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

Abstract

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.

FIG. 1.

Chromosome plot of copy number distribution over the S. boulardii genome. Using the window-averaged copy number for each ORF, the results have been plotted using the starting nucleotide position of the ORF. The x axis represents the length of each chromosome (Ch) and the y axis shows copy number. The circled areas indicate the three most prominent internal regions of low copy number on chromosomes I, VII, and XII. The rectangles indicate the ORFs deleted in S. cerevisiae BY4743 causing the appearance of overrepresented genes. The value for each chromosome is the copy number as revealed by CGH. The P values shown in brackets are estimates of the probability of obtaining a median chromosomal copy number of the magnitude observed, by chance, if ORF copy number values were uniformly distributed across the genome.

FIG. 2.

Copper sensitivity. Cells were plated out at the concentrations indicated onto minimal SD medium plates containing all required amino acids with or without CuSO₄. The plates were then incubated at 30°C for 3 days prior to examination. The experiment was performed in duplicate, and the images are a representation of the complete results.

FIG. 3.

Pseudohyphal switching upon nitrogen limitation. Shown are the photographs of the SLAD agar plates taken at a magnification of ×10 following inoculation by spreading with a loop and incubation at 30°C for up to 1 week. After 7 days (d), the edges of the colonies were compared.

FIG. 4.

Survival of yeast strains in the mouse gut. The different S. cerevisiae strains were distinguished from each other by plasmids conferring distinct antibiotic resistance phenotypes. CF-1 male mice were administered 10⁸ CFU of the appropriate yeast suspension by gastric gavage in 200 μl. (A) The percentage of yeast recovered in the feces at different times (1 to 72 h) after administration; n = 6 mice for each time point. (B) Percentage of yeast recovered from different gut compartments, 1 h or 3 h after administration of different yeast strains. Animals were euthanized by cervical dislocation, and the GI tract was removed and divided into the gut components as indicated. Aliquots of the mucosal homogenates and the flushing and fecal homogenates were spread onto YPD medium with appropriate antibiotics to determine the number of CFU of the yeast strains. For each strain and time point, n = 6 mice. (C) Percentage of viable cells recovered from the mice following inoculation and incubation for 1 or 3 h. An increase indicated continued cell growth and division following inoculation.

FIG. 5.

Acid tolerance of yeast strains. Appropriate dilutions of the cell suspensions incubated under different pH conditions at 37°C were spread on YPD agar, and the plates were incubated until colonies appeared. Survival is expressed as the number of CFU/ml observed following incubation.