New lineages of RNA viruses from clinical isolates of Rhizopus microsporus revealed by fragmented and primer-ligated dsRNA sequencing (FLDS) analysis.

Rhizopus microsporus is a species in the order Mucorales that is known to cause mucormycosis, but it is poorly understood as a host of viruses. Here, we examined 25 clinical strains of R. microsporus for viral infection with a conventional double-stranded RNA (dsRNA) assay using agarose gel electrophoresis (AGE) and the recently established fragmented and primer-ligated dsRNA sequencing (FLDS) protocol. By AGE, five virus-infected strains were detected. Then, full-length genomic sequences of 12 novel RNA viruses were revealed by FLDS, which were related to the families Mitoviridae, Narnaviridae, and Endornaviridae, ill-defined groups of single-stranded RNA (ssRNA) viruses with similarity to the established families Virgaviridae and Phasmaviridae, and the proposed family "Ambiguiviridae." All the characterized viruses, except a potential phasmavirid with a negative-sense RNA genome, had positive-sense RNA genomes. One virus belonged to a previously established species within the family Mitoviridae, whereas the other 11 viruses represented new species or even new genera. These results show that the fungal pathogen R. microsporus harbors diverse RNA viruses and extend our understanding of the diversity of RNA viruses in the fungal order Mucorales, division Mucoromycota. Identifying RNA viruses from clinical isolates of R. microsporus may expand the repertoire of natural therapeutic agents for mucormycosis in the future.IMPORTANCEThe diversity of mycoviruses in fungal hosts in the division Mucoromycota has been underestimated, mainly within the species Rhizopus microsporus. Only five positive-sense RNA genomes had previously been discovered in this species. Because current sequencing methods poorly complete the termini of genomes, we used fragmented and primer-ligated double-stranded RNA sequencing to acquire the full-length genomes. Eleven novel mycoviruses were detected in this study, including the first negative-sense RNA genome reported in R. microsporus. Our findings extend the understanding of the viral diversity in clinical strains of Mucoromycota, may provide insights into the pathogenesis and ecology of this fungus, and may offer therapeutic options.

Oral Isavuconazole Combined with Nebulized Inhalation and Bronchoscopic Administration of Amphotericin B for the Treatment of Pulmonary Mucormycosis: A Case Report and Literature Review.

Pulmonary mucormycosis (PM) is an invasive and potentially fatal fungal infection, with Rhizopus microsporus (R. microsporus) being the most common pathogen. The routine therapy for this infection includes surgery and antifungal agents. However, the therapeutic effects of single agents are unsatisfactory due to the rapid progression of mucormycosis, while not all patients can tolerate surgery. Innovative treatment methods like combination therapy await validations of their clinical efficacy. We report a case of PM that was diagnosed via metagenomics next-generation sequencing (mNGS) of black drainage fluid from the patient's lung. The patient eventually recovered and was discharged after a combination therapy of oral isavuconazole, inhaled amphotericin B, and local perfusion of amphotericin B through bronchoscopy, which may be a promising strategy for the treatment of PM, especially for cases where surgery is not possible. A retrospective study of 297 cases in a literature review highlights the different treatment methods used in clinical practice.

Deazaflavin metabolite produced by endosymbiotic bacteria controls fungal host reproduction.

The endosymbiosis between the pathogenic fungus Rhizopus microsporus and the toxin-producing bacterium Mycetohabitans rhizoxinica represents a unique example of host control by an endosymbiont. Fungal sporulation strictly depends on the presence of endosymbionts as well as bacterially produced secondary metabolites. However, an influence of primary metabolites on host control remained unexplored. Recently, we discovered that M. rhizoxinica produces FO and 3PG-F420, a derivative of the specialized redox cofactor F420. Whether FO/3PG-F420 plays a role in the symbiosis has yet to be investigated. Here, we report that FO, the precursor of 3PG-F420, is essential to the establishment of a stable symbiosis. Bioinformatic analysis revealed that the genetic inventory to produce cofactor 3PG-F420 is conserved in the genomes of eight endofungal Mycetohabitans strains. By developing a CRISPR/Cas-assisted base editing strategy for M. rhizoxinica, we generated mutant strains deficient in 3PG-F420 (M. rhizoxinica ΔcofC) and in both FO and 3PG-F420 (M. rhizoxinica ΔfbiC). Co-culture experiments demonstrated that the sporulating phenotype of apo-symbiotic R. microsporus is maintained upon reinfection with wild-type M. rhizoxinica or M. rhizoxinica ΔcofC. In contrast, R. microsporus is unable to sporulate when co-cultivated with M. rhizoxinica ΔfbiC, even though the fungus was observed by super-resolution fluorescence microscopy to be successfully colonized. Genetic and chemical complementation of the FO deficiency of M. rhizoxinica ΔfbiC led to restoration of fungal sporulation, signifying that FO is indispensable for establishing a functional symbiosis. Even though FO is known for its light-harvesting properties, our data illustrate an important role of FO in inter-kingdom communication.

