- Email: [email protected]
The indigenous gastrointestinal microflora
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9 Gucic, R., Dhandayuthaplni, S. and Dcrcric:, V. (1994) Mol. MM. l3,1057-1064 10 Let, B.Y. and Horwi~ MA. ( 199S)l. Cliti. Ivtrsr. 96.245-249 11 plum, G. and Clark-Cuniss, J.E. (1994) I&. Immun. 62. 476-478 12 Amida, 5. etd. (1993)Scienc~261,1454-1457 13 F&one, V. et al. (1995) Micrubrol. 141.1239-1245 14 ZhPng, Y. eta/. (1991) MO!. Micro&of. 5,381-391 15 Rcyrat, J., Be&t, F. and Gicqucl, B. (1995) Ptoc. Nat/. Acted. Sci. U. S. A. 92,8768-8772 16 Qcmcns, D.L., Lee, 6. and Horwia, MA. (1995)]. B~~&riol. 177,5644-5652 17 Wilson,T.M,dcLisk, G.W. and Collins, D.M. (1995) Mol. Micmbiol. l&1009-1015 18 Brennan, P.J. and Nikaido. H. (199.5) Annu. Rev. Biocbem. 64, 29-63 19 Yuan, Y. et al. (1995) Proc. N&l. A&. Sri. .!I. S. A. 92, 6630-66;4 20 Mundayoor, S., Crawford, J.T. and Shinnick, T.M. ( 1993 ) fnfict.
21 22 23 24
Immun. 61,2708-2712 Lcao, S.C. Ed ~11.(1995) Infect. Impnun. 63,4301-4306 Nonnan, E. et ~1. (199.5) Mol. Microbial. 16,7.55-760 Balasubramanian. V. P~JI. (I 996)]. Btirurrol. 178.27.3-179 Baulaid, A. Kremer. L. and Locht. C. (199611. flodmol. 178.
3091-3098 25 Falkow. 5. (1988)
Ret,. Infect. Da. 10. SLT+S276 26 Maurelli. A.T. ei RI. (1985) Infect. Immttn. 49, 164-171 27 Paxopclla. L. et ~1. ( 1994) Infpct. Immun. 62, 1313-l 3 19 28 S&h; D.W. ( 1964) in The h&obattcrr~: J Soctrccboob [Kubica. G.P. and Wavnc. L.G.. eds). DD. 925-946. Marcel Dekkcr 29 ihang, i. and Young. D:B. ( 1493) %&fs Muo&/. 1, 109-113 30 Zhana Y. er di. ( 19’!2) : Llure 3S&, 591-593 31 Wilson, T.M. and Collins, D.M. 11996) Mol. M~oh;of. 19, 1025-1034 32 Collins, D.M. CI al. (lY95) Pro<-. Naltl. Acud. Sci. U. S. A. 92. 8036-8040 33 Mahairas. G.C. PIJI. (199611. Bacreriol. 178, 1274-1282
The indigenous gastrointestinal microflora Rodney
D. Berg
T
he ‘indigenous’ microThe indigenous gastrointestinal (Cl) tract flora’ ;1s microorganisms that flora is composed of microflora has profound effects on the cannot colonize a particular microorganisms that inhabitat, except under abnormal anatomical, physiological and circumstances. habit body sites in which surimmunological development of the host. These terms are used only faces and cavities are open to The indigenous microflora stimulates the the environment. Thus, indighost immune system to respond more infrequently today; instead, indigenous flora and normal enous microorganisms norquickly to pathogen challenge and, mally inhabit the skin, oral through bacterial antagonism, inhibits flora are usually used intercavity, upper respiratory tract, colonization of the GI tract by overt changeably to describe the colgastrointestinal (Cl) tract, uriexogenous pathogens. Indigenous GI lection of microorganisms that nary tract and vagina. The term bacteria are also opportunistic pathogens normally inhabit the GI tract. ‘indigenous flora’ has been very and can translocate across the mucosal Nonetheless, the concepts fordifficult to define and is not barrier to cause systemic infection mulated by Dubos, Alexander clearly defined, even today. and Savage, in their attempts in debilitated hosts. Dubos et&’ conceptualized the to define and classify the indigR.D. Berg is in the Dept of Microbiology indigenous intestinal microenous GI microorganisms, have and Imrmmology, Louisima State University flora composition of a given been valuable in emphasizing Medical Cm&-r. Shrevepoti, LA 71106, USA. animal species as a combination the importance of considering k-l: +I 318 675 5762. far +I 318 675 S764, of the ‘autochthonous flora’ basic ecological principles when e-mail: [email protected] present during the evolution of des$+ng experiments to study the animal, the ‘normal flora’ consisting of microhost-microflora relationships. organisms so ubiquitous in a given community of aniIn a stable Gl ecosystem, all available habitats or mals that they become established in practically all its niches are occupied by indigenous microorganisms. members, and the %rue pathogens’, which are acquired Any transient species derived from food, water or even accidentally and are capable of persisting in tissues another part of the host Cl system or skin will not (Box 1). Taking into BEcount DuW concepts and the usually establish (i.e. colonize) and instead will pass ecthgkaI~ofAiexanderZ,Sa~redefind&e through the GI tract. Thus, a particular microbial ‘norma flora’ as composed of the autochthonous species might be indigenous to one habitat in the GI mkrootsnnirm that ?lazively co&nize 0 pilrhbr habitract but only a transient in another habitat after being tat or enviconmentaI nid3e and the ‘allochrbonoua shed horn its native site. In this context, habitats are cc*yrigk
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Box i. Ddlnlqg the gtmtfdade I~~~@Bww 1. Autochthonous 2. Normal
flora
flora
fBom
(defined
by Dubos
Microorganisms in all communities
present of
Micmorganisms
established
a
pathogens _.
--.-
_--.
Microorganisms acquired members of a community
Numud okra (redefined 1. Autochthonous (i.e. indigenous
2. Allochthonous (i.e. transient
flora
flora)
flora flora)
et 81..
in all members of a particular present in all communities
accidentally and therefore of an animal species.
and therefore commun:ty of these
not
normally
acqutred or even
physical spaces in the GI tract occupied normally by climax communities of indigenous microorganisms. These ecological principles explain why the population levels and species composition of the indigenous GI microflora of a particular host remain remarkably constam 3ver the passage of time and are not easily disru Ed, even by drastic dietary changes4. It is always difficult, and often impLssible, to determine whether or not a particular microorganism is truly indigenous to a particular host. Not only are the criteria for determining whether an organism is indigenous incomplete but there are severe limitations in the methodologies for culturing, quantifying and identifying indigenous micTobia1 species, especially since 99.9% of the indigenous GI microflora are obligate anaerob&. The methods employed to obtain specimens for culture have always been problematic, especially in humans. Various procedures, such as G: intubation and utilization of GI self-opening capsules, have been used in attempts to obtain accurc te samples from particular GI habitats, such as those regions close to the mucosal epithelium6. Specialized media containing exotic nutritional sources are required +c culture many of these fastidious microorganisms. As the majority of the indigenous GI microflora are obligate anaerobes, an anaerobic chamber (glove box) capable of reducing the proportion of atmospheric OL to -0.00 1% is also required. Furthermore, speciation of indigenous obligate anaerobes requires the use of gas-liquid chromatography to identify volatile fatty acids produced during fermentation. Even with these sophisticated techniques, only -60% of the bacterial forms observed during the microscopic examination of human fecai sampI= have been cuitured in the laboratory’. ~onofthelndlCyenousQIThe indigenous GI microflora does not appear spontaneous!y in newborn humans or animals; instead, certain microbes colonize particuiar intestinal habitats at various times after birth that are characteristic of that
.--
present
of an animal animals. present
in all
by Savage, 1977)’
Resident microorganisms present in ali communities of a par&Mar Properties: -Can grow anaerobically in the gastrointestinal tract. l Always present in GI tract of normal adults. l Colonize particular GI habitats or niches. l Maintain stable climax Gl populations. l Often associate intimately with GI mucosal epithelium. iJiicrr~u:ganisms ccTmunities
-
1965)’
during the evolution of an animal particular animal species.
species but not necessarily 3. True
-__
(UI) mkrofkm
transiently
and
animal
species.
