Digestive and Liver Disease 49 (2017) 1177–1184

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Digestive and Liver Disease

journal homepage: www.elsevier.com/locate/dld

Review Article

A consumer’s guide for probiotics: 10 golden rules for a correct use

Marco Toscano a , Roberta De Grandi a , Luca Pastorelli b,c , Maurizio Vecchi b,c ,
Lorenzo Drago a,d,∗
a
Laboratory of Clinical Microbiology, Department of Biomedical Science for Health, University of Milan, Milan, Italy
b
Department of Biomedical Science for Health, University of Milan, Milan, Italy
c
Gastroenterology and Gastrointestinal Endoscopy Unit, IRCCS Policlinico San Donato, San Donato Milanese, Italy
d
Laboratory of Clinical-Chemistry and Microbiology, IRCCS Galeazzi Institute, University of Milan, Milan, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Probiotics are used all over the world as their beneficial effects on the human organism have been widely
Received 15 February 2017 demonstrated. Certain probiotics can down-regulate production of pro-inflammatory cytokines and pro-
Received in revised form 19 July 2017 mote intestinal epithelial barrier functions, increasing an anti-inflammatory response and contributing
Accepted 20 July 2017
to the host’s overall health. The main mechanisms by which probiotic microorganisms can interact with
Available online 1 August 2017
the host are by modulating the immune system and the epithelial cell functions and interacting with
intestinal gut microbiota.
Keywords:
To date, hundreds of different microorganisms are used for the formulation of numerous probiotic
Anti-inflammatory response
Gut microbiota
products; therefore, it is very difficult to choose the best probiotic product for specific or more general
Multistrain products needs. Therefore, physicians are getting more and more confused due to the high number of commer-
cial products which are often lacking healthy effects on the host. Therefore, the aim of this paper is to
demonstrate the main characteristics that probiotic microorganisms and products should possess to have
a positive impact on the host’s health. To this purpose, this review suggests “10 golden rules” or “com-
mandments” that clinicians should follow to properly select the optimal probiotic product and avoid
misidentifications, mislabelling and “pie in the sky” stories.
© 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

1. Introduction
In the last few years, a growing interest in studying and using
probiotic microorganisms has been observed, not only for the treat-
ment of gastrointestinal diseases but also for the improvement of
overall human health. Indeed, several studies highlighting both the
systemic activity of probiotics and their beneficial role in ame-
liorating diabetes and allergic diseases management have been
published [1–4].
The leading mechanisms by which probiotics are thought to
be effective on the host’s health are their ability to modulate
the intestinal immune system, to improve intestinal physical
and immunological barrier functions [5,6] and displace potential
pathogenic microorganisms by competitive exclusion through the
production of antimicrobial peptides [7]. Recently, there has also
been a great interest in studying the effect of probiotics in modu-
lating the gut microbiota composition [8–11]. Commensal bacteria
belonging to intestinal microbiota protect the host from the action
of pathogens, regulate the host’s fat storage, stimulate intestinal
angiogenesis and the immune system and aid the digestion of
numerous dietary components [8,11]. However, gut microbiota is
often associated to many conditions of clinical interest, in partic-
ular dysbiosis [11]. Consequently, it is clear that the manipulation
of intestinal microbiota composition by means of probiotics may
be a promising approach to ensure the correct maintenance and
improvement of human health.
To date, hundreds of different intestinal microbial species are
used in the preparation of probiotic supplements and, for this rea-
son, it is very difficult to choose the probiotic product that is best
suited to a patient’s needs.
The common approach to this issue should be one that follows
a set of specific guidelines, which guarantee a probiotic product’s
quality and efficacy, such as:
• proper microbial species and strain identification of all microor-
ganisms contained in the product, with the deposit of all strains
in an international culture collection;
• characterization of the safety and probiotic efficacy of each strain;
• evaluation of the microbial beneficial effects on the human host.

∗ Corresponding author at: IRCCS Galeazzi Orthopaedic Institute, Milan, Italy. However, today it is very difficult to identify which probiotic
E-mail address: lorenzo.drago@unimi.it (L. Drago). formulation is the best one to improve the human health, mostly

http://dx.doi.org/10.1016/j.dld.2017.07.011
1590-8658/© 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1178 M. Toscano et al. / Digestive and Liver Disease 49 (2017) 1177–1184
Table 1
The 10 recommendations of probiotics.
