# Probiotic Bacteria Influence the Composition and  Function of the Intestinal Microbiota

Authors: Paul W. O'Toole; Jakki C. Cooney
Journal: Interdisciplinary Perspectives on Infectious Diseases
 (2008)

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## Abstract

Probiotics have a range of proposed health benefits for the consumer, which may include modulating the levels of beneficial elements in the microbiota. Recent investigations using molecular approaches have revealed a human intestinal microbiota comprising over 1000 phylotypes. Mechanisms whereby probiotics impact on the intestinal microbiota include competition for substrates, direct antagonism by inhibitory substances, competitive exclusion, and potentially host-mediated effects such as improved barrier function and altered immune response. We now have the microbial inventories and genetic blueprints to begin tackling intestinal microbial ecology at an unprecedented level of detail, aided by the understanding that dietary components may be utilized differentially by individual phylotypes. Controlled intervention studies in humans, utilizing latest molecular technologies, are required to consolidate evidence for bacterial species that impact on the microbiota. Mechanistic insights should be provided by metabolomics and other analytical techniques for small molecules. Rigorous characterization of interactions between the diet, microbiota, and probiotic bacteria will provide new opportunities for modulating the microbiota towards improving human health.


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## Body

## 1. Introductory Remarks

The history of microbiological
research has been dominated by investigations of the agents of human infectious
disease. Motivated by the desire to culture, characterize, and understand the
pathogenicity mechanisms of these organisms, several centuries of
microbiological research culminated in a broad range of antimicrobial
therapies, vaccines, and immunizations. In more recent years, similar
analytical methodologies have been applied to facilitate exploitation of
bacteria for industrial applications. Two related branches of microbiology—environmental
microbiology, and the study of intestinal commensals (a branch of the first in
purest terms)—lagged behind
until relatively recently. From the mid 1990s, a range of techniques allowed
environmental microbiologists to indentify soil microorganismsin situ, without resorting to
culture, based upon ribosomal small subunit RNA gene probes. A natural
extension of this approach was to sequence large numbers of cloned ribosomal
RNA gene amplicons, yielding catalogs of all the organisms (the microbiota)
present in complex samples. Latterly the field of metagenomics has provided
technical approaches to sequence large fractions of the entire microbial DNA
present in an ecological system. Coupled with the application of molecular
tools for studying commensal bacteria, many of which were originally developed
for studying pathogens, there is now an exciting nexus between technologies and
research foci whereby commensal bacteria may be studied in the context of
intestinal ecosystems. This review will summarize what is known about the effect
of introducing probiotic bacteria on the composition and activities of the microbiota,
with an emphasis on recent studies using culture-independent methods. The
likely mechanisms whereby commensals  exert their influence are discussed, and directions for future research are outlined.