?herefors not necessarily present in all of a single community of animals.
present in all members
particular habitat anLi animal host. In other words, there is bacterial succession’*‘. The fetus in utero is normally sterile and at birth becomes contaminated with a hetcroge.lcous collection of microorganisms from the birth canal and the immediate environment. Within days, many of these microorganisms are eliminated and successivcqualitative and qusnritativechanges occur in the GI microflora. Lactic acid bacteria and coliforms become the predominant misroorganisms in infant human ac-! animal GI tracts but, during weaning, the microflora changes drastically and obligately anaerobic bacteria become predominant. The indigenous GI microflora of the adult consists of climax communities of microorganisms that are remarkably stable. The indigenous bacteria are not just randomly distributed throughout the Gl tract of adults but are found at characteristic population levels in parricular regions of the tract (Fig. I ). The oral cavity contains an indigenous rnicrciflora of -200 SpecieQ. Saliva contams transient bacteria ( IO9 bacteria ml-‘) shed from oral surfaces, such as the tongue and cheeks. Gingival crevices and dental plaque, however, provide anaerobic environments with very low oxidation-reduction potentials, allowing theestablishment of a diverse anaerobic microflora. Dental plaque contains a very dense population of 10” bacteria g’. The human stomach and the upper two-thirds of the small intestine (duodenum and jejunum) contain only low numbers of microorganisms ( 103-I O4 bacteria ml-’ of gastri. or intestinal contents)9. Microbial numbers are restricted in these areas because of the low pH of stomach contenrs and the relatively swift flow (peristalsis) throlqgh the stomach and the small bowel. The principal microbial types in the upper small intestine are acid-tolerant lactobacilli and streptococci, which, unlike the majority of microorganisms found in food, survive passage &rough the stomach. Thus, the bacteria cultured from the stomach and upper small bowel ace considered transients from the oral cavity rather than indigenous micrwrganisms.
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because of the slow intestinal motility encountered here (transit times of up to 60 h) and the very low oxidation-reduction potentials“. (:onsequently. the large intestine 1012 skin bacteria harbors tremendous numbers of bacteria lot3 cells in the body (IO”‘-10” bacteria g-l intestinal contents), 1 014 GI bacteria comprising an estimated 400--500 species (Fig. 1)‘. It is important to emphasize that 99.9% of the indigenous GI microflora of the large intestine are obligately anaerobic bacteria. Thus, obligate anaerobes (e.g. Bactewid~s frugilis) are IOO- to 1000-fold more numerous than facultative anaerobes (e.g. En-berichia co/i and other coliformsl, the organisms commonly cultured as indicators of human fecal contamination. In addition to the characteristic vertical distributions of indigenous species from the Colon oral cavity to the colon, there are character(lo’“-lo”g-‘) istic horizontal distributions of indigenous (400-500 species, species from the GI lumen to the mucosal Ileum including the following: epithelium’. At least four major horizontal (1 Oe bacteria ml-l) Bact6mideS bacterial ha!Ctats have been suggested: the R3ptastreptococcus lumen of the GI tract, the mucus gel that EubacterUm overlies the epitheiium of the entire tract, the BiWoh3&wium deep layer of mucus gel in the intestinal crypts Ruminowccus and the immediate surfaces of the individual t3acilllls mucosal epitheiiai cells’*. Thus, certain miFuschacterium crobial types live freely in the lumen, some Clostridium colonize the intestinal crypts of Lieberkuhn Lacto&acillus and others interact intimately wito epnheiiai Enterococws ceil surfaces. The obligate anaerobes, which Enterobacter) are the numerically predominant indigenous bacteria, associate intimately with the gut wall w 1. Oistribution of Mis gestmintestinal (GI) microflora in humans. For every cell to form layers on the mucosai epithelium. in Me human bociy (lo*3 cells in total). there am ten viable indigenous bacteria in the GI tract. In addiion. the GI tract (lox4 bacteria) hf&ors lOO-foId more bacteria then the In summary, there are ten viable indigskin (10” bacteria).The microflora !xated in the cnlon are listed in descending order enow bacteria in the GI tract for every ceil ofprominence. in the human body: lOI total Gi bacteria compared with 10” total cells making up the human body. In contrast, the skin harbors 100-fold Recently, it has been discovered that Helicobncter pylori, a spiral-shaped, highly motile bacterium, is pres- fewer bacteria than the GI tract ( 1Or2 versus lOI bacent in the stomachs of a third to a half of the adult poputeria). The skin is a less hospitable place for bacterial lation of the world. Other species of Helicobacter may growth compared with the GI tract because of the lack also be present. It is has been suggested that Helicoof wateP. If we assume that one mutation in every batter might even be considered indigenous to the hu1 Or bacterial divisions is a viable mutation, 1Ot4 total man stomachr”. H. pyloti colonization of the stomach bacteria in the GI tract theoretically will produce IW antral surface does not produce disease symptoms in newly mutated viable bacteria at every division cycle. most people. In some individuals, however, it is a pathoIt is estimated that the bacteria in the GI tract divide gen because it clearly causes gastritis, gastric and/or every 20min (Ref. 11). This generation of large numduodenal ulcers, and is likely to be an important facbers of newly mutated bacteria at every division cycle tor in the development of gastric cancer. allows the indigenous GI microflora to adapt rapidly The distal small intestine (ileum) is considered a to GI environmental changes. ‘transition zone’ between the relatively sparse microflora of the upper bowel and the tremendous numbers Mod&uaexltostudyW@nousmkfo#ora-hoet of bacteria found in the large intestine9. Compared with dre upper small imesbe, the distal smaIl intestine (with It is difficult to devise meaningful experimental moddecreased peristalsis and lower oxidation-reduction els to delineate the complex interactions between the otentiala) maintains a more diverse microflora and indigenous bacterium and its host, when there are 400-500 species of indigenous GI bcteria, of which gi,h,, btscterial populations (1Oe bacteria ml-t intestinal contents). -40% have not been cultured in the laboratory. The The la inlathe (colon) ir the primary site of mi‘gptdree’ animal provides a ‘living test tube’, which crohkll~~tkminh umans and animals, probably can be colonized with one or more species of bacteria,
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-_ allowing the interactions between the host and this ‘sintplificd’ microflora to be studied. Germfree animals arc delivered by Cesarean section into sterile plastic-film isolators and maintaincd free of all bacteria, fungi, protozoa, viruses and other detectable life forms. The practice of introducing microorganisms into germfree animals is called association or colonization, whereas contamination is the accidental introduction of unwanted microorganisms. The germfree animal can be monoassociated with a single bacterium or polyassociated with multiple microorganisms. Association or colonization implies that the microorganism will persist in the CI tract without the need for periodic reintroduction of the mt
---. Box 2. cmgaammwtl~
Increased cecum size in germfree rodents and Decreased weight of intestinal wall l Decreased intestinal surface area *Thinner intestinal villi l Thinner lamina propria l Decreased size of liver, heart, adrenals, etc. l Decreased blood volume l
Physh@gkd/blochemW
conventional
1-m 0 Decreased 0 Decreased l Decreased j l Decreased
I
lympn node and spleen sue in germfree Peyer’s patch size serum gammaglobulin levels numbers of immunoglobulin-A producing l Decreased numbers of intraepithelial T cells 0 Decreased inflammatory response l Decreased blood clearance of microorganisms l Delayed immune response after antigenic challenge
or even
gnoto-
biotic mice, studies into the relationships between the indigenous GI microflora and the host have also been performed in vitro in anaerobic continuous-flow cultures inoculated with the whole cecal flora from mice”. Surprisingly, these continuous-flow cultures duplicate accurately the numerical relationships present in oioo among the complex microflora of the GI tract of animals and humans. For example, various indigenous bacterial types reach climax populations in anaerobic continuous-flow cultures similar to their climax population numbers in the mouse cecum or colon. These studies demonstrate that the population levels of the various members of the indiga~ous bacteria in the mouse c-cwn % * and colon are conttollcd primarily by mtabolic competition among these bacteriai3.
