# Probiotics and Gastrointestinal Infections

Authors: Robert A. Britton; James Versalovic
Journal: Interdisciplinary Perspectives on Infectious Diseases
 (2008)

---

## Abstract

Gastrointestinal infections are a major cause of morbidity and mortality
worldwide, particularly in developing countries. The use of probiotics to prevent and
treat a variety of diarrheal diseases has gained favor in recent years. Examples where
probiotics have positively impacted gastroenteritis will be highlighted. However, the overall
efficacy of these treatments and the mechanisms by which probiotics ameliorate gastrointestinal
infections are mostly unknown. We will discuss possible mechanisms by which probiotics could have a
beneficial impact by enhancing the prevention or
treatment of diarrheal diseases.


---

## Body

## 1. Introduction

Within the microbiota, individual
bacteria containing important genes may benefit the host in different ways. As
one considers the vast community of commensal microbes, subsets of these
organisms may have important physiologic benefits for the host in the context
of human nutrition and host:microbe interactions. Probiotics may stimulate
immunity, regulate immune signaling pathways, produce antipathogenic factors,
or induce the host to produce antipathogenic factors. Probiotics may produce
secreted factors that stimulate or suppress cytokines and cell-mediated
immunity. These factors may also interfere with key immune signaling pathways
such as theNF-κB and MAP kinase cascades. Probiotics may produce factors that
inhibit pathogens and other commensal bacteria, effectively enabling these
microbes to compete effectively for nutrients in complex communities. Microbes
that produce antipathogenic factors may represent sources of novel classes of
antimicrobial compounds, and these factors may be regulated by master
regulatory genes in particular classes of bacteria. Microbes can also regulate
signaling pathways in immune cells that result in the production of
antimicrobial factors by mammalian cells, effectively resulting in remodeling
of intestinal communities and prevention or treatment of infections.Gastrointestinal
infections are a major cause of morbidity and mortality worldwide. Studies
conducted in 2006 found that, globally, severe diarrhea and dehydration are
responsible each year for the death of 1,575,000 children under the age of
five. This represents 15% of the 10.5 million deaths per year of children in
this age group [1]. According to recent
estimates, acute gastroenteritis causes as many as 770,000 hospitalizations per
year in the United States [2]. Enteric pathogens include
viruses (rotaviruses, noroviruses) and bacteria such as different strains of
pathogenic Escherichia coli,
toxigenic Clostridium difficile, Campylobacter jejuni, and Vibrio cholerae. These
pathogens produce different types of toxins that can cause severe or
life-threatening dehydration and diarrhea. Despite medical advances in
diagnosis and treatment, the percent and number of hospitalized pediatric
patients less than 5 years of age with severe rotavirus infection significantly
increased when a recent time period (2001–2003) was compared to an earlier
time period (1993–1995) [3]. In addition to the typical
pattern of acute gastroenteritis, infectious agents such as enteropathogenic E. coli (EPEC) may cause persistent,
chronic diarrhea in children lasting longer than 1 week [4]. Such persistent infections may increase the risk of dehydration
and long-term morbidities. Importantly, the relative contributions of EPEC and
other bacterial pathogens to disease remains controversial to some extent. A recent
study highlighted that increased relative risk of gastrointestinal disease in
children was only demonstrable for enteric viruses [5].Recent studies have highlighted
long-term morbidities associated with gastroenteritis. Early childhood diarrhea
predisposes children to lasting disabilities, including impaired fitness,
stunted growth, and impaired cognition and school performance [6].
Along with this data, new research on maternal and child undernutrition
reported in The Lancet in January
2008 links poor nutrition with an increased risk for enteric infections in
children. Furthermore, irritable bowel syndrome (IBS), a costly and difficult
to treat condition that affects 20% of the United States population [7], has medical costs of up to
$30 billion per year, excluding prescription and over-the-counter drug costs [8]. IBS is precipitated by an
episode of acute gastroenteritis in up to 30% of all cases in prior studies [9]. Therefore, preventing or
treating acute gastroenteritis before long-term sequelae develop would
drastically reduce hospitalizations, disability-adjusted life years, and both
direct and indirect medical costs.Accurate diagnosis of acute
gastroenteritis is an ongoing challenge even in sophisticated academic medical
centers. In a pediatric patient population exceeding 4,700 children, less than
50% of stool samples that underwent complete microbiologic evaluation yielded a
specific diagnosis [10].
Enteric viruses represented the predominant etiologic agents in acute gastroenteritis
in children less than 3 years of age, and bacteria caused the majority of cases
of acute gastroenteritis in children older than 3 years of age [10]. The diagnostic challenges
with enteric viruses include the relative paucity of stool-based molecular or
viral antigen tests and the inability to readily culture most enteric viruses.
Bacterial pathogens may be difficult to identify (such as most strains of
disease-causing E. coli) because of the lack of specific assays
for these infections. The relative insensitivity of stool-based toxin assays
for the detection of toxigenic C.
difficile precludes accurate diagnosis. In a children’s hospital setting,
combination toxin antigen testing yielded sensitivity below 40% in pediatric
patients (J. Versalovic, unpublished data). The introduction of new molecular
assays for real-time PCR detection of toxin genes directly in stool has
markedly improved the ability to diagnose antimicrobial-associated diarrhea and
colitis due to toxigenic C. difficile [11]. In addition,
approximately 15–25% of cases of antimicrobial-associated diarrhea are caused
by C. difficile. The prevalence of
antimicrobial-associated diarrhea and gastrointestinal disease highlights the
importance of alternatives to antibiotic strategies for treatment. Furthermore,
antibiotics have limited utility for the treatment of gastroenteritis in
general. Antimicrobial agents are not generally recommended as prevention
strategies because of the problems of antibiotic resistance and
antimicrobial-associated disease. Thus, instead of suppressing bacterial
populations with antibiotics, can probiotics be used to remodel or shift microbial
communities to a healthy state [12]?


## 2. Probiotics

### 2.1. The Need for Mechanistic Details of Probiotic Action

The use
of probiotics to prevent and treat a wide variety of conditions has gained
favor in the past decade. This is in part due to a need to find alternatives to
traditional therapies such as antibiotics as well as the lack of good
treatments for GI ailments. While there are increasing reports of the efficacy
of probiotics in the treatment of diseases such as pouchitis [13, 14],
diarrhea [15–17],
and irritable bowel syndrome [18], the scientific basis for the
use of probiotics is just beginning to be understood. We will focus on the
potential applications for probiotics in the treatment of diarrheal disease. Several
examples will highlight how probiotics may be selected for and utilized against
pathogens causing gastroenteritis.The concept of using probiotic
microorganisms to prevent and treat a variety of human ailments has been around
for more than 100 years [19].
With the rise in the number of multidrug resistant pathogens and the
recognition of the role that the human microbiota plays in health and disease,
a recent expansion in the interest in probiotics has been generated. This
phenomenon is apparent in both the numbers of probiotic products being marketed
to consumers as well as the increased amount of scientific research occurring
in probiotics. Although many of the mechanisms by which probiotics benefit
human beings remain unclear, probiotic bacteria are being utilized more
commonly to treat specific diseases.Several definitions of what
constitutes a “probiotic” in the literature have been formulated. For this
review, we use the definition derived in 2001 by the Food and Agricultural
Organization (FAO) and the World Health Organization (WHO)—“Probiotics are
live microorganisms which when administered in adequate amount confer a health
benefit on the host.”  [20].
This definition is the currently accepted definition by the International
Scientific Association for Probiotics and Prebiotics (ISAPP)
(http://www.isapp.net/).