## 2. The Concepts of Probiotics and Prebiotics

The notion that certain intestinal
microorganisms might benefit the host derives historically from suggestions by Metchnikoff and others that putrefying bacteria that contribute to toxification and aging could be
deliberately replaced by fermentative organisms [1, 2]. In this context, some of the
fermentative bacteria, Metchnikoff was referring to, are what we now consider
as probiotic. Probiotic bacteria are live microorganisms which when
administered in adequate amount confer a health benefit on the host [3, 4]. Many microorganisms that are
considered probiotic have been traditionally used to preserve food products by
fermentation, and are present in the food in varying numbers, along with their
fermentation end products and other metabolites. Thus another operational definition
of the term probiotic requires the organism in question to be “consumed in
adequate amounts” to confer a benefit [5]. The host benefits that have
been attributed to consumption of probiotic microorganisms are diverse (reviewed
in [3, 6–8]; some major examples are listed
in Table 1), and have been substantiated to different degrees. Probiotic
bacteria are now included in a wide range of consumer formulations including
yoghurts, drinks, capsules, and dietary supplements, and they represent a
significant element in the modern functional foods market. Organisms used as
probiotic agents are frequently members of the genera Lactobacillus or Bifidobacterium,
but Escherichia coli, Bacillus subtilis, Saccharomyces boulardii, and Enterococcus faecium are also employed, among others [9]. Thus, an organism employed
as a probiotic agent may not necessarily be part of what is considered the
“normal microbiota.” Tannock distinguishes between allochthonous and
autochthonous species [10, 11].
Autochthonous means bacteria
both present and replicating in situ
in the human GI tract, as distinct from transiently passing through
(allochthonous). Bacteria administered as probiotic agents are not necessarily
autochthonous to the consuming animal, and indeed some Lactobacillus species may only be autochthonous for certain human
individual subjects, and possibly not the majority of subjects (see below).
Rate of growth of allochthonous lactobacilli may be a critically limiting step
preventing their establishment [12]. With regard to developing
probiotic strains for exploitation, it may prove easier to identify beneficial
traits in species that are autochthonous to the human consumer, as consumer
acceptance is likely to be easier if the probiotic ingredient in a functional
food (a food product with benefit to the consumer over and above inherent
nutrition) was first cultured from humans.Table 1
Beneficial properties reported for probiotic bacteria.
Host benefitMicrobial trait implicatedReference1Immune modulationStimulation of immunityEnhance T-cell numbers and activity levels[13]Dampening of inflammationPromote anti-inflammatory cytokine production[14]Pathogen burden reductionCompetitive exclusion[15]Direct antagonism[16, 17]Uncharacterised[18, 19]Improved gut barrier functionPromote gut barrier integrity[20]Reduced cancer riskDetoxification of carcinogenic metabolites[21]Reduced atopic allergy symptomsSuppression of hypersensitivity[22]Reduced cardiovascular disease riskCholesterol reduction by deconjugation of bile salts[23, 24]Production of anti-hypertensive peptides[25]Alleviation of dietary intoleranceCatabolism of dietary ingredients[26]Enhanced nutrient valueVitamin and co-factor production[27]Alleviation of IBS2 symptomsNot defined[28, 29]1Sample reference for each trait. See main text for review references,
2Irritable bowel syndrome.Related to the consumption of probiotic agents is the notion of dietary
adjustment to stimulate bacterial growth. A prebiotic compound is defined as “a
nondigestible food ingredient that beneficially affects the host by selectively
stimulating the growth and/or activity of one or a limited number of bacteria
in the colon and thus improves host health” [30, 31]. It follows from this definition
that the bacteria capable of metabolizing prebiotics should be restricted to a
small number of beneficial species or strains (reviewed in [32]). In practice, prebiotic compounds
must also be refractory to host digestive processes, and the combined catabolic
activities of bacteria higher up in the gastrointestinal tract, so that
prebiotic compounds are often oligosaccharides towards which probiotic bacteria
produce specific hydrolases [33]. Prebiotics are commonly found in,
or extracted from, plant material including fruits, cereal, and vegetables, but
are also present in human milk and colostrum [6]. The best characterized prebiotics
include inulin, fructooligosaccharide, galactooligosaccharide, xylooligosaccharide,
isomaltooligosaccharide, and lactulose (reviewed in [32]). Unravelling the health benefits
of prebiotics is a challenging task, because these compounds have parallel
direct effects on the host, and potentially on multiple members of the
microbiota. For example, β-glucans are unbranched polysaccharides with (1-4)
and (1-3)-linked β-D glucopyranosyl units, that are recognized as important
dietary ingredients (reviewed in [34]). β-glucans are components of plant
cell walls, and are abundant in the endosperm of cereals such as barley and
oatmeal. Consumption of β-glucans has attendant health benefits that are
recognized by health and regulatory bodies in several jurisdictions including
the US
[35]. These benefits include lowering of
blood cholesterol and lipoprotein [36], lowering of postprandial glucose
and insulin responses [37], and enhancement of antitumor
monoclonal antibodies [38]. Supplementation of mammalian diet
with β-glucan, or modification by prehydrolysis of in vitro
bacterial growth
medium, leads to increased
numbers and proportions of lactobacilli or bifidobacteria [39–41]. Furthermore, β-gluco-oligomers
promoted the growth of L. rhamnosus GG [40].
Barley supplementation of rat
diet [39] led to an increase in Lactobacillus numbers, a decrease in Bacteroides and coliforms, and an increase in the production
of butyrate. Butyrate is an important energy source, signalling metabolite, proliferation
stimulus for normal colonic epithelial cells, and anti-proliferative signal for
neoplastic colonocytes [42, 43], suggesting a potential direct
benefit from dietary ingredients or prebiotics that promote growth of
clostridia.