-t-IteNDs
IN
M~C:R~IUOL~~;Y
chamcterhtks
Decreased intestinal motility or peristalsis in gel#nfree rodents (i.e. time) l Decreased rate of villus epithetial cell renewal l Altered mucosal enzyme petterns (i.e. increasi;d trypsin. decreased l Lower pH of intestinal contents l Inw?ased oxygen levels (i.e. highi-r oxidatron-reduction potential) l Decreased basal metabolic rate 0 Decreased cardiac output l Decreased regional blood flow (to intestines, liver, etc.) l Deerea+nthesis of vitamin K and vitamin 8 complex l No bile acid transformation in intesttnes l Lack of shortchain fatty acids or coprostanol l
biotic animal sp&es of indigenous bacteria is also an ‘artificial’ model. Nonetheless, comparisons between germfree and conventional animals have revealed the important effects the indigenous GI microflora exerts on its host. These gnotobiotic models have also delineated certain important basic ecological principles operating in the GI tract. In an effort to provide a model that can be more than
rabbits
l
GI microflora during :he period animal acquired the inJigenous never he fully discounted. A gnotocolonized with only a few of the many
manipulated
rodant
Morphokecalc-
etc.) ot an indigenous before the germfree microorganism can
easily
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ofg4mherorkntswlthoutmIndlgscrow (al) mkrafkm compared with canveIluoMl with an Indigenous 01 mkrdlom
433
increased
g-glucuronidase)
rodents
lymphocytt?s
in lamina
Bacterial adherence to the inrestinal epithelium is also important in maintaining GI equilibrium of the indigenous micrcflora. For example, certain indigenous bacteria
in anaerobic
contrnuous-flow
cultures
asso-
ciate with
the glass wall of the flow culture vessel and with each other to form layers of bacteria that are necessary to maintain the proper ecological balance in the flow culture”. In other words, bacterial adherence to the culture vessel H-all and bacteria-bacteria adherence appears necessary to maintain the same pattern of population levels of various bacterial species found normally in the GI tract. It is suggested that bacterial association with the flow culture wall reduces bacteria1 elimination from the vessel, thereby allowing bacteria to maintain climax populations at lower multiplication rates I’. Adherence to the gut wall is also a very important attribute for a potential pathogen if it is to have any chance of becoming estabiished in the continuous-flow culture or in the GI tract in the presence of the indigenous GI microflora. I~elmlcrd)orcr-hodhltComparisons between germfree and conventional animals reveal the dramatic and profound effects the indigenous GI microflora exerts on the morphological, physiological, biochemical and immunological development of its host (Box 2) i4-i6. The most striking morphological abnormality of germfree rodents is a massive
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merabolic processes can be benettcial or harmful to the host (Box 3). For example, the indieenous GI microflora synthesizes I, vitamin BI1 and vitamin K. which are uti0sneikwlized by the host”. By contrast, deconiugation l BacterIaI antagonism (I.e. bacterial interference. coloniratlon resistance) of bile acids by bacteria in the upper bowel l ‘Prime’ host immune response to respond mom quickly to a pathogen l Malntain GI tract peristalsis end Intestinal mucosal lntegtity (contaminated bowel syndrome) can cause l Con& dietary pmcatclnogens and carcinogens to noncarcinogens decreased fat absorption, ieading to diarrhea l Synthesis of vitamin K and vitamin B complex and steatorrhea”. It is well known t!lat indigenous GI bac-teria transform certain dietary substance l Convert dietary noncarcinogens or ptecarclnogens to carcinogens l Ecological disruptions leading to intestinal overgrowth by indigenous bacteria to precarcinogens or carcinogens. Circuml Opportunistic infections (i.e. translocation from the Gl tract) stantial evidence suggests a strong link be. tween dietary factors, the metabolic activities of the indigenous Cl microflora and bowel distention of the cecum; the germfree cecum can be cancerLo. Conversely, some members of the GI microtenfold larger than the conventional rodent cecum. flora may be beneficial in converting precarcinogens Colonization of the germfree rodent with the whole and even carcinogens to inactive forms, although indigenous Gf microflora rapidly reduces the cecal these processes have not been defined. size to normal As mentioned above, the indigenous GI microflora Bmchemical and physiological parameters that differ stimulate the development of the immune defense system so that the host can respond more rapidly to pobetween germfree a?d conventional rodents have also tential pathogens - often a major determining factor been identified. For example, the time it takes for newly formed epithelial cells in the villus crypts to migrate as to whether the host will survive or succumb to ar the length of the ;illus and to he eventuallv sloushed infection. However, the most important beneficial rfi--t? th*- Gl lumen is increased fro111 two days in the feet ::f the indigenous GI microflora is to make it more conventional mouse to four days in the germfree mouse. difficult for exogenous pathogenic bacteria to establish Various physiological parameters, such as basal meta(colonize) in the Gl tract to cause disease. This phenombolic rate, cardiac output and regional blood flow to enon has been called ‘bacterial antagonism’2’, ‘bacterral the intestines and liver, are also reduced in germfree interference’12 or ‘colonization resistance’L.g.To estabrodents compared with conventional rodents. lish in the Gl tract, exogenous microorganisms must Many immunological parameters, such as the numcompete with the indigenous Gl microflora for carbon ber of lamina propria lymphocytes, the number of imand energy sources and also for adhesin sites on the munoglobulin A-producing plasma cells and the level intestinal mucosa. Disruption of the ecological equiof serum gammaglobulins, are decreased in the germlibrium of the GI tract often occurs when patients refree animal because of lack of antigenic stimulation by ceive oral antibiotics because antibiotic treatment usuan indigenous GI microflora*~*8. Secondary lymphatic ally eliminates only a portion of the large numbers of organs, such as the spleen and lymph nodes, are also indigenous bacterial species. The remaining indigenous underdeveloped in germfree animals. Thus, both serum bacteria can overgrow the intestine causing diarrhea or and cell-mediated immunity are depressed in germfree even translocste acrocz the intestinal epithelial barrier animals. The germfree animal mounts an effective imto cause infection in extraintestinal sites and lethal sepmune response following vaccination but it takes longer sis. Disruption of the ecology of the GI microflora also to do so than its conventional counterpart because it decreases colonization resistance, allowing the estabhas not been immunologically ‘primed’ by antigens of lishment of more pathogenic, exogenous bacteria. the indigenous microflorar6. Indigenous bacteria are continuously passing in low numbers from the GI tract across the mucosal barrier to Eenefkl&lamldetrbllentalef?ecte the mesenteric lymph nodes and other extraintestinal ‘The metabolic activity of the indigenous GI microflora sites, a process called translocationz4. in the healthy is potentially equal to that of the human liver. These host, __these _ __low numbers of translocating bacteria are usually killed en route or in sitrr in the mesenteric lymph I nodes (presumably by macrophages) and do not spread QueaknsforfLltureresearch to other sites, such as the liver, spleen or blood. In l WMdr of the species of i-s GI bacteria are most beneficial fact, the presence of indigenous transiocatinq bacteria and whkh are moat harmful to the host? in low numbers in the latnina propria and mesenteric *How doee the i-s GI tnlcmflom stimulate tha newborn lymph nodes could possibly be a ‘normal’ beneficial lmmunokolcel eyeten end how lmpottant 1sthis early antlgenic mechanism for stimulating the host immune system to 8tlmulatlon in letsr I&? I respond more quickly to exogenous pathogens. However, this hypothesis has not, as yet, been investigated. Ind@enous GI bacteria are opportunistic pathogens and translocating bacteria can cause life-threatening infections in debilitated patients, especiaiiy immunocompromised patients. The three major mechanisms
