In Vitro and In Vivo Interactions of TOR Inhibitor AZD8055 and Azoles against Pathogenic Fungi.

In the present study, in vitro and in vivo interactions of TOR inhibitor AZD8055 and azoles, including itraconazole, voriconazole, posaconazole and fluconazole, against a variety of pathogenic fungi were investigated. A total of 69 isolates were studied via broth microdilution checkerboard technique, including 23 isolates of Aspergillus spp., 20 isolates of Candida spp., 9 isolates of Cryptococcus neoformans complex, and 17 isolates of Exophiala dermatitidis. The results revealed that AZD8055 individually did not exert any significant antifungal activity. However, synergistic effects between AZD8055 and itraconazole, voriconazole or posaconazole were observed in 23 (33%), 13 (19%) and 57 (83%) isolates, respectively, including azole-resistant A. fumigatus strains and Candida spp., potentiating the efficacy of azoles. The combination effect of AZD8055 and fluconazole was investigated against non-auris Candida spp. and C. neoformans complex. Synergism between AZD8055 and fluconazole was observed in six strains (60%) of Candida spp., resulting in reversion of fluconazole resistance. Synergistic combinations resulted in 4-fold to 256-fold reduction of effective MICs of AZD8055 and azoles. No antagonism was observed. In vivo effects of AZD8055-azole combinations were evaluated by survival assay in Galleria mellonella model infected with A. fumigatus strain AF002, E. dermatitidis strain BMU00038, C. auris strain 383, C. albicans strain R15, and C. neoformans complex strain Z2. AZD8055 acted synergistically with azoles and significantly increased larvae survival (P < 0.05). In summary, the results suggested that AZD8055 combined with azoles may help to enhance the antifungal susceptibilities of azoles against pathogenic fungi and had the potential to overcome azole resistance issues. IMPORTANCE Limited options of antifungals and the emergence of drug resistance in fungal pathogens has been a multifaceted clinical challenge. Combination therapy represents a valuable alternative to antifungal monotherapy. The target of rapamycin (TOR), a conserved serine/threonine kinase from yeast to humans, participates in a signaling pathway that governs cell growth and proliferation in response to nutrient availability, growth factors, and environmental stimuli. AZD8055 is an orally bioavailable, potent, and selective TOR kinase inhibitor that binds to the ATP binding cleft of TOR kinase and inhibits both TORC1 and TORC2. Synergism between AZD8055 and azoles suggested that the concomitant application of AZD8055 and azoles may help to enhance azole therapeutic efficacy and impede azole resistance. TOR inhibitor with fungal specific target is promising to be served as combination regimen with azoles.

In vitro activities of 8 antifungal drugs against 126 clinical and environmental Exophiala isolates.

BACKGROUND: Exophiala is the main genus of black fungi comprising numerous opportunistic species. Data on antifungal susceptibility of Exophiala isolates are limited, while infections are potentially fatal. MATERIALS AND METHODS: In vitro activities of eight antifungal drugs (AMB, five azoles, two echinocandins) against 126 clinical (n = 76) and environmental (n = 47) isolates from around the world were investigated. E. oligosperma (n = 58), E. spinifera (n = 33), E. jeanselmei (n = 14) and E. xenobiotica (n = 21) were included in our dataset. RESULTS: The resulting MIC90 s of all strains were as follows, in increasing order: posaconazole 0.063 µg/ml, itraconazole 0.125 µg/ml, voriconazole and amphotericin B 1 µg/ml, isavuconazole 2 µg/ml, micafungin and caspofungin 4 µg/ml, and fluconazole 64 µg/ml. Posaconazole, itraconazole and micafungin were the drugs with the best overall activity against Exophiala species. Fluconazole could not be considered as a treatment choice. No significant difference could be found among antifungal drug activities between these four species, neither in clinical nor in environmental isolates. CONCLUSION: Antifungal susceptibility data for Exophiala spp. are crucial to improve the management of this occasionally fatal infection and the outcome of its treatment.

Dark-pigmented biodeteriogenic fungi in etruscan hypogeal tombs: New data on their culture-dependent diversity, favouring conditions, and resistance to biocidal treatments.

