Infección por Aspergillus flavus y Rhizopus oryzae complex en paciente con diabetes mellitus / Infection due to Aspergillus flavus and Rhizopus oryzae complex in a patient with diabetes mellitus

La sinusitis micótica es una condición patológica que puede presentarse en pacientes con diabetes mellitus y estar asociada a una crisis hiperglucémica. Es una entidad agresiva con complicaciones locales que incluyen afectación de la órbita y el sistema nervioso central, y compromiso vascular. A pesar del tratamiento quirúrgico y antimicótico, la mortalidad es de hasta el 75 %. Se describe el caso de una paciente con diagnóstico de cetoacidosis diabética y signos de oftalmoplejía unilateral que llevaron al estudio con resonancia magnética del sistema nervioso central; se encontraron signos de sinusitis, meningitis y cerebritis. Los estudios microbiológicos iniciales fueron negativos, y los biomarcadores galactomanano sérico y el antígeno de Cryptococcus también fueron negativos. Tras el manejo quirúrgico, se llegó a la identificación de Aspergillus flavus y Rhizopus spp. en el tejido de los senos paranasales. La paciente recibió tratamiento con posaconazol y, tras dos meses de seguimiento, había presentado mejoría clínica. La infección fúngica dual y la infección por A. flavus son entidades poco frecuentes y de relevancia clínica, sin casos presentados previamente en nuestro país por lo que este corresponde a un caso de interés clínico.

Fungal sinusitis is a pathology that can occur in patients with diabetes mellitus and be associated with a hyperglycemic crisis. It is an aggressive entity with local complications that include involvement of the orbit or the central nervous system, and vascular involvement. Despite surgical and antifungal treatment, mortality raises up to 75%. We report the case of a female patient with a diagnosis of diabetic ketoacidosis and signs of unilateral ophthalmoplegia, which led to the study with magnetic resonance imaging of the central nervous system, finding signs of sinusitis, meningitis, and cerebritis. Initial microbiological studies were negative, and biomarkers such as serum galactomannan and Cryptococcus antigen were also negative. After surgical management and the identification of Aspergillus flavus and Rhizopus spp. in sinus tissue, the patient received treatment with posaconazole and after two months of follow-up she presented clinical improvement. Dual fungal infection and infection by A. flavus are uncommon and clinically relevant entities, with no cases previously reported in our country, therefore this corresponds to a case of clinical interest.

Local strains Aspergillus oryzae KKB4 and Rhizopus oryzae KP1R1 as a reducing and detoxifying agents for deoxynivalenol

Aims@#Deoxynivalenol is a type B trichothecene produced by Fusarium graminearum that can cause serious health problems in human and livestock. The present study aimed to reduce and detoxify deoxynivalenol using a local strain Aspergillus oryzae KKB4 and Rhizopus oryzae KP1R1. @*Methodology and results@#Corn as solid substrate artificially inoculated with F. graminearum bio 163252 to produce deoxynivalenol. Deoxynivalenol contaminated corn then inoculated with A. oryzae KKB4 and R. oryzae KP1R1. During fermentation, a decrease in deoxynivalenol levels is analyzed including loss of dry matter and glucosamine content. Deoxynivalenol was extracted from the substrate by solid phase extraction and quantified using high-performance liquid chromatography. The reduction of deoxynivalenol by A. oryzae KKB4 and R. oryzae KP1R1 were 65.91% and 56.82%, respectively after ten days of fermentation. Toxicity analysis revealed that residues of deoxynivalenol were not toxic to growth of Saccharomyces cerevisiae cells. @*Conclusion, significance and impact of study@#Local strains A. oryzae KKB4 and R. oryzae KP1R1 were able to reduce and detoxify deoxynivalenol in solid substrates. This study provides supporting data to control mycotoxin that is critical for food and feed safety.

