Effect of Saccharomyces boulardii against experimental oral infection with Salmonella typhimurium and Shigella flexneri in conventional and gnotobiotic mice

A.C.P. RODRIGUES, R.M. NARDI, E.A. BAMBIRRA, E.C. VIEIRA AND J.R. NICOLI. 1996. Saccharomyces boulardii was shown to be capable of inhibiting multiplication of enteropathogenic bacteria in vitro and is currently used for its anti-diarrhoea properties. We studied the capacity of this yeast to antagonize Salmonella typhimurium and Shigella flexneri in the intestinal tract of conventional or gnotobiotic NMRI mice. Conventional animals were given daily 10 mg doses of S. boulardii , whereas germ-free animals were given a single 10 mg dose. Both groups were challenged orally 5 d later with the pathogenic bacteria (10⁸ or 10² viable cells, respectively). Control groups were treated with saline instead of S. boulardii. Mortality and/or histopathological data showed a protective effect against the pathogenic bacteria in yeast-treated mice. Saccharomyces boulardii colonized the digestive tract of gnotobiotic mice and the number of viable cells ranged around 10¹⁰ g^-1 of faeces. In experimental and control gnotobiotic animals, Salm. typhimurium and Sh. flexneri became rapidly established at a level of about 10¹⁰ viable cells g^-1 of faeces and remained at high levels until the animals died or were sacrificed. The protection against Salm. typhimurium and Sh. flexneri obtained in conventional and/or gnotobiotic mice previously associated with S. boulardii is not due to the reduction of the bacterial populations in the intestines.     

Prevention of Clostridium difficile induced mortality in gnotobiotic mice by Saccharomyces boulardii

Oral preventive treatment of gnotobiotic mice by Saccharomyces boulardii significantly decreased mortality following Clostridium difficile infection. A single S. boulardii ingestion protected 16% of mice, whereas 56% were protected when S. boulardii was given continuously in the drinking water. No direct antagonistic effect of the yeast on C. difficile numbers was detected, whereas a modulation of fecal cytotoxin production was demonstrated.     

Prevention of Clostridium difficile-induced experimental pseudomembranous colitis by Saccharomyces boulardii: a scanning electron microscopic and microbiological study

The ability of Saccharomyces boulardii to protect mice against intestinal pathology caused by toxinogenic Clostridium difficile was studied. Different regions of the intestine of experimental mice were prepared for observation by scanning electron microscopy or homogenized for C. difficile enumeration and quantification of toxin A by enzyme immunoassay and toxin B by cytotoxicity. The test group was treated for 6 d with an S. boulardii suspension in drinking water and challenged with C. difficule on day 4. The three control groups were: axenic mice, mice treated with only S. boulardii and mice only challenged with C. difficile. The results showed that: (i) 70% of the mice infected by C. difficile survived when treated with S. boulardii; (ii) the C. difficile-induced lesions on the small and large intestinal mucosa were absent or markedly less severe in S. boulardii-treated mice; and (iii) there was no decrease in the number of C. difficile but rather a reduction in the amount of toxins A and B in S. boulardii-treated mice.     

Molecular analysis of the digestive microbiota in a gnotobiotic mouse model during antibiotic treatment: Influence of Saccharomyces boulardii

