Colonic Bacterial Flora - Changing Understandings in the Molecular Age1

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Diet Induced Changes in the Colonic Environmentand 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 10111012 cells/g of intestinal contents; the number of speciespresent may exceed 500, although exact numbers remain to be defined, due in part to the fact that 30% ofmicroorganisms are culturable with current microbiologic methods. Molecular tools based on 16S rDNA sequencesimilarities 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 rDNAsequencing 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 thedynamics of this complex community, and its relationship to diet and human health. There is a need to understandboth the limitations of the current data and the importance of moving forward with the best possible molecular andepidemiologic 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 withmammals since the development of optical tools allowed forthe visualization of such microbes. Even before bacteria couldbe viewed by microscopy, infectious small particles (ani-malcules) had been suspected to cause various transmissiblehuman diseases (1). Modern microbiology has establishedthe close associations of bacteria with mammals not only as

a cause of disease, but also as part of an essential colonizingmicroflora (2,3).

Commensal bacterial communities are closely associatedwith the human skin, oral cavity, gastrointestinal tract and thefemale genital tract. The colon, in particular, is the home fora 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

human colon, although statistical extrapolations from the 16SrDNA sequencing of cloned amplicons derived from humanfecal community DNA from one patient suggest that there are150 operational taxonomic units (defined as differing in 16SrDNA sequence by 2%) (4,5). Future studies that utilize acombination 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 40species make up 99% of all isolates, with the Bacteroides-Prevotella group (a Gram-negative anaerobe) and Clostridiumspecies (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 ofanaerobes to aerobes having been used as a measure for nor-mal flora. The composition of the microflora changesthroughout the intestinal tract, with the highest microbialactivity observed in the proximal colon (2,6). The composi-tion of the microflora appears to be affected by changes insubstrate availability, pH and reduction potential. The totalnumber of microbes in the gastrointestinal tract seems to be

similar in different human populations and has been estimatedto be an order of magnitude higher than the number ofeucaryotic cells in the entire human body (8).

Host-associated bacteria with their metabolic contributionsto host physiology have clear trophic functions and play a rolein protecting the host against invasion by pathogenic species.Bacterial fermentation products such as short chain fatty acidscan be nutrients as well as growth signals for the intestinalepithelium, an example being butyrate with its pro-differenti-ation, anti-proliferation and anti-angiogenic effects oncolonocytes (911). Various bioactive molecules such as car-

1 Presented at the Experimental Biology meeting, April 1115 2003, SanDiego, 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 ofThe Journal of Nutrition has delegated supervision of both technical conformity tothe published regulations of The Journal of Nutrition and general oversight of thescientific merit of each article. The opinions expressed in this publication arethose 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 thesymposium publication are Jon A. Story, Department of Foods and Nutrition,Purdue University, West Lafayette, IN, and J. Glenn Morris, Jr., Department ofEpidemiology and Preventive Medicine, University of Maryland School of Medi-cine, Baltimore, MD.

2 To whom correspondence should be addressed.E-mail: [email protected].

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

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cinogenic xenobiotics, dietary phytoestrogens, and primarybile acids can be transformed by commensal bacteria (1214).The microflora facilitates the excretion of various toxic sub-stances and the exclusion of pathogenic microorganisms fromthe human host. Furthermore, the normal flora has beenshown to stimulate immune function through Peyers patchesand other gut-associated lymphoid tissue (GALT),3 which aredistributed 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 Crohns disease and ulcerative colitis(16,17). Specific host-microbe interactions have been reportedthat can regulate production and excretion of selective sugarsinto the intestinal lumen (18,19). This observation suggeststhat microbes have evolved synergistic mechanisms to influ-ence the colonic environment for their own benefit, andpotentially that of the host, by affecting epithelial host cellgene expression. Although interest in the role of the commen-sal microflora has been renewed by the development andrefinement of molecular analysis tools (reviewed by R. Gaskinset al. in an accompanying article in this issue) including theavailability of completed genome sequences (20 22), our un-

derstanding of the dynamics and physiologic functions of themicroflora is still in its infancy. In this paper we review recentadvances 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 ratherlaborious, time consuming and often inaccurate. It is alsolimited in scope, as a majority of the bacterial species presentin feces are not culturable using standard microbiologic tech-niques (19,23). Even among those that are culturable, species

identification by traditional identification methods is oftendifficult, if not impossible; only a limited number of specieshave been fully characterized, and biochemical identifica-tion systems may have minimal utility in differentiatingspecies (2,3).

