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1 Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, C.P. 486, 30161-970, Belo Horizonte, MG, Brazil
2 Departamento de Pediatria, Faculdade de Medicina, Universidade Federal de Minas Gerais, C.P. 486, 30161-970, Belo Horizonte, MG, Brazil
3 Centro de Desenvolvimento da Tecnologia Nuclear/Comissão Nacional de Energia Nuclear (CDTN/CNEN), Belo Horizonte, MG, Brazil
4 Faculdade de Medicina, UNIFENAS, Belo Horizonte, MG, Brazil
5 Departamento de Patologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, C.P. 486, 30161-970, Belo Horizonte, MG, Brazil
Correspondence
Jacques R. Nicoli
jnicoli{at}icb.ufmg.br
Received 16 January 2006
Accepted 2 November 2006
Abbreviations: sIgA, secretory IgA.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTSDISCUSSIONREFERENCES |
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S. boulardii, a non-pathogenic yeast, was isolated from lychee fruit in Indochina and grows at the unusually high temperature of 37 °C (McFarland & Bernasconi, 1993). It has been used for the treatment of different types of diarrhoeal diseases, such as antibiotic-associated diarrhoea (Bartlett, 1992; McFarland et al., 1995; Surawicz, 2003), Clostridium difficile-associated intestinal disease (Elmer et al., 1999; Surawicz et al., 2000; Surawicz, 2003), traveller's diarrhoea (Scarpignato & Rampal, 1995) and diarrhoea in HIV-infected patients (Born et al., 1993).
Many mechanisms of action have been proposed to explain S. boulardii protection and they have been summarized by Czerucka & Rampal (2002). It was demonstrated that this yeast modulates the immune system (Buts et al., 1990; Rodrigues et al., 2000), degrades C. difficile toxins A and B and their respective receptors on colonic mucosa (Pothoulakis et al., 1993; Castagliuolo et al., 1999; Qamar et al., 2001), inhibits cholera toxin action (Czerucka et al., 1994; Brandão et al., 1998; Neves et al., 2002), modulates the transduction pathway induced by enteropathogenic and enterohaemorrhagic Escherichia coli (Czerucka et al., 2000; Dahan et al., 2003), stimulates enzymic activities (Jahn et al., 1996) and fixes some enterobacteria on its surface (Gedek, 1999).
Until now S. boulardii is the only yeast commercialized as a probiotic for humans, but some authors have suggested the use of other yeast species or genera based essentially on in vitro assays and very few clinical trials (Kovacs & Berk, 2000; Kumura et al., 2004; Van der Aa Kühle et al., 2005). Genetically, Saccharomyces cerevisiae is virtually identical to S. boulardii, but they are metabolically very different (Fietto et al., 2004). This leads to the question whether other strains of Saccharomyces, as well as S. boulardii, possess biotherapeutic properties. Brazil is well known for its rich biodiversity in terms of animal, vegetal and microbial species, and yeast with probiotic properties certainly could be screened from Brazilian environmental and agro-industrial sources. Previous results obtained in our laboratory showed that S. cerevisiae strain 905, isolated from ‘cachaça’ production, was able to colonize and survive in the gastrointestinal tract of germ-free and conventional mice, and to protect them against experimental infections with Salmonella enterica serotype Typhimurium and C. difficile (Martins et al., 2005). Additional results obtained in the same study showed that this protective effect was not due to reduction of the intestinal populations of the pathogenic bacteria.
