Actual concept of "probiotics": Is it more functional to science or business?

Abstract

It is our contention that the concept of a probiotic as a living bacterium providing unspecified health benefits is inhibiting the development and establishment of an evidence base for the growing field of pharmacobiotics. We believe this is due in part to the current regulatory framework, lack of a clear definition of a probiotic, the ease with which currently defined probiotics can be positioned in the market place, and the enormous profits earned for minimum investment in research. To avoid this, we believe the following two actions are mandatory: international guidelines by a forum of stakeholders made available to scientists and clinicians, patient organizations, and governments; public research funds made available to the scientific community for performing independent rigorous studies both at the preclinical and clinical levels.

Key Words: Probiotics; Market; Regulations; Guidelines; Metanalysis

INTRODUCTION

Probiotics are generally defined as live microorganisms, preferentially of human origin, that upon ingestion in specific and sufficient numbers confer unspecified health benefits to the host. During the last twenty years the therapeutic potential of probiotic bacteria has been evaluated in a large number of basic, experimental and clinical studies[1-3] and their use in different clinical conditions has received considerable scientific and commercial attention.

Today probiotics represent a very big business. The global functional food market has been recently estimated at up to $50 billion annual share[4], while the world probiotic market is estimated at $15 billion. Today, this market is growing at a pace of 5%-30% depending on the country and product type[5]. The marketing agency Frost and Sullivan believes that the possibility to use salutistic indications on the label of the products containing probiotics, as permitted according to CE 1924/2006 rules, can further increase the consumer interest. Proper communication paired with effective marketing strategies will prove to be useful to this aim. Consumer acceptance varies greatly across Europe, with the most developed market in Northern European and Scandinavian countries, having a long traditional consumption of probiotic products[5]. The existing consumer confusion over the different probiotic strains as well as skepticism about their efficacy do not seem sufficient to counteract the salutistic propaganda of the media advertisements.

The regulatory status for probiotic products is not well established at international level yet. The United States Food and Drug Administration apply a conceptual distinction among “medical foods”, “dietary supplements”, “drugs” and “biological products” to probiotic products. The regulatory consequences that accompany a probiotic product that is categorized as a dietary supplement obviously dramatically differ from those that accompany a probiotic product categorized as a drug. If the probiotic product meets the definition of dietary supplement, the manufacturers may place the probiotic product on the market without any pre-market approval and may market the product with claims concerning the effect that the product has on the structure or function of the body. The European Commission has recognized probiotic bacteria as having the status of nutrients; in addition probiotics in powder, capsule or tablet form are in most European countries regarded as “food supplements” but with important differences: according to Bianca Herr of the Leatherhead Food International, in Italy and in Hungary probiotic products are widely accepted as food supplements, in Germany these products are accepted as food supplements in some cases but their acceptance as drugs depends on their concentration, while in Spain there is no specific legislation or guidance for probiotic products. Thus, in most cases, these products reach the market without being tested in the expensive three phases required for approval of a new drug. For these reasons not only big pharma and manufacturers of probiotics but also national pharmaceutical industries and even family farms are involved in this market. Also, the work of the European Food Safety Authority regarding claims made on food labeling and advertising concerning nutrition and health provides an important but very partial solution to the problem.

One would expect that the available scientific evidence is comparable to the size of this market; however, this is certainly not the case. Food And Agriculture Organization and World Health Organization defined the following characteristics of probiotic microorganisms: (1) probiotics should be taxonomically classified and deposited in an internationally recognized culture collection; (2) they have to remain viable and stable after culture, manipulation, and storage before consumption; (3) they have to survive to gastric acid and biliary and pancreatic digestion; (4) they have to induce a host response once ingested by adhering to gut epithelium or by other mechanisms; (5) they have to yield a functional or clinical benefit to the host when consumed; and (6) finally they have to be safe, not only regarding the assessment of side effects, but also in relation to antibiotic resistance patterns. In fact beneficial bacterial populations may play a role in the transfer of antibiotic resistance to pathogenic and opportunistic bacteria. These general rules are certainly meaningful but not sufficient as guidelines for this field. Although there are few international organizations that purport to be independent of industry, such as International Scientific Association for Probiotics and Prebiotics (ISAPP), whose mission is to engender and disseminate information on high quality, multidisciplinary, scientific investigation in the field of probiotics, in actual fact there is no organization, agency or scientific network able to (1) reduce the incredible confusion related to every aspect of probiotics; (2) direct the rudder of basic and experimental research on probiotics and, in the future, on pharmacobiotics (a fundamental goal is to move away from the restrictive and perhaps outdated term “probiotics” and over to the more inclusive term “pharmacobiotic or pharmabiotic”); and (3) propose well accepted guidelines for evaluating these products in controlled clinical trials. To date variability is the keyword and includes every aspect of probiotics: strain, dose, route of administration, trial methodology, endpoints and outcomes. A very large number of probiotic strains have been used in clinical studies for the treatment of the same clinical condition, and the same strain of probiotics has been used to treat very different disease states. In addition an incredible large range of doses [from 4.5 × 10² colony-forming units (CFUs) to 3.6 × 10¹² CFUs] of probiotics has been assayed in clinical trials. Furthermore, in different studies probiotics were administrated in a great variety of ways: capsules, powders, tablets, drops or yogurts. An equally great variability exists in methodology, endpoints and outcomes of clinical trials carried out so far, even limiting the analysis to a single clinical condition. As an example we summarized in Table 1, Table 2 and Table 3 the number of patients, duration of treatment, probiotic strains used, dose used and outcomes of clinical trials carried out on three adult clinical conditions in which probiotics have widely tested: irritable bowel syndrome, ulcerative colitis and Crohn’s disease. The tables end with the indication of published meta-analyses. Despite the existence of guidance[6] and recommendations[7] for probiotic use in these intestinal diseases, it seems clear from the tables that the lack of homogeneity of the published studies does not allow to draw final conclusions and to generate, through an evidence-based approach, true guidelines useful for adult patients. This is corroborated by meta-analysis studies that recognize the variety of species, strains and doses of probiotic used associated to an evident heterogeneity of study methodologies as main limitations in the field.

