Mucor germinans, a novel dimorphic species resembling Paracoccidioides in a clinical sample: questions on ecological strategy.

Dimorphism is known among the etiologic agents of endemic mycoses as well as in filamentous Mucorales. Under appropriate thermal conditions, mononuclear yeast forms alternate with multi-nucleate hyphae. Here, we describe a dimorphic mucoralean fungus obtained from the sputum of a patient with Burkitt lymphoma and ongoing graft-versus-host reactions. The fungus is described as Mucor germinans sp. nov. Laboratory studies were performed to simulate temperature-dependent dimorphism, with two environmental strains Mucor circinelloides and Mucor kunryangriensis as controls. Both strains could be induced to form multinucleate arthrospores and subsequent yeast-like cells in vitro. Multilateral yeast cells emerge in all three Mucor species at elevated temperatures. This morphological transformation appears to occur at body temperature since the yeast-like cells were observed in the lungs of our immunocompromised patient. The microscopic appearance of the yeast-like cells in the clinical samples is easily confused with that of Paracoccidioides. The ecological role of yeast forms in Mucorales is discussed.IMPORTANCEMucormycosis is a devastating disease with high morbidity and mortality in susceptible patients. Accurate diagnosis is required for timely clinical management since antifungal susceptibility differs between species. Irregular hyphal elements are usually taken as the hallmark of mucormycosis, but here, we show that some species may also produce yeast-like cells, potentially being mistaken for Candida or Paracoccidioides. We demonstrate that the dimorphic transition is common in Mucor species and can be driven by many factors. The multi-nucleate yeast-like cells provide an effective parameter to distinguish mucoralean infections from similar yeast-like species in clinical samples.

Whole-genome sequence of Mucor velutinosus NIH1002, a strain associated with disseminated disease.

The draft genome of Mucor velutinosus NIH1002, a 2011 isolate from a case of disseminated disease, was sequenced using PacBio long-read and HiSeq short-read technologies. The genome has 43 contigs, an N50 of 2.65 Mb, and 13,295 protein-coding genes. It is the most complete M. velutinosus genome to date.

Synergistic action of lactoferrin and its derived functional fragments as a promising therapeutic agent in combating mucormycosis.

Aim: Currently, we have limited armamentarium of antifungal agents against Mucorales. There is an urgent need to discover novel antifungal agents that are effective, safe and affordable. Materials & methods: In this study, the anti-Mucorale action of native lactoferrin (LF) and its functional fragments CLF, RR6 and LFcin against three common Mucorale species are reported. The synergistic action of LF with antifungal agents like amphotericin B, isavuconazole and posaconazole was analyzed using checkerboard technique. Results: All the three mucor species showed inhibition when treated with fragments. The checkerboard assay confirmed that native LF showed the best synergistic action against Mucorales in combination with Amphotericin B. Conclusion: These results highlight the potential therapeutic value of native LF against Mucorales.

Black fungus, or 'mucormycosis', is a dangerous fungal infection. Normally, it affects people with a weakened immune system. It is only treatable when diagnosed early. It spreads by breathing the fungus in, eating contaminated food or direct contact with an infected wound. There are not many medicines that can treat this type of fungus, so it is important to find new ones. In this study, we tested a natural protein called lactoferrin and some of its building blocks, called peptides, to see if they could stop the fungus from growing. Lactoferrin and its peptides could stop the fungus from growing, especially when used with a medicine called amphotericin B. This means that lactoferrin could potentially be a helpful treatment for this fungal infection.

Antifungal mechanism of phenyllactic acid against Mucor investigated through proteomic analysis.

The primary inhibitory targets of phenyllactic acid (PLA, including D-PLA and L-PLA) on Mucor were investigated using Mucor racemosus LD3.0026 isolated from naturally spoiled cherry, as an indicator fungi. The results demonstrated that the minimum inhibitory concentration (MIC) of PLA against Mucor was 12.5 mmol·L-1. Results showed that the growing cells at the tip of the Mucor were not visibly deformed, and there was no damage to the cell wall following PLA treatment; however, PLA damaged the cell membrane and internal structure. The results of isobaric tags for relative and absolute quantification (iTRAQ) indicated that the Mucor mitochondrial respiratory chain may be the target of PLA, potentially inhibiting the energy supply of Mucor. These results indicate that the antifungal mechanism of PLA against mold is independent of its molecular configuration. The growth of Mucor is suppressed by PLA, which destroys the organelle structure in the mycelium and inhibits energy metabolism.

