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Rev. sci. tech. Off. int. Epiz., 1989, 8 (2), 313-332. Bacterial interactions within the digestive tract R. DUCLUZEAU and P. RAIBAUD * Summary: Interactions between bacteria which constitute the microflora of the digestive tract may be antagonistic or synergic. Certain bacteria can form a barrier to prevent colonisation of the gut by bacteria of other species or other strains of the same species. This review of current research refers to experiments on gnotoxenic animals, with mention of Escherichia coli and species of Clostridium, Lactobacillus, Bacteroides and some other bacteria. K E Y W O R D S : Anaerobes - Bacteria - Bacterial antagonism - Digestive tract Enterobacteriaceae - Gnotobiotic animals - Intestinal flora - Research - Synergy. INTRODUCTION The digestive tract of human beings and animals is an enclave of the external environment which harbours a dense and complex bacterial population. At the steady state, attained in the weaned adult, the microbial flora of the distal parts of the digestive tract amounts to between 5.10 and 2.10 viable cells per gram of fresh contents. More than 190 species make up the human flora (20). Despite some individual variation, this flora remains remarkably stable within a given species. Within a healthy host, a dozen species (always the same ones) cohabitate at high population levels. Other species of the subdominant flora are present in smaller numbers (Fig. 1). 10 11 There is daily introduction of bacterial inocula into the digestive tract, carried in with food or the occasional ingestion of contaminated material. In most cases such inocula, which are often of considerable size, traverse the digestive tract without multiplying and without upsetting the balance of the ecosystem. A striking example of the stability of this microbial ecosystem is that of rats and mice given the same feed and reared on the same premises. The dominant faecal flora of the mice, as judged by optical microscopy of a drop of a 1:100 dilution placed between a slide and a cover slip, is very different from that of the rats, and this difference is permanent. Microbial interactions are responsible for maintaining and regulating the ecosystem of host and gut flora. In this article we shall review methods for studying bacterial interactions, the different types of interaction, their variants in relation to host and feeding, and what is known about their mechanisms. * L a b o r a t o r y for Microbial Ecology, I N R A , Centre de Recherches de J o u y , F-78350 Jouy-en-Josas, France. 314 Stomach D u o d e n u m , bile and pancreatic secretions Facultative Gram-positive anaerobes Jejunum Ileum Facultative anaerobes a n d strict anaerobes Caecum + colon Strict a n a e r o b e s Gram-positive a n d Gram-negative FIG. 1 D i a g r a m of the microbial flora of various c o m p a r t m e n t s o f t h e d i g e s t i v e tract o f h u m a n b e i n g s a n d rats T h e figures are logarithms of the n u m b e r of bacteria in each gram of fresh c o n t e n t s . M E T H O D S OF S T U D Y I N G BACTERIAL INTERACTIONS W I T H I N T H E DIGESTIVE TRACT The study of an interaction between two or more bacterial strains is likely to give rise to erroneous conclusions when results for strains cultured in artificial culture medium are followed by extrapolation to what may occur within the digestive tract of the living animal. There have been numerous statements that lactobacilli act within the digestive tract by destroying enterobacteria, thanks to their ability to produce lactic acid. What is overlooked is that this interaction between lactobacilli and enterobacteria has taken place in vitro in a medium rich in sugars (particularly lactose), in which case the lactic acid accumulates, whereas in the lower gut where interactions actually occur, there are limited amounts of fermentable sugars under normal conditions of feeding, and any lactic acid produced is rapidly absorbed across the intestinal mucosa. In order to examine microbial interactions inside the gut of animals, it is essential to use gnotoxenic animals, harbouring only known strains of intestinal bacteria. Such 315 animals are prepared by administering bacteria, usually by mouth, to axenic (germfree) animals. Axenic and gnotoxenic animals are kept in completely sterile units called isolators. They are given sterilised food and they breathe sterile air. Such equipment has now been developed for various species of animals, and even for newborn babies and adult human beings who need to be protected rigorously from a microbial environment. Animals or babies for axenic rearing may be obtained by sterile caesarean operation. In recent years they have been obtained more often by decontamination after birth by the normal route (15). Laboratory animals (rats and mice) are used most often for axenic studies, though sometimes other species are used in order to examine certain interactions. Thus axenic chicks are valuable for examining the factors involved in microbial interactions in newborn animals. They have the advantage of being easy to feed after hatching. Gnotoxenic quails and fowls are used to examine the influence of avian anatomy and physiology on the equilibrium of the flora in different compartments of the digestive tract. Axenic hares have been used to demonstrate the barrier effect of a complex flora on Clostridium difficile, which is highly pathogenic for this species (6). Another tool has been developed in our laboratory to study flora equilibrium in species difficult to experiment on for either ethical reasons (human beings) or economic and technical reasons (adult pigs). Axenic