A new, rapid multiplex PCR method identifies frequent probiotic origin among clinical Saccharomyces isolates.

An increasing number of infections originating from probiotic use are reported worldwide, with the majority of such cases caused by the yeast Saccharomyces 'boulardii', a subtype of S. cerevisiae. Reliably linking infectious cases to probiotic products requires unequivocal genotyping data, however, these techniques are often time-consuming and difficult to implement in routine diagnostics. This leads to a widespread lack of genetic data regarding the origin of Saccharomyces infections. We propose a quick and reliable PCR-based protocol for the identification of S. 'boulardii' based on a combined analysis of interdelta fingerprinting and microsatellite typing. By applying various typing methods and our proposed method to the clinical yeast collection of a Hungarian hospital we show that probiotic origin is common among clinical Saccharomyces, and that the new multiplex method enables rapid and unequivocal identification of probiotic yeast infections. This method can be applied for the identification of yeast infection sources, helping decisions on probiotic use.

Pseudozyma and other non-Candida opportunistic yeast bloodstream infections in a large stem cell transplant center.

Non-Candida opportunistic yeasts are emerging causes of bloodstream infection (BSI) in immunocompromised hosts. However, their clinical presentation, management, and outcomes in stem cell transplant (SCT) recipients are not well described. We report the first case to our knowledge of Pseudozyma BSI in a SCT recipient. He had evidence of cutaneous involvement, which has not been previously described in the literature. He became infected while neutropenic and receiving empiric micafungin, which is notable because Pseudozyma is reported to be resistant to echinocandins. He was successfully treated with the sequential use of liposomal amphotericin B and voriconazole. A review of the literature revealed nine reported instances of Pseudozyma fungemia. We performed a retrospective review of 3557 SCT recipients at our institution from January 2000 to June 2015 and identified four additional cases of non-Candida yeast BSIs. These include two with Cryptococcus, one with Trichosporon, and one with Saccharomyces. Pseudozyma and other non-Candida yeasts are emerging pathogens that can cause severe and disseminated infections in SCT recipients and other immunocompromised hosts. Clinicians should have a high degree of suspicion for echinocandin-resistant yeasts, if patients develop breakthrough yeast BSIs while receiving echinocandin therapy.

PH EFFECT ON ANTAGONISTIC ACTIVITY TOWARDS BACTERIA OF YEASTS ISOLATED FROM HUCUL DAIRY PRODUCTS AND GASTROINTESTINAL TRACT OF HUMAN.

The aim of this work was to study the influence of pH of medium on antagonistic ac- tivity of isolated from authentic Hucul dairy products and gastrointestinal tract (GIT) of Hucul long-livers yeasts towards potentially harmful for humans and animals bacteria. Among 52 tested yeast isolates 14 % yeasts showed considerable antagonistic activity towards Gram-positive bacteria Staphylococcus aureus and only 6 % of them inhibited growth of Gram negative bacteria belonging to genera Escherichia and Citrobacter Most ofyeasts with antagonistic activity (over 70 %) were isolatedfriom long-livers GIT There were identifed two optimal for antagonism areas of pH values of nutrient medium for tested yeasts being around 5.5 and 6.0 for Gram-positive bacteria and around 6.0 and 6.5 for Gram negative bacteria. It appeared that isolated fiom Hucul yogurt Saccharomyces pasterianus yeasts manifested their antagonistic activity in more acidic conditions com- pared to isolates fiom GIT.

Risk and safety of probiotics.

Probiotics have been used safely for years. Safety outcomes are inconsistently reported in published clinical trials. In 2011, a report released by the Agency for Healthcare Research and Quality concluded that, although the existing probiotic clinical trials reveal no evidence of increased risk, "the current literature is not well equipped to answer questions on the safety of probiotics in intervention studies with confidence." Critics point out that the preponderance of evidence, including the long history of safe probiotic use as well as data from clinical trials, and animal and in vitro studies all support the assumption that probiotics are generally safe for most populations. Theoretical risks have been described in case reports, clinical trial results and experimental models, include systemic infections, deleterious metabolic activities, excessive immune stimulation in susceptible individuals, gene transfer and gastrointestinal side effects. More research is needed to properly describe the incidence and severity of adverse events related to probiotics.