Rhizopus microsporus and Mucor racemosus coinfection following COVID-19 detected by metagenomics next-generation sequencing: A case of disseminated mucormycosis.

Mucormycosis is an invasive opportunistic fungal infection, which may be lethal and mostly affects patients with immunodeficiency or diabetes mellitus. Among Mucorales fungi, Rhizopus spp. is the most common cause of mucormycosis, followed by genera such as Mucor and Lichtheimia. Here we report a patient with severe COVID-19 infection who developed nasal pain, facial swelling, prominent black eschar on the nasal root. CT scan revealed pansinusitis along the maxillary, ethmoidal, and sphenoid sinuses. Mixed mold infection with Rhizopus microsporus and Mucor racemosus was detected by blood metagenomics next-generation sequencing (mNGS) and later nasal mucosa histological investigation confirmed mucormycosis. Severe COVID-19 infection led to the patient's thrombocytopenia and leukopenia. Later disseminated mucormycosis aggravated the infection and sepsis eventually resulted in death. It is the first case report of mucormycosis in which R. microsporus and M. racemosus as the etiologic agents were found simultaneously in one patient. COVID-19 infection combined with disseminated mucormycosisis can be fatal and mNGS is a fast, sensitive and accurate diagnostic method for fungi detection.

Miltefosine: A Repurposing Drug against Mucorales Pathogens.

Mucorales are a group of non-septated filamentous fungi widely distributed in nature, frequently associated with human infections, and are intrinsically resistant to many antifungal drugs. For these reasons, there is an urgent need to improve the clinical management of mucormycosis. Miltefosine, which is a phospholipid analogue of alkylphosphocholine, has been considered a promising repurposing drug to be used to treat fungal infections. In the present study, miltefosine displayed antifungal activity against a variety of Mucorales species, and it was also active against biofilms formed by these fungi. Treatment with miltefosine revealed modifications of cell wall components, neutral lipids, mitochondrial membrane potential, cell morphology, and the induction of oxidative stress. Treated Mucorales cells also presented an increased susceptibility to SDS. Purified ergosterol and glucosylceramide added to the culture medium increased miltefosine MIC, suggesting its interaction with fungal lipids. These data contribute to elucidating the effect of a promising drug repurposed to act against some relevant fungal pathogens that significantly impact public health.

Transcription activator-like effectors from endosymbiotic bacteria control the reproduction of their fungal host.

IMPORTANCE: Interactions between fungi and bacteria are critically important in ecology, medicine, and biotechnology. In this study, we shed light on factors that promote the persistence of a toxin-producing, phytopathogenic Rhizopus-Mycetohabitans symbiosis that causes severe crop losses in Asia. We present an unprecedented case where bacterially produced transcription activator-like (TAL) effectors are key to maintaining a stable endosymbiosis. In their absence, fungal sporulation is abrogated, leading to collapse of the phytopathogenic alliance. The Mycetohabitans TAL (MTAL)-mediated mechanism of host control illustrates a unique role of bacterial effector molecules that has broader implications, potentially serving as a model to understand how prokaryotic symbionts interact with their eukaryotic hosts.

Case Report and Literature Review of Prosthetic Cardiovascular Mucormycosis.

