Successful treatment of pulmonary mucormycosis caused by Rhizopus microsporus with posaconazole.

BACKGROUND: Mucormycosis is a rare fungal infection occurring chiefly in the lung or the rhino-orbital-cerebral compartment, particularly in patients with immunodeficiency or diabetes mellitus. Among Mucorales fungi, Rhizopus spp. are the most common cause of mucormycosis. CASE PRESENTATION: We report a case of pulmonary mucormycosis caused by Rhizopus microsporus in a young patient with diabetes but no other apparent risk factors. The diagnosis mainly relied on clinical manifestation, positive pulmonary tissue biopsy, and fungal culture. The patient was successfully treated with posaconazole oral suspension and remains asymptomatic at one-year follow-up. CONCLUSIONS: Pulmonary mucormycosis is a life-threatening condition and posaconazole is an effective treatment for pulmonary mucormycosis caused by Rhizopus microspores.

Impairment of Th cell response in Card9 knockout mice with cutaneous mucormycosis caused by Rhizopus arrhizus.

Card9 is a signalling adaptor protein in the downstream of many innate pattern recognition receptors (PRRs) and exerts a significant role in antifungal immunity. To date, Card9 deficiency has been reported to be related to increased susceptibility to many fungal infections. In this study, we established mucormycosis murine model of Rhizopus arrhizus (R. arrhizus) using wild-type (WT) mice and Card9 knockout (Card9-/- ) mice to investigate the antifungal effect of Card9 against R. arrhizus infection. Card9-/- mice were more susceptible to R. arrhizus infection than WT mice, which could be related to the impaired NF-κB pathway activation, local cytokine production and Th cell responses in Card9-/- mice.

Effect of liquid-state fermentation on the antioxidant and functional properties of raw and roasted buckwheat flours.

The influence of liquid-state fermentation (LSF) by selected lactic acid bacteria (LAB) and Rhizopus oligosporus fungi on the content of rutin and total phenolic compounds (TPC), antioxidant capacity measured by ABTS test, FRAP assay and photochemiluminescence technique, and the inhibitory activity against formation of fluorescent advanced glycation end-products (AGEs) in vitro of raw and roasted buckwheat flours was studied. LSF caused a slight, specific LAB-dependent increase in TPC and a decrease in rutin content. Fermented raw buckwheat flours contained higher amounts of rutin and TPC with one exception when the highest increase in TPC was noted in roasted flour fermented by fungi. A LAB-dependent difference in the antioxidant capacity of buckwheat flours was noted while the inhibitory activity of fermented flours against AGEs formation was generally reduced. It can be concluded that LSF with selected LAB and fungi may improve the antioxidant and functional properties of buckwheat flours.

Iron restriction inside macrophages regulates pulmonary host defense against Rhizopus species.

Mucormycosis is a life-threatening respiratory fungal infection predominantly caused by Rhizopus species. Mucormycosis has incompletely understood pathogenesis, particularly how abnormalities in iron metabolism compromise immune responses. Here we show how, as opposed to other filamentous fungi, Rhizopus spp. establish intracellular persistence inside alveolar macrophages (AMs). Mechanistically, lack of intracellular swelling of Rhizopus conidia results in surface retention of melanin, which induces phagosome maturation arrest through inhibition of LC3-associated phagocytosis. Intracellular inhibition of Rhizopus is an important effector mechanism, as infection of immunocompetent mice with swollen conidia, which evade phagocytosis, results in acute lethality. Concordantly, AM depletion markedly increases susceptibility to mucormycosis. Host and pathogen transcriptomics, iron supplementation studies, and genetic manipulation of iron assimilation of fungal pathways demonstrate that iron restriction inside macrophages regulates immunity against Rhizopus. Our findings shed light on the pathogenetic mechanisms of mucormycosis and reveal the role of macrophage-mediated nutritional immunity against filamentous fungi.

Effect on tomato plant and fruit of the application of biopolymer-oregano essential oil coatings.