### 2.2. Antipathogenic Activities

Perhaps
the most important scientific question regarding the use of probiotics in
medicine is the identification of mechanisms by which probiotics impact human
health. Several mechanisms have been implicated but most have not been
experimentally proven (Figure1). Here, we discuss possible mechanisms that are
relevant for the treatment of diarrheal diseases. We will highlight research
examples that support these putative mechanisms whenever possible.Figure 1
Probiotics and Beneficial Effects in the Intestine. Depiction of the interactions between beneficial
bacteria (left side), their secreted factors, pathogens, and the intestinal
mucosa (right side). Potential beneficial effects of probiotics are listed.
Only two host cell types are shown, intestinal epithelial cells and macrophages
although other cell types including dendritic cells, lymphocytes,
myofibroblasts, and neutrophils comprise the intestinal mucosa. The arrows
indicate the release and possible distribution of secreted factors derived from
probiotics.


### 2.3. Stimulation of Host Antimicrobial Defenses

Many probiotics have been shown to
produce antipathogenic compounds ranging from small molecules to bioactive
antimicrobial peptides. Most of these studies have focused on the in vitro
susceptibility of pathogens to products secreted by probiotic bacteria. In most
cases, the ability of an antimicrobial compound secreted by a probiotic
organism to inhibit the growth of a pathogen in vivo has not been demonstrated.
Conceptually, an antimicrobial compound produced by an organism would need to
be produced at a high enough level and in the right location in the intestinal
tract to exert a strong effect on a pathogen in vivo.An elegant proof of principle for
direct action of a probiotic-produced antimicrobial against a pathogen was
recently reported by Corr  et al. who demonstrated that production of the bacteriocin
Abp118 byLactobacillus salivarius was
sufficient to protect mice from disease by infection with Listeria monocytogenes [21].
To prove the action of the bacteriocin was directly responsible for the
protection of the mice, they generated a L.
salivarius strain that was unable to produce Abp118 and showed that this
mutant was incapable of protecting against L.
monocytogenes infection. Notably, they were able to express a gene that
confers immunity to the Abp118 bacteriocin within L. monocytogenes and showed that this strain was now resistant to
the probiotic effect of L. salivarius within
the mouse. This study provided clear evidence that a probiotic-derived
bacteriocin could function directly on a pathogen in vivo.


### 2.4. Pathogen Exclusion via Indirect Mechanisms

In addition to
producing antimicrobial compounds that act directly on pathogens, probiotics
may stimulate host antimicrobial defense pathways. The intestinal tract has a
number of mechanisms for resisting the effects of pathogens including the
production of defensins [22].
Defensins are cationic antimicrobial peptides that are produced in a number of
cell types including Paneth cells in the crypts of the small intestine and
intestinal epithelial cells. A deficiency in alpha-defensin production has been
correlated with ileal Crohn’s disease [23, 24]. Tissue
samples from patients with Crohn’s disease showed a lower level of
alpha-defensin production and extracts from these samples exhibited a reduced
ability to inhibit bacterial growth in vitro. Moreover, some pathogenic
bacteria have evolved mechanisms to inhibit the production or mechanism of
action of defensins (reviewed in [25]).Probiotics may act to stimulate
defensin activity via at least two mechanisms. First, probiotics may stimulate
the synthesis of defensin expression. This has been demonstrated for human beta
defensin 2 (hBD-2), whose expression is upregulated by the presence of several
probiotic bacteria via the transcription factorNF-κB [26, 27]. The
implication is that probiotic strains with this capability would strengthen
intestinal defenses by increasing defensin levels. This effect is also observed
with certain pathogenic bacteria and thus is not a specific property of
probiotic bacteria. Second, many defensins are produced in a propeptide form
that must be activated via the action of proteases. One well-characterized example
is the activation of the murine defensin cryptdin (an alpha-defensin that is
produced by Paneth cells) by the action of matrix metalloprotease 7 (MMP-7) [28]. Mice
defective for MMP-7 are more susceptible to killing by Salmonella. Evidence indicates that bacteria can stimulate the
production of MMP-7 in the intestine [29]. Thus,
one mechanism in which probiotics could participate in activating defensins is
by stimulating the production of MMPs in the intestinal tract. Alternatively,
probiotics could produce proteases that themselves activate defensins in the
intestinal lumen. Although there is no evidence yet to support this mechanism, a
subset of lactobacilli and streptococci encode MMP-like proteins in their
genomes (R. Britton, unpublished observation). These MMPs are not found in any
other bacteria and thus it will be interesting to determine what effect they
have on host cell function.


### 2.5. Immunomodulation

Rather than  directly inhibiting the growth or
viability of the pathogen, probiotics may compete for an ecological niche or,
otherwise, create conditions that are unfavorable for the pathogen to take hold
in the intestinal tract. There are many possible mechanisms for how pathogen
exclusion may take place. First, several probiotics have been demonstrated to
alter the ability of pathogens to adhere to or invade colonic epithelial cells
in vitro, for example, see [30, 31]. Second, probiotics could sequester essential
nutrients from invading pathogens and impair their colonization ability. Third,
probiotics may alter the gene expression program of pathogens in such a way as
to inhibit the expression of virulence functions [32].
Lastly, probiotics may create an unfavorable environment for pathogen
colonization by altering pH, the mucus layer, and other factors in the local
surroundings. It is important to note that although many of these possible
effects have been demonstrated in
vitro, the ability of probiotics to exclude pathogens in vivo remains to
be proven.


### 2.6. Enhancing Intestinal Barrier Function

Probiotics may have strain-dependent effects on the immune system. Different
strains representing differentLactobacillus species demonstrated contrasting effects with respect to proinflammatory
cytokine production by murine bone marrow-derived dendritic cells [33].
Specific probiotic strains counteracted the immunostimulatory effects of other
strains so that probiotics have the potential to yield additive or antagonistic
results. Interestingly, in this study, the anti-inflammatory cytokine IL-10 was
maintained at similar levels [31].
Different probiotic Lactobacillus strains
of the same species may also yield contrasting effects with respect to
immunomodulation. Human breast milk-derived Lactobacillus
reuteri strains either stimulated the key proinflammatory cytokine, human
tumor necrosis factor (TNF), or suppressed its production by human myeloid
cells [34].
The mechanisms of action may be due, not surprisingly, to contrasting effects
on key signaling pathways in mammalian cells. Probiotic strains such as Lactobacillus rhamnosus GG (LGG) may
activate NF-κB and the signal transducer and activator of transcription (STAT)
signaling pathways in human macrophages [35]. In
contrast, probiotic Lactobacillus strains may suppress NF-κB
signaling [36, 37] or MAP kinase-/c-Jun-mediated signaling [34].
Stimulation of key signaling pathways and enhancement of proinflammatory
cytokine production may be important to “prime” the immune system for defense
against gastrointestinal infections. Conversely, suppression of immune
signaling may be an important mechanism to promote homeostasis and tolerance to
microbial communities with many potential antigens, and these immunosuppressive
functions may promote healing or resolution of infections.