## 3. The Normal Microbiota of Humans and Animals

Until recently, the composition of the microbiota was examined by
relatively insensitive techniques. Culturing the bacteria was unrepresentative,
because a large proportion of the bacteria do not grow on standard laboratory
media [44]. Analysis by temperature gradient
gel electrophoresis provided one of the earliest insights into the uncharted
complexity of the microbiota [45]. Using denaturing gradient gel
electrophoresis (DGGE) of 16S rRNA gene amplicons, the same group later showed
that the colonic mucosal microbiota and faecal microbiota were different, and
the colonic mucosal microbiota was likely dependent on host factors [46]. Meanwhile fluorescent
hybridization of probes for 16S rRNA genes was being applied to determine
species identities, numbers, and proportions of intestinal bacteria [47, 48], exemplified by the studies of
Dore, Blaut and colleagues [49, 50]. These analyses highlighted
extensive inter-individual variation at phylotypes level (among northern
Europeans) [49], and some correlations of microbiota
with age, gender, and geographic origin but which varied between countries [50].Our current understanding of the intestinal
microbiota (reviewed in [51, 52]) has been significantly shaped by
culture-independent methods, in particular the sequencing of 16S rRNA gene amplicons,
either from clone libraries or direct pyrosequencing of the PCR product. A
consensus appears to be emerging in the literature of somewhere between 800 and
1000 bacterial phylotypes being present in the healthy human intestine [52]; the evidence for which will be
selectively presented here. A relatively small-scale investigation by Benno and colleagues in 2003
revealed an unexpectedly high number of novel phylotypes in 240 partially
sequenced 16S rRNA gene amplicons clones derived from six elderly individuals [53]. In a pioneering study, Relman
and colleagues applied the 16S rRNA molecular inventory-based approach, at a
much larger scale than previously published, on samples from both colonic sites
and faeces [54]; strikingly, the majority of the
sequences derived
corresponded to uncultivated species and novel microorganisms. The human
stomach, previously considered sterile except for infections with Helicobacter pylori, was revealed by 16S
rRNA gene library sequencing to be well populated by bacteria, based on
detection of 128 bacterial phylotypes from 23 gastric endoscopy samples [55]. Gill and colleagues [56] sequenced not just 16S rRNA genes,
but also randomly cloned bacterial DNA—so-called metagenomics, a concept developed for
environmental community analysis [57]. Gill et al. showed by this metagenomic approach that the bacteria in
the gut significantly expand the metabolic capabilities of the human gut [56]. By generating two to three 454
pyrosequencing runs per mouse cecum, Gordon and colleagues showed the existence
of an obesity-associated gut microbiome with increased capacity for energy
harvest [58]. Significantly, this balance of the
microbiota was borne out when investigating obese humans [59], showing a seminal link between
human obesity and changes in the microbiota. Furthermore, the complexity of the
microbiota in humans and 59 other mammalian species was shown to be linked to
phylogeny (of the mammal) and the composition of the diet [60]. Analysis of the metabolic
capability likely conferred by the microbial metagenome recently revealed 237
gene families commonly enriched in adult-type and 136 families in infant-type
microbiomes [61]. Thus, any consideration of the
effect of probiotics on the intestinal metagenome should ultimately include
analysis of the downstream effects upon the host of impacting on this metabolic
capability. A more fundamental consideration is that the genera whose members
are among the most commonly employed probiotics—Bifidobacterium and Lactobacillus—are not present
in the human gastrointestine at the high levels traditionally expected based on
culture-based approaches, being represented by 20 phylotypes (ca. 2%) and 36
phylotypes (ca. 3.6%), respectively [52]. Thus if probiotic bacteria impart
health benefits to the host under “natural conditions,” that is, in
individuals who have normal nonmanipulated numbers of probiotic bacteria, they accomplish
this despite being at much lower numbers than are achieved by consumption of
probiotic products.


