Typing of Saccharomyces cerevisiae Clinical Strains by Using Microsatellite Sequence Polymorphism
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微生物临床杂志 2005年第3期
Laboratoire de Parasitologie et Mycologie Medicale MNERT EA 2413, Faculte de Pharmacie, Universite de Montpellier I
Centre d'Etude sur le Polymorphisme des Micro-organismes (UMR CNRS-IRD 9926), Institut de Recherche pour le Developpement, Montpellier, France
ABSTRACT
It seems that S. cerevisiae, which was thought for about 30 years to be a nonpathogenic yeast, should now be considered an opportunistic pathogen. In this study, we estimated the discrimination ability of the microsatellite sequence amplification technique within a sample of clinical and reference S. cerevisiae strains and S. boulardii reference strains.
INTRODUCTION
Saccharomyces cerevisiae is a saprophyte of the digestive, respiratory, and genitourinary tracts (2-4, 17, 18, 25). However, with the progress in immunosuppressive therapies, there has been an increasing number of reports on yeast systemic infection cases involving Saccharomyces strains, particularly S. cerevisiae. The epidemiology of infections caused by Saccharomyces is still unknown. Some authors suggested a correlation between fungemia caused by Saccharomyces and treatment (24). Moreover, S. cerevisiae is usually considered to be responsible for infections. Nevertheless, some authors have suggested the role of other Saccharomyces species in systemic infections. Some of these infections may be due to treatment of diarrhea by S. boulardii (9, 20). It would thus be necessary to develop molecular markers for distinguishing various genotypes within S. cerevisiae.
Some epidemiological studies have shown intraspecific diversity within S. cerevisiae (11, 12). The present retrospective multicenter study exhibited polymorphism in microsatellite sequences of 69 clinical strains of S. cerevisiae by comparison to S. cerevisiae and S. boulardii reference strains. The aim of this work was to study genotypic polymorphism within clinical strains of S. cerevisiae compared to S. boulardii reference strains.
MATERIALS AND METHODS
In this study, 77 strains were tested; 69 came from different French medical centers (Table 1). These strains were isolated from different samples (Table 1). In parallel, five S. cerevisiae and three S. boulardii reference strains were tested (Table 1).
Yeast DNA was purified according to the protocol published by Querol et al. (22). The five microsatellite sequences used in our study have been previously described (10). These sequences are distributed in the S. cerevisiae genome (Table 2). PCR amplification was performed according to the protocol described by Durand et al. (8).
After heat denaturation, PCR fragments were separated by electrophoresis on a vertical polyacrylamide gel including formamide in the loading buffer.
The data were analyzed by factorial correspondence analysis with the PRAXIS-p.c., version 2.0, software package (Praxeme, R&D, Biometrie, C.N.R.S., Montpellier, France) (1, 6).
Factorial correspondence analysis was presented as a plane projection of the two most informative axes accounting for the genetic structure of the electrophoretic types. Allelic frequencies with each locus were calculated with the Genepop, version 1.2, software program (23).
RESULTS AND DISCUSSION
The microsatellite sequence polymorphism analysis gave reproducible results. Each locus tested was polymorphic, with 6, 11, 6, 6, and 5 different alleles for the microsatellite sequences of genes YKL 172W, YLR 177W, YKL 139W, YKR 072C, and YDR 289C, respectively. The fact that certain strains presented two different alleles for a given locus increased the number of patterns observed. In the present study, 12 pairs of different alleles were detected for the YKL 172W gene, 18 for YLR 177W, 11 for YKL 139W, 8 for YKR 072C, and 6 for YDR 289C. A predominant pair of alleles was found in microsatellite sequences of genes YKR 172W, YKR 072C, and YDR 289C, whereas for genes YLR 177W and YKL 139W, the allele pairs were distributed in a homogeneous way without any of them dominating (Table 3).
In the present work, 34 different alleles were observed and generated 64 distinct electrophoretic types. Among these 64 electrophoretic types, 58 represented a single strain, three represented two strains (electrophoretic types 26, 30, and 53), one represented four strains (electrophoretic type 14), one represented three strains (electrophoretic type 64), and one represented five strains which were isolated from the same patient (electrophoretic type 54). On the factorial correspondence analysis, electrophoretic types are projected in the most informative plane defined by axes 1 and 2, which explained 21.29% of the overall genetic variability.
