Accepted Manuscript

Title: A Consumer’s Guide for Probiotics: 10 Golden Rules

for a Correct Use

Authors: Marco Toscano, Roberta De Grandi, Luca Pastorelli,

Maurizio Vecchi, Lorenzo Drago

PII: S1590-8658(17)30986-6
DOI: http://dx.doi.org/doi:10.1016/j.dld.2017.07.011
Reference: YDLD 3499

To appear in: Digestive and Liver Disease

Received date: 15-2-2017

Revised date: 19-7-2017
Accepted date: 20-7-2017

Please cite this article as: Toscano Marco, De Grandi Roberta, Pastorelli Luca, Vecchi
Maurizio, Drago Lorenzo.A Consumer’s Guide for Probiotics: 10 Golden Rules for a
Correct Use.Digestive and Liver Disease http://dx.doi.org/10.1016/j.dld.2017.07.011

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TITLE PAGE

A Consumer’s Guide for Probiotics: 10 Golden Rules for a Correct Use

Marco Toscanoa, Roberta De Grandia, Luca Pastorelli b,c, Maurizio Vecchi b,c, Lorenzo Dragoa,d,*

a
Laboratory of Clinical Microbiology, Department of Biomedical Science for Health, University of

Milan, Via Mangiagalli 31, 20133, Milan, Italy;

b
Department of Biomedical Science for Health, University of Milan, Via Mangiagalli 31, 20133,

Milan, Italy;
c
Gastroenterology and Gastrointestinal Endoscopy Unit, IRCCS Policlinico San Donato, Via

Morandi 30, 20097, San Donato Milanese, Italy;

d
Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, Via

Galeazzi 4, 20164, Milan, Italy

*
Corresponding author at: IRCCS Galeazzi Orthopaedic Institute, Via Galeazzi 4, 20164, Milan,

Italy.

E-mail address: lorenzo.drago@unimi.it

Electronic word count: 4981

Abstract

Probiotics are used all over the world as their beneficial effects on the human organism have been

widely demonstrated. Certain probiotics can down-regulate production of pro-inflammatory

cytokines and promote intestinal epithelial barrier functions, increasing an anti-inflammatory

response and contributing to the host’s overall health. The main mechanisms by which probiotic

1
microorganisms can interact with the host are by modulating the immune system and the epithelial

cell functions and interacting with intestinal gut microbiota.

To date, hundreds of different microorganisms are used for the formulation of numerous probiotic

products; therefore, it is very difficult to choose the best probiotic product for specific or more

general needs. Therefore, physicians are getting more and more confused due to the high number of

commercial 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 “commandments” that clinicians should follow to properly select the optimal

probiotic product and avoid misidentifications, mislabelling and “pie in the sky” stories.

Keywords: multistrain products; gut microbiota; anti-inflammatory response

1. Introduction

In the last few years, a growing interest in studying and using probiotic microorganisms has been

observed, not only for the treatment 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 ameliorating 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 modulating the gut microbiota

composition [8-11]. Commensal bacteria belonging to intestinal microbiota protect the host from

2
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 particular 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 reason, 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 microorganisms 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.

However, today it is very difficult to identify which probiotic formulation is the best one to improve

the human health, mostly 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 microorganisms, 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 follow, in order to give an easy and immediate interpretation of

the probiotic(s) under consideration. The 10 “commandments” or “recommendations” clinicians

should never forget when dealing with probiotics are listed in Table 1.

2. 1st commandment: know the correct definition of probiotics

3
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 Lactobacillus

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]. Conversely, the term “pharma” is mainly related to pharmaceuticals, either

drugs or chemical components [13]. On the contrary, probiotics 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].

Recommendation: probiotics are live microorganisms acting as powerful biomodulators, 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 studying 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 enhancing the anti-inflammatory response [14-16]. The administration of heat-

4
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 function on the host [14]; DNA, lipopolysaccharides, peptidoglycans

and cell homogenates have all a strong immunomodulatory effect acting 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 organism 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 environment can be considered as probiotics and used in the formulation of

products [22]. Spores can survive transit across the stomach barrier 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 probiotic 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.

