A Unique Dual-Readout High-Throughput Screening Assay To Identify Antifungal Compounds with Aspergillus fumigatus.

Treatment of invasive mold infections is limited by the lack of adequate drug options that are effective against these fatal infections. High-throughput screening of molds using traditional antifungal assays of growth is problematic and has greatly limited our ability to identify new mold-active agents. Here, we present a high-throughput screening platform for use with Aspergillus fumigatus, the most common causative agent of invasive mold infections, for the discovery of novel mold-active antifungals. This assay detects cell lysis through the release of the cytosolic enzyme adenylate kinase and, thus, is not dependent on changes in biomass or metabolism to detect antifungal activity. The ability to specifically detect cell lysis is a unique aspect of this assay that allows identification of molecules that disrupt fungal cell integrity, such as cell wall-active molecules. We also found that germinating A. fumigatus conidia release low levels of adenylate kinase and that a reduction in this background allowed us to identify molecules that inhibit conidial germination, expanding the potential for discovery of novel antifungal compounds. Here, we describe the validation of this assay and proof-of-concept pilot screens that identified a novel antifungal compound, PIK-75, that disrupts cell wall integrity. This screening assay provides a novel platform for high-throughput screens with A. fumigatus for the identification of anti-mold drugs. IMPORTANCE Fungal infections caused by molds have the highest mortality rates of human fungal infections. These devastating infections are hard to treat and available antifungal drugs are often not effective. Therefore, the identification of new antifungal drugs with mold activity is critical. Drug screening with molds is challenging and there are limited assays available to identify new antifungal compounds directly with these organisms. Here, we present an assay suitable for use for high-throughput screening with a common mold pathogen. This assay has exciting future potential for the identification of new drugs to treat these fatal infections.

The Role of Zinc in Copper Homeostasis of Aspergillus fumigatus.

Copper is an essential metal ion that performs many physiological functions in living organisms. Deletion of Afmac1, which is a copper-responsive transcriptional activator in A. fumigatus, results in a growth defect on aspergillus minimal medium (AMM). Interestingly, we found that zinc starvation suppressed the growth defect of the Δafmac1 strain on AMM. In addition, the growth defect of the Δafmac1 strain was recovered by copper supplementation or introduction of the CtrC gene into the Δafmac1 strain. However, chelation of copper by addition of BCS to AMM failed to recover the growth defect of the Δafmac1 strain. Through Northern blot analysis, we found that zinc starvation upregulated CtrC and CtrA2, which encode membrane copper transporters. Interestingly, we found that the conserved ZafA binding motif 5'-CAA(G)GGT-3' was present in the upstream region of CtrC and CtrA2 and that mutation of the binding motif led to failure of ZafA binding to the upstream region of CtrC and upregulation of CtrC expression under zinc starvation. Furthermore, the binding activity of ZafA to the upstream region of CtrC was inversely proportional to the zinc concentration, and copper inhibited the binding of ZafA to the upstream region of CtrC under a low zinc concentration. Taken together, these results suggest that ZafA upregulates copper metabolism by binding to the ZafA binding motif in the CtrC promoter region under low zinc concentration, thus regulating copper homeostasis. Furthermore, we found that copper and zinc interact in cells to maintain metal homeostasis.

Nerolidol inhibits the LOX-1 / IL-1ß signaling to protect against the Aspergillus fumigatus keratitis inflammation damage to the cornea.

PURPOSE: Nerolidol, a naturally occurring sesquiterpene has both anti-microbial and anti-inflammatory properties. The current study aims to investigate the antifungal and the anti-inflammatory effects of nerolidol against mouse Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: The minimum inhibitory concentration (MIC) and cytotoxicity tests were used to study the antifungal ability. For in vivo and in vitro studies, the mouse corneas and the human corneal epithelial cells (HCECs) infected with A. fumigatus spores were intervented with nerolidol or phosphate buffer saline (PBS). Thereafter, the effect of the nerolidol on the response against inflammation was analyzed using the following parameters: recruitment of the neutrophils or macrophages and the expression of the lectin-type oxidized low density lipoprotein receptor-1 (LOX-1) and interleukin 1ß (IL-1ß). Techniques used were the slit lamp, immunofluorescence, myeloperoxidase (MPO) detection, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: Nerolidol directly inhibits the growth of A. fumigatus. The administration of nerolidol reduced the severity of fungal keratitis with infiltration of fewer inflammatory cells and reduced levels of the LOX-1, as well the anti-inflammatory cytokines such as IL-1ß were reduced compared with the PBS group. Additionally, in vitro studies showed that treatment with nerolidol inhibited the production of the LOX-1 / IL-1ß levels in A. fumigatus stimulated HCECs. CONCLUSION: Nerolidol attenuated the A. fumigatus keratitis inflammatory response by inhibiting the growth of A. fumigatus, reducing the recruitment of the neutrophils and the macrophages, and inhibiting the LOX-1/ IL-1ß signaling.

