Airway necrosis and granulation tissue formation caused by Rhizopus oryzae leading to severe upper airway obstruction: a case report.

Pulmonary Mucormycosis is a fatal infectious disease with high mortality rate. The occurrence of Mucormycosis is commonly related to the fungal virulence and the host's immunological defenses against pathogens. Mucormycosis infection and granulation tissue formation occurred in the upper airway was rarely reported. This patient was a 60-year-old male with diabetes mellitus, who was admitted to hospital due to progressive cough, sputum and dyspnea. High-resolution computed tomography (HRCT) and bronchoscopy revealed extensive tracheal mucosal necrosis, granulation tissue proliferation, and severe airway stenosis. The mucosal necrotic tissue was induced by the infection of Rhizopus Oryzae, confirmed by metagenomic next-generation sequencing (mNGS) in tissue biopsy. This patient was treated with the placement of a covered stent and local instillation of amphotericin B via bronchoscope. The tracheal mucosal necrosis was markedly alleviated, the symptoms of cough, shortness of breath, as well as exercise tolerance were significantly improved. The placement of airway stent and transbronchial microtube drip of amphotericin B could conduce to rapidly relieve the severe airway obstruction due to Mucormycosis infection.

Triple in silico targeting of IMPDH enzyme and RNA-dependent RNA polymerase of both SARS-CoV-2 and Rhizopus oryzae.

Aim: Mucormycosis has been associated with SARS-CoV-2 infections during the last year. The aim of this study was to triple-hit viral and fungal RNA-dependent RNA polymerases (RdRps) and human inosine monophosphate dehydrogenase (IMPDH). Materials & methods: Molecular docking and molecular dynamics simulation were used to test nucleotide inhibitors (NIs) against the RdRps of SARS-CoV-2 and Rhizopus oryzae RdRp. These same inhibitors targeted IMPDH. Results: Four NIs revealed a comparable binding affinity to the two drugs, remdesivir and sofosbuvir. Binding energies were calculated using the most abundant conformations of the RdRps after 100-ns molecular dynamics simulation. Conclusion: We suggest the triple-inhibition potential of four NIs against pathogenic RdRps and IMPDH, which is worth experimental validation.

Rhizopus arrhizus infection in mice causes brain histopathological alterations and exacerbates neuronal apoptosis.

Rhizopus arrhizus is a fungus that can cause central nervous system infections in animals, resulting in high morbidity and mortality, but the mechanism of injury is rarely reported. In this study, we investigated the mechanism of Rhizopus arrhizus damage to the central nervous system of mice by observing the clinical neurological symptoms and resolving the pathological changes in the ultrastructure of brain tissues, combined with the alteration of apoptosis-related genes and immunohistochemistry (IHC). The results showed that all the mice in the treated group died, the brain pyknosis of neurons, there were black mycelium aggregates around the blood vessels, and apoptotic vesicles were produced. The RT-qPCR results showed that, compared with the control group, the relative transcriptome levels of Caspase 8 and BcL-2 genes were significantly increased (P < 0.05), the relative transcriptome level of Caspase 9 gene was highly significant (P < 0.01), the relative transcriptome level of Caspase 3 and Bax gene was significantly decreased (P < 0.05), and the ratio of Bcl-2/Bax was significantly increased (P < 0.05) in the brains of the treated group. TUNEL staining showed that the rate of neuronal apoptosis in the treated group of mice was extremely significantly higher than that in the control group (P < 0.01). This study shows that Rhizopus arrhizus strain XMLO1 causes brain damage by triggering neuronal apoptosis. This study provided a theoretical basis for revealing the mechanism of Rhizopus arrhizus infection.

Development of a Strategy for L-Lactic Acid Production by Rhizopus oryzae Using Zizania latifolia Waste and Cane Molasses as Carbon Sources.