BACKGROUND: Oregano essential oil (EO) was incorporated into film-forming dispersions (FFDs) based on biopolymers (chitosan and/or methylcellulose) at two different concentrations. The effect of the application of the FFDs was evaluated on tomato plants (cultivar Micro-Tom) at three different stages of development, and on pre-harvest and postharvest applications on tomato fruit. RESULTS: The application of the FFDs at '3 Leaves' stage caused phytotoxic problems, which were lethal when the EO was applied without biopolymers. Even though plant growth and development were delayed, the total biomass and the crop yield were not affected by biopolymer-EO treatments. When the FFDs were applied in the 'Fruit' stage the pre-harvest application of FFDs had no negative effects. All FFDs containing EO significantly reduced the respiration rate of tomato fruit and diminished weight loss during storage. Moreover, biopolymer-EO FFDs led to a decrease in the fungal decay of tomato fruit inoculated with Rhizopus stolonifer spores, as compared with non-treated tomato fruit and those coated with FFDs without EO. CONCLUSION: The application of biopolymer-oregano essential oil coatings has been proven to be an effective treatment to control R. stolonifer in tomato fruit. © 2016 Society of Chemical Industry.

Mycelial Mattress from a Sporangia Formation-Delayed Mutant of Rhizopus stolonifer as Wound Healing-Enhancing Biomaterial.

A mycelial mattress of Rhizopus stolonifer obtained from a liquid static culture was utilized for wound dressing and biomedical use. Following screening of mutants induced by UV radiation, F6, exhibiting delayed sporangium formation was selected because its sporangium maturation exhibited a 5-day delay without significant loss of mycelial weight compared to the wild type. The sporangium-free mycelial mattress from the sporangiospore culture of F6 was treated with 1N sodium hydroxide NaOH at 85°C for 2 h to produce a sponge-like membrane named Rhizochitin. The trifluoroacetic acid hydrolysate of Rhizochitin contained 36% N-acetylglucosamine and 53% hexose respectively detected by the Elson-Morgen and phenol-sulfuric acid methods. Results indicated the wound area in rats covered with Rhizochitin was 40% less than that of the uncovered group. Rhizochitin decreased the expression of PDGF in the proliferation stage, increased the expression of TGF-ß in the inflammation and proliferation stages, and increased the expression of VEGF in the inflammation and proliferation stages. Rhizochitin inhibited secretion of matrix metalloproteinase-9 on days 1, 7, 9, and 12 and matrix metalloproteinase-2 on days 3, 7, 9, and 12. It was concluded that Rhizochitin has beneficial properties of biocompatible, biodegradable, and wound healing.

Adaptation to thermotolerance in Rhizopus coincides with virulence as revealed by avian and invertebrate infection models, phylogeny, physiological and metabolic flexibility.

Mucormycoses are fungal infections caused by the ancient Mucorales. They are rare, but increasingly reported. Predisposing conditions supporting and favoring mucormycoses in humans and animals include diabetic ketoacidosis, immunosuppression and haematological malignancies. However, comprehensive surveys to elucidate fungal virulence in ancient fungi are limited and so far focused on Lichtheimia and Mucor. The presented study focused on one of the most important causative agent of mucormycoses, the genus Rhizopus (Rhizopodaceae). All known clinically-relevant species are thermotolerant and are monophyletic. They are more virulent compared to non-clinically, mesophilic species. Although adaptation to elevated temperatures correlated with the virulence of the species, mesophilic strains showed also lower virulence in Galleria mellonella incubated at permissive temperatures indicating the existence of additional factors involved in the pathogenesis of clinical Rhizopus species. However, neither specific adaptation to nutritional requirements nor stress resistance correlated with virulence, supporting the idea that Mucorales are predominantly saprotrophs without a specific adaptation to warm blooded hosts.

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.

Clinical challenge: fatal mucormycotic osteomyelitis caused by Rhizopus microsporus despite aggressive multimodal treatment.

