Diet Induced Changes in the Colonic Environment

and Colorectal Cancer

Colonic Bacterial Flora: Changing Understandings in the Molecular Age1

Volker Mai and J. Glenn Morris, Jr.2
Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine,
Baltimore, MD 21201

ABSTRACT The human intestinal microbiota is a complex bacterial consortium that is critical to normal health.
The microflora is present at concentrations of 1011–1012 cells/g of intestinal contents; the number of species
present may exceed 500, although exact numbers remain to be defined, due in part to the fact that ⬍30% of
microorganisms are culturable with current microbiologic methods. Molecular tools based on 16S rDNA sequence
similarities such as fluorescent in-situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE),
quantitative dot blot hybridization, restriction fragment length polymorphism (RFLP) and large scale 16S rDNA
sequencing have helped to overcome limitations of conventional microbiological plating methods in studying the
fecal microflora composition. However, these tools are just now beginning to be applied to understand the

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dynamics of this complex community, and its relationship to diet and human health. There is a need to understand
both the limitations of the current data and the importance of moving forward with the best possible molecular and
epidemiologic techniques as we deal with these critical questions. J. Nutr. 134: 459 – 464, 2004.

KEY WORDS: ● intestinal microbiota ● molecular tools ● diet ● human disease

Bacteria have been known to be closely associated with
mammals since the development of optical tools allowed for
the visualization of such microbes. Even before bacteria could
be viewed by microscopy, infectious small particles (ani-
malcules) had been suspected to cause various transmissible
human diseases (1). Modern microbiology has established
the close associations of bacteria with mammals not only as
a cause of disease, but also as part of an essential colonizing
microflora (2,3).
Commensal bacterial communities are closely associated
with the human skin, oral cavity, gastrointestinal tract and the
female genital tract. The colon, in particular, is the home for
a complex consortium of microorganisms (primarily bacteria,
but also fungi and protozoa) that is critical for normal health.
The actual number of species that may be present is contro-
versial; it has been estimated that ⬎500 species coexist in the
1
Presented at the Experimental Biology meeting, April 11–15 2003, San
Diego, CA. The symposium was sponsored by the American Society for Nutri-
tional Sciences and supported in part by an educational grant from the Group
Danone and the Nutrition Science Research Group at the National Cancer Insti-
tute. The proceedings are published as a supplement to The Journal of Nutrition.
This supplement is the responsibility of the guest editors to whom the Editor of
The Journal of Nutrition has delegated supervision of both technical conformity to
the published regulations of The Journal of Nutrition and general oversight of the
scientific merit of each article. The opinions expressed in this publication are
those of the authors and are not attributable to the sponsors or the publisher,
editor, or editorial board of The Journal of Nutrition. The Guest Editors for the
symposium publication are Jon A. Story, Department of Foods and Nutrition,
Purdue University, West Lafayette, IN, and J. Glenn Morris, Jr., Department of
Epidemiology and Preventive Medicine, University of Maryland School of Medi-
cine, Baltimore, MD.
2
To whom correspondence should be addressed.
E-mail: jmorris@epi.umaryland.edu.
human colon, although statistical extrapolations from the 16S
rDNA sequencing of cloned amplicons derived from human
fecal community DNA from one patient suggest that there are
⬍150 operational taxonomic units (defined as differing in 16S
rDNA sequence by ⬍ 2%) (4,5). Future studies that utilize a
combination of conventional and molecular microflora anal-
ysis tools will help to better define the complexity of the
human microbiota. It has further been estimated that some 40
species make up ⬃99% of all isolates, with the Bacteroides-
Prevotella group (a Gram-negative anaerobe) and Clostridium
species (Gram-positive anaerobes) predominating (3,6,7).
In the colon of healthy humans, anaerobic species outnum-
ber aerobic ones by at least 10-fold, with the proportion of
anaerobes to aerobes having been used as a measure for “nor-
mal” flora. The composition of the microflora changes
throughout the intestinal tract, with the highest microbial
activity observed in the proximal colon (2,6). The composi-
tion of the microflora appears to be affected by changes in
substrate availability, pH and reduction potential. The total
number of microbes in the gastrointestinal tract seems to be
similar in different human populations and has been estimated
to be an order of magnitude higher than the number of
eucaryotic cells in the entire human body (8).
