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A special issue of Journal of Fungi (ISSN 2309-608X). This special issue belongs to the section "Fungal Cell Biology, Metabolism and Physiology".

Deadline for manuscript submissions: closed (15 July 2024) | Viewed by 7567

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Special Issue Editors

Dr. Célia F. Rodrigues

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Guest Editor

1. 1H-TOXRUN—One Health Toxicology Research Unit, CESPU-IUCS, Gandra, Portugal
2. LEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, University of Porto, Porto, Portugal
Interests: biofilms; fungal and bacterial infections; resistance to antimicrobials; microfluidics; pathogens detection; alternative therapies; surface functionalization of biomaterials
Special Issues, Collections and Topics in MDPI journals

Dr. Lucia Černáková

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Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University in Bratislava, 842 15 Bratislava, Slovakia
Interests: biofilm; Candida; MRSA; virulence; resistance; farnesol; photodynamic inactivation
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Fungal infections are an important and increasing global threat, carrying not only high morbidity and mortality rates, but also high healthcare costs. Without an effective response, it is predicted that 10 million people will die per year as a result of multidrug-resistant pathogens. A high percentage of the mortalities caused by fungi are known to be biofilm-related.

This Special Issue, "Fungal Biofilms", is intended to cover the state of fungal biofilm research, from virulence and pathogenicity, to new compounds with antibiofilm and antifungal activity. We welcome reviews and original research articles covering the development/evaluation/validation of recent studies, especially those regarding multidrug resistance.

Dr. Célia F. Rodrigues
Dr. Lucia Černáková
Guest Editors

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Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Journal of Fungi is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

fungi
infection
biofilm
Candida
Aspergillus
Cryptococcus
antifungal
resistance
matrix

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Published Papers (6 papers)

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22 pages, 3224 KiB

Open AccessArticle

Phenotypic and Genotypic Characterization of Resistance and Virulence Markers in Candida spp. Isolated from Community-Acquired Infections in Bucharest, and the Impact of AgNPs on the Highly Resistant Isolates

by Viorica Maria Corbu, Ana-Maria Georgescu, Ioana Cristina Marinas, Radu Pericleanu, Denisa Vasilica Mogos, Andreea Ștefania Dumbravă, Liliana Marinescu, Ionut Pecete, Tatiana Vassu-Dimov, Ilda Czobor Barbu, Ortansa Csutak, Denisa Ficai and Irina Gheorghe-Barbu

J. Fungi 2024, 10(8), 563; https://doi.org/10.3390/jof10080563 - 9 Aug 2024

Viewed by 312

Abstract

Background: This study aimed to determine, at the phenotypic and molecular levels, resistance and virulence markers in Candida spp. isolated from community-acquired infections in Bucharest outpatients during 2021, and to demonstrate the efficiency of alternative solutions against them based on silver nanoparticles (AgNPs). Methods: A total of 62 Candida spp. strains were isolated from dermatomycoses and identified using chromogenic culture media and MALDI-TOF MS, and then investigated for their antimicrobial resistance and virulence markers (VMs), as well as for metabolic enzymes using enzymatic tests for the expression of soluble virulence factors, their biofilm formation and adherence capacity on HeLa cells, and PCR assays for the detection of virulence markers and the antimicrobial activity of alternative solutions based on AgNPs. Results: Of the total of 62 strains, 45.16% were Candida parapsilosis; 29.03% Candida albicans; 9.67% Candida guilliermondii; 3.22% Candida lusitaniae, Candia pararugosa, and Candida tropicalis; and 1.66% Candida kefyr, Candida famata, Candida haemulonii, and Candida metapsilosis. Aesculin hydrolysis, caseinase, and amylase production were detected in the analyzed strains. The strains exhibited different indices of adherence to HeLa cells and were positive in decreasing frequency order for the LIP1, HWP1, and ALS1,3 genes (C. tropicalis/C. albicans). An inhibitory effect on microbial growth, adherence capacity, and on the production of virulence factors was obtained using AgNPs. Conclusions: The obtained results in C. albicans and Candida non-albicans circulating in Bucharest outpatients were characterized by moderate-to-high potential to produce VMs, necessitating epidemiological surveillance measures to minimize the chances of severe invasive infections. Full article