We report a rare case of aorto-bi-iliac prosthetic allograft mucormycosis in a 57-year-old immunocompetent patient in France. Outcome was favorable after surgery and dual antifungal therapy with liposomal amphotericin B and isavuconazole. In a literature review, we identified 12 other cases of prosthetic vascular or heart valve mucormycosis; mortality rate was 38%.

Invasive splenic mucormycosis due to Rhizopus microsporus during chemotherapy for acute monocytic leukemia: a case report and literature review.

Mucormycosis is a rare opportunistic fungal infection associated with high mortality that typically occurs in immunocompromised patients. It is difficult to diagnose owing to non-specific clinical manifestations, the serologic index, imaging features, and the limitations of diagnostic methods. The incidence of invasive splenic mucormycosis is extremely rare, with only a few cases documented in the literature. We report a survival case of invasive splenic mucormycosis involving the liver caused by Rhizopus microsporus in a patient during consolidation therapy for acute monocytic leukemia (AML-M5). The patient initially presented with recurrent fever and splenomegaly accompanied by multiple focal hypodensities unresponsive to empiric anti-infective treatment. Splenic mucormycosis was diagnosed by Contrast-Enhanced Ultrasonography (CEUS) and metagenomic next-generation sequencing (mNGS). However, surgical intervention carries a high risk due to the progressive involvement of the liver in invasive splenic mucormycosis. Fortunately, monotherapy with amphotericin B was effective, and the patient underwent allo-HSCT. This case aims to emphasize the importance of utilizing mNGS and CEUS for the timely diagnosis of mucormycosis to help clinicians identify splenic mucormycosis and initiate appropriate therapy as soon as possible to improve therapeutic efficacy and prognosis.

Chronic pulmonary mucormycosis caused by rhizopus microsporus mimics lung carcinoma in an immunocompetent adult: A case report.

BACKGROUND: Pulmonary mucormycosis is a rare but life-threatening invasive fungal infection that mostly affects immunocompromised patients. This disease usually develops acutely and progresses rapidly, often leading to a poor clinical prognosis. Chronic pulmonary mucormycosis is highly unusual in immunocompetent patients. CASE SUMMARY: A 43-year-old man, who was a house improvement worker with a long history of occupational dust exposure, presented with an irritating cough that had lasted for two months. The patient was previously in good health, without dysglycemia or any known immunodeficiencies. Chest computed tomography revealed a mass in the left lower lobe, measuring approximately 6 cm in diameter, which was suspected to be primary lung carcinoma complicated with obstructive pneumonia. Thoracoscopic-assisted left lower lobectomy was performed, and metagenomic next-generation sequencing detection, along with special pathological staining of surgical specimens, suggested Rhizopus microsporus infection. Postoperatively, the patient's respiratory symptoms were relieved, and no signs of recurrence were found during the six-month follow-up. CONCLUSION: This article reports a rare case of chronic pulmonary mucormycosis caused by Rhizopus microsporus in a middle-aged male without dysglycemia or immunodeficiency. The patient's surgical outcome was excellent, reaffirming that surgery remains the cornerstone of pulmonary mucormycosis treatment.

Volatile organic compounds mediated endogenous microbial interactions in Chinese baijiu fermentation.

Microorganisms release abundant volatile organic compounds, and these compounds have important roles in distant interactions and communication. However, the effects of microbial volatile organic compounds on microbial interactions remains unclear in traditional food fermentations. In this study, we applied high-throughput sequencing analysis to reveal the structure and succession of microbial community in the preparations of Qu starter for Chinese baijiu fermentation. The genus Weissella, Pediococcus, Lactobacillus, Saccharomycopsis, Saccharomyces, Candida and Rhizopus were dominant and varied in this preparation, and the abundant Rhizopus had a positive correlation with Lactobacillus in situ system. In addition, benzyl alcohol produced by Rhizopus microsporus could significantly promote the growth of Lactobacillus fermentum in a simulative baijiu fermentation under laboratory. Moreover, the addition of benzyl alcohol could enhance the activation of alanine, aspartate and glutamate metabolism and arginine biosynthesis for promoting the growth of Lactobacillus fermentum through transcriptional analysis. Therefore, this study revealed volatile organic compounds from microorganisms could meditate the endogenous microbial interactions in Chinese baijiu fermentation. Furthermore, it provides a potential approach to add or remove volatile organic compounds to regulate the microbial interactions, for improving traditional food fermentations.