### 2.7. Why Understanding Mechanisms Is Important?

The disruption of
epithelial barrier function and loss of tight junction formation in the
intestinal epithelium may contribute to pathophysiology and diarrheal symptoms
observed during infection with certain pathogens [38, 39]. Loss of
tight junctions can lead to increased paracellular transport that can result in
fluid loss and pathogen invasion of the submucosa. Pathogens may secrete
factors such as enterotoxins that may promote excessive apoptosis or necrosis
of intestinal epithelial cells, thereby disrupting the intestinal barrier.
Enteric pathogens may also cause effacing lesions at the mucosal surface due to
direct adherence with intestinal epithelial cells (e.g., EPEC). In contrast,
probiotics have been reported to promote tight junction formation and
intestinal barrier function [40, 41]. Although
the mechanisms of promoting barrier integrity are not well understood,
probiotics may counteract the disruption of the intestinal epithelial barrier
despite the presence of pathogens. Probiotics may also suppress toxin
production or interfere with the abilities of specific pathogens to adhere
directly to the intestinal surface. As a result, pathogens may have a
diminished ability to disrupt intestinal barrier function.


### 2.8. Important Considerations for the Use of Probiotics: Strain Selection and Microbial Physiology

An important
challenge in the field of probiotics is the identification of genes and
mechanisms responsible for the beneficial functions exerted by these microbes.
Successful identification of mechanistic details for how probiotics function
will have at least three important benefits. First, understanding mechanisms of
action will provide a scientific basis for the beneficial effects provided by
specific microbes. These breakthrough investigations will help move probiotics
from the status of dietary supplements to therapeutics. Second, understanding
mechanisms of probiosis and the gene products produced by probiotics will allow
for the identification of more potent probiotics or the development of
bioengineered therapeutics. As an example, the anti-inflammatory cytokine IL-10
was postulated to be a potential therapeutic for the treatment of inflammatory
bowel disease. To test this hypothesis, a strain ofLactococcus lactis engineered to produce and secrete IL-10 was
constructed and demonstrated to reduce colitis in a murine model [42].
Early clinical trials in patients with inflammatory bowel disease indicate some
relief from symptoms when treated with the IL-10 overproducing strain. Third,
the identification of gene products that are responsible for ameliorating
disease will allow researchers, industry, and clinicians to follow the production
of these products as important biomarkers during probiotic preparation. As
discussed below, the physiological state of microbes can be crucial to the
functions of probiotics. Thus, it will be important to be able to follow the
production of important bioactive molecules when culturing and processing
probiotics for applications in animals and humans.


### 2.9. Probiotics and Diarrhea

Probiotics
are considered to be living or viable microorganisms by definition. Unlike
small molecules that are stable entities, probiotics are dynamic microorganisms
and will change gene expression patterns when exposed to different
environmental conditions. This reality has two important implications for those
who choose to use these organisms to combat human or animal diseases. First,
probiosis is a strain-specific phenomenon. As defining a bacterial species is
challenging in this age of full genome sequencing, it is clear that probiotic
effects observed in vitro and in vivo are
strain specific. For example, modulation of TNF production by strains ofLactobacillus reuteri identified strains
that were immunostimulatory, immunoneutral, and immunosuppressive for TNF
production [34, 43].
These findings highlight the strain-specific nature of probiotic effects
exerted by bacteria. Thus, it is important for research groups and industry to
be cautious with strain handling and tracking so that inclusion of correct
strains is verified prior to administration in clinical trials.The second key point is that the
physiology of the probiotic strain is an important consideration. Being live
microorganisms, the proteins and secondary metabolites that are being produced
will change depending on growth phase. This feature raises a number of
important issues for the stability and efficacy of probiotic strains. First,
probiotics are subjected to numerous environmental stresses during production
and after ingestion by the host. Most notably, probiotics used to treat
intestinal ailments or whose mode of action is thought to be exerted in the
intestinal tract must be able to survive both acid and bile stress during
transit through the gut. The physiological state of the microbe is an important
characteristic that determines whether cells will be susceptible to different
types of environmental stress [44, 45]. For
example, exponentially growing cells of L.
reuteri are much more susceptible to killing by bile salts than cells in
stationary phase [45].
Thus, it is important to consider the physiological state of the cells in terms
of stress adaptation not only for survival in the host but also during
production. Second, the expression of bioactive molecules, which are most often
responsible for the health benefits exerted by probiotics, is often growth phase-dependent.
For example, our groups have been investigating the production of
immunomodulatory compounds and antimicrobial agents by strains of L. reuteri. In both cases, these
compounds are more highly expressed in the entry into and during stationary
phase (unpublished observation).


### 3. Probiotics and the Prevention and Treatment of Gastroenteritis—Examples

Commensal-derived probiotic
bacteria have been implicated as therapy for a range of digestive diseases,
including antibiotic-associated colitis,Helicobacter
pylori gastritis, and traveler’s diarrhea [46]. Probiotic formulations may include single strains or combinations of
strains. L. reuteri is indigenous to the
human gastrointestinal tract, is widely present in mammals, and has never been
shown to cause disease. In
human trials, probiotic treatment with L.
reuteri in small children with rotaviral gastroenteritis reduced the
duration of disease and facilitated patient recovery [15, 16], while in another study, it prevented
diarrhea in infants [17]. Despite the promising data from clinical trials, the primary molecular
mechanisms underlying the antipathogenic properties of L. reuteri remain unknown.Probiotics may be effective for the
prevention or treatment of infectious gastroenteritis. In the context of
disease prevention, several studies with different probiotic strains have
documented that these bacteria may reduce the incidence of acute diarrhea by 15–75%
depending on the study [17, 47–50]. Although
the relative impacts on disease incidence vary depending on the specific
probiotic strain and patient population, consistent benefits for disease
prevention have been demonstrated in multiple clinical studies. In one disease
prevention study [49],
supplementation with Bifidobacterium
lactis significantly reduced the incidence of acute diarrhea and rotavirus
shedding in infants. Studies that examined potential benefits of probiotics for
preventing antimicrobial-associated diarrhea have yielded mixed results [51–54]. One
prevention study reported a reduction in incidence of antimicrobial-associated
diarrhea in infants by 48% [52].Probiotics may also be incorporated in
treatment regimens for infectious gastroenteritis. Several meta-analyses of
numerous clinical trials with different probiotics documented reductions in
disease course of gastroenteritis that ranged from 17 to 30 hours [49, 50, 55]. Examined
another way, meta-analyses of probiotics used in clinical trials of
gastroenteritis noted significant reductions of incidence of diarrhea lasting
longer than 3 days (prolonged diarrhea). The incidence of prolonged diarrhea
was diminished by 30% or 60%, respectively, depending on the study [50, 56] (summarized
in [55]). The probiotic agent, LGG, contributed
to a significant reduction in rotavirus diarrhea by 3 days of treatment when
administered to children as part of oral rehydration therapy [57].
Recent data compilations of a large series of probiotics trials by the Cochrane
Database of Systematic Reviews (http://www.cochrane.org/) have yielded promising conclusions. As of 2008,
probiotics appear to be effective for preventing acute gastroenteritis in
children and may reduce duration of acute disease. Additionally, probiotics are
promising agents for preventing and treating antimicrobial-associated diarrhea,
although intention-to-treat analyses have not demonstrated benefits.


### 3.1.Clostridium difficile and Antibiotic-Associated Diarrhea

In what follows, we
highlight some possible mechanisms by which probiotics can be used to
ameliorate gastroenteritis. Because a number of infectious agents cause
diarrhea, colitis, and gastroenteritis, we will only focus on a few examples
with the idea that many of the mechanisms discussed can be extended to other
bacterial or viral causes of diarrhea.


