Review article: yeast as probiotics –Saccharomyces boulardii

Authors


Dr D. Czerucka, INSERM U526, 28 avenue de Valombrose, 06107 Nice cedex 2, France.
E-mail: czerucka@unice.fr

Abstract

Summary

Background

Probiotics are defined as live micro-organisms which confer a health benefit on the host. Although most probiotics are bacteria, one strain of yeast, Saccharomyces boulardii, has been found to be an effective probiotic in double-blind clinical studies.

Aims

To compare the main properties that differentiates yeast from bacteria and to review the properties of S. boulardii explaining its potential benefits as a probiotic.

Methods

The PubMed and Medline databases were searched using the keywords ‘probiotics’, ‘yeast’, ‘antibiotic associated diarrhea’, ‘Saccharomyces boulardii’,‘bacterial diarrhea’ and ‘inflammatory bowel disease’ in various combinations.

Results

Several clinical studies have been conducted with S. boulardii in the treatment and prevention of various forms of diarrhoea. Promising research perspectives have been opened in terms of maintenance treatment of inflammatory bowel diseases. The mechanism of S. boulardii’s action has been partially elucidated.

Conclusion

Saccharomyces boulardii is a strain of yeast which has been extensively studied for its probiotic effects. The clinical activity of S. boulardii is especially relevant to antibiotic-associated diarrhoea and recurrent Clostridium difficile intestinal infections. Experimental studies clearly demonstrate that S. boulardii has specific probiotic properties, and recent data has opened the door for new therapeutic uses of this yeast as an ‘immunobiotic’.

Introduction

The gastrointestinal (GI) microflora (‘microbiota’) is an extremely complex ecosystem that coexists in equilibrium with the host. When this equilibrium is disrupted, clinical disorders may occur. Microbiota plays a well-established role in infectious GI diseases. Recent research has linked intestinal microbiota disequilibrium to such GI disorders as antibiotic-associated diarrhoea (AAD), ulcers, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) and colon cancer. Furthermore, the microbiota has been proposed as a major regulator of the immune system outside the gut. Attempts have been made to improve the health status of affected individuals by modulating the indigenous intestinal flora using living microbial adjuncts called ‘probiotics’.

Probiotics have been defined as viable micro-organisms that (when ingested) have a beneficial effect in the prevention and treatment of specific pathological conditions.1 In fact, probiotics have been used for as long as people have eaten fermented foods. In the early 20th century, the Russian immunologist Elie Metchnikoff suggested that lactobacilli ingested in yogurt could have a positive influence on the normal microbial flora of the intestinal tract.2 He hypothesized that lactobacilli were important for human health and longevity. In recent years, the definition of a probiotic has changed, primarily because of the recognition that probiotic bacteria can influence the physiological outcomes, distant from the gut lumen. Moreover, the activation of local mucosal protective mechanisms and the modulation of adaptative immune effector functions can influence protection levels and the degree of inflammation at all mucosal sites. These observations shifted the concept of probiotics from a narrow range of dairy isolates that fermented milk and could ‘promote health’ to the concept of ‘immunobiotics’.3

Because viable and biologically active micro-organisms are usually required at the target site in the host, it is essential that the probiotic be able to withstand the host’s natural barriers against ingested micro-organisms. Most probiotic micro-organisms are bacteria. Strains of Lactobacillus acidophilus and Lactobacillus rhamnosus strain GG (formerly Lactobacillus casei) probably have the longest history of application as probiotics because of their health benefits. Currently used commercial probiotic products include Lactobacillus ssp., Bifidobacterium and even a few non-lactic acid bacteria.

Specificity of Yeast

Saccharomyces boulardii, a patented yeast preparation, is the only yeast probiotic that has been proven effective in double-blind studies.4 This yeast is used in many countries as both a preventive and therapeutic agent for diarrhoea and other GI disorders caused by the administration of antimicrobial agents. Saccharomyces boulardii possesses many properties that make it a potential probiotic agent, i.e. it survives transit through the GI tract, its temperature optimum is 37 °C, both in vitro and in vivo, it inhibits the growth of a number of microbial pathogens. However, S. boulardii belongs to the group of simple eukaryotic cells (such as fungi and algae) and, it thus differs from bacterial probiotics that are prokaryotes. Table 1 lists the main properties differentiating the yeast from the bacteria that account for the specificity of S. boulardii as a probiotic.