1st Knowing the correct definition of probiotics
2nd Microbial lysates, non-living bacteria and non-colonizing spores
cannot be considered probiotics
3rd Getting an exhaustive probiotic identikit
4th Monostrain or multistrains products: making the correct choice
5th Avoid antibiotic resistance genes in probiotic strains and products
6th Choose probiotic strains resistant to gastrointestinal environment
7th Probiotic strains must be able to colonize the gut
8th Preferring probiotics that are able to positively interact with gut
microbiota
9th Be sure about the safety of probiotic strains and evaluate the subject
health status before probiotic administration
10th Preferring probiotics with a demonstrated clinical efficacy
because of the lack of knowledge of specific probiotic features,
which should be considered.
The present paper aims to clearly inform Manufacturers and
Clinicians on the basic characteristics that probiotic microorgan-
isms, and above all, probiotic products should possess to be used
as positive bio-modulators of human health. This study also aims
to provide a useful and quick “instruction kit” for physicians to fol-
low, in order to give an easy and immediate interpretation of the
probiotic(s) under consideration. The 10 “commandments” or “rec-
ommendations” clinicians should never forget when dealing with
probiotics are listed in Table 1.
2. 1st commandment: know the correct definition of
probiotics
The classic definition of probiotics is “live microorganisms
which, when administered in adequate amounts, confer a health
benefit to the host” [12]. Interestingly, bacteria belonging to Lac-
tobacillus and Bifidobacterium genera are the most used probiotic
microorganisms in the food industry, due to their probiotic and
beneficial effects. However, also non-bacterial microorganisms,
such as Saccharomyces boulardii and Saccharomyces cerevisiae, are
often used as probiotics because of their postulated activities and
beneficial effects both at intestinal and systemic level.
Sometimes, probiotics are referred to as “biotherapeutics” or
“pharmacobiotics”, leading to a profound misunderstanding of
what probiotics are [13]. Indeed, the term “therapeutic” indicates
the handling of a disease and, therefore, should be referred only
to a drug, while probiotics can have a broader meaning [13]. Con-
versely, the term “pharma” is mainly related to pharmaceuticals,
either drugs or chemical components [13]. On the contrary, pro-
biotics are not drugs but, instead, they are “biomodulators” that
must be resistant to gastric juice and bile salts to arrive intact to
the intestinal environment and exert their beneficial activity on the
host’s organism [7]. Indeed, a probiotic microorganism should also
be able to positively influence the host’s health, leading to beneficial
effects after its intake [7].
2.1. Recommendation
Probiotics are live microorganisms acting as powerful biomod-
ulators, with a positive impact on human health. Indeed, the official
definition of probiotics given by FAO/WHO Committee is “live
microorganisms which, when administered in adequate amounts,
confer a health benefit to the host.”
3. 2nd commandment: microbial lysates, non-living
bacteria and non-colonizing spores cannot be considered
probiotics
In the last years, there was an increasing interest in study-
ing microbial components (e.g. proteins, lipids or nucleic acids)
and non-living bacteria for their beneficial effects on the human
organism. Several evidences highlighted the ability of dead cells
to induce a wide range of biological responses in the host enhanc-
ing the anti-inflammatory response [14–16]. The administration of
heat-killed bifidobacteria and lactobacilli, for instance, has been
observed to induce a pronounced increase in the production
of anti-inflammatory cytokines and modulate positively allergic
symptomatology [16,17]. Consequently, the immunostimulatory
effect of probiotics seems not to depend on the cells being alive but
it may be linked directly to the biological and physical nature of
specific microbial components. Indeed, there is also evidence that
metabolites and cell fractions of probiotics can exert a positive func-
tion on the host [14]; DNA, lipopolysaccharides, peptidoglycans and
cell homogenates have all a strong immunomodulatory effect act-
ing on the innate immune system and they have been demonstrated
to decrease the risk of atopic dermatitis onset in children [18–21].
Although microbial lysates have a beneficial impact on the host
health, they cannot be considered as probiotics; indeed, they are
effective on the organism only if continually administered not being
able to induce biological responses for a long period. Conversely,
only alive microorganisms can have a lasting impact on the organ-
ism being able to colonize the gastrointestinal environment and so
persist in the host.
Regarding microbial spore formers, instead, only spores that can
germinate in the gastrointestinal tract and colonize the environ-
ment can be considered as probiotics and used in the formulation
of products [22]. Spores can survive transit across the stomach bar-
rier and are more resistant than vegetative cells; moreover, they can
germinate in presence of nutrients and favourable environmental
conditions becoming able to colonize the gut and exert their probi-
otic effects [22]. However, if spores do not germinate they are not
active at intestinal level and no beneficial effects on the host will
be highlighted.
