EGF-mediated suppression of cell extrusion during mucosal damage attenuates opportunistic fungal invasion.

Severe and often fatal opportunistic fungal infections arise frequently following mucosal damage caused by trauma or cytotoxic chemotherapy. Interaction of fungal pathogens with epithelial cells that comprise mucosae is a key early event associated with invasion, and, therefore, enhancing epithelial defense mechanisms may mitigate infection. Here, we establish a model of mold and yeast infection mediated by inducible epithelial cell loss in larval zebrafish. Epithelial cell loss by extrusion promotes exposure of laminin associated with increased fungal attachment, invasion, and larval lethality, whereas fungi defective in adherence or filamentation have reduced virulence. Transcriptional profiling identifies significant upregulation of the epidermal growth factor receptor ligand epigen (EPGN) upon mucosal damage. Treatment with recombinant human EPGN suppresses epithelial cell extrusion, leading to reduced fungal invasion and significantly enhanced survival. These data support the concept of augmenting epithelial restorative capacity to attenuate pathogenic invasion of fungi associated with human disease.

Antifungal effects of thymol and salicylic acid on cell membrane and mitochondria of Rhizopus stolonifer and their application in postharvest preservation of tomatoes.

This study investigated effects of the simultaneous application of thymol and salicylic acid (SIMTSA) on the target sites of Rhizopus stolonifer, as well as the defenceenzymes of postharvest tomato, when applied as edible coating. SIMTSA induced the changes of ultrastructure and membrane integrity of R. stolonifer. When the concentrations of the fungistat increased, cells stained with propidium iodide and leakage of 260/280 nm-absorbing materials increased while ergosterol synthesis decreased, suggesting damage of cell membrane. Furthermore, SIMTSA treatment significantly reduced the citric acid content and the activities of enzymes related to the tricarboxylic acid cycle, and increased the mitochondrial membrane potential and the reactive oxygen species, indicating damage of mitochondrial-related functions. Moreover, SIMTSA edible coating increased the defence enzyme activities in tomato. Based on the results, SIMTSA can be used as a potential preservation method for tomato as it showed a targeted effect on the cell membrane and mitochondria of R. stolonifer.

Primary Cutaneous Mucormycosis in an Extremely Preterm Infant Successfully Treated with Liposomal Amphotericin B.

Cutaneous mucormycosis is a rare but often fatal invasive fungal infection that occurs most commonly in patients with diabetes, malignancy, and other immunocompromising conditions. We report an extremely preterm (<28 weeks) baby boy who developed polymicrobial sepsis and primary cutaneous mucormycosis within his first 10 days of life. He was successfully treated with medical management alone since he was not a candidate for surgery. Successful treatment of cutaneous mucormycosis without surgical debridement has been reported on only two other occasions. This case highlights the importance of rapid and thorough evaluation of skin lesions when evaluating preterm infants and other immunocompromised patients, even when other sources of infection have been identified.

Using immunohistochemistry to assess the accuracy of histomorphologic diagnosis of aspergillosis and mucormycosis.

BACKGROUND: Data on the accuracy of conventional histomorphologic diagnosis are limited, especially in mucormycosis. We therefore investigated the accuracy of histomorphologic diagnosis of mucormycosis and aspergillosis, using immunohistochemistry (IHC) tests for mucormycosis and aspergillosis. METHODS: Patients enrolled met the modified criteria for proven and probable mucormycosis (during a 22-year period) or invasive aspergillosis (during a 6-year period) and had formalin-fixed, paraffin-embedded tissues available. We first tested the diagnostic performance of IHC for mucormycosis and aspergillosis in proven cases. Then we determined the accuracy of histomorphologic diagnosis of probable cases, using the IHC tests. RESULTS: In 7 proven cases of mucormycosis, the sensitivity and specificity of mucormycosis IHC were 100% (95% confidence interval, 65%-100%) and 100% (68%-100%), respectively. In 8 proven cases of aspergillosis, and the sensitivity and specificity of aspergillosis IHC staining were 87% (53%-98%) and 100% (65%-100%), respectively. Of 23 probable mucormycosis cases, 20 (87%) were positive with mucormycosis IHC, 2 (9%) were positive with aspergillosis IHC (including 1 positive for both), and 2 were negative with both. Of 16 probable aspergillosis cases, 10 (63%) were positive with aspergillosis IHC, 4 (25%) were positive with mucormycosis IHC, and 2 (13%) were negative with both tests. CONCLUSIONS: Aspergillosis and mucormycosis seem not to be correctly diagnosed morphologically, because some of the probable cases showed either test with both antibodies or failure to stain with the homologous antibody. In the absence of fungal culture results, the IHC tests seem helpful in differentiating between aspergillosis and mucormycosis.