The electrophoretic type projection exhibited three distinct clusters (Fig. 1). Cluster I included only clinical strains of S. cerevisiae. Cluster II included reference strains of S. cerevisiae, and cluster III contained reference strains of S. boulardii. These three clusters were separated by axis 1, which was the most informative. The loci responsible for the structure of the sample were highlighted by the allelic frequencies indexed in Table 3. Allele pairs responsible for the structure of the sample were the least frequent (i.e., 177-30, 177-31, 13-32, 072-33, and 289-34).
Genotype differences between the electrophoretic types of cluster I were investigated with a second factorial correspondence analysis (data not shown). Genotypes were not linked to a geographical origin or anatomic site. No conclusions can thus be drawn at present, but it would be interesting to conduct a further study with a larger sample of isolates.
In this study the sample assessed was interesting, related exclusively to clinical S. cerevisiae strains. This sample should be increased, but it is very difficult to obtain strictly clinical strains of S. cerevisiae, which explains why the present sample was limited in size and spatiotemporally heterogeneous.
Sixty-four electrophoretic types for the 77 strains were found by the microsatellite sequence polymorphism technique. These results are in agreement with those of Zerva et al. by using restriction fragment length polymorphism (26). Joly et al. (11) with randomly amplified polymorphic DNA and Duarte et al. (7) with multilocus enzyme electrophoresis on S. cerevisiae found 48 types for 54 isolates and 32 types for 35 studied strains, respectively. Hennequin et al. (10) differentiated 52 types for 91 strains tested by the microsatellite technique. This could be explained by the high homogeneity of the clinical strain samples studied. In the work of Lewicka et al. (12), only 22 types for 52 strains by multilocus enzyme electrophoresis were observed. However, it appears that in all of these studies, there was high diversity in the clinical and nonclinical S. cerevisiae strains.
In the present study, the clinical strains of S. cerevisiae (cluster I) seemed to have genotypes distinct from those of the S. cerevisiae reference strains studied (cluster II). This is in agreement with the work of Clemons et al. (5) in which the clinical and nonclinical strains showed different degrees of virulence. These results are of epidemiological importance, because in the present work, environmental strains had electrophoretic profiles which differed from those of strains isolated from patients. The genotypes of the S. cerevisiae strains responsible for infection and those isolated from the environment were very different. Microsatellite typing also revealed a genotypic difference between the S. cerevisiae and S. boulardii strains tested. This is in agreement with Mallie et al. (13) and McFarland et al. (15), who differentiated S. cerevisiae from S. boulardii by multilocus enzyme electrophoresis.
However, some authors such as Molnar et al. (16), Perapoch et al., and McCullough et al. demonstrated, by randomly amplified polymorphic DNA and restriction fragment length polymorphism (14, 19), respectively, that S. cerevisiae and S. boulardii have identical genetic profiles. Moreover, Hennequin et al. (10) found the same profile for clinical Saccharomyces strains and S. boulardii strains. Nevertheless, no variations in the genotype of S. boulardii have ever been observed (10, 21), which is in agreement with the results of the present study. Indeed, the same electrophoretic type was found for the three S. boulardii reference strains tested. This work exhibited a difference between clinical strains of S. cerevisiae (cluster I) and reference strains of S. boulardii (cluster III). These results should be confirmed by a comparative study on a larger sample of S. cerevisiae and S. boulardii strains.
By the description of 64 different electrophoretic types for the 77 strains tested, the microsatellite sequence polymorphism technique highlighted significant intraspecific diversity within S. cerevisiae for both the clinical and reference strains. Microsatellite sequence polymorphism is a powerful molecular typing tool. The present work demonstrated a difference between clinical S. cerevisiae and S. boulardii strains, suggesting that S. boulardii was not responsible for the systemic fungal infections observed within the patients studied. It would be useful to carry out a comparative study with a broader sample of S. cerevisiae and S. boulardii in order to determine their roles in human pathological infections. This could help to clarify the transmission mechanisms of these fungi and identify the contamination risk factors so that more effective prevention measures can be implemented.