Influences of carbohydrate, nitrogen, and phosphorus sources on the citric acid production by fungal endophyte Aspergillus fumigatus P3I6.

Fungi are ecologically ubiquitous organisms on earth and regarded as one of the prolific sources of natural products. Fungal endophytes may provide essential prerequisite molecules to plant biochemical pathways which allow the efficient synthesis of primary and secondary metabolites. This study characterized the influences of various combinations of process parameters namely, carbohydrate, nitrogen, and phosphorus sources on citric acid (CA) production by the isolated fungal endophyte Aspergillus fumigatus P3I6 from Citrus microcarpa. Aspergillus fumigatus P3I6 had higher CA concentration of 9.2 (± 0.9) g L-1 and 9.0 (± 5.0 × 10-15) g L-1 when supplemented with sucrose and white refined sugar, respectively, than A. niger NRRL 599. Response Surface Methodology (RSM) had shown that A. fumigatus P3I6 produced the highest CA (23.8 g L-1) in Combination 4 (18.0% sucrose, 0.3 g L-1 ammonium sulfate, and 5.0 g L-1 dipotassium phosphate (K2HPO4)). Analysis of variance showed that when K2HPO4 concentrations were increased, CA content in fermentation media was significantly elevated. Hence, K2HPO4 was the most critical variable in the quadratic model (p < 0.05); however, sucrose concentration still has its role in production. Aside from using A. niger in most fermentation processes, this discovered fungal strain can be potentially used in biotechnological applications.

Fungicide-driven alterations in azole-resistant Aspergillus fumigatus are related to vegetable crops in Colombia, South America.

Aspergillus fumigatus resistant to azole as first-line therapy has been reported in azole-naïve patients. This worldwide resistance phenomenon has been linked to fungicide-driven alterations in the cyp51A gene and its promoter region (such as TR34/L98H and TR46/Y121F/T289A). Azole-resistant A. fumigatus related to the use of triazole fungicides in flower fields was recently reported In Colombia. The purpose of this study was to investigate the presence of azole-resistant A. fumigatus in soil samples from vegetable crops such as carrots, potatoes, maize, strawberries, and pea, and from prepared farming land surrounding the city of Bogotá. Species identification was based on sequencing of the ß-tubulin and calmodulin genes. All A. fumigatus strains were screened for azole resistance on agar supplemented with itraconazole or voriconazole. Among the 60 soil samples, 34 (56.6%) were positive for A. fumigatus and 15 samples exhibited strains (n = 18) that grew on agar supplemented with itraconazole or voriconazole. Triazole-resistant strains were isolated from soil samples associated with carrot, potato, maize, and pea crops. Sequencing of the cyp51A gene and its promoter region indicated polymorphism, mainly with the presence of TR46/Y121F/T289A (n = 8), TR34/L98H, and TR53. Eight resistant isolates exhibited cyp51A wild type without alterations in the promoter region. Our study showed evidence of dissemination of azole-resistant A. fumigatus, with high genetic diversity, in vegetable crops in Colombia. These data underline the need to determine the prevalence of azole resistance in A. fumigatus in clinical and environmental settings for other regions of Colombia as well as Latin America.

Antifungal efficacy of photodynamic therapy with TONS 504 for pathogenic filamentous fungi.