Mechanism of Adsorptive Removal of Methylene Blue Using Dried Biomass of Rhizopus oryzae.

Adsorption is an efficient way to remove synthetic dyes from industrial effluent. Here, we show mechanism of adsorptive removal of cationic dye methylene blue (MB) from its aqueous solution using dried biomass of Rhizopus oryzae as a biosorbent. The optimum pH and temperature for adsorption was found to be 7.0 and 28 °C, respectively. Scanning electron microscopy (SEM) of the biomass suggested distinct changes in surface topology post-MB adsorption, while Fourier transform infrared (FTIR) study indicated chemical interaction between the surface of the biomass and MB. Chemical modification of -OH and -C=O groups of biomass reduced the MB adsorption and corroborated with the FTIR analyses. Kinetics study revealed that the adsorption rate was fast initially and reached equilibrium at 4 h following a pseudo-second-order-kinetics. The adsorption isotherm followed Freundlich isotherm model with n value of 1.1615.The dried biomass of R. oryzae can be used as a potent biosorbent for the removal of MB.

Iron starvation induces apoptosis in Rhizopus oryzae in vitro.

Mortality associated with mucormycosis remains high despite current antifungals. Iron-starvation strategies have been shown to have promising activity against Mucorales. We hypothesized that iron starvation enhances apoptosis in Rhizopus oryzae. Apoptosis was characterized in R. oryzae transformed with RNAi plasmid targeting FTR1 expression (iron permease mutant) or empty plasmid grown in iron rich (0.125% FeCl3) and iron depleted media (YNB+1mM ferrozine and 1 mM ascorbic acid). Increased apoptosis was observed with dihydrorhodamine-123 and rhodamine-123 staining in the iron starved mutant FTR1 when compared to empty plasmid, followed by increased extracellular ATP levels. In addition, DNA fragmentation and metacaspase activity were prominent in FTR1. In contrast, Rhizopus strains grown in iron-rich medium displayed minimal apoptosis. Our results demonstrate a metacaspase dependent apoptotic process in iron deprived condition and further support the role of iron starvation strategies as an adjunct treatment for mucormycosis, a mechanism by which iron starvation affects R. oryzae.

Active invasion of bacteria into living fungal cells.

The rice seedling blight fungus Rhizopus microsporus and its endosymbiont Burkholderia rhizoxinica form an unusual, highly specific alliance to produce the highly potent antimitotic phytotoxin rhizoxin. Yet, it has remained a riddle how bacteria invade the fungal cells. Genome mining for potential symbiosis factors and functional analyses revealed that a type 2 secretion system (T2SS) of the bacterial endosymbiont is required for the formation of the endosymbiosis. Comparative proteome analyses show that the T2SS releases chitinolytic enzymes (chitinase, chitosanase) and chitin-binding proteins. The genes responsible for chitinolytic proteins and T2SS components are highly expressed during infection. Through targeted gene knock-outs, sporulation assays and microscopic investigations we found that chitinase is essential for bacteria to enter hyphae. Unprecedented snapshots of the traceless bacterial intrusion were obtained using cryo-electron microscopy. Beyond unveiling the pivotal role of chitinolytic enzymes in the active invasion of a fungus by bacteria, these findings grant unprecedented insight into the fungal cell wall penetration and symbiosis formation.

Novel fungal consortium for bioremediation of metals and dyes from mixed waste stream.