The first widely used molecular technique in microbialsystematics, which still required culturing of the respectivebacteria, was total genomic DNA hybridization. This ap-proach, which utilizes whole genomes rather than smallgenomic regions to determine the degree of similarity betweentwo 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 asfluorescent in-situ hybridization (FISH), denaturing gradient

gel electrophoresis (DGGE), quantitative dot blot hybridiza-tion, restriction fragment length polymorphism, and largescale 16S rDNA sequencing have helped to overcome limita-tions of conventional microbiological plating methods instudying the fecal microflora composition (2,24); they arereviewed in detail elsewhere in this issue. Microchips thatwould 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 themicrochip approach has been established, currently availablechips are still limited in scope. Microchips specifically de-signed to analyze the human fecal microflora should make it

possible to extend studies of the associations between diet,microflora, and health to large prospective cohort studies, anecessary next step in dealing with these questions.

In limited studies, molecular tools have been applied tostudy the development of the infant microflora (25,26) andchanges 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- andprobiotics on the human microflora composition (15,28) andthe effects of dietary interventions on the intestinal microflorain various animal models, as outlined below (29,30). Thesestudies have also underscored the degree of variability inherentto these types of complex systems (and assay systems). Forexample, a microflora study based on FISH analyses has showna large degree of variability among subjects and in individualsover time (31). Although some of this observed variation islikely 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 andmaintenance of the commensal microflora is a complex andmulti-factorial process that we do not fully understand. The

sequence in which bacteria settle a niche in the colon, due tochance encounters, nutrient supply, and immune surveillance,may well influence the ability of other bacteria to establishresidence.

Molecular studies have highlighted the diversity of the florawithin individuals. In this context, it is somewhat surprisingthat sequencing large numbers of16S rDNA clones derivedfrom fecal microflora communities has not resulted in largenumbers of identical or highly similar sequences, which mighthave been expected from prior work indicating the predomi-nance of a few genera in the microflora (5,7,32). Wilson andBlitchington 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 ofcloned 16S rDNAs (32). More recently, Suau et al. examined284 16S rDNA sequences from human intestinal microbiotaand 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 specieswere identified within these three groups. Similar observationswere 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 sequencesencoding 280 different peptide sequences (34).

These observations raise the possibility that the intestinalmicroflora contains large numbers of different species, each ofwhich is present only in low numbers. Bias introduced through

PCR or subcloning might affect such analyses (35); the extentof such potential distortions has not been thoroughly investi-gated. Future human microflora studies based on large numbersof cloned 16S rDNA sequences might well change currentconcepts of intestinal colonization and microflora composi-tion. It should be emphasized that sequencing studies of thehuman microflora have not reached the point of saturation. Itmay be necessary to sequence well over a thousand clones persample before reaching a point at which additional sequenceswill be unlikely to yield more unique sequences (unpublishedresults, C. Stine). A high proportion of sequences discoveredin sequencing studies have not previously been described.Both of the human studies summarized above found that onlya quarter of the sequences corresponded to known sequences

even when allowing for a 2% divergence between the observedand data base sequences.

3

Abbreviations used: CRC, colorectal carcinogenesis; DGGE, denaturinggradient gel electrophoresis; FISH, fluorescent in-situ hybridization; GALT, gut-associated lymphoid tissue; LAB, Lactic Acid Bacteria.

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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 shownclear differences between the fecal microflora composition andthe kinds of bacteria that are present at other anatomical sites,including bacteria in the cecum (36) and those associated withthe mucosa (37), differences in the human microflora at var-

ious anatomical sites are not well documented. It can beassumed that while the proportions and activities of the mi-croflora change with passage through the intestinal tract, mostviable as well as nonviable commensal intestinal bacteria willstill be detectable in feces with molecular methods. Until newnanotechnologies allowing for the convenient sampling ofmicroflora throughout the intestinal tract become more widelyavailable, feces remain the only realistic sample in large non-invasive studies. Before large human studies can be designed itneeds to be unequivocally established that analyzing micro-flora composition and activities in feces is representative ofimportant intestinal parameters.

Although the 16S rDNA based technologies continue toundergo refinement, it might be timely to develop standardized

protocols for the analysis of the intestinal microflora, whichwould facilitate an improved comparison of results across stud-ies. Such an effort could lead to the establishment of a singledatabase that contains the fecal bacterial profiles from subjectsacross various studies. Given their expense, studies to datehave generally involved small numbers of patients (or onepatient); establishment of a common database would facilitatecomparison of such data, allowing for a better understanding ofthe complexity and the variation of the human intestinalmicrobiota.