The pathogenic filamentous fungi Fusarium solani (F. solani) and Aspergillus fumigatus (A. fumigatus) are common causes of fungal keratitis. We have here evaluated the antifungal efficacy of photodynamic antimicrobial chemotherapy (PACT) with the novel chlorin derivative TONS 504 and a light-emitting diode (LED) with a wavelength of 660 nm for these fungal species. Isolated fungal spores were irradiated at LED energies of 10, 20, or 30 J/cm2 in the presence of TONS 504 at concentrations of 1 or 10 mg/L. As a control, spores were exposed to TONS 504 or LED radiation alone. The treated spores were then cultured on potato dextrose agar plates at 25 °C for 3 to 4 days before determination of colony formation as a measure of viability. Fungal growth was inhibited in a manner dependent on both LED energy and TONS 504 concentration. The inhibitory effect on F. solani was complete with TONS 504 at a concentration of 1 mg/L and LED irradiation at 30 J/cm2 as well as at a TONS 504 concentration of 10 mg/L and LED irradiation at 10, 20, or 30 J/cm2. In contrast, that on A. fumigatus was only partial at a TONS 504 concentration of 10 mg/L and LED irradiation at 20 or 30 J/cm2. The antifungal effect of PACT on A. fumigatus was thus inferior to that on F. solani. PACT with TONS 504 and an LED thus warrants further evaluation with regard to its potential effectiveness for the treatment of infectious fungal keratitis.

Alexidine Dihydrochloride Has Broad-Spectrum Activities against Diverse Fungal Pathogens.

Invasive fungal infections due to Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans constitute a substantial threat to hospitalized immunocompromised patients. Further, the presence of drug-recalcitrant biofilms on medical devices and emergence of drug-resistant fungi, such as Candida auris, introduce treatment challenges with current antifungal drugs. Worse, currently there is no approved drug capable of obviating preformed biofilms, which increase the chance of infection relapses. Here, we screened a small-molecule New Prestwick Chemical Library, consisting of 1,200 FDA-approved off-patent drugs against C. albicans, C. auris, and A. fumigatus, to identify those that inhibit growth of all three pathogens. Inhibitors were further prioritized for their potency against other fungal pathogens and their ability to kill preformed biofilms. Our studies identified the bis-biguanide alexidine dihydrochloride (AXD) as a drug with the highest antifungal and antibiofilm activity against a diverse range of fungal pathogens. Finally, AXD significantly potentiated the efficacy of fluconazole against biofilms, displayed low mammalian cell toxicity, and eradicated biofilms growing in mouse central venous catheters in vivo, highlighting its potential as a pan-antifungal drug.IMPORTANCE The prevalence of fungal infections has seen a rise in the past decades due to advances in modern medicine leading to an expanding population of device-associated and immunocompromised patients. Furthermore, the spectrum of pathogenic fungi has changed, with the emergence of multidrug-resistant strains such as C. auris High mortality related to fungal infections points to major limitations of current antifungal therapy and an unmet need for new antifungal drugs. We screened a library of repurposed FDA-approved inhibitors to identify compounds with activities against a diverse range of fungi in varied phases of growth. The assays identified alexidine dihydrochloride (AXD) to have pronounced antifungal activity, including against preformed biofilms, at concentrations lower than mammalian cell toxicity. AXD potentiated the activity of fluconazole and amphotericin B against Candida biofilms in vitro and prevented biofilm growth in vivo Thus, AXD has the potential to be developed as a pan-antifungal, antibiofilm drug.

The antifungal activity of extracts of Osmundea pinnatifida, an edible seaweed, indicates its usage as a safe environmental fungicide or as a food additive preventing post-harvest fungal food contamination.

In the present work, we explored the antifungal activity of the wild edible seaweed Osmundea pinnatifida (Rhodophyta) collected from the Portuguese coast, which is used as a food seasoning in Scotland, Ireland and Portugal. We performed a sequential extraction of the seaweed components with methanol, dichloromethane and n-hexane. These extracts showed an antifungal activity against Alternaria infectoria and Aspergillus fumigatus. The n-hexane fraction of the seaweed inhibited the sporulation of Alternaria infectoria at 30 µg mL-1 and induced a statistically significant (P < 0.001) decrease in ß-glucan content. Furthermore, liquid cultures of Aspergillus fumigatus supplemented with 10 µg mL-1 of the n-hexane fraction showed abnormal conidiophores, completely devoid of phialides and conidia associated with a decrease of 18.3% in the chitin content (P < 0.01). The n-hexane fraction analysis by GC-MS revealed that it includes palmitic acid (29.6%), phytol isomer 1 (12.8%), oleic acid (9.6%), stearic acid (6.2%) and d-(-)-tagatofuranose (4.1%), among other compounds present at lower concentrations. The present study reveals Osmundea pinnatifida as a promising source of biologically active compounds inhibiting fungal growth and conidiation, the main dispersal mechanism of filamentous fungi as Aspergillus fumigatus and Alternaria alternata, revealing its utility both as an environmental fungicide against fungal diseases and as a food preservative against fungal post-harvest food contamination.