## 4. Probiotics and Their Effect upon the Microbiota

There have been relatively few
studies which have rigorously characterized the effect upon the whole microbiota
of administering probiotic cultures, and until recently, all such studies
applied targeted analysis of specific groups of bacteria. In one of the
earliest investigations, Tannock and colleagues observed transient and modest
fluctuations in lactobacillus and bifidobacterium numbers following consumption
of a probioticL. rhamnosus strain
DR20 [62]. Lactobacilli and enterococci
were detected more frequently (among 10 subjects) and in higher numbers during
consumption. Interestingly, the presence of stable populations of lactobacilli
before the administration period precluded long-term colonization by the
administered probiotic strain [62]. Most subjects ceased
shedding the probiotic strain in faeces soon after its consumption stopped, but
the L. rhamnosus strain remained
detectable in faeces of one subject over 2 months after the test period. These
data suggest inter-host variables such as bacterium-host or bacterium-diet
interactions.Probiotics
and prebiotics are commonly applied in companion animals and production animals
[63, 64], and there have been some
studies of effects upon the microbiota. Administration of a cocktail containing
lactobacilli, bifidobacteria, enterococci, and pediococci improved weight gain
in broiler chickens, which was associated with an increase in numbers of Bifidobacterium spp., Lactobacilli, and Gram-positive cocci [65]. Administration of a
probiotic Enterococcus faecium strain
reduced E. faecalis numbers in the
intestines of weanling piglets, but total numbers of E. faecium remained unchanged, suggesting that the administered
strain had displaced part of a fixed number of niche sites occupied by the same
species [66]. Many investigations have
been published describing the effects of probiotic bacteria on human pathogens
(reviewed in [67]), some of which are normal
components of production animal microbiota. Enterobacteriaceae numbers were reduced
when a cocktail of two Lactobacillus strains was administered to pigs [68], and a five-strain probiotic
combination reduced Salmonella enterica serovar Typhimurium shedding in
pigs [69]. Although data from small
animal models for human probiotic strains must be interpreted with caution [70], it was interesting to note
from a recent study that administration of L.
casei and L. plantarum affected
the diversity of murine intestinal lactobacilli, but not the overall bacterial
community structure [71]. There was an increase in the
number of lactobacilli related to the acidophilus complex in the inoculated mice. These animal models provide an opportunity for
determining the effect of probiotic administration on the entire microbiota but
must ultimately be repeated in humans if that species is the desired host.Studies
in humans are currently few in number, and are often focused in nature. For
example, consumption of a commercial probiotic yoghurt reducedClostridium difficile-related diarrhoea
in hospitalized patients, but effects on the broader microbiota were not
studied [18]. Alterations in the fecal
microbiota have been reported in irritable bowel syndrome (IBS) [72, 73]. However, administration of a
multispecies probiotic supplementation which alleviated IBS had negligible effect
upon the composition of microbiota as measured by quantitative PCR with
group-specific primers [74]. However, this approach may
have missed changes in microbial composition within these groups. A follow-up
study reported stabilization of the microbiota over time [75], which was related to
amelioration of symptomatology that was absent from the placebo control group.
Alterations of the human intestinal microbiota have also been reported in
inflammatory bowel disease (IBD) [76–78]. Given the clinical impetus
to find simple non-medicinal solutions to IBD and IBS, one can anticipate
renewed vigor in studies of probiotic bacteria as agents for microbiota
modulation in these subjects. Probiotics also appear to be efficacious as
adjunct therapy for infectious diarrhea, with a recent meta-analysis revealing
reduction in risk and duration of diarrhea [79]. Most of the 23 studies
included in this analysis were descriptive rather than investigative of the
microbiological aspects, and future determination of the effects on the
microbiota wrought by probiotic intervention will be very informative. As
recently as 2006, the effect of probiotic administration in humans was still
being followed by bacteriological culture, but as concluded by the authors of
one such study, there was a clear case for culture-independent molecular
methods to be applied instead [80]. Community profiling by DGGE
showed that lactulose increased the levels of Bifidobacterium adolescentis in subjects consuming the prebiotic
lactulose, whereas the probiotic yeast S.
boulardii did not cause any significant universal changes in DGGE profiles [81].