As a biodegradable and renewable material, polylactic acid is considered a major environmentally friendly alternative to petrochemical plastics. Microbial fermentation is the traditional method for lactic acid production, but it is still too expensive to compete with the petrochemical industry. Agro-industrial wastes are generated from the food and agricultural industries and agricultural practices. The utilization of agro-industrial wastes is an important way to reduce costs, save energy and achieve sustainable development. The present study aimed to develop a method for the valorization of Zizania latifolia waste and cane molasses as carbon sources for L-lactic acid fermentation using Rhizopus oryzae LA-UN-1. The results showed that xylose derived from the acid hydrolysis of Z. latifolia waste was beneficial for cell growth, while glucose from the acid hydrolysis of Z. latifolia waste and mixed sugars (glucose and fructose) from the acid hydrolysis of cane molasses were suitable for the accumulation of lactic acid. Thus, a three-stage carbon source utilization strategy was developed, which markedly improved lactic acid production and productivity, respectively reaching 129.47 g/L and 1.51 g/L·h after 86 h of fermentation. This work demonstrates that inexpensive Z. latifolia waste and cane molasses can be suitable carbon sources for lactic acid production, offering an efficient utilization strategy for agro-industrial wastes.

Production of 11α-Hydroxysteroid Derivatives by Corynebacterium glutamicum Expressing the Rhizopus oryzae Hydroxylating System.

Hydroxylation of steroids has acquired special relevance for the pharmaceutical industry. Particularly, the 11α-hydroxylation of steroids is a process of biotechnological importance currently carried out at industrial scale for the production of contraceptive drugs and glucocorticoids. This process is performed by several fungal species including Rhizopus nigricans, Aspergillus ochraceus, Aspergillus niger, and Rhizopus oryzae that are used to produce by biotransformation hydroxylated steroids for pharmaceutical purposes (Wang et al., J Steroid Biochem Mol Biol 171:254-261, 2017). However, the development of more efficient biotransformation processes is essential since the steroidal derivatives obtained by the in vivo hydroxylation are often a mixture of hydroxylated compounds in different positions of the steroid molecule. This phenomenon is due to the large number of different CYPs contained in the fungal strains.The genes responsible for the 11α-hydroxylase activity in R. oryzae consisting in the cytochrome CYP509C12 and its redox partner, the reductase RoCPR1, have been chemically synthetized forming a synthetic operon named FUN optimized to be expressed in bacteria. To express this operon, we have selected the strain Corynebacterium glutamicum R31 that is a robust and GRAS bacterial strain widely used for industrial purposes. The synthetic operon has been cloned in the pECXK-99E vector, yielding pXKFUN plasmid, and transformed C. glutamicum R31 to generate C. glutamicum R31 (pXKFUN) strain. This strain is not a steroid degrader and can efficiently transport C19 and C21 steroids across the cytoplasmic membrane (García-Fernandez et al. Catalysts 316:1-12, 2017). C. glutamicum can be used as a clean host for steroid biotransformation, because it does not introduce additional undesired side reactions on the steroids, thus reducing the contamination of the final products (Felpeto-Santero et al., Microbiol Biotechnol 12:856-868, 2019). Here we show a proof of concept that C. glutamicum can be used as a suitable chassis to perform steroid biotransformation expressing eukaryotic cytochromes. The protocol below provides detailed information on steroid 11α-hydroxylations by Corynebacterium recombinant strain.

Production of Rhizopus oryzae lipase using optimized Yarrowia lipolytica expression system.