Subterranean Cultural Heritage sites are frequently subject to biological colonization due to the high levels of humidity, even in conditions of low irradiance and oligotrophy. Here microorganisms form complex communities that may be dangerous through mineral precipitation, through the softening of materials or causing frequent surface discolorations. A reduction of contamination's sources along with the control of microclimatic conditions and biocide treatments (overall performed with benzalkonium chloride) are necessary to reduce microbial growths. Dark discolorations have been recorded in the painted Etruscan tombs of Tarquinia, two of which have been analyzed to collect taxonomical, physiological, and ecological information. Eighteen dark-pigmented fungi were isolated among a wider culturable fraction: nine from blackening areas and nine from door sealings, a possible route of contamination. Isolates belonged to three major groups: Chaetothyriales, Capnodiales (Family Cladosporiaceae), and Acremonium-like fungi. Exophiala angulospora and Cyphellophora olivacea, a novelty for hypogea, were identified, while others need further investigations as possible new taxa. The metabolic skills of the detected species showed their potential dangerousness for the materials. Their tolerance to benzalkonium chloride-based products suggested a certain favouring effect through the decreasing competitiveness of less resistant species. The type of covering of the dromos may influence the risk of outer contamination. Fungal occurrence can be favoured by root penetration.

Melatonin confers heavy metal-induced tolerance by alleviating oxidative stress and reducing the heavy metal accumulation in Exophiala pisciphila, a dark septate endophyte (DSE).

BACKGROUND: Melatonin (MT), ubiquitous in almost all organisms, functions as a free radical scavenger. Despite several reports on its role as an antioxidant in animals, plants, and some microorganisms, extensive studies in filamentous fungi are limited. Based upon the role of melatonin as an antioxidant, we investigated its role in heavy metal-induced stress tolerance in Exophiala pisciphila, a dark septate endophyte (DSE), by studying the underlying mechanisms in alleviating oxidative stress and reducing heavy metal accumulation. RESULTS: A significant decrease in malondialdehyde (MDA) and oxygen free radical (OFR) in E. pisciphila was recorded under Cd, Zn, and Pb stresses as compared to the control. Pretreatment of E. pisciphila with 200.0 µM exogenous melatonin significantly increased the activity of superoxide dismutase (SOD) under Zn and Pb stresses. Pretreatment with 200.0 µM melatonin also lowered Cd, Zn, and Pb concentrations significantly. Melatonin production was enhanced by Cd, Cu, and Zn after 2 d, and melatonin biosynthetic enzyme genes, E. pisciphila tryptophan decarboxylase (EpTDC1) and serotonin N-acetyltransferase (EpSNAT1), were transcriptionally upregulated. The overexpression of EpTDC1 and N-acetylserotonin O-methyltransferase (EpASMT1) in Escherichia coli and Arabidopsis thaliana enhanced its heavy metal-induced stress tolerance. The overexpression of EpTDC1 and EpASMT1 reduced the Cd accumulation in the whole A. thaliana plants, especially in the roots. CONCLUSIONS: Melatonin conferred heavy metal-induced stress tolerance by alleviating oxidative stress, activating antioxidant enzyme SOD, and reducing heavy metal accumulation in E. pisciphila. Melatonin biosynthetic enzyme genes of E. pisciphila also played key roles in limiting excessive heavy metal accumulation in A. thaliana. These findings can be extended to understand the role of melatonin in other DSEs associated with economically important plants and help develop new strategies in sustainable agriculture practice where plants can grow in soils contaminated with heavy metals.

Chromoblastomycosis by Exophiala jeanselmei associated with squamous cell carcinoma.

Chromoblastomycosis is a subcutaneous, chronic, granulomatous mycosis that occurs more frequently in tropical and subtropical countries. Herein, we describe a case of a 90-year-old female patient with diagnosis of chromoblastomycosis by Exophiala jeanselmei with a 22-year evolution who developed a squamous cell carcinoma. In the meantime, She underwent two misdiagnoses and an unnecessary operation. This case is also the fifth case of E. jeanselmei caused CBM in history.

In vitro activity of olorofim (F901318) against fungi of the genus, Scedosporium and Rasamsonia as well as against Lomentospora prolificans, Exophiala dermatitidis and azole-resistant Aspergillus fumigatus.