Table 1.   Major differences between yeast and bacteria and their probiotic implications
 BacteriaYeastProbiotic implication
  1. LPS, lipopolysaccharide; LTA, lipoteichoic acid; PPM, phosphopetidomannan; LPM, phospholipomannan; TLR, Toll-like receptor; GI, gastrointestinal.

Presence in human flora99%<1% 
Cell size1 μm 10 μmStearic hindrance
Cell wallPeptidoglycan, LPS (Gram-negative-), LTA (Gram-positive)Chitin, mannose (PPM, PLM), glucanImmune response via TLRs, lectin receptors
Optimal growth conditions
 pH6.5–7.54.5–6.5Different sites of action in the GI tract
 Temperature (°C)10–8020–30
Resistance to antibioticsNoYesSafety in combination with antibiotherapy
Transmission of genetic material (e.g. resistance to antibiotics)YesNo

Yeast in microbial ecology

Commensal bacteria in the gut constitute a heterogenous microbial system containing approximately 1014 bacteria.5 Yeast are a part of the residual microflora that makes up <0.1% of microbiota. Most yeast isolates from the GI tract are Candida albicans, although Torulopsis glabratra and Candida tropicalis are occasionally recovered.6 Although yeast account for only a minority of the organisms making up the microbiota, their cell size is 10 times larger than that of bacteria (Figure 1) and they could represent a significant stearic hindrance for bacteria.

Figure 1.

 (a) Scanning electron micrographs of T84 cells exposed to Saccharomyces boulardii and Salmonella typhimurium. (b) Electron micrographs showing S. boulardii and S. typhimurium.

Microbial colonization of the human GI tract varies in number and species of bacteria as a function of environmental conditions.5 The low pH of the stomach, ranging from 2.5 to 3.5, is destructive to most microbes; it grows up towards the distal part of the GI tract. While the pH rises towards the distal part of the GI tract, the presence of aggressive intestinal fluids (e.g. bile and pancreatic juice) and the short transit time in the duodenum, creates a hostile environment, and the duodenum thus contains relatively few microbes. Yeast are found in the stomach and colon. The presence of yeast in such different conditions can be explained by their resistance to pH variation (Table 1). In fact, while yeast grows well at pH 7–8, optimal growth is observed between pH 4.5 and 6.5. Most yeast can grow at pH 3.0, and some species can tolerate highly acidic conditions with a pH as low as 1.5. Yeast are thus good candidates as probiotics because probiotics entering the GI tract must be resistant to local stresses such as the presence of GI enzymes, bile salts, organic acids and considerable variations of pH and temperature.

Impact of antibiotics on yeast

The development of antimicrobial resistance by the pathogenic bacteria associated with antibiotic treatment has become an important public health problem. The natural resistance of yeast to antibacterial antibiotics is thus, a major argument for their use in antibiotic-treated patients. Antimicrobial resistance occurs both vertically (inherent or natural resistance of bacterial species or genus) and horizontally because of the transfer of genes between bacteria. The mammalian GI tract provides favourable conditions for the transfer of genetic material between many species of bacteria.7 Resistance genes might be transferred not only between members of the resident gut flora, but also to and from transient bacterial probiotics. Recently, many investigators have speculated that commensal bacteria, including lactic bacteria, may act as reservoirs of antibiotic resistance genes similar to those found in human pathogens. Genes conferring resistance to tetracycline, erythromycin and vancomycin have been detected and characterized in Lactobacillus lactis, Enterococci and, recently, in Lactobacillus species isolated from fermented meat and milk products and in strains used as probiotics (for review see Refs8, 9). The main threat associated with these bacteria is that they might transfer resistance genes to pathogenic bacteria. No such transfer of genetic material occurs between bacteria and yeast, making yeast safe for use during antibiotic treatment.

