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Clinical Microbiology and Infection :... Jan 2020Our objective was to characterize the fungal microbiota on normal ocular surface of humans with the culture-based method and high-throughput sequencing approach.
OBJECTIVES
Our objective was to characterize the fungal microbiota on normal ocular surface of humans with the culture-based method and high-throughput sequencing approach.
METHODS
A total of 45 adults were recruited from an urban community, and 90 conjunctival swabs were obtained, one from each eye of each participant. One of the two swabs from each participant was randomly chosen and allocated to internal transcribed spacer (ITS) sequencing, and the other was subjected to conventional fungal cultivation.
RESULTS
Four filamentous fungi were isolated from the 45 samples using the culture-based method, Penicillium citrinum, Aspergillus niger, Phialophora and Trichoderma. In the other 45 samples, 18 samples were positive for PCR amplification and sent for subsequent ITS sequencing. A total of 518 703 valid reads were generated and assigned into 467 operational taxonomic units. Overall, 4 phyla and 94 genera were identified. Two phyla, Basidiomycota (78.67%) and Ascomycota (19.54%), and five genera, Malassezia (74.65%), Rhodotorula (1.93%), Davidiella (1.89%), Aspergillus (1.25%) and Alternaria (0.61%), which accounted for >80% of the fungal microbiome and presented in >80% of the individuals tested, constituted the possible 'core fungal taxa' on normal ocular surface.
CONCLUSIONS
The fungal microbiome on normal ocular surface of humans was identified using the high-throughput sequencing method, providing a basis for further investigations on the potential role of the fungal microbiota in ocular health and disease.
Topics: Adult; Aged; DNA, Intergenic; Eye; Female; Fungi; Healthy Volunteers; High-Throughput Nucleotide Sequencing; Humans; Male; Middle Aged; Mycobiome; Sequence Analysis, DNA
PubMed: 31128284
DOI: 10.1016/j.cmi.2019.05.011 -
Antimicrobial Agents and Chemotherapy Jul 2021Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by genera of melanized fungi: , , , , and . Melanin is a virulence factor known to influence...
Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by genera of melanized fungi: , , , , and . Melanin is a virulence factor known to influence antifungal susceptibility. A specific inhibitor of melanin biosynthesis is tricyclazole. The aim of this study was to evaluate the effect of melanin inhibition on antifungal susceptibility of chromoblastomycosis agents and describe the susceptibility profiles of some unusual CBM agents. Seventy-six clinical isolates, representing 13 species of the five main genera of CBM agents, were studied. The antifungal susceptibility testing was performed according to the M38-A2 protocol of CLSI (, 3rd ed., , 2017). In the melanin inhibition test, 16 mg/liter of tricyclazole was added to the medium used in the inoculum preparation and the susceptibility assay. CBM agents were less susceptible to amphotericin B than azoles and terbinafine. The unusual species showed similar susceptibility profiles to those of other species of the same genera. With tricyclazole exposure, MICs of terbinafine, posaconazole, and itraconazole for spp. significantly decreased ( < 0.05). For spp., this reduction was significant for posaconazole and itraconazole. For the other genera, there was a reduction in MICs of terbinafine and itraconazole; however, the statistical tests were not significant. Melanin inhibition can increase the antifungal susceptibility of most CBM agents to itraconazole and terbinafine, the main drugs used in the disease treatment. This increased susceptibility may open up new possibilities for therapy in refractory cases of CBM and/or cases caused by resistant fungal strains. Further studies are needed to confirm the same results .
Topics: Antifungal Agents; Ascomycota; Chromoblastomycosis; Humans; Itraconazole; Melanins; Microbial Sensitivity Tests; Terbinafine
PubMed: 33972246
DOI: 10.1128/AAC.00546-21 -
Journal of Fungi (Basel, Switzerland) Sep 2022Patients with chromoblastomycosis (CBM) suffer chronic tissue lesions that are hard to treat. Considering that biofilm is the main growth lifestyle of several pathogens...
Patients with chromoblastomycosis (CBM) suffer chronic tissue lesions that are hard to treat. Considering that biofilm is the main growth lifestyle of several pathogens and it is involved with both virulence and resistance to antimicrobial drugs, we have investigated the ability of CBM fungi to produce this complex, organized and multicellular structure. and conidial cells were able to adhere on a polystyrene abiotic substrate, differentiate into hyphae and produce a robust viable biomass containing extracellular matrix. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) showed the tridimensional architecture of the mature biofilms, revealing a dense network of interconnected hyphae, inner channels and amorphous extracellular polymeric material. Interestingly, the co-culture of each fungus with THP-1 macrophage cells, used as a biotic substrate, induced the formation of a mycelial trap covering and damaging the macrophages. In addition, the biofilm-forming cells of and were more resistant to the conventional antifungal drugs than the planktonic-growing conidial cells. The efflux pump activities of and biofilms were significantly higher than those measured in conidia. Taken together, the data pointed out the biofilm formation by CBM fungi and brought up a discussion of the relevance of studies about their antifungal resistance mechanisms.