## 5. Mechanisms of Probiotic Impact on the Microbiota

Figure1 shows a
schematic overview of the potential mechanisms whereby probiotic
micro-organisms might influence the intestinal microbiota. Consumption of probiotic cultures may modulate
the microbiota or change its metabolic properties by competition for nutritional
substrates. Gordon and colleagues have used transcriptional microarrays to show
that introducing a probiotic into the mouse gut changes the way the endogenous microbiota
metabolize the diet [82]. When germ-free mice that had been
monoassociated with Bacteroides
thetaiotaomicron were challenged with Bifidobacterium
animalis or Lactobacillus
casei, both interventions caused shifts in the gene expression pattern of
the B. thetaiotaomicron genome [82]. These differentially
expressed gene sets (i.e., in response to the two probiotics) did not overlap,
emphasizing that different probiotics elicit different responses. However, many
of the genes in B. thetaiotaomicron whose expression
was altered by presence of either probiotic strain were related to expansion of
the carbohydrate metabolizing capability of B. thetaiotaomicron. Thus, one of the ways in which
probiotics can impact upon the composition of the microbiota is apparently by
competing with them for substrate availability, and by altering the dynamics of
carbohydrate utilization by individual microbiota components. This competition
is probably not restricted to the intestine, since recent evidence indicates
that oral Bifidobacterium strains (B.
adolescentis) reduce vitamin K concentration, and may thus compete with Porphyromonas gingivalis in the
oral cavity [83].Figure 1
Schematic diagram illustrating potential or known mechanisms
whereby probiotic bacteria might impact on the microbiota. These mechanisms
include (1) competition for dietary ingredients as growth substrates, (2) bioconversion
of, for example, sugars into fermentation products with inhibitory properties, (3)
production of growth substrates, for example, EPS or vitamins, for other bacteria,
(4) direct antagonism by bacteriocins, (5) competitive exclusion for binding
sites, (6) improved barrier function, (7) reduction of inflammation, thus
altering intestinal properties for colonization and persistence within, and (8)
stimulation of innate immune response (by unknown mechanisms). IEC: epithelial cells,
DC: dendritic cells, T:T-cells. For further details, see main text.The application of
metabolic profiling methods to animal models has suggested another indirect way
in which probiotic bacteria might impact on the microbiota, namely, by
production of a significantly different microenvironment due to a diverse range
of metabolic pathway outcomes. In a recent study using germ-free mice colonized
by human baby microbiota and exposed to two lactobacillus strains, Nicholson
and colleagues observed microbiome modification, measured by selected culture regimes
[84]. This was accompanied by
changes in cecal concentrations of short-chain fatty acids, and marked changes
in fecal levels of diverse metabolites including choline, acetate, ethanol, a
range of putative N-acetylated metabolites (NAMs), unconjugated bile acids
(BAs), and tauro-conjugated bile acids. While a natural focus of these studies
is the effect of these metabolites upon the host [85], it is likely that such gross
changes in metabolic profile also impact upon intestinal microbiota
composition. As noted in Table 1, some probiotic bacteria also produce vitamins
[27], enhanced availability of
which may modulate the microbiota. In addition, exopolysaccharide produced by
probiotics including lactic acid bacteria [86] could act as a growth
substrate for selected components of the microbiota (see Figure 1).Probiotic bacteria
probably also impact on the general microbiota by direct antagonism. It has
been shown in several recent studies that they can modulate numbers of single
model organisms in experimental systems. For example, probioticL. salivarius strains inhibit the growth of H. pyloriin vitro in a strain-dependent manner [17], by mechanisms involving
lactic acid secretion, and another as yet uncharacterized mechanism (K. A. Ryan
and P. W. O’Toole, unpublished). Intestinal L. salivarius strains are distinguished by
production of a broad-spectrum bacteriocin Abp118 [87], but this is not likely to
contribute to antagonism to Gram-negative bacteria like H. pylori. However, production of this bacteriocin Abp118 was
identified as the mechanism whereby L. salivarius UCC118 eliminated Listeria monocytogenes infection in a
murine model, providing the first definitive mechanism for anti-infective
activity of a probiotic bacterium in
vivo [16]. Interestingly, both the
wild-type strain UCC118 and a bacteriocin-negative derivative were equally able
to suppress Salmonella Typhimurium
infection in the mouse model, suggesting that broader antimicrobial effects on
the Gram-negative components of the microbiota may occur. From an opposite perspective,
production of a bacteriocin-like substance by vaginal enterococci has been
linked to reduction in levels of commensal lactobacilli that is linked to
vaginosis [88]. Natural competition between
commensals and opportunistic pathogens may therefore be mediated by mechanisms
such as bacteriocin production, that can be exploited for using probiotics to
modulate the microbiota. Competitive exclusion (see Figure 1), whereby adherent
probiotic species occlude access of members of the microbiota to the epithelium
[89, 90], represents another way of
modulating the microbiota, although strong evidence for this occurring
in vivo is lacking.The most subtle
effects wrought by probiotics on the microbiota are potentially those that
operate by indirect mechanisms involving the host. Improvement of the
intestinal epithelium barrier function [91] might theoretically, for
example, impact on efficiency of invasion of pathogens, severity of subclinical
tissue damage, and release rates of host-derived micronutrients (see Figure 1),
that could translate into impacts on the microbiota. In an analogous manner,
pathological changes in intestinal epithelium might also favor growth of
certain members of the microbiota, if inflamed or damaged epithelial cells
differentially affect the microbiota. It is well established that some probiotics
can suppress inflammation by inhibiting proinflammatory cytokine production [92–94], and although the molecular
basis for this is not currently understood for probiotics, mechanisms and
molecules have recently been identified in commensals and pathogens [14, 95]. Reduction in gut
inflammation by probiotics could plausibly alter the gut environment
sufficiently to impact on the microbiota. Furthermore, some probiotic bacteria
have been reported to stimulate the innate immune system both in animal models
and in elderly subjects [96, 97], by an unknown mechanism.
Administration of probiotic bacteria could thus bolster innate immune activity
against transient pathogens, or non-commensal elements in the microbiota,
leading to subtle changes in long-term overall composition. However, more
studies are required to substantiate the mechanisms in the probiotic-host
interactions, and to investigate if they do in fact impact on the microbiota.