ACKNOWLEDGMENTS
We thank Biocodex laboratoires, A. Blancard, B. Couprie, F. Dromer, G. Galeazzi, C. Guiguen, M. D. Linas, and J. Reynes for supplying strains.
REFERENCES
Benzecri, J. P., and F. Benzecri1. 1980. Pratique de l'analyse des donnees. Analyse des correspondances. Dunot, Paris, France.
Berg, R., P. Bernasconi, D. Fowler, and M. Gautreaux. 1993. Inhibition of Candida albicans translocation from the gastrointestinal tract of mice by oral administration of Saccharomyces boulardii. J. Infect. Dis. 168:1314-1318.
Bonnay, S., J. Darchis, and P. Veyssier. 1991. Septicemie a Saccharomyces cerevisiae chez un cancereux. Med. Mal. Infect. 21:32-34.
Canafax, D. M., H. J. Mann, and S. H. Dougherty. 1982. Postoperative peritonitis due to Saccharomyces cerevisiae treated with ketoconazole. Drug Intell. Clin. Pharm. 16:698-699.
Clemons, K. V., J. H. McCusker, R. W. Davis, and D. A. Stevens. 1994. Comparative pathogenesis of clinical and nonclinical isolates of Saccharomyces cerevisiae. J. Infect. Dis. 169:859-867.
Cousteau, C., F. Renaud, C. Maillard, N. Pasteur, and B. Delay. 1991. Differential susceptibility to a trematode among genotypes of the Mytilus edulis/galloprovincialis complex. Genet. Res. 57:207-212.
Duarte, F. L., C. Pais, I. Spencer-Martins, and C. Leao. 1999. Distinctive electrophoretic isoenzyme profiles in. Saccharomyces sensu stricto. Int. J. Syst. Bacteriol. 49:1907-1913.
Durand, P., C. Sire, and A. Theron. 2000. Isolation of microsatellite markers in the digenetic trematode. Schistosoma mansoni from Guadeloupe island. Mol. Ecol. 9:997-998.
Hennequin, C., C. Kauffmann-Lacroix, A. Jobert, J. P. Viard, C. Ricour, J. L. Jacquemin, and P. Berche. 2000. Saccharomyces boulardii fungemia: the possible role of catheter. Eur. J. Clin. Microbiol. Infect. Dis. 16-20.
Hennequin, C., A. Thierry, G. F. Richard, G. Lecointre, H. V. Nguyen, C. Gaillardin, and B. Dujon. 2001. Microsatellite typing as a new tool for identification of Saccharomyces cerevisiae strains J. Clin. Microbiol. 39:551-559.
Joly, S. 2000. Ph.D. thesis. Universite des Sciences et Techniques du Languedoc, Universite Montpellier II, Montpelier, France.
Lewicka, K., M. Mallie, and J. M. Bastide. 1995. Genetic variability in the Saccharomyces Sensu Stricto complex revealed by multilocus enzyme electrophoresis. Int. J. Syst. Bacteriol. 45:538-543.
Mallie, M., P. van Nguyen, S. Bertout, C. Vaillant, and J. Bastide. 2001. Genotypic study of Saccharomyces boulardii compared to the Saccharomyces sensu stricto complex species. J. Mycol. Med. 11:19-25.
McCullough, M. J., K. V. Clemons, J. H. McCusker, and D. A. Stevens. 1998. Species identification and virulence attributes of Saccharomyces boulardii (nom. inval.). J. Clin. Microbiol. 36:2613-2617.
McFarland, L. V. 1996. Saccharomyces boulardii is not Saccharomyces cerevisiae. Clin. Infect. Dis. 22:200-201.
Molnar, O., R. Messner, H. Prillinger, U. Stahl, and E. Slavikova. 1995. Genotypic identification of Saccharomyces species using Random Amplified Polymorphic DNA Analysis. Syst. Appl. Microbiol. 18:136-145.
Morrison, V. A., R. J. Haake, and D. J. Weisdorf. 1993. The spectrum of non-Candida fungal infections following bone marrow transplantation. Medicine (Baltimore) 72:78-89.