(This article belongs to the Special Issue Fungal Biofilms, 2nd Edition)

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The experimental design (created with Biorender.com; accessed on 11 June 2024). Full article ">Figure 2
The distribution of arbitrary units by isolation sources of Candida spp. strains. Legend: AU1 = 1 arbitrary unit, AU2 = 2 arbitrary units, and AU0 = 0 arbitrary units. Full article ">Figure 3
Average MIC values for Candida spp. strains. Full article ">Figure 4
Adherence inhibition percentage (PICA%) values for AgNPs compared to the Candida spp. strains (* p < 0.05, ** p < 0.001, **** p < 0.0001) (Dunnett’s multiple comparisons test). Full article ">Figure 5
AgNPs’ effects on Candida spp. strains’ virulence factors (* p < 0.05, ** p < 0.001, *** p < 0.001, **** p < 0.0001) (Dunnett’s multiple comparisons test). (A) Caseinase production of Candida sp. strains, (B) Hemolysis production of Candida sp. strains, (C) Amylase production of Candida sp. strains, (D) Esculin Hydrolysis production of Candida sp. strains. Full article ">Figure 6
Extracellular NO content determined by the Griess reaction for AgNPs in the presence of C. albicans strains (Tukey’s method, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Full article ">Figure 7
Pearson correlation among extracellular NO content, adhesion inhibition percentage (PICA%), caseinase activity (%), amylase activity (%), and hemolysin (%) for C. albicans (A) and C. parapsilosis (B). Full article ">

20 pages, 72710 KiB

Open AccessArticle

Influence of Zinc on Histoplasma capsulatum Planktonic and Biofilm Cells

by Ana Carolina Moreira da Silva Pires, Angélica Romão Carvalho, Carolina Orlando Vaso, Maria José Soares Mendes-Giannini, Junya de Lacorte Singulani and Ana Marisa Fusco-Almeida

J. Fungi 2024, 10(5), 361; https://doi.org/10.3390/jof10050361 - 20 May 2024

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Abstract

Histoplasma capsulatum causes a fungal respiratory disease. Some studies suggest that the fungus requires zinc to consolidate the infection. This study aimed to investigate the influence of zinc and the metal chelator TPEN on the growth of Histoplasma in planktonic and biofilm forms. The results showed that zinc increased the metabolic activity, cell density, and cell viability of planktonic growth. Similarly, there was an increase in biofilm metabolic activity but no increase in biomass or extracellular matrix production. N′-N,N,N,N–tetrakis–2-pyridylmethylethane–1,2 diamine (TPEN) dramatically reduced the same parameters in the planktonic form and resulted in a decrease in metabolic activity, biomass, and extracellular matrix production for the biofilm form. Therefore, the unprecedented observations in this study highlight the importance of zinc ions for the growth, development, and proliferation of H. capsulatum cells and provide new insights into the role of metal ions for biofilm formation in the dimorphic fungus Histoplasma, which could be a potential therapeutic strategy. Full article

(This article belongs to the Special Issue Fungal Biofilms, 2nd Edition)

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19 pages, 2276 KiB

Open AccessArticle

Central Carbon Metabolism in Candida albicans Biofilms Is Altered by Dimethyl Sulfoxide

by Maria Fernanda Cordeiro Arruda, Romeu Cassiano Pucci da Silva Ramos, Nicoly Subtil de Oliveira, Rosimeire Takaki Rosa, Patrícia Maria Stuelp-Campelo, Luiz Fernando Bianchini, Silas Granato Villas-Bôas and Edvaldo Antonio Ribeiro Rosa