### 3.1.1. The Potential Role of Probiotics in Treating CDAD

An
estimated 500,000–3,000,000 cases ofClostridium
difficile-associated diarrhea (CDAD) occur annually with related health
care costs exceeding $1 billion per year [58–60]. CDAD occurs primarily in patients that have undergone
antibiotic therapy in a health care setting, indicating that alterations in the
intestinal microbiota are important for the initiation of CDAD. In a small but
increasing number of cases, more severe complications will occur including
pseudomembranous colitis and toxic megacolon. Moreover, the emergence of metronidazole-resistant
strains of C. difficile has
diminished the efficacy of metronidazole, and vancomycin- and
metronidazole-induced cecitis reinforces the need for new therapies for the
treatment and prevention of CDAD [61, 62].Approximately
10–40% of patients treated for an initial bout of CDAD will show recurrent
disease, often with multiple episodes [63]. Such recurrences are often refractory to existing
therapies including antibiotic therapy. Patients with recurrent CDAD had a
marked decrease in the diversity of organisms in their fecal microbiota while
patients that were free of recurrent disease had a normal microbiota [64]. Thus, therapies that restore a normal microbiota or
suppress C. difficile growth while
allowing the repopulation of the intestine with a favorable microbiota may be
important to resolve infections and maintain intestinal health.


### 3.1.2. Eradication ofC. difficile through the Production of Antimicrobial Compounds

Probiotic organisms have been used to treat recurrentC. difficile in the past and in a few
cases have showed a modest effect in ameliorating recurrent disease [63]. This application has been somewhat controversial and at
this time the use of probiotics in ameliorating CDAD is not recommended [65]. However, the organisms tested were not specifically
isolated for the treatment of CDAD and, therefore, may have not been the
appropriate strains to be used to prevent recurrent CDAD. In what follows, we
outline potential mechanisms in which carefully selected or engineered
probiotics could be used in the treatment of C. difficile and the eradication of this pathogen.


### 3.1.3. Competitive Exclusion ofC. difficile Using Probiotics

CDAD is currently treated by the use
of antimicrobial agents that are effective againstC. difficile, most often vancomycin or metranidazole. Because these
drugs are broad-spectrum antibiotics, they likely play a role in recurrent
disease by suppressing the normal intestinal microbiota. Using antimicrobial
compounds that target C. difficile while
allowing restoration of resident organisms would be one possible mechanism to
prevent recurrent CDAD.


### 3.1.4. Probiotics andC. difficile Spore Germination

As mentioned above, CDAD is usually an
infection that is acquired in the hospital or other health care setting.Therefore, a
probiotic that could competitively excludeC.
difficile could be administered prior to entry into the hospital.
Unfortunately, little is known about how and where C. difficile colonizes the intestine. Once this information is
known, strategies for blocking colonization with probiotics can be developed.Nonetheless, a promising probiotic
approach using non-toxigenicC. difficile has been described. Using a hamster model of C. difficile infection, Gerding et al. demonstrated a protective
effect of populating the hamster with strains of C. difficile that are unable to produce toxin prior to challenge
with a virulent toxin-producing strain [66].
Colonization of the intestinal tract by the nontoxigenic strain appeared to be
required for protection. Currently, this probiotic approach is under
investigation for use in humans (http://www.viropharma.com/).


### 3.2. EnterohemorrhagicE. coli

A likely
contributor to the difficulty in eradicatingC. difficile from the intestine is the ability of the organism to develop
stress-resistant spores. The identification of probiotic strains that can
prevent either spore formation or the germination of spores in the intestinal
tract provides a promising avenue to combat CDAD. Recent work on spore
germination has provided in vitro assays in which inhibitory activities
of probiotics can be tested [67].Germination of spores in the laboratory requires the presence of
bile acids, with taurocholate and cholate demonstrating the best activity [67].
Thus, bile acids could play a role in signaling to C. difficile that spores are in the correct location of the gut to
germinate. Sorg and Sonenshein have recently proposed a mechanism by which the
reduction in the intestinal microbiota could lead to efficient spore
germination and overgrowth of C.
difficile [67].
They found that the bile acid deoxycholate (DOC) was able to induce spore
germination but that subsequent growth was inhibited due to toxic effects of
DOC on vegetative C. difficile. Their
work suggests a model in which a reduction in the concentration of DOC in the
intestine, due to the disruption of the normal microbiota, removes this key
inhibitor of C. difficile growth. DOC
is a secondary bile acid produced from dehydroxylation of cholate by the enzyme
7α-dehydroxylase, an activity that is produced by members of the intestinal
microbiota. While it is unclear whether or not antibiotic therapy reduces the
level of DOC in the intestine, it is tempting to speculate that providing
probiotic bacteria capable of producing 7α-dehydroxylase may prevent intestinal
overgrowth by C. difficile while the
normal microbiota is being reestablished.


### 3.2.1. Toxin Sequestration and Removal

EnterohemorrhagicE. coli (EHEC) infections cause sporadic
outbreaks of hemorrhagic colitis throughout the world (~100,000 cases per year
in the United States) [68].
Most infections result in the development of bloody diarrhea but a subset
(~5–10%) of EHEC patients (mostly children) will develop the life-threatening
condition hemolytic uremic syndrome (HUS) [69, 70].
HUS is the leading cause of kidney failure in children. EHEC, which likely
evolved from an EPEC strain [71],
also produces attaching and effacing lesions on host epithelial cells and
reduces intestinal epithelial barrier function. In addition, EHEC strains are
characterized by the expression of Shiga toxin (Stx) genes, and thus they can
be labeled as Shiga-toxin-producing E.
coli (STEC). Currently, only supportive therapy for EHEC infection is
available since antibiotic therapy may increase the risk of developing HUS,
and therefore, novel therapies must be developed. One promising alternative
therapeutic may be the use of probiotics to treat EHEC infections.


### 3.2.2. Inhibition of Toxin Production by EHEC—Identification of Strains That Repress the Lytic Functions of Lambda

Shiga toxins are
ribosome-inactivating proteins that inhibit protein synthesis by removing a
specific adenine residue from the 28S rRNA of the large ribosomal subunit [72].
Shiga toxin is required for the development of HUS and recent work has
indicated that EHEC strains mutated for Shiga toxin production fail to cause
disease in a germfree mouse model [73].
Indeed, injection of Shiga toxin with LPS directly into mice is sufficient to
generate a HUS-like disease in the kidneys of mice [74].
Therefore, Shiga toxin is an important mediator of HUS and therapies aimed at
neutralizing its activity are expected to reduce or eliminate this life-threatening
complication although current attempts at Shiga toxin neutralization have been
unsuccessful [75].As a possible mechanism for treating
EHEC disease and reducing the incidence of HUS cases, Paton et al. have
generated “designer probiotics” in which the oligosaccharide receptor (Gb3) for Stx is expressed on the cell surface of
an E. coli strain [76–78]. This
probiotic strain was shown to be capable of neutralizing Stx in vitro. As a
proof-of-concept, mice that were challenged with a STEC strain were protected
by administration of the probiotic expressing the Gb3  receptor  [79].
The protective effect was observed even when the strains were formalin-killed
prior to use, supporting the hypothesis that toxin sequestration and removal
was the mechanism by which the mice were protected. Similar results have been
obtained using bacteria-expressing receptors for toxins produced by other
diarrheal pathogens including enterotoxigenic E. coli (most common cause of traveler’s diarrhea) and Vibrio cholerae.


### 3.2.3. Inhibition of Pathogen Adherence and Strengthening of Intestinal Barrier Functions

Stx genes are carried on lambdoid prophages and are usually
located in a late transcribed region of the virus, near the lytic genes [80].
Since no mechanism for toxin secretion has been identified, the location of Stx
near the lytic genes suggests
that phage activation and cell lysis are responsible for Stx production and
release. This genetic juxtaposition suggests that therapeutics that suppress
the lytic decision of lambda in vivo would greatly reduce or eliminate complications
caused by systemic release of Stx.