PubMed: 36135688
DOI: 10.3390/jof8090963 -
Frontiers in Microbiology 2017is a dematiaceous fungus able to cause chromoblastomycosis, phaeohyphomycosis and mycetoma. All these fungal diseases are extremely difficult to treat and often...
is a dematiaceous fungus able to cause chromoblastomycosis, phaeohyphomycosis and mycetoma. All these fungal diseases are extremely difficult to treat and often refractory to the current therapeutic approaches. Therefore, there is an urgent necessity to develop new antifungal agents to combat these mycoses. In this context, the aim of the present work was to investigate the effect of 1,10-phenanthroline-5,6-dione (phendione) and its metal-based derivatives [Ag(phendione)]ClO = ([Ag(phendione)]) and [Cu(phendione)](ClO).4HO = ([Cu(phendione)]) on crucial physiological events of conidial cells. Using the CLSI protocol, we have shown that phendione, [Ag(phendione)] and [Cu(phendione)] were able to inhibit fungal proliferation, presenting MIC/IC values of 12.0/7.0, 4.0/2.4, and 5.0/1.8 μM, respectively. [Cu(phendione)] had fungicidal action and when combined with amphotericin B, both at sub-MIC (½ × MIC) concentrations, significantly reduced (~40%) the fungal growth. Cell morphology changes inflicted by phendione and its metal-based derivatives was corroborated by scanning electron microscopy, which revealed irreversible ultrastructural changes like surface invaginations, cell disruption and shrinkages. Furthermore, [Cu(phendione)] and [Ag(phendione)] were able to inhibit metallopeptidase activity secreted by conidia by approximately 85 and 40%, respectively. Ergosterol content was reduced (~50%) after the treatment of conidial cells with both phendione and [Ag(phendione)]. To different degrees, all of the test compounds were able to disturb the conidia-into-mycelia transformation. Phendione and its Ag and Cu complexes may represent a promising new group of antimicrobial agents effective at inhibiting growth and morphogenesis.
PubMed: 28194139
DOI: 10.3389/fmicb.2017.00076 -
International Biodeterioration &... 2017Water-damaged buildings can lead to fungal growth and occupant health problems. Green building materials, derived from renewable sources, are increasingly utilized in...
Water-damaged buildings can lead to fungal growth and occupant health problems. Green building materials, derived from renewable sources, are increasingly utilized in construction and renovations. However, the question as to what fungi will grow on these green compared to non-green materials, after they get wet, has not been adequately studied. By determining what fungi grow on each type of material, the potential health risks can be more adequately assessed. In this study, we inoculated green and non-green pieces of ceiling tile, composite board, drywall, and flooring with indoor dust containing a complex mixture of naturally occurring fungi. The materials were saturated with water and incubated for two months in a controlled environment. The resulting fungal microbiomes were evaluated using ITS amplicon sequencing. Overall, the richness and diversity of the mycobiomes on each pair of green and non-green pieces were not significantly different. However, different genera dominated on each type of material. For example, spp. had the highest relative abundance on green and non-green ceiling tiles and green composite boards, but spp. dominated the non-green composite board. In contrast, spp. dominated green and non-green flooring samples. Green gypsum board was dominated by spp. and spp., but non-green gypsum board by spp. These data suggest that water-damaged green and non-green building materials can result in mycobiomes that are dominated by fungal genera whose member species pose different potentials for health risks.
PubMed: 29681691
DOI: 10.1016/j.ibiod.2017.07.018 -
Journal of Clinical Immunology May 2024Autosomal recessive CARD9 deficiency can underly deep and superficial fungal diseases. We identified two Japanese patients, suffering from superficial and invasive...
Autosomal recessive CARD9 deficiency can underly deep and superficial fungal diseases. We identified two Japanese patients, suffering from superficial and invasive Candida albicans diseases, carrying biallelic variants of CARD9. Both patients, in addition to another Japanese and two Korean patients who were previously reported, carried the c.820dup CARD9 variant, either in the homozygous (two patients) or heterozygous (three patients) state. The other CARD9 alleles were c.104G > A, c.1534C > T and c.1558del. The c.820dup CARD9 variant has thus been reported, in the homozygous or heterozygous state, in patients originating from China, Japan, or South Korea. The Japanese, Korean, and Chinese patients share a 10 Kb haplotype encompassing the c.820dup CARD9 variant. This variant thus originates from a common ancestor, estimated to have lived less than 4,000 years ago. While phaeohyphomycosis caused by Phialophora spp. was common in the Chinese patients, none of the five patients in our study displayed Phialophora spp.-induced disease. This difference between Chinese and our patients probably results from environmental factors. (161/250).