Genetic Manipulation in Mucorales and New Developments to Study Mucormycosis.

The study of the Mucoralean fungi physiology is a neglected field that the lack of effective genetic tools has hampered in the past. However, the emerging fungal infection caused by these fungi, known as mucormycosis, has prompted many researchers to study the pathogenic potential of Mucorales. The main reasons for this current attraction to study mucormycosis are its high lethality, the lack of effective antifungal drugs, and its recent increased incidence. The most contemporary example of the emergence character of mucormycosis is the epidemics declared in several Asian countries as a direct consequence of the COVID-19 pandemic. Fortunately, this pressure to understand mucormycosis and develop new treatment strategies has encouraged the blossoming of new genetic techniques and methodologies. This review describes the history of genetic manipulation in Mucorales, highlighting the development of methods and how they allowed the main genetic studies in these fungi. Moreover, we have emphasized the recent development of new genetic models to study mucormycosis, a landmark in the field that will configure future research related to this disease.

Systemic mucormycosis caused by Rhizopus microsporus in a captive bottlenose dolphin.

A 6-year-old female bottlenose dolphin (Tursiops truncatus) kept in dolphinarium died after a 3.5-month period of lethargy and inappetence despite antibiotics and supportive care. At necropsy, gross findings included diffuse varying-sized nodules in the lungs and scattered nodules throughout the heart, spleen, mesenteric and hilar lymph node and kidney. Microscopically, the lesions were characterised by disseminated fungal pyogranulomas with numerous intralesional Mucor-like fungi. The fungi structures were demonstrated by Periodic acid-Schiff and Gomori methenamine silver stain. Molecular analyses of the fungi were Rhizopus microsporus by PCR sequencing 18S ribosomal RNA gene. Ziehl-Neelsen stain failed to show acid-fast bacterial infection. Based on pathological and molecular examination, systemic granulomatous mucormycosis was diagnosed. To our knowledge, this is the first reported case of systemic mucormycosis caused by Rhizopus microsporus in bottlenose dolphin.

Heterologous expression and biochemical characterization of a cold-active lipase from Rhizopus microsporus suitable for oleate synthesis and bread making.

OBJECTIVES: Cold-active lipases which show high specific activity at low temperatures are attractive in industrial applications in terms of product stability and energy saving. We aimed to identify novel cold-active lipase suitable for oleates synthesis and bread making. RESULTS: A novel lipase gene (RmLipA) from Rhizopus microsporus was cloned and heterologously expressed in Pichia pastoris. The encoding sequence displayed 75% identity to the lipase from R. niveus. The highest extracellular lipase activity of 7931 U/mL was achieved in a 5-L fermentation. The recombinant enzyme (RmLipA) was optimally active at pH 8.0 and 20-25 °C, respectively, and stable over a wide pH range of 2.0-11.0. The enzyme was a cold-active lipase, exhibiting > 80% of its maximal activity at 0 °C. RmLipA was a sn-1,3 regioselective lipase, and preferred to hydrolyze pNP esters and triglycerides with relatively long chain fatty acids. RmLipA synthesized various oleates using oleic acid and different alcohols as substrates (> 95%). Moreover, it significantly improved the quality of bread by increasing its specific volume (21.7%) and decreasing its crumb firmness (28.6%). CONCLUSIONS: A novel cold-active lipase gene from R. microsporus was identified, and its application potentials were evaluated. RmLipA should be a potential candidate in oleates synthesis and bread making industries.

Mucormycosis after Coronavirus disease 2019 infection in a heart transplant recipient - Case report and review of literature.