Assessing the efficacy of the ovicidal fungus Mucor circinelloides in reducing coccidia parasitism in peacocks.

The biological control of gastrointestinal (GI) parasites using predatory fungi has been recently proposed as an accurate and sustainable approach in birds. The current study aimed to assess for the first time the efficacy of using the native ovicidal fungus Mucor circinelloides (FMV-FR1) in reducing coccidia parasitism in peacocks. For this purpose, an in vivo trial was designed in the resident peacock collection (n = 58 birds) of the São Jorge Castle, at Lisbon, Portugal. These animals presented an initial severe infection by coccidia of the genus Eimeria (20106 ± 8034 oocysts per gram of feces, OPG), and thus received commercial feed enriched with a M. circinelloides suspension (1.01 × 108 spores/kg feed), thrice-weekly. Fresh feces were collected every 15 days to calculate the coccidia shedding, using the Mini-FLOTAC technique. The same bird flock served simultaneously as control (t0 days) and test groups (t15-t90 days). The average Eimeria sp. shedding in peacocks decreased up to 92% following fungal administrations, with significant reduction efficacies of 78% (p = 0.004) and 92% (p = 0.012) after 45 and 60 days, respectively. Results from this study suggest that the administration of M. circinelloides spores to birds is an accurate solution to reduce their coccidia parasitism.

A Biomimetic Approach to Premyrsinane-Type Diterpenoids: Exploring Microbial Transformation to Enhance Their Chemical Diversity.

Premyrsinane-type diterpenoids have been considered to originate from the cyclization of a suitable 5,6- or 6,17-epoxylathyrane precursor. Their biological activities have not been sufficiently explored to date, so the development of synthetic or microbial approaches for the preparation of new derivatives would be desirable. Epoxyboetirane A (4) is an 6,17-epoxylathyrane isolated from Euphorbia boetica in a large enough amount to be used in semi-synthesis. Transannular cyclization of 4 mediated by Cp2TiIIICl afforded premyrsinane 5 in good yield as an only diasteroisomer. To enhance the structural diversity of premyrsinanes so their potential use in neurodegenerative disorders could be explored, compound 5 was biotransformed by Mucor circinelloides NRRL3631 to give rise to hydroxylated derivatives at non-activated carbons (6-7), all of which were reported here for the first time. The structures and absolute configurations of all compounds were determined through extensive NMR and HRESIMS spectroscopic studies.

Dissecting Holistic Metabolic Acclimatization of Mucor circinelloides WJ11 Defective in Carotenoid Biosynthesis.

Mucor circinelloides WJ11 is a lipid-producing strain with industrial potential. A holistic approach using gene manipulation and bioprocessing development has improved lipid production and the strain's economic viability. However, the systematic regulation of lipid accumulation and carotenoid biosynthesis in M. circinelloides remains unknown. To dissect the metabolic mechanism underlying lipid and carotenoid biosynthesis, transcriptome analysis and reporter metabolites identification were implemented between the wild-type (WJ11) and ΔcarRP WJ11 strains of M. circinelloides. As a result, transcriptome analysis revealed 10,287 expressed genes, with 657 differentially expressed genes (DEGs) primarily involved in amino acid, carbohydrate, and energy metabolism. Integration with a genome-scale metabolic model (GSMM) identified reporter metabolites in the ΔcarRP WJ11 strain, highlighting metabolic pathways crucial for amino acid, energy, and nitrogen metabolism. Notably, the downregulation of genes associated with carotenoid biosynthesis and acetyl-CoA generation suggests a coordinated relationship between the carotenoid and fatty acid biosynthesis pathways. Despite disruptions in the carotenoid pathway, lipid production remains stagnant due to reduced acetyl-CoA availability, emphasizing the intricate metabolic interplay. These findings provide insights into the coordinated relationship between carotenoid and fatty acid biosynthesis in M. circinelloides that are valuable in applied research to design optimized strains for producing desired bioproducts through emerging technology.