Table 1 Results of clinical trials with probiotics in irritable bowel syndrome.

Ref.	Patients (n)	Duration of therapy	Probiotic strains	Dose (CFU/d)	Outcomes
Maupas et al[88]	34	1 mo	Saccharomyces boulardii	9 × 109	Improved stool number and consistency
Gade et al[89]	54	1 mo	Paraghurt (Streptococcus faecium)	1 × 10¹²	Improved symptoms
Halpern et al[90]	18	4 mo	Lactobacillus acidophilus	2 × 1010	Improved symptoms
O’Sullivan et al[91]	25	1 mo	Lactobacillus GG	1 × 1010	No benefit
Nobaek et al[92]	60	1 mo	Lactobacillus plantarum 299V Pro-Viva®	5 × 107	Improved global symptoms
Niedzielin et al[93]	40	1 mo	Lactobacillus plantarum 299V Pro-Viva®	2 × 1010	Improved global symptoms
Kim et al[94]	25	2 d-IBS	VSL3®	9 × 10¹¹	Reduced bloating
Tsuchiya et al[95]	68	3 mo	Lactobacillus acidophilus	1.5 × 106	Improved symptoms
			Lactobacillus helveticus	1.3 × 109
			Bifidobacterium	4.95 × 109
O’Mahony et al[96]	80	2 mo	Bifidobacterium longum subspecies infantis vs Lactobacillus salivarius	1 × 1010	B. infantis: improved global symptoms and anti-inflammatory cytokine profileLactobacillus salivarius: no benefit
Kajander et al[97]	103	6 mo	Mixture (2 strains of Lactobacillus rhamnosus, Bifidobacterium breve, Propionibacterium freudenreichii)	8-9 × 109	Improved global symptoms
Bittner et al[98]	25	0.5 mo	29 bacteria + prebiotic Prescript-Assist®	2.6 × 108	Improved wellbeing
Sen et al[99]	12	1 mo	Lactobacillus plantarum 299V Pro-Viva®	5 × 107	No benefit; Study design flawed
Bausserman et al[100]	50	1.5 mo	Lactobacillus GG	2 × 1010	No benefit
Niv et al[101]	39	6 mo	Lactobacillus GG	2 × 108	No benefit Francis severity IBS score
Kim et al[102]	48	1 or 2 mo	VSL3®	8 × 109	Reduced flatulence, retarded colonic transit
Whorwell et al[103]	362	1 mo	Bifidobacterium longum subspecies infantis 35 624 in 3 doses	1 × 106	Improved global symptoms
				1 × 108
				1 × 1010
Long et al[104]	60	0.5 mo	Bifidobacterium	2 × 108	Symptoms resolved
Gawrońska et al[105]	104	1 mo	Lactobacillus GG	6 × 109	Reduced frequency of pain
Moon et al[106]	34	1 mo	Bifidobacterium subtilis, Streptococcus faecium	750 mL/d, CFU/d not given	Reduced frequency pain
Marteau et al[107]	116	1 mo	Lactibiane® (4 strains of Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus lactis, Streptococcus thermophilus)	1 × 1010	Reduced painIncreased colonic transit in those with constipation
Simrén et al[108]	76	1.5 mo	Lactobacillus plantarum 299V	2 × 109	No benefit
Simrén et al[109]	118	2 mo	Lactobacillus paracasei ssp paracasei	2 × 1010	No benefit
Guyonnet et al[110]	274	1.5 mo	Bifidobacterium animalis, Streptococcus thermophilus and Lactobacillus bulgaricus	1.25 × 1010 1.2 × 109	Improved bloating and constipation
Drouault-Holowacz et al[111]	116	1 mo	Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus lactis, Streptococcus thermophilus	1 × 1010	Not significant in relieving symptoms
Sinn et al[112]	40	1 mo	Lactobacillus acidophilus	2 × 108	Improved abdominal pain and discomfort
Enck et al[113]	297	1 mo	Escherichia coli, Enterococcus faecalis	4.5 × 102	Improvement in pain
Hun et al[114]	44	2 mo	Bacillus coagulans	8 × 108	Improvement abdominal pain and bloating
Dolin et al[115]	61	2 mo	Bacillus coagulans	2 × 109	Diminution of diarrhea
Ligaarden et al[116]	16	1 mo	Lactobacillus plantarum	1010/L	Worsening of symptoms
Moayyedi et al[117]	19 randomised controlled trials in 1650 patients				Probiotics appear to be efficacious but the magnitude of benefit and the most effective strains are uncertain

CFU: Colony-forming unit; IBS: Irritable bowel syndrome.