mice are dosed with the faecal flora of the subject under study. The flora which becomes established in the mice, at least during the first weeks, is very similar to that of the donor, and very different from the flora usually associated with mice. One result of such experiments is the finding that absorption of antibiotics by mice having a human flora is closely similar to that which occurs in persons receiving the same antibiotic. It provides a rare example of direct experimentation on the human flora by using an interposed axenic animal (1). The techniques of rearing gnotoxenic animals have been simplified considerably during recent years, but they are not yet available in every laboratory. This partly explains the paucity of currently available information on the mechanisms of bacterial interactions, especially since the techniques of counting gut bacteria and identifying their metabolites remain highly complex. The dominant bacteria are mostly anaerobes. They all grow in the absence of air, but some can be manipulated in the presence of air, rendering the counting procedure relatively simple. By contrast, others are killed by even brief contact (ten minutes in some cases) with the oxygen of air. The sample has to be transferred as quickly as possible to an anaerobic chamber in order to count this sort of bacteria. The chamber contains a mixture of reduced gases (10% H , 5% CO and 85% N ), rendered permanently free from oxygen by means of a catalyst (palladium oxide). In order to separate the numerous bacterial metabolites, the standard techniques of chromatography and electrophoresis are used, while enzymatic or immuno-enzymatic techniques are available for assay purposes. In brief, the study of such interactions requires a well-equipped laboratory located at a multidisciplinary research centre. 2 2 2 BACTERIAL INTERACTIONS AFFECTING THE POPULATION LEVELS OF DIFFERENT STRAINS IN T H E ECOSYSTEM Factors governing the establishment of a bacterial strain in the digestive tract When a bacterial strain is ingested by an axenic animal, it becomes established in the digestive tract within 12-24 hours in most cases. In other words, the inoculum 316 has multiplied and the bacterial population has reached its peak in the various parts of the tract, at a level which remains stable (with rare exceptions) as long as the animal remains associated with this single bacterial strain. If the bacterial strain does not become established, the physico-chemical conditions of the digestive medium may not be right for its multiplication, or the temperature may be too high for strictly psychrophilic bacteria, or too low for strict thermophiles. Alternatively, the oxidation-reduction potential may be too low for aerophiles, or even too high for strict anaerobes. The bacterium may have nutritional requirements not provided for in the diet, or bactericidal or bacteriostatic factors may be present in the digestive tract. A given bacterial strain which is capable of developing within a gnotoxenic animal becomes established at population levels which vary according to location and the food ingested by the host. Stomach, rumen and crop are the first reservoirs where the food bolus spends some hours, and where the bacteria may multiply. The population level depends on the doubling time under the prevailing physico-chemical conditions, and the emptying rhythm of the particular reservoir. By contrast, most of the small intestine is no place for bacterial proliferation in a healthy host, simply because the intestinal transit is too rapid to allow time for bacterial division. Proliferation occurs in the small intestine only when there is adhesion to the gut wall, or mechanical incidents lead to stasis of ingesta, with pathological consequences. Alimentary stasis is the rule at the lower end of the small intestine (ileum) and in the large intestine (caecum, colon and rectum), and all gut bacteria have sufficient time to multiply there. The population level will depend essentially on substrates available to the bacteria for division, and once again the rhythm of emptying of the large intestine. Different bacterial strains do not attain the same population levels in the large intestine of gnotoxenic animals; they vary from more than 10 viable bacterial cells per gram of fresh contents in the case of Bacteroides, to 5 x 10 in the case of C. difficile. 11 8 Bacterial antagonism When two strains of bacteria are given together or successively, there are two possible outcomes. Both bacteria may develop as well together as they do apart, and the resulting equilibrium is usually stable. Alternatively, there may be an interaction between the two bacterial strains, one dominating the other, possibly eliminating it completely, regardless of the order in which they are introduced. This observation invalidates the old theory of the first occupant, according to which the first strain to arrive "occupies the site", precluding proliferation of any other bacteria introduced subsequently. When a greater number of strains is introduced into a gnotoxenic animal, there is a veritable chain reaction, in which each change in balance between two strains brings about changes in the populations of the other strains. The observer witnesses only the consequence of these many interactions, which we call an "integrated mechanism". Such integrated mechanisms attain their greatest degree of complexity in a holoxenic host, that is, a host reared from birth in an uncontrolled microbial environment. In such animals the population levels of different strains which can be counted selectively vary from 10 to 10 per gram. If a strain which reaches a population level of 10 in a holoxenic host reaches 10 or more in a gnotoxenic host, both receiving the same food, one may conclude that bacterial interactions have been responsible for the difference in population levels. 