Best Practice & Research Clinical Gastroenterology 30 (2016) 17e25

Contents lists available at ScienceDirect

Best Practice & Research Clinical

Gastroenterology

Probiotics: definition, scope and mechanisms of

action
Gregor Reid, Professor of Microbiology and Immunology, and
Surgery a, b, *
a
Canadian Center for Human Microbiome and Probiotic Research, Lawson Health Research Institute,
London, Ontario, Canada
b
Department of Microbiology and Immunology, Division of Urology, Department of Surgery, Western
University, London, Ontario, Canada

a b s t r a c t
Keywords:
Probiotics For a subject area of science, medicine and commerce to be so
Terminology recently defined and investigated, few can compare to probiotics
Mechanisms for the controversy they have incited. Barely a paper is published
Guidelines without the use of a different definition, or challenging the most
used one, or proposing a different nuance of it. The situation has
become even more surreal with the European Food and Safety
Authority banning the word probiotic for use on labels. The reit-
eration of the FAO/WHO definition by the world's leading group of
probiotic experts, should provide relative consistency in the near
future, but what are the causes of these aberrations? This review
will discuss the rationale for the definition, and the scope of the
subject area and why alternatives emerge. While mechanisms of
action are not widely proven, in vitro and some in vivo experiments
support several. Ultimately, the goal of any field or product is to be
understood by lay people and experts alike. Probiotics have come a
long way in 100 years since Metchnikoff and 10 years since their
globalization, but their evolution is far from over.
© 2015 Elsevier Ltd. All rights reserved.

* Canadian Research & Development Centre for Probiotics, Lawson Health Research Institute, 268 Grosvenor St., N6A 4V2,
London, Ontario, Canada. Tel.: þ1 519 646 6100x65256.
E-mail address: gregor@uwo.ca.

http://dx.doi.org/10.1016/j.bpg.2015.12.001
1521-6918/© 2015 Elsevier Ltd. All rights reserved.
18 G. Reid / Best Practice & Research Clinical Gastroenterology 30 (2016) 17e25

Introduction

Definitions for many, are necessary evils, which describe the meaning of a term, and in some cases
every object the term describes. But, with differences of opinion of the definition of definition itself,
one can appreciate why readers will have already tuned out.
In defining probiotics as “Live microorganisms which when administered in adequate amounts
confer a health benefit on the host”, the Expert Panel commissioned in 2001 by the Food and Agri-
culture Organization of the United Nations, at Argentina's request, and supported by the World Health
Organization, provided clarity and distinctiveness to the term probiotic [1]. While initially this was in
relation to food, the Panel was cognisant of the potential for probiotics to be used under many regu-
latory categories. The wording was carefully chosen to encompass all real or envisaged ways of
delivering various types of microbes to a host. Therefore, words like consumption and ingested were
too restrictive, and ‘administered’ was chosen. The Panel was clear that there is no such entity as a dead
probiotic. If dead organisms convey a benefit, they should be referred to by a different term. Yet to this
day, authors continue to misunderstand the term, even when they include the actual reference to the
FAO/WHO definition [2]. In this particular example, the author states “Probiotic applications can be
either mono or multiple strains, or even in combination with prebiotic, immunostimulants such as
synbiotics and synbiotism, and in live or dead forms.” It behoves editors and journals to enforce the
proper use of the definition, but this is rarely done.
The conferring of a health benefit has been criticized as being too vague or requiring further clar-
ification. In fact, a Working Group of FAO/WHO was convened in 2002 to provide such clarification [3].
More recently, the FAO/WHO definition has been re-evaluated and supported [4]. Clearly, a benefit to
the host must be realized and shown to be above and beyond that placebo, yet the majority of products
on the market have not undergone such appropriate testing and verification.
In this review, reasons for misunderstanding probiotic will be explored, as well as mechanisms
whereby their effects are conveyed.

Why the confusion?