The present study is targeted towards development of a three member fungal consortium for effective removal of metals [Cr(6+) and Cu(2+)] and dyes [AB and PO] from mixed waste streams. Initial studies using individual fungal strain showed that Aspergillus lentulus was best for Cu(2+) and AB removal, Aspergillus terreus for Cr(6+) removal whereas, Rhizopus oryzae was best for PO removal. Based on the complementary pollutant affinities and positive interactions, a consortium comprising all three strains was developed. Consortium removed 100% Cr(6+) and 81.60% Cu(2+) from metal mixture which was significantly higher than that achieved individually by A. lentulus (Cr(6+): 83.11%; Cu(2+): 67.32%), A. terreus (Cr(6+): 95.57%; Cu(2+): 65.77%) or R. oryzae (Cr(6+): 25.34%; Cu(2+): 30.20%). Further, 98.0% AB and 100.0% PO was removed after 48 h by the consortia. Unlike individual strains, consortium's performance was unaltered irrespective of the complexity of metal-dye mixtures, thereby establishing its superiority.

Effects of fengycin from Bacillus subtilis fmbJ on apoptosis and necrosis in Rhizopus stolonifer.

The lipopeptide antibiotic fengycin, produced by Bacillus subtilis, strongly inhibits growth of filamentous fungi. In this study, we evaluated the effects of fengycin treatment on apoptosis and necrosis in Rhizopus stolonifer by means of cell staining and epifluorescence microscopy. At fengycin concentrations less than 50 µg/ml, treated fungal cells demonstrated a dose-dependent increase in apoptosis-associated markers compared with the untreated control. These markers included chromatin condensation, reactive oxygen species accumulation, mitochondrial membrane potential depolarization, phosphatidylserine externalization, and the occurrence of DNA strand breaks. These results showed that fungal cells were impaired in a number of important functions and entered apoptosis upon treatment with low concentrations of fengycin. In contrast, high concentrations (>50 µg/ml) induced necrosis, indicating that the fungicidal action of fengycin operates via two modes: apoptosis at low concentrations and necrosis at high concentrations. Additionally, the apoptotic effect that we have shown suggests that lower concentrations of fengycin than previously thought may be effective for food preservation.

Effects of 7-hydroxycalamenene isolated from Croton cajucara essential oil on growth, lipid content and ultrastructural aspects of Rhizopus oryzae.

The leaves and bark of Croton cajucara, a shrub from the Amazon region, have been used in folk medicine to treat diabetes, malaria, and gastrointestinal and liver disorders. The essential oil from the leaves, rich in linalool, presented antileishmanial and antimicrobial activities. A chemotype of this species was found with an essential oil rich in 7-hydroxycalamenene. During our studies of the C. cajucara essential oil, we isolated 7-hydroxycalamenene at > 98 % purity. The minimum inhibitory concentration of 7-hydroxycalamenene against Absidia cylindrospora, Cunninghamella elegans, Mucor circinelloides, Mucor circinelloides f. circinelloides, Mucor mucedo, Mucor plumbeus, Mucor ramosissimus, Rhizopus microsporus, Rhizopus oryzae, and Syncephalastrum racemosum ranged from 19.53 to 2500 µg/mL. The reference drug used, amphotericin B, presented a minimum inhibitory concentration ranging from 0.085 µg/mL to 43.87 µg/mL. 7-Hydroxycalamenene also altered spore differentiation and total lipid content. Ultrastructural analysis by transmission electron microscopy showed significant alterations in the cellular structure of R. oryzae.

Preparation of immobilized whole cell biocatalyst and biodiesel production using a packed-bed bioreactor.

Rhizopus oryzae NBRC 4697 was selected from among promising candidates as a biocatalyst for biodiesel production. This microorganism was immobilized on to polyurethane foam coated with activated carbon for reuse, and, for biodiesel production. Vacuum drying of the immobilized cells was found to be more efficient than natural or freeze-drying processes. Although the immobilized cells were severely inhibited by a molar ratio of methanol to soybean oil in excess of 2.0, stepwise methanol addition (3 aliquots at 24-h feeding intervals) significantly prevented methanol inhibition. A packed-bed bioreactor (PBB) containing the immobilized whole cell biocatalyst was then operated under circulating batch mode. Stepwise methanol feeding was used to mitigate methanol inhibition of the immobilized cells in the PBB. An increase in the feeding rate (circulating rate) of the reaction mixture barely affected biodiesel production, while an increase in the packing volume of the immobilized cells enhanced biodiesel production noticeably. Finally, repeated circulating batch operation of the PBB was carried out for five consecutive rounds without a noticeable decrease in the performance of the PBB for the three rounds.