Cell wall components are determinants in the immune response

Substantial differences in the cell wall composition of bacteria and yeast have an impact on their antigenic responses. All bacteria contain a high-molecular weight sugar associated with protein that forms a rigid structure called peptidoglycan. Gram-negative and Gram-positive bacteria differ in the lipid concentration of their cell wall. Gram-negative bacteria contain up to 20% lipids composed of lipopolysaccharide (LPS) while Gram-positive organisms have much fewer lipids in their cell walls but contain lipoteichoic acids (LTA).10 The yeast cell wall consists of at least two layers. The outer layer contains a combination of mannose associated with either protein [phosphopetidomannan (PPM), commonly termed mannan] or lipid [phospholipomannan (PLM)]. The inner layer is composed of chitin and 1,3-β- and 1,6-β-glucan.11 In living species, the first line of defence against microbial aggression is innate immunity.12 Innate immunity relies on the recognition of pathogen-associated molecular pattern (PAMP) antigens by specific proteins referred to as pattern-recognition receptors (PRRs). Peptidoglycan, LPS and LTA, which are present in bacteria, and PLM, PPM and glycan, which are present in yeast, are all PAMPs and are recognized by different PPRs and thus can account for different responses of these micro-organisms as ‘immunobiotics’.12

Specificity of S. boulardii

The non-pathogenic yeast S. boulardii was isolated from litchis in Indochina and is not a part of the autochthonous flora. It has been prescribed since mid-20th century, providing empirical evidence of its efficacy as an adjuvant agent for the treatment of diarrhoea and the prevention of AAD. Starting in the 1980s, research was conducted to evaluate the benefits of S. boulardii for the host organism and to determine its mechanisms of action. In particular, studies have investigated this yeast’s effect in bacterial infections, its effects on the mucosa and, more recently, its immunomodulatory properties. Saccharomyces boulardii is the only yeast whose effect has been evaluated in double-blind clinical studies.

Saccharomyces boulardii was initially identified as a separate species of the hemiascomycete genus Saccharomyces.13 In 1994, Cardinali and Martini14 classified S. boulardii outside of S. cerevisiae species using comparative electrophoretic karyotyping and multivariate analysis of the polymorphism, observed in pulsed-field gel electrophoresis (PFGE). However, the rapid development of molecular phylogenetics in recent years, had led to changes in the classification of many yeast species.15 Typing using four molecular techniques [species-specific polymerase chain reaction (PCR), randomly amplified polymorphic DNA–PCR, restriction fragment length polymorphic analysis of rDNA spacer region and PFGE] classified S. boulardii within the species S. cerevisiae.16 Recently, Edwards-Ingram et al.17 using comparative genomic hybridization for whole-genome analysis, also concluded that S. cerevisiae and S. boulardii are the members of the same species.

However, genetically S. boulardii differs from other S. cerevisiae. Hennequin et al.18 identified a unique and specific microsatellite allele characterizing S. boulardii that distinguishes it from other strains of S. cerevisiae. Recently, pertinent characteristics of the S. boulardii genome such as trisomy of chromosome IX and altered copy number of individual genes have been revealed by comparative genome hybridization using oligonucleotide-based microarrays coupled with a rigorous statistical analysis.17 Authors suggest that overexpression of gene related to protein synthesis and stress responses could be contributing to the increased growth rate and better survival of S. boulardii in acid pH.

In fact, metabolically and physiologically, S. boulardii differs considerably from S. cerevisiae, particularly, when it concerns as concerns growth yield and resistance to temperature and acidic stresses.19 Whereas most S. cerevisiae strains grow and metabolize at a temperature of 30 °C, S. boulardii is a thermotolerant yeast that grows optimally at 37 °C, i.e. the physiological temperature of the host. Recent studies have demonstrated that S. boulardii appears to be more resistant than the S. cerevisiae strain W303 when exposed to a simulated gastric environment.19

However, the overexpression of genes in S. boulardii did not correlate with increased adherence to epithelial cells or transit through mouse gut.17 Pharmacokinetic studies performed in man and rat, have shown that, after repeated administration, S. boulardii achieves steady-state concentrations in the colon within 3 days and is cleared from the stools 2–5 days after discontinuation.20, 21

Resistance to antibiotics

As S. boulardii is naturally resistant to antibiotics, it can be prescribed to patients receiving antibiotics.22 Research on the administration of S. boulardii to patients suffering from recurrent Clostridium difficile infections has shown that the faecal yeast count is significantly higher in patients who do not relapse compared to patients that do.21 The efficiency of S. boulardii treatment thus appears correlated with the faecal yeast concentration.