In recent decades, invasive infections caused by fungal pathogens have been reported with increasing frequency. Concurrently, the rates of detected resistance mechanisms against commonly used antifungal agents in fungi are increasing. The need for novel antifungal drugs is thus imminent. In this study, the novel drug olorofim (F901318) was tested for its antifungal activity against the human fungal pathogens Lomentospora prolificans (n = 20), Scedosporium aurantiacum (n = 2), Scedosporium apiospermum (n = 6), Rasamsonia argillacea species complex (n = 23), Exophiala dermatitidis (n = 10) and azole-resistant Aspergillus fumigatus (ARAF) (n = 25) in an in vitro broth microdilution assay according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations. Whilst olorofim was ascertained to be effective against R. argillacea species complex [minimum inhibitory concentrations (MICs) of ≤0.008 mg/L], Scedosporium spp. (MICs of 0.032-0.5 mg/L), L. prolificans (MICs of 0.032-0.5 mg/L) and ARAF (MICs of ≤0.008-0.032 mg/L), the drug had an MIC of >4 mg/L against E. dermatitidis. These data demonstrate the antifungal activity of olorofim against a broad range of filamentous fungal pathogens.

A screening of the MMV Pathogen Box® reveals new potential antifungal drugs against the etiologic agents of chromoblastomycosis.

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by traumatic implantation of many species of black fungi. Due to the refractoriness of some cases and common recurrence of CBM, a more effective and less time-consuming treatment is mandatory. The aim of this study was to identify compounds with in vitro antifungal activity in the Pathogen Box® compound collection against different CBM agents. Synergism of these compounds with drugs currently used to treat CBM was also assessed. An initial screening of the drugs present in this collection at 1 µM was performed with a Fonsecaea pedrosoi clinical strain according to the EUCAST protocol. The compounds with activity against this fungus were also tested against other seven etiologic agents of CBM (Cladophialophora carrionii, Phialophora verrucosa, Exophiala jeanselmei, Exophiala dermatitidis, Fonsecaea monophora, Fonsecaea nubica, and Rhinocladiella similis) at concentrations ranging from 0.039 to 10 µM. The analysis of potential synergism of these compounds with itraconazole and terbinafine was performed by the checkerboard method. Eight compounds inhibited more than 60% of the F. pedrosoi growth: difenoconazole, bitertanol, iodoquinol, azoxystrobin, MMV688179, MMV021013, trifloxystrobin, and auranofin. Iodoquinol produced the lowest MIC values (1.25-2.5 µM) and MMV688179 showed MICs that were higher than all compounds tested (5 - >10 µM). When auranofin and itraconazole were tested in combination, a synergistic interaction (FICI = 0.37) was observed against the C. carrionii isolate. Toxicity analysis revealed that MMV021013 showed high selectivity indices (SI ≥ 10) against the fungi tested. In summary, auranofin, iodoquinol, and MMV021013 were identified as promising compounds to be tested in CBM models of infection.

Microbicidal activity of N-chlorotaurine can be enhanced in the presence of lung epithelial cells.

BACKGROUND: N-chlorotaurine (NCT) is an endogenous active chlorine compound that can be used as an antiseptic and anti-infective in different body regions. Recently, tolerability of inhaled NCT has been demonstrated in humans so that it is of interest for future treatment of cystic fibrosis. In the present study, we tested the bactericidal and fungicidal activity of NCT in different lung cell culture models. METHODS: Bacteria (Staphylococcus aureus, Pseudomonas aeruginosa) and fungi (Candida albicans, Exophiala dermatitidis) were co-incubated with lung epithelial cell cultures, and after 4 h NCT was added. After different incubation times, aliquots were removed and quantitative cultures were performed. RESULTS: NCT at the therapeutically applied concentration of 1% (55 mM) completely killed the test pathogens within 15 - 30 min at 20 °C and at 37 °C. Killing by 0.3% NCT lasted up to 4 h dependent on the pathogen at 20 °C and up to 1 h at 37 °C. 0.1% NCT was the threshold concentration for killing since this amount of oxidation capacity was consumed by reactions with the organic compounds of the medium within 3 h (20 °C) and 0.5 h (37 °C). CONCLUSIONS: NCT in therapeutic concentration demonstrated its microbicidal activity in the presence of lung epithelial cells. Remarkably, particularly the fungicidal activity was higher under these conditions than in phosphate buffer. This can be explained by formation of the stronger microbicidal monochloramine in equilibrium by transchlorination. The results suggest the suitability of NCT as inhalation medication in the lung.

Exophiala angulospora infection in hatchery-reared lumpfish (Cyclopterus lumpus) broodstock.