Efficacy of an oral lipid nanocrystal formulation of amphotericin B (MAT2203) in the neutropenic mouse model of pulmonary mucormycosis.

Invasive mucormycosis (IM) is associated with high mortality and morbidity. MAT2203 is an orally administered lipid nanocrystal formulation of amphotericin B, which has been shown to be safe and effective against other fungal infections. We sought to compare the efficacy of MAT2203 to liposomal amphotericin B (LAMB) treatment in a neutropenic mouse model of IM due to Rhizopus arrhizus var. delemar or Mucor circinelloides f. jenssenii DI15-131. In R. arrhizus var. delemar-infected mice, 15 mg/kg of MAT2203 qd was as effective as 10 mg/kg of LAMB in prolonging median survival time vs placebo (13.5 and 16.5 days for MAT2203 and LAMB, respectively, vs 9 days for placebo) and enhancing overall survival vs placebo-treated mice (40% and 45% for MAT2203 and LAMB, respectively, vs 0% for placebo). A higher dose of 45 mg/kg of MAT2203 was not well tolerated by mice and showed no benefit over placebo. Similar results were obtained with mice infected with M. circinelloides. Furthermore, while both MAT2203 and LAMB treatment resulted in a significant reduction of ~1.0-2.0log and ~2.0-2.5log in Rhizopus delemar or M. circinelloides lung and brain burden vs placebo mice, respectively, LAMB significantly reduced tissue fungal burden in mice infected with R. delemar vs tissues of mice treated with MAT2203. These results support continued investigation and development of MAT2203 as a novel and oral formulation of amphotericin for the treatment of mucormycosis.

Ibrexafungerp is efficacious in a neutropenic murine model of pulmonary mucormycosis as monotherapy and combined with liposomal amphotericin B.

Ibrexafungerp (formerly SCY-078) is the first member of the triterpenoid class that prevents the synthesis of the fungal cell wall polymer ß-(1,3)-D-glucan by inhibiting the enzyme glucan synthase. We evaluated the in vivo efficacy of ibrexafungerp against pulmonary mucormycosis using an established murine model. Neutropenic mice were intratracheally infected with either Rhizopus delemar or Mucor circinelloides. Treatment with placebo (diluent control), ibrexafungerp (30 mg/kg, PO BID), liposomal amphotericin B (LAMB 10 mg/kg IV QD), posaconazole (PSC 30 mg/kg PO QD), or a combination of ibrexafungerp plus LAMB or ibrexafungerp plus PSC began 16 h post-infection and continued for 7 days for ibrexafungerp or PSC and through day 4 for LAMB. Ibrexafungerp was as effective as LAMB or PSC in prolonging median survival (range: 15 days to >21 days) and enhancing overall survival (30%-65%) vs placebo (9 days and 0%; P < 0.001) in mice infected with R. delemar. Furthermore, median survival and overall percent survival resulting from the combination of ibrexafungerp plus LAMB were significantly greater compared to all monotherapies (P ≤ 0.03). Similar survival results were observed in mice infected with M. circinelloides. Monotherapies also reduce the lung and brain fungal burden by ~0.5-1.0log10 conidial equivalents (CE)/g of tissue vs placebo in mice infected with R. delemar (P < 0.05), while a combination of ibrexafungerp plus LAMB lowered the fungal burden by ~0.5-1.5log10 CE/g compared to placebo or any of the monotherapy groups (P < 0.03). These results are promising and warrant continued investigation of ibrexafungerp as a novel treatment option against mucormycosis.

Safety evaluation of an extension of use of the food enzyme triacylglycerol lipase from the non-genetically modified Mucor circinelloides strain AE-LMH.

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is produced with the non-genetically modified Mucor circinelloides strain AE-LMH by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in three food manufacturing processes. Subsequently, the applicant requested to extend its use to include two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of five food manufacturing processes. The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.845 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (784 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 928. Based on the data provided for the previous evaluation and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Molecular mechanisms that govern infection and antifungal resistance in Mucorales.