Safety and packaging

Except for several sporadic reports of fungaemia, in patients with severe general or intestinal disease who had an indwelling catheter,23S. boulardii is considered to be a safe and well-tolerated treatment. The origin of these cases of fungaemia remains unclear, but is likely related to catheter colonization.23, 24 Presence of such catheters is thus, a contraindication for the administration of S. boulardii.

Saccharomyces boulardii is administered in a lyophilized form, and is prepared, packaged and controlled as such. Therefore, lyophilized S. boulardii is clearly distinct from dietary probiotic products which contain diverse strains of micro-organisms and are used either in animals to improve zootechnical yields or in healthy humans (often in form of yogurt) to strengthen host physiology in the absence of any pathological context. Saccharomyces boulardii can be considered, an example of a ‘probiotic drug’.

S. boulardii IN PLACEBO-CONTROLLED CLINICAL TRIALS

Antibiotic-associated diarrhoea

Antibiotic-associated diarrhoea is a common complication of antibiotic use. Surawicz et al.25 evaluated the efficacy of S. boulardii administered during treatment and continued for 2 weeks, after the end of course in 180 hospitalized patients receiving antibiotics belonging to various classes. The incidence of diarrhoea was significantly reduced in patients receiving S. boulardii (10% vs. 22% in placebo, P = 0.038). The same authors carried out a similar study, focusing on β-lactam antibiotics and prolonging the follow-up period for 7 weeks, after the drug had been stopped.26Saccharomyces boulardii (at the dosage of 1 g/day) or placebo was administered to 193 patients from the beginning of antibiotic treatment and continued 3 days after the end of the course. In this study, S. boulardii mediated a significant preventive effect on the occurrence of diarrhoea (7% vs. 15%, P = 0.02). Similar results had been obtained by Adam et al.27 in a study of 388 out-patients taking tetracycline or β-lactam antibiotics together with S. boulardii at a dosage of 200 mg/day. The frequency of diarrhoea was 18% in the placebo-treated group, compared to 5% in the S. boulardii-treated group (P < 0.001). A recent study investigated, the impact on AAD of S. boulardii in children with otitis media and/or respiratory tract infections.28 Children received antibiotics plus 250 mg of S. boulardii (n = 132) or placebo (n = 137) orally, twice daily for the duration of antibiotic treatment. Analyses included data from 246 children and show that patients receiving S. boulardii, had a lower prevalence of diarrhoea than those who received the placebo (8% vs. 23%); RR, 0.3 (95% CI: 0.2–0.7); NNT, 7 (95% CI: 5–15). In a meta-analysis of probiotics for the prevention of AAD, D’Souza et al.29 concluded that two types of probiotics (S. boulardii and S. lactobacilli) have the potential to be used in that situation. Nevertheless, S. boulardii is the probiotic that has been the most extensively studied with four large-scale placebo-controlled clinical studies showing a significant efficacy for preventing AAD (Table 2).

Table 2.   Large-scale randomized trials of Saccharomyces boulardii in the prevention of antibiotic-associated diarrhoea (AAD)
ProbioticsPatientsNAntibiotic treatmentEffect (% of diarrhoea)
S. boulardii vs. placebo
P-valueReference
S. boulardiiHospitalized patients180Various10 vs. 220.03824
S. boulardiiHospitalized patients193β-Lactam7 vs. 150.0225
S. boulardiiOut-patients388β-Lactam or cyclin5 vs. 180.00126
S. boulardiiChildren in-patients and out-patients246Various8 vs. 23RR = 0.327