[Cutaneous mucormycosis caused by Rhizopus microsporus]. / Mucormycose cutanée à Rhizopus microsporus.

BACKGROUND: Mucormycosis are rare fungal infections occurring chiefly in the lung or the rhinocerebral compartment, particularly in patients with immunodeficiency or mellitus diabetes. We report the case of an elderly patient with cutaneous mucormycosis caused by Rhizopus microsporus. PATIENTS AND METHODS: An 89-year-old man presented a skin lesion of the forearm rapidly becoming inflammatory and necrotic. The patient had been treated for 2months with oral corticosteroids for idiopathic thrombocytopenia. Histological and mycological examination of the skin biopsy revealed the presence of a filamentous fungus, R. microsporus. The outcome was unfavorable, despite prescription of high-dose liposomal amphotericin B. DISCUSSION: Mucormycosis are infrequent opportunistic infections caused by angio-invasive fungi belonging to the Mucorales order. Cutaneous presentations are rare, and in rare cases the species R. microsporus is isolated in clinical samples. Diagnosis is based on histological examination highlighting the characteristic mycelium within infected tissue, together with ex vivo mycological identification using morphological and molecular methods. Treatment consists of liposomal amphotericin B combined with debridement surgery. CONCLUSION: R. microsporus is a marginal fungal species rarely isolated in clinical practice, and even less in dermatology departments. This clinical case report highlights the severity of infection with this fungus, particularly in the absence of early surgery.

Enhanced fumaric acid production from brewery wastewater and insight into the morphology of Rhizopus oryzae 1526.

The present work explores brewery wastewater as a novel substrate for fumaric acid production employing the filamentous fungal strain Rhizopus oryzae 1526 through submerged fermentation. The effects of different parameters such as substrate total solid concentrations, fermentation pH, incubation temperature, flask shaking speed, and inoculum size on the fungal morphologies were investigated. Different morphological forms (mycelium clumps, suspended mycelium, and solid/hairy pellets) of R. oryzae 1526 were obtained at different applied fermentation pH, incubation temperature, flask shaking speed, and inoculum size. Among all the obtained morphologies, pellet morphology was found to be the most favorable for enhanced production of fumaric acid for different studied parameters. Scanning electron microscopic investigation was done to reveal the detailed morphologies of the pellets formed under all optimized conditions. With all the optimized growth conditions (pH 6, 25 °C, 200 rpm, 5% (v/v) inoculum size, 25 g/L total solid concentration, and pellet diameter of 0.465 ± 0.04 mm), the highest concentration of fumaric acid achieved was 31.3 ± 2.77 g/L. The results demonstrated that brewery wastewater could be used as a good substrate for the fungal strain R. oryzae 1526 in submerged fermentation for the production of fumaric acid.

Biomineralization mechanism of gold by zygomycete fungi Rhizopus oryzae.

In recent years, there has been significant progress in the biological synthesis of nanomaterials. However, the molecular mechanism of gold biomineralization in microorganisms of industrial relevance remains largely unexplored. Here we describe the biosynthesis mechanism of gold nanoparticles (AuNPs) in the fungus Rhizopus oryzae . Reduction of AuCl(4)(-) [Au(III)] to nanoparticulate Au(0) (AuNPs) occurs in both the cell wall and cytoplasmic region of R. oryzae . The average size of the as-synthesized AuNPs is ~15 nm. The biomineralization occurs through adsorption, initial reduction to Au(I), followed by complexation [Au(I) complexes], and final reduction to Au(0). Subtoxic concentrations (up to 130 µM) of AuCl(4)(-) in the growth medium increase growth of R. oryzae and induce two stress response proteins while simultaneously down-regulating two other proteins. The induction increases mycelial growth, protein yield, and AuNP biosynthesis. At higher Au(III) concentrations (>130 µM), both mycelial and protein yield decrease and damages to the cellular ultrastructure are observed, likely due to the toxic effect of Au(III). Protein profile analysis also confirms the gold toxicity on R. oryzae at high concentrations. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis shows that two proteins of 45 and 42 kDa participate in gold reduction, while an 80 kDa protein serves as a capping agent in AuNP biosynthesis.

