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Fungal spoilage is one of the causes of consequential losses in the dairy industry. In this context, the use of bioprotective cultures can be an alternative or a complementary approach to be considered. Lactic acid bacteria (LAB) and propionibacteria, as well as some fungal species, can exhibit antifungal activities with large differences in activity between strains. Therefore, it is necessary to develop high-throughput screening methods to test a large number of strains and find the most efficient ones. In the present study, we developed a miniaturized high-throughput screening technique to rapidly detect antifungal activities in a cheese-like model. This model, distributed in a 24-well plate, consisted of 5-fold concentrated whole milk ultrafiltration retentate (final fat concentration of 45%), rennet (0.03%) and inoculated with a mesophilic lactic commercial starter and a pH indicator. Each well of the plate could be considered as a miniature cheese of ~2 g. Potent antifungal isolates were cultured in two dairy media; (i) a 10%-reconstituted low heat skim milk supplemented with 45% anhydrous milk fat (LH) and (ii) a 6-fold concentrated milk ultrafiltration permeate sterilized by 0.22 μm filtration and complemented with 10 g/l yeast extract and a pH indicator (UF). After cultivation, cultures (100 µl) were deposited on the miniature cheese surfaces followed by inoculation in duplicate with 50 spores or cells of 4 different fungal targets (1 fungi/plate), e.g., Mucor racemosus, Galactomyces geotrichum, Penicillium commune and Yarrowia lipolytica, and incubation at 12°C for up to 15 days. We screened 505 bacterial isolates belonging to Lactobacillus, Lactococcus, Pediococcus, Leuconostoc and Propiobacterium genera and 198 fungal isolates belonging to 28 genera. This high-throughput screening for antifungal activity revealed that 52 and 216 bacteria, and, 53 and 89 fungi, inhibited at least one fungal target after cultivation in UF and LH, respectively. Among the 4 tested fungal targets, P. commune was the most frequently inhibited fungus while only few isolates were able to inhibit M. racemosus or Y. lipolytica. This method opens new possibilities to screen microorganisms for antifungal activities. These results also underline the importance of the culture and screening media used on the expression of antifungal activities by bacteria or fungi.

Following antifungal treatment, Candida albicans, and other human pathogenic fungi can undergo microevolution, which leads to the emergence of drug resistance. However, the capacity for microevolutionary adaptation of fungi goes beyond the development of resistance against antifungals. Here we used an experimental microevolution approach to show that one of the central pathogenicity mechanisms of C. albicans, the yeast-to-hyphae transition, can be subject to experimental evolution. The C. albicans cph1Δ/efg1Δ mutant is nonfilamentous, as central signaling pathways linking environmental cues to hyphal formation are disrupted. We subjected this mutant to constant selection pressure in the hostile environment of the macrophage phagosome. In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation. In addition, the evolved mutant exhibited hyper-virulence in a murine infection model and an altered cell wall composition compared to the cph1Δ/efg1Δ strain. Moreover, the transcriptional regulation of hyphae-associated, and other pathogenicity-related genes became re-responsive to environmental cues in the evolved strain. We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery. This mutation was responsible for the reconnection of the hyphal growth program with environmental signals in the evolved strain and was sufficient to bypass Efg1/Cph1-dependent filamentation. These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure., Author Summary Pathogenic microbes often evolve complex traits to adapt to their respective hosts, and this evolution is ongoing: for example, microorganisms are developing resistance to antimicrobial compounds in the clinical setting. The ability of the common human pathogenic fungus, Candida albicans, to switch from yeast to hyphal (filamentous) growth is considered a central virulence attribute. For example, hyphal formation allows C. albicans to escape from macrophages following phagocytosis. A well-investigated signaling network integrates different environmental cues to induce and maintain hyphal growth. In fact, deletion of two central transcription factors in this network results in a mutant that is both nonfilamentous and avirulent. We used experimental evolution to study the adaptation capability of this mutant by continuous co-incubation within macrophages. We found that this selection regime led to a relatively rapid re-connection of signaling between environmental cues and the hyphal growth program. Indeed, the evolved mutant regained the ability to filament and its virulence in vivo. This bypass of central transcription factors was based on a single nucleotide exchange in a gene encoding a component of the general transcription regulation machinery. Our results show that even a complex regulatory network, such as the transcriptional network which governs hyphal growth, can be remodeled via microevolution.