The probiotic Saccharomyces boulardii is a non-pathogenic yeast that has been proven efficient in the prevention of antimicrobial-associated diarrhea and of Clostridium difficile associated colitis. We evaluated the influence of the administration of S. boulardii on the composition of the fecal microbiota in a human microbiota-associated mouse model. This evaluation was run before, during and after a 7-day oral treatment with amoxicillin clavulanic acid. Predominant groups of bacteria were quantified with fluorescence in situ hybridization combined with flow cytometry using group-specific 16S rRNA targeted oligonucleotide probes designed for the Eubacteria, Bacteroides-Porphyromonas-Prevotella, Clostridium coccoides-Eubacterium rectale, Faecalibacterium prausnitzii, Clostridium histolyticum, Lactobacillus-Enterococcus and Enterobacteriaceae groups and Bifidobacterium species. S. boulardii did not quantitatively alter the total anaerobic microbiota nor the dominant bacterial groups. During the antibiotic treatment in the two groups of mice receiving the yeast or not, the level of Enterobacteriaceae and Bacteroides groups increased when the C. coccoides-E. rectale group decreased dramatically. After the antibiotic treatment was discontinued, the return to the initial level was reached more rapidly in the S. boulardii-treated mice than in the control mice (p<0.05) for the C. coccoides-E. rectale and Bacteroides-Porphyromonas-Prevotella groups. This quicker recovery of normal intestinal microbiota equilibrium after antibiotic therapy could be a mechanism for S. boulardii preventive effect on antibiotic-associated diarrhea in humans.     

The antagonistic effect of Saccharomyces boulardii on Candida albicans filamentation, adhesion and biofilm formation

The dimorphic fungus Candida albicans is a member of the normal flora residing in the intestinal tract of humans. In spite of this, under certain conditions it can induce both superficial and serious systemic diseases, as well as be the cause of gastrointestinal infections. Saccharomyces boulardii is a yeast strain that has been shown to have applications in the prevention and treatment of intestinal infections caused by bacterial pathogens. The purpose of this study was to determine whether S. boulardii affects the virulence factors of C. albicans . We demonstrate the inhibitory effect of live S. boulardii cells on the filamentation (hyphae and pseudohyphae formation) of C. albicans SC5314 strain proportional to the amount of S. boulardii added. An extract from S. boulardii culture has a similar effect. Live S. boulardii and the extract from S. boulardii culture filtrate diminish C. albicans adhesion to and subsequent biofilm formation on polystyrene surfaces under both aerobic and microaerophilic conditions. This effect is very strong and requires lower doses of S. boulardii cells or concentrations of the extract than serum-induced filamentation tests. Saccharomyces boulardii has a strong negative effect on very important virulence factors of C. albicans , i.e. the ability to form filaments and to adhere and form biofilms on plastic surfaces.     

布拉酵母菌对胃黏膜上皮细胞损伤的保护作用及机制

目的研究布拉酵母菌对胃黏膜上皮细胞损伤的保护作用。方法建立人胃黏膜上皮细胞GES-1单层细胞模型,并将其分为对照组,布拉酵母菌上清培养组,阿司匹林处理组(18.32mmol/L),阿司匹林+布拉酵母菌上清液组。采用流式细胞术检测各组细胞凋亡率;采用免疫荧光、Western blot方法检测胃黏膜上皮细胞中Occludin和ZO-1蛋白、JNK蛋白、ERK蛋白、磷酸化JNK(p-JNK)蛋白、磷酸化ERK(p-ERK)蛋白表达水平。结果流式细胞术显示阿司匹林+布拉酵母菌上清液组凋亡率低于阿司匹林处理组,差异有统计学意义(P0.05);免疫荧光结果显示,阿司匹林+布拉酵母菌上清液组Occludin蛋白、ZO-1蛋白表达较阿司匹林处理组增加,Western blot结果显示,阿司匹林+布拉酵母菌上清液组的该两种蛋白表达量较阿司匹林处理组增加,阿司匹林+布拉酵母菌上清液组p-ERK、p-JNK蛋白表达较阿司匹林处理组减少。结论布拉酵母菌对阿司匹林造成的胃黏膜上皮细胞损伤有保护作用,其机制可能通过抑制MAPK信号通路中ERK、JNK的激活而上调Occludin和ZO-1蛋白的表达。     

Modulation of Clostridium difficile induced mortality as a function of the dose and the viability of the Saccharomyces boulardii used as a preventative agent in gnotobiotic mice

The ability of viable and nonviable Saccharomyces boulardii to protect gnotobiotic mice from Clostridium difficile induced mortality was tested. With the exception of irradiated S. boulardii, which retained some activity, only viable yeast protected the mice from lethality. The survival of C. difficile infected mice was dependent on the dose of the yeast provided in the drinking water.     