Clostridium difficile accounts for 20–25% of AAD in hospitalized patients and about 10% of AAD in community patients.30Clostridium difficile is responsible for 95% of pseudomembranous colitis. Saccharomyces boulardii is the sole probiotic that has proven a significant efficacy in treating relapsing C. difficile-associated diarrhoea. In a randomized, placebo-controlled trial, McFarland et al.31 evaluated the effect of S. boulardii (1 g/day for 28 days) and placebo as adjunctive therapy to metronidazole or vancomycin in 124 patients. It was the first episode of C. difficile infection in 64 cases and a relapse in 60 cases. In this study, after the administration of S. boulardii, the authors observed a 50% reduction of recurrences in patients who had previously experienced a first relapse of C. difficile infection. Surawicz et al.32 obtained similar results, but only in patients receiving high-dose vancomycin.

Infectious diarrhoea

Traveller’s diarrhoea

Traveller’s diarrhoea is a well-known public health problem, particularly among travellers to developing countries. Enterotoxinogenic Escherichia coli, Shigellae and Salmonellae account for about 80% of cases with an identified pathogen in acute diarrhoea in travelers.33 Kollaritsch et al.34 evaluated the efficacy of S. boulardii for the prevention of diarrhoea in 1016 travellers visiting various countries in the world. The incidence of diarrhoea was 40% in patients receiving placebo, 34% in patients receiving S. boulardii 250 mg/day (P = 0.019) and 29% in patients receiving S. boulardii 1 g/day (P < 0.005). In a meta-analysis of probiotics for the prevention of traveller’s diarrhoea analysing 12 different studies, McFarland35 concluded that two probiotics, S. boulardii and a mixture of L. acidophilus and Bifidobacterium bifidum, had significant efficacy.

Acute diarrhoea in children

In children, infectious diarrhoea represents a public health problem and in the developing countries, several million children die of dehydration every year. In a meta-analysis assessing the efficacy of probiotics in the treatment and prevention of acute infectious diarrhoea, Szajewska et al.36 have demonstrated that there is evidence of a clinically significant benefit of probiotics, Lactobacillus GG showing the most consistent effect. Since then, Kurugol et al.37 have investigated the effect of S. boulardii in a double-blind randomized study involving 200 children. The duration of diarrhoea was significantly reduced (4.7 vs. 5.5 days, P = 0.03) as well as the number of days of hospitalization (2.9 vs. 3.9 days, P < 0.001). Saccharomyces boulardii has also been shown to be efficient, in reducing the number of children with prolonged diarrhoea (three of 44 vs. 12 of 44; RR 0.25; 95% CI: 0.1–0.8) in a double-blind, randomized study.38 Finally, preliminary results suggest that, S. boulardii might be effective in preventing the occurrence of new episodes of diarrhoea in a 2-month long-term follow-up.39

Tube-feeding-associated diarrhoea

Diarrhoea is a common complication in critically ill patients receiving enteral nutrition. The addition of S. boulardii to nutrient supplements administered to patients receiving enteral nutrition decreased the incidence of diarrhoea. A 50% reduction in the number of treatment days with diarrhoea was reported in one study.40 For patients suffering from moderate-to-severe burns receiving enteral nutrition, a reduction in the number of days with diarrhoea was accompanied by an increase in the mean number of calories tolerated per day.41 In a multicentre, randomized, double-blind, placebo-controlled trial involving 128 critically ill tube-fed patients,42 treatment with S. boulardii reduced the mean percentage of days with diarrhoea per feeding days from 19% to 14% (OR = 0.67, 95% CI: 0.50–0.90, P = 0.0069). The improvement of diarrhoea was more important in patients with high risk for diarrhoea (up to 42% vs. 25% in the whole study population).