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promoting bacterial translocation from the GI tract in animal models are: (I) increased bacterial populations (i.e. intestinal overgrowth) in the GI tract following disruption of the GI ecology by oral antibiotics, protein malnutrition, shock and other conditions, (2) physical damage to the mucosal barrier, such as occurs with ischemialreperfusion injury during endotoxic or hemorrhagic shock or (3) decreased immune defenses resulting from immunosuppressive drugs or disease syndromes, such as cancer and AIDSzs-Z8. Indigenous bacteria have been cultured directly from the mesenteric lym$ nodes of certain classes of patients, such as those with cancer, bowel obstruction, Crohn’s disease or hemorrhagic shockz9-“. Although i:>digenous obligate anaerobes (e.g. B. fiagi/is) cause human disease, the indigenous Enterobacteriaceae (e.g. E. coli, EnZero6acfer and Proterrs) translocate much more readily than the ob!igate anaerobes in animal models and in humans. In fact, these Enterobacteriaceae are the leading cause of seprd:emia in hospitalized patients’ More research is required to delineate the pathogenesrs of ir.digenctis bacterial translocation from the GI tract in order to devise prc: - (-tative strategies against these opportunistic infections. The complex ecological mechanisms operating between the indigenous microflora and its host and among the various members of the indigenous GI microflora are only beginning to be understood. Recent renewed interes- in the use of probiotics for both humans and otl.cr animals has increased the impetus for examining these ecological relationships. Fuller” defines a probiotic as ‘live microbial supplements that beneficially affect the host by improving its microbtcll balance’. Oral, live Saccharomyces boukwdii is at present prescribed for the treatment of CIostridium difficile-associated colitis3’. Strains of Lactobacillus and Bifidobacteria are also currently used as probiotics, although their beneficial effects are controversia19. Probiotics may eventually be of use for improving nutrition, renewing or even bolstering colonization resistance, decreasing the risk of bowel cancer and stimulating canspecific, or even specific, immunity to certain pathogens. At present, it is premature to attempt TO manipulate the indigenous GI microflora to realize these benefits until we discern the basic mechanisms that maintain the ecological equ;librium among the variolls Tembers of the indigenous GI microflora and learn much more concerning the complex relationships between the indigenous microflora and its host. 1 Dubos,R~al.(1965)1.E*P.Med.
122,67-76
2 Alc&der, M. (1971) &&zl Ecology, John Wiley SC Sons 3 Savaae. Ecofm of tf~e Gut __ D.C. I1 9n) in Micr&al (Clark, R.T.J. and Bauchop, T., eds), pp.--27;1-3 10, Academic 4
Press Dresar, B.S. and Hill, M.J. pp. 9-2.5, Acachic Press
(1974)
in Humun
lnzestinu/
J Moore, W.E.C. and Hokkman, L.V. (1974) A@ %I-979 6 Cla&, RT.J. (1977) in Micmbkrl &oIogy ofthe 7
M-o/.
Flora, 27,
Gut (Clarke, R.T.J. and Baucbop, T.. eds), pp. l-33, Academic Press Savage, D.C (I 977) in Humm Infesti& Micm&ra in He&
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, .kdd--1.
iv. Lc
8 Hardie, J.M. and Bowdcn, G.H. (1974) in The NIJ~: -I ,‘&cro&tI Fbru of.Wdv (Skmner, F.A. and Carl, J.G.. eds). pp. 47-83. Academic Press 9 Tannock. G.W. (19951 Normal M~~oflora, Chapman & Hall 10 her, M.J. (1993) Trelrds M~crobiul. 1.2S5-2i9 11 Freter. R. (1992) in Probiotics (Fuller, R., ed.), pp. 1 I 1-144, Chapman & Hall 12 Trexler, P.C. and Barry, E.D. (1958) Lab. Anim. Gre 8,75-77 13 Freter. R. et af. (19831 fttfect. Immun. 39.686-703 14 Plea&s, J.R. (1968) in fhc Cerm/ree A&ul m Rescurcb (&ares, M.E., ed.), pp. 113-125, Academic Press 15 Coatcs, M.E. and Fuller, R. ( 1977) in Microbial Fcnlogy of the Cut (Clarke, R. 1 .J. and bauchop, T., eds), pp. 31 r-346. Academic Press 16 Wostmann, B.S. (1968) in 7%e Gwnfree Aninrcll m Research (&ares. M.E., ed,), op. 197-209, Academic Press 17 Bealmear, P.M. (1981) in Immunologrcal Defects 5; Laboratory Animals (Gershwin, IM.E. and Merchant, B., eds), pp. 261-350. Plenum 18 Berg, R.D. (I 98 3) in Humun Intestinal Microflora in Health and Disease (Hentges, D.J., ed.), pp 101-126. Academic Press 19 Drasar, B.S. and Hill. M.-r. (1974) in Human Intestinal Now, pp. 173-183, Academic Press 20 Gorbach, S.L. and Goldin, B.R. (1990) Rev. /feet. DE. 12, 252-26 1 21 Fretr:,R.(1956)j.Exp.Med. 104,411-418 22 Dubos. R. 119631 Am.]. Dis. child. 105,6434X 23 Van der Waaij, D. et al. (1971)]. Hyg. 6?, 405-441 24 Berg, R.D. and Garhngton, A.W. (1979) Infect. Immun. 23, 403-411 25 Berg. R.D. ( 1983) in The fntestinol Microfrom in He& otzd D&&se (Hentges. D., ed.., pp. 333-352. Academrc Press 2s Benz. R.D. ( 1985) m. Anti. 34.1-I 6 27 Bcr& R.D. i1992j in krohotics (I&Y, R., ed.), pp. 55-85, Chapman & Hall 28 Berg, R.D. (1995) Trends Microbid. 3,149-l (4 29 Vmcent, P. erul. (1988)]. Infect. Da. 158,1395-1396 30 Deitch, EA. (1989) Arch. Surg. 124,69!+701 31 Ambrose, M.S. et al. (1084) Br. 1. Sq. 71,623-625 32 Rush, B.F. tiuf. (1988) Amr. Surg. 217,549-554 33 Steffen, E.K., Berg, R.D. and De&h, E.A. (1988)]. Infect. Dis. 157.1032-1037 34 Full& R. ( 1992) m Prolnotits (Fuller, R., cd.), pp. l- 8, Chapman & Hall 35 Surawirz, CM. et ul. (lY89) Gustromferology %,981-988
Letters to the Editor Trends in Microbiology welcomes correspondence. Letters (not more than 500 words) may relate to topics raised in earlier issues of the Journal or to other matters of general interest to microbiolog&ts. Please send letters to: Carollne Ash, Editor. Trends In A#cfvbio/~, Elsevler Trends Journals, 68 Hills Road, CambrIdge, UK CB2 1LA. fan: +44 1223 444630, e-mail: timlsevier.co.uk
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9 Gucic, R., Dhandayuthaplni, S. and Dcrcric:, V. (1994) Mol. MM. l3,1057-1064 10 Let, B.Y. and Horwi~ MA. ( 199S)l. Cliti. Ivtrsr. 96.245-249 11 plum, G. and Clark-Cuniss, J.E. (1994) I&. Immun. 62. 476-478 12 Amida, 5. etd. (1993)Scienc~261,1454-1457 13 F&one, V. et al. (1995) Micrubrol. 141.1239-1245 14 ZhPng, Y. eta/. (1991) MO!. Micro&of. 5,381-391 15 Rcyrat, J., Be&t, F. and Gicqucl, B. (1995) Ptoc. Nat/. Acted. Sci. U. S. A. 92,8768-8772 16 Qcmcns, D.L., Lee, 6. and Horwia, MA. (1995)]. B~~&riol. 177,5644-5652 17 Wilson,T.M,dcLisk, G.W. and Collins, D.M. (1995) Mol. Micmbiol. l&1009-1015 18 Brennan, P.J. and Nikaido. H. (199.5) Annu. Rev. Biocbem. 64, 29-63 19 Yuan, Y. et al. (1995) Proc. N&l. A&. Sri. .!I. S. A. 92, 6630-66;4 20 Mundayoor, S., Crawford, J.T. and Shinnick, T.M. ( 1993 ) fnfict.