3.1. Recommendation
Although microbial components have been observed to have
a beneficial impact on the host’s immune system and health,
they cannot be considered as probiotics, as well as non-colonizing
spores.
4. 3rd commandment: get an exhaustive probiotic identikit
In the last few decades, advances in molecular biology and
microbiology techniques have allowed a clear identification and
characterization of bacterial strains, avoiding any confusion about
probiotic identity [23]. Identification and tracking of individual
strains is essential when microorganisms are used for the formu-
lation of probiotic products; to this purpose, phylogenetic analysis
is the most powerful tool for bacterial taxonomic classification, as
ribosomal RNA sequences provide detailed information for a com-
parative identification of probiotic microorganisms [24]. When a
microorganism is used for the formulation of food supplements or
other products for human consumption, its full characterization at
both genomic and physiological level is critical. Each microorgan-
ism contained in probiotic products must be identified at species
and strain level, according to the International Code of Nomencla-
ture and its microbial genome must be completely sequenced. This
allows the identification of every single gene involved in bacte-
rial metabolism and its function. Consequently, the safety of food
products and, above all, of commercial probiotic strains should be
evaluated before their launch on the market. Generally, the guide-
lines require:
 M. Toscano et al. / Digestive and Liver Disease 49 (2017) 1177–1184
1) minimum concentration of 109 CFU of live microorgan-
isms/daily dose on labels (even less if the usefulness of this
lower dose has been clearly shown and supported at scientific
experimental level);
2) identification of each probiotic strain by integrating phenotypic
and genotypic characterization;
3) absence of pathogenic factors.
Commercial probiotic products currently on the market are
not always correctly labelled, as many microorganisms which are
claimed to be contained may not be present or their amount may
be lower than that declared on the label. In 2002, Weese et al.
detected several deficiencies in the labels of numerous Canadian
commercial probiotics for human oral intake, finding that bacteria
contained were improperly identified in 43% of analysed products
and the content of 25% of products were misspelled [25]. Drago et al.
also performed a quality assessment of the main probiotic products
available on the Italian market in 2011 [26], obtaining results simi-
lar to those of Weese. Indeed, in the Italian study, authors observed
that 42% of analysed products did not contain the declared bac-
terial amount of at least one of the labelled strains. Moreover, no
viable microorganisms were detected in 17% of products and a con-
tamination due to Enterococcus faecium was found in 8% [26]. The
presence of a non-declared microorganism, which might poten-
tially hold numerous pathogenic traits, represents a serious risk for
the health of the host.
These studies highlight the need for specific legislation, which
mandates the accurate identification and characterization of pro-
biotic strains in commercial products, as well as the careful testing
of all commercial products available on the market.
4.1. Recommendation
Only a well, fully characterized microbial strain should be used
for the formulation of probiotic products. Claimed dosage, genetic
typing and traceability are the main tools for a new probiotic.
5. 4th commandment: monostrain or multistrain products:
making the correct choice
Hundreds of different probiotic products are available on the
market. These products differ for excipients, amount and species
of microorganisms and their activity on the host’s organism. The
main difference among probiotic formulations is the presence of
one or more microorganisms in the same product [27]; however,
to date, it is difficult to understand which kind of formulation is
better. Many scientific studies have claimed that probiotic mixtures
have beneficial effects against a wide range of diseases affecting the
host, thus suggesting that the combination of different probiotic
microorganisms in the same product can confer greater protection
against several intestinal pathogens compared to monostrain prod-
ucts [28–30]. However, sometimes when a probiotic mixture has a
stronger effect than a mono-strain product, this is not due to the
real synergistic activity of all strains, but only to the amount of
specific strain/s contained in the product. [27]. The only way two
or more microbial strains can be more effective on the host than
a single strain is acting in a synergistic way, mutually enhancing
the activity of each strain and leading to a greater probiotic effect.
In a recent study, Drago et al. showed that a combination of Lac-
tobacillus salivarius LS01 and Bifidobacterium breve BR03 increased
the immunomodulatory activity of each single strain in peripheral
blood mononuclear cells of asthmatic patients [28]. In particular,
a significant increase in Interleukin 10 (IL-10) production, as well
as a reduction of the pro-inflammatory cytokines Interleukin 13
(Il-13) and Interleukin 17 (IL-17), were observed when LS01 and
1179
BR03 were used in combination rather than alone. Consequently,
these two bacterial strains showed promising probiotic features
and their combination may lead to a greater beneficial effect on the
human organism, enhancing anti-inflammatory immune response
and improving host’s overall health.