Samples from moribund lumpfish were collected in a marine hatchery in Scotland in 2015. Black nodules were noted on the skin, and gills and fungal hyphae were extensively distributed in musculature and internal organs. Multifocal chronic inflammatory lesions displaced structures in all affected organs. Mortalities commenced on completion of spawning in May and were evenly distributed over the second year in the temperature range 11-15°C. The main systemic infection causing agent was initially identified based on morphological characteristics as an Exophiala species. Ribosomal DNA (rDNA) ITS regions of the isolates were subsequently sequenced confirming the isolates belonged to Exophiala genus. All isolates fell in a single phylogenetic cluster, which is represented by Exophiala angulospora. Fish were treated with either formalin or Bronopol or a combination of both, but there was no effect on the pattern or numbers of mortalities. Isolates were also tested against three different concentrations of Latrunculin A, Amphotericin B and Itraconazole with no success. It is of utmost importance to increase the knowledge on pathogen-host interactions to successfully develop sustainable control methods.

Invasive Fungal Infection Caused by Exophiala dermatitidis in a Patient After Lung Transplantation: Case Report and Literature Review.

This report describes a case of invasive Exophiala dermatitidis infection after double lung transplantation in a 76-year-old man. After thoracotomy, the patient's wound showed dehiscence with purulent secretion. The black yeast was isolated from cultures taken from the wound, and species identification was confirmed by sequence analysis of the internal transcribed spacer (ITS-S2) region. The results of the susceptibility testing showed voriconazole as the most active drug. Despite adaptation of the antifungal therapy the clinical condition worsened, and the patient died. In addition, we evaluated the fungicidal activity of antiseptics towards E. dermatitidis and aimed to provide a brief literature review of previously reported infections caused by this rare fungus. To the best of our knowledge, this is the first report of a rapidly progressing invasive fungal infection with E. dermatitidis originating from a colonized wound after lung transplantation.

In vitro interactions between 17-AAG and azoles against Exophiala dermatitidis.

BACKGROUND: Exophiala dermatitidis causes a variety of illnesses in humans which are always refractory to available treatment modalities. Hsp90 governs crucial stress responses, cell wall repair mechanisms and antifungal resistance in pathogenic fungi. Thus, targeting Hsp90 with specific inhibitors holds considerable promise as combination strategy. OBJECTIVES: To investigate the antifungal effect of 17-AAG alone or combined with azoles against E. dermatitidis. METHODS: In vitro interactions of 17-AAG, a Hsp90 inhibitor, and azoles including itraconazole, voriconazole and posaconazole against E. dermatitidis were evaluated via broth microdilution chequerboard technique, adapted from the CLSI M38-A2 method. A total of 18 clinical strains were studied. Candida parapsilosis (ATCC22019) was included to ensure quality control. RESULTS AND CONCLUSIONS: 17-AAG alone exhibited minimal antifungal activity against all tested isolates. However, synergistic effects between 17-AAG and posaconazole, itraconazole or voriconazole were observed against 15 (83.3%), 12 (66.7%) and 1 (5.6%) isolates of E. dermatitidis, respectively. The effective working ranges of 17-AAG in synergistic combinations were mostly within 2-8 µg/mL. No antagonism was observed. In conclusion, harnessing fungal Hsp90 with 17-AAG might prove a potential antifungal regimen for E. dermatitidis infections. However, due to the host toxicity of 17-AAG, more efforts are needed to develop fungal specific Hsp90 inhibitors.

In vitro activities of antifungal drugs against environmental Exophiala isolates and review of the literature.

Exophiala is a genus of black fungi isolated worldwide from environmental and clinical specimens. Data on antifungal susceptibility of Exophiala isolates are limited and the methodology on susceptibility testing is not yet standardised. In this study, we investigated in vitro antifungal susceptibilities of environmental Exophiala isolates. A total of 87 Exophiala isolated from dishwashers or railway ties were included. A CLSI M38-A2 microdilution method with modifications was used to determine antifungal susceptibility for fluconazole, voriconazole, posaconazole, itraconazole, amphotericin B and terbinafine. Minimum inhibitory concentration (MIC) values were determined visually at 48 hours, 72 hours and 96 hours. MIC-0 endpoint (complete inhibition of growth) was used for amphotericin B and azoles, except fluconazole, for which MIC-2 endpoint (~50% inhibition compared to growth control) was used. Both MIC-0 and MIC-1 (~80% inhibition compared to growth control) results were analysed for terbinafine to enable comparison with previous studies. Fungal growth was sufficient for determination of MICs at 48 hours for all isolates except two Exophiala dermatitidis strains. At 72 hours, most active antifungal agents according to GM MIC were voriconazole and terbinafine, followed by posaconazole, itraconazole and amphotericin B in rank order of decreasing activity. While amphotericin B displayed adequate in vitro activity despite relatively high MICs, fluconazole showed no meaningful antifungal activity against Exophiala.