SUMMARYThe World Health Organization has established a fungal priority pathogens list that includes species critical or highly important to human health. Among them is the order Mucorales, a fungal group comprising at least 39 species responsible for the life-threatening infection known as mucormycosis. Despite the continuous rise in cases and the poor prognosis due to innate resistance to most antifungal drugs used in the clinic, Mucorales has received limited attention, partly because of the difficulties in performing genetic manipulations. The COVID-19 pandemic has further escalated cases, with some patients experiencing the COVID-19-associated mucormycosis, highlighting the urgent need to increase knowledge about these fungi. This review addresses significant challenges in treating the disease, including delayed and poor diagnosis, the lack of accurate global incidence estimation, and the limited treatment options. Furthermore, it focuses on the most recent discoveries regarding the mechanisms and genes involved in the development of the disease, antifungal resistance, and the host defense response. Substantial advancements have been made in identifying key fungal genes responsible for invasion and tissue damage, host receptors exploited by the fungus to invade tissues, and mechanisms of antifungal resistance. This knowledge is expected to pave the way for the development of new antifungals to combat mucormycosis. In addition, we anticipate significant progress in characterizing Mucorales biology, particularly the mechanisms involved in pathogenesis and antifungal resistance, with the possibilities offered by CRISPR-Cas9 technology for genetic manipulation of the previously intractable Mucorales species.

H3K4 methylation regulates development, DNA repair, and virulence in Mucorales.

Mucorales are basal fungi that opportunistically cause a potentially fatal infection known as mucormycosis (black fungus disease), which poses a significant threat to human health due to its high mortality rate and its recent association with SARS-CoV-2 infections. On the other hand, histone methylation is a regulatory mechanism with pleiotropic effects, including the virulence of several pathogenic fungi. However, the role of epigenetic changes at the histone level never has been studied in Mucorales. Here, we dissected the functional role of Set1, a histone methyltransferase that catalyzes the methylation of H3K4, which is associated with the activation of gene transcription and virulence. A comparative analysis of the Mucor lusitanicus genome (previously known as Mucor circinelloides f. lusitanicus) identified only one homolog of Set1 from Candida albicans and Saccharomyces cerevisiae that contains the typical SET domain. Knockout strains in the gene set1 lacked H3K4 monomethylation, dimethylation, and trimethylation enzymatic activities. These strains also showed a significant reduction in vegetative growth and sporulation. Additionally, set1 null strains were more sensitive to SDS, EMS, and UV light, indicating severe impairment in the repair process of the cell wall and DNA lesions and a correlation between Set1 and these processes. During pathogen-host interactions, strains lacking the set1 gene exhibited shortened polar growth within the phagosome and attenuated virulence both in vitro and in vivo. Our findings suggest that the histone methyltransferase Set1 coordinates several cell processes related to the pathogenesis of M. lusitanicus and may be an important target for future therapeutic strategies against mucormycosis.

Invasive Sinusitis Presenting with Orbital Complications in COVID Patients: Is Mucor the Only Cause?

The second wave of COVID pandemic was associated with an outbreak of Mucormycosis. The mortality rate of Mucormycosis reaches 50-80% in cases with orbital and intracranial extension (Fadda in Acta Otorhinolaryngol Ital 41:43-50, 2021). In this outbreak we found that few of these patients had bacterial invasive sinusitis mimicking fungal sinusitis. Amphotericin the only effective drug against Mucormycosis is highly toxic and expensive and not indicated in bacterial sinusitis. Our aim was to  determine the exact etiologic agent, predisposing factors and outcome of treatment of COVID associated invasive sinusitis presenting with orbital complications. It is a retrospective observational study done in 33 patients with orbital complications in COVID associated invasive sinusitis. Demographic details of the patients and clinical presentation were documented. Rhinological examination was done and a nasal swab was taken for KOH mount along with Gram`s stain and Culture and Sensitivity. All Patients underwent radiological evaluation by contrast enhanced computed tomography (CECT) or MRI. Liposomal Amphotericin B was started. Surgical debridement done. Amphotericin-B was stopped in cases reported negative for fungal elements and antibiotics administered for two weeks. Outcome of treatment was documented. A total of 33 patients were included in the study. 48.5% patients were found to have bacterial infection and 27.3% patient's fungal infections and 24.2% mixed infections.Eschar formation, necrotic tissue, erosion of the lamina papyracea was seen in both Klebsiella (33.3%) and Staphylococcal infections (16.6%) similar to Mucor and mixed infections. Persistent opthalmoplegia and deterioration of vision was associated with Mucor and mixed infections. However improvement in proptosis, ptosis, ophthalmoplegia, and vision was observed in cases associated with bacterial invasive sinusitis. Invasive bacterial sinusitis was under diagnosed during second wave of COVID. Identification of invasive bacterial sinusitis can help in de-escalation of treatment.