Seeing the light: Shifting from wild rhizomes to extraction of active ingredients from above-ground parts of Paris polyphylla var. yunnanensis.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried rhizomes of Paris polyphylla var. yunnanensis are widely used in traditional Chinese medicine (TCM) as hemostatic, antitumor, and antimicrobial agents. More than 70 Chinese patent medicines are based on P. polyphylla var. yunnanensis rhizomes. Steroidal saponins are considered as the main active ingredients of these rhizomes. However, wild populations of P. polyphylla var. yunnanensis are greatly threatened due to the illegal wild harvest and over-utilization of the rhizomes. In contrast, the renewable above-ground parts (leaves and stems) of P. polyphylla var. yunnanensis are usually thrown away as waste material, whether from wild or cultivated material. AIM OF THE STUDY: The aim of this study was to use HPLC analyses of chemical constituents and bioactive assays to assess whether the above-ground parts could be an alternative source of active ingredients to the rhizomes of P. polyphylla var. yunnanensis. MATERIALS AND METHODS: The saponin components of the rhizomes and above-ground parts of P. polyphylla var. yunnanensis were analyzed by HPLC-UV. The total saponins extracted from the rhizomes and above-ground parts of P. polyphylla var. yunnanensis were evaluated for their hemostatic, cytotoxic, and antimicrobial activities by using the rabbit blood in vitro based on turbidimetric method, MTT assay method, and a dilution antimicrobial susceptibility test method, respectively. RESULTS: Four bioactive spirostanol saponins (paris saponins I, II, VI, and VII) were detected in the total saponins from the rhizomes and above-ground parts of P. polyphylla var. yunnanensis, which indicated they should have similar pharmacological properties. The bioactive assays revealed that both the parts of P. polyphylla var. yunnanensis exhibited the same hemostatic, cytotoxic, and antimicrobial effects. CONCLUSION: Our results revealed that based on saponin content in the above-ground parts of P. polyphylla var. yunnanensis and the requirements stipulated in 2015 of Chinese Pharmacopoeia, the above-ground parts (especially its leaves) can be an alternative and more sustainable source of active ingredients compared to the rhizomes.

Isolation and characterization of antibiotic producing bacterial strains from red soil of Himalayan region of Pakistan.

The emergence of multi drug resistant microbial pathogens has become a global health challenge and set a dire requirement of searching new effective antimicrobials. Soil is an ultimate reservoir of biologically active micro flora, which harbors trillions of microbial strains producing compounds of commercial interest. Hence aim of the present study was an attempt to isolate and identify the antibiotic producing microbial strains from the red soil of Himalayan an unexplored region of Pakistan. In this study from 10 different soil samples only one bacterial strain was isolated capable of antimicrobial activity. Strain was identified by biochemical characteristics and final identification was done by API 20 NE kit which showed 99% homology with P. aeruginosa. Hence the strain was identified as P. aeruginosa S2. Antibacterial and antifungal activity of the P. aeruginosa S2 showed that Staphylococcus aureus was extremely sensitive to it with a zone of inhibition of 42mm. Staphylococcus epidermidis, Enterobacter aerogenes, Aspergillus fumigatus and Candida albicans were also inhibited by the isolated strain. Effect of Glycerol, Copper sulphate (CuSo4), Sodium sulphate (Na2SO4) and Glycerol on antibiotic production was also evaluated by supplementing growth media with these chemicals. Pseudomonas aeruginosa was grown in bulk quantity using solid state fermentation and crude extract was prepared using organic solvents and subjected to silica gel column chromatography for purification of active compound. Purified compound showed antibacterial against human pathogens. The unexplored Kashmir Himalayas are of great significance because of its richness in biodiversity and need to be explored for isolation and characterization of native microbes for biologically active secondary metabolites. This untouched region may be considered as hub of new antimicrobials and may have applications in natural product-based drug discovery.

Application of a low molecular weight antifungal protein from Penicillium chrysogenum (PAF) to treat pulmonary aspergillosis in mice.