Table 2 Results of clinical trials with probiotics in ulcerative colitis.

Ref.	Patients (n)	Duration of therapy	Probiotic strains	Dose (CFU/d)	Outcomes
Kruis et al[118]	120	12 wk	Escherichia coli Nissle 1917	50 × 1010	Maintaining the remission (similar to 5-ASA)
Rembacken et al[119]	116	1 yr	Escherichia coli Nissle 1917	5 × 1010	Induction of remission (similar to 5-ASA); maintaining of relapses (similar to 5-ASA)
Venturi et al[120]	20	1 yr	VSL3®	5 × 10¹¹	Maintaining the remission
Ishikawa et al[121]	21	1 yr	Milk with bifidobacteria	10 × 108	Maintaining the remission
Guslandi et al[122]	25	4 wk	Saccharomyces boulardii	250 mg × 3	Induction of remission
Kruis et al[123]	327	1 yr	Escherichia coli Nissle 1917	2.5-25 × 109	Induction of remission (5-ASA better than probiotic)
Tursi et al[124]	90	8 wk	Balsalazide/VSL3®	900 × 108	Induction of remission
Cui et al[125]	30	8 wk	Bifidobacteria	1.26 g/d	Maintaining of remission
Kato et al[126]	20	12 wk	Bifidobacterium-fermented milk vs placebo	109	CDAI lower in Bifidobacterium fermented milk that in placebo
Furrie et al[127]	18	4 wk	Bifidobacterium longum + prebiotic (Synergy 1)	4 × 10¹¹	Induction of remission
Bibiloni et al[128]	32	6 wk	VSL3®	1800 billion × 2	Induction of remission
Zocco et al[129]	187	12 mo	Lactobacillus GG vs mesalazina	18 × 109	No difference between the treatment groups
Henker et al[130]	34	12 mo	Escherichia coli Nissle 1917	5 × 1010	Maintenance of remission
Miele et al[131]	29	12 mo	VSL3®	450-1800 × 109	Induction of remission (92.8% in treated with VSL3® and 36.4% in the placebo group)
Sood et al[132]	147	12 wk	VSL3®	3.6 × 10¹²	Induction of remission (42.9% against 15.7% in the placebo group)
Matthes et al[133]	57	4 wk	Escherichia coli Nissle 1917	10-40 × 108	Induction of remission
Sang et al[134]	13 RCTs				Heterogenity between the studies in their methodology and results

5-ASA: 5-aminosalicylic acid; CDAI: Crohn's disease activity index; CFU: Colony-forming unit; RCTs: Randomised controlled trials.

Table 3 Results of clinical trials with probiotics in patients with Crohn’s disease.

Ref.	Patients (n)	Duration of therapy	Probiotic strains	Dose (CFU/d)	Outcomes
Malchow et al[135]	24	3 mo	Escherichia coli Nissle 1917	2.5 × 1010	Maintaining the remission
Guslandi et al[136]	32	6 mo	Saccharomyces boulardii	1 g	Postsurgical prevention of CD recurrence (relapse rate probiotic+ 5-ASA vs 5-ASA alone)
Prantera et al[137]	45	1 yr	Lactobacillus GG	12 × 109	Postsurgical prevention of CD recurrence (no effects)
Schultz al[138]	11	6 mo	Lactobacillus GG	2 × 109	Probiotics are not superior to placebo in maintaining remission
Bousvaros et al[139]	75	1 yr	Lactobacillus GG	2 × 1010	Probiotics are not superior to placebo in maintaining remission
Marteau et al[140]	98	6 mo	Lactobacillus johnsonii	4 × 109	Postsurgical prevention of CD recurrence (recurrence rate decreased vs placebo)
Chermesh et al[141]	30	24 mo	Synbiotic 2000 (Pediococcus pentoseceus, Lactobacillus raffinolactis, Lactobacillus paracasi susp paracsei, Lactobacillusplantarum 2362) and 4 fermentable fibers vs placebo	10¹¹	Postsurgical prevention of CD recurrence (NS)
Van Gossum et al[142]	70	12 wk	Lactobacillus johnsonii or placebo	1010	Postsurgical prevention of CD recurrence (NS)
Rolfe et al[143]	7 RCTs				No benefit of probiotics in the maintenance of remission of CD
Rahimi et al[144]	8 RCTs				None benefit for probiotic treatment in the maintenance of clinical remission of CD

RCTs: Randomised controlled trials; CD: Crohn's disease; 5-ASA: 5-aminosalicylic acid; CFU: Colony-forming unit; NS: Not significant.

This would not be accepted in clinical pharmacology. No drug can be approved for the market with a defined clinical indication without sufficient knowledge of its mode of action, pharmacokinetic parameters, toxicological features, tolerability and effectiveness. In addition this knowledge will be substantially increased by post-marketing surveillance. By contrast, probiotics are commonly commercialized and prescribed for specific clinical indications in the absence of any conclusive proof concerning their putative pharmacological properties. Finally, it should be remembered that the safety of probiotics should not be considered a foregone datum: in abdominal surgery, translocation of bacteria from the gastrointestinal tract through the mucosa could occur[8], and probiotic treatment has been associated with increased mortality in patients with acute pancreatitis[9].