### 3.3. Rotavirus

A key
interaction of EHEC, as well as EPEC, with the intestinal epithelium is the
formation of attaching and effacing lesions on the surface of the epithelium [81].
This interaction is brought about by factors secreted directly from the bacterium
into the host cell, where a redistribution of the actin cytoskeleton occurs.
EHEC and EPEC infection also induces a loss of tight junction formation and
reduction of the intestinal epithelial barrier by inducing the rearrangement of
key tight junction proteins including occludin [82, 83].
Therapies that would either disrupt this interaction of EHEC/EPEC with the
intestinal epithelium or inhibit the loss of barrier function should ameliorate
disease.Probiotics have shown some success
inhibiting adhesion, A/E lesion formation and enhancing barrier function in
response to EHEC infection in vitro.
Johnson-Henry et al. tested the ability ofLactobacillus
rhamnosus GG to prevent loss of barrier integrity and formation of A/E
lesions induced by EHEC infection of cell culture in vitro [40].
They found that pretreatment of intestinal epithelial cells in vitro with LGG
was sufficient to reduce the number of A/E lesions and to prevent loss of
barrier function as measured by transepithelial resistance, localization of
tight junction proteins, and barrier permeability assays. Importantly, live LGG
was required for these effects as heat-killed bacteria were not effective in
preventing EHEC effects on epithelial cells.Enteric viruses including noroviruses
and rotavirus represent major causes of gastroenteritis, especially in young
children. Rotavirus infection results in acute gastroenteritis with
accompanying dehydration and vomiting mainly in children 3–24 months of age.
Human rotavirus primarily infects intestinal epithelial cells of the distal small
intestine, resulting in enterotoxin-mediated damage to intestinal barrier
function. Recent studies indicate that probiotics may reduce the duration and
ameliorate disease due to rotavirus infection ([84];
G. Preidis and J. Versalovic, unpublished data). Probiotics promoted intestinal
immunoglobulin production and appeared to reduce the severity of intestinal
lesions due to rotavirus infection in a mouse model. These findings and related
investigations suggest that probiotics may diminish the severity and duration
of gastrointestinal infections by mechanisms independent of direct pathogen
antagonism. Probiotics may also promote healing and homeostasis by modulating
cytokine production and facilitating intestinal barrier function.


## 2.1. The Need for Mechanistic Details of Probiotic Action

The use
of probiotics to prevent and treat a wide variety of conditions has gained
favor in the past decade. This is in part due to a need to find alternatives to
traditional therapies such as antibiotics as well as the lack of good
treatments for GI ailments. While there are increasing reports of the efficacy
of probiotics in the treatment of diseases such as pouchitis [13, 14],
diarrhea [15–17],
and irritable bowel syndrome [18], the scientific basis for the
use of probiotics is just beginning to be understood. We will focus on the
potential applications for probiotics in the treatment of diarrheal disease. Several
examples will highlight how probiotics may be selected for and utilized against
pathogens causing gastroenteritis.The concept of using probiotic
microorganisms to prevent and treat a variety of human ailments has been around
for more than 100 years [19].
With the rise in the number of multidrug resistant pathogens and the
recognition of the role that the human microbiota plays in health and disease,
a recent expansion in the interest in probiotics has been generated. This
phenomenon is apparent in both the numbers of probiotic products being marketed
to consumers as well as the increased amount of scientific research occurring
in probiotics. Although many of the mechanisms by which probiotics benefit
human beings remain unclear, probiotic bacteria are being utilized more
commonly to treat specific diseases.Several definitions of what
constitutes a “probiotic” in the literature have been formulated. For this
review, we use the definition derived in 2001 by the Food and Agricultural
Organization (FAO) and the World Health Organization (WHO)—“Probiotics are
live microorganisms which when administered in adequate amount confer a health
benefit on the host.”  [20].
This definition is the currently accepted definition by the International
Scientific Association for Probiotics and Prebiotics (ISAPP)
(http://www.isapp.net/).


## 2.2. Antipathogenic Activities

Perhaps
the most important scientific question regarding the use of probiotics in
medicine is the identification of mechanisms by which probiotics impact human
health. Several mechanisms have been implicated but most have not been
experimentally proven (Figure1). Here, we discuss possible mechanisms that are
relevant for the treatment of diarrheal diseases. We will highlight research
examples that support these putative mechanisms whenever possible.Figure 1
Probiotics and Beneficial Effects in the Intestine. Depiction of the interactions between beneficial
bacteria (left side), their secreted factors, pathogens, and the intestinal
mucosa (right side). Potential beneficial effects of probiotics are listed.
Only two host cell types are shown, intestinal epithelial cells and macrophages
although other cell types including dendritic cells, lymphocytes,
myofibroblasts, and neutrophils comprise the intestinal mucosa. The arrows
indicate the release and possible distribution of secreted factors derived from
probiotics.


## 2.3. Stimulation of Host Antimicrobial Defenses

Many probiotics have been shown to
produce antipathogenic compounds ranging from small molecules to bioactive
antimicrobial peptides. Most of these studies have focused on the in vitro
susceptibility of pathogens to products secreted by probiotic bacteria. In most
cases, the ability of an antimicrobial compound secreted by a probiotic
organism to inhibit the growth of a pathogen in vivo has not been demonstrated.
Conceptually, an antimicrobial compound produced by an organism would need to
be produced at a high enough level and in the right location in the intestinal
tract to exert a strong effect on a pathogen in vivo.An elegant proof of principle for
direct action of a probiotic-produced antimicrobial against a pathogen was
recently reported by Corr  et al. who demonstrated that production of the bacteriocin
Abp118 byLactobacillus salivarius was
sufficient to protect mice from disease by infection with Listeria monocytogenes [21].
To prove the action of the bacteriocin was directly responsible for the
protection of the mice, they generated a L.
salivarius strain that was unable to produce Abp118 and showed that this
mutant was incapable of protecting against L.
monocytogenes infection. Notably, they were able to express a gene that
confers immunity to the Abp118 bacteriocin within L. monocytogenes and showed that this strain was now resistant to
the probiotic effect of L. salivarius within
the mouse. This study provided clear evidence that a probiotic-derived
bacteriocin could function directly on a pathogen in vivo.