PAF, a small antifungal protein from Penicillium chrysogenum, inhibits the growth of several pathogenic filamentous fungi, including members of the Aspergillus genus. PAF has been proven to have no toxic effects in vivo in mice by intranasal application. To test its efficacy against invasive pulmonary aspergillosis (IPA), experiments were carried out in mice suffering from IPA. Adult mice were immunosuppressed and then infected with Aspergillus fumigatus. After stable infection, the animals were inoculated with PAF intranasally at a concentration of 2.7 mg/kg twice per day. At this concentration-which is highly toxic in vitro to A. fumigatus-the mortality of the animals was slightly delayed but finally all animals died. Histological examinations revealed massive fungal infections in the lungs of both PAF-treated and untreated animal groups. Because intranasally administered PAF was unable to overcome IPA, modified and combined therapies were introduced. The intraperitoneal application of PAF in animals with IPA prolonged the survival of the animals only 1 day. Similar results were obtained with amphotericin B (AMB), with PAF and AMB being equally effective. Combined therapy with AMB and PAF-which are synergistic in vitro-was found to be more effective than either AMB or PAF treatment alone. As no toxic effects of PAF in mammals have been described thus far, and, moreover, there are so far no A. fumigatus strains with reported inherent or acquired PAF resistance, it is worth carrying out further studies to introduce PAF as a potential antifungal drug in human therapy.

Pharmacodynamics of Voriconazole in Children: Further Steps along the Path to True Individualized Therapy.

Voriconazole is the agent of choice for the treatment of invasive aspergillosis in children at least 2 years of age. The galactomannan index is a routinely used diagnostic marker for invasive aspergillosis and can be useful for following the clinical response to antifungal treatment. The aim of this study was to develop a pharmacokinetic-pharmacodynamic (PK-PD) mathematical model that links the pharmacokinetics of voriconazole with the galactomannan readout in children. Twelve children receiving voriconazole for treatment of proven, probable, and possible invasive fungal infections were studied. A previously published population PK model was used as the Bayesian prior. The PK-PD model was used to estimate the average area under the concentration-time curve (AUC) in each patient and the resultant galactomannan-time profile. The relationship between the ratio of the AUC to the concentration of voriconazole that induced half maximal killing (AUC/EC50) and the terminal galactomannan level was determined. The voriconazole concentration-time and galactomannan-time profiles were both highly variable. Despite this variability, the fit of the PK-PD model was good, enabling both the pharmacokinetics and pharmacodynamics to be described in individual children. (AUC/EC50)/15.4 predicted terminal galactomannan (P= 0.003), and a ratio of >6 suggested a lower terminal galactomannan level (P= 0.07). The construction of linked PK-PD models is the first step in developing control software that enables not only individualized voriconazole dosages but also individualized concentration targets to achieve suppression of galactomannan levels in a timely and optimally precise manner. Controlling galactomannan levels is a first critical step to maximizing clinical response and survival.

Mitochondrial dynamics in the pathogenic mold Aspergillus fumigatus: therapeutic and evolutionary implications.

Mitochondria within eukaryotic cells continuously fuse and divide. This phenomenon is called mitochondrial dynamics and crucial for mitochondrial function and integrity. We performed a comprehensive analysis of mitochondrial dynamics in the pathogenic mold Aspergillus fumigatus. Phenotypic characterization of respective mutants revealed the general essentiality of mitochondrial fusion for mitochondrial genome maintenance and the mold's viability. Surprisingly, it turned out that the mitochondrial rhomboid protease Pcp1 and its processing product, s-Mgm,1 which are crucial for fusion in yeast, are dispensable for fusion, mtDNA maintenance and viability in A. fumigatus. In contrast, mitochondrial fission mutants show drastically reduced growth and sporulation rates and increased heat susceptibility. However, reliable inheritance of mitochondria to newly formed conidia is ensured. Strikingly, mitochondrial fission mutants show a significant and growth condition-dependent increase in azole resistance. Parallel disruption of fusion in a fission mutant partially rescues growth and sporulation defects and further increases the azole resistance phenotype. Taken together, our results indicate an emerging dispensability of the mitochondrial rhomboid protease function in mitochondrial fusion, the suitability of mitochondrial fusion machinery as antifungal target and the involvement of mitochondrial dynamics in azole susceptibility.

Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies.