It is clear from the literature and conference presentations around the world, that either people
want to define probiotics themselves, or they misunderstand what probiotics are. Examples of the
former include the FDA which still uses the term biotherapeutics, ostensibly because no one has
taken the time to update the field. Terms such as biotherapeutics and pharmacobiotics appear to
emerge as a means of specifying an activity rather than being an alternative to the term probiotic
and its definition. The very use of the word therapeutic indicates treatment of a disease, and would
be specific for only a drug, whereas probiotics can be foods, supplements, drugs, medical foods,
medical devices or cosmetics. The term pharma implies pharmaceutical, either as a drug or a
chemical component. Factors secreted by probiotic strains have been referred to, by some, as
‘postbiotics’.
The term ‘Novel’ probiotics [5] is simply a qualifier rather than a new definition. The term ‘psy-
chobiotics’ has been propagated since 2012 [6] defined as “a live organism that, when ingested in
adequate amounts, produces a health benefit in patients suffering from psychiatric illness”. There are a
number of issues with this. Firstly, creating a sub-category of probiotic per se is not necessarily a
problem, and in this case, it should be cited as such. Secondly, when introducing a new term and
definition, it should be clear and encompassing. In the case of psychobiotics, it seems premature, and
requires the type of clarification undertaken for probiotics. For example, must the organism be
ingested, or could signalling molecules from bacteria in the oral cavity, skin, urogenital tract influence
the brain? Do the patients have to be suffering, and to what extent suffering, or could they be at risk of a
psychiatric illness? Would organisms influencing the brain but not psychiatric illness be termed
psychobiotics? Such clarifications would be helpful.
Others are dissatisfied with the definition because it does not specify health benefits and is driven
by corporate profits [7]. Ironically, the authors believe that the concept of probiotics is inhibiting the
field of phamacobiotics. In fact, how probiotics are regulated, sold and marketed has nothing to do with
the definition. As already stated, probiotics, unlike pharmacobiotics, can be sold as foods and
 G. Reid / Best Practice & Research Clinical Gastroenterology 30 (2016) 17e25 19

supplements. One could argue that the term pharmacobiotics is also confusing and almost all pharma
agents are non-living chemical entities, and thus should not include living organisms even if a
component of that organism is responsible for a specific effect on the host. With respect to not defining
health benefits, this is actually an advantage of the probiotic definition, since it does not limit living
microbes function to benefit the host. The term ‘health’ is defined by the WHO as “a state of complete
physical, mental and social well-being and not merely the absence of disease or infirmity”, which is
draconian and untenable by any human. Thus it is beyond the challenge of any probiotic experts to try
to fit specific benefits within this framework. Rather, the onus is on the person or company to show
that a probiotic induces a certain benefit that can be described and measured.
Other examples of a case being made for specificity might be to add the phrase reduces the duration
an ailment. But too is vague, and at best would be a sub-category.
Many regulatory agencies have added to the confusion. Although serving an important purpose of
policing the safety of products that act upon the human body, invariably, their mandate and processes
are antiquated and they still state that only drugs can cure, treat, mitigate and prevent disease. In fact,
foods have been known to do this for years, even if not with the immediate effect of drugs. Different
claims are permitted for categories of products within the regulatory system, and therefore a probiotic
food is not allowed to claim prevention of disease. Often, the best claims a food can obtain involve
structure and function, but for consumers this results in a lack of adequate information and clarity. A
Canadian Clinical Guide has been created to fill this consumer gap [8]. It contains a table of probiotic
products sold in Canada (many are also sold elsewhere), and based upon peer-reviewed publications
which they list, the guide puts each product in a box based upon levels of evidence. This helps con-
sumers and practitioners determine for themselves if a product has been tested in humans and found
to have merit.
Canada is arguably at the forefront of regulating probiotics, having already permitted informative
claims on some products. The situation in Europe is the polar opposite. The bureaucrats decided to
use a committee within the European Food Safety Authority (EFSA) to adjudicate probiotic claims.
Composed of almost a whole committee of people with no expertise in probiotics, decision were
made arbitrarily and without adequate guidelines or feedback. In one instance, the committee
completely misunderstood the importance of bacterial ascension from the rectum to vagina and the
requirement of the urogenital tract for nutrition [9]. In another case, they criticized an excellent peer-
reviewed study on a probiotic that prevented antibiotic-associated diarrhea [10], somehow
concluding years later that some patients might have known they were receiving the probiotic, when
no effort was made to interview the patients and see if this might have been true. Overall, with no
claims being permitted, the European Union (EU) essentially decided to ban the use of the word
probiotic on labels, as it implies conferring a health benefit, and in their view none of the products
had been shown to do that.
The fundamental problem lies not only with the EU and EFSA's interpretation of policies they
created, but also in their understanding of the definition of probiotics and the regulatory system they
advocate. With an emphasis on safety for well-defined chemicals that treat disease, there is little room
for biological agents (bacteria, fungi) whose batches are not 100 percent reproducible, and whose
activity may function not through the host per se, but through interactions with other microbes in the
host, which no committee can control or truly measure risk versus benefit. Except, the track record for
probiotic safety and lack of risk compared to drugs is exemplary. In the USA, the benefits of probiotics
cannot be assessed in disease unless the foods are registered as drugs. Thus, adding to the evidence of
benefits to satisfy EFSA and FDA is prohibitory.
The net effect has been confusion in Europe and false marketing in the USA, with web sites stating
probiotics for acne, chronic bowel problems, celiac disease, inflammation, allergies, yeast infections,
acid reflux, stress and many other diseases and conditions, with an insertion “These statements have
not been evaluated by the Food and Drug Administration. This product is not intended to diagnose,
treat, cure or prevent any disease.” Since agencies appear to lack the resources to force companies
to retract these claims, consumers may believe them. Certainly, without searching peer-reviewed
papers, there is no way to identify the products tested in, and shown to benefit, humans. If the label
doesn't even allow ‘probiotic’, what do regulators think consumers can do to check studies on the
product?
20 G. Reid / Best Practice & Research Clinical Gastroenterology 30 (2016) 17e25

