Yeasts are able to inhibit growth of disease-associated fungi CURRENT STATUS: POSTED

Background Fungal sepsis is often caused by non-albicans Candida or other species. These disease-associated species have strong virulence and often show resistance to the commonly used antifungal treatments. Therefore, finding new inhibitory agents nowadays is increasingly urgent. Results Our screening revealed that although the pathogenic fungi were much more tolerant to yeast-produced bioactive agents than the non-disease-associated yeasts, growth of Kodamaea ohmeri and Candida tropicalis could be inhibited by Metschnikowia andauensis , while Cryptococcus albidus can be controlled by Pichia anomala and Candida tropicalis. The size of the inhibitory zone formed by yeasts was depended on media, pH and temperature. However, extensive studies were carried out, we failed to find inhibitory yeast against Pichia kudriavzevii, suggesting that it must have high natural resistance. Conclusions Certain yeast species can contribute to the future solutions of problems caused by fungal resistance and can be good candidates for finding new bioactive agents which inhibit growth of disease-associated fungi.

Background Fungal sepsis is often caused by non-albicans Candida or other species. These diseaseassociated species have strong virulence and often show resistance to the commonly used antifungal treatments. Therefore, finding new inhibitory agents nowadays is increasingly urgent.
Results Our screening revealed that although the pathogenic fungi were much more tolerant to yeastproduced bioactive agents than the non-disease-associated yeasts, growth of Kodamaea ohmeri and Candida tropicalis could be inhibited by Metschnikowia andauensis , while Cryptococcus albidus can be controlled by Pichia anomala and Candida tropicalis. The size of the inhibitory zone formed by yeasts was depended on media, pH and temperature. However, extensive studies were carried out, we failed to find inhibitory yeast against Pichia kudriavzevii, suggesting that it must have high natural resistance.
Conclusions Certain yeast species can contribute to the future solutions of problems caused by fungal resistance and can be good candidates for finding new bioactive agents which inhibit growth of disease-associated fungi.

Background
Fungaemia is associated with substantial morbidity and mortality of immuno-compromised persons.
Studies have demonstrated that fungal sepsis can quite often be caused by non-albicans Candida species. Pichia kudriavzevii (is the teleomorph of the Candida krusei) was isolated from neonates and hospitalized patients [1, 2, reviewed in 3]. It is supposed to be the fifth most common cause of candidemia. Kodamaea ohmeri cells (is the teleomorph of Candida guillermondii) were isolated from infant and neonate or wound lesions and blood in several cases [reviewed in 3 and 4, 5, 6]. Candida tropicalis is one of the most common colonizer in tropical countries. Its infections involve gastrointestinal invasions or arthritis [reviewed in 7], while Cryptococcus albidus was isolated from transplant recipient and lesion [8,9].
Successful infection of the mentioned above species can be in connection with their dimorphisms (ability to morphological switch), polymorphisms of their virulence-related genes and possibly with their resistance to the commonly used antifungal agents [5,10,11,12,13,14].
Because of these problems, we wanted to investigate whether cell division of the disease-associated species mentioned above can be inhibited by bioactive agents produced by yeasts or not. Well-known antagonistic species and species not studied for biological control were equally tested. Our screening revealed the species that were able to inhibit cell division of infectious fungi and shed light on that size of the inhibitory zones produced by the yeasts, strongly depended on media, pH and temperature. Our data suggested that Pichia kudriavzevii must have strong inherited resistance to the yeast-produced antifungal agents.
As the Table 1 shows, growth of Kodamaea ohmeri   (Fig.1a) and Candida tropicalis  could be inhibited by M. andauensis cells , while Cryptococcus albidus (2-1365) was controlled by P. anomala  and C. tropicalis . Other test species were not able to form inhibitory zone on the lawns of disease-associated species, in turn they were effective in the case of non-disease-associated yeast lawns, which were used as control (Table 1). Among the non-diseaserelated species, the Saccharomycopsis crataegensis  and Wickerhamomyces orientalis ) cells were especially sensitive, because almost all test strains were able to inhibit their growth (Table 1). Interestingly, in some cases, growth stimulation around of the lawn (indicated with S in the Table 1, Fig.1b) or co-occurence of inhibitory-and stimulation zones could also be observed (indicated with I-S in the Table 1, Fig.1c).

