Total transcriptome response for tyrosol exposure in Aspergillus nidulans.

Although tyrosol is a quorum-sensing molecule of Candida species, it has antifungal activity at supraphysiological concentrations. Here, we studied the effect of tyrosol on the physiology and genome-wide transcription of Aspergillus nidulans to gain insight into the background of the antifungal activity of this compound. Tyrosol efficiently reduced germination of conidia and the growth on various carbon sources at a concentration of 35 mM. The growth inhibition was fungistatic rather than fungicide on glucose and was accompanied with downregulation of 2199 genes related to e.g. mitotic cell cycle, glycolysis, nitrate and sulphate assimilation, chitin biosynthesis, and upregulation of 2250 genes involved in e.g. lipid catabolism, amino acid degradation and lactose utilization. Tyrosol treatment also upregulated genes encoding glutathione-S-transferases (GSTs), increased specific GST activities and the glutathione (GSH) content of the cells, suggesting that A. nidulans can detoxify tyrosol in a GSH-dependent manner even though this process was weak. Tyrosol did not induce oxidative stress in this species, but upregulated "response to nutrient levels", "regulation of nitrogen utilization", "carbon catabolite activation of transcription" and "autophagy" genes. Tyrosol may have disturbed the regulation and orchestration of cellular metabolism, leading to impaired use of nutrients, which resulted in growth reduction.

KI and WU Polyomaviruses: Seroprevalence Study and DNA Prevalence in SARS-CoV-2 RNA Positive and Negative Respiratory Samples.

The aim of this work was to study the possible co-infection of KI and WU polyomavirus (KIPyV and WUPyV, respectively) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in respiratory samples and to detect the seroprevalence of KIPyV and WUPyV. A total of 1030 nasopharyngeal samples were analyzed from SARS-CoV-2 RNA positive (n = 680) and negative (n = 350) adults and children (age: 1 day to 94.2 years) collected from August 2020 to October 2021. KIPyV DNA was detected in two SARS-CoV-2-positive samples (2/680, 0.29%) and in three SARS-CoV-2-negative samples (3/350, 0.86%). WUPyV DNA was observed in one-one samples from both groups (1/680, 0.15% vs. 1/350, 0.29%). We did not find an association between SARS-CoV-2 and KIPyV or WUPyV infection, and we found low DNA prevalence of polyomaviruses studied after a long-term lockdown in Hungary. To exclude a geographically different distribution of these polyomaviruses, we studied the seroprevalence of KIPyV and WUPyV by enzyme-linked immunosorbent assay among children and adults (n = 692 for KIPyV and n = 705 for WUPyV). Our data confirmed that primary infections by KIPyV and WUPyV occur mainly during childhood; the overall seropositivity of adults was 93.7% and 89.2% for KIPyV and WUPyV, respectively. Based on our data, we suggest that the spread of KIPyV and WUPyV might have been restricted in Hungary by the lockdown.

The Neosartorya fischeri Antifungal Protein 2 (NFAP2): A New Potential Weapon against Multidrug-Resistant Candida auris Biofilms.

Candida auris is a potential multidrug-resistant pathogen able to persist on indwelling devices as a biofilm, which serve as a source of catheter-associated infections. Neosartorya fischeri antifungal protein 2 (NFAP2) is a cysteine-rich, cationic protein with potent anti-Candida activity. We studied the in vitro activity of NFAP2 alone and in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against C. auris biofilms. The nature of interactions was assessed utilizing the fractional inhibitory concentration index (FICI), a Bliss independence model, and LIVE/DEAD viability assay. NFAP2 exerted synergy with all tested antifungals with FICIs ranging between 0.312-0.5, 0.155-0.5, 0.037-0.375, 0.064-0.375, and 0.064-0.375 for fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. These results were confirmed using a Bliss model, where NFAP2 produced 17.54 µM2%, 2.16 µM2%, 33.31 µM2%, 10.72 µM2%, and 111.19 µM2% cumulative synergy log volume in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. In addition, biofilms exposed to echinocandins (32 mg/L) showed significant cell death in the presence of NFAP2 (128 mg/L). Our study shows that NFAP2 displays strong potential as a novel antifungal compound in alternative therapies to combat C. auris biofilms.

Candida biofilm production is associated with higher mortality in patients with candidaemia.

BACKGROUND: Candidaemia is a common life-threatening disease among hospitalised patients, but the effect of the Candida biofilm-forming ability on the clinical outcome remains controversial. OBJECTIVE: The aim was to determine the impact of biofilms, specifically focusing on biofilm mass and metabolic activity, on the mortality in candidaemia. PATIENTS/METHODS: The clinical data of patients (n = 127) treated at the University of Debrecen, Clinical Centre, between January 2013 and December 2018, were investigated retrospectively. Biofilm formation was assessed using the crystal violet and XTT assays, measuring the biofilm mass and metabolic activity, respectively. Isolates were classified as low, intermediate and high biofilm producers both regarding biofilm mass and metabolic activity. The susceptibility of one-day-old biofilms to fluconazole, amphotericin B, anidulafungin, caspofungin and micafungin was evaluated and compared to planktonic susceptibility. RESULTS: Intermediate/high biofilm mass was associated with significantly higher mortality (61%). All Candida tropicalis, Candida parapsilosis and Candida glabrata isolates originating from fatal infections were intermediate/high biofilm producers, whereas this ratio was 85% for Candida albicans. Solid malignancy was associated with intermediate/high biofilm producers (P = .043). The mortality was significantly higher in infections caused by Candida strains producing biofilms with intermediate/high metabolic activity (62% vs. 33%, P = .010). The ratio of concomitant bacteraemia was higher for isolates forming biofilms with low metabolic activity (53% vs 28%, P = .015). CONCLUSIONS: This study provides evidence that the Candida biofilms especially with intermediate/high metabolic activity are related to higher mortality in candidaemia.