3 3 11 9 317 In contrast, when a strain introduced into the gastro-intestinal tract of a holoxenic host fails to become established, although it can become established in an axenic host, this provides evidence of interactions between the strains already established and that recently introduced (voluntarily or involuntarily) into the gastro-intestinal tract. Such a case is more precisely referred to as the consequence of a microbial barrier, or resistance to colonisation. Establishment of a bacterial strain in the digestive tract is defined as permanent colonisation following a single administration of the bacterial inoculum. A microbiological procedure is available to demonstrate the establishment of a strain, or by contrast its passive transit. This utilises a marker of transit consisting of spores of a strictly thermophilic species of Bacillus, which fail to germinate during passage through the host, and can resist all inhibitory factors without losing viability. These bacteria can be counted selectively in faeces at their optimum growth temperature (60°C), which precludes the growth of all other bacteria of the digestive tract. An inoculum of Bacillus spores is combined with a roughly equivalent number of viable cells of the strain under study for colonisation. If the elimination curve of this strain is parallel to that of Bacillus spores, it can be concluded that the strain, like the marker, has undergone passive transit (Fig. 2). The barrier flora has had only a bacteriostatic effect on the target bacterium, which is consequently eliminated passively through peristaltic movements. Certain bacteria disappear more rapidly from the digestive tract than the transit marker, which indicates that they are being partly destroyed during transit. By contrast, others may persist in small numbers after disappearance of the marker, which indicates that they are continuing to multiply at a low rate, sufficient to compensate for evacuation; this is the "healthy carrier" state (8). In the case of daily administration of cultures of a strain which fails to become established but is unaffected by the resident bacteria, the faecal population level of the strain reflects the number of viable bacteria ingested. This is a case of apparent establishment, which differs from the true establishment described above. hours FIG. 2 Demonstration of the drastic type of barrier effect Comparative transit of Bacillus subtilis spores (marker of inert transit) and the digestive tract of holoxenic mice. By Ducluzeau et al. \ Shigella flexneri in 318 At the Laboratory for Microbial Ecology at Jouy-en-Josas, we employ various criteria to define the effect of a microbial barrier. When a strain, which we call the target strain, confronts a barrier formed by one or more other strains, which we call inhibitory strains, there will be one of several outcomes. The barrier effect exercised by the inhibitory strains is called drastic if the target strain is eliminated completely, in the same way as Bacillus spores. It is called permissive if the target strain is maintained within the gastro-intestinal tract at a population level lower than that attainable in the absence of a barrier - it is suppressed but not eliminated by the inhibitory strains. Other effects depend on the order in which the strains are given. The barrier effect is called curative if the target strain is eliminated or suppressed by the inhibitory strains, regardless of the order of administration. It is called preventive if the effect occurs solely when the target strain is given after the inhibitory strains. Depending on the type of strains present, the barrier effect may be intraspecific when target and inhibitory strains belong to the same species, or interspecific when they belong to different species. These terms account for the different types of effect manifested by the microbial barrier. Examples of the intraspecific barrier effect The intraspecific barrier effect between strains of E. coli has been studied by DuvalIflah et al. (17, 19). In gnotoxenic mice, a plasmid-free strain of E. coli exerted a general barrier effect, most often permissive, on target strains of E. coli which differed from the inhibitory strain by possessing one or more plasmids, introduced by conjugation. The role of such a barrier effect is important when the suppressed target strains are carrying plasmids coding for resistance to antibiotics or the production of enterotoxic substances. Duval-Iflah et al. (18) also showed that such an inhibitory strain, given to newborn babies just a few hours old, was capable of suppressing E. coli strains carrying resistance plasmids (Fig. 3). Some uninoculated babies acquired plasmid-free strains of E. coli spontaneously (for they do occur, even in a maternity unit of a hospital), and these had the same effect as the ones inoculated experimentally. Experimentally, Duval-Iflah et al. (17) demonstrated that the inhibitory strain could have a curative barrier effect, and not just preventive, against the same target strains when maintained for a month in the gastro-intestinal tract of gnotoxenic mice. This property disappeared after the first subculture in broth culture of the "adapted" strain. Other intraspecific, permissive effects have been observed by Ducluzeau et al. (16) between strains of E. coli sensitive and resistant to a bacteriophage, between strains of Lactobacillus acidophilus which differed only in trehalose fermentation (13), and also