Microbial enhance of chitosan production by Rhizopus arrhizus using agroindustrial substrates.

This study investigated the potential of Rhizopus arrhizus UCP 402 for producing chitosan using corn steep liquor and honey as agroindustrial nitrogen and carbon sources. A complete factorial design was used to assess the improved biomass and chitosan production. The results were evaluated using Pareto charts (Statistica 7.0 software). The chitosan obtained was characterized by X-ray diffraction. The cristallinity index (I(C)), and infrared spectroscopy (FTIR) were used to evaluate the degree of deacetylation (DD %). The morphological aspects of the R. arrhizus were evaluated by measuring the diameter of the colonies by light microscopy. The results obtained showed higher biomass and chitosan yields (20.61 g/L and 29.3 mg/g), respectively, in the selected assays. The characterization of the macromolecular arrangement of chitosan showed a crystallinity index compatible with the literature, and the infrared peaks confirmed a degree of 86%. The experimental data obtained suggest that adding honey to corn steep liquor is a promising way to improve microbiological chitosan production.

A combination of heat treatment and Pichia guilliermondii prevents cherry tomato spoilage by fungi.

This study investigated the effectiveness of heat treatment and Pichia guilliermondii, either alone or in combination, to combat postharvest fungal spoilage in cherry tomato fruit. In vitro experiments demonstrated that heat treatment at 38 degrees C significantly inhibited mycelial growth of three different pathogens (Botrytis cinerea, Alternaria alternata and Rhizopus stolonifer Ehrenb). In vivo experiments unveiled that either heat treatment or P. guilliermondii reduced decay caused by these pathogens. Furthermore, a combination of heat treatment followed by the application of P. guilliermondii (H+P) provided the best efficacy in prevention of cherry tomato from fungal spoilage. Following, H+P treatment, electronic nose detected a reduction of volatility in cherry tomato fruit odor, an indicator of preserving fruit's freshness. Scanning electron microscopy unveiled that heat treatment at 38 degrees C for 24h inhibited hyphae growth and spore germination of R. stolonifer Ehrenb while P. guilliermondii multiplied rapidly on fruit wounds, and its cells had a strong capability of adhesion to the hyphae of R. stolonifer Ehrenb. However, heat treatment also seriously injured P. guilliermondii, therefore P. guilliermondii should be applied after heat treatment. A combination of heat treatment and P. guilliermondii is one of the most effective techniques at controlling postharvest fungal spoilage in cherry tomato fruit.

Antifungal activity of colistin against mucorales species in vitro and in a murine model of Rhizopus oryzae pulmonary infection.

In immunosuppressed hosts, mucormycosis is a life-threatening infection with few treatment options. We studied the activity of colistin (polymyxin E) against Mucorales species in vitro and in a murine model of pulmonary Rhizopus oryzae infection. Colistin exhibited fungicidal activity in vitro against Mucorales spores and mycelia. At the colistin MIC, initial R. oryzae hyphal damage was followed by rapid regrowth; however, regrowth was prevented by combining colistin with a subinhibitory concentration of amphotericin B. Using electron microscopy and FM4-64 staining, we demonstrated that colistin disrupts R. oryzae cytoplasmic and vacuolar membranes, resulting in the leakage of intracellular contents. The prophylactic intranasal treatment of immunosuppressed mice with colistimethate significantly reduced the mortality rate and pulmonary fungal burden resulting from inhalational challenge with R. oryzae spores, whereas intraperitoneal colistimethate treatment had no effect. We conclude that colistin has modest in vitro and in vivo fungicidal activity against Mucorales spp. Further studies are warranted to assess the use of this drug in the prevention and treatment of mucormycosis.