Topics: Humans; CARD Signaling Adaptor Proteins; Founder Effect; Male; Female; Candidiasis, Chronic Mucocutaneous; Haplotypes; Mutation; Asia, Eastern; Alleles; Candida albicans; Adult; Pedigree; Asian People
PubMed: 38758287
DOI: 10.1007/s10875-024-01724-7 -
Fungal Biology Aug 2017Dark septate endophytes (DSEs) are melanin-enriched ascomycetous fungi that are abundant in stressed environments. However, little is known about their physiology and...
Dark septate endophytes (DSEs) are melanin-enriched ascomycetous fungi that are abundant in stressed environments. However, little is known about their physiology and metabolism, and DSE genes have not been functionally characterized yet. Therefore developing molecular genetic tools to investigate the biological function of genes of interest in DSEs is of major significance. We investigated Agrobacterium tumefaciens-mediated transformation (ATMT) efficiency in eight DSE strains belonging to Cadophora sp., Cadophora malorum, Leptodontidium sp., Phialophora mustea, and Cladosporium cladosporioides. ATMT efficiency was DSE-dependent and ranged from 0.6 to 125 %. We further focused on the effect of mycelium ageing on ATMT efficiency. Leptodontidium sp. Me07, Leptodontidium sp. Pr30, and C. cladosporioides CBS 101367 were significantly more transformed using 15-d-old mycelium (44.5, 6.9, and 1.1 %, respectively) as compared to 2-d-old mycelium (121, 28.7, and 25.1, respectively), whereas P. mustea Pr29 was more transformed using young mycelium (21.5 % compared to 5.3 % for the old mycelium). Finally, we focused on the effect of melanin content on ATMT efficiency. Melanin content in mycelium ranged from 0.9 to 3.2 mg g DW. Tricyclazole negatively modulated melanin content, while copper positively modulated it. However there was no correlation between hyphal melanin content and ATMT efficiency.
Topics: Agrobacterium tumefaciens; Ascomycota; Copper; Endophytes; Melanins; Thiazoles; Transformation, Genetic
PubMed: 28705394
DOI: 10.1016/j.funbio.2017.04.004 -
Veterinary Dermatology Oct 2022Phaeohyphomycosis was diagnosed in a 6-year-old, male castrated Dachshund on immunosuppressive treatment. The fungus was identified by culture and PCR as Phialophora...
Phaeohyphomycosis was diagnosed in a 6-year-old, male castrated Dachshund on immunosuppressive treatment. The fungus was identified by culture and PCR as Phialophora americana. This is the first reported case of infection with this pathogen in a dog. The infection was successfully managed medically, without surgical intervention.
Topics: Animals; Dog Diseases; Dogs; Male; Phaeohyphomycosis; Phialophora
PubMed: 35641851
DOI: 10.1111/vde.13096 -
Plant Disease Oct 2020Maize (Zea mays L.) is one of the most important commodities, and Brazil is the second-largest maize exporter country in the world. In April 2019, the period of the...
Maize (Zea mays L.) is one of the most important commodities, and Brazil is the second-largest maize exporter country in the world. In April 2019, the period of the second crop maize (safrinha), it was observed black decayed lesions on roots and wilting of some maize plants, causing a "sudden death" in a commercial area in the west of Paraná state, Brazil (Figure 1A-C). Symptomatic root and stalk were collected, and tissues surface disinfected with 70% ethanol for 30 s, 1.5% NaOCl for 1 min and rinsed three times in sterile distilled water, slices of necrotic tissues were transferred to potato dextrose agar (PDA) medium and grown for 7 days at 27 ± 1ºC with a photoperiod of 12 h. Pure cultures were obtained through monosporic isolation. The fungal morphology is alike Gaeumannomyces radicicola, which is a synonym of Phialophora radicicola var. radicicola, Harpophora radicicola, P. zeicola, H. zeicola and G. graminis var. maydis (Hernández-Restrepo et al. 2016). Colonies on PDA showed flat, white to light gray at first (Fig. 1D), turning gray to black with age (Fig. 1E). Colony diameter approximately 5.2 cm on PDA in the dark after 7 days at 27ºC. Conidiophores with slightly thickened wall, mostly branched, varying in dimensions, with a range of 57.5-166.5 (avg. 128.7 μm) × 2.9-5.9 (avg. 4.2 μm) n = 25 (Fig. 1H-J). The conidia showed lunate-shaped with rounded ends, produced successively at the apex of phialide, 3.3-9.7 (avg. 6.6 μm) × 1.5-3.6 μm (avg. 2.5 μm), n = 100 (Fig. 1G-J). Morphological characteristics were comparable to the description of this specie (Cain 1952; Gams 2000; McKeen 1952). The total genomic DNA of a representative isolate, LEMIDPRZm 19-01 was extracted and the partial large subunit (28S nrDNA; LSU), internal transcribed spacer nrDNA including the intervening 5.8S nrDNA (ITS), and part of the