Moreover, in multi-strain products, it is also essential that all
microbial strains do not compete for nutrients and energy sources
and do not inhibit the growth of other strains [27–31]. Different
strains used in the same formulation, indeed, can act as antago-
nists, not only inhibiting probiotic activity of other microorganisms,
but also slowing microbial growth rate, as demonstrated by Vitali
et al. The authors, indeed, evaluated the growth compatibility of
three Bifidobacterium strains (Bifidobacterium infantis Y1, B. breve
Y8 and Bifidobacterium longum Y10) contained in a probiotic prod-
uct, observing a significant inhibition of the growth rate induced
on B. longum Y10 when co-cultured with the other 2 strains [32].
5.1. Recommendation
It is fundamental that research on multistrain probiotic formula-
tions is based on those microorganisms showing in vitro synergistic
and symbiotic activities to enhance the possibility of providing
more clinically effective probiotic formulations and justify, in this
way, the use of multistrain products with respect to monostrain
ones.
6. 5th commandment: avoid antibiotic resistance genes in
probiotic strains and products
The intensive use of probiotic products in association with
massive antibiotic therapies might establish, over time, a dan-
gerous reservoir of antibiotic resistance determinants in probiotic
microorganisms [33]. Generally, we can consider antibiotic resis-
tance among probiotic bacteria a positive feature, as these
microorganisms are able to restore intestinal eubiosis without
being influenced by a concomitant antibiotic therapy. However,
when antibiotic resistance can be transferred to pathogenic bac-
teria by means of resistant genes and determinants, a serious
risk for human health may arise, as pathogens can become resis-
tant to antimicrobial agents, nullifying antibiotic therapies and
further contributing to the spread of antibiotic resistance. Pro-
biotic bacteria, indeed, as any other microorganism, can develop
antibiotic resistance and resistant mutants if subjected to antibi-
otic selective pressure. The European Food Safety Authority (EFSA)
document recommends that commercial microbial strains used
as food supplements should not harbour any transferable antibi-
otic resistance and, consequently, the determination of Minimum
Inhibitory Concentrations (MICs) of the main antimicrobial agents
must be performed for each strain [34]. The antibiotic resistance
can be transferred in microbial communities by means of integrons,
conjugative plasmids, transposons, insertional elements and lytic
or temperate bacteriophage [34,35]. Interestingly, resistant gene
transfer occurring at high frequency has also been observed among
lactobacilli as well as from these microorganisms to pathogenic
bacteria, thus strengthening the hypothesis that probiotic bacte-
ria can act as an important reservoir of antimicrobial resistant
genes that are potentially transferable to any microorganism in the
intestinal environment [33].
The development of antibiotic resistance among pathogenic
microorganisms, such as Staphylococcus aureus, Pseudomonas
aeruginosa, Streptococcus pneumoniae and Klebsiella pneumoniae,
is leading to a severe health and economic burden worldwide
[36]. Optimizing antibiotic dosage and reducing underdosage is
fundamental to preventing the selection of resistant subpopula-
tions of bacteria during antimicrobial therapy [37]; however, it
1180 M. Toscano et al. / Digestive and Liver Disease 49 (2017) 1177–1184
is also essential to avoid probiotic products and other food sup-
plements containing microbial strains with transferable antibiotic
resistance genes to reduce the spread of antibiotic resistance. One
of the main problems existing in the probiotic market is the pres-
ence of numerous microbial strains that although do not adhere
to EFSA guidelines, they are widely used in the formulation of
probiotic products. In 2013, Drago et al. performed a phenotypic
and genotypic antibiotic susceptibility characterization of the main
microorganisms used for the formulation of Italian and European
probiotic products [38]. The authors showed that 24% of isolated
strains were resistant to erythromycin; 67% were resistant to gen-
tamicin; and 9.5% of all tested bacterial strains were resistant to
tetracycline. The real problem was not the antibiotic resistance
observed in the probiotic bacteria contained in these products, but
more so, the detection of several antimicrobial resistance genes,
such as ermB which confers resistance to erythromycin; tetW and
tetS which are involved in resistance to tetracycline; and aadA,
aph3-III, ant6-I and aac-aphI which mediate resistance to amino-
glycosides [38]. The numerous antibiotic resistance traits found
in many Italian and European probiotic strains may be the con-
sequence of an extensive use of antibiotics, which has created a
positive selective pressure for point mutations and the acquisi-
tion of mobile genetic elements encoding antimicrobial resistance
and leading to the spread of a variety of antimicrobial resistance
determinants. An example of testing antibiotic resistance genes
is recently followed by Lactobacillus kefiri LKF01 (DSM32079), a
new probiotic strain isolated from kefir grains [39]. This strain
showed sensitivity to erythromycin, gentamicin, penicillin, clin-
damycin and ampicillin and resistance only to ciprofloxacin and
tetracycline, but no resistance genes have been detected. Keeping
this in mind, the careful selection of a probiotic product is defini-
tively mandatory. In particular, attention must be paid not only
to the number of viable bacteria contained in products and the
number of microbial strains used for its formulation, but also to
the antimicrobial susceptibility profile of each strain, in order to
avoid the serious risk of potential antibiotic genes transfer between
intestinal microorganisms.