Host-associated bacteria with their metabolic contributions
to host physiology have clear trophic functions and play a role
in protecting the host against invasion by pathogenic species.
Bacterial fermentation products such as short chain fatty acids
can be nutrients as well as growth signals for the intestinal
epithelium, an example being butyrate with its pro-differenti-
ation, anti-proliferation and anti-angiogenic effects on
colonocytes (9 –11). Various bioactive molecules such as car-

0022-3166/04 $8.00 © 2003 American Society for Nutritional Sciences.

459
460
cinogenic xenobiotics, dietary phytoestrogens, and primary
bile acids can be transformed by commensal bacteria (12–14).
The microflora facilitates the excretion of various toxic sub-
stances and the exclusion of pathogenic microorganisms from
the human host. Furthermore, the normal flora has been
shown to stimulate immune function through Peyer’s patches
and other gut-associated lymphoid tissue (GALT),3 which are
distributed throughout the gastrointestinal tract (15). The
commensal microflora is involved in the regulation of gastro-
intestinal immune tolerance, disturbances of which can con-
tribute to diseases such as Crohn’s disease and ulcerative colitis
(16,17). Specific host-microbe interactions have been reported
that can regulate production and excretion of selective sugars
into the intestinal lumen (18,19). This observation suggests
that microbes have evolved synergistic mechanisms to influ-
ence the colonic environment for their own benefit, and
potentially that of the host, by affecting epithelial host cell
gene expression. Although interest in the role of the commen-
sal microflora has been renewed by the development and
refinement of molecular analysis tools (reviewed by R. Gaskins
et al. in an accompanying article in this issue) including the
availability of completed genome sequences (20 –22), our un-
derstanding of the dynamics and physiologic functions of the
microflora is still in its infancy. In this paper we review recent
advances in understanding the associations between diet, mi-
croflora, and health, in the context of the increasing availabil-
ity of molecular tools.
The molecular age of microflora analysis
Traditionally, the stool flora has been analyzed by micro-
biological culture techniques. However, this approach is rather
laborious, time consuming and often inaccurate. It is also
limited in scope, as a majority of the bacterial species present
in feces are not culturable using standard microbiologic tech-
niques (19,23). Even among those that are culturable, species
identification by traditional identification methods is often
difficult, if not impossible; only a limited number of species
have been fully characterized, and biochemical identifica-
tion systems may have minimal utility in differentiating
species (2,3).
The first widely used molecular technique in microbial
systematics, which still required culturing of the respective
bacteria, was total genomic DNA hybridization. This ap-
proach, which utilizes whole genomes rather than small
genomic regions to determine the degree of similarity between
two microbes, formed the basis for molecular microbial phy-
logeny before the advent of the 16S rDNA revolution. Mo-
lecular tools based on 16S rDNA sequence similarities such as
fluorescent in-situ hybridization (FISH), denaturing gradient
gel electrophoresis (DGGE), quantitative dot blot hybridiza-
tion, restriction fragment length polymorphism, and large
scale 16S rDNA sequencing have helped to overcome limita-
tions of conventional microbiological plating methods in
studying the fecal microflora composition (2,24); they are
reviewed in detail elsewhere in this issue. Microchips that
would allow for more efficient identification of bacterial spe-
cies present in complex communities are currently under de-
velopment in various laboratories. While the feasibility of the
microchip approach has been established, currently available
chips are still limited in scope. Microchips specifically de-
signed to analyze the human fecal microflora should make it
3 a quarter of the sequences corresponded to known sequences
Abbreviations used: CRC, colorectal carcinogenesis; DGGE, denaturing
gradient gel electrophoresis; FISH, fluorescent in-situ hybridization; GALT, gut-
associated lymphoid tissue; LAB, Lactic Acid Bacteria.
SYMPOSIUM
possible to extend studies of the associations between diet,
microflora, and health to large prospective cohort studies, a
necessary next step in dealing with these questions.