Only a few cases based on studies regarding pediatric population formal meta-analyses have been used to generate clinical guidelines. These studies demonstrated beneficial effects of probiotics in acute diarrhea of children. These effects are strain- and dose-dependent, being generally greater with doses > 10¹⁰ CFUs, highly significant for watery diarrhea and viral gastroenteritis but less so for invasive bacterial diarrhea, more evident when the treatment is started early in the course of disease, and more evident in children living in developed than in developing countries[10]. In May 2008, probiotics were for the first time included in a guideline document named “the guidelines for the management of acute gastroenteritis” and produced by a Committee of the European Society for Pediatric Infectious Diseases[11]; this guideline document was developed through an evidence based systematic review approach that incorporates tables of evidence with their grading. The guidelines state that only the use of probiotic strains with proven efficacy and in appropriate doses is suggested for the management of acute diarrhea in European children as an adjunct to rehydration therapy. The evidence of efficacy regards only two strains: Lactobacillus rhamnosus GG was rated as 1A and Saccharomyces boulardii was rated as 2B, corresponding to the level of evidence based respectively on meta-analysis of randomised controlled trials (RCTs) and properly designed RCTs of appropriate size. This evidence is actually confined only to the prevention/treatment of childhood acute gastroenteritis and of antibiotic-associated diarrhea. In the few conditions in which selected probiotic bacteria have shown a proven efficacy competitive mechanisms or mechanisms of restoration of bacteria flora seem to be involved. No final evidence is available in other conditions or diseases in which probiotic agents are largely used. It appears evident that the tremendous dichotomy between the huge market of probiotic products and the insufficient knowledge of probiotic-based therapies. This would be unacceptable for any other pharmacological treatment.

We believe that some important fields of research exist that should be encouraged due to the possibility of getting information of incommensurate value in the near future. These fields of investigation will possibly permit development of a new concept of “probiotic agents”[12,13], and should be adequately investigated.

A NEW CONCEPT OF “PROBIOTICS”

The relationship between probiotic agents and innate immune system

In recent years there have been tremendous advances in our understanding of the structure and function of signal receptors, and the pivotal role of pattern recognition receptors (PRRs) and cells of the innate immune system in processing bacterial and food components is now well established[14-17]. PRRs include trans-membrane Toll-like receptors (TLRs) and Dectin-I; endosomal PRRs (TLR 3, 7/8 and 9); and cytosolic nucleotide oligomerization domain (NOD)-like receptors: (NOD1 and NOD2), Rig-1-like RNA helicase receptor (retinoic acid-inducible gene-1 and iron-regulated surface determinant sensors). The cells involved are dendritic cells (DC), intraepithelial lymphocytes, macrophages, neutrophils and enterocytes. At this level microorganisms are recognized only as microorganism-associated molecular patterns (MAMPs). MAMPs are first recognized by a PRR, and activation of the receptor by binding of the MAMP sequentially activates intracellular molecules such as the cytoplasmic adapter molecule MyD88, leading to the activation of transcription factors including nuclear factor-κ B (NFκB) and activator protein-1 (AP-1), which are required for gene transcription and cytokine synthesis. The different receptors of the innate immune system are obviously only able to process specific molecular components of microorganisms and foods, whereas the recognition of a whole bacterium or food does not appear possible although simultaneous activation of several PRR’s may be characteristic of a specific organism or food and lead to a different outcome than activation by single PRR. For example, studies on host mucosal gene expression following exposure to different whole bacteria have demonstrated up-regulation of different gene networks for each organism. Networks stimulated by these probiotic bacteria included cell proliferation, Th-1/Th2 balance, control of blood pressure, tissue development, water and ion regulation and wound healing. Major host differences were noted in the stimulated transcriptosome. The pathways stimulated by the whole organism corresponded to pathways stimulated by known pharmacological preparations. However, the specific molecules of the bacteria that caused these effects are currently unknown[18]. Further, whether the bacterium is alive or dead does not seem relevant for the recognition of a molecular pattern by specific PRRs. The accessibility of MAMPs for PRRs and the presence of other microbial effector molecules, such as toxins produced by pathogens, have a pivotal role in the modulation of host immune response. Other important factors determining the host response are host-derived direct or indirect negative regulators of PRR signaling.

To date pathogenic, probiotic and commensal bacteria are considered to induce different levels of immune response: a strong host response stimulated by pathogens, an intermediate response induced by probiotics and finally a homeostatic control of the response is triggered by commensal bacteria. An important exception to this concerns a restricted number of commensal bacteria, the prototype of which is the segmented Filamentous Bacteria (SFB), which could largely recapitulate and orchestrate a broad spectrum of B and T cell responses[19,20]. SFB colonized mice had low levels of ATP, suggesting that host sensing of SFB does not involve TLR or NOD receptors[21]. We have recently showed that the progressive penetration of the holdfast segments of these bacteria within the specialized epithelial cells of the terminal ileum could permit an impressive presentation of bacterial antigens directly to the lymphocytes contained in the lymphoid packets characteristic of the M cells and to antigen presenting cells[22].