Pulmonary Mucormycosis: Beyond Classic COVID-19-Associated Fungal Infections.

Coronavirus disease 2019 (COVID-19) is often linked to a broad range of opportunistic bacterial and fungal infections. The second wave of the COVID-19 pandemic has witnessed an unprecedented surge in mucormycosis cases, predominantly in India, while the disease remained relatively rare in Europe. The authors describe the case of a 62-year-old female patient admitted to the hospital for consolidation therapy with chemotherapy as a part of the treatment protocol for acute myeloid leukemia. During hospitalization, she was diagnosed with nosocomial COVID-19, which later progressed to respiratory deterioration. COVID-19 with bacterial superinfection was presumed, leading to the initiation of empirical antibiotic therapy. A bronchoscopy was performed several days later due to a lack of improvement, revealing an infection by the Rhizopus microsporus complex. Despite antifungal treatment, the patient experienced an unfavorable clinical course and ultimately died. Given the high index of suspicion required to diagnose pulmonary mucormycosis, which can lead to delays in appropriate treatment and increase the burden of disease, the authors are aiming to enhance its awareness.

Nitrogen-doped carbon quantum dots as a novel treatment for black fungal bone infections (Mucormycosis): in vitro and in vivo study.

Most fungal bone and joint infections (arthritis) are caused by Mucormycosis (Mucor indicus). These infections may be difficult to treat and may lead to chronic bone disorders and disabilities, thus the use of new antifungal materials in bone disorders is vital, particularly in immunocompromised individuals, such as those who have contracted coronavirus disease 2019 (COVID-19). Herein, we reported for the first time the preparation of nitrogen-doped carbon quantum dots (N/CQDs) and a nitrogen-doped mesoporous carbon (N/MC) using a quick micro-wave preparation and hydrothermal approach. The structure and morphology were analysed using X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM) and surface area analyser. Minimum inhibitory concentration (MIC), disc diffusion tests, minimum fungicidal concentration (MFC) and antifungal inhibitory percentages were measured to investigate the antifungal activity of N/CQDs and N/MC nanostructures. In addition to the in vivo antifungal activity in rats as determined by wound induction and infection, pathogen count and histological studies were also performed. According to in vitro and in vivo testing, both N/CQDs with small size and N/MC with porous structure had a significant antifungal impact on a variety of bone-infecting bacteria, including Mucor infection. In conclusion, the present investigation demonstrates that functional N/CQDs and N/MC are effective antifungal agents against a range of microbial pathogenic bone disorders in immunocompromised individuals, with stronger and superior fungicidal activity for N/CQDs than N/MC in vitro and in vivo studies.

Production of polyunsaturated fatty acids in pork backfat fermented by Mucor circinelloides.