In limited studies, molecular tools have been applied to
study the development of the infant microflora (25,26) and
changes in the human microflora during aging, with the sug-
gestion that complexity increases with age (with a correspond-
ing decrease in the number of Bifidobacteria) (6,27). These
tools have also been used to evaluate the effects of pre- and
probiotics on the human microflora composition (15,28) and
the effects of dietary interventions on the intestinal microflora
in various animal models, as outlined below (29,30). These
studies have also underscored the degree of variability inherent
to these types of complex systems (and assay systems). For
example, a microflora study based on FISH analyses has shown
a large degree of variability among subjects and in individuals
over time (31). Although some of this observed variation is
likely due to differences in environmental factors such as diet,
other factors, including host genetics and the potential con-
tribution of chance, cannot be neglected. Establishment and
maintenance of the commensal microflora is a complex and
multi-factorial process that we do not fully understand. The
sequence in which bacteria settle a niche in the colon, due to
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chance encounters, nutrient supply, and immune surveillance,
may well influence the ability of other bacteria to establish
residence.
Molecular studies have highlighted the diversity of the flora
within individuals. In this context, it is somewhat surprising
that sequencing large numbers of16S rDNA clones derived
from fecal microflora communities has not resulted in large
numbers of identical or highly similar sequences, which might
have been expected from prior work indicating the predomi-
nance of a few genera in the microflora (5,7,32). Wilson and
Blitchington undertook direct amplification and partial se-
quencing of cloned 16S genes from community DNA ex-
tracted from a human fecal sample. Fifty isolated clones had 27
distinct sequences and gave an estimated 59% coverage of
cloned 16S rDNAs (32). More recently, Suau et al. examined
284 16S rDNA sequences from human intestinal microbiota
and found 82 molecular species from three dominant groups:
Clostridia coccoides-like, C. leptum-like, and Bacteroides (5).
However, many different rather than a few dominant species
were identified within these three groups. Similar observations
were made in pigs, where 16S rDNA sequences analysis de-
tected 375 different phylotypes in 24 pigs (33) and chapero-
nine-60 analysis detected 398 different nucleotide sequences
encoding 280 different peptide sequences (34).
These observations raise the possibility that the intestinal
microflora contains large numbers of different species, each of
which is present only in low numbers. Bias introduced through
PCR or subcloning might affect such analyses (35); the extent
of such potential distortions has not been thoroughly investi-
gated. Future human microflora studies based on large numbers
of cloned 16S rDNA sequences might well change current
concepts of intestinal colonization and microflora composi-
tion. It should be emphasized that sequencing studies of the
human microflora have not reached the point of saturation. It
may be necessary to sequence well over a thousand clones per
sample before reaching a point at which additional sequences
will be unlikely to yield more unique sequences (unpublished
results, C. Stine). A high proportion of sequences discovered
in sequencing studies have not previously been described.
Both of the human studies summarized above found that only
even when allowing for a 2% divergence between the observed
and data base sequences.
 COLONIC BACTERIAL FLORA
Due to relative ease of stool collection most of the intesti-
nal microflora studies have been performed under the assump-
tion that feces contain a representative sample of the preva-
lent intestinal microflora. Although some studies have shown
clear differences between the fecal microflora composition and
the kinds of bacteria that are present at other anatomical sites,
including bacteria in the cecum (36) and those associated with
the mucosa (37), differences in the human microflora at var-
ious anatomical sites are not well documented. It can be
assumed that while the proportions and activities of the mi-
croflora change with passage through the intestinal tract, most
viable as well as nonviable commensal intestinal bacteria will
still be detectable in feces with molecular methods. Until new
nanotechnologies allowing for the convenient sampling of
microflora throughout the intestinal tract become more widely
available, feces remain the only realistic sample in large non-
invasive studies. Before large human studies can be designed it
needs to be unequivocally established that analyzing micro-
flora composition and activities in feces is representative of
important intestinal parameters.