Mucormycosis is an invasive fungal infection (IFI) due to several species of saprophytic fungi, occurring in patients with underlying co-morbidities (including organ transplantation). During the ongoing Coronavirus disease 2019 (COVID-19) pandemic, there have been increasing reports of bacterial and fungal co-infections occurring in COVID-19 patients, including COVID-19 associated pulmonary aspergillosis (CAPA). We describe a case of mucormycosis occurring after COVID-19, in an individual who received a recent heart transplant for severe heart failure. Two months after heart transplant, our patient developed upper respiratory and systemic symptoms and was diagnosed with COVID-19. He was managed with convalescent plasma therapy and supportive care. Approximately three months after COVID-19 diagnosis, he developed cutaneous mucormycosis at an old intravascular device site. He underwent extensive surgical interventions, combined with broad-spectrum antifungal therapy. Despite the aggressive therapeutic measures, he died after a prolonged hospital stay. In this case report, we also review the prior well-reported cases of mucormycosis occurring in COVID-19 patients and discuss potential mechanisms by which COVID-19 may predispose to IFIs. Similar to CAPA, mucormycosis with COVID-19 may need to be evaluated as an emerging disease association. Clinicians should be vigilant to evaluate for invasive fungal infections such as mucormycosis in patients with COVID-19 infection.

Stable and reproducible homologous recombination enables CRISPR-based engineering in the fungus Rhizopus microsporus.

Mucormycosis is a lethal and emerging disease that has lacked a genetic model fulfilling both high virulence and the possibility of performing stable and reproducible gene manipulation by homologous recombination (HR). Here, we developed a new methodology to successfully perform HR in Rhizopus microsporus. We isolated an uracil auxotrophic recipient strain and optimized the critical steps in the genetic transformation of this fungus. This was followed by an adaptation of a plasmid-free CRISPR-Cas9 system coupled with microhomology repair templates. We reproducibly generated stable mutants in the genes leuA and crgA, encoding a 3-isopropylmalate dehydratase and an ubiquitin ligase, respectively. Our new genetic model showed that mutations in the gene pyrF, a key virulence gene in several bacterial and fungal pathogens, correlated with an avirulent phenotype in an immunocompetent murine host. This was reverted by gene complementation, showing the broad possibilities of our methodology.

Rhizopus microsporus lung infection in an immunocompetent patient successfully treated with amphotericin B: A case report.

BACKGROUND: Rhizopus microsporus (R. microsporus) lung infection is an invasive fungal disease with high mortality that is increasingly common in immunocompromised patients. However, it is very rare in immunocompetent patients. Here, we present the case of a 19-year-old girl who developed R. microsporus lung infection without any known immunodeficiency. CASE SUMMARY: The patient presented to our hospital because of hemoptysis and irritative cough without expectoration. She was first treated for community-acquired pneumonia until the detection of R. microsporus in bronchoalveolar lavage fluid by metagenomics next-generation sequencing (mNGS). After a combination therapy of intravenous inhalation and local airway perfusion of amphotericin B, she eventually recovered, with significant absorption of lung infections. CONCLUSION: Early diagnosis and treatment are very important for pulmonary mucormycosis. Compared to fungal culture, mNGS is a relatively precise and convenient method to obtain pathogenic results. A combination therapy of intravenous inhalation and local airway perfusion of amphotericin B may be a promising strategy for the treatment of pulmonary mucormycosis in the future.

Gastrointestinal mucormycosis due to Rhizopus microsporus following Streptococcus pyogenes toxic shock syndrome in an HIV patient: a case report.

BACKGROUND: Gastrointestinal (GI) mucormycosis is a rare and often deadly form of mucormycosis. Delayed diagnosis can lead to an increased risk of death. Here, we report a case of GI mucormycosis following streptococcal toxic shock syndrome in a virologically suppressed HIV-infected patient. CASE PRESENTATION: A 25-year-old Thai woman with a well-controlled HIV infection and Grave's disease was admitted to a private hospital with a high-grade fever, vomiting, abdominal pain, and multiple episodes of mucous diarrhea for 3 days. On day 3 of that admission, the patient developed multiorgan failure and multiple hemorrhagic blebs were observed on all extremities. A diagnosis of streptococcal toxic shock was made before referral to Siriraj Hospital - Thailand's largest national tertiary referral center. On day 10 of her admission at our center, she developed feeding intolerance and bloody diarrhea due to bowel ischemia and perforation. Bowel resection was performed, and histopathologic analysis of the resected bowel revealed acute suppurative transmural necrosis and vascular invasion with numerous broad irregular branching non-septate hyphae, both of which are consistent with GI mucormycosis. Peritoneal fluid fungal culture grew a grayish cottony colony of large non-septate hyphae and spherical sporangia containing ovoidal sporangiospores. A complete ITS1-5.8S-ITS2 region DNA sequence analysis revealed 100% homology with Rhizopus microsporus strains in GenBank (GenBank accession numbers KU729104 and AY803934). As a result, she was treated with liposomal amphotericin B. However and in spite of receiving appropriate treatment, our patient developed recurrent massive upper GI bleeding from Dieulafoy's lesion and succumbed to her disease on day 33 of her admission. CONCLUSION: Diagnosis of gastrointestinal mucormycosis can be delayed due to a lack of well-established predisposing factors and non-specific presenting symptoms. Further studies in risk factors for abdominal mucormycosis are needed.