The aim of the present work was to examine the effects of S. cerevisiae strain 905 on the translocation of Salm. Typhimurium, as well as its effect on some aspects of the immune system, in gnotobiotic and conventional mice in order to investigate the possible mechanism of protection.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Micro-organisms. The yeasts used in this work were from the yeast bank of Dr Carlos A. Rosa, Laboratory of Yeast Ecology and Biotechnology, UFMG. The identity of strain 905 was determined as S. cerevisiae using the computer program YEASTCOMPARE (Ciriello & Lachance, 2001). Salm. Typhimurium of human origin and E. coli B41 isolates were provided by the Laboratory of Microbial Ecology and Physiology, Department of Microbiology, UFMG. Salm. Typhimurium and E. coli B41 (0101 : K99 : F41) were maintained at –70 °C in brain heart infusion (BHI) broth (Difco) containing 20 % glycerol. S. cerevisiae 905 was maintained at –86 °C in medium containing 1 % yeast extract, 2 % peptone, 25 % glycerol. The identity of the bacteria was regularly confirmed by Gram staining and using the API 20E identification kit (BioMérieux). The purity of yeast cultures was routinely confirmed by inoculating onto BHI agar (Difco) supplemented with 100 mg cycloheximide l–1, as well as by microscopic examination of faecal dilution from yeast mono-associated gnotobiotic mice.
Mono-association and treatment. Yeast were grown in YPG (1 % yeast extract, 2 % peptone, 2 % glucose) broth for 24 h at 37 °C and 150 r.p.m. The culture was then concentrated tenfold by centrifugation to obtain 9.0 log c.f.u. ml–1 of S. cerevisiae 905. For mono-association (experimental gnotobiotic group), a single dose of 0.1 ml was administered to germ-free mice by intragastric intubation 10 days before the analyses or the experimental challenge with pathogenic bacteria. For treatment (experimental conventional group), the same dose was administered daily to conventional mice, 10 days before the analyses or the challenge and, in this last case, during all the remaining experimental period of infection. As a control, germ-free (control gnotobiotic group) and conventional (control conventional group) mice were submitted to inoculation with 0.9 % saline according to the same schedule as the respective experimental groups.
Yeast translocation. S. cerevisiae 905 translocation to Peyer's patches, mesenteric lymph nodes, liver and spleen of gnotobiotic and conventional mice was determined after 10 days of yeast mono-association or treatment, respectively. Mice were sacrificed and organs were aseptically collected, weighed, and macerated in sterile PBS (1 : 10, w/v). Serial tenfold dilutions were prepared and 100 µl aliquots were plated onto Sabouraud dextrose agar (Difco). Colonies were counted after incubation at 37 °C for 48 h.
Clearance. Control and experimental gnotobiotic groups were tested for their capacity to clear a Gram-negative bacterium injected systemically. Each mouse was injected with 0.2 ml suspension containing 108 c.f.u. E. coli B41 ml–1 in sterile PBS in the tail vein after ether anaesthesia. Blood samples were taken from the retro-orbital plexus shortly after the injection (time 0) and 15, 30, 60 and 90 min after the injection. Tenfold serial dilutions of 0.02 ml blood were plated and cultured onto MacConkey agar (Difco). Dishes were incubated at 37 °C for 24 h and colonies were counted. Results are the number of c.f.u. expressed as a percentage of the counts obtained at time 0 (100 %).
Histological and morphometrical examinations. After sacrifice by cervical dislocation, tissue samples from the liver of control and experimental gnotobiotic mice were fixed in buffered 4 % formaldehyde and processed for paraffin embedding. The histopathological sections (3–5 µm) were stained with haematoxylin–eosin. The slides were coded and examined by a single pathologist, who was unaware of the experimental conditions of each sample. For morphometrical examination of the liver, images were obtained with a JVC TK-1270/RGB microcamera and the KS 300 software built in a Kontron Elektronick/Carl Zeiss image analyser (Oberkohen). Küpffer cells were counted for each of 100 hepatocytes in each of 12 fields of view of the samples.
Immunoglobulin determination. After sacrifice, the small intestine of gnotobiotic and conventional mice from both experimental and control groups was removed, and the contents were withdrawn, weighed and suspended at 500 mg intestinal contents per 2.0 ml PBS. After centrifugation at 2000 g for 30 min at 4 °C, the supernatant fluid was collected and kept frozen at –70 °C until use. Serum samples were obtained after blood collection from the axillary plexus. Immunoglobulin levels in intestinal fluid and serum were evaluated by capture ELISA using goat anti-mouse IgA, IgG and IgM (Sigma) and horseradish peroxidase-conjugated goat anti-mouse IgA, IgG and IgM (Sigma). Colour was developed with o-phenylenediamine (Sigma) and absorbance at 492 nm was determined with an ELISA plate reader (Bio-Rad). The concentrations of each immunoglobulin were determined using the respective purified mouse standard (Southern Biotechnology).