Oriol, A., J. M. Ribera, J. Arnal, F. Milla, M. Batlle, and E. Feliu Saccharomyces cerevisiae septicemia in a patient with myelodysplastic syndrome Am. J. Hematol. 43:325-326, 1993.
Perapoch, J., A. M. Planes, A. Querol, V. Lopez, I. Martinez-Bendayan, R. Tormo, F. Fernandez, G. Peguero, and S. Salcedo. 2000. Fungemia with Saccharomyces cerevisiae in two newborns, only one of whom had been treated with ultra-levura. Eur. J. Clin. Microbiol. Infect. Dis. 19:468-470.
Piarroux, R. L., K. Millon, O. Bardonnet, Vagner, and H. Koenig. 1999. Are live Sacharomyces yeasts harmful to patients Lancet i:1851-1852.
Posteraro, B., M. Sanguinetti, G. D'Amore, L. Masucci, G. Morace, and G. Fadda. 1999. Molecular and epidemiological characterization of vaginal Saccharomyces cerevisiae isolates. J. Clin. Microbiol. 37:2230-2235.
Querol, A., E. Barrio, T. Huerta, and D. Ramon. 1992. Molecular monitoring of wine fermentations conducted by active dry yeast strains. Appl. Environ. Microbiol. 58:2948-2953.
Raymond, M., and F. Rousset. 1995. Genepop (V1.2): a population genetics software for exact tests and eumenicism. J. Hered. 86:248-249.
Riquelme, A. J., M. A. Calvo, A. M. Guzman, M. S. Depix, P. Garcia, C. Perez, M. Arrese, and J. A. Labarca. 2003. Saccharomyces cerevisiae fungemia after Saccharomyces boulardii treatment in immunocompromised patients. J. Clin. Gastroenterol. 36:41-43.
Sobel, J. D., J. Vazquez, M. Lynch, C. Meriwether, and M. J. Zervos. 1993. Vaginitis due to Saccharomyces cerevisiae: epidemiology, clinical aspects, and therapy. Clin. Infect. Dis. 16:93-99.
Zerva, L., R. J. Hollis, and M. A. Pfaller. 1996. In vitro susceptibility testing and DNA typing of Saccharomyces cerevisiae clinical isolates. J. Clin. Microbiol. 34:3031-3034.(J. Y. Malgoire, S. Bertou)
Centre d'Etude sur le Polymorphisme des Micro-organismes (UMR CNRS-IRD 9926), Institut de Recherche pour le Developpement, Montpellier, France
ABSTRACT
It seems that S. cerevisiae, which was thought for about 30 years to be a nonpathogenic yeast, should now be considered an opportunistic pathogen. In this study, we estimated the discrimination ability of the microsatellite sequence amplification technique within a sample of clinical and reference S. cerevisiae strains and S. boulardii reference strains.
INTRODUCTION
Saccharomyces cerevisiae is a saprophyte of the digestive, respiratory, and genitourinary tracts (2-4, 17, 18, 25). However, with the progress in immunosuppressive therapies, there has been an increasing number of reports on yeast systemic infection cases involving Saccharomyces strains, particularly S. cerevisiae. The epidemiology of infections caused by Saccharomyces is still unknown. Some authors suggested a correlation between fungemia caused by Saccharomyces and treatment (24). Moreover, S. cerevisiae is usually considered to be responsible for infections. Nevertheless, some authors have suggested the role of other Saccharomyces species in systemic infections. Some of these infections may be due to treatment of diarrhea by S. boulardii (9, 20). It would thus be necessary to develop molecular markers for distinguishing various genotypes within S. cerevisiae.
Some epidemiological studies have shown intraspecific diversity within S. cerevisiae (11, 12). The present retrospective multicenter study exhibited polymorphism in microsatellite sequences of 69 clinical strains of S. cerevisiae by comparison to S. cerevisiae and S. boulardii reference strains. The aim of this work was to study genotypic polymorphism within clinical strains of S. cerevisiae compared to S. boulardii reference strains.
MATERIALS AND METHODS
In this study, 77 strains were tested; 69 came from different French medical centers (Table 1). These strains were isolated from different samples (Table 1). In parallel, five S. cerevisiae and three S. boulardii reference strains were tested (Table 1).