Bioprospection and characterization of the amylolytic activity by filamentous fungi from Brazilian Atlantic Forest / Bioprospecção e caracterização da atividade amilolítica de fungos filamentosos da Mata Atlântica Brasileira

Abstract Filamentous fungi are widely diverse and ubiquitous organisms. Such biodiversity is barely known, making room for a great potential still to be discovered, especially in tropical environments - which are favorable to growth and species variety. Filamentous fungi are extensively applied to the production of industrial enzymes, such as the amylases. This class of enzymes acts in the hydrolysis of starch to glucose or maltooligosaccharides. In this work twenty-five filamentous fungi were isolated from samples of decomposing material collected in the Brazilian Atlantic Forest. The two best amylase producers were identified as Aspergillus brasiliensis and Rhizopus oryzae. Both are mesophilic, they grow well in organic nitrogen-rich media produce great amounts of glucoamylases. The enzymes of A. brasiliensis and R. oryzae are different, possibly because of their phylogenetical distance. The best amylase production of A. brasiliensis occurred during 120 hours with initial pH of 7.5; it had a better activity in the pH range of 3.5-5.0 and at 60-75°C. Both fungal glucoamylase had wide pH stability (3-8) and were activated by Mn2+. R. oryzae best production occurred in 96 hours and at pH 6.5. Its amylases had a greater activity in the pH range of 4.0-5.5 and temperature at 50-65ºC. The most significant difference between the enzymes produced by both fungi is the resistance to thermal denaturation: A. brasiliensis glucoamylase had a T50 of 60 minutes at 70ºC. The R. oryzae glucoamylase only had a residual activity when incubated at 50°C with a 12 min T50.

Resumo Fungos filamentosos são organismos amplamente diversificados e ubíquos. Esta biodiversidade ainda é pouco caracterizada, desta forma, há um grande potencial a ser descoberto, sobretudo em biomas tropicais, que favorecem o crescimento e diversificação de espécies. Fungos filamentosos são extensivamente utilizados para a produção industrial de enzimas, como as amilases. Esta classe de enzimas atua na hidrólise do amido em glicose ou maltooligossacarídeos. Neste trabalho 25 cepas de fungos filamentosos foram isoladas a partir de amostras de material em decomposição coletados na Mata Atlântica Brasileira. As duas cepas que produziram mais amilases foram identificadas como Aspergillus brasiliensis e Rhizopus oryzae. Ambos os fungos são mesofílicos, crescem bem em meio de cultivo rico em nitrogênio orgânico, e produziram grande quantidade de glucoamilase. As enzimas de A. brasiliensis e R. oryzae possuem características distintas, possivelmente devido à distância filogenética das espécies. A produção de amilase mais expressiva de A. brasiliensis ocorreu em 120 horas de cultivo e pH inicial de 7,5; possui maior atividade em temperaturas entre 60-75ºC e pH entre 3,5-5,0. Ambas glucoamilases fúngicas obtiveram ampla estabilidade de pH (3-8) e foram ativadas por Mn2+. A melhor produção de R. oryzae ocorreu em 96 horas de cultivo e pH 6,5. Suas amilases são mais ativas na faixa de pH de 4,0-5,5 e temperatura entre 50-60ºC. A diferença mais significativa dentre as enzimas produzidas pelos fungos selecionados é a resistência à desnaturação térmica, tendo a glucoamilase de A. brasiliensis um T50 de 60 minutos a 70ºC, já a glucoamilase de R. oryzae somente obteve atividade residual quando incubada a 50°C, com um T50 de apenas 12 minutos.

Kinetics Study of Extracellular Detergent Stable Alkaline Protease from Rhizopus oryzae

In this study, extracellular alkaline protease was produced from Rhizopus oryzae in submerged fermentation using dairy waste (whey) as a substrate. Fermentation kinetics was studied and various parameters were optimized. The strain produced maximum protease at initial medium pH of 6.0 medium depth of 26 mm, inoculum size of 2% at incubation temperature of 35ºC for 168 h of fermentation. Alkaline protease was purified to homogeneity by ammonium sulphate fractionation followed by sephadex G-100 chromatography. The molecular mass of alkaline protease was 69 kDa determined by 10% SDS-PAGE. The optimum pH and temperature of alkaline protease was 9.0 and 40ºC, respectively. Metal profile of the enzyme showed that the enzyme was non-metallic in nature. The Km , Kcat , Vmax and Kcat/Km values of purified protease were 7.0 mg/mL, 3.8 x102 S-1, 54.30 µmol/min and 54.28 s-1 mg -1.mL respectively, using casein as substrate. The purified alkaline protease had stability with commercial detergents. .

Pyomyositis with rhino-orbital mucormycosis in type II diabetes mellitus.

