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SUPPLEMENT ARTICLE
How Science Will Help Shape Future Clinical
Applications of Probiotics
Gregor Reid
Canadian Research and Development Centre for Probiotics, Lawson Health Research Institute, and Department of Microbiology and Immunology
and Department of Surgery, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Canada
The recent increased interest in probiotics among clinicians has many causes, primarily the concern about
the limitations of the current armamentarium of pharmaceutical agents. Although probiotics have been used
mostly in dietary supplements and foods to maintain health, scientific and clinical studies are recognizing the
potential of some probiotics to be therapeutic in function. Scientific breakthroughs in understanding the
source and composition of the human microbiota, the key nutritional factors that influence these microbes,
and their immunomodulatory effects; the creation of disease-targeted recombinant strains; the isolation and
characterization of signaling molecules that can modulate microbial biofilms and infectious processes; and
advances in biomedical engineering that will provide new delivery systems for probiotics will shape the future
of clinical applications of probiotics. In time and with rigorous documentation, some probiotics will likely
find an important place in medical practice.
WHAT ARE PROBIOTICS?
In 2001, an expert-panel report from the United
Nations and the World Health Organization finally provided a definition of probiotics that embraced the
breadth of their applications and emphasized the need
for clinical documentation of the benefits they confer
[1]. This definition is “live microorganisms which when
administered in adequate amounts confer a health benefit on the host” [1, p. 5]. The subsequent guidelines
published jointly by both international organizations
[2] and later endorsed by the International Scientific
Association for Probiotics and Prebiotics [3] gave scientific, clinical, and manufacturing standards that need
to be met for products to be called “probiotic.” Such
is the nature of these guidelines that they provide the
means for developing new strains and new applications.
In this short review, 5 examples are provided to illustrate how scientific advances will likely lead to new and
Reprints or correspondence: Dr. Gregor Reid, Canadian Research and
Development Centre for Probiotics, F2-116 Lawson Health Research Institute, 268
Grosvenor St., London, Ontario, N6A 4V2 Canada ([email protected]).
Clinical Infectious Diseases 2008; 46:S62–6
2008 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2008/4603S2-0003$15.00
DOI: 10.1086/523340
S62 • CID 2008:46 (Suppl 2) • Reid
potentially exciting breakthroughs that will have farreaching benefits for many humans and animals.
THE ORIGIN OF OUR MICROBIOTA
More than 500 million years ago, Earth was inhabited
only by single-cell microbes. It can be argued that humans thus emerged from bacteria and that bacteria
actually “permitted” this to occur, in the sense that they
could have eradicated the human race on many occasions but did not. There is no question that microbes
influence health, as the study of infectious disease pathogens has long shown. However, less well understood
are the beneficial microbes, their origin before they colonized humans, and their role in retention of good
health in a host.
With 125% of humans born by cesarean delivery
and, therefore, not exposed as newborns to the microbiota of the mother’s vaginal and rectal areas, one
would assume that the microbial composition of humans varies profoundly. Given that the microbiota tend
to stabilize within the first year of life and that, thereafter, it is difficult for exogenous organisms to take a
foothold [4, 5], it is critical to determine the source of
the organisms that humans inherit.
Elegant animal studies have been performed on Bacteroides thetaiotaomicron and Bacteroides fragilis, show-
ing their importance in the development of healthy intestinal
and immune systems, respectively [6, 7]. In humans, however,
where do these organisms come from? In a study of the microbiota of 2 newborns, B. thetaiotaomicron was detected early
in life in 1 child [8]. This anaerobe is found in the intestine,
but how would it be passed to a baby born by cesarean delivery,
and what would be the consequence of it not being present?