between strains of Lactobacillus fermenti in which the inhibitory strain possessed plasmids. Recently an intraspecific interaction between a toxinogenic strain of C. difficile and a spontaneous non-toxinogenic mutant strain has been reported by Corthier and Muller (4). The non-toxinogenic mutant had a preventive barrier effect against the toxinogenic parent strain. This interaction is important because it enables mice to be protected from an otherwise lethal inoculation of the toxinogenic strain. Examples of the interspecific barrier effect Many experimental models have been developed by using gnotoxenic animals, or animals inoculated with a mixed but unknown flora and reared in isolators like gnotobionts, in order to examine interspecific barrier effects against various target strains, and in particular potential pathogens, such as Clostridia, enterobacteria (Salmonella and Shigella), staphylococci and Candida yeasts. As far as we know, 319 Strain of E. coli Strains of E. coli spontaneously introduced voluntarily at birth FIG. present in faeces 3 Intraspecific barrier effect b e t w e e n E. coli strains in t h e digestive tract o f a n e w b o r n i n f a n t (by Duval-Iflah et al., 18) only one paper has demonstrated an interspecific barrier effect in the newborn. Hudault et al. (25) found that a strain of Lactobacillus casei was eliminated from the gastro-intestinal tract of an infant reared in an isolator because of suspected severe immunodeficiency. However, immunological tests showed that the infant was not affected, and this raised the problem of leaving the isolator. Experimental procedure was devised to inoculate four strains representing the four species most commonly found in the faeces of holoxenic newborn, namely one strain each of E. coli, Streptococcus, Bifidobacterium and Bacteroides. Before these four strains were introduced, however, the infant was first inoculated with a strain of L. casei, marketed under the name L. acidophilus, and stated to be capable of establishment in the human gastro-intestinal tract. This strain did become established in the infant, producing D- and L-lactic acids, which changed faecal pH to about 5.5. After a single dose of the four strains was given, L. casei was eliminated from the faeces within a few 320 days. Since the strain of E. coli was the first to become established in the digestive tract of the infant, Hudault et al. believed that it had been responsible for a curative barrier effect against L. casei (Fig. 4). In fact, such an interaction was reproduced in gnotoxenic mice. We describe this case in detail because it demonstrates that in vivo studies may give results directly opposite to those obtained in vitro. Such an interaction may explain why lactobacilli, including L. acidophilus, are not among the dominant flora of the newborn, whether they are fed on mother's milk or substitute milk, because E. coli is the first to become installed in the gastro-intestinal tract (28). Another type of interspecific interaction involving two strains, of E. coli and Staphylococcus pyogenes, was described by Ducluzeau and Raibaud (12). This Inoculation of L. casei Inoculation of a complex flore of h u m a n origin FIG. 4 Interspecific barrier effect in a n e w b o r n i n f a n t Elimination of a strain of Lactobacillus casei by a complex flora in a gnotoxenic infant. By Hudault et al. (25) 321 interaction may result from the fact that staphylococci seldom reach high population levels, although they can do so in gnotoxenic animals. Attempts to recover the bacteria actually responsible for a precise barrier effect from the dominant flora of an animal or an adult human being encounter a major technical difficulty due to the large number of strains and mixtures of strains to be tested, and the frequent impossibility of culturing certain strains of strict anaerobes. This is the reason why very little work has been done on the complete identification of a barrier flora. Corpet et al. (2) and Yurdusev et al. (31) have described the inhibitory effect of strict anaerobes isolated from the faeces of holoxenic piglets against a target strain of Clostridium perfringens (Fig. 5). Ducluzeau et al. (10) obtained two strains of Clostridia, extremely sensitive to oxygen, from the dominant flora of holoxenic mice which were capable of inhibiting the growth of Shigella flexneri in FIG. 5 Example of the interspecific barrier effect of a known combination of bacteria Drastic type of barrier formed b y a combination of Fusobacterium s p . (F), Clostridium s p . (C) and Bacteroides s p . (B) against various strains of Clostridium perfringens isolated from rat ( P ) , pig ( P ) a n d hare ( P ) . By Yurdusev et al. (31) R p L 322 gnotoxenic mice, though only in the presence of E. coli. There are various reports of the inhibitory effect of mixtures of strains cultured together, but without information on precise numbers or the exact nature of the mixture. Freter et al. (23) described an interaction between a target strain of E. coli and a collection of 100 inhibitory strains of strict anaerobes from the faecal flora of holoxenic mice. They failed to show that all 100 strains were required for an inhibitory effect, or that they actually became implanted in animals. Hudault (26) similarly obtained a mixture (only partly determined) of fusiform Clostridia from the caecum of holoxenic mice, which was active against a target strain of C. perfringens. Much research has followed that of Nurmi and Rantala (27) to find strains present in the faeces of adult fowls, capable of inhibiting the growth of salmonella in the digestive tract of chicks. Nurmi and Rantala obtained an effective