Pichia kudriavzevii was highly resistant
Our screening suggested that Pichia kudriavzevii  can have strong resistance against yeasts (Table 1). To learn whether it is true or not, further test strains belonging to different species and originated from different regions of the World were investigated on the Pichia kudriavzevii  lawns. Our data confirmed the strong resistance of Pichia kudriavzevii (Table 2), since altogether 50 strains belonging 35 species were not able to inhibit its growth on complete and minimal media ( Table 2). In contrast, Saccharomycopsis crataegensis cells (11-463) (used as control) could be inhibited by several yeast species ( Table 2).

Size of inhibitory zone can strongly depend on media, pH and temperature
Our earlier data suggested that medium and culture conditions can have strong impact on biocontrol activity (see Saccharomycopsis crataegensis- Table 2). Thus, we repeated our experiments with one of the disease-associated species (Cryptococcus albidus) (2-1365) applying minimal (EMMA) and complete (YPA) media, different pH and temperature and using further test strains. Our data confirmed that culture conditions can strongly influence antagonistic effect of the test strains (Table   3). Consequently, modifying of the culture conditions could lead to finding further antagonistic species, such as e.g. Candida insectorum  against Cryptococcus albidus (Table 3).

Discussion
Non-albicans Candida or other species including Pichia kudriavzevii, Kodamaea ohmeri Candida tropicalis or Cryptococcus albidus have been more frequently isolated from hospitalized patients [1,2,3,4,7,9]. These species seem to be very virulent and often show resistance to the commonly used antifungal treatments [5,7,13,14]. Thus, consequences of these fungal infections can be very serious, especially in children, neonates or immune-compromised patients. Accordingly, finding new inhibitory agents is increasingly urgent.
In order to identify yeast species which can have inhibitory effect against disease-associated fungi, screening of yeasts on Pichia kudriavzevii, Kodamaea ohmeri, Candida tropicalis, Cryptococcus albidus lawns were carried out. Our data showed that growth of Kodamaea ohmeri and Candida tropicalis could be inhibited by Metschnikowia andauensis, while Cryptococcus albidus can be controlled by Pichia anomala and Candida tropicalis (Table 1, Fig.1a). It means that bioactive agents of these inhibitory test strains well worth examining and yeasts can be attractive possibilities in the future solution of fungal resistance problems. Although, certain enzymes and proteins produced by these yeasts are partly known (11,17,23,24,25), we do not know exactly, which inhibitory agent was effective against the disease-associated strains mentioned above. To identify them precisely, further studies are required. Our tests shed also light that pathogenic fungi are much more tolerant to bioactive agents than the non-disease-associated yeast, such as e.g. Saccharomycopsis crataegensis and Wickerhamomyces orientalis ( Table 1). The antagonistic effects were often dependent on media, pH and temperature (  (Table 3). In contrast, application of minimal and complex media and 50 different test strains (belonging to 35 species) did not lead to success in the case of Pichia kudriavzevii, because we failed to find inhibitory yeast against it ( Table 2). Causes of its high resistance are not known and require further studies. We suppose that it can be an inherited speciesspecific feature of Pichia kudriavzevii, because our strains  were isolated from nature and did not meet earlier with antifungal medicaments. Its high tolerance is in good agreement with multidrug resistance of the clinical isolates [2].
Our experiments shed also light on complexity of the action of bioactive agents, since growth stimulation was noticed in certain lawns (Fig.1 b) (Tables 1, 3), similarly to the previous experiences [28,30]. Co-appearance of inhibitory-and stimulation zones was more interesting and unexpected ( Fig.1 c). The latter phenomenon suggests a sophisticated mechanism of action and can indicate that effect of the bioactive agent produced by M. andauensis might be concentration dependent.

Conclusion 6
Taken together, this study demonstrates that yeasts can be good candidates for finding new bioactive agents which can inhibit growth of disease-associated fungi. These bioactive agents can contribute to the future solutions of problems of fungal resistance.

Taxonomic position
PCR and sequencing methods were used for identification of the strains. Taxonomic position of the yeast species were identified by analysis of D1/D2 domain of 26S rDNA [15] (Table 2).

Spot assay for growth inhibition
Cells of the overnight culture (YPL incubated at 28 o C) were harvested and cell suspension was prepared in sterile water (final cell density was OD 595 =1). EMMA minimal and YPA complete media were flooded with 1mL of the cell suspension. After drying of the cell suspension in sterile box (lawn), yeast strains to be tested for antagonistic capacity (test-strain) were streaked or dropped (10 ul of cell suspension, OD 595 =1) onto the surface of agar plates and were incubated at the indicated temperature. Appearances of inhibitory zones were investigated after 3-10 days. The results are coming from three separate experiments.

Ethics approval and consent to participate
Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
Data of this study are included in this published article.

Competing of interest
The authors declare no conflict of interest.