Treatment of Aspergillus fumigatus with echinocandins such as caspofungin inhibits the synthesis of cell wall ß-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2 and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca(2+)-calcineurin signaling pathways. A. fumigatus mutants with the chs gene (encoding chitin synthase) deleted (ΔAfchs) were tested for their response to these agonists to determine the chitin synthase enzymes that were required for the compensatory upregulation of chitin synthesis. Only the ΔAfchsG mutant was hypersensitive to caspofungin, and all other ΔAfchs mutants tested remained capable of increasing their chitin content in response to treatment with CaCl2 and CFW and caspofungin. The resulting increase in cell wall chitin content correlated with reduced susceptibility to caspofungin in the wild type and all ΔAfchs mutants tested, with the exception of the ΔAfchsG mutant, which remained sensitive to caspofungin. In vitro exposure to the chitin synthase inhibitor, nikkomycin Z, along with caspofungin demonstrated synergistic efficacy that was again AfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of caspofungin. In addition, intrahyphal hyphae were formed in response to echinocandin treatment. These in vitro data demonstrate that A. fumigatus has the potential to survive echinocandin treatment in vivo by AfChsG-dependent upregulation of chitin synthesis. Chitin-rich cells may, therefore, persist in human tissues and act as the focus for breakthrough infections.

Carum copticum and Thymus vulgaris oils inhibit virulence in Trichophyton rubrum and Aspergillus spp.

Emergence of drug-resistant strains has demanded for alternative means of combating fungal infections. Oils of Carum copticum and Thymus vulgaris have long been used in ethnomedicine for ailments of various fungal infections. Since their activity has not been reported in particular against drug-resistant fungi, this study was aimed to evaluate the effects of oils of C. copticum and T. vulgaris on the growth and virulence of drug-resistant strains of Aspergillus spp. and Trichophyton rubrum. The gas chromatography-mass spectrometry analysis revealed thymol constituting 44.71% and 22.82% of T. vulgaris and C. copticum, respectively. Inhibition of mycelial growth by essential oils was recorded in the order of thymol > T. vulgaris > C. copticum against the tested strains. RBC lysis assay showed no tested oils to be toxic even up to concentration two folds higher than their respective MFCs. Thymol exhibited highest synergy in combination with fluconazole against Aspergillus fumigatus MTCC2550 (FICI value 0.187) and T. rubrum IOA9 (0.156) as determined by checkerboard method. Thymol and T. vulgaris essential oil were equally effective against both the macro and arthroconidia growth (MIC 72 µg/mL). A > 80% reduction in elastase activity was recorded for A. fumigatus MTCC2550 by C. copticum, T. vulgaris oils and thymol. The effectiveness of these oils against arthroconidia and synergistic interaction of thymol and T. vulgaris with fluconazole can be exploited to potentiate the antifungal effects of fluconazole against drug-resistant strains of T. rubrum and Aspergillus spp.

Carum copticum and Thymus vulgaris oils inhibit virulence in Trichophyton rubrum and Aspergillus spp

Emergence of drug-resistant strains has demanded for alternative means of combating fungal infections. Oils of Carum copticum and Thymus vulgaris have long been used in ethnomedicine for ailments of various fungal infections. Since their activity has not been reported in particular against drug-resistant fungi, this study was aimed to evaluate the effects of oils of C. copticum and T. vulgaris on the growth and virulence of drug-resistant strains of Aspergillus spp. and Trichophyton rubrum. The gas chromatography-mass spectrometry analysis revealed thymol constituting 44.71% and 22.82% of T. vulgaris and C. copticum, respectively. Inhibition of mycelial growth by essential oils was recorded in the order of thymol > T. vulgaris > C. copticum against the tested strains. RBC lysis assay showed no tested oils to be toxic even up to concentration two folds higher than their respective MFCs. Thymol exhibited highest synergy in combination with fluconazole against Aspergillus fumigatus MTCC2550 (FICI value 0.187) and T. rubrum IOA9 (0.156) as determined by checkerboard method. Thymol and T. vulgaris essential oil were equally effective against both the macro and arthroconidia growth (MIC 72 µg/mL). A > 80% reduction in elastase activity was recorded for A. fumigatus MTCC2550 by C. copticum, T. vulgaris oils and thymol. The effectiveness of these oils against arthroconidia and synergistic interaction of thymol and T. vulgaris with fluconazole can be exploited to potentiate the antifungal effects of fluconazole against drug-resistant strains of T. rubrum and Aspergillus spp.

Steam-treatment-based soil remediation promotes heat-tolerant, potentially pathogenic microbiota.