mixture of cultivable strains, but its composition was not elucidated. It would, of course, be difficult to develop such a mixture for field use, since it would be necessary to achieve uniform output. Hudault et al. (24) succeeded in obtaining a known mixture of fourteen strictly anaerobic strains and two facultative anaerobes from a million-fold dilution of faeces from holoxenic fowls, which had a partial barrier effect against Salmonella typhimurium in holoxenic chicks. The principal characteristics of interspecific barriers Despite the small number of adequately described examples, it is possible to discern certain common characteristics of the barrier flora present in the dominant flora of adult animals, composed mostly of strict anaerobes. The known barriers all arise from mixtures of bacteria acting synergically: three (or even just two) in barriers against C. perfringens of porcine origin (31), several in barriers against C. perfringens in mice (26) (Fig. 6) or S. flexneri in mice (10). In each case studied, each barrier strain inoculated alone has had only a weak effect or no effect at all against the target strain. Days FIG. 6 E f f e c t o f s y n e r g y b e t w e e n bacteria exerting a barrier effect Elimination of C. perfringens (CP) b y a combination of clostridial strains (strains 10, C3 and C1) obtained from t h e caecum of holoxenic mice. By H u d a u l t et al. (26) 323 Such barrier effects are specific in the sense that certain target strains are sensitive and others are not. This specificity is unrelated to the taxonomic position of the target strain. For example, C. perfringens type A and S. flexneri are inhibited to the same extent by a simplified barrier of murine origin, whereas C. perfringens type C is not. Similarly, a two-strain barrier from piglets is more active against type C than against type A of C. perfringens. The ability to exert a barrier effect is an exclusive property of certain strains of bacteria which again bears no relation to taxonomic position. In the case of the antiC. perfringens barrier mentioned above, it is not possible to substitute haphazardly the active strains of Bacteroides and Fusobacterium by other strains belonging to the same genus, let alone the same species, isolated from the same animals. However, such barriers may be regarded as superfluous, for there are many strains within the flora of a holoxenic animal which are capable of exerting the same barrier effect on the same target. It is obviously important during the course of evolution for animals to maintain a constant barrier in the digestive tract against a major pathogen present in the environment, and it is easy to understand the value of selection over the course of time of a range of defence systems capable of compensating for a deficiency in any one system (Fig. 7). The curative or preventive, drastic or permissive nature of Il a n d I2 a r e the first a n d second inoculations of C. difficile ( C . d . ) a n d t h e transit m a r k e r ( T . m . ) — spores of a strictly thermophilic variety of Bacillus subtilis. T h e figures indicate t h e l o g of the n u m ber of viable cells or spores inoculated. The solid line shows t h e n u m b e r of viable cells of C . d . , a n d t h e b r o k e n line t h e n u m b e r of spores of t h e transit m a r k e r . The first t w o inocula of C . d . u n d e r w e n t drastic elimination, m o r e rapid t h a n that of t h e m a r k e r . At the 66th d a y of the experiment the barrier effect ceased a b r u p t l y , without obvious change in t h e e n v i r o n m e n t . 1 0 FIG. 7 V a r i a b i l i t y o f t h e barrier e f f e c t Kinetics of the viable cell c o u n t of Clostridium difficile in faeces from gnotoxenic mice h a r b o u r i n g a flora of h u m a n faecal origin 324 these microbial barriers is a characteristic of the constituent strains. In certain cases of a purely preventive barrier, it can be shown that the persistence of target strains implanted first is due to an adaptation phenomenon. Mutant strains resistant to the barrier develop progressively within the population of strains sensitive to a barrier established in an axenic animal, and the resistant strains become dominant (14). The mechanism of such selection remains unknown and the phenomenon is not widespread. Synergic effects Interactions affecting the population level are not exclusively the result of antagonistic forces, for synergic forces also exist. Thus the establishment of strictly anaerobic strains extremely oxygen-sensitive (EOS) in the gastro-intestinal tract of gnotoxenic animals is impossible unless other strains resistant to oxygen are already present, altering the oxidation-reduction potential of the gut to a level compatible with the establishment of EOS strains. There are other examples of a more specific interaction between two strains. Thus Dubos et al. (7) showed that a strain of C. difficile permitted the establishment of a strain of Clostridium perenne in the gut of gnotoxenic mice fed a semi-synthetic diet. In this case, the strain of C. difficile hydrolysed a particular dipeptide, ß-aspartic-e-lysine, introduced into the diet from heated casein. This dipeptide was not absorbed and it inhibited the development of C. perenne when chelated with dietary copper within the gut. In the rumen, Fonty et al. (21) showed that the development of cellulolytic strains, mainly Bacteroides succinogenes, Ruminococcus flavefaciens and R. albus, was dependant upon prior establishment of other bacterial strains. Ducluzeau et al. (11) examined the synergy between an auxotrophic mutant of E. coli requiring diaminopimelic acid (DAP) (utilised as a recipient in