AIDS

A randomized, double-blind trial covering 35 patients with AIDS-related diarrhoea showed the efficacy of S. boulardii 3 g/day given for 7 days in resolving diarrhoea. Sixty-one percentage of the patients were diarrhoea-free after 1 week of treatment with S. boulardii vs. 12% in the placebo group.43

Inflammatory bowel diseases

Three preliminary studies have evaluated the effect of S. boulardii in patients with IBD. A double-blind study of 20 patients suffering from Crohn’s disease with moderate activity found that the addition of S. boulardii to conventional therapy with sulfasalazine or mesalazine (mesalamine) and corticosteroids significantly reduces bowel movements.44 Similarly, a single-blind study of 32 patients with Crohn’s disease of the ileum or colon who had been in remission for ≥3 months45 showed that 6-month maintenance therapy with mesalazine 500 mg twice daily plus S. boulardii 500 mg/day was significantly more effective in preventing relapse than mesalazine 500 mg three times daily (P = 0.04). Finally, an open pilot study evaluated 25 patients with a clinical flare-up of left-sided, mild-to-moderate ulcerative colitis receiving ≥3 months’ maintenance therapy with mesalazine. The addition of S. boulardii 250 mg three times daily for 4 weeks to the mesalazine regimen resulted in therapeutic success according to Rachmilewitz’s Clinical Activity Index (i.e. stool frequency, blood in stool) in 68% of the patients.46 Further placebo-controlled studies are needed to support these preliminary results.

Irritable bowel syndrome

In a double-blind, placebo-controlled study in patients presenting with diarrhoea-predominant IBS,47S. boulardii treatment resulted in a decrease of the daily number of stools (P < 0.05) and an improvement of the consistency of the stools (P < 0.05).

EXPERIMENTAL EFFECT

Effect on enteric pathogens

Several studies using animal models or cell models indicated that S. boulardii may exert a beneficial effect against various enteric pathogens such as C. difficile, Vibrio cholerae, Salmonella, Shigella and E. coli. Saccharomyces boulardii appeared to act by two main mechanisms: (i) production of factors that neutralized bacterial toxins and (ii) modulation of the host cell signalling pathway implicated in proinflammatory response during bacterial infection (reviewed in Ref.48 and Figure 2).

Figure 2.

 Proposed model for the mechanism of action of Saccharomyces boulardii against Vibrio cholerae (a), Clostridium difficile (b) and pathogenic Escherichia coli (EPEC and EHEC) infections (c). (Panel a) Saccharomyces boulardii produces a 120 kDa protein that exerts an effect on intestinal mucosa and inhibits cholera toxin (CT)-stimulated adenylate cyclase (AC) and chloride secretion. Saccharomyces boulardii also binds CT. (Panel b) Saccharomyces boulardii acts on intestinal mucosa and decreases phosphorylation of MLC implicated in the control of tight junctions as well as activation of MAPK and NF-κB implicated in the synthesis of proinflammatory cytokine IL-8 and TNF-α. (Panel c) Saccharomyces boulardii secretes a protease (>50 kDa) that lyses C. difficile toxins A and B and a protein (<10 kDa) that inhibits the signalling pathway implicated in IL-8 synthesis. Saccharomyces boulardii stimulates antitoxin A IgA production.

Neutralization of bacterial toxins

The antitoxin action of S. boulardii was demonstrated in cases of C. difficile infection. Toxin A, a 308 kDa protein, is a major virulence factor of C. difficile. Injection of toxin A into rodent intestines caused fluid secretion, increased mucosal permeability, mucosal damage and release inflammatory mediators. Oral administration of S. boulardii to rats before the addition of toxin A to the intestinal loop reduced toxin A-induced intestinal secretion and permeability.49 Further investigation demonstrated that the addition of toxin A mixed with S. boulardii-filtered supernatant decreased toxin A-induced secretion. Two fractions were identified in S. boulardii supernatant: a fraction enriched in a 54 kDa serine protease that acted by proteolysis of both the toxin A and its receptor50 and, another fraction (<10 kDa) that exerted an anti-inflammatory effect.51

Another antitoxin factor produced by S. boulardii was described in cases of cholera toxin (CT). Anatomopathological studies on the small intestine of rats receiving V. cholerae alone and rats pre-treated for 5 days with S. boulardii have shown that the yeast prevents the morphological damage caused by V. cholerae.52 Vidon et al.53 demonstrated that the addition of S. boulardii to the intestinal loops of rats treated by CT decreased CT-induced fluid and sodium secretion by 50%. In vitro studies demonstrated that S. boulardii produces a 120 kDa protein that inhibits CT-stimulated chloride secretion by reducing the formation of cyclic AMP.54, 55

Saccharomyces boulardii also synthesized a phosphatase that can dephosphorylate endotoxins such as LPS from E. coli O55B5 and can partially inactivated its cytotoxic effects.56 This mechanism may account for the protection afforded in cases of sepsis.

