Download Bacterial interactions within the digestive tract

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).
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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.
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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
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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.
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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
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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.
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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.
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9
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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
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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,
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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
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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)
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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
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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)
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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
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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
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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
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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. This requires a more onerous
research effort, but sooner or later it will lead to an understanding of the true
mechanisms of bacterial interactions and, at a later stage, improvement in the
relationships between the host and its gastro-intestinal flora.
*
**
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