BACKGROUND: Mucormycosis is an invasive mycotic disease caused by fungi in the zygomycetes class. Although ubiquitous in the environment, zygomycetes are rarely known to cause invasive disease in immunocompromised hosts with a high mortality even under aggressive antifungal and surgical therapy. Clinically, mucormycosis frequently affects the sinus occasionally showing pulmonary or cerebral involvement. However skeletal manifestation with Rhizopus microsporus (RM) osteomyelitis leading to emergency surgical proximal femoral resection with fatal outcome has not been described yet. CASE PRESENTATION: We report the case of a 73-year-old male suffering from myelodysplastic syndrome with precedent bone marrow transplantation. Six months after transplantation he consulted our internal medicine department in a septic condition with a four week history of painful swelling of the right hip. Radiography, computed tomography and magnetic resonance imaging revealed multiple bone infarcts in both femurs. In the right femoral head, neck and trochanteric region a recent infarct showed massive secondary osteomyelitis, breaking through the medial cortex. Emergency surgical proximal femoral resection was performed due to extensive bone and soft tissue destruction. Microbiological and basic local alignment search tool (BLAST) analysis revealed RM. Amphotericin B and posaconazole treatment with septic revision surgery was performed. However the disease ran a rapid course and was fatal two months after hospital admission. CONCLUSION: This alarming result with extensive RM osteomyelitis in the proximal femur of an immunocompromised patient may hopefully warn medical staff to perform early imaging and aggressive surgical supported multimodal treatment in similar cases.

Can host receptors for fungi be targeted for treatment of fungal infections?

The invasion and stimulation of normally non-phagocytic host cells, such as epithelial and endothelial cells, is a key step in the pathogenesis of many fungal infections. In most cases, host cell invasion and/or stimulation of a proinflammatory response is induced when proteins or carbohydrates on the fungal cell surface bind to receptors on the host cell. Although many of these fungal-host cell interactions have only been investigated in vitro, the therapeutic efficacy of blocking the host cell receptors for Candida albicans and Rhizopus oryzae has been demonstrated in experimental animal models of infection. We summarize recent studies of the fungal receptors on normally non-phagocytic host cells and the therapeutic implications of blocking these receptors.

Rhizopus oryzae hyphae are damaged by human natural killer (NK) cells, but suppress NK cell mediated immunity.

Mucormycosis has a high mortality and is increasingly diagnosed in hematopoietic stem cell transplant (HSCT) recipients. In this setting, there is a growing interest to restore host defense to combat infections by adoptively transferring donor-derived immunocompetent cells. Natural killer (NK) cells exhibit antitumor and antiinfective activity, but the interaction with Mucormycetes is unknown. Our data demonstrate that both unstimulated and IL-2 prestimulated human NK cells damage Rhizopus oryzae hyphae, but do not affect resting conidia. The damage of the fungus is mediated, at least in part, by perforin. R. oryzae hyphae decrease the secretion of immunoregulatory molecules by NK cells, such as IFN-γ and RANTES, indicating an immunosuppressive effect of the fungus. Our data indicate that NK cells exhibit activity against Mucormycetes and future research should evaluate NK cells as a potential tool for adoptive immunotherapy in HSCT.

In vitro control of post-harvest fruit rot fungi by some plant essential oil components.

Eight substances that are main components of the essential oils from three Mediterranean aromatic plants (Verbena officinalis, Thymus vulgaris and Origanum vulgare), previously found active against some phytopathogenic Fungi and Stramenopila, have been tested in vitro against five etiological agents of post-harvest fruit decay, Botrytis cinerea, Penicillium italicum, P. expansum, Phytophthora citrophthora and Rhizopus stolonifer. The tested compounds were ß-fellandrene, ß-pinene, camphene, carvacrol, citral, o-cymene, γ-terpinene and thymol. Citral exhibited a fungicidal action against P. citrophthora; carvacrol and thymol showed a fungistatic activity against P. citrophthora and R. stolonifer. Citral and carvacrol at 250 ppm, and thymol at 150 and 250 ppm stopped the growth of B. cinerea. Moreover, thymol showed fungistatic and fungicidal action against P. italicum. Finally, the mycelium growth of P. expansum was inhibited in the presence of 250 ppm of thymol and carvacrol. These results represent an important step toward the goal to use some essential oils or their components as natural preservatives for fruits and foodstuffs, due to their safety for consumer healthy and positive effect on shelf life extension of agricultural fresh products.

Food grade fungal stress on germinating peanut seeds induced phytoalexins and enhanced polyphenolic antioxidants.