J. Fungi 2024, 10(5), 337; https://doi.org/10.3390/jof10050337 - 8 May 2024

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Abstract

The effect of dimethyl sulfoxide (DMSO) on fungal metabolism has not been well studied. This study aimed to evaluate, by metabolomics, the impact of DMSO on the central carbon metabolism of Candida albicans. Biofilms of C. albicans SC5314 were grown on paper discs, using minimum mineral (MM) medium, in a dynamic continuous flow system. The two experimental conditions were control and 0.03% DMSO (v/v). After 72 h of incubation (37 °C), the biofilms were collected and the metabolites were extracted. The extracted metabolites were subjected to gas chromatography–mass spectrometry (GC/MS). The experiment was conducted using five replicates on three independent occasions. The GC/MS analysis identified 88 compounds. Among the 88 compounds, the levels of 27 compounds were markedly different between the two groups. The DMSO group exhibited enhanced levels of putrescine and glutathione and decreased levels of methionine and lysine. Additionally, the DMSO group exhibited alterations in 13 metabolic pathways involved in primary and secondary cellular metabolism. Among the 13 altered pathways, seven were downregulated and six were upregulated in the DMSO group. These results indicated a differential intracellular metabolic profile between the untreated and DMSO-treated biofilms. Hence, DMSO was demonstrated to affect the metabolic pathways of C. albicans. These results suggest that DMSO may influence the results of laboratory tests when it is used as a solvent. Hence, the use of DMSO as a solvent must be carefully considered in drug research, as the effect of the researched drugs may not be reliably translated into clinical practice. Full article

(This article belongs to the Special Issue Fungal Biofilms, 2nd Edition)

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Means of the relative abundances (<1.4) of the C. albicans SC5314 intracellular metabolites which showed statistically significant differences (p ≤ 0.05) between the control and DMSO groups. ACC = 1-aminocyclopropane-1-carboxylic acid. Full article ">Figure 2
Means of the relative abundances (<14) of the C. albicans SC5314 intracellular metabolites which showed statistically significant differences (p ≤ 0.05) between the control and DMSO groups. Full article ">Figure 3
Means of the relative abundances (<250) of the C. albicans SC5314 intracellular metabolites which showed statistically significant differences (p ≤ 0.05) between the control and DMSO groups. 10,13-DMTDA = 10,13-dimethylthetradecanoic acid; GABA = 4-aminobutyric acid. Full article ">Figure 4
Metabolic pathways with their activity altered (p ≤ 0.05) by the presence of 0.03% (v/v) DMSO in C. albicans SC5314 biofilm samples. Pathways with an activity score (AS) less than 0 (zero) represent those downregulated by DMSO, and pathways with an AS greater than 0 (zero) represent those upregulated. Full article ">Scheme 1
Proposed shifts in the metabolic network of C. albicans SC5314 altered by 0.03% (v/v) DMSO and assessed by GC/MS. Upregulated pathways and increased metabolites are represented in red, and downregulated pathways and reduced metabolites are depicted in blue. ⊗ = inhibition. Full article ">

14 pages, 2389 KiB

Open AccessArticle

Died or Not Dyed: Assessment of Viability and Vitality Dyes on Planktonic Cells and Biofilms from Candida parapsilosis

by Betsy Verónica Arévalo-Jaimes and Eduard Torrents

J. Fungi 2024, 10(3), 209; https://doi.org/10.3390/jof10030209 - 11 Mar 2024

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Abstract

Viability and vitality assays play a crucial role in assessing the effectiveness of novel therapeutic approaches, with stain-based methods providing speed and objectivity. However, their application in yeast research lacks consensus. This study aimed to assess the performance of four common dyes on C. parapsilosis planktonic cells as well as sessile cells that form well-structured biofilms (treated and not treated with amphotericin B). Viability assessment employed Syto-9 (S9), thiazole orange (TO), and propidium iodide (PI). Metabolic activity was determined using fluorescein diacetate (FDA) and FUN-1. Calcofluor white (CW) served as the cell visualization control. Viability/vitality percentage of treated samples were calculated for each dye from confocal images and compared to crystal violet and PrestoBlue results. Heterogeneity in fluorescence intensity and permeability issues were observed with S9, TO, and FDA in planktonic cells and biofilms. This variability, influenced by cell morphology, resulted in dye-dependent viability/vitality percentages. Notably, PI and FUN-1 exhibited robust C. parapsilosis staining, with FUN-1 vitality results comparable to PrestoBlue. Our finding emphasizes the importance of evaluating dye permeability in yeast species beforehand, incorporating cell visualization controls. An improper dye selection may lead to misinterpreting treatment efficacy. Full article