## 2.4. Pathogen Exclusion via Indirect Mechanisms

In addition to
producing antimicrobial compounds that act directly on pathogens, probiotics
may stimulate host antimicrobial defense pathways. The intestinal tract has a
number of mechanisms for resisting the effects of pathogens including the
production of defensins [22].
Defensins are cationic antimicrobial peptides that are produced in a number of
cell types including Paneth cells in the crypts of the small intestine and
intestinal epithelial cells. A deficiency in alpha-defensin production has been
correlated with ileal Crohn’s disease [23, 24]. Tissue
samples from patients with Crohn’s disease showed a lower level of
alpha-defensin production and extracts from these samples exhibited a reduced
ability to inhibit bacterial growth in vitro. Moreover, some pathogenic
bacteria have evolved mechanisms to inhibit the production or mechanism of
action of defensins (reviewed in [25]).Probiotics may act to stimulate
defensin activity via at least two mechanisms. First, probiotics may stimulate
the synthesis of defensin expression. This has been demonstrated for human beta
defensin 2 (hBD-2), whose expression is upregulated by the presence of several
probiotic bacteria via the transcription factorNF-κB [26, 27]. The
implication is that probiotic strains with this capability would strengthen
intestinal defenses by increasing defensin levels. This effect is also observed
with certain pathogenic bacteria and thus is not a specific property of
probiotic bacteria. Second, many defensins are produced in a propeptide form
that must be activated via the action of proteases. One well-characterized example
is the activation of the murine defensin cryptdin (an alpha-defensin that is
produced by Paneth cells) by the action of matrix metalloprotease 7 (MMP-7) [28]. Mice
defective for MMP-7 are more susceptible to killing by Salmonella. Evidence indicates that bacteria can stimulate the
production of MMP-7 in the intestine [29]. Thus,
one mechanism in which probiotics could participate in activating defensins is
by stimulating the production of MMPs in the intestinal tract. Alternatively,
probiotics could produce proteases that themselves activate defensins in the
intestinal lumen. Although there is no evidence yet to support this mechanism, a
subset of lactobacilli and streptococci encode MMP-like proteins in their
genomes (R. Britton, unpublished observation). These MMPs are not found in any
other bacteria and thus it will be interesting to determine what effect they
have on host cell function.


## 2.5. Immunomodulation

Rather than  directly inhibiting the growth or
viability of the pathogen, probiotics may compete for an ecological niche or,
otherwise, create conditions that are unfavorable for the pathogen to take hold
in the intestinal tract. There are many possible mechanisms for how pathogen
exclusion may take place. First, several probiotics have been demonstrated to
alter the ability of pathogens to adhere to or invade colonic epithelial cells
in vitro, for example, see [30, 31]. Second, probiotics could sequester essential
nutrients from invading pathogens and impair their colonization ability. Third,
probiotics may alter the gene expression program of pathogens in such a way as
to inhibit the expression of virulence functions [32].
Lastly, probiotics may create an unfavorable environment for pathogen
colonization by altering pH, the mucus layer, and other factors in the local
surroundings. It is important to note that although many of these possible
effects have been demonstrated in
vitro, the ability of probiotics to exclude pathogens in vivo remains to
be proven.


## 2.6. Enhancing Intestinal Barrier Function

Probiotics may have strain-dependent effects on the immune system. Different
strains representing differentLactobacillus species demonstrated contrasting effects with respect to proinflammatory
cytokine production by murine bone marrow-derived dendritic cells [33].
Specific probiotic strains counteracted the immunostimulatory effects of other
strains so that probiotics have the potential to yield additive or antagonistic
results. Interestingly, in this study, the anti-inflammatory cytokine IL-10 was
maintained at similar levels [31].
Different probiotic Lactobacillus strains
of the same species may also yield contrasting effects with respect to
immunomodulation. Human breast milk-derived Lactobacillus
reuteri strains either stimulated the key proinflammatory cytokine, human
tumor necrosis factor (TNF), or suppressed its production by human myeloid
cells [34].
The mechanisms of action may be due, not surprisingly, to contrasting effects
on key signaling pathways in mammalian cells. Probiotic strains such as Lactobacillus rhamnosus GG (LGG) may
activate NF-κB and the signal transducer and activator of transcription (STAT)
signaling pathways in human macrophages [35]. In
contrast, probiotic Lactobacillus strains may suppress NF-κB
signaling [36, 37] or MAP kinase-/c-Jun-mediated signaling [34].
Stimulation of key signaling pathways and enhancement of proinflammatory
cytokine production may be important to “prime” the immune system for defense
against gastrointestinal infections. Conversely, suppression of immune
signaling may be an important mechanism to promote homeostasis and tolerance to
microbial communities with many potential antigens, and these immunosuppressive
functions may promote healing or resolution of infections.


## 2.7. Why Understanding Mechanisms Is Important?

The disruption of
epithelial barrier function and loss of tight junction formation in the
intestinal epithelium may contribute to pathophysiology and diarrheal symptoms
observed during infection with certain pathogens [38, 39]. Loss of
tight junctions can lead to increased paracellular transport that can result in
fluid loss and pathogen invasion of the submucosa. Pathogens may secrete
factors such as enterotoxins that may promote excessive apoptosis or necrosis
of intestinal epithelial cells, thereby disrupting the intestinal barrier.
Enteric pathogens may also cause effacing lesions at the mucosal surface due to
direct adherence with intestinal epithelial cells (e.g., EPEC). In contrast,
probiotics have been reported to promote tight junction formation and
intestinal barrier function [40, 41]. Although
the mechanisms of promoting barrier integrity are not well understood,
probiotics may counteract the disruption of the intestinal epithelial barrier
despite the presence of pathogens. Probiotics may also suppress toxin
production or interfere with the abilities of specific pathogens to adhere
directly to the intestinal surface. As a result, pathogens may have a
diminished ability to disrupt intestinal barrier function.


## 2.8. Important Considerations for the Use of Probiotics: Strain Selection and Microbial Physiology

An important
challenge in the field of probiotics is the identification of genes and
mechanisms responsible for the beneficial functions exerted by these microbes.
Successful identification of mechanistic details for how probiotics function
will have at least three important benefits. First, understanding mechanisms of
action will provide a scientific basis for the beneficial effects provided by
specific microbes. These breakthrough investigations will help move probiotics
from the status of dietary supplements to therapeutics. Second, understanding
mechanisms of probiosis and the gene products produced by probiotics will allow
for the identification of more potent probiotics or the development of
bioengineered therapeutics. As an example, the anti-inflammatory cytokine IL-10
was postulated to be a potential therapeutic for the treatment of inflammatory
bowel disease. To test this hypothesis, a strain ofLactococcus lactis engineered to produce and secrete IL-10 was
constructed and demonstrated to reduce colitis in a murine model [42].
Early clinical trials in patients with inflammatory bowel disease indicate some
relief from symptoms when treated with the IL-10 overproducing strain. Third,
the identification of gene products that are responsible for ameliorating
disease will allow researchers, industry, and clinicians to follow the production
of these products as important biomarkers during probiotic preparation. As
discussed below, the physiological state of microbes can be crucial to the
functions of probiotics. Thus, it will be important to be able to follow the
production of important bioactive molecules when culturing and processing
probiotics for applications in animals and humans.


## 2.9. Probiotics and Diarrhea

Probiotics
are considered to be living or viable microorganisms by definition. Unlike
small molecules that are stable entities, probiotics are dynamic microorganisms
and will change gene expression patterns when exposed to different
environmental conditions. This reality has two important implications for those
who choose to use these organisms to combat human or animal diseases. First,
probiosis is a strain-specific phenomenon. As defining a bacterial species is
challenging in this age of full genome sequencing, it is clear that probiotic
effects observed in vitro and in vivo are
strain specific. For example, modulation of TNF production by strains ofLactobacillus reuteri identified strains
that were immunostimulatory, immunoneutral, and immunosuppressive for TNF
production [34, 43].
These findings highlight the strain-specific nature of probiotic effects
exerted by bacteria. Thus, it is important for research groups and industry to
be cautious with strain handling and tracking so that inclusion of correct
strains is verified prior to administration in clinical trials.The second key point is that the
physiology of the probiotic strain is an important consideration. Being live
microorganisms, the proteins and secondary metabolites that are being produced
will change depending on growth phase. This feature raises a number of
important issues for the stability and efficacy of probiotic strains. First,
probiotics are subjected to numerous environmental stresses during production
and after ingestion by the host. Most notably, probiotics used to treat
intestinal ailments or whose mode of action is thought to be exerted in the
intestinal tract must be able to survive both acid and bile stress during
transit through the gut. The physiological state of the microbe is an important
characteristic that determines whether cells will be susceptible to different
types of environmental stress [44, 45]. For
example, exponentially growing cells of L.
reuteri are much more susceptible to killing by bile salts than cells in
stationary phase [45].
Thus, it is important to consider the physiological state of the cells in terms
of stress adaptation not only for survival in the host but also during
production. Second, the expression of bioactive molecules, which are most often
responsible for the health benefits exerted by probiotics, is often growth phase-dependent.
For example, our groups have been investigating the production of
immunomodulatory compounds and antimicrobial agents by strains of L. reuteri. In both cases, these
compounds are more highly expressed in the entry into and during stationary
phase (unpublished observation).