Yeast DNA was purified according to the protocol published by Querol et al. (22). The five microsatellite sequences used in our study have been previously described (10). These sequences are distributed in the S. cerevisiae genome (Table 2). PCR amplification was performed according to the protocol described by Durand et al. (8).
After heat denaturation, PCR fragments were separated by electrophoresis on a vertical polyacrylamide gel including formamide in the loading buffer.
The data were analyzed by factorial correspondence analysis with the PRAXIS-p.c., version 2.0, software package (Praxeme, R&D, Biometrie, C.N.R.S., Montpellier, France) (1, 6).
Factorial correspondence analysis was presented as a plane projection of the two most informative axes accounting for the genetic structure of the electrophoretic types. Allelic frequencies with each locus were calculated with the Genepop, version 1.2, software program (23).
RESULTS AND DISCUSSION
The microsatellite sequence polymorphism analysis gave reproducible results. Each locus tested was polymorphic, with 6, 11, 6, 6, and 5 different alleles for the microsatellite sequences of genes YKL 172W, YLR 177W, YKL 139W, YKR 072C, and YDR 289C, respectively. The fact that certain strains presented two different alleles for a given locus increased the number of patterns observed. In the present study, 12 pairs of different alleles were detected for the YKL 172W gene, 18 for YLR 177W, 11 for YKL 139W, 8 for YKR 072C, and 6 for YDR 289C. A predominant pair of alleles was found in microsatellite sequences of genes YKR 172W, YKR 072C, and YDR 289C, whereas for genes YLR 177W and YKL 139W, the allele pairs were distributed in a homogeneous way without any of them dominating (Table 3).
In the present work, 34 different alleles were observed and generated 64 distinct electrophoretic types. Among these 64 electrophoretic types, 58 represented a single strain, three represented two strains (electrophoretic types 26, 30, and 53), one represented four strains (electrophoretic type 14), one represented three strains (electrophoretic type 64), and one represented five strains which were isolated from the same patient (electrophoretic type 54). On the factorial correspondence analysis, electrophoretic types are projected in the most informative plane defined by axes 1 and 2, which explained 21.29% of the overall genetic variability.
The electrophoretic type projection exhibited three distinct clusters (Fig. 1). Cluster I included only clinical strains of S. cerevisiae. Cluster II included reference strains of S. cerevisiae, and cluster III contained reference strains of S. boulardii. These three clusters were separated by axis 1, which was the most informative. The loci responsible for the structure of the sample were highlighted by the allelic frequencies indexed in Table 3. Allele pairs responsible for the structure of the sample were the least frequent (i.e., 177-30, 177-31, 13-32, 072-33, and 289-34).
Genotype differences between the electrophoretic types of cluster I were investigated with a second factorial correspondence analysis (data not shown). Genotypes were not linked to a geographical origin or anatomic site. No conclusions can thus be drawn at present, but it would be interesting to conduct a further study with a larger sample of isolates.
In this study the sample assessed was interesting, related exclusively to clinical S. cerevisiae strains. This sample should be increased, but it is very difficult to obtain strictly clinical strains of S. cerevisiae, which explains why the present sample was limited in size and spatiotemporally heterogeneous.
Sixty-four electrophoretic types for the 77 strains were found by the microsatellite sequence polymorphism technique. These results are in agreement with those of Zerva et al. by using restriction fragment length polymorphism (26). Joly et al. (11) with randomly amplified polymorphic DNA and Duarte et al. (7) with multilocus enzyme electrophoresis on S. cerevisiae found 48 types for 54 isolates and 32 types for 35 studied strains, respectively. Hennequin et al. (10) differentiated 52 types for 91 strains tested by the microsatellite technique. This could be explained by the high homogeneity of the clinical strain samples studied. In the work of Lewicka et al. (12), only 22 types for 52 strains by multilocus enzyme electrophoresis were observed. However, it appears that in all of these studies, there was high diversity in the clinical and nonclinical S. cerevisiae strains.