largest subunit of the RNA polymerase II gene (RPB1) were amplified and sequenced, as following by Hernández-Restrepo et al. (2016) and Klaubauf et al. (2014). The primers to LSU - NL1 (O'Donnel, 1993) and LR5 (Vilgalys; Hester, 1990); ITS - ITS5 and ITS4 (White et al., 1990); and RPB1 - RPB1F and RPB1R (Klaubauf et al., 2014) were used in this study. The gene sequences of LSU (MT123866), ITS (MT114427), and RPB1 (MT123867) were deposited in GenBank and showed 99.67%, 99.75%, and 100% identity with type material G. radicicola CBS 296.53 (KM484962, KM484845, and KM485061). A multi-locus phylogenetic analysis based on Bayesian Inference showed the isolate LEMIDPRZm 19-01 in the G. radicicola clade (Fig. 2). To confirm pathogenicity, ex vivo assays were performed with mycelial PDA discs of 5 mm from a 7-day-old culture using detached roots (adapted method by Degani et al., 2019), on wounded and unwounded stalk and leaves, each treatment consisted of five replications. PDA discs without fungal were used in negative tissue controls. Pathogenicity tests were also conducted in vivo, two experiments performed: i) the stalk tissue was inoculated by sterilized toothpick grown on PDA with fungal mycelium and the leaves inoculated as ex vivo assay, and toothpick without fungal mycelium was used to stalk negative control, whereas PDA discs without fungal were used in the tested leaves; ii) 6 mycelial PDA discs/500 mL were placed on potato dextrose broth (PDB) media and it remained in agitation for 10 days to obtain a mycelial suspension. Subsequently, the mycelial was crushed to soil infestation, and 50 mL from this suspension were dropped in each 2 L maize pot with soil sterilization 10 days after emergence. Maize pots with soil sterilization without mycelium fungal were used as negative controls. Four replications (maize pots), for each treatment, were used in both tests. Experiments were repeated twice. In the ex vivo assay, all inoculated tissues with and without wounds showed necrotic lesions (Fig. 1K-N). In the first in vivo assay, stalk rot symptoms, including wilting of the inoculated plants causing premature plant death, were observed within 6 days (Fig. 1O-Q). In the second in vivo assay, inoculated plants had inferior growth than compared with plant control. Sixty days after inoculation, the plants were removed from the pots and it was observed a roots degeneration with symptoms of necrosis (Fig. 1R-U). No symptoms were detected in the control treatments and the pathogen was re-isolated from symptomatic tissues confirming Koch's postulate for all assays. So far, to our knowledge, the pathogen distribution was reported solely in the west area of Paraná state, but it may become a potential threat to Brazilian maize production. Further monitoring is necessary to better understand the epidemiology of this pathogen to address a strategy for disease control. The pathogen has already been detected in Canada, South Africa, and China. To our knowledge, this is the first report of G. radicicola in Brazil, as well as in South America.
PubMed: 33026305
DOI: 10.1094/PDIS-03-20-0556-PDN -
FEMS Microbiology Letters Jul 2019Dark septate endophytes (DSEs) are widely distributed worldwide and can promote plant growth. Therefore, they are considered potentially important plant allies,...
Dark septate endophytes (DSEs) are widely distributed worldwide and can promote plant growth. Therefore, they are considered potentially important plant allies, especially in stressful environments. Previous studies have reported that DSEs cohabit roots with other microorganisms such as ectomycorrhizal (ECM), endophytic and pathogenic fungi/oomycetes. However, interactions between different DSE species have not yet been reported, and studies on the interactions between DSEs and other fungi are scarce. Using a simple and reproducible pairwise growth assay in vitro, we studied the synergistic/antagonistic interactions between eight DSEs, two ECM fungi and three root pathogens. Most of the DSE/DSE outcomes were neutral. Interestingly, we identified several DSE strains acting in synergy with other strains, as well as strains that could potentially act as biocontrol agents. Notably, three metal-tolerant DSE strains, namely, Cadophora sp., Leptodontidium sp. and Phialophora mustea, could decrease the growth of the root phytopathogens Pythium intermedium, Phytophthora citricola and Heterobasidion annosum. The present data are discussed in the general context of the use of fungal consortia as inocula in the tree-based phytomanagement of marginal lands.
Topics: Endophytes; Fungi; Mycorrhizae; Phenotype; Plant Roots; Symbiosis
PubMed: 31295350
DOI: 10.1093/femsle/fnz158