It should also to be remembered that interactions between PRR and ligands are not as specific as those between antigens and antibodies, and ligands for PRR such as TLRs are generally present in repetitive structures to increase avidity.

Therefore, some very important and specific questions concerning immune-mediated probiotic activity are: (1) Are whole live bacteria essential to promote biological effects on the immune system? (2) Can the concept of probiotics be extended to include bacterial-derived molecular bioactive components? (3) Moreover, can probiotic molecules be also produced by non-probiotic bacteria? and (4) Finally, can probiotics be genetically manipulated to synthesize specific bioactive molecules?

Probiotic molecules

Bacterial DNA: Bacterial genomic DNA of probiotics in VSL-3™ induced a remarkable strain-specific immune response in humans as evaluated by the release of interleukin (IL)-1β, IL-6 and IL-10. Total bacterial DNA from faeces increased the Th-1 cytokine IL-1β more than IL-10 compared to DNA from the probiotic bacteria which had the reverse effect. However, total DNA from faeces, after being given a course of the probiotic bacteria, produced a greater stimulation of IL-10 compared to IL-1[23]. Notably, the respective role of IL-1β and IL-6 in the beginning and maintenance of a Th17 response is well known[24,25]. An important and provocative study[26] showed that in a mouse irritable bowel disease model the protective effects of probiotics contained in VSL-3 are mediated by their DNA rather than by their ability to colonize the gut mucosa. TLR 9 signaling is essential in mediating the anti-inflammatory effects of probiotics. TLR-9 is an endosomal TLR which is known to interact with bacterial DNA upon bacterial lysis. The authors suggested that DNA-TLR9 signaling resulted in the differentiation of naive cluster of differentiation-4 (CD4) T lymphocytes into regulatory T cells, mediating the protective action. Another example of the immunomodulatory capacity of probiotic DNA is represented by DNA of Lactobacillus rhamnosus GG that induces B-cell proliferation and activates DCs[27]. More recently, the effects on the Th1/Th2 balance by genomic DNA of different probiotic bacteria (Lactobacillus rhamnosus GG, Lactobacillus gasseri, Bifidobacterium bifidum, Bifidobacterium longum) were compared with the effects of live bacteria by using peripheral blood mononuclear cells from healthy subjects and from patients with an allergy against the house dust mite[28]. Compared with live Lactobacilli, bacterial DNA inhibited IL-4 and IL-5 secretion in a similar way, and based on the maximal effects achieved with Lactobacilli and their DNA, more than 50% of these effects seem to be due to their DNA (Table 4).

Table 4 Reference studies concerning the probiotic role of bacterial DNA.

Ref.	Outcomes
Lammers et al[23]	Bacterial DNA from faeces collected after VSL-3 administration modulated a decrease of IL-1β and an increase of IL-10
Rachmilewitz et al[26]	Study in a mouse IBD model: protective effects of probiotics contained in VSL-3 are mediated by their DNA and TLR9 signaling mediates anti-inflammatory effect
Iliev et al[27]	Lactobacillus rhamnosus GG DNA induces B-cell proliferation and activate DC
Ghadimi et al[28]	Bacterial DNA inhibited IL-4 and IL-5 secretion in different Lactobacilli
Ménard et al[30]	Study from 5 bifidobacterial strains: unmethylated CpG motifs are specific to bacterial DNA by activating TLR9

IL: Interleukin; IBD: Irritable bowel disease; TLR: Toll-like receptor.

The immunomodulatory activity of DNA is characterized by unmethylated CpG motifs which can activate innate immune responses through binding to TLR9 and triggers the translocation of NFκB and AP-1 from the cytoplasm to the nucleus thereby up-regulating gene expression pathways. Stimulatory oligodeoxynucleosides contain the CpG within a flanking region to give a motif of Pur-p-Pur-p-CpG-p-Pyr-p-Pyr. Typically more than one CpG is present in the immunostimulatory oligodeoxynucleoside and maximal effect occurs if they are separated by 1-2 base pairs. A 5’ TpC and pyrimidine rich 3’ ends also increases the immunostimulatory effects. In terms of a potential therapeutic, the in-vivo degradation can be decreased by synthesizing a phosporthiorate backbone which increases the stimulatory activity of the motif[29]. A very recent study based on entire genome sequences from five bifidobacterial strains[30] showed that Bifidobacterium genomes contained several CpG motifs and biologically active sequences previously identified in Lactobacilli. These bioactive sequences induced the production of monocyte chemotactic protein-1 and tumor necrosis factor-α (TNF-α) through a pattern of TLR-9 stimulation of macrophages. An inter- and intra-species investigation of 71 strains of Bifidobacteria of various origins showed that these bioactive DNA sequences were highly conserved in the genus. The results of these studies clearly suggest the necessity of further investigation.

MOLECULAR PRESENT AT THE BACTERIAL SURFACE

Bacterial cells wall molecules are potential probiotic ligands that can interact with PRRs and induce signaling pathways resulting in probiotic effects (Table 5).

Table 5 Reference studies concerning the probiotic role of molecules presented at the bacterial surface.