21 22 23 24
Immun. 61,2708-2712 Lcao, S.C. Ed ~11.(1995) Infect. Impnun. 63,4301-4306 Nonnan, E. et ~1. (199.5) Mol. Microbial. 16,7.55-760 Balasubramanian. V. P~JI. (I 996)]. Btirurrol. 178.27.3-179 Baulaid, A. Kremer. L. and Locht. C. (199611. flodmol. 178.
3091-3098 25 Falkow. 5. (1988)
Ret,. Infect. Da. 10. SLT+S276 26 Maurelli. A.T. ei RI. (1985) Infect. Immttn. 49, 164-171 27 Paxopclla. L. et ~1. ( 1994) Infpct. Immun. 62, 1313-l 3 19 28 S&h; D.W. ( 1964) in The h&obattcrr~: J Soctrccboob [Kubica. G.P. and Wavnc. L.G.. eds). DD. 925-946. Marcel Dekkcr 29 ihang, i. and Young. D:B. ( 1493) %&fs Muo&/. 1, 109-113 30 Zhana Y. er di. ( 19’!2) : Llure 3S&, 591-593 31 Wilson, T.M. and Collins, D.M. 11996) Mol. M~oh;of. 19, 1025-1034 32 Collins, D.M. CI al. (lY95) Pro<-. Naltl. Acud. Sci. U. S. A. 92. 8036-8040 33 Mahairas. G.C. PIJI. (199611. Bacreriol. 178, 1274-1282
The indigenous gastrointestinal microflora Rodney
D. Berg
T
he ‘indigenous’ microThe indigenous gastrointestinal (Cl) tract flora’ ;1s microorganisms that flora is composed of microflora has profound effects on the cannot colonize a particular microorganisms that inhabitat, except under abnormal anatomical, physiological and circumstances. habit body sites in which surimmunological development of the host. These terms are used only faces and cavities are open to The indigenous microflora stimulates the the environment. Thus, indighost immune system to respond more infrequently today; instead, indigenous flora and normal enous microorganisms norquickly to pathogen challenge and, mally inhabit the skin, oral through bacterial antagonism, inhibits flora are usually used intercavity, upper respiratory tract, colonization of the GI tract by overt changeably to describe the colgastrointestinal (Cl) tract, uriexogenous pathogens. Indigenous GI lection of microorganisms that nary tract and vagina. The term bacteria are also opportunistic pathogens normally inhabit the GI tract. ‘indigenous flora’ has been very and can translocate across the mucosal Nonetheless, the concepts fordifficult to define and is not barrier to cause systemic infection mulated by Dubos, Alexander clearly defined, even today. and Savage, in their attempts in debilitated hosts. Dubos et&’ conceptualized the to define and classify the indigR.D. Berg is in the Dept of Microbiology indigenous intestinal microenous GI microorganisms, have and Imrmmology, Louisima State University flora composition of a given been valuable in emphasizing Medical Cm&-r. Shrevepoti, LA 71106, USA. animal species as a combination the importance of considering k-l: +I 318 675 5762. far +I 318 675 S764, of the ‘autochthonous flora’ basic ecological principles when e-mail: [email protected] present during the evolution of des$+ng experiments to study the animal, the ‘normal flora’ consisting of microhost-microflora relationships. organisms so ubiquitous in a given community of aniIn a stable Gl ecosystem, all available habitats or mals that they become established in practically all its niches are occupied by indigenous microorganisms. members, and the %rue pathogens’, which are acquired Any transient species derived from food, water or even accidentally and are capable of persisting in tissues another part of the host Cl system or skin will not (Box 1). Taking into BEcount DuW concepts and the usually establish (i.e. colonize) and instead will pass ecthgkaI~ofAiexanderZ,Sa~redefind&e through the GI tract. Thus, a particular microbial ‘norma flora’ as composed of the autochthonous species might be indigenous to one habitat in the GI mkrootsnnirm that ?lazively co&nize 0 pilrhbr habitract but only a transient in another habitat after being tat or enviconmentaI nid3e and the ‘allochrbonoua shed horn its native site. In this context, habitats are cc*yrigk
n-M
0 I996 ebwk
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Box i. Ddlnlqg the gtmtfdade I~~~@Bww 1. Autochthonous 2. Normal
flora
flora
fBom
(defined
by Dubos
Microorganisms in all communities
present of
Micmorganisms
established
a
pathogens _.
--.-
_--.
Microorganisms acquired members of a community
Numud okra (redefined 1. Autochthonous (i.e. indigenous
2. Allochthonous (i.e. transient
flora
flora)
flora flora)
et 81..
in all members of a particular present in all communities
accidentally and therefore of an animal species.
and therefore commun:ty of these
not
normally
acqutred or even
physical spaces in the GI tract occupied normally by climax communities of indigenous microorganisms. These ecological principles explain why the population levels and species composition of the indigenous GI microflora of a particular host remain remarkably constam 3ver the passage of time and are not easily disru Ed, even by drastic dietary changes4. It is always difficult, and often impLssible, to determine whether or not a particular microorganism is truly indigenous to a particular host. Not only are the criteria for determining whether an organism is indigenous incomplete but there are severe limitations in the methodologies for culturing, quantifying and identifying indigenous micTobia1 species, especially since 99.9% of the indigenous GI microflora are obligate anaerob&. The methods employed to obtain specimens for culture have always been problematic, especially in humans. Various procedures, such as G: intubation and utilization of GI self-opening capsules, have been used in attempts to obtain accurc te samples from particular GI habitats, such as those regions close to the mucosal epithelium6. Specialized media containing exotic nutritional sources are required +c culture many of these fastidious microorganisms. As the majority of the indigenous GI microflora are obligate anaerobes, an anaerobic chamber (glove box) capable of reducing the proportion of atmospheric OL to -0.00 1% is also required. Furthermore, speciation of indigenous obligate anaerobes requires the use of gas-liquid chromatography to identify volatile fatty acids produced during fermentation. Even with these sophisticated techniques, only -60% of the bacterial forms observed during the microscopic examination of human fecai sampI= have been cuitured in the laboratory’. ~onofthelndlCyenousQIThe indigenous GI microflora does not appear spontaneous!y in newborn humans or animals; instead, certain microbes colonize particuiar intestinal habitats at various times after birth that are characteristic of that
.--
present
of an animal animals. present
in all
by Savage, 1977)’
Resident microorganisms present in ali communities of a par&Mar Properties: -Can grow anaerobically in the gastrointestinal tract. l Always present in GI tract of normal adults. l Colonize particular GI habitats or niches. l Maintain stable climax Gl populations. l Often associate intimately with GI mucosal epithelium. iJiicrr~u:ganisms ccTmunities
-
1965)’
during the evolution of an animal particular animal species.
species but not necessarily 3. True
-__
(UI) mkrofkm
transiently
and
animal
species.
