Projects / Programmes source: ARIS

Evaluation of antifungal resistance in emerging opportunistic yeast pathogens during invasive growth.

Research activity

Code Science Field Subfield
7.00.00  Interdisciplinary research     

Code Science Field
B230  Biomedical sciences  Microbiology, bacteriology, virology, mycology 

Code Science Field
1.07  Natural Sciences  Other natural sciences 
Candida glabrata, Saccharomyces cerevisiae, antifungal susceptibility, invasion, agar-invasion, assay, microsatellites, virulence, pathogen, clinical, adhesion, assay, probiotic, resistance, proteins, efflux pumps, SDS-PAGE, real-time PCR, microtiter plate, hydrophobicity, immunocompromised, immunosupresive, fluconazole, azoles, antifungal
Evaluation (rules)
source: COBISS
Researchers (1)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  26541  PhD Jure Zupan  Biotechnology  Head  2011 - 2013  88 
Organisations (1)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  65,900 
With the exponential growth of world population, population aging, increased use in catheters, broad spectrum antimicrobial therapy or prophylaxis, immunosuppressives, and chemotherapeutic drugs, there has been an increasing number of reports on systemic infections involving opportunistic pathogens. Candida glabrata has become the second causative agent of human candidiasis and is associated with as high mortality rate as with Candida albicans. Saccharomyces cerevisiae, on the other hand, has for centuries been used in numerous biotechnological applications and has been considered non-pathogenic. In the last few decades, however, it has been implicated in quite a number of invasive infections in immunocompromised adults and newborns. Moreover, Saccharomyces boulardii (nom. inval.), which has been licensed for use in Europe as a probiotic agent, was reported to be responsible for 40% of invasive Saccharomyces infections. It is obvious that extensive study is needed to better understand C. glabrata and S. cerevisiae virulence traits, which separate them from non-pathogenic strains. In the project we are proposing an idea to combine quantitative agar-invasion assay with standard antifungal susceptibility method M27-A2. The resulted method would give the information about the pathogen's antifungal resistance and its invasiveness in a single assay, which is planed to be simple, reproducible and reliable. We will use the proposed method to study the resistance mechanisms of C. glabrata and S. cerevisiae during the invasive growth. We will isolate cell wall proteins from invasively growing cells of C. glabrata and S. cerevisiae and examine the correlation of their expression to invasiveness and antifungal susceptibility. In addition, we will evaluate the risk of probiotic S. boulardii strains by determining their invasiveness and antifungal susceptibility. Next objective of the project is to determine the influence of various immunosuppressives, chemotherapeutic drugs and antibiotics on the stimulation/repression of resistance to azoles and on the invasiveness of C. glabrata and S. cerevisiae. Finally, we will perform a clustering of clinical and non-clinical C. glabrata, S. cerevisiae and S. boulardii strains based on microsatellite typing and examine the possibility to use the method to identify probiotic, invasive and/or azole-resistant strains.
Significance for science
Fungal infections are extremely widespread problem for people in general and especially in elderly population and in children. The problem originates from the fact that we have very limited number of efficient antifungals, as these organisms are eukaryotes and are therefore structurally similar to human cells, which means that the drug impairs pathogen and human cell at the same time. In addition, microorganisms continuously adapt to antibiotics and develop resistance to them. Antifungals therefore represent an evolving area that needs a support of research community. Antifungal agent fluconazole (Diflucan or Trican), which is the main focus of the project, is produced by one of the leading pharmaceutical companies, Pfizer, and is listed on 92th place of the best-selling medical substances with annual income from the sale of $ 125 billion (source: SDI / Verispan, VONA, for the whole of 2008). Even bigger profits bring immunosuppressives, which are involved in the project because of their putative synergistic effect on the sensitivity of yeast to azoles (tacrolimus and mycophenolate mofetil with 2,000 million dollars per year). Knowledge of virulence factors of pathogenic micro-organisms is therefore crucial. Unfortunately, however, virulence factors are not equally present in all strains of the same species, and therefore the identification of pathogenic yeast to the level of species is not enough for proper decision making during medical treatment. New types of approaches, such as the method, which was developed in the project, are therefore always welcome. The method evaluates currently known virulence factors of yeast in one assay – growth at elevated body temperature (above 37 ° C), invasiveness and resistance to antimycotics – and in that way exposes potentially problematic strains. To our knowledge, there are no similar methods published so far. It is evident from the results of the project that the differences between the strains are extremely large, so in the hydrophobicity of the cell wall, which is directly correlated to the adhesion, as well as in the invasiveness and in the antimycotic resistance. Based on the results of the project, we plan to publish four scientific papers, which will contribute to the development of this area. Methodological article that describes a method for testing the virulence factors of pathogenic strains has been written already and is prepared for publication.
Significance for the country
From the start of the project, we actively cooperated with the Institute of Immunology and Microbiology of the Faculty of Medicine of the University of Ljubljana, which represented an important link between the school, routine microbiology and patients. This is particularly important because there is a lack of such links in Slovenia. In addition, we used antifungal agents and medical agents that are currently most often used in Slovenia in such cases of infections, while the synergistic and antagonistic effects of those drugs are not known. Caspofungin is currently very successful drug and replaces other antifungals because of mild effects on humans and at the same time low minimum inhibitory concentration. The latter was also confirmed by the results of the project. In the case of azole antifungals (fluconazole and itraconazole) we showed some antagonistic and synergistic effects when the antimycotics were used together with certain immunosuppressives. This information will be extremely useful in future projects, in which we can translate in vitro testing of the observed phenomena into in vivo environment and come closer to the understanding of the mechanisms that are responsible for the development of resistance in some strains and consequently to treatment failure. The area of pathogenic yeasts in Slovenia is poorly developed despite the fact that there are limited number of successful antifungals and that they represent high cost. Development in this area is necessary and needs stronger support, if not in production at least in acquiring new knowledge.
Most important scientific results Annual report 2012, final report, complete report on dLib.si
Most important socioeconomically and culturally relevant results Annual report 2011, 2012, final report, complete report on dLib.si
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