Although the 16S rDNA based technologies continue to
undergo refinement, it might be timely to develop standardized
protocols for the analysis of the intestinal microflora, which
would facilitate an improved comparison of results across stud-
ies. Such an effort could lead to the establishment of a single
database that contains the fecal bacterial profiles from subjects
across various studies. Given their expense, studies to date
have generally involved small numbers of patients (or one
patient); establishment of a common database would facilitate
comparison of such data, allowing for a better understanding of
the complexity and the variation of the human intestinal
microbiota.
Effects of diet on microflora composition
There have been several studies suggesting that each indi-
vidual harbors his or her own distinctive pattern of intestinal
microflora composition. This pattern tends to remain constant
across time, although there may be some increase in species
diversity with age (27,38). While studies are limited, it appears
that overall dietary patterns (as seen among persons living in
different geographic areas) and intake of various nutrients can
influence general patterns of fecal microflora (39 – 41). At the
same time, and in keeping with prior comments about the
variability of results, in subjects in a feeding study the same
diet may have very different effects on the microflora, possibly
due to differential effects of the diet on the individual’s un-
derlying microflora composition, and/or underlying genetic
differences (Mai et al., this issue).
Most molecular studies of the associations between nutri-
ents and microflora composition have been done with supple-
ments that supply either viable bacteria (probiotics), oligosac-
charides that can selectively enhance the growth of
“beneficial” bacteria (prebiotics), or a combination of both
(synbiotics). The effectiveness of these supplements in mod-
ulating the intestinal microflora toward a composition with
increased proportions of bacteria that are thought to be ben-
eficial, such as Bifidobacteria and Lactic Acid Bacteria (LAB),
is well-established (15,28,42). Other studies have focused on
differences in the fecal microflora of neonates that are fed
either breast milk or formula (25,26). These studies have
shown that feeding breast milk results in the development of
an intestinal microflora that has increased proportions of Bi-
fidobacteria.
It is important to note that labeling nonpathogenic com-
mensal bacteria as either beneficial or detrimental remains
461
highly speculative. For instance, although it is widely assumed
that Bifidobacteria and LAB are beneficial whereas high num-
bers of Clostridia and Bacteroides are detrimental, labeling
them as such is not necessarily supported by rigorous data. In
fact, one study showed that although LAB were inversely
associated with colorectal carcinogenesis (CRC), a positive
association with CRC was observed not only for Bacteroides
but also for Bifidobacterium species (38). In vitro studies that
investigate the effects of specific bacteria on cancer cell lines,
which already have undergone a variety of genetic alterations
and which do not resemble the physiologic and immunologic
conditions that are found in the colon, and animal studies that
evaluate the effects of microflora changes in either germ free,
rodent-flora or human-flora associated rodents should be in-
terpreted very carefully. The complexity and the dynamics of
the human microflora, which we know very little about, and its
potential species or even strain specific interactions with the
human host including its immune system cannot be effectively
studied in any simplified model system. Thus, claims of asso-
ciations between specific commensal bacterial species or
strains and human health will have to be established in human
feeding/intervention studies or large epidemiologic studies.
The available molecular tools should now allow for such
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studies. Model systems should be continued to be utilized a) to
establish if and how intestinal microflora composition and
activity can be modulated by complex diets, and b) to
strengthen the hypothesis that specific commensal bacteria or
microflora profiles are associated with specific diseases includ-
ing carcinogenesis.
Unfortunately, studies of specific interactions between diets
that differ in macronutrient content and microflora composi-
tion have not yet been carefully investigated with 16S rDNA
based molecular tools. Early ecological studies have suggested
that fecal microflora differences in populations with varying
dietary habits contribute to the observed differences in health
in these groups. Studies based on conventional culturing tech-
niques have indicated that the protein and fat content of the
diet as well as the nature of the carbohydrates (simple sugars
vs. complex carbohydrates) does affect microflora composition
and activity (41,43,44). Animal studies support the hypothesis
that the intestinal microflora can be modified by diet and
antibiotic administration in mice and chicken (29,45). How-
ever, as mentioned above, studies that evaluate effects of
dietary interventions on the intestinal microflora often use
different methods and thus have to be interpreted with cau-
tion. For instance, food restriction, arguably one of the most
effective dietary interventions, has been shown in one study to
have little effect on the microflora of rats as measured by
conventional anaerobic culture, cellular fatty acid profile and
PCR (46). In contrast, we have observed that food restriction
and diet composition both strongly affect the microflora com-
position as judged by DGGE (47). Standardized 16S rDNA
based molecular tools should now be used, preferably in com-
bination with conventional methods, to study in depth in both
human and animal models the natural variation in the micro-
flora and interactions between diet and the composition and
activities of the intestinal microflora.