genetic manipulation) and various strains of Clostridium possessing DAP in their cell wall. The presence of certain of these Clostridia in the gut provided the E. coli strain with enough DAP to enable it to become established. It is highly probable, though not yet proved, that synergy between strains is essential for building up the complex flora of a holoxenic animal. Depending on the extent to which pioneer strains become established, inhibitory factors disappear, growth factors appear, and the flora become diversified until a state of equilibrium governed by bacterial antagonism is reached, its expression depending on the host and its environment. M E T A B O L I C INTERACTIONS BETWEEN BACTERIAL STRAINS PRESENT I N T H E DIGESTIVE T R A C T This type of interaction does not imply a profound modification in the population levels of strains which interact. The toxinogenic strain of C. difficile studied by Corthier et al. (3) killed 100% of gnotoxenic mice, but failed to kill any when certain strains of E. coli or Bifidobacterium bifidum had been established previously in the gnotoxenic mice. However, these strains had no antagonistic effect on the C. difficile population. Assay of the concentration of cytotoxin formed by the target strain within intestinal contents revealed a pronounced diminution when the inhibitory strains were present in this ecosystem. This was a case of inhibition of cytotoxin production by C. difficile without a notable effect on its population level (Fig. 8). A similar phenomenon was observed by Duval-Iflah et al. (17) in gnotoxenic piglets first inoculated with a non-toxinogenic strain of E. coli and then with a toxinogenic strain 325 Clostridium difficile Mortality log10 n o . of C. d./g faeces C . : controls S.b.: treated * p < 0.001 vs controls (log10 Cytoxin titre dilution) a: mice which died b : mice which survived M o r t a l i t y rate, faecal colonisation by C. difficile a n d cytotoxin titre in g n o t o x e n i c mice t w o days after orogastric i n o c u l a t i o n of 5 x 10 C. difficile. T r e a t e d mice (S.b.) received unrestricted a m o u n t s of a solu tion containing 5 x 1 0 Saccharomyces boulardii per ml, starting four days before inoculation, while the controls (C) received plain w a t e r . Faecal colonisation by C. difficile was n o t influenced by t r e a t m e n t , b u t the cytotoxin titre was s o m e 1,000 times lower in surviving mice protected b y S. boulardii. T h e mortality r a t e levelled off after cessation of t r e a t m e n t (lasting for 10 days). By C o r t h i e r et al. (3). 7 9 FIG. 8 Example of metabolic interaction of E. coli. The non-toxinogenic strain protected the piglet without bringing about a major diminution in the population level of the toxinogenic strain. However, in these experiments the amount of toxin was not measured, and it was therefore impossible to verify that metabolic interaction took place. But there is also evidence of metabolic synergy. For example, methane-forming bacteria fail to produce methane within the ruminant digestive tract unless other bacteria are present to provide the necessary hydrogen and carbon precursors. In gnotoxenic chicks fed starch and lactose, Szylit et al. (30) found that a strain of Clostridium and a strain of Veillonella produced more volatile fatty acids in the presence of an amylolytic strain of Lactobacillus than in its absence. Production of lactic acid by the Lactobacillus and its subsequent utilisation by the strict anaerobes seems to provide a model of enhanced utilisation of dietary carbon substrates. Dabard and Dubos (5) demonstrated that the death of newborn hares was due to a synergic action between two toxinogenic Clostridia, C. difficile and C. perfringens. Sacquet et al. (29) described another type of metabolic synergy resulting from the production of a bacterial metabolite of ß-muricholic acid, one of two bile acids 326 produced by the liver of rats, mainly in the form of a taurine derivative. The metabolite in question, co-muricholic acid, differs from the ß form in the position of the hydroxyl group attached to the carbon 6 part of the molecule. It was produced by a strain of Clostridium when associated with a strain of Bacteroides. In this case, the Bacteroides hydrolysed tauro-ß-muricholic acid, the taurine derivative present within the rat's caecum, rendering it accessible to the enzyme system of Clostridium which deals with ß-muricholic acid. EFFECT OF T H E HOST A N D ITS D I E T O N T H E EXPRESSION OF B A C T E R I A L INTERACTIONS The fact that there are numerous differences in the composition of the gastro intestinal flora according to species of animal implies a major role for the host. However, very little is known about this role. It may be that the type of diet, the type of feed and the secretions and excretions of the host affect the early sequences of bacterial colonisation, and it is known that these differ from one animal species to another. The evolution of physiological and dietary parameters of the host, which also differ between species, probably influences the evolution of bacterial interactions, which favour some strains and exclude others. Many authors have observed that axenic mice are good recipients for bacterial flora from different sources (pig, rat, chick, man), in that many of the functions fulfilled by the flora in its original host are also carried out in recipient mice. Nevertheless, when a recipient mouse, reared in an isolator, is placed among a group of holoxenic mice, it soons acquires the flora of the group. This shows that the strains present in holoxenic mice are capable of exerting drastic and curative barrier effects