Oral consumption of potential probiotic Saccharomyces boulardii strain Unique 28 in patients with acute diarrhoea: a clinical report.

This study was conducted to evaluate the efficacy and safety of Saccharomyces boulardii strain Unique 28 in patients suffering from acute diarrhoea. A total of 25 patients (average age 30.72±4.38 years) with symptoms of acute diarrhoea (≥3 loose motion in last 24 hours for <7 days) were included upon informed consent and ethical committee approval. All subjects were assigned to consume S. boulardii strain Unique 28 (5×109 cfu/capsule) twice a day for a duration of 10 days. Primary outcome measures such as duration of diarrhoea, frequency of defaecation, abdominal pain and consistency of stool were analysed on day 1, 3, 6 and 10 of the study. Secondary outcome measures were evaluated by assessment of incidence and type of adverse events (blood pressure and pulse rate), physical examination and clinical laboratory tests (complete blood count, glutamic-pyruvic transaminase, serum creatinine, stool examination and microscopy and these tests were performed on day 1 and 10 of the study. The results of the present study indicate that the mean duration of diarrhoea decreased from 34.20±4.25 to 9.40±3.00 (P<0.0001) min per day, frequency of defaecation decreased from 7.04±0.84 to 1.76±0.52 (P<0.0001) times a day, abdominal pain decreased from 3.28±1.06 (severe) to 0.72±0.50 (absent) (P<0.0001) and consistency of stool improved from 3.80±0.50 (watery) to 1.32±0.47 (soft) (P<0.0001). In addition, no significant changes in safety parameters were observed during treatment. Therefore, the present study concludes that S. boulardii strain Unique-28 might be useful in alleviating the symptoms of diarrhoea without any adverse effects.

[Candida and Saccharomyces spp. fungal associations in fecal microbiocenosis of diabetes patients and healthy subjects].

The article is devoted to analysis of pathogenic and diagnostic significance of Candida and Saccharomyces co-existence in diabetic patients. These transient fungi are known to be present in fecal microbiocenosis of both healthy subjects and patients with diabetes mellitus. However, their overall occurrence is significantly increased in the disease and the structure of the biocenosis undergoes alteration. These data confirm the role of yeast-like fungi in pathogenesis of diabetes. The diagnostic value of detection of monospecific and mixed populations of Candida and Saccharomyces spp. is not very high, but their presence in feces, especially in women, may be regarded as a sign of disturbed carbohydrate metabolism.

Estudios de la viabilidad y la vitalidad frente al congelado de la levadura probiótica Saccharomyces boulardii: efecto del preacondicionamiento fisiológico / Studies of viability and vitality after freezing of the probiotic yeast Saccharomyces boulardii: physiological preconditioning effect

El objetivo de este estudio fue evaluar la vitalidad y la viabilidad de la levadura probiótica Saccharomycesboulardii después de su congelación y descongelación, y el efecto del preacondicionamiento fisiológico sobredichas propiedades. Los resultados indican que la velocidad específica de crecimiento (0,3 h–1) y la biomasa(2-3 × 108 células/ml) de S. boulardii obtenida en frascos agitados tanto a 28 como a 37 °C fueron semejantes.El cultivo de esta levadura en biorreactores en lote, utilizando glucosa o melaza de caña de azúcar como fuentede carbono, alcanzó rendimientos de 0,28 g de biomasa/g azúcar consumida tras 10 h de cultivo a 28 °C,obteniéndose resultados similares en fermentaciones en lote alimentado. Además, en los cultivos en lote, lavitalidad de las células en fase de crecimiento exponencial fue mayor que la de las células en fase estacionaria.En cambio, en el lote alimentado, la vitalidad de las células fue semejante a la del lote en fase estacionaria.La supervivencia a la congelación a –20 °C y posterior descongelación de las células de fermentaciones en loteen fase de crecimiento exponencial fue del 0,31% y la de fase estacionaria del 11,5%. El pretratamiento de estalevadura en medios de actividad de agua (aw) 0,98 aumentó 10 veces la supervivencia al congelado de las célulasalmacenadas a –20 °C durante 2 meses. La exposición de las levaduras a medios de aw reducida y/o elproceso de congelación/descongelación afectaron negativamente la vitalidad celular. Se concluye que condicionesde estrés como las estudiadas en este trabajo disminuyen la vitalidad de S. boulardii. Además, la cepaestudiada presentó buena tolerancia a las sales biliares aun a bajos valores de pH del cultivo(AU)