Intestinal microflora composition and health
At the beginning of the last century Metchnikoff suggested
that the use of live bacteria in fermented milk products such as
yogurt could increase longevity and improve health by detox-
ifying putrefactive substances (48). In the more recent past
interest in the potential of improving human health through
modifications of the intestinal microflora has reemerged and
462 SYMPOSIUM
various dairy products that are commercially available claim
such effects. It has, however, been difficult to establish the
existence of associations between specific microbes and health.
Studies that attempt to associate complex diets with changes
in the microflora and disease are virtually nonexistent.
The recent literature on the efficacy of probiotic interven-
tions supports the hypothesis that changes in the microflora
induced by the consumption of probiotics can reduce the
frequency and severity of diarrhea, as well as atopic disease in
infants (49 –52). Crohn’s disease, inflammatory bowel disease,
and gastrointestinal cancers are thought to be associated with
the microflora composition and recent data supports such
association (23,53–55). Although epidemiologic studies do
not support an inverse association between intake of fer-
mented milk products and colorectal cancer (56,57), these
studies are limited in scope because they neither differentiate
between viable and nonviable products nor between the spec-
ificity of the consumed bacterial strains.
Many potential mechanisms have been suggested to medi-
ate the proposed associations between microflora and human
health (58), but only a few of them have been well established.
The commensal microflora might participate in a) excluding
pathogenic organisms from colonizing the gut through
strengthening the barrier function or competing for attach-
ment sites; b) interacting with the intestinal immune system,
contributing to the regulation of immune function including
tolerance; c) producing either beneficial or harmful fermenta-
tion end products (butyrate, acetaldehyde) that might change
intestinal pH; d) facilitating the metabolic conversion and
uptake of beneficial dietary components (phytoestrogens, vi-
tamins); e) transforming and/or excreting toxic substances
(bile acids, nitroseamines, heterocyclic amines); and f) gener-
ating fecal bulk that might decrease transit time and lower
exposure of the intestinal lumen to toxic substances. More
specific interactions have recently emerged, including the mo-
lecular communication between B. thetaiotaomicron and the
intestinal epithelium (19) and the proposed associations be-
tween enterotoxic B. fragilis (increased risk) (59,60) or ente-
rotoxigenic E.coli (decreased risk) (61) and colorectal carci-
nogenesis.
Diet, microflora and colon cancer
Colorectal cancer is one of the four leading carcinomas in
the United States today; NCI/SEER estimates are that 147,500
new patients with this disease will be diagnosed with this
disease in 2003, with 57,100 deaths (62). While a portion
(perhaps as much as 35%) of the predisposition to developing
colon cancer is attributable to heredity, the majority of risk
appears to come from nonhereditary environmental factors.
Colon cancer is much less common in the underdeveloped
countries/regions of Africa, Asia, and South or Central Amer-
ica than in developed Western societies [age standardized
incidence of 9.91 vs. 37.30 cases/100,0000 population/y
(IARC, Globocan)], consistent with the hypothesis that there
are lifestyle or other factors within these societies that increase
the risk of CRC (63). The importance of environmental
factors is further supported by the observation that risk in-
creases among low-risk population groups after movement to
developed countries (and, presumably, assumption of the hab-
its of these counties); for example, in one study of Chinese
men, rates of colorectal cancer increased as much as twofold
after migration from Shanghai to Los Angeles or Hawaii (63).