Mucormycosis is a devastating infection caused by fungi from the order Mucorales. Rhino-Orbital (RO) and Rhino-cerebral (RC) are two forms of the disease which are uncommon, acute, and aggressive infections caused by these fungi occurring in several immune - compromised states including diabetes which is the most common (60-80%) predisposing factor. Because of its rapid progression and high mortality, early recognition and aggressive treatment offer the only chance to increase the survival rate. We reported a case of Rhino-Orbital Mucormycosis (ROM) caused by Rhizopus oryzae (R. arrhizus) that developed in a 39 year old male patient with type 11 diabtes mellitus (DM) and pyomyositis. He was successfully managed with liposomal amphotericin B, nasal endoscopic surgery and correction of underlying predisposing factors.

Functional Properties of Filamentous Fungi Isolated from the Indonesian Fermented Dried Cassava, with Particular Application on Poultry

The study aimed to evaluate the probiotic properties, antioxidant activity and fermentative capacity of Acremonium charticola and Rhizopus oryzae isolated from the Indonesian fermented dried cassava, with particular application on poultry. A. charticola inhibited the growth of Escherichia coli and Aspergillus flavus. A. charticola and R. oryzae grew in potato dextrose agar (PDA) adjusted to pH 3 and 8 or in PDA supplemented with bile salt up to 0.8%. After soaking for 8 hr, the survival rate of A. charticola in the simulated gastric juice (pH 2) and bile solutions (2% bile salt) was lower than that of R. oryzae. A. charticola and R. oryzae exhibited strong antioxidant activities. Compared to unfermented cassava pulp (control), the fibre content of cassava pulp tended to be lower after fermentation with A. charticola for 14 days. The populations of A. charticola and R. oryzae were significantly higher in fermented cassava pulp than in unfermented one. Coliform was higher in cassava pulp fermented with R. oryzae or A. charticola + R. oryzae compared to control after 7 days of fermentation, however, the bacteria were not different between A. charticola-fermented cassava pulp and control. Lactic acid bacteria (LAB) were higher in A. charticola- and R. oryzae-fermented cassava pulp than those in control, however, no difference of LAB was observed between A. charticola + R. oryzae-fermented cassava pulp and control. In conclusion, A. charticola exhibited antibacterial, antifungal and antioxidant activity, gastrointestinal persistence and fermentative capacity that may be beneficial for poultry industry.

Increase in Endoglucanase Productivity and Mycelial Stability of Rhizopus oryzae by Classical Mutagenesis.

Aim: To develop a mutant strain with high endoglucanase productivity and optimization of some cultivation parameters. Place and Duration of Study: Microbiology Research Laboratory, Department of Zoology, Molecular Biology & Genetics, Presidency University, College Street, Kolkata: 700 073, India, between Aug, 2010 and March 2011. Methodology: The wild strain of Rhizopus oryzae PR7 MTCC 9642 was subjected to classical mutagenesis by suspending 5 hyphal discs (0.5 cm) in 10ml of N-methyl-N'-nitro- N-nitrosoguanidine (MNNG) solutions of various concentrations (125-1000μg). The in situ cellulolytic activity of the colonies of the mutant strains on the plates were measured by using alcoholic iodine solution and the highest enzyme producing mutant was selected. The mutant strain was later cultivated in presence of various domestic wastes at various pH, temperature, time. The morphological alteration was also checked by staining with fluorescent dye. Results: Out of 50 mutants, strain A7 was selected that showed about 33% increase in endoglucanase synthesis utilizing orange bagasse as sole carbon source in a shake flask screen. The strain was found to have the same pH and temperature optima, but could achieve highest level of enzyme production earlier than that by its wild counterpart. Being a dimorphic fungus, the wild type strain of Rhizopus oryzae, showed a transformation to yeast like pelleted form, whereas the mutant strain A 7 showed persistent filamentous structure indicating the achievement of a structural stability in presence of environmental stress. Conclusion: The present mutant strain could ferment orange bagasse and showed an increased production of endoglucanase with minimized time consumption with greater mycelial stability against various environmental stresses. These achievements will definitely add economy in industrial production of endoglucanse at a nominal cost.

Soft Rot of Rhizopus oryzae as a Postharvest Pathogen of Banana Fruit in Korea

Soft rot on banana fruit caused by Rhizopus oryzae was identified for the first time in Korea. Colonies were white to light brown and formed numerous sporangiospores. Optimum temperature for mycelial growth was 30degrees C. Sporangia were globose and 30~200 microm. Sporangiophores were usually straight, 8~20 microm, and rhizoids usually in groups of 3~5. Columella were globose to sub-globose and 90~110 microm. Sporangiospores were sub-globose or oval and 4~10 microm. Based on its mycological characteristics, molecular analysis, and pathogenicity to host plants, this fungus was identified as Rhizopus oryzae Went & Prisen Geerligs. This is the first report of soft rot on banana caused by Rhizopus oryzae in Korea.