Perhaps these organisms come from the environment and are
passed to the baby by handling or kissing or are received by
the baby putting objects in his or her mouth. Without basic
microbiological studies, we cannot be sure. To date, there have
been no studies that compare the gut microbiota between infants born vaginally and infants born by cesarean section or
between infants breast-fed and infants fed with formula, and
there has been no long-term follow-up to determine whether
and how these differences may affect health outcomes. Such
studies would be difficult to undertake, and some people might
think they do not have value because, epidemiologically, there
have not been major differences noted between the health and
longevity of adults in one category of infants and those in
another category. Nevertheless, just as Barker’s [9] long-term
studies unexpectedly discovered a link between maternal health
during gestation and adulthood illness, so too might microbiota
differences in species types and microbial numbers lead to insight into the critical problems that face the current health care
system because of syndrome X. Already, a link between microbes has been reported for obesity [10] and ankylosing spondylitis [11]. Whether probiotic intervention can alter such clinical outcomes remains to be seen.
The finding of lactobacilli in breast milk [12] has led to the
suggestion that M or dendritic cells sample the microbiota,
pick up these strains, and then transfer them into the mammary
duct, through which they pass to the baby. If that were true,
presumably Bacteroides species could be transferred likewise.
Several molecular techniques have been developed to identify
and track changes in microbiota in human, animal, and environmental samples [13–15]. Although it is a tedious task,
studies are needed to determine where the constituents of our
microbiota come from in the environment. The “hygiene hypothesis” suggests that lack of exposure to certain organisms
could be partly responsible for increased rates of some chronic
diseases, such as allergies [16]. If we understood the types of
organisms whose inheritance significantly enhanced health, appropriate strategies could be developed for delivery of probiotics in a more systematic way, rather than in the random
method used to date. Such approaches have major ethical considerations and are unlikely to occur in the next 10 years.
However, just as human genomics will lead to interventions in
utero and at different stages in life (e.g., performance of prophylactic mastectomy for women at high risk of cancer and
death), so too might selective use of probiotics be applied to
alter progression of disease.
DIETARY FACTORS
Genetically, humans are virtually the same as they were at the
end of Paleolithic times ∼20,000 years ago, yet the development
of agriculture and domestication of animals ∼10,000 years ago
and the Industrial Revolution 200 years ago brought new dietary practices and pressures [17].
Historical and anthropological studies claim that huntergatherers were generally healthy, fit, and largely free of the
degenerative cardiovascular diseases common in modern societies. Life spans were short, but this was because of infectious
disease, predators, and fighting, rather than heart disease or
diabetes. Since those times, the energy requirements of an enlarged brain have coincided with a reduction in the size and
energy requirements of the digestive system, which leads to the
postulate that changes in food staples and food-processing procedures have altered crucial nutritional characteristics of our
ancestral diet—namely, glycemic load, fatty-acid level, macronutrient balance, trace nutrient density, acid-base balance,
sodium-potassium balance, and fiber content [17–19].
Prebiotics are nondigestible substances that provide a beneficial physiological effect on the host by selectively stimulating
the favorable growth or activity of a limited number of indigenous bacteria [1]. Ancient humans ate many sources of prebiotics (e.g., plant roots) and used fermentation as a means of
food preservation and preparation. To design new foods either
to deliver probiotic or prebiotic products or to intentionally
and specifically modify the intestinal microbiota as a means of
lowering rates of chronic disease, we might want to assess a
maternal diet supplemented with prebiotics or fermented foods
and plant roots and see whether it alters mammary levels of
Lactobacillus organisms or other species. Also, such diets could
be tested during weaning to see whether they modify the gut
microbiota or susceptibility to asthma and allergy. Studies of
babies are difficult for ethical reasons, and it is impossible to
know exactly what the Paleolithic diet was and whether it was
advantageous to the health of babies. Nevertheless, diet and
microbes influence the fundamental aspects of immunity and
development, yet we know so little about how we acquire bacteria and which ones should be supplemented to achieve shortand long-term good health. In due course, new symbiotic products (i.e., mixtures of probiotics and prebiotics) will be
developed to improve the acquisition of healthy gut microbiota,
assuming that such microbiota exist.
ALTERING THE COURSE OF DISEASE
There are many examples of pharmaceutical and dietary interventions that have fundamentally altered the course of disease. It is difficult to know the extent to which this applies to
The Future of Probiotics • CID 2008:46 (Suppl 2) • S63
probiotics, but their use in alleviating diarrhea is at least 1
indication of their potential. This application could have a
profound effect on the quality of life of people with HIV infection or AIDS, among whom chronic diarrhea is commonplace and often contributes to death.