Ref.	Outcomes
Mazmanian et al[33]	Bacterial capsular PSA elaborated by Bacteroides fragilis activates CD4+ and elicits cytokine production
Mazmanian et al[35]	Purified PSA suppress pro-inflammatory IL-17 production and protects from inflammatory disease by induction of IL-10
Ryu et al[36]	Purified LTA from Gram-positive bacteria has lower potency in the stimulation of Toll-like receptor-2 pathway to induce pro-inflammatory molecules.
Grangette et al[37]	Modified LTA is able to induce secretion of anti-inflammatory IL-10
Benz et al[39]	Lipoproteins and glycoproteins at the cell surface are attractive candidates as probiotic molecules
Schlee et al[40]	Flagellins of the Escherichia coli Nissle 1917 induces expression of human β-defensin 2
Matsumoto et al[83]	Purified PSPG-I from Lactobacillus casei Shirota has anti-inflammatory actions in chronic intestinal inflammatory disorders

PSA: Polysaccharide A; IL: Interleukin; LTA: Lipotheichoic acid; PSPG: Polysaccharide-peptidoglycan.

The immune system is able of recognizing any biological polymer constituting the bacterial cell wall and presenting it to T cells. Most probiotics are typically Gram-positive bacteria, in which the cell wall is composed of a thick peptidoglycan layer with proteins, theicoic acids and polysaccharides[31]. However few Gram-negative probiotics exist, such as Escherichia coli strain Nissle 1917; in this case the cell wall is composed of a thin peptidoglycan layer and an outer membrane which contains lipopolysaccharides (LPS) that is further decorated with the proteins and polysaccharides[32].

Although adaptive immune responses have been considered the territory of antigenic proteins or glycoproteins, whereas carbohydrates were considered as not recognized by the adaptive immune system, recent studies have revolutionized this assumption. Bacterial wall polysaccharides and glycolipids are now considered perhaps the more attractive targets in the research for immunomodulatory molecules. Interestingly, the bacterial capsular polysaccharide A (PSA), the most immunodominant among the zwitterionic polysaccharides elaborated by Bacteroides fragilis, a commensal Gram-negative anaerobe that colonizes the mammalian lower gastrointestinal tract, has been demonstrated to be the archetypal bacterial molecule capable of mediating development of the host immune system[33]. PSA presented by intestinal DCs activates CD4+ T cells and elicits appropriate cytokine production. Bacteroides species are among the earliest colonizing and most represented microorganisms of the gut microbiota[34], and they are not considered probiotic species. More recently Mazmanian et al[35] showed that the Bacteroides fragilis-produced PSA protects mice from experimental colitis induced by Helicobacter hepaticus: purified PSA is required to suppress pro-inflammatory IL-17 production by intestinal T cells, and it also protects from inflammatory disease by induction of IL-10-producing CD4+ T cells. Therefore, although bacteria may have developed polysaccharide capsules known to be not recognized by the immune system, it may be that the host not only tolerates but also has evolved to require cooperation by commensal bacteria for its health. Strikingly, the finding that PSA from Bacteroides fragilis is a natural anti-inflammatory molecule that promotes health, so clearly performing important probiotic activities, is not produced by a probiotic bacteria, provides a fundamental platform for the discovery of new biomolecules having important probiotic effects independently from their bacterial derivation.

Polysaccharides synthesized extracellularly (exopolysaccharides, EPSO) also represent attractive candidates as probiotic effector molecules interacting witch PRRs. EPSO are produced by both probiotic and symbiotic bacteria, and also potentially pathogenic bacteria, but they have not yet been studied in detail.

On the other hand, lipoteichoic acid (LTA) is considered the major immunostimulating component of the cell wall of Gram-positive bacteria via TLR 2 (most of the known probiotics, Lactobacilli and Bifidobacteria, are Gram-positive bacteria), in the same way as LPS is the major immunostimulating component in the cell wall of Gram-negative bacteria via TLR 4. Two important concepts concerning LTA have emerged in recent years: the first concerns the much lower potency in the stimulation of TLR 2 pathway to induce pro-inflammatory molecules by using purified LTA from a probiotic strains of Lactobacillus plantarum in comparison with a pathogenic strain of Staphylococcus aureus[36]; the second very important concept is related to the possible modification of LTA molecules to induce a substantial reduction in D-alanine content with a marked increase in glucose substitutions[37]. These modified LTA may be candidates as probiotic effector molecules able to induce secretion of anti-inflammatory IL-10.

On the other hand, LPS synthesized by Gram-negative bacteria of the gut microbiota have been recently involved in the development of inflammation, obesity and type 2 diabetes induced by a high fat diet[38]. If confirmed, these findings open up a new possible role in this field not only for a direct bacterial competition by live probiotics, but also for research into non-immunostimulating molecules competing with LPS for the TLR 4 pathway.

Finally, both lipoproteins and glycoproteins present at the cell surface are also attractive candidates as probiotic molecules for their interactions with TLR 2 receptors, but to date their role is unexplored even in pathogenic bacteria[39]. However flagellins of the Escherichia coli Nissle 1917 induce the expression of human β-defensin 2, an inducible antimicrobial peptide[40].

Recombinant probiotics: Colonizing (e.g., Streptococcus gondii, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus acidophilus) as well as non-colonizing (e.g., Lactobacillus lactis) bacterial species have been investigated both as live vaccine vehicles (acting as carriers for protective antigens) and as active producers of molecules with known pharmacological properties.