against strains originating from other animal species. The work of Yurdusev et al. has demonstrated a host effect in the expression of the barrier effect produced by two inhibitory strains against a target strain of C. perfringens. This barrier effect was of the drastic type in gnotoxenic mice, but only permissive in rats harbouring the same inhibitory strains (in which the level of colonisation was the same), receiving the same feed. Another example of the host role is provided by Ducluzeau and Raibaud (14), in which a strain of S. flexneri was eliminated by a strain of E. coli when the Shigella strain had been present in gnotoxenic mice for just a week. After the Shigella strain had been present in gnotoxenic mice for three months, it was no longer eliminated by the E. coli strain. Thus within three months the host had selected a population of S. flexneri which differed from the original one. The effect of the diet on the expression of bacterial interactions is as little understood as the host effect, but is just as important. Sacquet et al. (29) demonstrated clearly that a change of diet could result in profound changes in bacterial metabolites within the gut, and consequently in bacterial interactions. To return to the experimental models devised by Yurdusev et al. (31), the simple act of sterilising a mouse diet in an autoclave instead of by irradiation altered the drastic barrier effect exerted by two inhibitory strains against a target strain and made it a permissive barrier. In this case the expression of the barrier effect was changed, and there was no direct effect of autoclaved feed on the population level of inhibitory strains, for these remained unchanged (Fig. 9). 327 Days P = Clostridium perfringens B = barrier strains (Clostridium C , Fusobacterium a n d Bacteroides B ) 2 • = g r o u p of rats receiving first an irradiated then an autoclaved diet O = g r o u p of rats receiving first an autoclaved then an irradiated diet FIG. 9 I n f l u e n c e o f t h e m e t h o d o f sterilising t h e diet o f g n o t o x e n i c rats o n t h e barrier e f f e c t e x e r t e d b y t h e t h r e e strictly a n a e r o b i c s t r a i n s o f Clostridium perfringens (by Yurdusev et al., 31) M E C H A N I S M S OF B A C T E R I A L I N T E R A C T I O N S The lack of fundamental knowledge is particularly striking in regard to the ways in which bacterial interactions occur in the gastro-intestinal tract. It is far easier to study these interactions within a culture tube or fermentor ! Mechanisms of synergy between bacterial strains are probably multiple, as two examples have shown. In the interaction between C. difficile and C. perenne, the mechanism is the hydrolysis of ß-aspartic-e-lysine by the former. In the case of interaction between a strain of Clostridium and a strain of Bacteroides, the mechanism is hydrolysis of the tauro-ßmuricholic amide bond. However, we are a long way from understanding all the synergy mechanisms which are doubtless involved in the sequences of establishment of bacteria in the gastro-intestinal tract, although it can be assumed that they involve 328 a disappearance of inhibitory factors, or the production of growth factors. Precise knowledge of these factors would be of great value in order to understand and perhaps modify at birth the phenomena which give rise to the complex flora of adults. We do not know much about the mechanisms of bacterial antagonism within the gastro-intestinal tract either. Do the various types of antagonism demonstrated in vitro occur in animals? The answer is probably no in most cases. Lactic acid is a powerful bactericide in vitro. In a monogastric animal provided with intestinal lactase, a small amount of lactic acid is produced in the large intestine only when the capacity of the small intestine to hydrolyse lactic acid is saturated. In the newborn inoculated with a strain of L. casei, L-lactic acid is present in the faeces, even though E. coli has eliminated L. casei. Many authors maintain that volatile fatty acids are responsible for bacterial antagonism both in vitro and in vivo. Yet when the mechanisms are studied in vivo, it is found that this does not apply. Thus Hudault et al. (24) demonstrated that there was no cause-effect relationship between the caecal content of volatile fatty acids and the elimination of S. typhimurium from the gastro-intestinal tract of mice and gnotoxenic chicks. The production of a non-absorbable antibiotic of the bacitracin type by a strain of Bacillus was demonstrated by Ducluzeau et al. (9) in the gut of gnotoxenic mice. It was sufficient to inhibit a sensitive strain. But when a Lactobacillus was introduced into the ecosystem, the Bacillus no longer produced antibiotic and the sensitive strain coexisted with it and the Lactobacillus. As far as we know, no strain which produces an antibiotic in vivo, detectable by standard gel diffusion tests, has as yet been isolated from the gastro-intestinal tract. Bacteriocin production is often invoked to explain intraspecific interactions. However, a plasmid-free strain of E. coli which can inhibit in vivo plasmid-carrying target strains is sensitive in vitro to the colicine produced by one of the target strains. The role of trypsin-resistant microcines has yet to be demonstrated. One of the more advanced hypotheses is that of Freter (22). He first showed that the barrier effect exerted by a collection of 100 inhibitory strains against a target strain of E. coli was reproducible in a system for continuous culture which he devised. Next he showed that within this system E. coli was no longer able to utilise growth factors present in the dialysed culture medium