Extra-cellular isoamylase production by Rhizopus oryzae in solid-state fermentation of agro wastes

Extra-cellular isoamylase was produced by Rhizopus oryzae PR7 in solid-state fermentations of various agro wastes, among which millet, oat, tapioca, and arum (Colocasia esculenta) showed promising results. The highest amount of enzyme production was obtained after 72 h of growth at 28°C. The optimum pH for enzyme production was - 8.0. Among the various additives tested, enzyme production increased with ions such as Ca2+, Mg2+ and also with cysteine, GSH, and DTT. The enzyme synthesis was reduced in the presence of thiol inhibitors like Cu2+ and pCMB. The surfactants like Tween-40, Tween-80 and Triton X-100 helped in enhancing the enzyme activity. The production could be further increased by using the combinations of substrates. The ability to produce high amount of isoamylase within a relatively very short period and the capability of degrading wastes could make the strain suitable for commercial production of the enzyme.

First Report of Rhizopus oryzae as a Postharvest Pathogen of Apple in Korea

Soft rot in apple caused by Rhizopus oryzae was found for the first time in Korea. A detailed description of the specimen is given along with its internal transcribed spacer rDNA sequence. The fungus was identified as Rhizopus oryzae based on the mycological characteristics, molecular data, and pathogenicity testing.

Soft Rot on Cucumis melo var. makuwa Caused by Rhizopus oryzae

Rhizopus oryzae is reported for the first time on Cucumis melo L. var. makuwa Makino. A detailed description of this Korean specimen is given, along with its rDNA internal transcribed spacer sequence. On the basis of mycological characteristics and molecular data, the fungus was identified as R. oryzae Went & Prinsen Geerligs.

KINETIC STUDY OF PALMITIC ACID ESTERIFICATION CATALYZED BY Rhizopus oryzae RESTING CELLS / Estudio cinético de la esterificación del ácido palmítico catalizado por células en reposo de Rhizopus oryzae

In the present study, a kinetic model for the biocatalytic synthesis of esters using Rhizopus oryzae resting cells is proposed. The kinetic study has been made in a range of 30-50 °C and atmospheric pressure. The Influence of operating variables, water content, pH, amount of mycelium was studied. Different values of temperature, initial mycelium concentration and acid/alcohol molar ratio were tested. Initial rates were estimated from the slope of the concentration of palmitic acid, or their corresponding ester at conversions of less than 10%, versus time and reported as mmol l-1 min -1. The values of kinetic constants were computed using the freeware program SIMFIT (http://www.simfit.man.ac.uk).

En el presente estudio, un modelo cinético para la síntesis de esteres usando Rhizopus oryzae resting cells es propuesto. El estudio cinético fue realizado en un rango de temperatura de 30-50 ºC a presión atmosférica reducida. La influencia de las variables de operación tales como temperatura, pH y contenido de agua fueron estudiadas. Diferentes valores de concentración de micelio y relación molar de ácido/alcohol son ensayadas, Las velocidades iníciales se estimaron de la curva de concentración de acido palmítico, y su correspondiente conversión a ester en menos del 10%, frente a tiempo y reportadas en mmol I-1 min -1. Los valores de las constantes cinéticas fueron calculados usando el programa freeware SIMFIT (http://www.simfit.man.ac.uk).

Zigomicosis cutánea primaria por Rhyzopus oryzae en una niña con leucemia linfoblástica aguda tipo B / Primary cutaneous zygomycosis by Rhyzopus oryzae in a girl diagnosed with an acute lymphoblastic leukemia B type