## 6. Knowledge Gaps and Concluding Remarks

There has been a rapid recent
accumulation of sequence-based information on the composition of the gut
microbiota. However, for pragmatic reasons of sample collection facility, this
is largely based on fecal analysis, and the microbiota of the colon and small
intestine will be different from feces. Studies of the small intestine are
particularly warranted because probiotics are proportionally more numerous
there, and may exert significant biological activity at this site.There is adequate
information in the literature to support the hypothesis that administration of
probiotic cultures in high doses to human subjects will impact on the
intestinal microbiota. A comprehensive intervention study, supporting this
hypothesis by deep compositional and functional metagenomics approaches, and
supplemented by metabolomics, is not currently available (June 2008). In this
hypothetical study, mechanisms whereby changes in the microbiota that were achieved could
be inferred to a degree by global transcriptional analysis, but definitive linkages
between bacterial gene products and effects upon the microbiota could be
impossible to establish because of the regulatory issues surrounding human
trials with genetically modified organisms. As noted above, proof of principle
may be established in animal models, but ultimately these studies must be
validated in human subjects. There remains the intriguing question of the role,
if any, of the relatively small numbers of potentially probiotic organisms as
part of the microbiota in ostensibly healthy individuals. Do these organisms
contribute to maintenance of health—or avoidance of
disease? Is the level of candidate probiotic organisms in the microbiota
critical, and does its importance vary with age? As noted above, there is
reasonable evidence that changes in the microbiota accompany disease states
like IBD and IBS, conditions whose prevalence increases with aging. There are attractive
hygiene-related hypotheses suggesting that depletion of probiotic commensal microbiota
in early life may be responsible for the dramatic rise in diseases involving
immune dysregulation [98]. The challenge now is to rigorously
tackle the interplay of diet, microbiota, and host factors in tractable
experiments that will elucidate the key elements in determining outcomes of
this interplay, and allow its manipulation.



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