In one study, 40 HIV-infected patients who were taking a
stavudine- and/or didanosine-based HAART regimen were prospectively randomized to receive micronutrients or placebo
twice daily for 12 weeks. The absolute CD4 cell count increased
by an average of 24% in the group receiving micronutrients,
compared with a 0% change in the placebo group (P p .01)
[20]. This study did not assess the impact of the micronutrients
on the gut microbiota or immunity per se, and it would be
interesting to perform such an assessment, especially with prebiotics added to the micronutrients. In a second randomized,
placebo-controlled study of 24 subjects with HIV infection or
AIDS, daily ingestion of probiotic Lactobacillus rhamnosus GR1 and Lactobacillus reuteri RC-14, supplemented in yogurt, led
to rapid resolution of diarrhea, flatulence, and nausea, as well
as a small increase in CD4 cell counts [21]. For the vast majority
of patients with AIDS who cannot gain access to antiretroviral
therapies or who cannot take them because they have insufficient food intake to avoid the major adverse effects of the
drugs, the use of relatively simple foods and supplements could
have significant benefits. The increase in CD4 cell counts found
after consumption of nutrients and probiotics needs to be further confirmed in larger studies, but this approach could affect
a significant number of patients with AIDS.
Another major disease that affects a large amount of the
world’s population is diabetes. Some preliminary data suggest
that probiotics may lengthen the time to onset of type I diabetes
[22]. The mechanisms are unclear, but, given the anti-inflammatory effects [23] of the probiotics used in that study, it seems
feasible that modulation of immunity plays a role. Integration
of human IL-10 genes into lactococci has led to the creation
of an organism that appears able to alleviate inflammatory
processes in the gut of humans [24]. The link between antiinflammatory lactic acid bacteria and alteration of the course
of diabetes is still far from proven, but any condition influenced
so greatly by diet is surely also affected by the microbiota in
the gut, and alteration of these organisms is at least worthy of
investigation.
During the crisis of severe acute respiratory syndrome, sales
of certain probiotics increased substantially in Hong Kong, apparently because of the perception that they could enhance
immunity. In fact, there are a number of studies that show
improved recovery from respiratory infections with use of orally
administered probiotics [25–30]. This implies modulation of
immunity and distant site effects of ingested microbes. The use
of probiotics to mitigate or augment the treatment of avian
influenza or other major respiratory infections is worth invesS64 • CID 2008:46 (Suppl 2) • Reid
tigating, especially since antiviral therapies may not work for,
or be available to, the populations of people infected. As a first
step, studies are needed to understand whether and how probiotic organisms alter susceptibility of the respiratory tract to
disease.
BIOFILMS AND SIGNALING
The growing rates of antibiotic resistance and the realization
that biofilm formation makes it more difficult for antibiotics
to eradicate infections have led to studies of new approaches
to managing infectious biofilms. These include disruption or
penetration of biofilms by beneficial microbes or alteration of
the environment to restore a noninfectious biofilm. The relatively short interval during which a vaginal microbiota can
change from one dominated by lactobacilli to one dominated
by anaerobic gram-negative pathogens that cause bacterial vaginosis provides a good model system to study these issues.
The failure of metronidazole to cure bacterial vaginosis in
some cases [31] and the finding of clue cells covered in dense
biofilms within 3 weeks after treatment would suggest an inability of the drug to eradicate biofilms. This is particularly
likely given that major resistance of planktonic vaginosis bacteria to metronidazole has not been reported. An in vitro study
has shown that Gardnerella vaginalis biofilms can be penetrated
by L. rhamnosus GR-1, leading to rapid disruption and death
of the pathogens [32]. This correlates with human studies in
which Gardnerella species are displaced [33] and bacterial vaginosis is cured [34] by probiotic lactobacilli. Other studies
have shown that this Lactobacillus strain can also prevent Candida albicans biofilms from forming, and it can kill the yeast
in vitro [35]. In the near future, fluorescent in situ hybridization
and confocal laser microscopy, used successfully to study complex oral biofilms, will be used to better understand how gramnegative and gram-positive bacteria interact in biofilms and are
affected by different nutrients [36].