Yarrowia lipolytica is an alternative yeast for heterologous protein production. Based on auto-cloning vectors, a set of 18 chromogenic cloning vectors was developed, each containing one of the excisable auxotrophic selective markers URA3ex, LYS5ex, and LEU2ex, and one of six different promoters: the constitutive pTEF, the phase dependent hybrid pHp4d, and the erythritol-inducible promoters from pEYK1 and pEYL1 derivatives. These vectors allowed to increase the speed of cloning of the gene of interest. In parallel, an improved new rProt recipient strain JMY8647 was developed by abolishing filamentation and introducing an auxotrophy for lysine (Lys-), providing an additional marker for genetic engineering. Using this cloning strategy, the optimal targeting sequence for Rhizopus oryzae ROL lipase secretion was determined. Among the eight targeting sequences, the SP6 signal sequence resulted in a 23% improvement in the lipase activity compared to that obtained with the wild-type ROL signal sequence. Higher specific lipase activities were obtained using hybrid erythritol-inducible promoters pHU8EYK and pEYL1-5AB, 1.9 and 2.2 times, respectively, when compared with the constitutive pTEF promoter. Two copy strains produce a 3.3 fold increase in lipase activity over the pTEF monocopy strain (266.7 versus 79.7 mU/mg).

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

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.

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.

Selective pertraction of dicarboxylic acids from simulated Rhizopus oryzae fermentation broths.

Fumaric, malic and succinic acids have been selectively separated by facilitated pertraction with Amberlite LA-2, using n-heptane as liquid membrane. The feed phase consisted on viscous aqueous solution with similar mixture of carboxylic acids and viscosity as those of Rhizopus oryzae fermentation broth. Due to the differences between the acidities and molecule size of these acids, it is possible to selectively recover fumaric acid from the initial solution. The pH-gradient between the feed and stripping phases, as well as carrier concentration in the liquid membrane represent the main process parameters influencing the pertraction selectivity. Among them, Amberlite LA-2 concentration exhibits the most important control on the selectivity factor S, the maximum value of S being reached for carrier concentration of 30 g/l. The increase of feed phase viscosity amplified the magnitude of these influences on pertraction selectivity, due to the hindrance of acids diffusion towards the region where their reaction with Amberlite LA-2 occurs, effect more important for malic acid. Therefore, by modifying the viscosity from 1 to 24 cP, the maximum value of selectivity factor was increased from 12 to 18.8.

Primary cutaneous mucormycosis due to Rhizopus arrhizus in an immunosuppressed patient with paroxysmal nocturnal haemoglobinuria.

OBJECTIVE: We present a case of primary cutaneous mucormycosis in a patient with bone marrow failure secondary to paroxysmal nocturnal haemoglobinuria (PNH). CLINICAL CASE: A 60-year-old male patient with a history of PNH, complicated to a severe aplastic anaemia, presented to the emergency department complaining of papules on the lower limbs that rapidly turned into necrotic plaques within 2 months. Histopathological examination showed granulomatous and suppurative dermatitis with tissue necrosis and the presence of non-septate hyphae. Molecular identification was achieved by amplification and sequencing of the 18S-ITS1-5.8S-ITS2-28S rRNA region using the polymerase chain reaction. The sequence showed 100% identity with Rhizopus arrhizus. The patient received treatment with liposomal amphotericin B and surgical debridement. Nonetheless, the patient suffered from severe low red blood cells and platelets and also underwent septic shock; he died 6 days after admission to the hospital. CONCLUSION: Mucormycosis in the setting of immunosuppression is challenging. Upon suspicion of a diagnosis, immediate treatment is required. Adjunctive therapies may be considered; however, the case fatality rate remains high.

Antifungal Activity and Mechanism of Electron Beam Irradiation Against Rhizopus oryzae.

Electron beam irradiation is a physical fungicidal technique that has emerged as a potential application in China. However, its antifungal activity and mechanism against Rhizopus oryzae have not been reported. Thus, this study aimed to investigate the antifungal activity and mechanism of electron beam irradiation of R. oryzae. The antifungal activity analysis showed that the D10 value and complete elimination dose of R. oryzae irradiated by electron beam were 1.73 kGy and 8.08 kGy, respectively. Electron beam irradiation has a strong inhibitory effect on the filamentous biomass of R. oryzae. To reveal the antifungal mechanism of electron beam against R. oryzae, this study analyzed the dynamic changes in the cell wall, cell membrane, and oxidative stress induced by different irradiation doses. The results showed that electron beam irradiation destroyed the cell wall structure of R. oryzae, increasing chitinase activity and decreasing chitin content. Cell membrane integrity is disrupted, increasing relative conductivity, decreasing pH values, and decreasing soluble protein content. Electron beam irradiation causes oxidative stress in cells, increasing H2O2 content, decreasing antisuperoxide anion activity, decreasing DPPH free radical scavenging activity, and inhibiting defense enzyme (CAT and SOD) activity. This phenomenon indicates that electron beams can cause structural damage to and metabolic dysfunction of cells and disorders of redox homeostasis, which may be the main cause of growth inhibition and cell death in R. oryzae.