In respect to the use of these microorganisms as carriers for antigens, the most complete studies have been carried out with the 50 kDa carboxy-terminal fragment of tetanus toxin[41]; this approach has now been extended to additional antigens eg the B subunit of cholera toxin[1-3].

Transfected bacteria have also been used to deliver cytokines, but this technique was recently used to investigate other biological properties. Steidler et al[42] chose to construct recombinant Lactobacillus lactis strains secreting murine IL-10. These authors demonstrated that these recombinant strains were able to prevent and treat inflammation in two murine models of colitis. Significantly, the same effects were obtained with much lower doses of IL-10 than those required when IL-10 itself was used as a drug. The same authors further constructed a safe (no antibiotic resistance markers and a chromosomally integrated transgene) strain of Lactobacillus lactis secreting IL-10 of human origin[43]. Authorization by a local ethical committee to carry out a phase 1 clinical study on voluntary patients has been obtained in the Netherlands[44]. In this study, Crohn’s disease patients were treated with recombinant Lactobacillus lactis (LL-THY 12) in which the thymidylate synthase gene was replaced with a synthetic sequence encoding mature human IL-10. The oral administration of this strain was safe and a decrease in disease activity was observed. The authors concluded that the use of genetically modified bacteria for mucosal delivery of therapeutic proteins is a feasible strategy in human beings. This strategy avoids systemic side effects and appears suitable as maintenance treatment for chronic intestinal diseases. Novel therapeutic strategies for acute and chronic colitis based on recombinant probiotics were also assessed by the generation and in vivo evaluation of Lactobacillus lactis strains secreting bioactive murine trefoil factors (TFF)[45]. The authors demonstrated that intragastric administration of this bacterial strain, but not of purified TFF, led to prevention and healing in the acute dextran sodium sulfate (DSS)-induced murine model of colitis, and was similarly effective in reducing established chronic DSS colitis. It has also to be remembered that production and mucosal delivery of different bioactive molecules such as allergens, digestive enzymes and single-chain Fw antibodies have been achieved using lactic acid bacteria[3]. Targeted diseases included vaginal candidiosis[46], dental caries[47], allergies[48-50], autoimmune diseases[51,52], human papillomaviruses-induced tumors[53] and pancreatic insufficiency[54]. More recently, Rosberg-Cody et al[55] investigated whether a recombinant strain of Lactobacillus paracasei, previously isolated from the human gastrointestinal tract, expressing conjugated linoleic acid (CLA) isomerase from Propionibacterium acnes, could influence the fatty acid composition of different tissues in the mouse. Ingestion of the Lactobacillus paracasei strain expressing CLA isomerase was associated with a 4-fold increase (P > 0.001) in t10c12 CLA in adipose tissues of the mice when compared with animals that received the non-CLA producing isogenic strain. These data show that a single gene encoding CLA isomerase expressed by an intestinal bacterium can influence the fatty acid composition of the host adipose tissue. This t10c12 CLA isomer is also associated with decreased body fat and increased lean body mass in various animal species[56-60] and, to some extents, human beings[61-65]. It is also well known that t10c12 CLA isomer is the most potent isomer in terms of potential to prevent cell proliferation and induce apoptosis in cancer cells[66-69]; notably, when the microbially derived t10c12 CLA was incubated with SW480 colon cancer cells for 5 d, cell viability was decreased by 92%[70], and it is possible that a CLA-producing probiotic will be able to keep colon cancer cells in check. Although commensal Bifidobacterium and Lactobacillus species from the gastrointestinal tract have been shown to produce CLA in vitro[71-73], the majority of these studies have demonstrated the production of c9t11 CLA from linoleic acid, while only a few bacteria such as Propionibacterium acnes[74] , the rumen bacterium Megasphera elsodenii[75], and the human derived Lactobacillus rhamnosus PL60 and Lactobacillus plantarum PL62[76,77] have been reported to produce t10c12 CLA. Modulation of fatty acid production by bacteria may represent very important probiotic activity and recombinant probiotics may become useful for this in the near future.

Recombinant probiotics may be linked not only to the addition of one or more genes but also to the deletion of one or more genes. In fact, to study the molecular mechanisms involved in the induction and repression of intestinal inflammation, Mohamadzadeh et al[78] have recently deleted the phosphoglycerol transferase gene that plays a key role in LTA biosynthesis in Lactobacillus acidophilus NCK 56.

The results of these authors show that the Lactobacillus acidophilus LTA^- not only down regulated IL-12 and TNF-α, which are known pro-inflammatory cytokines, but also significantly enhanced IL-10 production by DC and controlled the regulation of co-stimulatory DC functions, resulting in their inability to induce CD 4+ T cell activation. The treatment of mice with Lactobacillus acidophilus LTA^-, compared with Lactobacillus acidophilus LTA⁺, significantly counteracted DSS-induced colitis. These authors concluded that directed alteration of cell-surface components of Lactobacillus acidophilus represents a potential new strategy for the treatment of inflammatory intestinal disorders.