Effects of monocolonization with<Emphasis Type="Italic">Escherichia coli</Emphasis> strains O6K13 and nissle 1917 on the development of experimentally induced acute and chronic intestinal inflammation in germ-free immunocompetent and immunodeficient mice

Germ-free immunocompetent (BALB/c) and immunodeficient (SCID) mice were colonized either by E. coli O6K13 or by E. coli strain Nissle 1917 and intestinal inflammation was induced by administering 2.5% dextran sulfate sodium (DSS) in drinking water. Controls were germ-free mice which demonstrated only mild inflammatory changes after induction of an acute intestinal inflammation with DSS as compared with conventional mice in which acute colitis of the colon mucosa similar to human ulcerative colitis is elicited. In mice monocolonized with the nonpathogenic E. coli Nissle 1917 the inflammatory disease did not develop (damage grade 0) while animals monocolonized with uropathogenic E. coli O6K13 exhibited inflammatory changes similar to those elicited in conventionally reared mice (damage grade 3). In the chronic inflammation model, immunocompetent BALB/c mice monocolonized with E. coli Nissle 1917 showed no conspicuous inflammatory changes of the colon mucosa whereas those monocolonized with E. coli O6K13 developed colon inflammation associated with marked infiltration of inflammatory cells. In contrast to germ-free immunodeficient SCID mice that died after application of DSS, the colon mucosa of SCID mice monoassociated with E. coli Nissle 1917 exhibited only moderate inflammatory changes which were less pronounced than changes of colon mucosa of SCID mice monoassociated with E. coli O6K13.     

Genotypic and physiological characterization of Saccharomyces boulardii, the probiotic strain of Saccharomyces cerevisiae

Saccharomyces boulardii, a yeast that was isolated from fruit in Indochina, has been used as a remedy for diarrhea since 1950 and is now a commercially available treatment throughout Europe, Africa, and South America. Though initially classified as a separate species of Saccharomyces, recent publications have shown that the genome of S. boulardii is so similar to Saccharomyces cerevisiae that the two should be classified as conspecific. This raises the question of the distinguishing molecular and phenotypic characteristics present in S. boulardii that make it perform more effectively as a probiotic organism compared to other strains of S. cerevisiae. This investigation reports some of these distinguishing characteristics including enhanced ability for pseudohyphal switching upon nitrogen limitation and increased resistance to acidic pH. However, these differences did not correlate with increased adherence to epithelial cells or transit through mouse gut. Pertinent characteristics of the S. boulardii genome such as trisomy of chromosome IX, altered copy number of a number of individual genes, and sporulation deficiency have been revealed by comparative genome hybridization using oligonucleotide-based microarrays coupled with a rigorous statistical analysis. The contributions of the different genomic and phenotypic features of S. boulardii to its probiotic nature are discussed.     

布拉酵母菌在成人感染性腹泻治疗中的作用研究

目的评价布拉酵母菌在感染性腹泻治疗中的作用及影响因素。方法收集浙江省龙游县人民医院自2010年5月至2012年10月共36例感染性腹泻病例,其中18例患者治疗应用左氧氟沙星联合布拉酵母菌,对照组18例患者单用左氧氟沙星,观察患者症状的改善状况和治愈疗程的差异。结果左氧氟沙星联合布拉酵母菌治疗患者大便次数在疗程第2~4天较单用左氧氟沙星患者明显缩短,两组比较差异有统计学意义（P 〈 0. 05）;联合治疗组平均止泻时间为（3. 85±1. 74） d,单用左氧氟沙星治疗组的平均止泻时间为（4. 89 ± 1. 67）d,两组比较差异有统计学意义〈0. 05）。结论感染性腹泻患者联合应用布拉酵母菌,有助于平衡肠道菌群,及早康复。     