Determination of glucosamine and N-acetyl glucosamine in fungal cell walls.

A new method was developed to determine glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) in materials containing chitin and chitosan, such as fungal cell walls. It is based on two steps of hydrolysis with (i) concentrated sulfuric acid at low temperature and (ii) dilute sulfuric acid at high temperature, followed by one-step degradation with nitrous acid. In this process, chitin and chitosan are converted into anhydromannose and acetic acid. Anhydromannose represents the sum of GlcN and GlcNAc, whereas acetic acid is a marker for GlcNAc only. The method showed recovery of 90.1% of chitin and 85.7-92.4% of chitosan from commercial preparations. Furthermore, alkali insoluble material (AIM) from biomass of three strains of zygomycetes, Rhizopus oryzae, Mucor indicus, and Rhizomucor pusillus, was analyzed by this method. The glucosamine contents of AIM from R. oryzae and M. indicus were almost constant (41.7 +/- 2.2% and 42.0 +/- 1.7%, respectively), while in R. pusillus, it decreased from 40.0 to 30.0% during cultivation from 1 to 6 days. The GlcNAc content of AIM from R. oryzae and R. pusillus increased from 24.9 to 31.0% and from 36.3 to 50.8%, respectively, in 6 days, while it remained almost constant during the cultivation of M. indicus (23.5 +/- 0.8%).

Use of Rhizopus oligosporus produced from food processing wastewater as a biosorbent for Cu(II) ions removal from the aqueous solutions.

Dried biomass of Rhizopus oligosporus produced from food processing wastewater was used as an adsorbent for copper ions in water. The adsorption process was carried out in a batch process and the effects of contact time (1-48 h), initial pH (2.0-6.0), initial metal ion concentration (20-100 mg L(-1)) and temperature (20-38 degrees C) on the adsorption were investigated. Experimental results showed that the maximum adsorption capacity was achieved at pH 5.0 and adsorbed Cu(II) ion concentration was increased with increasing initial metal concentration and contact time. The isothermal data could be described well by the Langmuir equations and monolayer capacity had a mean value of 79.37 mg g(-1). A pseudo-second order reaction model provided the best description of the data with a correlation coefficient 0.99 for different initial metal concentrations. Thermodynamic parameters indicated that biosorption of Cu(II) on R. oligosporus dried biomass was exothermic and spontaneous. To observe the copper pellets on the biosorbent surface after biosorption SEM was used and copper was characterized by EDX. The results of FTIR analyses indicated that amide I and hydroxyl groups of adsorbent played important role in binding Cu(II).

Burkholderia rhizoxinica sp. nov. and Burkholderia endofungorum sp. nov., bacterial endosymbionts of the plant-pathogenic fungus Rhizopus microsporus.

Several strains of the fungus Rhizopus microsporus harbour endosymbiotic bacteria for the production of the causal agent of rice seedling blight, rhizoxin, and the toxic cyclopeptide rhizonin. R. microsporus and isolated endobacteria were selected for freeze-fracture electron microscopy, which allowed visualization of bacterial cells within the fungal cytosol by their two parallel-running envelope membranes and by the fine structure of the lipopolysaccharide layer of the outer membrane. Two representatives of bacterial endosymbionts were chosen for phylogenetic analyses on the basis of full 16S rRNA gene sequences, which revealed that the novel fungal endosymbionts formed a monophyletic group within the genus Burkholderia. Inter-sequence similarities ranged from 98.94 to 100%, and sequence similarities to members of the Burkholderia pseudomallei group, the closest neighbours, were 96.74-97.38%. In addition, the bacterial strains were distinguished from their phylogenetic neighbours by their fatty acid profiles and other biochemical characteristics. The phylogenetic studies based on 16S rRNA gene sequence data, together with conclusive DNA-DNA reassociation experiments, strongly support the proposal that these strains represent two novel species within the genus Burkholderia, for which the names Burkholderia rhizoxinica sp. nov. (type strain, HKI 454T=DSM 19002T=CIP 109453T) and Burkholderia endofungorum sp. nov. (type strain, HKI 456T=DSM 19003T=CIP 109454T) are proposed.