Mucormycosis in Africa: Epidemiology, diagnosis and treatment outcomes.

Mucorales fungi cause mucormycosis, an invasive and rapidly progressive disease which increasingly affects mostly immunocompromised but also immunocompetent individuals. The objective of this study was to highlight the epidemiology, diagnostic modalities, treatment and overall survival of mucormycosis in Africa. We searched for relevant publications in PubMed, Google Scholar and African Journal Online databases covering the period 1960-2022. A total of 147 articles were identified, of which 66 were included in the review, detailing 408 individual cases from 12 African countries; 330 (80.9%) from North Africa, 63 (15.4%) from Southern Africa, seven (1.7%) from East Africa, seven (1.7%) from West Africa and a single case (0.2%) from Central Africa. The most frequently described clinical forms were rhino-orbital-cerebral (n = 307, 75.2%) and gastrointestinal (n = 51, 12.5%). Diabetes mellitus, COVID-19, malignancies and neutropaenia were the commonest underlying risks in 203 (49.8%), 101 (24.8%), 65 (15.9%) and 53 (13.0%) cases respectively. Most cases, 296 (72.5%) were diagnosed by histopathology. Fungal aetiology was identified in 38 (9.3%), of which the commonest was Rhizopus oryzae/arrhizus (27/38, 71.1%). Of the 408 cases, 334 (81.9%) patients received antifungal therapy, while 244 (59.8%) had surgery. In cases with a specified outcome, survival rate was 59.1% (228/386). Based on case reporting, a substantial burden of mucormycosis occurs in North Africa but the disease is rarely reported in most of the sub-Saharan region. Establishing a comprehensive registry for standardised data collection could improve understanding of the epidemiology of mucormycosis in the region.

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.

Exploring the potential of Rhizopus oryzae AUMC14899 as a novel endophytic fungus for the production of L-tyrosine and its biomedical applications.

BACKGROUND: A significant threat to the public's health is the rise in antimicrobial resistance among numerous nosocomial bacterial infections. This may be a detriment to present initiatives to enhance the health of immune-compromised patients. Consequently, attention has been devoted to exploring new bioactive compounds in the field of drug discovery from endophytes. Therefore, this study is the first on the production of L-tyrosine (LT) as a promising bio-therapeutic agent from endophytic fungi. RESULTS: A new endophytic fungal isolate has been identified for the first time as Rhizopus oryzae AUMC14899 from Opuntia ficus-indica (L.) and submitted to GenBank under the accession number MZ025968. Separation of amino acids in the crude extract of this fungal isolate was carried out, giving a higher content of LT, which is then characterized and purified. LT exhibited strong antibacterial and anti-biofilm activities against multidrug-resistant Gram-negative and Gram-positive bacteria. The recorded minimum inhibitory concentration (MIC) values ranged from 6 to 20 µg/ml. In addition, LT caused a strong reduction in biofilm formation and disrupted the preformed biofilm. Moreover, results indicated that LT supported cell viability, evidencing hemocompatibility and no cytotoxicity. CONCLUSION: Our findings suggest that LT has potential as a therapeutic agent due to its potential antibacterial, anti-biofilm, hemocompatibility, and lack of cytotoxic activities, which may also increase the range of therapy options for skin burn infections, leading to the development of a novel fungal-based drug.

Glutaraldehyde-crosslinked Rhizopus oryzae whole cells show improved catalytic performance in alkene epoxidation.

