SARS-CoV-2 Post-Infection and Sepsis by Saccharomyces cerevisiae: A Fatal Case Report-Focus on Fungal Susceptibility and Potential Virulence Attributes

Abstract

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for approximately 6.8 million deaths worldwide, threatening more than 753 million individuals. People with severe coronavirus disease-2019 (COVID-19) infection often exhibit an immunosuppression condition, resulting in greater chances of developing co-infections with bacteria and fungi, including opportunistic yeasts belonging to the Saccharomyces and Candida genera. In the present work, we have reported the case of a 75-year-old woman admitted at a Brazilian university hospital with an arterial ulcer in the left foot, which was being prepared for surgical amputation. The patient presented other underlying diseases and presented positive tests for COVID-19 prior to hospitalization. She received antimicrobial treatment, but her general condition worsened quickly, leading to death by septic shock after 4 days of hospitalization. Blood samples collected on the day she died were positive for yeast-like organisms, which were later identified as Saccharomyces cerevisiae by both biochemical and molecular methods. The fungal strain exhibited low minimal inhibitory concentration values for the antifungal agents tested (amphotericin B, 5-flucytosine, caspofungin, fluconazole and voriconazole), and it was able to produce important virulence factors, such as extracellular bioactive molecules (e.g., aspartic peptidase, phospholipase, esterase, phytase, catalase, hemolysin and siderophore) and biofilm. Despite the activity against planktonic cells, the antifungals were not able to impact the mature biofilm parameters (biomass and viability). Additionally, the S. cerevisiae strain caused the death of Tenebrio molitor larvae, depending on the fungal inoculum, and larvae immunosuppression with corticosteroids increased the larvae mortality rate. In conclusion, the present study highlighted the emergence of S. cerevisiae as an opportunistic fungal pathogen in immunosuppressed patients presenting several severe comorbidities, including COVID-19 infection.

Keywords: COVID-19; Tenebrio molitor; biofilm formation; fungal infection; hydrolytic enzymes.

Figure 1

Microscopic (A) and macroscopic (B) aspects of S. cerevisiae HUPE-Sc1 strain grown for 48 h at 37 °C on Sabouraud dextrose agar. (C) Phylogenetic neighbor-joining dendrogram generated from a genetic similarity matrix based on comparison of ITS1-5.8S-ITS2 gene sequences from HUPE-Sc1 strain and type strains belonging to the Saccharomyces genus; sequences were obtained from GenBank database.

Figure 2

Extracellular molecules produced by S. cerevisiae HUPE-Sc1 strain by means of agar plates containing specific substrates to detect: caseinase, aspartic peptidase, phospholipase, esterase and phytase (A), and catalase activity demonstrated by the formation of bubbles after the contact of S. cerevisiae HUPE-Sc1 strain with H₂O₂, which correspond to the hydrolysis of H₂O₂ in water and molecular oxygen (B). The results represent means ± standard deviation of three independent experiments.

Figure 3

Extracellular molecules produced by S. cerevisiae HUPE-Sc1 strain by means of agar plates containing specific substrates to detect hemolysin and siderophores (A). Lysis of fresh erythrocytes after 3 h and 24 h of incubation at 37 °C by different inocula of S. cerevisiae HUPE-Sc1 strain (10⁶, 10⁷ and 10⁸ cells) (B) and different protein amounts of the cell-free culture supernatant in Sabouraud (C). SDS-PAGE demonstrating the hydrolysis of hemoglobin (Hg) by the enzymes present in the culture supernatant (Sob) of HUPE-Sc1 strain (2.5, 5 and 10 μg of proteins); the dashed boxes highlight intact hemoglobin (upper box) and hydrolyzed hemoglobin (lower box) (D). HUPE-Sc1 strain growth capacity in different nutrient sources (Sabouraud, FBS, blood [100%] and diluted blood [2%]) after incubation of 10⁴ yeasts/mL at 37 °C for 24 h as demonstrated by CFU counts (E) and by spot inoculum of 10 μL of each system on SDA (F). The results represent means ± standard deviation of three independent experiments. The symbol (*) indicates p values < 0.05 (one-way ANOVA, Tukey’s multiple comparison).

Figure 4

Biofilm formation by S. cerevisiae HUPE-Sc1 strain over a polystyrene surface. Three biofilm parameters were measured: biomass (absorbance at 590 nm), viability (absorbance at 492 nm) and extracellular matrix (absorbance at 530 nm). The results represent means ± standard deviation of three independent experiments.

Figure 5

Biomass (A) and viability (B) of biofilm formed by S. cerevisiae HUPE-Sc1 strain over a polystyrene surface exposed to different concentrations of antifungals: amphotericin B (AMB), 5-flucytosine (5-FLU), caspofungin (CSF), fluconazole (FLC) and voriconazole (VRC). The results were assessed spectroscopically (biomass at 590 nm and viability at 492 nm) and expressed as the mean of metabolic and biomass percentages compared to untreated biofilms (control), which correspond to 100% (blue line). The graphs exhibit the mean ± standard deviation of three independent experiments. The dashed box represents the concentrations of CSF that caused statistically significant reduction in cell viability in relation to the respective control (p < 0.05; one-way ANOVA analysis of variance and Dunnett’s multiple comparison test).

Figure 6

Survival curves of T. molitor larvae infected with different inoculum sizes of S. cerevisiae HUPE-Sc1 strain (10⁴, 10⁵, 10⁶ and 10⁷ fungi/larvae) (A) and effect of the treatment with corticosteroids (100 μg) on larvae survival after injection of 10⁶ fungi/larva (B). In both cases, groups of 10 larvae were infected with indicated systems, repeated three times and pooled together in order to build survival curves with 30 animals. Negative controls were composed by T. molitor larvae injected only with PBS or corticosteroids (100 μg).