Experimental infection. Salm. Typhimurium was grown in BHI broth (Difco) at 37 °C for 18–24 h under aerobic conditions. Mice were inoculated intragastrically with 0.1 ml bacterial suspension containing 2.0 and 4.0 log c.f.u. for gnotobiotic (control and experimental groups) and conventional mice (control and experimental groups), respectively.
Salm. Typhimurium translocation. Bacteria translocation to mesenteric lymph nodes, liver and spleen was determined after 3 days of challenge for gnotobiotic mice (control and experimental groups), and after 5 and 10 days of challenge for conventional mice (control and experimental groups). After sacrifice, the organs were aseptically collected, weighed, and macerated in sterile PBS (1 : 10, w/v). Serial tenfold dilutions were prepared and 100 µl aliquots were plated onto MacConkey agar (Difco). Colonies were counted after incubation at 37 °C for 24 h.
Statistical analysis. For obtaining data on the translocation, clearance, immunoglobulin production and histology, all groups consisted of 4 to 11 animals. The results were expressed as the mean of at least two independent experiments. The data were statistically analysed using the Student's t-test at a probability level of 0.05. Statistical analyses were performed using the programme Sigma Stat (Jandel Scientific Software; version 1.0).
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RESULTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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show that S. cerevisiae strain 905 translocated in low levels to mesenteric lymph nodes and/or Peyer's patches, and was not found in liver or spleen of experimental gnotobiotic and conventional mice. The presence of the yeast in these lymphoid organs did not result in an increase in the organ weight : body weight ratios (data not shown).
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). However, translocation to the liver was noted only in control gnotobiotic animals (Fig. 2). In conventional mice, translocation of Salmonella to mesenteric lymph nodes, spleen and liver was detected in control animals but not in experimental mice, both after 5 and 10 days of challenge (Fig. 3).
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The clearance capacity for E. coli B41 from the bloodstream in experimental gnotobiotic mice was higher than that observed for control animals (P<0.05) (Fig. 4). This improved clearance was not due to an increase in the size of livers of experimental gnotobiotic mice, as hepatomegalia was not observed.
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. shows the influence of S. cerevisiae 905 on levels of IgA in intestinal fluid and serum of gnotobiotic mice. A statistically significant difference (P<0.05) between control and experimental groups was observed for levels of secretory IgA (sIgA) in the intestinal content (Fig. 5b), and serum IgA (Fig. 5a) and IgM in serum (5973±1342 ng ml–1 control gnotobiotic mice versus 10 457–666 ng ml–1 experimental gnotobiotic mice, P=0.014, n=4 animals in each group). For the other determinations, a discrete but not statistically significant difference was observed: intestinal IgM (56±16 ng ml–1 control mice versus 69±3 ng ml–1 experimental mice, P=0.21, n=4 animals in each group); serum IgG (16 633±6060 ng ml–1 control mice versus 20 278±8670 ng ml–1 experimental mice, P=0.32. n=9 animals in each group); intestinal IgG (225±86 ng ml–1 control mice versus 268±48 ng ml–1 experimental mice, P=0.32, n=6 animals in each group). In conventional mice an increase in total IgG and IgM was observed only in the intestinal content (IgG 123±6 ng ml–1 control mice versus 181±23 ng ml–1 experimental mice, P=0.16, n=4 animals in each group; IgM 12±2 ng ml–1 control mice versus 21±9 ng ml–1 experimental mice, P=0.38. n=4 animals in each group), but these differences were not statistically significant. No alterations were observed in the levels of immunoglobulins in serum of conventional mice (data not shown).