What can consumers and patients look for?

The failure or most medical schools to adequately teach future physicians about the human
microbiome and applications of probiotics and prebiotics, has left a void that has serious implications
for patient care. While continuing medical education courses and conferences have started to offer
lectures on these topics, widespread ignorance or misunderstanding prevails. Admittedly, this is a
perception that is not easy to prove. But, 19% of Irish doctors surveyed recommended probiotics for
autism when there are no data on this [11]; New Zealand general practitioners showed a lack of
knowledge in the use and indications for probiotic therapy [12]; even gastroenterology specialists have
not received training in this area [13]. Interestingly, patients appear to be familiar with probiotics [14]
and in the Republic of Georgia 94% of doctors use probiotics either as monotherapy (22%) or in com-
bination (78%) to treat acute diarrhea [15]. The latter is perhaps not surprising as probiotic use has been
widespread for many years in the former Soviet Republic, and is even part of their space program [16].
A survey of American gastroenterologists in 2010 reported 93% had patients taking probiotics most
often for irritable bowel syndrome [17], so there are exceptions and perhaps a trend to more physician
engagement. This is important as patients generally rely heavily on product recommendations from
doctors, and the growing literature on how the microbiome affects health [18], disease treatment [19]
and symptomatology [20], how probiotics can improve and/or supplement care [21e24], makes it
imperative that the knowledge gap for physicians be closed, and effective courses introduced to
medical programs.
Realising that regulators are unable or unwilling to police all products on the market, and con-
sumers have an almost impossible task of tracking which strains are in which products and what
human studies had been done on that formulation, a ‘tree’ system was proposed to categorize pro-
biotics [25]. Strain designation and end of shelf life viability count were minimal requirements, and the
lowest category would have undergone minimal human studies that showed a benefit, for example in
offsetting lactose intolerance. A middle category designation would be given to products in which at
least two randomized controlled studies of suitable sample size to show a difference, had been pub-
lished in peer-reviewed journals. This could include probiotics that counter adverse effects of antibi-
otics or restore microbial homeostasis to the vagina or mouth. The top category including recombinant
strains and species not previously used in foods and supplements, was intended for products targeting
vulnerable populations, such as premature infants and frail elderly. Such proposals can only be
incorporated if regulators and companies are willing to embrace them. Currently, there seems no
willingness to do so, but hopefully the idea has added to the intent of providing consumers more
information on which products meet probiotic status.
Many consumers and patients appear to be influenced by the viable count and number of strains
present in products, invariably believing that the more the better. Companies oblige by selling multi-
species products, but in almost all cases the compatibility of the strains have not been tested. In a rare
exception, L. reuteri RC-14 was added to L. rhamnosus GR-1 to provide activity against Gram positive
uropathogens to compliment the effects against Gram negative bacteria [26,27]. This step-wise
approach is the ideal way to create a multi-strain probiotic whose whole is better than the individ-
ual parts, for the stated purpose. In a seminal study, a product labelled probiotic containing 15 bacterial
strains was investigated. There was significant cross inhibition of growth amongst the strains, sug-
gesting that together they would be less effective at inhibiting pathogens such as Clostridium difficile
[28]. In other words, such products would be less effective than single strains. In essence, this approach
represents companies simply putting a group of strains into a capsule, sachet or table, and calling them
probiotic, when in fact they should not use this term. At the very least, these multi-strain products
should be tested in humans and any benefits they provide should be stated and supported by peer-
reviewed publication.