6.1. Recommendation
The safety of commercial microbial strains should be assessed
before their launch on the market to evaluate not only the presence
or absence of virulence factors, but also their ability of acquiring and
transferring antibiotic resistance-genetic elements. Furthermore,
all microorganisms contained in probiotic products should have a
susceptible profile with no antibiotic resistance genes.
7. 6th commandment: choose probiotic strains resistant to
gastrointestinal environment
The stomach’s low pH and the secretion of bile salts in the
duodenum make the gastrointestinal tract (GIT) a hostile envi-
ronment for microbial growth [40]. Generally, the viability of
microorganisms at gastric juice is species- and strain-specific;
Bifidobacterium animalis subsp. lactis, for example, was observed
to possess the highest acid tolerance among the Bifidobacterium
species, which generally shows a very low resistance to acidic
conditions [40]. These probiotic bacteria have several protective
mechanisms, which allow them to have an adaptive response to
low pH exposure [41,42], such as the ability to exclude protons from
inside by increasing H+-ATPase activity [43]. The same mechanism
is involved in the acid tolerance of Lactococcus lactis subsp. Lactis,
which can significantly increase the biosynthesis of cell membrane-
bound H+-ATPase when incubated under acidic conditions [44].
Resistance to bile salts is also an essential criterion to consider
when testing a probiotic microorganism for its ability to survive in
the small intestine. Bile salt hydrolase (BSH) activity allows bacte-
ria to be resistant to toxic conjugated bile salts and remain viable
in the gastrointestinal tract [45]. BSH activity has been detected in
probiotic microorganisms belonging to the Lactobacillus, Bifidobac-
terium and Enterococcus genera, which are generally located in the
intestinal environment [46–50]. BSH enzymes seem to play a piv-
otal role in tolerance to bile salts, protecting bacterial cells from the
toxicity of protonated form of bile salts through the formation of
the weaker unconjugated counterparts [51].
Therefore, resistance to low pH and ability to hydrolyse bile
salts are fundamental features in the selection of probiotic strains
for human consumption. Sahadeva et al. demonstrated the abil-
ity of several probiotic microorganisms contained in cultured milk
drinks to resist low pH and tolerate different bile concentrations
[52]. In particular, all probiotic strains remained viable after three
hours of incubation under acidic conditions (pH 3.0) and growth
was not inhibited even when bacteria were subjected to 2% of
bile [50]. Moreover, in a recent study Toscano et al. evaluated the
probiotic characteristics of three Bifidobacterium strains (B. breve
M-16 V, B. longum subsp. infantis M-63 and B. longum subsp. longum
BB536), reporting the strong ability of these strains to resist acidic
conditions, as shown by a high rate of viability after one hour
of incubation in gastric solution [45]. Furthermore, the Bifidobac-
terium strains tested in the aforementioned study were also able to
grow in the presence of bile salts, but only B. breve M-16V and B.
longum subsp. longum BB536 were capable of hydrolysing bile salts,
confirming that BSH activity is closely species- and strain-specific
[45].
7.1. Recommendation
Only probiotic products containing microbial strains able to
resist gastrointestinal conditions should be considered for human
consumption.
8. 7th commandment: probiotic strains must be able to
colonize the gut
As discussed before, probiotics play a pivotal role in maintain-
ing intestinal microbial eubiosis, mainly by inhibiting the growth of
pathogenic bacteria. This inhibition can be due to the production of
inhibitory compounds, such as bacteriocins and organic acids, or to
the competitive adhesion to the intestinal epithelium [53]. Indeed,
probiotics can block the adherence of pathogens competing for the
same intestinal receptor or inducing an increased production of
mucin that inhibits adhesion of several harmful microorganisms,
such as enteropathogenic Escherichia coli [54]. Moreover, the ability
to adhere, colonize the gut and survive over time in the intestinal
environment is a fundamental feature for probiotic microorgan-
isms, which allows them to exert their beneficial activities.