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DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Like other probiotics, such as S. boulardii (Rodrigues et al., 1996), Bifidobacterium longum (Silva et al., 2004) and Lactobacillus acidophilus (Moura et al., 2001), S. cerevisiae 905 was rapidly eliminated from the digestive tract of mammals harbouring a complex intestinal microbiota (2–3 days) and for this reason daily ingestion of these micro-organisms was necessary to maintain artificially high viable levels in the gastrointestinal ecosystem of conventional animals. On the other hand, its implantation was possible in germ-free animals using a single dose. Gnotobiotic mice were used in the present study to obtain a simplified in vivo ecosystem allowing the observation of ecological interactions between the probiotic, bacterial pathogen and the host. The results were compared with those obtained using a similar experimental design but with animals harbouring an indigenous gastrointestinal microbiota (conventional).
In mice, Salm. Typhimurium is described as a facultative intracellular parasite that grows primarily inside the macrophages of liver and spleen. It has been shown that within 30 min of infection, invasive Salm. Typhimurium entered M cells found within the follicle-associated epithelium (FAE) of Peyer's patches. At 60 min, internalized bacteria were cytotoxic for the M cells and the dead cell formed a gap in the FAE, which allowed bacteria to invade adjacent enterocytes (Jones & Falkow, 1996) or rapidly migrate to a number of sites in the body, including the spleen and liver, where they replicated inside phagocytic cells. This dissemination stimulates the formation of neutrophil-rich and mononuclear-cell-rich microabscesses, which subsequently evolve into granulomas containing activated mononuclear cells.
The development of an immune response toward foreign antigens of microbial and viral origin provides a means through which potentially pathogenic organisms are recognized and controlled. However, this response cannot be exacerbated to the point of causing tissue damage and disease. There is an extensive body of literature addressing the possible health benefits associated with the consumption of probiotics. Probiotic micro-organisms may exert their effects through immunomodulation, particularly by regulation of pro- and anti-inflammatory cytokine levels (Rodrigues et al., 2000; Silva et al., 2004) to obtain an efficient, but not excessive response during an infectious aggression.
As observed with S. boulardii (Rodrigues et al., 2000), S. cerevisiae strain 905 translocated in low levels to mesenteric lymph nodes and/or Peyer's patches, but not to liver or spleen. The translocation is involved in the pathogenesis of invasive intestinal bacterial infection, but under normal conditions, a physiological translocation also occurs and would be responsible for the modulation of the host immune system by the indigenous microbiota (Berg, 1996). Translocation of a probiotic limited to local lymphoid organs, such as Peyer's patches and mesenteric lymph nodes, could bring about antigen presentation events, triggering immune responses in situ and systemically. In addition, before the development of T-cell-mediated responses, innate immunity events could be triggered by the presence of the probiotic in the gut-associated lymphoid tissues. Consequently, the low level of limited translocation observed for S. cerevisiae strain 905 could be beneficial in priming the host immune response to improve defence against overt or opportunist pathogens.
The reduced translocation of Salm. Typhimurium observed in mice treated with S. cerevisiae 905 could explain the lower number of hepatic inflammatory foci in these animals 28 days after the challenge with the enteropathogenic bacteria as observed in our previous study (Martins et al., 2005). The translocation of Salm. Typhimurium to liver and spleen of control animals, without detection of the pathogen in lymph nodes (Fig. 3a
), could be explained by more physical damage to the intestinal epithelium in this animal group. As described by Berg (1995), translocation to liver could occur via intracellular passage (lymphatic route, via lymph nodes) or extracellular passage (vascular route, due to physical damage to the intestinal epithelium). Reduced levels of translocation have been also observed in various animal models (burn injury, liver injury, experimental immunosuppression, abdominal infection) using probiotic products based on bacteria (Chiva et al., 2002; Gun et al., 2005) and yeast (Peret et al., 1998; Herek et al., 2004). It seems that all the components of the host immune system – including mucosal, cell-mediated and humoral immunities – are involved in controlling bacterial translocation. sIgA is thought to inhibit the close association of pathogenic bacteria with the mucosal epithelium, and so to reduce bacterial penetration. Cell-mediated immunity in the lamina propria and in lymphoid organs provides a second line of host defence against bacterial translocation. Serum immunoglobulins probably act as opsonins to increase the effectiveness of phagocytosis by macrophages and polymorphonuclear leukocytes, and subsequent killing of translocating bacteria. In the case of successful translocation, the reticuloendothelial system (RES) plays a key role in removing micro-organisms from the circulatory system, and hepatic phagocytosis by Küppfer cells accounts for more than 80 % of the RES function (Saba, 1970). Glucan and other Saccharomyces extracts are important RES stimulators. Proliferation of pluripotent haematopoietic stem cells, granulocyte-macrophage colony-forming cells and erythroid colony-forming cells in murine bone marrow has been demonstrated after glucan administration (Patchen & McVittie, 1982). Furthermore, after glucan administration, there is an increased production of interleukin 1 either by a direct effect on macrophages or indirectly by an increase on colony stimulating factor(s) production from glucan-stimulated T-cells (Hamuro et al., 1982).