Modification of host cell signalling

Enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) shares a common pathogenic mechanism characterized by bacterial adhesion to the intestinal mucosa and subsequent changes in the integrity of tight-junction permeability and activation of signalling pathways (mitogen activated protein kinase (MAPK) and the transcription factor NF-κB) that stimulated IL-8 synthesis. In vitro studies have demonstrated that exposure of cells to S. boulardii before the addition of bacteria prevents the EPEC- and EHEC-induced decrease in transepithelial resistance and IL-8 secretion, suggesting that the yeast exerted a preventive effect.57, 58 The observation that the yeast did not modify the number of adherent bacteria prompted research on the effects of S. boulardii on host cell signalling. The yeast has been shown to abolish phosphorylation of the myosin light chain (MLC) that is associated with a cytoskeletal protein involved in intercellular tight-junctions control. Saccharomyces boulardii also inhibits EPEC- or EHEC-induced NF-κB DNA-binding activity and activation of MAP kinases.57, 58 TNF-α synthesis is controlled by MAP kinase and NF-κB. Recently, Dalmasso et al.59 demonstrated that S. boulardii delays EHEC-induced apoptosis; this can be partially explained by the reduced TNF-α synthesis observed in the presence of yeast. Thus, in vitro, S. boulardii modified the signalling pathways implicated in proinflammatory cytokine synthesis.

Production of antitoxin factors as well as modification of proinflammatory responses of the host cell by S. boulardii do not exclude the possibility that other mechanisms may also account for the protective effect of S. boulardii in bacterial infection (Figure 2). Saccharomyces boulardii cell walls present binding properties for CT and EHEC.52, 60 In cases of C. difficile infection, an inhibitory effect of S. boulardii on C. difficile adhesion has also been reported61 as well as production of antitoxin IgA62 (Figure 2c).

Effect on intestinal immune factors

Oral ingestion of S. boulardii causes an increase in secretory IgA and the secretory component in the rat small intestine. Buts et al.63 found that growing rats which were given high doses of S. boulardii (0.5 mg/g body weight, three times a day) had an 80% increase in the secretory component of crypt cells and a 69% increase in villus cells. The mean secretory IgA level in the intestinal lumen was increased by 57%, and the polymeric immunoglobulin receptor concentration in crypt cells increased by 63% in S. boulardii-treated rats.

Trophic effect of S. boulardii on intestinal mucosa

Stimulation of brush-border membrane (BBM) enzymes by S. boulardii was first described in biopsies of human volunteers.64 This study showed significant increases in the specific and total activity of sucrase-isomaltase (+82%), lactase (+77%) and maltase-glucoamylase (+75%) after 8 days of oral treatment by yeast compared to the baseline biopsies. These observations were confirmed in growing rats65 and in two other studies conducted in rats after partial resection of the small bowel.66, 67 At last, oral administration of S. boulardii improved dissaccharidase activities, enhanced the absorption of d-glucose coupled to Na+ by the symport glucose/Na+ and expression of the sodium-glucose cotransporter-1 (SLGT-1) in the brush-border of the remaining intestinal segments. Saccharomyces boulardii cells contain substantial amounts of polyamines (673 nmol/100 mg of lyophilized preparation of S. boulardii); in light of the well-known physiological effects of polyamines on cell maturation, enzyme expression and membrane transport mechanisms, Buts et al.68 implicated polyamines as S. boulardii’s mediator of trophic effect. Recently, Schneider et al.69 investigated the effect of S. boulardii administration on short-chain fatty acids (SFCA) faecal concentration in patients on total enteral nutrition (TEN). They demonstrated that treatment with S. boulardii significantly increased total faecal SFCAs levels in TEN patients (150 ± 27.2 vs. 107 ± 18.2 mmol/kg), whereas no modification of SFCA was observed in controls. At the end of treatment with S. boulardii these TEN patients had higher faecal butyrate (16.0 ± 4.4 vs. 10.1 ± 2.9). In these patients S. boulardii did not modify faecal flora. Total SCFAs remained high 9 days after treatment was discontinued. The authors conclude from this study that S. boulardii-induced increase of faecal SCFAs concentration (especially butyrate) may explain the preventive effect of this yeast on TEN-induced diarrhoea.