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

5
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 formulation 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 comparative 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

microorganism contained in probiotic products must be identified at species and strain level,

according to the International Code of Nomenclature and its microbial genome must be completely

sequenced. This allows the identification of every single gene involved in bacterial 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 guidelines require:

1) minimum concentration of 109 CFU of live microorganisms/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 JS 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 similar to those of

6
Weese. Indeed, in the Italian study, authors observed that 42% of analysed products did not contain

the declared bacterial amount of at least one of the labelled strains. Moreover, no viable

microorganisms were detected in 17% of products and a contamination due to Enterococcus

faecium was found in 8% [26]. The presence of a non-declared microorganism, which might

potentially 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 probiotic strains in commercial products, as well as the careful testing of all

commercial products available on the market.

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 products [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. (2015) showed that a combination of Lactobacillus

7
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 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 antagonists, not only inhibiting probiotic activity of

other microorganisms, but also slowing microbial growth rate, as demonstrated by Vitali et al.

(2003). The authors, indeed, evaluated the growth compatibility of three Bifidobacterium strains

(Bifidobacterium infantis Y1, Bifidobacterium breve Y8 and Bifidobacterium longum Y10)

contained in a probiotic product, observing a significant inhibition of the growth rate induced on B.

longum Y10 when co-cultured with the other 2 strains [32].

Recommendation: It is fundamental that research on multistrain probiotic formulations 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 dangerous reservoir of antibiotic resistance determinants in probiotic

microorganisms [33]. Generally, we can consider antibiotic resistance among probiotic bacteria a

positive feature, as these microorganisms are able to restore intestinal eubiosis without being

8
influenced by a concomitant antibiotic therapy. However, when antibiotic resistance can be

transferred to pathogenic bacteria by means of resistant genes and determinants, a serious risk for

human health may arise, as pathogens can become resistant to antimicrobial agents, nullifying

antibiotic therapies and further contributing to the spread of antibiotic resistance. Probiotic bacteria,

indeed, as any other microorganism, can develop antibiotic resistance and resistant mutants if

subjected to antibiotic selective pressure. The European Food Safety Authority (EFSA) document

recommends that commercial microbial strains used as food supplements should not harbour any

transferable antibiotic 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 bacteria 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

subpopulations of bacteria during antimicrobial therapy [37]; however, it is also essential to avoid

probiotic products and other food supplements 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 presence 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

9
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

gentamicin; 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 aminoglycosides [38]. The

numerous antibiotic resistance traits found in many Italian and European probiotic strains may be

the consequence of an extensive use of antibiotics, which has created a positive selective pressure

for point mutations and the acquisition 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, clindamycin 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 definitively 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.

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.

10
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 environment 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 bacteria 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, Bifidobacterium and Enterococcus genera, which are generally located in the

intestinal environment [46-50]. BSH enzymes seem to play a pivotal 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. (2011) demonstrated the

ability 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. (2015)

11
evaluated the probiotic characteristics of three Bifidobacterium strains (Bifidobacterium breve M-

16V, Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium 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

Bifidobacterium 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].

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 maintaining 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 microorganisms, 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 natural environment [55]. Biofilm

formation is a developmental process that has been compared to differentiation in multicellular

organisms. 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

12
production 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 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 bacterial strain tested, and this is the reason for

many conflicting data about fecal recovery of probiotic bacteria. Indeed, Dommels et al. (2009)

demonstrated that Lactobacillus reuteri DSM 17938 and Lactobacillus rhamnosus GG were

detected in fecal samples of volunteers after 3 weeks of probiotic daily consumption, suggesting a

strong colonization ability for both Lactobacillus strains [57]. Conversely, in a previous study,

Prilassnig et al. (2007) were not able to detect strains belonging to Lactobacillus acidophilus,

Lactobacillus gasseri, Lactobacillus rhamnosus and Lactobacillus casei, which were administered

daily to healthy individuals [58]. These data highlight the fact that various probiotic

microorganisms show different characteristics and properties 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 adhesion and regulate paracellular diffusion to the host’s tissues, but is also

fundamental to discriminate between commensal microorganisms and pathogens, adjusting finely

the immune response to pathogenic bacteria [59]. In this context, probiotics are fundamental 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

proteins after exposure to pathogens and restoring disrupted epithelial barrier by enhancing cell

regeneration [59].