Biofilm production is another important mechanism probiotic
bacteria use to colonize the intestinal environment. Biofilms are
surface-associated communities of microorganisms embedded in
an extracellular polymeric matrix that virtually exist in every natu-
ral environment [55]. Biofilm formation is a developmental process
that has been compared to differentiation in multicellular organ-
isms. A typical biofilm forms when bacteria adsorb to a surface
and become attached, growing further and dividing into two layers
under the control of specific biofilm genes [55]. Biofilm produc-
tion is now considered one of the main bacterial survival strategies
necessary to increase access to nutrients, maintain the activity
of extracellular enzymes and protect microorganisms from the
action of antibiotic and pathogens [56]. Generally, the production of
 M. Toscano et al. / Digestive and Liver Disease 49 (2017) 1177–1184
biofilm and the related adhesion to intestinal cells are both strictly
species- and strain-dependent.
Several studies have assessed the ability of probiotic bacteria
to reach the gut and colonize it by evaluating the faecal recovery
of these microorganisms. As stated before, the ability to adhere
and colonize the intestine highly depends on the specific bacte-
rial strain tested, and this is the reason for many conflicting data
about fecal recovery of probiotic bacteria. Indeed, Dommels et al.
demonstrated that Lactobacillus reuteri DSM 17938 and Lactobacil-
lus rhamnosus GG were detected in fecal samples of volunteers after
3 weeks of probiotic daily consumption, suggesting a strong colo-
nization ability for both Lactobacillus strains [57]. Conversely, in
a previous study, Prilassnig et al. were not able to detect strains
belonging to Lactobacillus acidophilus, Lactobacillus gasseri, L. rham-
nosus and Lactobacillus casei, which were administered daily to
healthy individuals [58]. These data highlight the fact that various
probiotic microorganisms show different characteristics and prop-
erties to intestinal colonization and they further suggest the need
for a better choice of the best-suited strains. More interestingly, a
strong connection between probiotics and the gut barrier has been
observed. The gut barrier is essential to prevent bacterial adhe-
sion and regulate paracellular diffusion to the host’s tissues, but
is also fundamental to discriminate between commensal microor-
ganisms and pathogens, adjusting finely the immune response to
pathogenic bacteria [59]. In this context, probiotics are fundamen-
tal to implement the activity of intestinal microbiota in maintaining
the integrity of the gut barrier by influencing the secretion of mucus
and chloride, preventing the rearrangement of tight junction pro-
teins after exposure to pathogens and restoring disrupted epithelial
barrier by enhancing cell regeneration [59].
8.1. Recommendation
A microorganism with a strong in vitro probiotic activity but
unable to adhere and colonize the intestinal environment is not a
good candidate for probiotic product formulation.
9. 8th commandment: prefer probiotics that are able to
positively interact with gut microbiota
To date, few data exist regarding the ability of probiotics to
modulate intestinal microbiota composition. A recent review high-
lighted the lack of evidence showing a real impact of probiotic
microorganisms on intestinal microbiota composition, in terms of
diversity and richness of intestinal bacterial composition [60,61].
However, numerous factors can put into doubt the conclusions
drawn from different studies on gut microbiota, such as the use
of different probiotic strains either alone or in combination; the
duration of probiotic administration; the low-resolution methods
used to study intestinal microbiota composition; the small sample
size with low statistical power; and the possible inter-individual
variation in susceptibility toward the probiotic strain used [60].
Nevertheless, in a previous study based on a culture-dependent
approach, the authors of this paper demonstrated that the admin-
istration of the probiotic strain L. salivarius LS01 to patients affected
by atopic dermatitis (AD) could improve the classic symptoms asso-
ciated with AD, as well as lead to a significant decrease in intestinal
staphylococcal load [62]. Even if this result highlights the poten-
tial ability of probiotics to influence directly the gut microbiota
composition, other studies are needed to confirm these promising
data.
Interestingly, the impact of probiotics on gut microbiota may
not involve only changes in intestinal bacterial composition but
also in intestinal bacterial metabolism [63]. Indeed, in one study,
the administration of L. acidophilus was observed to have induced
1181
a significant decrease of ␤-glucosidase, nitroreductase and azore-
ductase activity in healthy volunteers [63]. All these enzymes are
closely associated to intestinal bacteria and are not produced by
the human host; indeed, 30 days after the end of probiotic oral
intake, enzyme activities were observed to return to baseline lev-
els. Furthermore, also the L. casei strain GG has the same effect
on gut microbiota metabolism, being able to reduce faecal ␤-
glucuronidase in the host [64]. Further studies with microbiota
composition modification as the primary outcome should be car-
ried out to clarify the impact and interaction of probiotics with
human gut microbiota, since this is influenced by a vast array of
host and environmental variables, thus making it difficult to state
clear and meaningful hypotheses on this issue.