Within dense multispecies biofilms, it is clear that several
communication tools are used. Quorum-sensing molecules,
such as autoinducers, provide information that helps microbe
populations modulate growth, expression of virulence factors,
and formation of biofilms [37–39]. The discovery of a signal
produced by L. reuteri RC-14, which significantly down-regulates exotoxin production by Staphylococcus aureus [40], is the
cusp of a new and exciting field that will see many signaling
molecules identified that potentially can be used to prevent and
treat infections in the future. Likewise, factors that influence
host cells—such as those produced by B. thetaiotaomicron,
which affect Paneth cells [6], and those produced by lactobacilli
that induce mucin production [41] and down-regulate inflammatory processes [42]—will in due course become part of the
new biotechnological approach to patient care. Using an Affymetrix gene array, we have recently shown that L. rhamnosus
GR-1 instillation into the vagina is associated with up-regulation of some host defense factors (authors’ unpublished data).
By the combination of human gene-array studies with the application of defined, genome-sequenced probiotic organisms,
it will be possible to more optimally target specific probiotic
strains to specific patients or conditions.
DELIVERY MECHANISMS
Advances in biomedical engineering will prove to be equally
important to molecular biology in terms of the developing
systems that deliver bacteria and/or nutritional factors to the
host. These will include encapsulating probiotics, such that they
rehydrate at specific sites, and encasing prebiotics in nanoaggregates that protect against stomach acid and deliver their
payload when the pH reaches 7.4 [43]. Potentially, such nanoencapsulation will also allow delivery in foods such as biscuits,
whereas targeted, water-protected macrocapsules containing
probiotic organisms may prove useful in animal food pellets
and perhaps in liquids, which currently cannot be used because
of problems with shelf stability. At the macromolecular level,
it will soon be possible to coat capsules with biosensors that
detect the optimal conditions for release of probiotic contents.
Likewise, recombinant strains will be created to respond to
specific triggers in a host (e.g., a pathogen’s toxin) and produce
factors to counteract them. Such sensors are already being described, such as the repressor FucR in B. thetaiotaomicron,
which responds to l-fucose availability [44].
The Given Imaging’s PillCam is a good example of engineering technology in medicine. This tiny capsule is swallowed,
and, as it passes through the host, it takes photographs of the
mucosal tissues. In time, such devices will be controlled remotely, will have sensors and sampling devices, and will deliver
payloads at the desired site. Such science fiction will become
reality within 10 years.
In summary, molecular, nano, biochemical, microbiological,
immunological, and engineering sciences hold the key to future
advances in the clinical application of probiotic and prebiotic
products. To make these breakthroughs of value to patients,
companies in the food, biotechnology, engineering, and pharmaceutical industries will have to learn from each other with
regard to production of strains and their delivery-suitable formulations, while traditionally bureaucratic and slow-moving
regulatory agencies will have to become visionary, proactive,
and more adept at processing these new developments. Unless
this happens, the great strides made in science may never reach
the people who need it the most.
Acknowledgments
The assistance of the International Scientific Association for Probiotics
and Prebiotics is appreciated.
Financial support. Ontario Ministry of Agriculture and Food.
Supplement sponsorship. This article was published as part of a sup-
plement entitled “Developing Probiotics as Foods and Drugs: Scientific and
Regulatory Challenges,” sponsored by the Drug Information Association,
the National Institutes of Health National Center for Complementary and
Alternative Medicine (1R13AT003805-01 to Patricia L. Hibberd), the California Dairy Research Foundation, Chr. Hansen, the Dannon Company,
General Mills, Institut Rosell, and Yakult International.
Potential conflicts of interest. G.R. has received recent research funding from Wyeth Aherst and Otsuka, owns patents licensed to Chr. Hansen
for probiotics for women’s health, and owns patents on the use of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 for urogenital
health.
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