Mechanisms of action

A common question is how do probiotics work? Given that different strains and product formu-
lations exist, there is no single answer. The perception is that mechanisms of action of drugs are all
known, and thus the same should apply for probiotics. Certainly, pharmaceutical agents like Warfarin
 G. Reid / Best Practice & Research Clinical Gastroenterology 30 (2016) 17e25 21

prescribed to reduce the risk of cardiac arrest decrease the body's ability to form blood clots by blocking
the formation of vitamin Kedependent clotting factors. Such knowledge was acquired by animal and
human studies and was arguably made easier to prove because of the specific mode of action. But this is
not necessarily a trait of all drugs. There are conflicting data on cardiovascular disease prevention using
highly studied aspirin, whose mechanisms are presumed to be known [29]. So, proving cause and effort
is not simple.
Bacterial strains such as L. reuteri SD2112 (ATCC 55730) and L. reuteri RC-14 are different genetically
and functionally [27], with the former producing reuterin believed to be important to inhibit patho-
gens in the gut [30] and the latter producing biosurfactants that inhibit attachment of uropathogens
[31]. This further emphasizes the need to not use the general term probiotics, when describing
mechanisms of action, but try to specify the strains. Having said that, there are general benefits
ascribed to probiotics, with a shared mechanism of creating a more favourable gut environment, and
supporting a healthy digestive tract and a healthy immune system [4]. This conclusion was based upon
high-quality meta-analyses and activity against infectious diarrhoea, antibiotic-associated diarrhoea,
intestinal transit, irritable bowel syndrome and other conditions. This does not mean the mechanisms
are the same for each condition, nor that precise mechanisms have been proven. Efforts to do that are
sparse in humans mostly because of difficulty in using recombinant strains, but one rat study nicely
showed that a bacteriocin produced by L. salivarius UCC118 was responsible for inhibition of Listeria
monocytogenes [32].
A study of atopic dermatitis patients showed that 12 weeks of treatment with L. salivarius LS01 and
Bifidobacterium breve BR03 provided benefits through reducing gut microbial translocation, and acti-
vating an immune response as detected by improved T-helper cell (Th)17/regulatory T cell (Treg) and
Th1/Th2 (P ¼ 0.028) ratios [33]. This certainly suggested mechanistic pathways, even if strain knock-
outs were not assessed. Other human studies support the concept of improved barrier function pre-
venting disease [34].
The manipulation and control of the immune system by probiotics is hard to evaluate and make
general conclusions. The same strain might enhance antimicrobial activity and the Th1 response
[35,36] as well as increase anti-inflammatory Th2 response [37,38] or regulatory activity [39]
depending on the host and perhaps delivery method. As a review elegantly showed, probiotic
strains such as L. rhamnosus GG have multiple mechanisms to interact with the host, and likewise there
are many ways for the host to respond [40]. Arguably, the strain can assess the niche into which it is
placed, and react accordingly. This means it could relieve inflammation and stimulate Th1 responses,
both of which might appear to be contradictory mechanisms. Specificity is still evident in that L.
rhamnosus GG reduces the duration of diarrhea [41] but does not alleviate constipation [42].
An intriguing aspect of probiotic strains is the ability of some to confer effects distant to the site of
administration. This may occur through actual transfer of the organisms, for example from the gut to
the mammary glands of lactating women [43]. Or, it may be due to production of molecules that are
adsorbed across the intestine [44], or that influence host compounds directly or indirectly. Examples of
the latter include molecules that increase the adiponectin/leptin ratio following cardiac ligation with
improvements in cardiac re-modelling [45], and in colostrum with implications of lowering the risk of
gestational diabetes mellitus [46]. The lowering of blood cholesterol is another example, with mech-
anisms including reducing deoxycholic acid (DCA) levels in the intestine [47], deconjugation by bile salt
hydrolase activity [48], production of compounds that inhibit 3-hydroxy-3-methylglutaryl coenzyme
A, and through assimilation of cholesterol [49]. The ability of probiotic strains to reduce the severity
and duration of respiratory tract infections illustrates a distant site effect likely mediated by
enhancement in immune mediators [50].
A notable and consistent finding in probiotic studies is that relatively few organisms, compared to
the numbers in the niche into which they are entering, can convey benefits. This is true for the highly
populated intestinal tract in which a mere fraction (109e1010 per mL) of probiotics are added. Inter-
estingly, microscopic analysis of the healthy vagina shows a sparse number of lactobacilli, suggesting
that numbers per se are not the critical issue. Indeed, competition experiments have shown that small
proportions of lactobacilli can displace and compete against larger numbers of pathogens on surfaces
[51,52]. The production of biosurfactant compounds could be one such mechanism of displacement
[31,53], but clearly several possibilities exist [54].
22 G. Reid / Best Practice & Research Clinical Gastroenterology 30 (2016) 17e25