## 3. Probiotics and the Prevention and Treatment of Gastroenteritis—Examples

Commensal-derived probiotic
bacteria have been implicated as therapy for a range of digestive diseases,
including antibiotic-associated colitis,Helicobacter
pylori gastritis, and traveler’s diarrhea [46]. Probiotic formulations may include single strains or combinations of
strains. L. reuteri is indigenous to the
human gastrointestinal tract, is widely present in mammals, and has never been
shown to cause disease. In
human trials, probiotic treatment with L.
reuteri in small children with rotaviral gastroenteritis reduced the
duration of disease and facilitated patient recovery [15, 16], while in another study, it prevented
diarrhea in infants [17]. Despite the promising data from clinical trials, the primary molecular
mechanisms underlying the antipathogenic properties of L. reuteri remain unknown.Probiotics may be effective for the
prevention or treatment of infectious gastroenteritis. In the context of
disease prevention, several studies with different probiotic strains have
documented that these bacteria may reduce the incidence of acute diarrhea by 15–75%
depending on the study [17, 47–50]. Although
the relative impacts on disease incidence vary depending on the specific
probiotic strain and patient population, consistent benefits for disease
prevention have been demonstrated in multiple clinical studies. In one disease
prevention study [49],
supplementation with Bifidobacterium
lactis significantly reduced the incidence of acute diarrhea and rotavirus
shedding in infants. Studies that examined potential benefits of probiotics for
preventing antimicrobial-associated diarrhea have yielded mixed results [51–54]. One
prevention study reported a reduction in incidence of antimicrobial-associated
diarrhea in infants by 48% [52].Probiotics may also be incorporated in
treatment regimens for infectious gastroenteritis. Several meta-analyses of
numerous clinical trials with different probiotics documented reductions in
disease course of gastroenteritis that ranged from 17 to 30 hours [49, 50, 55]. Examined
another way, meta-analyses of probiotics used in clinical trials of
gastroenteritis noted significant reductions of incidence of diarrhea lasting
longer than 3 days (prolonged diarrhea). The incidence of prolonged diarrhea
was diminished by 30% or 60%, respectively, depending on the study [50, 56] (summarized
in [55]). The probiotic agent, LGG, contributed
to a significant reduction in rotavirus diarrhea by 3 days of treatment when
administered to children as part of oral rehydration therapy [57].
Recent data compilations of a large series of probiotics trials by the Cochrane
Database of Systematic Reviews (http://www.cochrane.org/) have yielded promising conclusions. As of 2008,
probiotics appear to be effective for preventing acute gastroenteritis in
children and may reduce duration of acute disease. Additionally, probiotics are
promising agents for preventing and treating antimicrobial-associated diarrhea,
although intention-to-treat analyses have not demonstrated benefits.


## 3.1.Clostridium difficile and Antibiotic-Associated Diarrhea

In what follows, we
highlight some possible mechanisms by which probiotics can be used to
ameliorate gastroenteritis. Because a number of infectious agents cause
diarrhea, colitis, and gastroenteritis, we will only focus on a few examples
with the idea that many of the mechanisms discussed can be extended to other
bacterial or viral causes of diarrhea.


## 3.1.1. The Potential Role of Probiotics in Treating CDAD

An
estimated 500,000–3,000,000 cases ofClostridium
difficile-associated diarrhea (CDAD) occur annually with related health
care costs exceeding $1 billion per year [58–60]. CDAD occurs primarily in patients that have undergone
antibiotic therapy in a health care setting, indicating that alterations in the
intestinal microbiota are important for the initiation of CDAD. In a small but
increasing number of cases, more severe complications will occur including
pseudomembranous colitis and toxic megacolon. Moreover, the emergence of metronidazole-resistant
strains of C. difficile has
diminished the efficacy of metronidazole, and vancomycin- and
metronidazole-induced cecitis reinforces the need for new therapies for the
treatment and prevention of CDAD [61, 62].Approximately
10–40% of patients treated for an initial bout of CDAD will show recurrent
disease, often with multiple episodes [63]. Such recurrences are often refractory to existing
therapies including antibiotic therapy. Patients with recurrent CDAD had a
marked decrease in the diversity of organisms in their fecal microbiota while
patients that were free of recurrent disease had a normal microbiota [64]. Thus, therapies that restore a normal microbiota or
suppress C. difficile growth while
allowing the repopulation of the intestine with a favorable microbiota may be
important to resolve infections and maintain intestinal health.


## 3.1.2. Eradication ofC. difficile through the Production of Antimicrobial Compounds

Probiotic organisms have been used to treat recurrentC. difficile in the past and in a few
cases have showed a modest effect in ameliorating recurrent disease [63]. This application has been somewhat controversial and at
this time the use of probiotics in ameliorating CDAD is not recommended [65]. However, the organisms tested were not specifically
isolated for the treatment of CDAD and, therefore, may have not been the
appropriate strains to be used to prevent recurrent CDAD. In what follows, we
outline potential mechanisms in which carefully selected or engineered
probiotics could be used in the treatment of C. difficile and the eradication of this pathogen.


## 3.1.3. Competitive Exclusion ofC. difficile Using Probiotics

CDAD is currently treated by the use
of antimicrobial agents that are effective againstC. difficile, most often vancomycin or metranidazole. Because these
drugs are broad-spectrum antibiotics, they likely play a role in recurrent
disease by suppressing the normal intestinal microbiota. Using antimicrobial
compounds that target C. difficile while
allowing restoration of resident organisms would be one possible mechanism to
prevent recurrent CDAD.


## 3.1.4. Probiotics andC. difficile Spore Germination

As mentioned above, CDAD is usually an
infection that is acquired in the hospital or other health care setting.Therefore, a
probiotic that could competitively excludeC.
difficile could be administered prior to entry into the hospital.
Unfortunately, little is known about how and where C. difficile colonizes the intestine. Once this information is
known, strategies for blocking colonization with probiotics can be developed.Nonetheless, a promising probiotic
approach using non-toxigenicC. difficile has been described. Using a hamster model of C. difficile infection, Gerding et al. demonstrated a protective
effect of populating the hamster with strains of C. difficile that are unable to produce toxin prior to challenge
with a virulent toxin-producing strain [66].
Colonization of the intestinal tract by the nontoxigenic strain appeared to be
required for protection. Currently, this probiotic approach is under
investigation for use in humans (http://www.viropharma.com/).