The effects of food grade fungus Rhizopus oligosporus stress on phytochemicals and phytoalexins of germinating peanut seeds were investigated by comparing the metabolic profiles of ungerminated (UG), germinated (G), and germinated seeds under fungal stress (GS). Three types of peanut seeds with different skin color (red, reddish brown, and black) were compared in the process. The polyphenolic contents were analyzed and correlated with antioxidant capacity for specific free radicals including peroxyl radical ROO(•) (ORAC), hydroxyl radical HO(•) (HORAC), superoxide radical O(2)(•-) (SORAC), and DPPH radical. The polyphenolic fingerprints analyzed by HPLC and LC-MS(n) showed that phenolic acids (coumaric, sinapinic, and ferulic acids derivatives) were the major group of phenolic compounds in ungerminated seeds. G or GS increased the level of phenolic acids, phytoalexins, and antioxidant capacity values in reddish and red peanuts but not in black peanuts. From the LC-MS(n) spectral data, 45 compounds were identified tentatively in the germinated peanuts, including 14 coumaric acids, 3 ferulic acids, 4 sinapinic acids, 2 hydroxybenzoic acids, 1 caffeic acid, 2 flavonoids, and 19 stilbenoids derivatives. Reddish brown germinated peanuts produced the highest amount of phytoalexins after GS with 55 compounds detected. Forty-five of these compounds were suggested as stilbenoid phytoalexins derivatives. The high content of phytoalexins may enhance the bioactivity of peanut seeds as functional food ingredients.

The effects of fungal stress on the antioxidant contents of black soybeans under germination.

Black soybeans were germinated with or without Rhizopus oligosporus for 3 days. The samples collected from each day were freeze-dried and extracted with chloroform-methanol-water mixture to simultaneously obtain organic phase (lipophilic extract) and aqueous phase (hydrophilic extract). In the lipophilic extract, α-, γ-, and δ-tocopherol were drastically decreased by 51%, 97%, and 88%, respectively, after 3 day germination under stress. On the other hand, the lipid peroxide concentration was increased in the fungi stressed, germinating beans from day 2 onward, while it was continually decreased in the nonstressed germinating counterpart. The change of antioxidant capacity (ORACoil value) was correlated to the changes of tocopherols and lipid peroxide concentrations. In nonstressed germinatng beans, ORACoil value dropped by 38% after 3 days, while in the fungi-stressed germinating beans, ORACoil was decreased by 80%. In hydrophilic extract, the antioxidant activity, measured by ORAChydro assay, is about 80 times higher than the ORACoil values, but there is no significant change before and after germination (either with or without stress). The total phenolic content in the hydrophilic extract increased only slightly by 9% in germinated beans and 15% in fungus-stress germinated samples, respectively. The advantage of stress germination of soybeans in enriching phytoalexins is traded off by loss of tocopherols.

[Perineural spread by fungal cells. Case report and literature review]. / Infiltración perineural por células fúngicas. Presentación de un caso y revisión de la literatura.

The perineural spread by fungal cells during the progression of the infection could be an important prognostic factor, especially in mycoses localized in the rhino-orbito-cerebral and pulmonary areas. We present a clinical case of a 73-year-old male, with diabetes and acute myelomonocytic leukaemia that began with tumefaction on the left side of his face, spreading to the sinus with invasion of the soft tissues and fistulae in the oral cavity. Clinical examination showed cerebral involvement. The histopathological sections and the histochemical techniques showed perineural involvement by fungal cells. Although the patient was diagnosed and treated in a short period of time, he died due to the infection. We would like to alert that perineural spread could be a retrograde way of mycoses dissemination, particularly in infections located in areas rich in neural cells.

Efficacy of liposomal amphotericin B combined with gamma interferon or granulocyte-macrophage colony-stimulating factor for treatment of systemic zygomycosis in mice.

Granulocyte-macrophage colony-stimulating factor enhanced the efficacy of liposomal amphotericin B (LAMB) in a murine model of disseminated infection by Rhizopus oryzae, significantly prolonging survival and reducing tissue burden. The use of gamma interferon (IFN-gamma) alone was ineffective, and IFN-gamma combined with LAMB did not improve the results obtained with LAMB alone.

Multidrug-resistant endosymbiotic bacteria account for the emergence of zygomycosis: A hypothesis.