The aim of this study was to evaluate the vitality and viability of the probiotic yeast Saccharomyces boulardiiafter freezing/thawing and the physiological preconditioning effect on these properties. The results indicatethat the specific growth rate (0.3/h–1) and biomass (2-3 × 108 cells/ml) of S. boulardii obtained in flasks shakenat 28 °C and at 37 °C were similar. Batch cultures of the yeast in bioreactors using glucose or sugar-canemolasses as carbon sources, reached yields of 0.28 g biomass/g sugar consumed, after 10 h incubation at 28 °C;the same results were obtained in fed batch fermentations. On the other hand, in batch cultures, the vitality ofcells recovered during the exponential growth phase was greater than the vitality of cells from the stationaryphase of growth. Vitality of cells from fed-batch fermentations was similar to that of stationary growing cellsfrom batch fermentations. Survival to freezing at –20 °C and subsequent thawing of cells from batch cultureswas 0.31% for cells in exponential phase of growth and 11.5% for cells in stationary phase. Pre-treatment ofthis yeast in media with water activity (aw) 0.98 increased the survival to freezing of S. boulardii cells stored at–20 °C for 2 months by 10 fold. Exposure of the yeast to media of reduced aw and/or freezing/thawing processnegatively affected cell vitality. It was concluded that stress conditions studied herein decrease vitality ofS. boulardii. Besides, the yeast strain studied presented good tolerance to bile salts even at low pH values(AU)

Saccharomyces boulardii fungaemia in an intensive care unit patient treated with caspofungin.

We describe a case of Saccharomyces boulardii fugaemia in a critically ill patient with septic shock treated with a probiotic agent containing this yeast. We attributed this fugaemia to gut translocation. Our use of caspofugin yielded excellent results.

Invasive infections due to Saccharomyces boulardii (Ultra-levure).

(1) Saccharomyces boulardii, a yeast closely related to Saccharomyces cerevisiae, is marketed in France for adjuvant treatment of diarrhoea, although it has no proven efficacy. (2) Invasive infections due to Saccharomyces boulardii have occurred. Most of the patients had gastrointestinal disorders, an intravenous catheter, or ongoing antibiotic treatment. Only 25% of patients were immunosuppressed. (3) Unless the value of this type of treatment based on live microorganisms is demonstrated in methodologically sound clinical trials, it is best to avoid prescribing it to fragile patients.

Invasive Saccharomyces infection: a comprehensive review.

BACKGROUND: Saccharomyces cerevisiae (also known as "baker's yeast" or "brewer's yeast") is mostly considered to be an occasional digestive commensal. However, since the 1990s, there have been a growing number of reports about its implication as an etiologic agent of invasive infection. A particular feature of such infections is their association with a probiotic preparation of Saccharomyces boulardii (a subtype of S. cerevisiae) for treatment various diarrheal disorders. METHODS: We collected published case reports, through May 2005, of invasive Saccharomyces infection by use of a Medline query. Epidemiological and clinical charts and therapeutic strategies were analyzed. RESULTS: We found 92 cases of Saccharomyces invasive infection. Predisposing factors were similar to those of invasive candidiasis, with intravascular catheter and antibiotic therapy being the most frequent. Blood was the most frequent site of isolation (for 72 patients). S. boulardii accounted for 51.3% of fungemias and was exclusively isolated from blood. Compared with patients infected with S. cerevisiae, patients infected with S. boulardii were more frequently immunocompetent and had a better prognosis. Saccharomyces invasive infection was clinically indistinguishable from an invasive candidiasis. Overall, S. cerevisiae clinical isolates exhibited low susceptibility to amphotericin B and azole derivatives. However, global outcome was favorable in 62% of the cases. Treatment with intravenous amphotericin B and fluconazole, in combination with central vascular catheter removal, were effective therapeutic options. CONCLUSION: Saccharomyces organisms should now be added to the growing list of emerging fungal pathogens. Special caution should be taken regarding the use of S. boulardii probiotic preparations.