In the present study, the clinical strains of S. cerevisiae (cluster I) seemed to have genotypes distinct from those of the S. cerevisiae reference strains studied (cluster II). This is in agreement with the work of Clemons et al. (5) in which the clinical and nonclinical strains showed different degrees of virulence. These results are of epidemiological importance, because in the present work, environmental strains had electrophoretic profiles which differed from those of strains isolated from patients. The genotypes of the S. cerevisiae strains responsible for infection and those isolated from the environment were very different. Microsatellite typing also revealed a genotypic difference between the S. cerevisiae and S. boulardii strains tested. This is in agreement with Mallie et al. (13) and McFarland et al. (15), who differentiated S. cerevisiae from S. boulardii by multilocus enzyme electrophoresis.
However, some authors such as Molnar et al. (16), Perapoch et al., and McCullough et al. demonstrated, by randomly amplified polymorphic DNA and restriction fragment length polymorphism (14, 19), respectively, that S. cerevisiae and S. boulardii have identical genetic profiles. Moreover, Hennequin et al. (10) found the same profile for clinical Saccharomyces strains and S. boulardii strains. Nevertheless, no variations in the genotype of S. boulardii have ever been observed (10, 21), which is in agreement with the results of the present study. Indeed, the same electrophoretic type was found for the three S. boulardii reference strains tested. This work exhibited a difference between clinical strains of S. cerevisiae (cluster I) and reference strains of S. boulardii (cluster III). These results should be confirmed by a comparative study on a larger sample of S. cerevisiae and S. boulardii strains.
By the description of 64 different electrophoretic types for the 77 strains tested, the microsatellite sequence polymorphism technique highlighted significant intraspecific diversity within S. cerevisiae for both the clinical and reference strains. Microsatellite sequence polymorphism is a powerful molecular typing tool. The present work demonstrated a difference between clinical S. cerevisiae and S. boulardii strains, suggesting that S. boulardii was not responsible for the systemic fungal infections observed within the patients studied. It would be useful to carry out a comparative study with a broader sample of S. cerevisiae and S. boulardii in order to determine their roles in human pathological infections. This could help to clarify the transmission mechanisms of these fungi and identify the contamination risk factors so that more effective prevention measures can be implemented.
ACKNOWLEDGMENTS
We thank Biocodex laboratoires, A. Blancard, B. Couprie, F. Dromer, G. Galeazzi, C. Guiguen, M. D. Linas, and J. Reynes for supplying strains.
REFERENCES
Benzecri, J. P., and F. Benzecri1. 1980. Pratique de l'analyse des donnees. Analyse des correspondances. Dunot, Paris, France.
Berg, R., P. Bernasconi, D. Fowler, and M. Gautreaux. 1993. Inhibition of Candida albicans translocation from the gastrointestinal tract of mice by oral administration of Saccharomyces boulardii. J. Infect. Dis. 168:1314-1318.
Bonnay, S., J. Darchis, and P. Veyssier. 1991. Septicemie a Saccharomyces cerevisiae chez un cancereux. Med. Mal. Infect. 21:32-34.
Canafax, D. M., H. J. Mann, and S. H. Dougherty. 1982. Postoperative peritonitis due to Saccharomyces cerevisiae treated with ketoconazole. Drug Intell. Clin. Pharm. 16:698-699.
Clemons, K. V., J. H. McCusker, R. W. Davis, and D. A. Stevens. 1994. Comparative pathogenesis of clinical and nonclinical isolates of Saccharomyces cerevisiae. J. Infect. Dis. 169:859-867.
Cousteau, C., F. Renaud, C. Maillard, N. Pasteur, and B. Delay. 1991. Differential susceptibility to a trematode among genotypes of the Mytilus edulis/galloprovincialis complex. Genet. Res. 57:207-212.
Duarte, F. L., C. Pais, I. Spencer-Martins, and C. Leao. 1999. Distinctive electrophoretic isoenzyme profiles in. Saccharomyces sensu stricto. Int. J. Syst. Bacteriol. 49:1907-1913.
Durand, P., C. Sire, and A. Theron. 2000. Isolation of microsatellite markers in the digenetic trematode. Schistosoma mansoni from Guadeloupe island. Mol. Ecol. 9:997-998.
Hennequin, C., C. Kauffmann-Lacroix, A. Jobert, J. P. Viard, C. Ricour, J. L. Jacquemin, and P. Berche. 2000. Saccharomyces boulardii fungemia: the possible role of catheter. Eur. J. Clin. Microbiol. Infect. Dis. 16-20.