?herefors not necessarily present in all of a single community of animals.
present in all members
particular habitat anLi animal host. In other words, there is bacterial succession’*‘. The fetus in utero is normally sterile and at birth becomes contaminated with a hetcroge.lcous collection of microorganisms from the birth canal and the immediate environment. Within days, many of these microorganisms are eliminated and successivcqualitative and qusnritativechanges occur in the GI microflora. Lactic acid bacteria and coliforms become the predominant misroorganisms in infant human ac-! animal GI tracts but, during weaning, the microflora changes drastically and obligately anaerobic bacteria become predominant. The indigenous GI microflora of the adult consists of climax communities of microorganisms that are remarkably stable. The indigenous bacteria are not just randomly distributed throughout the Gl tract of adults but are found at characteristic population levels in parricular regions of the tract (Fig. I ). The oral cavity contains an indigenous rnicrciflora of -200 SpecieQ. Saliva contams transient bacteria ( IO9 bacteria ml-‘) shed from oral surfaces, such as the tongue and cheeks. Gingival crevices and dental plaque, however, provide anaerobic environments with very low oxidation-reduction potentials, allowing theestablishment of a diverse anaerobic microflora. Dental plaque contains a very dense population of 10” bacteria g’. The human stomach and the upper two-thirds of the small intestine (duodenum and jejunum) contain only low numbers of microorganisms ( 103-I O4 bacteria ml-’ of gastri. or intestinal contents)9. Microbial numbers are restricted in these areas because of the low pH of stomach contenrs and the relatively swift flow (peristalsis) throlqgh the stomach and the small bowel. The principal microbial types in the upper small intestine are acid-tolerant lactobacilli and streptococci, which, unlike the majority of microorganisms found in food, survive passage &rough the stomach. Thus, the bacteria cultured from the stomach and upper small bowel ace considered transients from the oral cavity rather than indigenous micrwrganisms.
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because of the slow intestinal motility encountered here (transit times of up to 60 h) and the very low oxidation-reduction potentials“. (:onsequently. the large intestine 1012 skin bacteria harbors tremendous numbers of bacteria lot3 cells in the body (IO”‘-10” bacteria g-l intestinal contents), 1 014 GI bacteria comprising an estimated 400--500 species (Fig. 1)‘. It is important to emphasize that 99.9% of the indigenous GI microflora of the large intestine are obligately anaerobic bacteria. Thus, obligate anaerobes (e.g. Bactewid~s frugilis) are IOO- to 1000-fold more numerous than facultative anaerobes (e.g. En-berichia co/i and other coliformsl, the organisms commonly cultured as indicators of human fecal contamination. In addition to the characteristic vertical distributions of indigenous species from the Colon oral cavity to the colon, there are character(lo’“-lo”g-‘) istic horizontal distributions of indigenous (400-500 species, species from the GI lumen to the mucosal Ileum including the following: epithelium’. At least four major horizontal (1 Oe bacteria ml-l) Bact6mideS bacterial ha!Ctats have been suggested: the R3ptastreptococcus lumen of the GI tract, the mucus gel that EubacterUm overlies the epitheiium of the entire tract, the BiWoh3&wium deep layer of mucus gel in the intestinal crypts Ruminowccus and the immediate surfaces of the individual t3acilllls mucosal epitheiiai cells’*. Thus, certain miFuschacterium crobial types live freely in the lumen, some Clostridium colonize the intestinal crypts of Lieberkuhn Lacto&acillus and others interact intimately wito epnheiiai Enterococws ceil surfaces. The obligate anaerobes, which Enterobacter) are the numerically predominant indigenous bacteria, associate intimately with the gut wall w 1. Oistribution of Mis gestmintestinal (GI) microflora in humans. For every cell to form layers on the mucosai epithelium. in Me human bociy (lo*3 cells in total). there am ten viable indigenous bacteria in the GI tract. In addiion. the GI tract (lox4 bacteria) hf&ors lOO-foId more bacteria then the In summary, there are ten viable indigskin (10” bacteria).The microflora !xated in the cnlon are listed in descending order enow bacteria in the GI tract for every ceil ofprominence. in the human body: lOI total Gi bacteria compared with 10” total cells making up the human body. In contrast, the skin harbors 100-fold Recently, it has been discovered that Helicobncter pylori, a spiral-shaped, highly motile bacterium, is pres- fewer bacteria than the GI tract ( 1Or2 versus lOI bacent in the stomachs of a third to a half of the adult poputeria). The skin is a less hospitable place for bacterial lation of the world. Other species of Helicobacter may growth compared with the GI tract because of the lack also be present. It is has been suggested that Helicoof wateP. If we assume that one mutation in every batter might even be considered indigenous to the hu1 Or bacterial divisions is a viable mutation, 1Ot4 total man stomachr”. H. pyloti colonization of the stomach bacteria in the GI tract theoretically will produce IW antral surface does not produce disease symptoms in newly mutated viable bacteria at every division cycle. most people. In some individuals, however, it is a pathoIt is estimated that the bacteria in the GI tract divide gen because it clearly causes gastritis, gastric and/or every 20min (Ref. 11). This generation of large numduodenal ulcers, and is likely to be an important facbers of newly mutated bacteria at every division cycle tor in the development of gastric cancer. allows the indigenous GI microflora to adapt rapidly The distal small intestine (ileum) is considered a to GI environmental changes. ‘transition zone’ between the relatively sparse microflora of the upper bowel and the tremendous numbers Mod&uaexltostudyW@nousmkfo#ora-hoet of bacteria found in the large intestine9. Compared with dre upper small imesbe, the distal smaIl intestine (with It is difficult to devise meaningful experimental moddecreased peristalsis and lower oxidation-reduction els to delineate the complex interactions between the otentiala) maintains a more diverse microflora and indigenous bacterium and its host, when there are 400-500 species of indigenous GI bcteria, of which gi,h,, btscterial populations (1Oe bacteria ml-t intestinal contents). -40% have not been cultured in the laboratory. The The la inlathe (colon) ir the primary site of mi‘gptdree’ animal provides a ‘living test tube’, which crohkll~~tkminh umans and animals, probably can be colonized with one or more species of bacteria,
I1
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-_ allowing the interactions between the host and this ‘sintplificd’ microflora to be studied. Germfree animals arc delivered by Cesarean section into sterile plastic-film isolators and maintaincd free of all bacteria, fungi, protozoa, viruses and other detectable life forms. The practice of introducing microorganisms into germfree animals is called association or colonization, whereas contamination is the accidental introduction of unwanted microorganisms. The germfree animal can be monoassociated with a single bacterium or polyassociated with multiple microorganisms. Association or colonization implies that the microorganism will persist in the CI tract without the need for periodic reintroduction of the mt
---. Box 2. cmgaammwtl~
Increased cecum size in germfree rodents and Decreased weight of intestinal wall l Decreased intestinal surface area *Thinner intestinal villi l Thinner lamina propria l Decreased size of liver, heart, adrenals, etc. l Decreased blood volume l
Physh@gkd/blochemW
conventional
1-m 0 Decreased 0 Decreased l Decreased j l Decreased
I
lympn node and spleen sue in germfree Peyer’s patch size serum gammaglobulin levels numbers of immunoglobulin-A producing l Decreased numbers of intraepithelial T cells 0 Decreased inflammatory response l Decreased blood clearance of microorganisms l Delayed immune response after antigenic challenge
or even
gnoto-
biotic mice, studies into the relationships between the indigenous GI microflora and the host have also been performed in vitro in anaerobic continuous-flow cultures inoculated with the whole cecal flora from mice”. Surprisingly, these continuous-flow cultures duplicate accurately the numerical relationships present in oioo among the complex microflora of the GI tract of animals and humans. For example, various indigenous bacterial types reach climax populations in anaerobic continuous-flow cultures similar to their climax population numbers in the mouse cecum or colon. These studies demonstrate that the population levels of the various members of the indiga~ous bacteria in the mouse c-cwn % * and colon are conttollcd primarily by mtabolic competition among these bacteriai3.