## 3.2. EnterohemorrhagicE. coli

A likely
contributor to the difficulty in eradicatingC. difficile from the intestine is the ability of the organism to develop
stress-resistant spores. The identification of probiotic strains that can
prevent either spore formation or the germination of spores in the intestinal
tract provides a promising avenue to combat CDAD. Recent work on spore
germination has provided in vitro assays in which inhibitory activities
of probiotics can be tested [67].Germination of spores in the laboratory requires the presence of
bile acids, with taurocholate and cholate demonstrating the best activity [67].
Thus, bile acids could play a role in signaling to C. difficile that spores are in the correct location of the gut to
germinate. Sorg and Sonenshein have recently proposed a mechanism by which the
reduction in the intestinal microbiota could lead to efficient spore
germination and overgrowth of C.
difficile [67].
They found that the bile acid deoxycholate (DOC) was able to induce spore
germination but that subsequent growth was inhibited due to toxic effects of
DOC on vegetative C. difficile. Their
work suggests a model in which a reduction in the concentration of DOC in the
intestine, due to the disruption of the normal microbiota, removes this key
inhibitor of C. difficile growth. DOC
is a secondary bile acid produced from dehydroxylation of cholate by the enzyme
7α-dehydroxylase, an activity that is produced by members of the intestinal
microbiota. While it is unclear whether or not antibiotic therapy reduces the
level of DOC in the intestine, it is tempting to speculate that providing
probiotic bacteria capable of producing 7α-dehydroxylase may prevent intestinal
overgrowth by C. difficile while the
normal microbiota is being reestablished.


## 3.2.1. Toxin Sequestration and Removal

EnterohemorrhagicE. coli (EHEC) infections cause sporadic
outbreaks of hemorrhagic colitis throughout the world (~100,000 cases per year
in the United States) [68].
Most infections result in the development of bloody diarrhea but a subset
(~5–10%) of EHEC patients (mostly children) will develop the life-threatening
condition hemolytic uremic syndrome (HUS) [69, 70].
HUS is the leading cause of kidney failure in children. EHEC, which likely
evolved from an EPEC strain [71],
also produces attaching and effacing lesions on host epithelial cells and
reduces intestinal epithelial barrier function. In addition, EHEC strains are
characterized by the expression of Shiga toxin (Stx) genes, and thus they can
be labeled as Shiga-toxin-producing E.
coli (STEC). Currently, only supportive therapy for EHEC infection is
available since antibiotic therapy may increase the risk of developing HUS,
and therefore, novel therapies must be developed. One promising alternative
therapeutic may be the use of probiotics to treat EHEC infections.


## 3.2.2. Inhibition of Toxin Production by EHEC—Identification of Strains That Repress the Lytic Functions of Lambda

Shiga toxins are
ribosome-inactivating proteins that inhibit protein synthesis by removing a
specific adenine residue from the 28S rRNA of the large ribosomal subunit [72].
Shiga toxin is required for the development of HUS and recent work has
indicated that EHEC strains mutated for Shiga toxin production fail to cause
disease in a germfree mouse model [73].
Indeed, injection of Shiga toxin with LPS directly into mice is sufficient to
generate a HUS-like disease in the kidneys of mice [74].
Therefore, Shiga toxin is an important mediator of HUS and therapies aimed at
neutralizing its activity are expected to reduce or eliminate this life-threatening
complication although current attempts at Shiga toxin neutralization have been
unsuccessful [75].As a possible mechanism for treating
EHEC disease and reducing the incidence of HUS cases, Paton et al. have
generated “designer probiotics” in which the oligosaccharide receptor (Gb3) for Stx is expressed on the cell surface of
an E. coli strain [76–78]. This
probiotic strain was shown to be capable of neutralizing Stx in vitro. As a
proof-of-concept, mice that were challenged with a STEC strain were protected
by administration of the probiotic expressing the Gb3  receptor  [79].
The protective effect was observed even when the strains were formalin-killed
prior to use, supporting the hypothesis that toxin sequestration and removal
was the mechanism by which the mice were protected. Similar results have been
obtained using bacteria-expressing receptors for toxins produced by other
diarrheal pathogens including enterotoxigenic E. coli (most common cause of traveler’s diarrhea) and Vibrio cholerae.


## 3.2.3. Inhibition of Pathogen Adherence and Strengthening of Intestinal Barrier Functions

Stx genes are carried on lambdoid prophages and are usually
located in a late transcribed region of the virus, near the lytic genes [80].
Since no mechanism for toxin secretion has been identified, the location of Stx
near the lytic genes suggests
that phage activation and cell lysis are responsible for Stx production and
release. This genetic juxtaposition suggests that therapeutics that suppress
the lytic decision of lambda in vivo would greatly reduce or eliminate complications
caused by systemic release of Stx.


## 3.3. Rotavirus

A key
interaction of EHEC, as well as EPEC, with the intestinal epithelium is the
formation of attaching and effacing lesions on the surface of the epithelium [81].
This interaction is brought about by factors secreted directly from the bacterium
into the host cell, where a redistribution of the actin cytoskeleton occurs.
EHEC and EPEC infection also induces a loss of tight junction formation and
reduction of the intestinal epithelial barrier by inducing the rearrangement of
key tight junction proteins including occludin [82, 83].
Therapies that would either disrupt this interaction of EHEC/EPEC with the
intestinal epithelium or inhibit the loss of barrier function should ameliorate
disease.Probiotics have shown some success
inhibiting adhesion, A/E lesion formation and enhancing barrier function in
response to EHEC infection in vitro.
Johnson-Henry et al. tested the ability ofLactobacillus
rhamnosus GG to prevent loss of barrier integrity and formation of A/E
lesions induced by EHEC infection of cell culture in vitro [40].
They found that pretreatment of intestinal epithelial cells in vitro with LGG
was sufficient to reduce the number of A/E lesions and to prevent loss of
barrier function as measured by transepithelial resistance, localization of
tight junction proteins, and barrier permeability assays. Importantly, live LGG
was required for these effects as heat-killed bacteria were not effective in
preventing EHEC effects on epithelial cells.Enteric viruses including noroviruses
and rotavirus represent major causes of gastroenteritis, especially in young
children. Rotavirus infection results in acute gastroenteritis with
accompanying dehydration and vomiting mainly in children 3–24 months of age.
Human rotavirus primarily infects intestinal epithelial cells of the distal small
intestine, resulting in enterotoxin-mediated damage to intestinal barrier
function. Recent studies indicate that probiotics may reduce the duration and
ameliorate disease due to rotavirus infection ([84];
G. Preidis and J. Versalovic, unpublished data). Probiotics promoted intestinal
immunoglobulin production and appeared to reduce the severity of intestinal
lesions due to rotavirus infection in a mouse model. These findings and related
investigations suggest that probiotics may diminish the severity and duration
of gastrointestinal infections by mechanisms independent of direct pathogen
antagonism. Probiotics may also promote healing and homeostasis by modulating
cytokine production and facilitating intestinal barrier function.


## 4. Concluding Remarks

Probiotics may provide an important
strategy for the prevention and treatment of gastrointestinal infections.
Specific bacteria derived from human microbial communities may have key
features that establish these microbes as primary candidates for probiotic
therapies. These beneficial microbes may have different effects within the host
such as prevention of pathogen proliferation and function. Probiotics may also
stimulate the host’s immune function and mucosal barrier integrity. By working
via different mechanisms of probiosis, probiotics may yield effects at
different steps in the process. Probiotics may prevent disease from occurring
when administered prophylactically. Probiotics may also suppress or diminish
severity or duration of disease in the context of treatment. As our knowledge
of the human microbiome advances, rational selection of probiotics based on
known mechanisms of action and mechanisms of disease will facilitate
optimization of strategies in therapeutic microbiology. Ultimately, we expect that
probiotics will help to promote stable, diverse, and beneficial microbial
communities that enhance human health and prevent disease.



---

*Source: 290769-2009-02-04.xml*