BACKGROUND: Existing methods for alkene epoxidation are based on lipase-catalysed perhydrolysis. However, the inactivation of the expensive lipase enzyme is problematic for enzymatic epoxidation at large scales due to the use of hydrogen peroxide and peracids at high concentrations in the reaction. The immobilisation of whole cells appears to be a promising approach to alleviate this problem. RESULTS: A green oxidation system containing hydrogen peroxide, Na3C6H5O7, an acyl donor, and glutaraldehyde (GA)-crosslinked cells of Rhizopus oryzae was developed for the epoxidation of alkenes. GA-crosslinked cells of Rhizopus oryzae were adopted as a biocatalyst into the epoxidation system. A variety of alkenes were oxidised with this system, with a 56-95% analytical yield of the corresponding epoxides. The catalytic performance of the crosslinked treated cells was substantially improved compared to that of the untreated cells and the initial reaction rate increased from 126.71 to 234.72 mmol/L/h, retaining 83% yields even after four batches of reactions. The addition of 3.5 mmol Na3C6H5O7 not only acts as an acid-trapping reagent to eliminate the negative effect of the carboxylic acid on the alkene oxide but also forms a saturated salt solution with the aqueous phase, affecting the concentration of H2O2 in the three phases and thus the epoxidation reaction. Organic solvents with a logP value > 0.68 were good at producing hydroxy peracids; however, this method is only suitable for oxidation in a two-liquid phase. CONCLUSIONS: Compared with other lipase biocatalysts, the GA-crosslinked whole-cell biocatalyst is inexpensive, readily available, and highly stable. Therefore, it can be considered promising for industrial applications.

Alteration of Chain-Length Selectivity and Thermostability of Rhizopus oryzae Lipase via Virtual Saturation Mutagenesis Coupled with Disulfide Bond Design.

Rhizopus oryzae lipase (ROL) is one of the most important enzymes used in the food, biofuel, and pharmaceutical industries. However, the highly demanding conditions of industrial processes can reduce its stability and activity. To seek a feasible method to improve both the catalytic activity and the thermostability of this lipase, first, the structure of ROL was divided into catalytic and noncatalytic regions by identifying critical amino acids in the crevice-like binding pocket. Second, a mutant screening library aimed at improvement of ROL catalytic performance by virtual saturation mutagenesis of residues in the catalytic region was constructed based on Rosetta's Cartesian_ddg protocol. A double mutant, E265V/S267W (with an E-to-V change at residue 265 and an S-to-W change at residue 267), with markedly improved catalytic activity toward diverse chain-length fatty acid esters was identified. Then, computational design of disulfide bonds was conducted for the noncatalytic amino acids of E265V/S267W, and two potential disulfide bonds, S61C-S115C and E190C-E238C, were identified as candidates. Experimental data validated that the variant E265V/S267W/S61C-S115C/E190C-E238C had superior stability, with an increase of 8.5°C in the melting temperature and a half-life of 31.7 min at 60°C, 4.2-fold longer than that of the wild-type enzyme. Moreover, the variant improved the lipase activity toward five 4-nitrophenyl esters by 1.5 to 3.8 times, exhibiting a potential to modify the catalytic efficiency. IMPORTANCE Rhizopus oryzae lipase (ROL) is very attractive in biotechnology and industry as a safe and environmentally friendly biocatalyst. Functional expression of ROL in Escherichia coli facilitates effective high-throughput screening for positive variants. This work highlights a method to improve both selectivity and thermostability based on a combination of virtual saturation mutagenesis in the substrate pocket and disulfide bond prediction in the noncatalytic region. Using the method, ROL thermostability and activity to diverse 4-nitrophenyl esters could be substantially improved. The strategy of rational introduction of multiple mutations in different functional domains of the enzyme is a great prospect in the modification of biocatalysts.

Screening of potential antigens from whole proteome and development of multi-epitope vaccine against Rhizopus delemar using immunoinformatics approaches.