The taxonomic position of Saccharomyces boulardii as evaluated by sequence analysis of the D1/D2 domain of 26S rDNA, the ITS1-5.8S rDNA-ITS2 region and the mitochondrial cytochrome-c oxidase II gene

A taxonomic study was carried out on eight strains of Saccharomyces boulardii. Morphological and physiological characteristics were consistent with those of Saccharomyces cerevisiae. Sequences of the D1/D2 domain of the 26S rDNA were identical for all strains examined and had a similarity value of 100% compared to sequences of the type strain of S. cerevisiae (CBS 1171T) and strain S288c. For all S. boulardii isolates was found the exact same ITS1-5.8S rDNA-ITS2 sequence, which displayed a close resemblance with the sequences published for S288c (99.9%), CBS 1171(T) (99.3%) and other S. cerevisiae strains. Sequence analysis of the mitochondrial cytochrome-c oxidase II gene (COX2) also resulted in identical sequences for the S. boulardii isolates and comparisons with available nucleotide sequences revealed close relatedness to strains of S. cerevisiae including S288c (99.5%) and CBS 1171(T) (96.6%). The electrophoretic karyotypes of the S. boulardii strains appeared quite uniform and although very typical of S. cerevisiae, they formed a cluster separate from strains of this species. The results of the present study strongly indicate a close relatedness of S. boulardii to S. cerevisiae and thereby support the recognition of S. boulardii as a member of S. cerevisiae and not as a separate species.     

布拉酵母对小儿重症肺炎患儿 肠黏膜屏障功能的影响

目的探讨布拉酵母对小儿重症肺炎患儿肠黏膜屏障功能的影响。方法将90例重症肺炎患儿按随机数字表分为对照组和观察组各45例。对照组患儿采用常规治疗,观察组患儿在常规治疗的同时加用布拉酵母治疗1周。比较两组患儿治疗前后血浆D-乳酸及肠型脂肪酸结合蛋白变化情况、胃肠功能障碍及腹泻发生率以及患儿不良反应。结果治疗1周后,两组患儿血浆D-乳酸和肠型脂肪酸结合蛋白水平均低于治疗前(均P0.01),且不同时间点观察组指标水平下降幅度大于对照组(均P0.01)。观察组患儿胃肠功能障碍发生率为6.7%、对照组为22.2%;观察组患儿腹泻发生率为15.6%,对照组为33.3%,二者比较差异均有统计学意义(均P0.05)。观察组患儿无药物不良反应发生。结论布拉酵母对重症肺炎患儿肠黏膜屏障具有保护作用,能减少胃肠功能障碍及腹泻发生率。     

布拉酵母菌散联合蒙脱石散对轮状病毒肠炎患儿细胞免疫功能的影响及疗效观察

目的探讨布拉酵母菌散联合蒙脱石散对轮状病毒肠炎患儿细胞免疫功能的影响及疗效。方法76例婴幼儿轮状病毒肠炎随机分为观察组和对照组。两组患儿均予以补液、抗病毒、纠正水电解质及酸碱平衡紊乱等常规治疗。观察组患儿加用布拉酵母菌散剂和蒙脱石散治疗,对照组患儿予以单纯的蒙脱石散治疗。观察两组患儿治疗后临床疗效及不良反应,并比较两组患者治疗后1个月T淋巴细胞亚群的变化。结果治疗72h后,观察组患儿的临床总有效率为94．74％,明显高于对照组的78．95％（χ2=4．15,P〈0．05）,两组患儿治疗中未发现明显药物不良反应。治疗后1个月,观察组患儿CD4＋及CD4＋／CD8＋比值较治疗前明显上升,CD8＋较治疗前明显下降（P〈0．05）。而对照组患儿治疗前后CD4＋、CD8＋、CD4＋／CD8＋比值无明显变化（P〉0．05）。结论布拉酵母菌散联合蒙脱石散治疗婴幼儿轮状病毒肠炎具有较好的疗效,安全性较好,与增强患儿的细胞免疫功能密切相关。     