Effects of diet on microflora composition

There have been several studies suggesting that each indi-

vidual harbors his or her own distinctive pattern of intestinalmicroflora composition. This pattern tends to remain constantacross time, although there may be some increase in speciesdiversity with age (27,38). While studies are limited, it appearsthat overall dietary patterns (as seen among persons living indifferent geographic areas) and intake of various nutrients caninfluence general patterns of fecal microflora (39 41). At thesame time, and in keeping with prior comments about thevariability of results, in subjects in a feeding study the samediet may have very different effects on the microflora, possiblydue to differential effects of the diet on the individual s un-derlying microflora composition, and/or underlying geneticdifferences (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 ofbeneficial bacteria (prebiotics), or a combination of both(synbiotics). The effectiveness of these supplements in mod-ulating the intestinal microflora toward a composition withincreased 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 ondifferences in the fecal microflora of neonates that are fedeither breast milk or formula (25,26). These studies haveshown that feeding breast milk results in the development ofan 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

highly speculative. For instance, although it is widely assumedthat Bifidobacteria and LAB are beneficial whereas high num-bers of Clostridia and Bacteroides are detrimental, labelingthem as such is not necessarily supported by rigorous data. Infact, one study showed that although LAB were inverselyassociated with colorectal carcinogenesis (CRC), a positiveassociation with CRC was observed not only for Bacteroidesbut 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 alterationsand which do not resemble the physiologic and immunologicconditions that are found in the colon, and animal studies thatevaluate 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 ofthe human microflora, which we know very little about, and itspotential species or even strain specific interactions with thehuman host including its immune system cannot be effectivelystudied in any simplified model system. Thus, claims of asso-ciations between specific commensal bacterial species orstrains and human health will have to be established in humanfeeding/intervention studies or large epidemiologic studies.

The available molecular tools should now allow for suchstudies. Model systems should be continued to be utilized a) toestablish if and how intestinal microflora composition andactivity can be modulated by complex diets, and b) tostrengthen the hypothesis that specific commensal bacteria ormicroflora profiles are associated with specific diseases includ-ing carcinogenesis.

Unfortunately, studies of specific interactions between dietsthat differ in macronutrient content and microflora composi-tion have not yet been carefully investigated with 16S rDNAbased molecular tools. Early ecological studies have suggestedthat fecal microflora differences in populations with varyingdietary habits contribute to the observed differences in healthin these groups. Studies based on conventional culturing tech-

niques have indicated that the protein and fat content of thediet as well as the nature of the carbohydrates (simple sugarsvs. complex carbohydrates) does affect microflora compositionand activity (41,43,44). Animal studies support the hypothesisthat the intestinal microflora can be modified by diet andantibiotic administration in mice and chicken (29,45). How-ever, as mentioned above, studies that evaluate effects ofdietary interventions on the intestinal microflora often usedifferent methods and thus have to be interpreted with cau-tion. For instance, food restriction, arguably one of the mosteffective dietary interventions, has been shown in one study tohave little effect on the microflora of rats as measured byconventional anaerobic culture, cellular fatty acid profile andPCR (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 rDNAbased molecular tools should now be used, preferably in com-bination with conventional methods, to study in depth in bothhuman and animal models the natural variation in the micro-flora and interactions between diet and the composition andactivities of the intestinal microflora.

Intestinal microflora composition and health

At the beginning of the last century Metchnikoff suggestedthat the use of live bacteria in fermented milk products such asyogurt 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 throughmodifications of the intestinal microflora has reemerged and

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various dairy products that are commercially available claimsuch effects. It has, however, been difficult to establish theexistence of associations between specific microbes and health.Studies that attempt to associate complex diets with changesin the microflora and disease are virtually nonexistent.

The recent literature on the efficacy of probiotic interven-tions supports the hypothesis that changes in the microflorainduced by the consumption of probiotics can reduce the

frequency and severity of diarrhea, as well as atopic disease ininfants (49 52). Crohns disease, inflammatory bowel disease,and gastrointestinal cancers are thought to be associated withthe microflora composition and recent data supports suchassociation (23,5355). Although epidemiologic studies donot support an inverse association between intake of fer-mented milk products and colorectal cancer (56,57), thesestudies are limited in scope because they neither differentiatebetween 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 humanhealth (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 throughstrengthening the barrier function or competing for attach-ment sites; b) interacting with the intestinal immune system,contributing to the regulation of immune function includingtolerance; c) producing either beneficial or harmful fermenta-tion end products (butyrate, acetaldehyde) that might changeintestinal pH; d) facilitating the metabolic conversion anduptake 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 lowerexposure of the intestinal lumen to toxic substances. Morespecific 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 inthe United States today; NCI/SEER estimates are that 147,500new patients with this disease will be diagnosed with thisdisease in 2003, with 57,100 deaths (62). While a portion(perhaps as much as 35%) of the predisposition to developingcolon cancer is attributable to heredity, the majority of riskappears to come from nonhereditary environmental factors.