when hydrogen sulphide was present. Under such conditions, the population level of E. coli in the continuous culture dialysate was the same as in the digestive tract. When glucose was introduced into the dialysate, or when H S was eliminated by shaking, growth of E. coli resumed. Freter drew conclusions from these findings that he believed could explain many interactions occurring in vivo. In the presence of certain bacterial metabolites or in the presence of H S, certain strains were short of nutrients and their population level remained low in the gastro-intestinal tract. Nevertheless, this hypothesis does not explain the interactions described by Yurdusev et al. (31), which have not been reproduced in the continuous culture system of Freter. Such interactions have been reproduced in a static incubation system of faecal suspension from gnotoxenic mice. These in vitro experiments have shown that there was neither depletion of growth factors from the culture medium, nor the appearance of diffusible inhibitory substances. However, there was a highly significant inverse correlation between the number of live cells of the two inhibitory strains present in the suspension at the start of incubation, and the number of divisions which the target strain of C. perfringens could undergo during culture. A hypothesis developed from in vivo results which could be reproduced in vitro is that growth of the target strain becomes static in the presence of adequate numbers of live cells of the inhibitory strains. If this bacteriostasis is complete, the barrier effect is of the drastic type, and the target strain is eliminated at the same rate as spores of a strictly thermophilic 2 2 329 bacillus. The origin of this bacteriostasis may be the synergic production by the two inhibitory strains of one or more bacteriostatic substances, capable of reaching the target strain without accumulating in the gut. This hypothesis differs from that of Freter in regard to the nature of the bacteriostatic substances. They may be substances capable of attachment to specific receptors on the target strain, produced by inhibitory strains from substrates present in the large intestine. Here the distant prospect of "missile" drugs comes into view. CONCLUSION Bacterial interactions, which may be antagonistic or synergic, play a primordial role in maintaining the ecosystem of the host and the bacterial flora of its gastro intestinal tract. They are responsible for the complexity of this bacterial flora and the mechanisms under which, once established, it can oppose the establishment of bacterial strains ingested by the host every day. If such strains should be pathogenic bacteria, it is easy to grasp the value of continuous maintenance of the microbial barriers. An immediate application of this concept has been to examine the impact of antibiotics ingested by the host on its flora, in order to select those which upset the ecosystem least. No new antibacterial drug is now marketed without exhaustive studies in gnotoxenic animals of its impact on the gut flora. Of course, there is also a barrier effect against the non-pathogenic bacteria which arrive, sometimes in large numbers, in the digestive tract with food. This is particularly the case with lactobacilli in fermented foods. Such an input, even when massive, is not followed by lasting colonisation. This explains the stability of the complex flora of an adult, and the barrier effect is one of the elements contributing to the homeostasis of an individual. There is at present an obstacle to the multiplication of practical applications of our knowledge of the gut flora. This is the impossibility of implanting a given strain, the value of which has been determined from gnotoxenic models, into the dominant flora of a holoxenic individual. It provides an example of the adverse effect of a barrier. We have seen that such an implantation can succeed only at the moment of birth, which limits its value. One solution might be the continuous administration of large numbers of the useful bacterium. It would continue to be metabolised during transit, and could imitate the effect of an implanted flora. This could form a basis for certain "probiotics" in animal feeding. This type of effect has been demonstrated in human beings who ingest large amounts of Saccharomyces boulardii (an "ultrayeast") or lactic fermented products. But it is clear that the major advances will occur when we increase our knowledge of interaction mechanisms, thus leading to regulation of the ecosystem. Research in progress raises the hope of genetic localisation of the factors which confer on a given strain an ecological advantage, enabling it to persist in the dominant flora of a given host. The techniques of genetic manipulation may make it possible to transfer these characteristics to bacteria recognised as being the most useful, even though our lack of knowledge of the genetics of strict anaerobes will create technical difficulties. Finally, it is still true that much remains to be done in the field of gnotoxenics. Numerous theories, including those concerning the role of lactic enzymes and the role of Bifidobacterium in the gut flora, have been formulated from the results of experiments conducted in vitro. It still has to be proved that the same applies in vivo, 330 and this can be done by using gnotoxenic animals. There is always the risk that the theory may not correspond with reality. The only rewarding course is to identify the partners of in vivo bacterial interaction, and then to attempt to reproduce the interaction in vitro in order to elucidate its mechanism. 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