It is well known that Küppfer cells represent one of the largest populations of tissue macrophages and, together with neutrophils, have been implicated as being responsible for the clearance of bacteria from the bloodstream (Hirakata et al., 1991; Gregory et al., 1996). The higher number of Küpffer cells in liver from experimental gnotobiotic mice was also observed when germ-free mice were mono-associated with Lactobacillus delbrueckii UFV-H2B20 (Neumann et al., 1998) and S. boulardii (Rodrigues et al., 2000), and this increase was simultaneous with a higher clearance of systematically injected pathogenic bacterium. When tested in the present study, the clearance capacity of E. coli B41 from the bloodstream in experimental gnotobiotic mice was higher than that observed for control gnotobiotic animals. Taken together, the results suggest that, in this experimental model, the higher number of Küppfer cells in the liver of experimental gnotobiotic mice was responsible for the improved clearance observed when compared with control gnotobiotic animals.
To assess if a humoral immune response could be related to the protective effect observed against Salm. Typhimurium, we determined immunoglobulin concentrations in the intestinal fluid and serum of yeast-treated mice. S. cerevisiae strain 905 enhanced the local (intestinal fluid) as well as the systemic (serum) production of this immunoglobulin. sIgA represents an immune barrier against the adhesion of pathogens to the intestinal mucosa, binding either to the surface of bacteria and trapping them in the mucous layer of the intestinal epithelia, or to bacterial surface molecules that mediate adhesion to epithelial cells, thus preventing translocation to internal organs (Hajishengallis et al., 1992). The ability to stimulate the production of sIgA is not the same for different micro-organisms, and in gnotobiotic mice Gram-negative bacteria and yeast generally induce a higher stimulation than Gram-positive bacteria (Moreau et al., 1982; Neumann et al., 1998; Rodrigues et al., 2000).
As expected and observed with other probiotics (Rodrigues et al., 1996; Silva et al., 2004), the protection offered by S. cerevisiae 905 against the pathogenic challenge was different between conventional and gnotobiotic animals due to the complementary protective effect from the indigenous intestinal microbiota and from the biotherapeutic agent in the first case. Germ-free animals are generally more susceptible to experimental infection due to both the lack of ecological competition in the gastrointestinal lumen and to the underdeveloped immune system in the internal compartment.
The present findings suggest that S. cerevisiae 905 may have potential as a probiotic as it reduces the translocation levels of Salm. Typhimurium. Host immunomodulation by the yeast may be one explanation for its protection against Salm. Typhimurium, and against C. difficile infections as observed in our previous study (Martins et al., 2005). Probiotic treatment before experimental infection in animal models may not necessarily reproduce all the mechanisms operating in humans, and the extrapolation from animal data to therapeutic effects in humans must be interpreted cautiously. However, probiotic efficacy against experimental pathogenic challenge can only be predicted by such extrapolations, and the use of this model prior to human trials was recommended by the Food and Agriculture Organization of the United Nations & World Health Organization (2002).
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ACKNOWLEDGEMENTS |
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) and experimental (
) gnotobiotic mice. The results are expressed as the percentage of viable cells (ml blood)–1 in relation to time 0 (100 %). Each point represents the mean of 11 mice from 2 representative experiments. Vertical bars representSDs of the means. *Indicates statistically significant difference between mono-associated and germ-free mice as evaluated by Student's t-test (P<0.05), n=11 animals in each group.