Although the extensive use of Aspergillus-active antifungals has been recently associated with an increase in zygomycosis in several cancer centers, the frequency of this opportunistic mycosis began to rise earlier, since the mid 1990s. The reasons for that emergence are unclear. Recent evidence suggests that endosymbiotic bacteria of Rhizopus species produce toxins that enhance fungal pathogenicity. We postulate that, although Zygomycetes appear equally ubiquitous and virulent to Aspergillus, zygomycosis was rare in the past in immunosuppressed patients specifically because of the widespread use of antibacterials in this patient population. Such use may have resulted in inhibition of endosymbiotic, toxin-producing bacteria and led indirectly in attenuation of Zygomycetes virulence. Thus, the growing rates of antimicrobial resistance over the past decade selected for multidrug-resistant endosymbiotic bacteria of Zygomycetes, which could facilitate the emergence of zygomycosis. This hypothesis, if true, will be the first paradigm of modulation of virulence of opportunistic fungi by antibacterials.

Effect of individual amino acids and glucose on activation and germination of Rhizopus oligosporus sporangiospores in tempe starter.

AIM: To understand the conditions promoting activation and germination of spores, and to contribute to the control of tempe starters. METHODS AND RESULTS: Using microscopic counts of fluorescent labelled spores, the following results were obtained: (1) L-alanine plays an important role (of the same order as that of peptone) in stimulation of germination of dormant spores. Alanine can satisfy the requirements of carbon as well as nitrogen for spore germination; (2) L-proline, on the other hand, inhibits alanine uptake presumably by blocking/congesting transporters of spore cells, resulting in apparent low viability on agar media; (3) L-leucine and L-isoleucine slightly favour spore germination while L-arginine and L-lysine do not have any stimulating effect; (4) The stimulatory role of glucose was only evident in the presence of phosphate (in minimal medium); when glucose is used in the absence of phosphate, either alone or in combination with single amino acids its role is hardly distinguishable; (5) Phosphate plays a facilitating role in spore germination. CONCLUSIONS: Glucose and amino acids play important roles in activation and germination of sporangiospores of Rhizopus oligosporus in tempe starter (stored for 12 months). The ability and rate of germination of dormant/old sporangiospores of R. oligosporus, depend on their ability for uptake of individual amino acids and/or glucose. SIGNIFICANCE AND IMPACT OF STUDY: New light was shed on the counteractive role of proline and the stimulating effect of phosphate. Soybeans subjected to traditional preparation for tempe making are heavily leached; germination of starter spores on such beans is sub-optimal, and bean processing could be optimized.

Identification of the asexual state of Rhizopus species on histologic tissue sections in a patient with rhinocerebral mucormycosis.

Mucormycosis is an infection caused by a group of fungi in the order Mucorales in the phylum Zygomycota. The most well-known form of this disease is rhinocerebral mucormycosis, which usually develops in diabetic or immunocompromised patients. The fungal hyphal elements are easily detected in biopsy specimens by direct or histologic examination. However, the confirmatory identification of the genus or species requires culture of the specimen. This article presents a case of rhinocerebral mucormycosis in which presumptive identification of the genus was made without microbiologic cultures and was based on the extraordinarily rare appearance of fungal sporangia and sporangiospores in histologic tissue sections. Identification of these structures allowed an early and accurate diagnosis of rhinocerebral invasive mucormycosis.

[Nutritional evaluation of rice flour fermented with Rhizopus oligosporus]. / Avaliação nutricional de farinha de arroz fermentada com Rhizopus oligosporus.

In order to increase the proteic content of rice meal, fermentation with Rhizopus oligosporus was performed. During fermentation, samples were taken at the times of 0,20,30,40,50,60,70 and 80 hours. These samples were oven dried and further analysed. The amino acid composition of rice meal had lysine and threonine the most limiting ones. After fermentation the lysine content increase and the more limiting were the sulfur amino acids methionine and cystine (76.04%), threonine (91.03%) and lysine (97,04%. With the aim of verifying the biological value of the protein a bioassay was carried out. The fermented rice meal presented a higher digestibility value and the net protein utilization for the fermented rice meal lower than for the non fermented one.