Species identification and virulence attributes of Saccharomyces boulardii (nom. inval.)

Abstract

Saccharomyces boulardii (nom. inval.) has been used for the treatment of several types of diarrhea. Recent studies have confirmed that S. boulardii is effective in the treatment of diarrhea, in particular chronic or recurrent diarrhea, and furthermore that it is a safe and well-tolerated treatment. The aim of the present study was to identify strains of S. boulardii to the species level and assess their virulence in established murine models. Three strains of S. boulardii were obtained from commercially available products in France and Italy. The three S. boulardii strains did not form spores upon repeated testing. Therefore, classical methods used for the identification of Saccharomyces spp. could not be undertaken. Typing by using the restriction fragment length polymorphisms (RFLPs) of the PCR-amplified intergenic transcribed spacer regions (including the 5. 8S ribosomal DNA) showed that the three isolates of S. boulardii were not separable from authentic isolates of Saccharomyces cerevisiae with any of the 10 restriction endonucleases assessed, whereas 9 of the 10 recognized species of Saccharomyces could be differentiated. RFLP analysis of cellular DNA with EcoRI showed that all three strains of S. boulardii had identical patterns and were similar to other authentic S. cerevisiae isolates tested. Therefore, the commercial strains of S. boulardii available to us cannot be genotypically distinguished from S. cerevisiae. Two S. boulardii strains were tested in CD-1 and DBA/2N mouse models of systemic disease and showed intermediate virulence compared with virulent and avirulent strains of S. cerevisiae. The results of the present study show that these S. boulardii strains are asporogenous strains of the species S. cerevisiae, not representatives of a distinct and separate species, and possess moderate virulence in murine models of systemic infection. Therefore, caution should be advised in the clinical use of these strains in immunocompromised patients until further study is undertaken.

FIG. 1

Photograph of the ethidium bromide-stained, UV-transilluminated, MaeI-digested PCR products after electrophoresis within a 3% agarose gel. The DNA from the PCR had been first purified by the Wizard PCR Preps purification system prior to overnight digestion with the 10 U of restriction endonuclease MaeI. Molecular size markers are in lanes M, and their corresponding sizes (in base pairs) are given on the left of the figure. Lanes: 1, S. boulardii Sb 48; 2, S. boulardii Sb 49; 3, S. boulardii Sb It; 4, S. cerevisiae ATCC 52530; 5, S. cerevisiae ATCC 26108; 6, S. cerevisiae YJM128, a clinical isolate (14, 30); 7, S. cerevisiae ItB9, a clinical isolate (28); 8, S. cerevisiae ItB8, a clinical isolate (28); 9, S. bayanus ATCC 76515; 10, S. paradoxus ATCC 76856; 11, S. paradoxus/S. cerevisiae hybrid YJM508 (29); 12, S. bayanus/S. cerevisiae hybrid YJM334 (29).

FIG. 2

Photograph of the ethidium bromide-stained, UV-transilluminated, HaeIII-digested PCR products after electrophoresis within a 3% agarose gel. The DNA from the PCR had been first purified by the Wizard PCR Preps purification system prior to overnight digestion with the 10 U of restriction endonuclease HaeIII. Molecular size markers are in lane M, and their corresponding sizes (in base pairs) are given on the left of the figure. Lane assignments are the same as those for Fig. 1.

FIG. 3

Representative photograph of UV-transilluminated, ethidium bromide-stained agarose gel. RFLPs generated by EcoRI digestion of S. cerevisiae DNA are shown. Molecular size markers are in lanes M, and their corresponding sizes (in kilobases) are given on the left of the figure. Lane assignments are the same as those for Fig. 1.