-t-IteNDs
IN
M~C:R~IUOL~~;Y
chamcterhtks
Decreased intestinal motility or peristalsis in gel#nfree rodents (i.e. time) l Decreased rate of villus epithetial cell renewal l Altered mucosal enzyme petterns (i.e. increasi;d trypsin. decreased l Lower pH of intestinal contents l Inw?ased oxygen levels (i.e. highi-r oxidatron-reduction potential) l Decreased basal metabolic rate 0 Decreased cardiac output l Decreased regional blood flow (to intestines, liver, etc.) l Deerea+nthesis of vitamin K and vitamin 8 complex l No bile acid transformation in intesttnes l Lack of shortchain fatty acids or coprostanol l
biotic animal sp&es of indigenous bacteria is also an ‘artificial’ model. Nonetheless, comparisons between germfree and conventional animals have revealed the important effects the indigenous GI microflora exerts on its host. These gnotobiotic models have also delineated certain important basic ecological principles operating in the GI tract. In an effort to provide a model that can be more than
rabbits
l
GI microflora during :he period animal acquired the inJigenous never he fully discounted. A gnotocolonized with only a few of the many
manipulated
rodant
Morphokecalc-
etc.) ot an indigenous before the germfree microorganism can
easily
----
ofg4mherorkntswlthoutmIndlgscrow (al) mkrafkm compared with canveIluoMl with an Indigenous 01 mkrdlom
433
increased
g-glucuronidase)
rodents
lymphocytt?s
in lamina
Bacterial adherence to the inrestinal epithelium is also important in maintaining GI equilibrium of the indigenous micrcflora. For example, certain indigenous bacteria
in anaerobic
contrnuous-flow
cultures
asso-
ciate with
the glass wall of the flow culture vessel and with each other to form layers of bacteria that are necessary to maintain the proper ecological balance in the flow culture”. In other words, bacterial adherence to the culture vessel H-all and bacteria-bacteria adherence appears necessary to maintain the same pattern of population levels of various bacterial species found normally in the GI tract. It is suggested that bacterial association with the flow culture wall reduces bacteria1 elimination from the vessel, thereby allowing bacteria to maintain climax populations at lower multiplication rates I’. Adherence to the gut wall is also a very important attribute for a potential pathogen if it is to have any chance of becoming estabiished in the continuous-flow culture or in the GI tract in the presence of the indigenous GI microflora. I~elmlcrd)orcr-hodhltComparisons between germfree and conventional animals reveal the dramatic and profound effects the indigenous GI microflora exerts on the morphological, physiological, biochemical and immunological development of its host (Box 2) i4-i6. The most striking morphological abnormality of germfree rodents is a massive
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merabolic processes can be benettcial or harmful to the host (Box 3). For example, the indieenous GI microflora synthesizes I, vitamin BI1 and vitamin K. which are uti0sneikwlized by the host”. By contrast, deconiugation l BacterIaI antagonism (I.e. bacterial interference. coloniratlon resistance) of bile acids by bacteria in the upper bowel l ‘Prime’ host immune response to respond mom quickly to a pathogen l Malntain GI tract peristalsis end Intestinal mucosal lntegtity (contaminated bowel syndrome) can cause l Con& dietary pmcatclnogens and carcinogens to noncarcinogens decreased fat absorption, ieading to diarrhea l Synthesis of vitamin K and vitamin B complex and steatorrhea”. It is well known t!lat indigenous GI bac-teria transform certain dietary substance l Convert dietary noncarcinogens or ptecarclnogens to carcinogens l Ecological disruptions leading to intestinal overgrowth by indigenous bacteria to precarcinogens or carcinogens. Circuml Opportunistic infections (i.e. translocation from the Gl tract) stantial evidence suggests a strong link be. tween dietary factors, the metabolic activities of the indigenous Cl microflora and bowel distention of the cecum; the germfree cecum can be cancerLo. Conversely, some members of the GI microtenfold larger than the conventional rodent cecum. flora may be beneficial in converting precarcinogens Colonization of the germfree rodent with the whole and even carcinogens to inactive forms, although indigenous Gf microflora rapidly reduces the cecal these processes have not been defined. size to normal As mentioned above, the indigenous GI microflora Bmchemical and physiological parameters that differ stimulate the development of the immune defense system so that the host can respond more rapidly to pobetween germfree a?d conventional rodents have also tential pathogens - often a major determining factor been identified. For example, the time it takes for newly formed epithelial cells in the villus crypts to migrate as to whether the host will survive or succumb to ar the length of the ;illus and to he eventuallv sloushed infection. However, the most important beneficial rfi--t? th*- Gl lumen is increased fro111 two days in the feet ::f the indigenous GI microflora is to make it more conventional mouse to four days in the germfree mouse. difficult for exogenous pathogenic bacteria to establish Various physiological parameters, such as basal meta(colonize) in the Gl tract to cause disease. This phenombolic rate, cardiac output and regional blood flow to enon has been called ‘bacterial antagonism’2’, ‘bacterral the intestines and liver, are also reduced in germfree interference’12 or ‘colonization resistance’L.g.To estabrodents compared with conventional rodents. lish in the Gl tract, exogenous microorganisms must Many immunological parameters, such as the numcompete with the indigenous Gl microflora for carbon ber of lamina propria lymphocytes, the number of imand energy sources and also for adhesin sites on the munoglobulin A-producing plasma cells and the level intestinal mucosa. Disruption of the ecological equiof serum gammaglobulins, are decreased in the germlibrium of the GI tract often occurs when patients refree animal because of lack of antigenic stimulation by ceive oral antibiotics because antibiotic treatment usuan indigenous GI microflora*~*8. Secondary lymphatic ally eliminates only a portion of the large numbers of organs, such as the spleen and lymph nodes, are also indigenous bacterial species. The remaining indigenous underdeveloped in germfree animals. Thus, both serum bacteria can overgrow the intestine causing diarrhea or and cell-mediated immunity are depressed in germfree even translocste acrocz the intestinal epithelial barrier animals. The germfree animal mounts an effective imto cause infection in extraintestinal sites and lethal sepmune response following vaccination but it takes longer sis. Disruption of the ecology of the GI microflora also to do so than its conventional counterpart because it decreases colonization