Molecular Dialogues between Early Divergent Fungi and Bacteria in an Antagonism versus a Mutualism.

Fungal-bacterial symbioses range from antagonisms to mutualisms and remain one of the least understood interdomain interactions despite their ubiquity as well as ecological and medical importance. To build a predictive conceptual framework for understanding interactions between fungi and bacteria in different types of symbioses, we surveyed fungal and bacterial transcriptional responses in the mutualism between Rhizopus microsporus (Rm) (ATCC 52813, host) and its Mycetohabitans (formerly Burkholderia) endobacteria versus the antagonism between a nonhost Rm (ATCC 11559) and Mycetohabitans isolated from the host, at two time points, before and after partner physical contact. We found that bacteria and fungi sensed each other before contact and altered gene expression patterns accordingly. Mycetohabitans did not discriminate between the host and nonhost and engaged a common set of genes encoding known as well as novel symbiosis factors. In contrast, responses of the host versus nonhost to endobacteria were dramatically different, converging on the altered expression of genes involved in cell wall biosynthesis and reactive oxygen species (ROS) metabolism. On the basis of the observed patterns, we formulated a set of hypotheses describing fungal-bacterial interactions and tested some of them. By conducting ROS measurements, we confirmed that nonhost fungi increased production of ROS in response to endobacteria, whereas host fungi quenched their ROS output, suggesting that ROS metabolism contributes to the nonhost resistance to bacterial infection and the host ability to form a mutualism. Overall, our study offers a testable framework of predictions describing interactions of early divergent Mucoromycotina fungi with bacteria.IMPORTANCE Animals and plants interact with microbes by engaging specific surveillance systems, regulatory networks, and response modules that allow for accommodation of mutualists and defense against antagonists. Antimicrobial defense responses are mediated in both animals and plants by innate immunity systems that owe their functional similarities to convergent evolution. Like animals and plants, fungi interact with bacteria. However, the principles governing these relations are only now being discovered. In a study system of host and nonhost fungi interacting with a bacterium isolated from the host, we found that bacteria used a common gene repertoire to engage both partners. In contrast, fungal responses to bacteria differed dramatically between the host and nonhost. These findings suggest that as in animals and plants, the genetic makeup of the fungus determines whether bacterial partners are perceived as mutualists or antagonists and what specific regulatory networks and response modules are initiated during each encounter.

A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host.

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. A btl19-13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.

Rhizoferrin Glycosylation in Rhizopus microsporus.

Rhizopus spp. are the most common etiological agents of mucormycosis, causing over 90% mortality in disseminated infections. The diagnosis relies on histopathology, culture, and/or polymerase chain reaction. For the first time, the glycosylation of rhizoferrin (RHF) was described in a Rhizopus microsporus clinical isolate by liquid chromatography and accurate tandem mass spectrometry. The fermentation broth lyophilizate contained 345.3 ± 13.5, 1.2 ± 0.03, and 0.03 ± 0.002 mg/g of RHF, imido-RHF, and bis-imido-RHF, respectively. Despite a considerable RHF secretion rate, we did not obtain conclusive RHF detection from a patient with disseminated mucormycosis caused by the same R. microsporus strain. We hypothesize that parallel antimycotic therapy, RHF biotransformation, and metabolism compromised the analysis. On the other hand, the full profile of posaconazole metabolites was retrieved by our in house software CycloBranch.