Combination of Amphotericin B and Terbinafine against Melanized Fungi Associated with Chromoblastomycosis.

Our in vitro studies showed that a combination of amphotericin B and terbinafine had synergistic effects against the majority of melanized fungi associated with chromoblastomycosis (CBM) and similar infections, including those with Cladophialophora carrionii, Cladophialophora arxii, Exophialadermatitidis, Exophialaspinifera, Fonsecaea monophora, Fonsecaea nubica, Fonsecaea pedrosoi, and Phialophora verrucosa. This drug combination could provide an option for the treatment of severe or unresponsive cases of CBM, particularly in cases due to species of Fonsecaea and Cladophialophora.

Lonafarnib synergizes with azoles against Aspergillus spp. and Exophiala spp.

Farnesylation, which is catalyzed by farnesyltransferase, promotes membrane association of the modified protein and protein-protein interactions, and plays an important role in a number of physiological processes of pathogenic fungi, including stress response, environmental adaption and virulence. Lonafarnib is an orally bioavailable nonpeptide tricyclic farnesyltransferase inhibitor with excellent pharmacokinetic and safety profile. In the present study, we investigated the in vitro activities of lonafarnib alone or combined with azoles, including itraconazole, voriconazole, and posaconazole, against 22 strains of Aspergillus spp. and 18 strains of Exophiala dermatitidis via broth microdilution checkerboard technique. Lonafarnib alone was inactive against all isolates tested. However, synergistic effects between lonafarnib and itraconazole were observed in 86% Aspergillus strains and 94% E. dermatitidis strains. In addition, lonafarnib/posaconazole combination also exhibited synergism against 59% of Aspergillus strains and 100% E. dermatitidis strains. However, synergistic effects of lonafarnib/voriconazole were only observed in 32% Aspergillus strains and 28% E. dermatitidis strains. The effective working ranges of lonafarnib were 2-4 µg/ml and 1-4 µg/ml against Aspergillus isolates and E. dermatitidis isolates, respectively. No antagonism was observed in all combinations. This study demonstrated that lonafarnib could enhance the in vitro antifungal activity of itraconazole, posaconazole and voriconazole against Aspergillus spp. and E. dermatitidis, suggesting that azoles, especially itraconazole and posaconazole, combined with farnesyltransferase inhibitor might provide a potential strategy to the management of Aspergillus and Exophiala infections. However, further studies are warranted to elucidate the underlying mechanism and to investigate the potential of reliable and safe application in clinical practice.

In vitro interactions between IAP antagonist AT406 and azoles against planktonic cells and biofilms of pathogenic fungi Candida albicans and Exophiala dermatitidis.

In vitro interactions of AT406, a novel IAP antagonist, and azoles including itraconazole, voriconazole, and fluconazole against planktonic cells and biofilms of Candida albicans and Exophiala dermatitidis were assessed via broth microdilution checkerboard technique. AT406 alone exhibited limited antifungal activity. However, synergistic effect between AT406 and fluconazole was observed against both planktonic cells and biofilms of C. albicans, including one fluconazole-resistant strain. Moreover, synergism was also demonstrated between AT406 and itraconazole against both planktonic cells and biofilms of E. dermatitidis. No interaction was observed between AT406 and voriconazole. No antagonism was observed in all combinations.

Management of an Outbreak of Exophiala dermatitidis Bloodstream Infections at an Outpatient Oncology Clinic.

We report the presentation and management of 17 cases of Exophiala dermatitidis and Rhodotorula mucilaginosa bloodstream infections caused by a compounded parenteral medication at an oncology clinic. Twelve patients were asymptomatic. All central venous catheters were removed and antifungal therapy, primarily voriconazole, was administered to patients. Three patients died.

Synergistic Effects of Tacrolimus and Azoles against Exophiala dermatitidis.

In vitro interactions of tacrolimus, a calcineurin inhibitor, and azoles, including itraconazole, voriconazole, and posaconazole, against planktonic cells and biofilms of Exophiala dermatitidis were assessed via a broth microdilution checkerboard technique. A total of 16 clinical isolates were studied. The results revealed favorable synergistic inhibitory activity between tacrolimus and itraconazole, voriconazole, or posaconazole against 68.8%, 87.5%, and 100% of tested strains of planktonic E. dermatitidis, respectively.However, limited synergism was observed against biofilms of E. dermatitidis No antagonism was observed in all combinations.