resistance, allowing the estabhas not been immunologically ‘primed’ by antigens of lishment of more pathogenic, exogenous bacteria. the indigenous microflorar6. Indigenous bacteria are continuously passing in low numbers from the GI tract across the mucosal barrier to Eenefkl&lamldetrbllentalef?ecte the mesenteric lymph nodes and other extraintestinal ‘The metabolic activity of the indigenous GI microflora sites, a process called translocationz4. in the healthy is potentially equal to that of the human liver. These host, __these _ __low numbers of translocating bacteria are usually killed en route or in sitrr in the mesenteric lymph I nodes (presumably by macrophages) and do not spread QueaknsforfLltureresearch to other sites, such as the liver, spleen or blood. In l WMdr of the species of i-s GI bacteria are most beneficial fact, the presence of indigenous transiocatinq bacteria and whkh are moat harmful to the host? in low numbers in the latnina propria and mesenteric *How doee the i-s GI tnlcmflom stimulate tha newborn lymph nodes could possibly be a ‘normal’ beneficial lmmunokolcel eyeten end how lmpottant 1sthis early antlgenic mechanism for stimulating the host immune system to 8tlmulatlon in letsr I&? I respond more quickly to exogenous pathogens. However, this hypothesis has not, as yet, been investigated. Ind@enous GI bacteria are opportunistic pathogens and translocating bacteria can cause life-threatening infections in debilitated patients, especiaiiy immunocompromised patients. The three major mechanisms
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promoting bacterial translocation from the GI tract in animal models are: (I) increased bacterial populations (i.e. intestinal overgrowth) in the GI tract following disruption of the GI ecology by oral antibiotics, protein malnutrition, shock and other conditions, (2) physical damage to the mucosal barrier, such as occurs with ischemialreperfusion injury during endotoxic or hemorrhagic shock or (3) decreased immune defenses resulting from immunosuppressive drugs or disease syndromes, such as cancer and AIDSzs-Z8. Indigenous bacteria have been cultured directly from the mesenteric lym$ nodes of certain classes of patients, such as those with cancer, bowel obstruction, Crohn’s disease or hemorrhagic shockz9-“. Although i:>digenous obligate anaerobes (e.g. B. fiagi/is) cause human disease, the indigenous Enterobacteriaceae (e.g. E. coli, EnZero6acfer and Proterrs) translocate much more readily than the ob!igate anaerobes in animal models and in humans. In fact, these Enterobacteriaceae are the leading cause of seprd:emia in hospitalized patients’ More research is required to delineate the pathogenesrs of ir.digenctis bacterial translocation from the GI tract in order to devise prc: - (-tative strategies against these opportunistic infections. The complex ecological mechanisms operating between the indigenous microflora and its host and among the various members of the indigenous GI microflora are only beginning to be understood. Recent renewed interes- in the use of probiotics for both humans and otl.cr animals has increased the impetus for examining these ecological relationships. Fuller” defines a probiotic as ‘live microbial supplements that beneficially affect the host by improving its microbtcll balance’. Oral, live Saccharomyces boukwdii is at present prescribed for the treatment of CIostridium difficile-associated colitis3’. Strains of Lactobacillus and Bifidobacteria are also currently used as probiotics, although their beneficial effects are controversia19. Probiotics may eventually be of use for improving nutrition, renewing or even bolstering colonization resistance, decreasing the risk of bowel cancer and stimulating canspecific, or even specific, immunity to certain pathogens. At present, it is premature to attempt TO manipulate the indigenous GI microflora to realize these benefits until we discern the basic mechanisms that maintain the ecological equ;librium among the variolls Tembers of the indigenous GI microflora and learn much more concerning the complex relationships between the indigenous microflora and its host. 1 Dubos,R~al.(1965)1.E*P.Med.
122,67-76
2 Alc&der, M. (1971) &&zl Ecology, John Wiley SC Sons 3 Savaae. Ecofm of tf~e Gut __ D.C. I1 9n) in Micr&al (Clark, R.T.J. and Bauchop, T., eds), pp.--27;1-3 10, Academic 4
Press Dresar, B.S. and Hill, M.J. pp. 9-2.5, Acachic Press
(1974)
in Humun
lnzestinu/
J Moore, W.E.C. and Hokkman, L.V. (1974) A@ %I-979 6 Cla&, RT.J. (1977) in Micmbkrl &oIogy ofthe 7
M-o/.
Flora, 27,
Gut (Clarke, R.T.J. and Baucbop, T.. eds), pp. l-33, Academic Press Savage, D.C (I 977) in Humm Infesti& Micm&ra in He&
REVIEWS
--
und DiSeuSC !hIIgCS,
D.J.? Cd.). ),,I
55-i
, .kdd--1.
iv. Lc
8 Hardie, J.M. and Bowdcn, G.H. (1974) in The NIJ~: -I ,‘&cro&tI Fbru of.Wdv (Skmner, F.A. and Carl, J.G.. eds). pp. 47-83. Academic Press 9 Tannock. G.W. (19951 Normal M~~oflora, Chapman & Hall 10 her, M.J. (1993) Trelrds M~crobiul. 1.2S5-2i9 11 Freter. R. (1992) in Probiotics (Fuller, R., ed.), pp. 1 I 1-144, Chapman & Hall 12 Trexler, P.C. and Barry, E.D. (1958) Lab. Anim. Gre 8,75-77 13 Freter. R. et af. (19831 fttfect. Immun. 39.686-703 14 Plea&s, J.R. (1968) in fhc Cerm/ree A&ul m Rescurcb (&ares, M.E., ed.), pp. 113-125, Academic Press 15 Coatcs, M.E. and Fuller, R. ( 1977) in Microbial Fcnlogy of the Cut (Clarke, R. 1 .J. and bauchop, T., eds), pp. 31 r-346. Academic Press 16 Wostmann, B.S. (1968) in 7%e Gwnfree Aninrcll m Research (&ares. M.E., ed,), op. 197-209, Academic Press 17 Bealmear, P.M. (1981) in Immunologrcal Defects 5; Laboratory Animals (Gershwin, IM.E. and Merchant, B., eds), pp. 261-350. Plenum 18 Berg, R.D. (I 98 3) in Humun Intestinal Microflora in Health and Disease (Hentges, D.J., ed.), pp 101-126. Academic Press 19 Drasar, B.S. and Hill. M.-r. (1974) in Human Intestinal Now, pp. 173-183, Academic Press 20 Gorbach, S.L. and Goldin, B.R. (1990) Rev. /feet. DE. 12, 252-26 1 21 Fretr:,R.(1956)j.Exp.Med. 104,411-418 22 Dubos. R. 119631 Am.]. Dis. child. 105,6434X 23 Van der Waaij, D. et al. (1971)]. Hyg. 6?, 405-441 24 Berg, R.D. and Garhngton, A.W. (1979) Infect. Immun. 23, 403-411 25 Berg. R.D. ( 1983) in The fntestinol Microfrom in He& otzd D&&se (Hentges. D., ed.., pp. 333-352. Academrc Press 2s Benz. R.D. ( 1985) m. Anti. 34.1-I 6 27 Bcr& R.D. i1992j in krohotics (I&Y, R., ed.), pp. 55-85, Chapman & Hall 28 Berg, R.D. (1995) Trends Microbid. 3,149-l (4 29 Vmcent, P. erul. (1988)]. Infect. Da. 158,1395-1396 30 Deitch, EA. (1989) Arch. Surg. 124,69!+701 31 Ambrose, M.S. et al. (1084) Br. 1. Sq. 71,623-625 32 Rush, B.F. tiuf. (1988) Amr. Surg. 217,549-554 33 Steffen, E.K., Berg, R.D. and De&h, E.A. (1988)]. Infect. Dis. 157.1032-1037 34 Full& R. ( 1992) m Prolnotits (Fuller, R., cd.), pp. l- 8, Chapman & Hall 35 Surawirz, CM. et ul. (lY89) Gustromferology %,981-988
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