Pork backfat (PB) contains excessive saturated fatty acids (SFAs), but lacks polyunsaturated fatty acids (PUFAs). Excessive SFAs can be used as a substrate for the growth of certain microorganisms that convert them into PUFAs and monounsaturated fatty acids (MUFAs), and the added value of PB can be enhanced. In this study, Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189 were co-cultured for conversion of PB into fermented pork backfat (FPB) with high level of PUFAs. Our results showed that the content of γ-linolenic acid (GLA) and linoleic acid (LA) in the surface of FPB reached 9.04 ± 0.14 mg/g and 107.31 ± 5.16 mg/g for 7-day fermentation, respectively. To convert the internal SFAs of PB, ultrasound combined with papain was used to promote the penetrative growth of M. circinelloides into the internal PB, and the GLA level in the third layer of fat reached 2.58 ± 0.31 mg/g FPB. The internal growth of M. circinelloides in PB was promoted by adjusting the oxygen rate and ventilation rate through the wind velocity sensor. When the oxygen rate is 2 m/s and the ventilation rate is 18 m3/h, the GLA level in the third layer of fat reached 4.13 ± 1.01 mg/g FPB. To further improve the level of PUFAs in PB, FPB was produced by M. circinelloides at 18 °C. The GLA content on the surface of FPB reached 15.73 ± 1.13 mg/g FPB, and the GLA yield in the second and third layers of fat reached 8.68 ± 1.77 mg/g FPB and 6.13 ± 1.28 mg/g FPB, the LA yield in the second and third layers of fat reached 105.45 ± 5.01 mg/g FPB and 98.46 ± 4.14 mg/g FPB, respectively. These results suggested that excessive SFAs in PB can be converted into PUFAs and provided a new technique for improving PUFAs in FPB. KEY POINTS: • This article achieved the conversion of PUFAs in pork backfat by Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189. • This article solved the internal growth of M. circinelloides CBS277.49 in pork backfat by ultrasound combined with papain. • This article proposed an innovative of promoting the internal growth of M. circinelloides and increasing the PUFAs production by oxygen ventilation in pork backfat.

Silkworm pupae protein co-degradation by magnetic nanoparticles immobilized proteinase K and Mucor circinelloides aspartic protease for further utilization of sericulture by-products.

Silkworm pupae, by-product of sericulture industry, is massively discarded. The degradation rate of silkworm pupae protein is critical to further employment, which reduces the impact of waste on the environment. Herein, magnetic Janus mesoporous silica nanoparticles immobilized proteinase K mutant T206M and Mucor circinelloides aspartic protease were employed in the co-degradation. The thermostability of T206M improved by enhancing structural rigidity (t1/2 by 30 min and T50 by 5 °C), prompting the degradation efficiency. At 65 °C and pH 7, degradation rate reached the highest of 61.7%, which improved by 26% compared with single free protease degradation. Besides, the immobilized protease is easy to separate and reuse, which maintains 50% activity after 10 recycles. Therefore, immobilized protease co-degradation was first applied to the development and utilization of silkworm pupae resulting in the release of promising antioxidant properties and reduces the environmental impact by utilizing a natural and renewable resource.

First insights on the susceptibility of native coccidicidal fungi Mucor circinelloides and Mucor lusitanicus to different avian antiparasitic drugs.

BACKGROUND: The combined application of predatory fungi and antiparasitic drugs is a sustainable approach for the integrated control of animal gastrointestinal (GI) parasites. However, literature addressing the possible interference of antiparasitic drugs on the performance of these fungi is still scarce. This research aimed to assess the in vitro susceptibility of six native coccidicidal fungi isolates of the species Mucor circinelloides and one Mucor lusitanicus isolate to several antiparasitic drugs commonly used to treat GI parasites' infections in birds, namely anthelminthics such as Albendazole, Fenbendazole, Levamisole and Ivermectin, and anticoccidials such as Lasalocid, Amprolium and Toltrazuril (drug concentrations of 0.0078-4 µg/mL), using 96-well microplates filled with RPMI 1640 medium, and also on Sabouraud Agar (SA). RESULTS: This research revealed that the exposition of all Mucor isolates to the tested anthelminthic and anticoccidial drug concentrations did not inhibit their growth. Fungal growth was recorded in RPMI medium, after 48 h of drug exposure, as well as on SA medium after exposure to the maximum drug concentration. CONCLUSIONS: Preliminary findings from this research suggest the potential compatibility of these Mucor isolates with antiparasitic drugs for the integrated control of avian intestinal parasites. However, further in vitro and in vivo studies are needed to confirm this hypothesis.