Trypanosoma cruzi: influence of predominant bacteria from indigenous digestive microbiota on experimental infection in mice

To verify the influence of some predominant components from indigenous microbiota on systemic immunological responses during experimental Chagas disease, germ-free NIH Swiss mice were mono-associated with Escherichia coli, Enterococcus faecalis, Bacteroides vulgatus or Peptostreptococcus sp. and then infected with the Y strain of Trypanosoma cruzi. All the mono-associations predominantly induced a Th1 type of specific immune response to the infection by T. cruzi. A direct correlation was observed between a higher survival rate and increased IFN-gamma and TNF-alpha production (P<0.05) in E. faecalis-, B. vulgatus-, and Peptostreptococcus-associated mice. Moreover, higher levels of anti-T. cruzi IgG1 and anti-T. cruzi IgG2a were also found in mono-associated animals after infection. On the other hand, with the exception of E. faecalis-associated mice, mono-association induced a lower IL-10 production after infection (P<0.05) when compared with germ-free animals. Interestingly, spleen cell cultures from non-infected germ-free and mono-associated mice spontaneously produced higher levels (P<0.05) of IL-10 than cultures from infected mono-associated mice, except again for E. faecalis-associated animals. In conclusion, the presence of the components of the indigenous microbiota skews the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi in gnotobiotic mice. However, the degree of increase in production of cytokines depends on each bacterial component.     

Characterization of clostridia isolated from faeces of limited flora mice and their effect on caecal size when associated with germ-free mice

115 strains of clostridia were accumulated from 3 separate isolations from the faeces of 1 limited flora (LF) mouse produced by inoculation of germ-free mice with chloroform-treated faeces of conventional mice. These strains were divided into 36 types on the basis of conventional biochemical characteristics. There was some variation in types isolated on the 3 occasions. Although 3 groups of polyassociated (PA) mice were produced from mixing 46 monoassociated mice, each inoculated with 1 of 46 strains of 36 bacterial types, the caecal size of PA mice was still greater than that of the control mice prepared by inoculation of chloroform-treated faeces of an LF mouse. After PA mice of each group were housed together, the caecal size became smaller and was only slightly larger than the control.     

Interactions between gut-associated lymphoid tissue and colonization levels of indigenous, segmented, filamentous bacteria in the small intestine of mice.

Unlike most other indigenous bacteria, segmented filamentous bacteria (SFB) are potent activators of the mucosal immune system. SFB are strongly anchored to the epithelial cells of the small intestine where they have a preference for mucosal lymphoid epithelium. Since SFB are only present in high numbers shortly after weaning, it was investigated whether an SFB-induced immune reaction results in the removal of these bacteria from the small intestine. A correlation was found between age and colonization levels in the small intestines of SFB monoassociated Swiss mice. Five-week-old athymic BALB/c (nu/nu) mice showed lower colonization levels than their heterozygous littermates, but the opposite was found at the age of 12 weeks. However, SFB inoculation of germfree Swiss mice resulted in higher colonization levels in 5-week-old mice when compared with 4-month-old mice. We conclude that SFB colonization levels in the small intestine are likely influenced by the activity of the mucosal immune system. However, an additional age-dependent factor that modulates SFB colonization levels cannot be excluded.     