We investigated microbiota in surface and subsurface soil from a site, above steam-treated deep sub-soil originally contaminated with chlorinated solvents. During the steam treatment, the surface soil reached temperatures c. 30 degrees C higher than the temperature in untreated soil; whereas the subsurface soil, at a depth of about 40 cm, reached a temperature c. 45 degrees C higher than untreated soil. The soil was examined prior to, during, and 6, 12, 14, 20 and 31 months after treatment. Numbers of bacteria cultivable at 42 degrees C increased significantly in subsurface soil. Similarly, substrate utilization in ECOLOG plates, incubated at 42 degrees C, increased from less than 10% of available carbon sources in the untreated soil to more than 60% of the available carbon sources in the steam-treated soil. Aspergillus fumigatus was quantified as an example ofheat-tolerant fungi normally found in compost. These organisms are rarely detected in Danish soils but high numbers (c. 10(5) hyphal forming units g(-1)) occurred in the treated soil up to 31 months after the steam-treatment. We conclude that steam-treatment leads to changes of the microbial communities. Some changes are temporary while others can last for years after termination of the steam-treatment; reflecting different strategies that soil microorganisms follow.

Effect of physical and chemical properties of oil palm empty fruit bunch, decanter cake and sago pith residue on cellulases production by Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2.

The effect of cultivation condition of two locally isolated ascomycetes strains namely Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 were compared in submerged and solid state fermentation. Physical evaluation on water absorption index, solubility index and chemical properties of lignin, hemicellulose and cellulose content as well as the cellulose structure on crystallinity and amorphous region of treated oil palm empty fruit bunch (OPEFB) (resulted in partial removal of lignin), sago pith residues (SPR) and oil palm decanter cake towards cellulases production were determined. Submerged fermentation shows significant cellulases production for both strains in all types of substrates. Crystallinity of cellulose and its chemical composition mainly holocellulose components was found to significantly affect the total cellulase synthesis in submerged fermentation as the higher crystallinity index, and holocellulose composition will increase cellulase production. Treated OPEFB apparently induced the total cellulases from T. asperellum UPM1 and A. fumigatus UPM2 with 0.66 U/mg FPase, 53.79 U/mg CMCase, 0.92 U/mg ß-glucosidase and 0.67 U/mg FPase, 47.56 U/mg and 0.14 U/mg ß-glucosidase, respectively. Physical properties of water absorption and solubility for OPEFB and SPR also had shown significant correlation on the cellulases production.

Impact of in vivo triazole and echinocandin combination therapy for invasive pulmonary aspergillosis: enhanced efficacy against Cyp51 mutant isolates.

Previous studies examining combination therapy for invasive pulmonary aspergillosis (IPA) have revealed conflicting results, including antagonism, indifference, and enhanced effects. The most commonly employed combination for this infection includes a mold-active triazole and echinocandin. Few studies have evaluated combination therapy from a pharmacodynamic (PD) perspective, and even fewer have examined combination therapy against both wild-type and azole-resistant Cyp51 mutant isolates. The current studies aim to fill this gap in knowledge. Four Aspergillus fumigatus isolates were utilized, including a wild-type strain, an Fks1 mutant (posaconazole susceptible and caspofungin resistant), and two Cyp51 mutants (posaconazole resistant). A neutropenic murine model of IPA was used for the treatment studies. The dosing design included monotherapy with posaconazole, monotherapy with caspofungin, and combination therapy with both. Efficacy was determined using quantitative PCR, and results were normalized to known quantities of conidia (conidial equivalents [CE]). The static dose, 1-log kill dose, and associated PD target area under the curve (AUC)/MIC ratio were determined for monotherapy and combination therapy. Monotherapy experiments revealed potent activity for posaconazole, with reductions of 3 to 4 log10 Aspergillus CE/ml with the two "low"-MIC isolates. Posaconazole alone was less effective for the two isolates with higher MICs. Caspofungin monotherapy did not produce a significant decrease in fungal burden for any strain. Combination therapy with the two antifungals did not enhance efficacy for the two posaconazole-susceptible isolates. However, the drug combination produced synergistic activity against both posaconazole-resistant isolates. Specifically, the combination resulted in a 1- to 2-log10 decline in burden that would not have been predicted based on the monotherapy results for each drug. This corresponded to a reduction in the free-drug posaconazole AUC/MIC ratio needed for stasis of up to 17-fold. The data suggest that combination therapy using a triazole and an echinocandin may be a beneficial treatment strategy for triazole-resistant isolates.