Moreover, efforts have been devoted to improve the efficacy of probiotic bacteria as delivery systems; in this context cell wall mutants of Lactobacillus plantarum and Lactobacillus lactis defective in alanine racemase (alr gene) were constructed[79,80]: each of these mutants behaved as a substantially improved antigen delivery system compared with its wild-type counterpart[81]. The potency of the Lactobacillus plantarum Alr^- mutant was further confirmed using a weak immunogen, such as Helicobacter pylori urease B, as a protective antigen; a significant reduction of the Helicobacter pylori load in the mouse stomach was achieved after immunization with the recombinant mutant Lactobacillus plantarum strain in contrast to results obtained with its wild-type counterpart[82].

Any gene coding for an active molecule, potentially useful for human health, may be used to generate recombinant probiotic bacteria; in this context, an impressive number of options are available to be investigated in in vitro and in vivo studies. It is worthy of note, however, that several gene products need glycosylation, phosphorylation or other more complex chemical changes; these may require the enzymatic machinery of eukaryotic cells. Thus, although current available genomic information should greatly facilitate the generation of useful recombinant probiotics, several technical issues and biologically limiting factors have to be taken attentively in consideration. In any case, the use of rapidly evolving genomic technology will surely help to evolve this intriguing and fascinating field and we can expect that from the present pioneering status we will soon progress to the generation of innovative therapeutic tools.

CONCLUSION

We are convinced that, even if as mentioned above there is a very large amount of work to be performed in this field, the available evidence is already enough to move from the actual concept of probiotics to novel and very promising pharmacobiotic strategies. In fact, probiotic molecules and recombinant probiotics may represent an unlimited resource for innovative therapeutics. The following questions arise from the present analysis of available knowledge: (1) Why the therapeutic potential of probiotic molecules and recombinant probiotics has been neglected so far? (2) Why important studies showing that whole live bacteria are not needed for probiotic activity have not received adequate attention by the scientific community? (3) Why molecules such as polysaccharide-peptidoglycan (PSPG)-I from Lactobacillus shirota, which have demonstrated to be able to suppress inflammation in chronic intestinal inflammatory disorders via inhibition of IL-6, have not been extensively investigated yet? IL-6 plays a pivotal role both in activation and sustainment of Th 17 response as well as a crucial role as a proinflammatory IL in Th 17 and Th1 cell responses. Thus the dose dependent pharmacological inhibition of IL-6 levels could have a crucial clinical impact, as suggested by animal studies[83] . Based on these considerations, (4) Why, after identification of adequate drug delivery strategies (in fact, there may be major challenges with formulation and delivery in single cases), the clinical effectiveness of PSPG-I has not been assessed yet? and (5) Why has only a phase I study has been performed with recombinant probiotics? These are crucial questions with important implications. Thus these questions should be discussed at international level by a forum involving different players including, basic researchers, clinicians (gastroenterologists, pediatrics, allergologists, pneumologists, etc.), lawgivers and regulatory agencies, and probiotics pharma. Although there are already international organizations that declare to be independent of the industry, such as the aforementioned ISAPP, which tackle these issues, these have within them industry advisory committee members and have not been able until now to pull the current outdated concept of probiotics to the more inclusive concept of pharmabiotics.

Although guidelines and recommendations substantiating the evidence for beneficial effects of probiotics in different clinical conditions of adult patients have been published[6,7,84-87], the only clinical conditions in which strains of whole live probiotics have been shown to be effective thus far are acute gastroenteritis and antibiotic-associated diarrhoea. It seems therefore that live probiotics can exert a competitive action and can have a role in restoration of intestinal flora. However, a specific role in the cure of chronic and/or autoimmune diseases has not been conclusively demonstrated. Despite this, an entire world involving probiotic molecules and recombinant probiotics is ready to be investigated. In any case, if specific live probiotic strains have been or will be found effective in specific concentrations for specific disease conditions should they still categorized as food supplements? To what extent does the market influence the national regulatory laws in this area? We think that gut microbiota and probiotic bacteria represent an inexhaustible mine from which countless molecules of potential value for human health can be obtained and investigated. If this does not happen, we risk going on discussing whether a live strain is better than another without ever reaching any definitive conclusion for many years. Even if single RCTs demonstrate a level of evidence 1a, but the findings are not confirmed by other authors in order to remove any doubt about the therapeutic role of that strain in the given clinical condition at that specific doses and route of administration, it does not solve the problem and continues to maintain doubts about the role of probiotic therapy. In addition, it should be underlined that clinical studies are almost always sponsored by companies and results of rigorous RCTs are restricted to the strains of company interest. Who needs to maintain the “status quo” without moving the research to a plot of real pharmacobiotic strategies? Is the huge market based on “easy” trade of live microorganisms involved? We do not want to be unpleasant to anybody, but we think that opening an international forum on this important issue would be of great benefit to both physicians and patients. If to tell the story of salutistic products through well-made advertisements in the media induces big gains without big expenses, we fear that hardly anyone will decide to invest in this area. This way the birth of the pharmacobiotic era will turn away more and more. The resources that are available to us are often sacrificed by humans on the altar of interests and market strategies: among the chief concerns of the scientific community is the need to denounce all those situations in which scientific rigor is sacrificed to commercial interests. To avoid this, the following two actions are mandatory: (1) international guidelines by the forum of players made available to scientists and clinicians, patient organizations, and lawgivers; and (2) public research funds made available to the scientific community for performing independent rigorous studies both at preclinical and clinical levels.