(This article belongs to the Special Issue Fungal Biofilms, 2nd Edition)

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Figure 1
Viability and vitality staining of C. parapsilosis 11103595 overnight cultures. (A) Viability assessment of C. parapsilosis 11103595 planktonic cells using S9 (green) + PI (red) and TO (green) + PI (red) staining. Dyes were compared with CW (blue) merged image for cell visualization control. White arrows in the S9 + PI row highlight some of the cells that were not stained with S9 but were visible with CW. (B) Vitality evaluation using FDA (green) and FUN-1 (green and red) staining. CW (blue) merged image was used as a cell visualization control. White arrows in the FDA row indicate high intensity only in a dead cell. White arrow in the zoomed in image of the FUN-1 row shows a green non-metabolically active cell, while the red arrow shows a cell with red cylindrical intravacuolar structures (CIVSs), indicating metabolic activity. Confocal images were processed using ImageJ v1.54f. The scale bar of 10 µm is consistent for all cases, except in the FUN-1 zoomed-in image where it represents a 5 µm length. Images were taken at 100×. S9 = Syto9, PI = propidium iodide, TO = thiazole orange, CW = calcofluor white, FDA = fluorescein diacetate. Full article ">Figure 2
Total biomass and metabolic activity of C. parapsilosis 11103595 biofilms treated with amphotericin B (AmB). (A) Total biomass quantification (cell biomass and extracellular matrix) with crystal violet (CV) assay and (B) metabolic activity evaluation by PrestoBlue assay after 20 h treatment with 2.5 μg/mL of AmB. Biofilm results experiments were conducted in triplicate. Numbers after the symbol ↓ indicate the percentage of decrease in the mean value with respect to the control. Data are represented as mean ± standard deviation. Asterisks indicate statistically significant differences versus control in an unpaired t-test (*: p-value < 0.05, ***: p-value < 0.001). RFUs = relative fluorescence units. Full article ">Figure 3
Viability staining of C. parapsilosis 11103595 biofilms treated with amphotericin B (AmB). (A) Viability assessment using S9 (green live cells) + PI (red dead cells) dye with the respective biomass quantification. (B) Viability assessment using TO (green live cells) + PI (red dead cells) dyes with the respective biomass quantification. Each dye combination was compared with the CW (gray) merged image and the respective biomass correction. Biomass quantifications were performed from images at a 10× magnification with the plugin COMSTA2 from ImageJ software v1.54f. Data are represented as mean ± standard deviation from n ≥ 3 replicates. Asterisks indicate statistically significant differences versus control (***: p-value < 0.001; ****: p-value < 0.0001). Numbers after the symbol ↓ indicate the percentage of decrease in the mean value with respect to the control. Z-stack of biofilm top layers from confocal images at a 63× magnification were created using ImageJ v1.54f. The scale bar of 10 µm is consistent for all cases. S9 = Syto9, PI = propidium iodide, TO = thiazole orange, CW = calcofluor white. Full article ">Figure 4
Vitality staining of C. parapsilosis 11103595 biofilms treated with amphotericin B (AmB). (A) Vitality assessment using FDA (green metabolically active cells) dye with the respective biomass quantification. (B) Vitality assessment using FUN-1 (green metabolically unactive cells and green/red metabolically active cells) with the respective biomass quantification. Each dye was compared with the CW (gray) merged image and the respective biomass correction. Biomass quantifications were performed from images at a 10× magnification with the plugin COMSTA2 from ImageJ software v1.54f. Data are represented as mean ± standard deviation from n ≥ 3 replicates. Asterisks indicate statistically significant differences versus control (*: p-value < 0.05). Numbers after the symbol ↑ indicate the percentage of increase in the mean value with respect to the control, while numbers after the symbol ↓ indicate the percentage of decrease in the mean value with respect to the control. Z-stack of biofilm top layers from confocal images at a 63× magnification were created using ImageJ v1.54f. The scale bar of 10 μm is consistent for all cases. CW = calcofluor white, FDA = fluorescein diacetate. Full article ">