9.1. Recommendation
Even if there are few and conflicting data on the impact of probi-
otics on gut microbiota composition, only probiotic products with a
strong intestinal activity should be considered and used for improv-
ing human health.
10. 9th commandment: be sure about the safety of
probiotic strains and evaluate the subject health status
before probiotic administration
The safety of probiotics is another important aspect we must
consider in choosing probiotic microorganisms for human con-
sumption. Generally, in vitro assays are useful to exclude potential
pathogenic microorganisms from being used as probiotics. How-
ever, a virulence factor may be down-regulated under conditions
used in safety assays, thus being undetectable [65]. For this reason,
in vivo models are often more useful than in vitro ones, as virulence
is mediated by several factors, including direct interaction with
the host. Also, screening of microbial genome for the presence of
virulence and pathogenic factors is very helpful to predict the pos-
sibility of non-active safety risk determinants [65]. Even if the oral
intake of probiotics is generally safe, as there are no side effects due
to their consumption, a problematic case of the use of Lactobacillus
bacteremia on an 11-month-old male patient with short gut syn-
drome has been described [66]. The patient had fever and hypoxia
and he received probiotic L. rhamnosus GG to treat rotavirus-related
diarrhea. However, the compromised condition of the patient likely
allowed the lactobacillus strain to reach the bloodstream, probably
translocating across the intestinal epithelium, where it acted as a
pathogen [66]. Similarly, a lactobacillus bacteremia was observed in
an immunodepressed 17-year-old male. This patient was affected
by ulcerative colitis (UC) and he took L. rhamnosus GG to treat a
flare-up of the disease, even if there were no clinical data to sup-
port probiotic efficacy in pediatric patients with UC. However, the
probiotic strain was able to exceed intestinal barrier and reach the
bloodstream, causing bacteremia [67]. Both case reports highlight
the importance of considering the health status of individuals, the
severity of the disease and other risk factors before administer-
ing probiotics, especially during conditions in which physical and
immunological gut barrier may be severely compromised, in order
to minimize all potential side effects and harmful consequences on
the host.
10.1. Recommendation
Generally, the real risk in using probiotic products is related
more to a compromised health status of the patient than to the
microbial strain used in the probiotic product.
1182 M. Toscano et al. / Digestive and Liver Disease 49 (2017) 1177–1184
11. 10th commandment: prefer probiotics with a
demonstrated clinical efficacy
All probiotic characteristics analysed in the previous sections of
this paper are fundamental for a microorganism to exert a benefi-
cial role in the human organism, counteracting pathogenic bacteria
in the gastrointestinal tract and promoting the overall health of the
host. However, it is also extremely important to evaluate the effi-
cacy of probiotic strains in improving human diseases, and above
all, improving the symptoms associated with a given disease. A
recent meta-analysis on probiotics highlighted how these microor-
ganisms can positively contribute to intestinal mucosal integrity,
exerting a pivotal role in reducing the risk of necrotizing entero-
colitis (NEC) in neonates [68]. Moreover, feeding newborns with
human milk, which is rich in probiotic bacteria, reduces the inci-
dence of NEC and thus has a protective effect on infants’ health
[69,70].
Probiotics also appear to be able to improve glucose metabolism
in patients with type 2 diabetes mellitus. A recent meta-analysis
of randomized, controlled trials showed that administration of
probiotics led to a significant reduction in homeostasis model
assessment of insulin resistance (HOMA-IR), as previously observed
in patients with non-alcoholic fatty liver disease [71,72]. A high-
fasting blood glucose (FBG) can lead to numerous diseases, which
include, in addition to diabetes, kidney and cardiovascular dis-
eases. Interestingly, probiotics can lower FBG levels in adults
[73].
Moreover, a recent study highlighted the positive clinical effect
of probiotics, in particular L. salivarius LS01, in children affected by
moderate/severe atopic dermatitis [74]. This specific Lactobacillus
strain, indeed, was able to significantly reduce SCORAD (Scoring
atopic dermatitis), the score used to define the severity of atopic
dermatitis, and itch intensity, leading to a general increase in qual-
ity of life after 8 weeks of probiotic oral intake. More interestingly,
the positive impact of L. salivarius LS01 persisted also after the end
of probiotic administration, suggesting the ability of this strain to
produce permanent beneficial effects in the host’s organism [72].