Colon cancer is much less common in the underdevelopedcountries/regions of Africa, Asia, and South or Central Amer-ica than in developed Western societies [age standardizedincidence of 9.91 vs. 37.30 cases/100,0000 population/y(IARC, Globocan)], consistent with the hypothesis that thereare lifestyle or other factors within these societies that increasethe risk of CRC (63). The importance of environmentalfactors is further supported by the observation that risk in-creases among low-risk population groups after movement todeveloped countries (and, presumably, assumption of the hab-its of these counties); for example, in one study of Chinesemen, rates of colorectal cancer increased as much as twofoldafter 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. Earlyepidemiological observations suggested that diet could explain

up to 90% of colorectal cancer risk (64). Although this levelof 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 totalfat or saturated fatty acids and red meat as causal (with redmeat having the greatest potential impact) and fruits andvegetables and, possibly, fiber, as protective (63,65 67). How-

ever, the evidence for many of these food groups does notreach the level of being conclusive (65).

In one study of associations between microflora composi-tion and colorectal carcinogenesis (initiated in 1971 but notcompleted until 1995 because of technical difficulties in char-acterizing stool microflora), stool samples from rural SouthAfricans were compared with those from rural Japanese (bothgroups consuming a native diet), and three groups eating awestern-style diet, including Japanese-Hawaiians, whites fromHawaii and the continental U.S., and patients with a historyof recent polyp removal (38). While each subject had his orher own unique pattern, the general fecal microflora patternseen in the rural Japanese was distinctive from that of theother groups, as was that of the rural South Africans; compo-

sition of microflora was similar among the group with polypsand the Japanese-Hawaiians (with increased concentrations ofBacteriodes and Bifidobacterium species), while whites had pat-terns somewhere between these high risk persons and therural low risk groups. At the species level, 13 species weresignificantly associated with a high risk of colon cancer andthe western diet, while 6 species were associated with a lowrisk and the native diets. The numbers in the study wererelatively small (total n 88 for all groups), and more tradi-tional microbiologic techniques were used for characterizationof fecal microflora. Nonetheless, these data support the hy-pothesis that patterns of fecal microflora differ among groupsfrom different geographic areas, who are consuming differenttypes 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 arole in intestinal carcinogenesis in APCMin mice, because thefrequency of intestinal polyps was not different in germfreemice when compared with controls (68). However, their studydoes not exclude the possibility that specific bacterial strainsmight have either beneficial or detrimental effects on carci-nogenesis in this cancer model. In fact, Schauer et al. havereported that inoculation of APCMin mice with Citrobacterrodentii increases intestinal polyp frequency (69).

Utilization of molecular tools in future studies

The scope of studies of the associations between microfloraand human health has so far been limited to observationalstudies, controlled short term feeding studies and small-scaleintervention studies. Although these studies have contributedsignificantly to our understanding of the human intestinalmicroflora, we are still lacking a comprehensive understandingof the strength of the associations between diet and microfloraand the degree of variation within and among individuals.

Animal studies, which have the advantage of being wellcontrolled for environmental and genetic factors, should bedesigned to evaluate microflora stability and the effects ofdietary interventions on microflora composition and disease.Various existing models would allow for such studies with IBD

or markers of intestinal carcinogenesis as the relevant endpoints. Although details from such studies will not be directly

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applicable to humans, such studies could strengthen the hy-pothesis that the effect of diet on health is modulated througheffects on the microflora. Due to important differences inintestinal anatomy and physiology as well as in immune sur-veillance between humans and rodents, it is questionable thatassociating rodents with a human fecal microflora would bemore informative than observing changes in the normal ro-dent microflora.

Molecular tools have the potential for allowing analysis offecal samples from a large number of subjects. These tools needto be validated and standardized and should then be utilized tobuild a database of the human intestinal microbiota, whichwill form the basis for determining the degree to which themicroflora can be influenced by dietary changes. A concerted,multidisciplinary effort, incorporating modern molecular toolsfor the microflora analysis in the setting of well-designedprospective studies, is needed to advance our knowledge of thecomplex interactions between host and microflora to the pointthat we can design effective dietary interventions. Ultimately,this should lead to clinical intervention studies to determine ifdiet-induced microflora changes can reduce the risk of majorgastrointestinal diseases such as Crohns disease or colorectal

cancer.

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