Hennequin, C., A. Thierry, G. F. Richard, G. Lecointre, H. V. Nguyen, C. Gaillardin, and B. Dujon. 2001. Microsatellite typing as a new tool for identification of Saccharomyces cerevisiae strains J. Clin. Microbiol. 39:551-559.
Joly, S. 2000. Ph.D. thesis. Universite des Sciences et Techniques du Languedoc, Universite Montpellier II, Montpelier, France.
Lewicka, K., M. Mallie, and J. M. Bastide. 1995. Genetic variability in the Saccharomyces Sensu Stricto complex revealed by multilocus enzyme electrophoresis. Int. J. Syst. Bacteriol. 45:538-543.
Mallie, M., P. van Nguyen, S. Bertout, C. Vaillant, and J. Bastide. 2001. Genotypic study of Saccharomyces boulardii compared to the Saccharomyces sensu stricto complex species. J. Mycol. Med. 11:19-25.
McCullough, M. J., K. V. Clemons, J. H. McCusker, and D. A. Stevens. 1998. Species identification and virulence attributes of Saccharomyces boulardii (nom. inval.). J. Clin. Microbiol. 36:2613-2617.
McFarland, L. V. 1996. Saccharomyces boulardii is not Saccharomyces cerevisiae. Clin. Infect. Dis. 22:200-201.
Molnar, O., R. Messner, H. Prillinger, U. Stahl, and E. Slavikova. 1995. Genotypic identification of Saccharomyces species using Random Amplified Polymorphic DNA Analysis. Syst. Appl. Microbiol. 18:136-145.
Morrison, V. A., R. J. Haake, and D. J. Weisdorf. 1993. The spectrum of non-Candida fungal infections following bone marrow transplantation. Medicine (Baltimore) 72:78-89.
Oriol, A., J. M. Ribera, J. Arnal, F. Milla, M. Batlle, and E. Feliu Saccharomyces cerevisiae septicemia in a patient with myelodysplastic syndrome Am. J. Hematol. 43:325-326, 1993.
Perapoch, J., A. M. Planes, A. Querol, V. Lopez, I. Martinez-Bendayan, R. Tormo, F. Fernandez, G. Peguero, and S. Salcedo. 2000. Fungemia with Saccharomyces cerevisiae in two newborns, only one of whom had been treated with ultra-levura. Eur. J. Clin. Microbiol. Infect. Dis. 19:468-470.
Piarroux, R. L., K. Millon, O. Bardonnet, Vagner, and H. Koenig. 1999. Are live Sacharomyces yeasts harmful to patients Lancet i:1851-1852.
Posteraro, B., M. Sanguinetti, G. D'Amore, L. Masucci, G. Morace, and G. Fadda. 1999. Molecular and epidemiological characterization of vaginal Saccharomyces cerevisiae isolates. J. Clin. Microbiol. 37:2230-2235.
Querol, A., E. Barrio, T. Huerta, and D. Ramon. 1992. Molecular monitoring of wine fermentations conducted by active dry yeast strains. Appl. Environ. Microbiol. 58:2948-2953.
Raymond, M., and F. Rousset. 1995. Genepop (V1.2): a population genetics software for exact tests and eumenicism. J. Hered. 86:248-249.
Riquelme, A. J., M. A. Calvo, A. M. Guzman, M. S. Depix, P. Garcia, C. Perez, M. Arrese, and J. A. Labarca. 2003. Saccharomyces cerevisiae fungemia after Saccharomyces boulardii treatment in immunocompromised patients. J. Clin. Gastroenterol. 36:41-43.
Sobel, J. D., J. Vazquez, M. Lynch, C. Meriwether, and M. J. Zervos. 1993. Vaginitis due to Saccharomyces cerevisiae: epidemiology, clinical aspects, and therapy. Clin. Infect. Dis. 16:93-99.
Zerva, L., R. J. Hollis, and M. A. Pfaller. 1996. In vitro susceptibility testing and DNA typing of Saccharomyces cerevisiae clinical isolates. J. Clin. Microbiol. 34:3031-3034.(J. Y. Malgoire, S. Bertou)