Anti-inflammatory effect of S. boulardii

The beneficial effect of S. boulardii on intestinal inflammation was investigated in two murine models of colitis: CD45RBhi CD4+ T cell-restored SCID mice (chronic inflammation); and 2,4,6-trinitrobenzoic sulfonic acid (TNBS)-treated mice (acute colitis).70, 71 In both models, S. boulardii afforded protection from histological damage, suppressed NF-κB activation, and inhibited proinflammatory cytokine gene expression.72, 73

Several molecules have been hypothesized to play a role in this anti-inflammatory effect of S. boulardii. A small (<1 kDa) heat-stable and water-soluble anti-inflammatory molecule termed Saccharomyces anti-inflammatory factor (SAIF) has been identified in the yeast supernatant.74 Butyrate has been shown to inhibit the inflammatory response via inhibition of NF-κB75 and, butyric acid synthesis is increased in patients on enteral nutrition receiving S. boulardii.69 Another study has suggested that S. boulardii stimulates PPAR-γ expression and reduces the response of human colon cells to proinflammatory cytokines.76 Finally, administration of S. boulardii to rats with castor oil-induced diarrhoea modulates expression of the iNOS molecule that has also been implicated in the control of acute colitis.77

Saccharomyces boulardii has been shown to modify the migratory behaviour of lymphocytes.73 This effect was described in the chronic model of IBD based on injection of naive CD4+ CD45RBhi T lymphocytes that leads to the development of severe colitis characterized by infiltration of pathogenic IFN-γ-producing CD4+ T cells within the colon mucosa.70 In animals treated with S. boulardii, the inhibition of inflammation was correlated with a decrease of IFN-γ-producing CD4+ T cells within the colonic mucosa and an enrichment of IFN-γ-producing T cells in the mesenteric lymph nodes (mLN). Further research demonstrated that S. boulardii supernatant modifies the capacity of endothelial cells to adhere to leucocytes, allowing better cell rolling and adhesion. This last finding suggests, new pathways of investigation to improve our understanding of the action of S. boulardii. Identification of molecule(s) that inhibit(s) NF-κB activation and modify(ies) T lymphocyte cell rolling is primordial.

Conclusion

The antidiarrhoeal effect of lyophilized S. boulardii has been investigated in several forms of diarrhoeal diseases. The clinical efficacy of the yeast has been clearly demonstrated for both the prevention of AAD and the treatment of recurrent C. difficile disease. The superiority of probiotic yeast over probiotic bacteria in these indications is probably because of the natural resistance of yeast to antibacterial antibiotics, which leaves intact their viability and probiotic properties.

Data on acute gastroenteritis and on traveller’s diarrhoea are accumulating. The clinical relevance of S. boulardii in inflammatory bowel diseases needs further investigation.

Investigations designed to elucidate the mechanisms of action of S. boulardii have demonstrated the existence of additional mechanisms: release in vivo of substances that inhibit certain bacterial toxins and/or their pathogenic effects; trophic effects; antisecretory activity and immunostimulatory effects on the intestinal mucosa. The recent discovery of an anti-inflammatory effect opens the door to new clinical applications.

In conclusion, the activity of probiotics is strain-dependent, and in this context S. boulardii is the only yeast probiotic presenting its own specificity.

Acknowledgements

Declaration of personal and funding interests: Dorota Czerucka has served as speaker, a consultant for Biocodex and has received research funding from Biocodex. Patrick Rampal has served as speaker, a consultant for Biocodex and has received research funding from Biocodex. This study was funded in part by Biocodex.

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