17 pages, 1447 KiB

Open AccessArticle

High-Throughput Screening of the Repurposing Hub Library to Identify Drugs with Novel Inhibitory Activity against Candida albicans and Candida auris Biofilms

by Olabayo H. Ajetunmobi, Gina Wall, Bruna Vidal Bonifacio, Lucero A. Martinez Delgado, Ashok K. Chaturvedi, Laura K. Najvar, Floyd L. Wormley, Jr., Hoja P. Patterson, Nathan P. Wiederhold, Thomas F. Patterson and Jose L. Lopez-Ribot

J. Fungi 2023, 9(9), 879; https://doi.org/10.3390/jof9090879 - 27 Aug 2023

Cited by 3 | Viewed by 1658

Abstract

Candidiasis is one of the most frequent nosocomial infections affecting an increasing number of at-risk patients. Candida albicans remains the most frequent causative agent of candidiasis, but, in the last decade, C. auris has emerged as a formidable multi-drug-resistant pathogen. Both species are fully capable of forming biofilms, which contribute to resistance, increasing the urgency for new effective antifungal therapies. Repurposing existing drugs could significantly accelerate the development of novel therapies against candidiasis. Here, we have screened the Repurposing Hub library from the Broad Institute, containing over 6000 compounds, in search for inhibitors of C. albicans and C. auris biofilm formation. The primary screen identified 57 initial hits against C. albicans and 33 against C. auris. Confirmatory concentration-dependent assays were used to validate the activity of the initial hits and, at the same time, establish their anti-biofilm potency. Based on these results, ebselen, temsirolimus, and compound BAY 11-7082 emerged as the leading repositionable compounds. Subsequent experiments established their spectrum of antifungal activity against yeasts and filamentous fungi. In addition, their in vivo activity was examined in the murine models of hematogenously disseminated C. albicans and C. auris infections. Although promising, further in vitro and in vivo studies are needed to confirm their potential use for the therapy of candidiasis and possibly other fungal infections. Full article

(This article belongs to the Special Issue Fungal Biofilms, 2nd Edition)

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14 pages, 3605 KiB

Open AccessReview

Interactions between Bacteria and Aspergillus fumigatus in Airways: From the Mycobiome to Molecular Interactions

by Anne Debourgogne, Lorra Monpierre, Khadeeja Adam Sy, Isabel Valsecchi, Jean-Winoc Decousser and Françoise Botterel

J. Fungi 2023, 9(9), 900; https://doi.org/10.3390/jof9090900 - 1 Sep 2023

Viewed by 1513

Abstract

Interactions between different kingdoms of microorganisms in humans are common but not well described. A recent analysis of the mycobiome has described the presence of different fungi and their positive and/or negative interactions with bacteria and other fungi. In chronic respiratory diseases, these different microorganisms form mixed biofilms to live inside. The interactions between Gram-negative bacteria and filamentous fungi in these biofilms have attracted more attention recently. In this review, we analyse the microbiota of the respiratory tract of healthy individuals and patients with chronic respiratory disease. Additionally, we describe the regulatory mechanisms that rule the mixed biofilms of Aspergillus fumigatus and Gram-negative bacteria and the effects of this biofilm on clinical presentations. Full article

(This article belongs to the Special Issue Fungal Biofilms, 2nd Edition)

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