The possible role of diet as a risk factor for colon cancer has
been examined in a number of epidemiologic studies. Early
epidemiological observations suggested that diet could explain
up to 90% of colorectal cancer risk (64). Although this level
of involvement has not been supported by subsequent re-
search, there remains a strong consensus that diet is an im-
portant component of the total risk profile. Several diet com-
ponents consistently emerge from these studies, including total
fat or saturated fatty acids and red meat as causal (with red
meat having the greatest potential impact) and fruits and
vegetables and, possibly, fiber, as protective (63,65– 67). How-
ever, the evidence for many of these food groups does not
reach the level of being conclusive (65).
In one study of associations between microflora composi-
tion and colorectal carcinogenesis (initiated in 1971 but not
completed until 1995 because of technical difficulties in char-
acterizing stool microflora), stool samples from rural South
Africans were compared with those from rural Japanese (both
groups consuming a “native” diet), and three groups eating a
western-style diet, including Japanese-Hawaiians, whites from
Hawaii and the continental U.S., and patients with a history
of recent polyp removal (38). While each subject had his or
her own unique pattern, the general fecal microflora pattern
seen in the rural Japanese was distinctive from that of the
other groups, as was that of the rural South Africans; compo-
sition of microflora was similar among the group with polyps
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and the Japanese-Hawaiians (with increased concentrations of
Bacteriodes and Bifidobacterium species), while whites had pat-
terns somewhere between these “high risk” persons and the
rural “low risk” groups. At the species level, 13 species were
significantly associated with a high risk of colon cancer and
the western diet, while 6 species were associated with a low
risk and the native diets. The numbers in the study were
relatively small (total n ⫽ 88 for all groups), and more tradi-
tional microbiologic techniques were used for characterization
of fecal microflora. Nonetheless, these data support the hy-
pothesis that patterns of fecal microflora differ among groups
from different geographic areas, who are consuming different
types of diets, and who might be expected to have different
cancer risks.
Some animal studies support the hypothesis that changes in
the microflora contribute to intestinal carcinogenesis. In con-
trast, Dove et al. have argued that microflora might not play a
role in intestinal carcinogenesis in APCMin mice, because the
frequency of intestinal polyps was not different in germfree
mice when compared with controls (68). However, their study
does not exclude the possibility that specific bacterial strains
might have either beneficial or detrimental effects on carci-
nogenesis in this cancer model. In fact, Schauer et al. have
reported that inoculation of APCMin mice with Citrobacter
rodentii increases intestinal polyp frequency (69).
Utilization of molecular tools in future studies
The scope of studies of the associations between microflora
and human health has so far been limited to observational
studies, controlled short term feeding studies and small-scale
intervention studies. Although these studies have contributed
significantly to our understanding of the human intestinal
microflora, we are still lacking a comprehensive understanding
of the strength of the associations between diet and microflora
and the degree of variation within and among individuals.
Animal studies, which have the advantage of being well
controlled for environmental and genetic factors, should be
designed to evaluate microflora stability and the effects of
dietary interventions on microflora composition and disease.
Various existing models would allow for such studies with IBD
or markers of intestinal carcinogenesis as the relevant end
points. Although details from such studies will not be directly
 COLONIC BACTERIAL FLORA
applicable to humans, such studies could strengthen the hy-
pothesis that the effect of diet on health is modulated through
effects on the microflora. Due to important differences in
intestinal anatomy and physiology as well as in immune sur-
veillance between humans and rodents, it is questionable that
associating rodents with a human fecal microflora would be
more informative than observing changes in the normal ro-
dent microflora.
Molecular tools have the potential for allowing analysis of
fecal samples from a large number of subjects. These tools need
to be validated and standardized and should then be utilized to
build a database of the human intestinal microbiota, which
will form the basis for determining the degree to which the
microflora can be influenced by dietary changes. A concerted,
multidisciplinary effort, incorporating modern molecular tools
for the microflora analysis in the setting of well-designed
prospective studies, is needed to advance our knowledge of the
complex interactions between host and microflora to the point
that we can design effective dietary interventions. Ultimately,
this should lead to clinical intervention studies to determine if
diet-induced microflora changes can reduce the risk of major
gastrointestinal diseases such as Crohn’s disease or colorectal
cancer.
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