A wealth of data regarding the role of probiotic supplementation
in the treatment of inflammatory bowel diseases (IBD) has been
generated through the years; indeed, IBD appears to originate from
an inappropriate and exaggerated immune response towards com-
mensal bacteria and several alterations in gut microbiome profiling
were detected in both ulcerative colitis (UC) and Crohn’s disease,
compared to healthy controls [75]. Despite the researchers’ great
interest and engagement in modulating IBD inflammatory response
by administering selected strain/s of microorganisms, only two
probiotics have been accepted so far by international guidelines
for specific indications in IBD management, based on evidence
from clinical trials [76]. One is a probiotic formulation of Strepto-
coccus thermophilus, B. longum, B. breve, B. infantis, L. acidophilus,
Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus
delbrueckii subsp. bulgaricus which has been demonstrated to be
clinically effective in maintaining antibiotic-induced remission in
pouchitis and in preventing the occurrence of this clinical entity
[77,78], although to date it is still not clear which strain or strains
between those contained in the aforementioned probiotic prod-
uct are the most effective in improving the patients’ health. It is
worth noting that the term “pouchitis” refers to the inflammation
of the ileal pouch in UC patients who underwent restorative proc-
tocolectomy with ileal pouch-anal anastomosis, and it is thought
to originate from the bacterial overgrowth inside the pouch and it
usually responds to antibiotic therapies [79]; indeed, it represents
a different model of inflammation compared to UC. Nonetheless,
European guidelines on the management of UC include a second
probiotic and state that the E. coli strain Nissle 1917 is a valid alter-
native for mesalamine, according to clinical trials demonstrating
the equivalence between E. coli Nissle 1917 and mesalamine, in
maintaining remission in mild to moderate UC [80–82].
Interestingly, when considering the potential harmfulness of
other strains of E. coli, the demonstrated anti-inflammatory effects
of E. coli Nissle is a clear example that the efficacy of probiotics is
closely species- and strain-specific and, for this reason, different
species belonging to the same bacterial genus can have different
activities and, consequently, they are not all effective in the same
human disease conditions. For example, L. acidophilus was observed
to be effective in the prevention and treatment of several gastroin-
testinal diseases, including irritable bowel syndrome, necrotizing
enterocolitis, antibiotic-associated diarrhea and Helicobacter pylori
infection, while L. plantarum and B. infantis showed no efficacy
towards the above-mentioned diseases and conditions [83].
Also the role of probiotics in Irritable Bowel Syndrome (IBS) and
functional disorders is noteworthy, as numerous evidences high-
light their ability to reduce the inflammation by decreasing the
number of intestinal pathogenic microorganisms and restoring a
normal colonic fermentation [84–86]. Moreover, probiotics abil-
ity to reduce visceral hypersensitivity and influence positively the
host’s immune response is essential to counteract the low-grade
inflammation and/or immune dis-reactivity usually associated to
IBS patients [84].
In support of that, different studies underlined the significant
reduction in the frequency and intensity of abdominal pain follow-
ing the oral intake of a lactobacillus-based probiotics [87,88].
11.1. Recommendation
In the choice of the best probiotic species or strain to use in the
presence of a given disease, much attention should be paid towards
those microorganisms/strains for which clinical efficacy has been
already demonstrated.
12. Conclusion
Probiotics represent a promising approach to improving human
health as well as helping in the prevention and treatment of several
diseases of clinical interest. However, it is essential to clearly under-
stand which characteristics a microorganism must possess to be
used in the formulation of probiotic products, since, to date, numer-
ous probiotics available on the market have no beneficial effects on
human health and, above all, might contain some pathogenic and
harmful traits.
Furthermore, clear legislation regulating probiotic production,
as well as strict safety controls of all phases during the manufac-
turing of probiotics are needed to guarantee that only beneficial
and well-characterized microorganisms can be used for human
consumption.
The present review does not have the presumption to be con-
sidered as a complete and precise analysis of the current scientific
literature, but it should provide physicians with help when pre-
scribing probiotics and in understanding the main differences that
exist between numerous probiotic products that are currently
available on the market.
We are convinced that these simple and clear 10 command-
ments could be a great help to physicians, industries and users
to better understand this interesting but complicated field, called
“Probiotics.”
Conflict of interest
None declared.
 M. Toscano et al. / Digestive and Liver Disease 49 (2017) 1177–1184
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