Ascaridia galli (Nematoda): development and survival in gnotobiotic chickens

Infection and growth rates of the fowl nematode Ascaridia galli were compared in germ-free, conventional, and other gnotobiotic chickens. Uniform experimental infections induced in chickens at 7–12 days of age showed that significantly fewer (P < 0.01) worms established themselves in germ-free chickens than in chickens with conventional flora. Numbers of worms established in monoassociated hosts (Bacillus subtilis, B. cereus, and Penicillium sp.) were intermediate between those in grossly contaminated and germ-free hosts. Mean worm lengths were shorter from germ-free than conventional hosts but differences were not statistically significant. These experiments demonstrate that some A. galli larvae may establish themselves and grow for 14 or 21 days postinoculation in a germ-free environment.     

Evaluation of the Pathogenicity of the Bacteroides fragilis Toxin Gene Subtypes in Gnotobiotic Mice

Enterotoxigenic Bacteroides fragilis (ETBF) strains produce a metalloprotease toxin (BFT) related to diarrheal disease in animals, young children, and adults. Three different isoforms of the enterotoxin, designated BFT-1, BFT-2, and BFT-3, have been identified and sequenced. In the present study, the pathogenicity of the ETBF strains carrying bft-1 or bft-2 was evaluated. Each toxin gene subtype of ETBF (bft-1 or bft-2) was intragastrically monoassociated to germ-free mice during 10 days and histopathological data from intestines and liver compared with those from mice monoassociated to a non-enterotoxigenic B. fragilis. Histopathological alterations were observed in all groups of animals related to ETBF. These alterations were characterized mainly by ulceration, edema, and inflammatory infiltration in intestine. However, these lesions were slightly more severe in mice monoassociated with bft-2 subtype. No alteration or lesion was observed in animals associated with the non-enterotoxigenic B. fragilis. In conclusion, strains harboring bft-1 or bft-2 gene subtypes were able to induce histopathological alterations in intestine of a gnotobiotic mice model and it could explain the effect produced for the enterotoxin.     

Antagonistic effect of Lactobacillus acidophilus, Saccharomyces boulardii and Escherichia coli combinations against experimental infections with Shigella flexneri and Salmonella enteritidis subsp. typhimurium in gnotobiotic mice

Lactobacillus acidophilus, Saccharomyces boulardii and Escherichia coli are probiotic strains used individually to protect against enteropathogenic agents. In order to determine if a synergistic effect of the individual protective mechanisms ordinarily attributed to each of these biotherapeutic agents is possible, we orally administered Lact. acidophilus H2B20, S. boulardii and E. coli EMO (LSE) to germfree mice. Ten days after colonization of the digestive tract, groups of animals associated (experimental) or not (control) with LSE were challenged orally with streptomycin resistant (Sfr) or streptomycin sensitive (Sfs) Shigella flexneri strains or Salmonella enteritidis subsp. typhimurium. Bacterial counts in faeces from experimental mice showed that the Sfr strain was eliminated 11 d after challenge while Sfs and S. enteritidis subsp. typhimurium colonized the digestive tract and continued to be present at high population levels (108 CFU g-1 of faeces), which is similar to that observed in control animals. All possible di- and monoassociations of the three probiotics with gnotobiotic mice were also performed before experimental oral infection with Sfr. The data showed that antagonism was obtained only when E. coli EMO was present. Different sensitivity of Sh. flexneri Sfr and Sfs to E. coli EMO antagonism could be explained by the different generation times between Sfr and Sfs, as shown by colonization kinetic experiments in the digestive tract of gnotobiotic mice.     

Molecular and physiological comparisons between Saccharomyces cerevisiae and Saccharomyces boulardii

In this paper, comparative molecular studies between authentic Saccharomyces cerevisiae strains, related species, and the strain described as Saccharomyces boulardii were performed. The response of a S. boulardii strain and a S. cerevisiae strain (W303) to different stress conditions was also evaluated. The results obtained in this study show that S. boulardii is genetically very close or nearly identical to S. cerevisiae. Metabolically and physiologically, however, it shows a very different behavior, particularly in relation to growth yield and resistance to temperature and acidic stresses, which are important characteristics for a microorganism to be used as a probiotic.