13
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 highlighted 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 administration of the probiotic strain Lactobacillus salivarius LS01 to

patients affected by atopic dermatitis (AD) could improve the classic symptoms associated with

AD, as well as lead to a significant decrease in intestinal staphylococcal load [62]. Even if this

result highlights the potential 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 Lactobacillus acidophilus was observed to have induced a significant decrease of

β-glucosidase, nitroreductase and azoreductase 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

14
baseline levels. Furthermore, also the Lactobacillus casei strain GG has the same effect on gut

microbiota metabolism, being able to reduce faecal β-glucuronidase in the host [64]. Further studies

with microbiota composition modification as the primary outcome should be carried 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.

Recommendation: Even if there are few and conflicting data on the impact of probiotics on gut

microbiota composition, only probiotic products with a strong intestinal activity should be

considered and used for improving 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 consumption. Generally, in vitro assays are useful to exclude potential

pathogenic microorganisms from being used as probiotics. However, 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 possibility 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 syndrome has been described [66]. The patient had

fever and hypoxia and he received probiotic Lactobacillus 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-

15
old male. This patient was affected by ulcerative colitis (UC) and he took Lactobacillus rhamnosus

GG to treat a flare-up of the disease, even if there were no clinical data to support 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

administering 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.

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.

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 beneficial 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 efficacy 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 microorganisms can positively contribute to intestinal mucosal

integrity, exerting a pivotal role in reducing the risk of necrotizing enterocolitis (NEC) in neonates

[68]. Moreover, feeding newborns with human milk, which is rich in probiotic bacteria, reduces the

incidence 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

16
diabetes, kidney and cardiovascular diseases. Interestingly, probiotics can lower FBG levels in

adults [73].

Moreover, a recent study highlighted the positive clinical effect of probiotics, in particular

Lactobacillus 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 quality 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 commensal 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

Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium

infantis, Lactobacillus 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 product 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

17
UC patients who underwent restorative proctocolectomy 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 Escherichia coli strain Nissle 1917 is a valid alternative 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, Lactobacillus acidophilus was observed to be

effective in the prevention and treatment of several gastrointestinal diseases, including irritable

bowel syndrome, necrotizing enterocolitis, antibiotic-associated diarrhea and Helicobacter pylori

infection, while Lactobacillus plantarum and Bifidobacterium 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 highlight their ability to reduce the inflammation by decreasing

the number of intestinal pathogenic microorganisms and restoring a normal colonic fermentation

[84-86]. Moreover, probiotics ability 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 following the oral intake of a lactobacillus-based probiotics [87, 88].

18
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

understand which characteristics a microorganism must possess to be used in the formulation of

probiotic products, since, to date, numerous 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 manufacturing 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 considered as a complete and precise

analysis of the current scientific literature, but it should provide physicians with help when

prescribing 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 commandments could be a great help to

physicians, industries and users to better understand this interesting but complicated field, called

“Probiotics.”

Conflict of interest
All authors have no conflict of interest.

19
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28
Table 1 The 10 Recommendations of probiotics

1st Knowing the correct definition of probiotics

2st Microbial lysates, non-living bacteria and non-colonizing

spores cannot be considered probiotics

3st Getting an exhaustive probiotic identikit

4st Monostrain or multistrains products: making the correct

choice

5st Avoid antibiotic resistance genes in probiotic strains and

products

6st Choose probiotic strains resistant to gastrointestinal

environment

7st Probiotic strains must be able to colonize the gut

8st Preferring probiotics that are able to positively interact with

gut microbiota.

9st Be sure about the safety of probiotic strains and evaluate the

subject health status before probiotic administration

10st Preferring probiotics with a demonstrated clinical efficacy

29