Mucormycosis is a deadly fungal disease mainly caused by Rhizopus oryzae (strain 99-880), also known as Rhizopus delemar. Previously, mucormycosis occurs in immunocompromised patients of diabetes mellitus, cancer, organ transplant, etc. But there was a drastic increase in mucormycosis cases in the ongoing COVID-19 pandemic. Despite several available therapies and antifungal treatments, the mortality rate of mucormycosis is about more than 50%. Currently, there is no vaccine available in the market for mucormycosis that urgently needs to develop a potential vaccine against mucormycosis with high efficacy. In the present study, we have screened 4 genome-derived predicted antigens (GDPA) through sequential filtration of the whole proteome of R. delemar using different benchmarked bioinformatics tools. These 4 GDPA along with 4 randomly selected experimentally reported antigens (ERA) were sourced for prediction of B- and T- cell epitopes and utilized in designing of two potential multi-epitope vaccine candidates which can induce both innate and adaptive immunity against R. delemar. Besides these, comparative immune simulation studies and in silico cloning were performed using L. lactis as an expression system for their possible uses as oral vaccines. This is the first multi-epitope vaccine designed against R. delemar through systematic pipelined reverse vaccinology and immunoinformatic approaches. Although the wet-lab based experimental validation of designed vaccines is required before testing in the preclinical model, the current study will significantly help in reducing the cost of experimentation as well as improving the efficacy of vaccine therapy against mucormycosis and other pathogenic diseases.Communicated by Ramaswamy H. Sarma.

A unique case of Rhizopus oryzae brain abscess treated with intracavitary amphotericin.

Here we present a case of a poorly controlled diabetic who developed extensive rhinocerebral mucormycosis. Systemic and intrathecal amphotericin were not able to improve his life threatening infection. Therefore, salvage therapy with intracavitary amphotericin B deoxycholate was used to instill antifungal therapy directly into the patient's brain abscess. For proper dosing of intracavitary amphotericin B deoxycholate, we devised a formula which can be theoretically applied for all intracavitary therapies. Unfortunately, the patient's family withdrew care 6 days after starting intracavitary amphotericin and efficacy of this therapy could not be evaluated.

Purification, Characterization, and Application of Endoglucanase from Rhizopus oryzae as Antibiofilm Agent.

The enzyme endoglucanase is responsible for the depolymerization of cellulose. This study focuses on characterization and purification of endoglucanase from Rhizopus oryzae MTCC 9642 through a simple size exclusion method and its effective application as an antibiofilm agent. Extracellular ß-1,4-endoglucanase, an enzyme that catalyzes the hydrolysis of carboxymethyl cellulose, was found to be synthesized by Rhizopus oryzae MTCC 9642. The enzyme was purified up to homogeneity simply by size exclusion process through ultrafiltration and gel chromatography. The molecular weight of purified enzyme protein was estimated to be 39.8 kDa and it showed the highest substrate affinity towards carboxymethyl-cellulose with Km and Vmax values of 0.833 mg ml-1 and of 0.33 mmol glucose min-1 mg-1protein, respectively. The purified enzyme exhibited optimal activity at pH 6 with a broad stability range of pH 3-8. The most preferred temperature was 35 °C and 50% of activity could be retained after the thermal exposure at 40 °C for 25 min. The purified enzyme protein was inactivated by Cu2+, while the activity could be enhanced by the addition of exogenous thiols. Since biofilm is a challenge for health sector, with the aim of eradicating the biofilm, the purified endoglucanase was used to remove biofilm produced by two nosocomial bacteria. As predicted by in silico molecular docking interaction, the purified enzyme could effectively degrade biofilm architecture of bacterial strains S. aureus and P. aeruginosa by 76.52 ± 6.52% and 61.67 ± 8.76%, respectively. The properties of purified enzyme protein, as elucidated by in vitro and in silico characterization, may be favourable for its commercial applications as a potent antibiofilm agent.