In Vivo Applicability of Neosartorya fischeri Antifungal Protein 2 (NFAP2) in Treatment of Vulvovaginal Candidiasis.

As a consequence of emerging numbers of vulvovaginitis cases caused by azole-resistant and biofilm-forming Candida species, fast and efficient treatment of this infection has become challenging. The problem is further exacerbated by the severe side effects of azoles as long-term-use medications in the recurrent form. There is therefore an increasing demand for novel and safely applicable effective antifungal therapeutic strategies. The small, cysteine-rich, and cationic antifungal proteins from filamentous ascomycetes are potential candidates, as they inhibit the growth of several Candida spp. in vitro; however, no information is available about their in vivo antifungal potency against yeasts. In the present study, we investigated the possible therapeutic application of one of their representatives in the treatment of vulvovaginal candidiasis, Neosartorya fischeri antifungal protein 2 (NFAP2). NFAP2 inhibited the growth of a fluconazole (FLC)-resistant Candida albicans strain isolated from a vulvovaginal infection, and it was effective against both planktonic cells and biofilm in vitro We observed that the fungal cell-killing activity of NFAP2 is connected to its pore-forming ability in the cell membrane. NFAP2 did not exert cytotoxic effects on primary human keratinocytes and dermal fibroblasts at the MIC in vitro. In vivo murine vulvovaginitis model experiments showed that NFAP2 significantly decreases the number of FLC-resistant C. albicans cells, and combined application with FLC enhances the efficacy. These results suggest that NFAP2 provides a feasible base for the development of a fundamental new, safely applicable mono- or polytherapeutic topical agent for the treatment of superficial candidiasis.

Interaction between Different Pharmaceutical Excipients in Liquid Dosage Forms-Assessment of Cytotoxicity and Antimicrobial Activity.

Nowadays, the safety of parabens as pharmaceutical preservatives is debated. Recent studies investigated their interference with the oestrogen receptors, nevertheless their carcinogenic activity was also proved. That was the reason why the re-evaluation of the biocompatibility and antimicrobial activity of parabens is required using modern investigation methods. We aimed to test the cytotoxic, antifungal and antibacterial effect of parabens on Caco-2 cells, C. albicans, C. parapsilosis, C. glabrata, E. coli, P. aeruginosa and S. aureus. Two complex systems (glycerol-Polysorbate 20; ethanol-Capryol PGMC™) were formulated to study-with the MTT-assay and microdilution method, respectively-how other excipients may modify the biocompatibility and antimicrobial effect of parabens. In the case of cytotoxicity, the toxicity of these two systems was highly influenced by co-solvents and surfactants. The fungi and bacteria had significantly different resistance in the formulations and in some cases the excipients could highly modify the effectiveness of parabens both in an agonistic and in a counteractive way. These results indicate that with appropriate selection, non-preservative excipients can contribute to the antimicrobial safety of the products, thus they may decrease the required preservative concentration.

Commercial strain-derived clinical Saccharomyces cerevisiae can evolve new phenotypes without higher pathogenicity.

SCOPE: Saccharomyces cerevisiae is one of the most important microbes in food industry, but there is growing evidence on its potential pathogenicity as well. Its status as a member of human mycobiome is still not fully understood. METHODS AND RESULTS: In this study, we characterize clinical S. cerevisiae isolates from Hungarian hospitals along with commercial baking and probiotic strains, and determine their phenotypic parameters, virulence factors, interactions with human macrophages, and pathogenicity. Four of the clinical isolates could be traced back to commercial strains based on genetic fingerprinting. Our observations indicate that the commercial-derived clinical isolates have evolved new phenotypes and show similar, or in two cases, significantly decreased pathogenicity. Furthermore, immunological experiments revealed that the variability in human primary macrophage activation after coincubation with yeasts is largely donor and not isolate dependent. CONCLUSION: Isolates in this study offer an interesting insight into the potential microevolution of probiotic and food strains in human hosts. These commensal yeasts display various changes in their phenotypes, indicating that the colonization of the host does not necessarily impose a selective pressure toward higher virulence/pathogenicity.

Serum interleukin-6 levels in murine models of Candida albicans infection.

Two Balb/C mouse models of Candida infection were used to detect serum interleukin-6 (IL-6) responses. The first model used systemic infection by Candida albicans ATCC 10231 strain infected through the lateral tail vein of mice without any specific pretreatment. The median Candida burdens of the kidneys were 1.5 × 106 CFU/ml 24 h postinoculation (p.i.) and 1.2 × 107 CFU/ml 72 h p.i., while median serum IL-6 levels were 479.3 pg/ml and 934.5 pg/ml, respectively. The Candida burden showed significant correlation with serum IL-6 24 h p.i. (R2 = 0.6358; P = 0.0082) but not 72 h p.i.The second model was a mouse vaginitis model applying intravaginal inoculation of mice pretreated with subcutaneous estradiol-valerate (10 mg/ml) 3 days before infection. Candida cell count in vaginal lavage fluid was 2.8 × 106 CFU/ml 24 h p.i. and 1.4 × 108 CFU/ml 72 h p.i. Serum IL-6 response was detected in 4 of 15 mice 24 h p.i. and 9 of 15 mice 72 h p.i. Even the responders had low IL-6 serum levels (mean values 29.9 pg/ml and 60.1 pg/ml, respectively) not correlating with Candida cell count in vaginal lavage fluid.In conclusion, serum IL-6 had strong relationship with systemic C. albicans infection while the local C. albicans infection of the vagina led to partial, prolonged and limited serum IL-6 response.