La Zigomicosis es una infección infrecuente causada por hongos oportunistas integrantes del orden Mucorales, que se presenta en pacientes de alto riego como en: leucemia, linfomas con neutropenia prolongada, cetoacidosis diabética, malnutrición severa, ruptura de la integridad de la barrera cutánea y terapia inmunosupresora. Se presenta un caso de Zigomicosis cutánea en una paciente pediátrica con leucemia linfoblástica aguda de tipo B, con severa neutropenia y tratamiento con corticoides. A los cinco días de su hospitalización desarrolló en el antebrazo (zona de punción venosa), una lesión indurada, eritematosa, que progresó y ulceró. A partir de exudados y biopsias del tejido subcutáneo se realizaron exámenes microscópicos directos con KOH, cultivos en agar Sabouraud y estudio histológico a través de técnicas convencionales de hematoxilinaeosina y PAS. Los análisis de los materiales clínicos revelaron la presencia de hifas hialinas, no tabicadas, gruesas, compatibles con un Zygomycete. En todos los tubos se obtuvo abundante desarrollo de un hongo filamentoso, identificado como Rhizopus oryzae. Posteriormente a la escisión quirúrgica y tratamiento con anfotericina B se obtuvo una evolución favorable del paciente hasta el presente.

Zygomycosis is an infrequent infection caused by opportunistic fungi which belong to the order Mucorales and which is present in high risk patients diagnosed with : leukemia, lymphomas with prolonged neutropenia, diabetic cetoacidosis, severe malnutrition, rupture of the entire cutaneous barrier and immunesuppressing therapy. This paper deals with a case of cutaneous Zygomycosis in a pediatric patient diagnosed with acute lymphoblastic leukemia B type, suffering a severe neutropenia and corticosteroid treatment. On the fifth day of hospitalization, her forearm (venous puncture zone) showed an indured, erimatose lesion which progressed and ulcerated. Collection of exudates and biopsies of subcutaneous tissue served to carry out direct microsco-pic examinations with KOH, cultures in Sabouraud Agar and a histologic study through conventional hematoxilin-eosin and PAS techniques. Analyses of the clinical materials revealed the presence of hyaline, not septated and broad hyphae suitable to a Zygomycete. In all the tubes there was an abundant development of filamentous fungus identified as Rhizopus oryzae. After the surgical scission and treatment with anfotericine B, the patient showed a favorable evolution up to now.

The Study of Enzymatic Activity of Mutant Strain Rhizopus oryzae with L-Malic Acid Accumulation / 微生物学通报

A stable and efficient L-Malic acid accumulation mutant strain Rhizopus oryzae ME-M15 was discovered occasionally in the mutation breading for fumaric acid producers. Rhizopus oryzae ME-M15 gave a L-Malic acid output of 16.3 g/L on average after fermentation for 96 hours, more than 3 times than that of the parent strain ME-F10. In addition, other metabolites such as ethanol and fumaric acid were re-markably decreased in accordance with the depressed activity of the cytosolic isoenzyme of fumarase and alcohol dehydrogenase in strain ME-M15, while the activity of the pyruvate carboxylase had no significant difference.

The Screening of Alcohol Dehydrogenase (ADH) Mutant of Rhizopus oryzae / 微生物学通报

During L-lactic acid fermentation with Rhizopus oryzae,there existed a branch pathway by which pyruvate was transformed to eth- anol catalyzed by pyruvate decarboxylase(PDC)and alcohol dehydrogenase(ADH),thus decreasing the flux of pyruvate to lactic acid.In this study,the spores of Rhizopus oryzae AS3.3462 mutagenized with nitrosoguanidine(NTG),the appropriate dosage was 0.15 mg/mL and the lethal rate was 70%～80%.Two mutants,named mut-1 and mut-2,with decreased ADH activity were screened out by yeast peptone dextrose(YPD)agar medium containing allyl alcohol.These two mutants had decreased ADH activities of 41.63% and 50.29% compared with the parent strain.The fermentation behavior after 72h showed that the yields of ethanol produced by mut-1 and mut-2 were 4.87g/L and 6.56g/L respectively,while the wild type strain was 28.9g/L,and the lactate concentrations of mut-1 and mut-2 also increased from 40.31g/L to 54.45g/L and 44.07 g/L,respectively.It is also found that mut-1 and mut-2 had a high reducing sugar consumption rate and biomass accumulation than its present strain

Studyies on the Breeding and Cultivation of L-Lactic Acid Producing Strain / 微生物学通报

In order to obtain higher L-lactic acid yield industrial strain, the original strain Rhizopus oryzae PW352 was mutated by means of N+ ions implantation and a mutant strain Rhizopus oryzae RE3303 was obtained. Its lactic acid yield was increased by 75% than that of the original one. The acid producing condition was optimized by orthogonal design. The concentration of L-lactic acid reached to 131～136g/L and the conversion rate of glucose was as high as 86%～90% under the optimum condition.