Molecular tools for differentiating probiotic and clinical strains of Saccharomyces cerevisiae.

The subtype of the Saccharomyces cerevisiae yeast species known as S. cerevisiae Hansen CBS 5926 was formerly believed to be a separate species, Saccharomyces boulardii. It is widely considered non-pathogenic and is used as a probiotic agent for treatment and prevention of diarrhea. The biological properties of Saccharomyces spp. show considerable intraspecies variability and the beneficial properties of probiotic yeasts are considered strain-specific. Septicemia and fungemia caused by S. boulardii have recently been described in both immunocompromised and immunocompetent patients receiving biotherapy with this yeast. It cannot be distinguished from other S. cerevisiae strains by phenotypic criteria, so identification of these infections requires molecular typing. To identify the most effective approach for distinguishing S. boulardii, we typed 35 isolates of S. cerevisiae, of which 27 were from various clinical specimens and 8 were isolates of S. boulardii (6 obtained from probiotic preparations and 2 from clinical specimens) using four different molecular methods, two based on PCR-restriction enzyme analysis or sequencing of rDNA spacer regions, the third based on microsatellite polymorphism analysis of the S. cerevisiae genes YKL139w and YLR177w, and the last based on hybridization analysis with retrotransposon Ty917. Several clinical isolates appeared to be identical to one or more other isolates with the first three methods used, whereas with the Ty917 hybridization method all of the isolates tested appeared to be very heterogeneous. The eight S. boulardii isolates were clearly distinguishable from the clinical S. cerevisiae isolates only with Ty917 hybridization and microsatellite DNA analyses. In the latter method, the eight S. boulardii isolates exhibited an allelic variant at one of loci tested that was not shared with any other strain. Our results suggest that microsatellite polymorphism analysis of the YKL139w and YLR177w genes, as well as the analysis by Ty917 hybridization, are the most useful tools for a correct identification of S. boulardii strains.

Saccharomyces boulardii induced sepsis: successful therapy with voriconazole after treatment failure with fluconazole.

Saccharomyces boulardii is frequently used for prevention and treatment of all forms of diarrhoea. We report the case of a 19-y-old white male with an underlying severe neurological disease, who developed a fungal sepsis after prophylactic application of a drug containing S. boulardii. Initial treatment with fluconazole was not successful. After the application of voriconazole, the sepsis resolved completely. This is the first clinical report of a successful treatment of Saccharomyces sepsis with voriconazole.

Large-scale screening of selected Candida maltosa, Debaryomyces hansenii and Pichia anomala killer toxin activity against pathogenic yeasts.

The killer activity of selected crude toxins produced by nine Candida maltosa, Debaryomyces hansenii and Pichia anomala strains was tested at 37 degrees C against 383 strains belonging to 19 pathogenic species of 10 genera (Candida, Clavispora, Cryptococcus, Filobasidiella, Issatchenkia, Kluyveromyces, Pichia, Saccharomyces, Stephanoascus and Trichosporon). The broad killer spectra exhibited by all crude toxins may lead to the development of new antimycotic agents against medically important yeasts.

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

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.

The K3 type killer strains of genus Saccharomyces for wine production.

Using simultaneous fusion of protoplasts of three strains the auxotrophic K3 killer designated MH1 was prepared at a very low frequency and further employed for the transfer of the K3 killer character into a commercial wine yeast. A novel Saccharomyces Bratislava 1K3 strain with desirable technological and anti-yeast killer abilities was thus constructed. Attempts to prepare double K1/K3 killers were made. The obtained fusion products contained M1 dsRNA and were able to produce only the K1 type killer toxin.