In the case of distant sites to which the probiotic itself does not travel, like the brain, the molecules
they produce, such as neurochemicals [55], or that they stimulate the host to product, appear to reach
the brain at least through the vagus nerve system [56]. Potentially, this is the most intriguing mech-
anistic area of probiotic research, as it relates to the organ controlling much of what we do. It seems
feasible that radically changing the gut microbiota through transplantation or administration of pro-
biotics strains producing specific factors, could not only alter mood and memory [57], but influence
what we eat and when [58], and how slowly or rapidly pathological changes occur.

Practice points

 Many ‘probiotic’ products are sold, but only those tested in humans should be recom-
mended, for example those listed in the Clinical Guide of Probiotic Supplements.
 As microbes are living entities with somewhat complex components, they can interact with
the host and other microbes in different ways. Thus, effects can be strain specific but not
necessarily only conferring a single health benefit.
 A number of mechanisms whereby probiotic organisms confer their effects have been
described including through the response of the host and other microbes.
 Educational courses on probiotics and the microbiome should be a requirement for all
medical schools, and encouraged for practicing physicians and surgeons.

Research agenda

 Sampling human tissues and analysing the metatranscriptome is key to understanding how
probiotic organisms are influencing the host environment.
 Time-course microbiota and metabolomics samples collected from well-defined clinical co-
horts, such as patients at risk of type 1 diabetes or with early stage dementia, should help
identify triggers of physiological and endocrinological change, and determine the influence
of probiotic intervention.
 Studies are needed to align probiotic therapy with drugs and dietary intake.

Summary

In summary, with microbes being such a large physical part of the gastrointestinal tract, as well as
other sites, it is vitally important that specialists appreciate their existence, and consider what role they
might have in health and disease. It will not be long before rapid diagnostic tools become available to
inform about the contents of the microbiome, and while guidance will accompany results, it will make
life much easier for interpreters of the findings, if they have some knowledge of this field. Unlike much
or clinical microbiology that has linked one pathogen with a disease, the novelty of the microbiome are
is that it will be composition and functionality of multiple organisms that likely correlate with health.
The application of probiotics as therapy or health maintenance remedies will require thorough
documentation of the strain(s) and product formulation and mechanisms of action.
If computer guided sampling can be done within the gut in real time and without content evacu-
ation, and RNA analysed, it will be possible to learn how probiotics work at different sites. Conjoint
metabolomic and proteomic studies can then identify molecules mediating effects. The ability to grow
currently unculturable organisms and manipulate them through recombination, will further improve
 G. Reid / Best Practice & Research Clinical Gastroenterology 30 (2016) 17e25 23

our armamentarium for patient care. Thus, the future will see more microbial species added to food
and administered as supplements and drugs, that target a multitude of conditions. Microbes have
always been a major component of the human body; it's just that humans are finally appreciating their
value.

Conflict of interest

The author has no conflict of interest with respect to the content of this review.

Acknowledgements

Our work is supported by a grant from NSERC, but it played no part in the preparation of this paper.

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