are present in the
intestine in far greater numbers than the clostridia,
it seems unlikely that the latter can be quanti-
tatively of main importance in the nuclear
dehydrogenation of bile acids in man. As Goddard
et al. 18 9 have pointed out, it would be necessary
to screen 104 nonspore-forming anaerobes
unsuccessfully before accepting the clostridia as
the only organisms capable of carrying out these
reactions.
There is obviously a need for more effective
methods of identifying microflora components
capable of steroid aromatization reactions and the
products of these reactions. Considering the suc-
cesses of radiolabeling techniques in revealing
many fine points of metabolism of both neutral
and acid steroids, it seems a bit surprising that
similar techniques have not yet been employed to
search for aromatized steroids. While it seems
unlikely that fecal carcinogens resulting from
microbial action could be detected as mutagens in
human populations at risk, it should nevertheless
be investigated. Also, site-specific animal models,
such as those described by Wheeler et al., s 4 7 > S 4 8
may also prove useful in investigating steroid
aromatization and its effects. Reddy et a l . 4 2 2 ' 4 2 4
feel that such flora mediated reactions are unlikely
to yield polycyclic aromatic hydrocarbons; they
believe that products that act as colon tumor
promoters and not as complete carcinogens will be
produced. Narisawa et al. 3 7 9 reported an increase
in adenomas among rats receiving an intrarectal
dose of TV-methyl-TV'-nitro-A^-nitroso guanidine
(MNNG) and taurodeoxycholic acid or lithocholic
acid as promoters (compared to the MNNG con-
Equilin Equilenin
 trol group). Animals given bile acids produced no
tumors; thus, it appears that these bile acids
(taurodeoxycholate is deconjugated to deoxy-
cholate in the large bowel), when present in high
concentration in the lower gut, act as colon tumor
promoters to an established large bowel animal
carcinogen.
F. Bile Pigment Metabolism
Conjugated bilirubin (diglucuronide), derived
from protoporphyrin IX by cleavage and reduction
respectively of the a and y methylene bridges,
respectively, is excreted into the duodenum and
passes to the colon, where it is reduced by
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bacterial action to a mixture of urobilinogens. In
turn, these are partially absorbed and reexcreted
by the liver and kidney. Estimations of urinary
and fecal "urobilinogen" are commonly employed
as measures of hepatic function and hemoglobin
turnover.3 ° 5 The intestinal microflora are wholly
responsible for bile pigment reduction; uro-
bilinogen is absent in germfree animals and
eliminated by broad-spectrum antibiotic therapy.
Fahmy et al. 1 5 9 studied the reduction of
various bile pigments by a number of intestinal
microorganisms. Of 12 anaerobes and facultative
anaerobes studied in pure culture, only two (viz.,
Bacteroides fragilis and Clostridium G62) were
capable of reducing bilirubin as effectively as
mixed human fecal flora (ca. 25 to 40% reduction
in 48 hr). Escherichia coli had a synergistic effect
on bilirubin reduction by Clostridium G62,
increasing mean urobilinogen formation by 20%.
Reduction of conjugated bilirubin by Qostridium
G62 appeared to be carried out more effectively
than that of free bilirubin.
A number of bile pigments, in addition to
biliverdin and free and conjugated bilirubin, were
incubated with mixed human fecal bacteria. No
effect was noted on biliverdin while natural
(+)- and /-urobilin, free and conjugated bilirubin,
and (-)-stercobilin were reduced to (-)-sterco-
bilinogen. Mesobilirubin was reduced to a mixture
of i- and (+)-urobilins and mesobiliviolin to a
chromogen of an unknown nonurobilinoid pig-
ment. Both (+)-urobilin and /-urobilin were
reduced (20 to 22%) by Clostridium G62 to an
unknown levorotatory pigment. Stercobilin was
reduced (45%) to stercobilinogen by the G62
strain. The reduction of free bilirubin (but not
conjugated bilirubin) to stercobilinogen was
observed in cell free (soluble) extracts of Clos-
tridium G62. The same extract had no effect on
mesobilirubin.
Mesobilirubin, while reduced to urobilinogen in
a yield resembling bilirubins, gave a mixture of /-
and (+)-urobilinogens but never (-)-sterco-
bilinogen. This difference in comparison to bili-
rubin may be due to in vitro manipulation of the
bacteria but, nevertheless, is in sharp contrast.
Other investigators 542 ' 543 have occasionally
observed the conversion of mesobilirubin to
(-)-stercobilinogens, although i- and (+)-uro-
bilinogens were frequently the major products.
Fahmy et al. 1 5 9 propose a mechanism (Figure 21)
for bilirubin hydrogenation which, while
impossible for the reductionof mesobilirubin, will
lead to an intermediate precursor of (-)-sterco-
bilinogen. These findings, while incomplete in
many respects, seem to support the scheme shown
in Figure 22.
ACKNOWLEDGMENTS
The author wishes to thank Mrs. Masako
Kanazawa and Mrs. Pat Hernandez for typing and
proofreading the manuscript and Mr. J. Schwartz,
Literature Scientist at Dynapol, for obtaining
photocopied literature not available locally. Some
of the author's colleagues have critically reviewed
the text and he also wishes to extend his thanks to
them: Drs. T. M. Parkinson, N. M. Weinshenker,
and W. L. Leonard. Professor Marvin L. Speck of
North Carolina State University, Raleigh kindly
supplied information on the current status of
"Sweet Acidophilus" Milk.
January 1977 315
 M P P M M V

1:4 1:6
Hydroge nation Bilirubin Hydrogenation
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Tetrahydro-i/j-ethylidene
intermediate

+8H

(-)-Stercobilinogen

FIGURE 21. Formation of tetrahydro-bis-ethylidene intermediate in the

conversion of bilirubin to (-)-stercobilinogen. (Taken from Fahmy, K., Gray, C.
H., and Nicholson, D. C.Biochim. Biophys. Acta, ISA, 85,1972.)

316 Critical Reviews in Food Science and Nutrition

 M V M P P M M V

M V M P P M M V M ,dC

M V M P P M M E M V M P P M M E M V M P P

Monovinyl (+1 urabilinogm H

Dthydrobilirubin tCj,H 3( N 4 O,) Mooovinyl {+, orobilin H 40

E M
1
P P M M E E M P P M
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H H

Gt.ucobitin H 3 ,
IC 33 H 4() N 4 O S »

ME M P P M M E ME MP PM ME

H H H H

it) Dihydromejobilirubm - H 4 , bila mc*obilivk>lin H4(

ME M
1
P P M M E ME MP PM ME

H H H H

(.Urobitinogen) (±1 - IRR + SS) f-Urobilin H 4 2

ME M P P
M E M P P

H H H H H H

-)- Half-stMcobilinogen-H4| Stable mnobiiiviolin H.,

V - CH-CH2
M-CHj
E M P P M M E P -CH 2 CH 2 CO 2 H

ri M P P M

(-) - StereobilinoeMi-H4t
(-) - Starcobilin H

FIGURE 22. Interrelationships of the bile pigments showing proposed reductive pathways (arrows). The structures on
the right and (-)-half-stercobilin in the lower center probably represent oxidation products of the proposed biological
intermediates. (Taken from Watson, C. J., Weimer, M., Moscowitz, A., Lightner, D. A., Petryka, Z. J., and Davis, E.,
Biochem. Med., 2,484,1969. With permission.)

January 1977 317

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336 Critical Reviews in Food Science and Nutrition