In vitro combination with doxycycline plus antifungals against clinical Mucorales pathogens.

PURPOSE: Since systematic antifungals for mucormycosis showed variable MICs depending on strains, effective and safe antifungal therapy was still needed. This study is aimed to evaluate the in vitro activity of doxycycline combined with antifungal therapy against dominant Mucorales pathogens. METHODS: Multidrug susceptibility testing was performed with doxycycline and antifungals, including itraconazole, posaconazole, and amphotericin, in 21 isolates of 8 dominant Mucorales pathogens. RESULTS: The fractional inhibitory concentration index according to M38 showed one Rhizopus arrhizus isolate synergic (∑FICI = 0.375) and other isolates in addition (0.5 < ∑FICI < 4). CONCLUSIONS: Doxycycline was found to have in vitro advantages in combined antifungal treatment over antifungals alone.

First Report of Mucor irregularis Causing Flower Rot on Selenicereus undatus in China.

Dragon fruit (Selenicereus undatus (Haw.) D.R.Hunt is a famous tropical fruit (Korotkova et al. 2017). In May 2021, a flower rot disease was found on Dragon fruit in a field (21˚19'42''N, 110˚28'32''E), Zhanjiang, Guangdong Province, China. The incidence rate was approximately 30% (n=500 investigated plants from about 30 hectares). Flower rot was evident, and was light brown, watery, soft, and covered with white mycelia. The pathogen could continue to infect the fruit during the fruit ripening stage with about 20% rot rate. Ten samples of symptomatic flowers were collected in the field. Margins of the diseased tissue were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Three representative isolates (HUM-1,HUM-2, and HUM-3) by single-spore isolation were randomly selected for further study. Colonies on PDA were circular with massive aerial hyphae, white to ochraceous in color. Nonseptate hyphae were hyaline. Sporangiophores arose from hyphae. Sporangiospores were hyaline, smooth-walled, mostly subspherical to ellipsoidal, and measured 3.15 to 6.55 µm × 1.35 to 2.85 µm (n =50). Morphological characteristics of isolates were consistent with the description of Mucor irregularis (Lima et al. 2018). Molecular identification was done using the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) used to amplify the internal transcribed spacer (ITS) region and large subunit (LSU) with ITS1/ITS4 and LR0R1/LR5 (Vilgalys et al. 1990). The amplicons were sequenced and the sequences were deposited in GenBank with accession numbers ITS, OL376751-OL376753, and LSU, OM672239-OM672241. BLAST analysis of these sequences revealed a 100% identity with M. irregularis in GenBank. The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered with M. irregularis (the type strain CBS 103.93).The pathogenicity was tested through in vivo experiments. Nine healthy flowers of Dragon fruit were inoculated with 3-day-old mycelial plugs (5 × 5 mm) of isolates, while another five healthy flowers were treated with PDA plugs (controls). Those plugs were embedded inside the calyxes, and each flower was inoculated with one plug in one calyx. Besides, the inoculated and control flowers (n = 5) were sprayed with a spore suspension (1 × 105 per mL) of the three isolates individually and sterile distilled water, respectively, until run-off (Feng and Li. 2019). The plants were grown in pots in a greenhouse at 28°C, with relative humility approximately 80%. The test was repeated three times. After 3 days of incubation, rot symptoms developed on the inoculated flowers, which were similar to those observed on the naturally samples in the field. The control flowers remained healthy. The fungus was reisolated from the inoculated flowers and confirmed as M. irregularis by morphology and ITS analysis. M. irregularis was reported as a pathogen causing human skin diseases and post-harvest diseases of crop (Álvarez et al. 2011; Lima et al. 2018; Wang et al. 2022). This is the first report of M. irregularis causing flower rot of Dragon fruit and reduce yield in China. This research can provide a theoretical basis for the fruit industry to maintain yield.

Rhino-Orbital-Cerebral Mycosis and Extranodal Natural Killer or/and T-Cell Lymphoma, Nasal Type.

Background: Extranodal natural killer/T-cell lymphoma, nasal type is a syndrome of middle face destruction with an association to Epstein-Barr virus. Fungi have been recovered from the diseased tissue now and then but were often seen as a lymphoma-associated secondary infection. However, there are ENKTL-NT cases with the recoveries of fungi and complete recovery with antifungal therapy, which are quite similar to rhino-orbital-cerebral mycosis (ROCM) that often confuses the physicians. Methods: We searched Medline for English-language manuscripts limited to "human" and "case reports," "letters," "reviews," and "clinical conferences" from 1966 to 2022. We used MeSH terms "lymphoma, extranodal nk-t-cell" [MeSH Terms] or "lethal midline granuloma" [MeSH Terms], in combination with MeSH terms "microbiology" [subheading] or "microbiology" [all fields] or "fungi" [all fields] or "fungi" [MeSH Terms] for ENKTL-NT with infections. We used MeSH terms "Mycoses" in combination with "Nose" [Mesh] OR "Orbital Diseases" [Mesh] for rhino-orbital-cerebral fungal infections. Results: We appraised 149 included articles and extracted references related to ENKTL-NT and/or ROCM. Themes and subcategories were subsequently derived. Our findings revealed that ROCM and ENKTL-NT are characterized by progressive and destructive ulcers in the midline face or rhino-orbital structures. ROCM is mainly caused by fungi in the order of Mucorales, and ENKTL-NT is usually associated with Epstein-Barr virus and sometimes fungi. Radiologically, both are characterized by non-specific features of sinusitis, soft tissue infection, and necrosis. Pathologically, ROCM and ENKTL-NT share the same characteristics of inflammation, necrosis, and granuloma. ROCM is characterized by the detection of fungi in tissue, while ENKTL-NT is typically positive for NK/T-cell markers and cytotoxic granule-associated proteins, proliferation, and vascular damage of angioinvasion, which could be incited by Mucor irregularis and Rhizopus arrhizus in patients and mice. Conclusion: ENKTL-NT and ROCM share many similarities in clinical presentations, radiology, and histopathology, and might have the same etiology. This may explain why the two diseases are tangled together in the reported cases, and suggests the role that the fungi may play in the development of these ENKTL-NT/ROCM diseases. The reason why ENKTL-NT and ROCM are sometimes confused is that the main pathogens of ROCM, Mucor irregularis and Rhizopus arrhizus, are the fungal causative agents of ENKTL-NT.

A thermal adaptation landscape related to virulence in Mucor irregularis transcriptional profiles.

OBJECTIVES: Our study aimed to better understand the different thermal adaptation in Mucor irregularis (M. irregularis) strains under high temperature and the involved virulence-related genes, and to offer more appropriate explanation for the diverse pathogenicity of M. irregularis in human infections. METHODS: M. irregularis isolates were incubated at 30 and 35°C for Illumina HiSeq technology (RNA-seq), as well as the virulence difference detected through Galleria mellonella infection models. We verified their transcriptional profile with RT-PCR and analysed differentially expressed genes with GO and KEGG annotations. RESULTS: All 25 isolates formed the biggest colonies at 28°C and did not grow at 37°C, while were differently inhibited at 22 and 35°C. Six selected M. irregularis displayed virulence in sync with their growth condition at high temperature. From the outcomes of RNA-seq, a total of 1559 differentially expressed genes (FC ≥ 2, FDR < 0.05) were obtained, of which 1021 genes were upregulated, and 538 genes were downregulated. Cell wall structure genes related to Ras-like and GH16 proteins, influx-efflux pumps consist of transmembrane proteins as ABC and MFS proteins, and metabolic genes as DGKɛ and Hsfs, seem to be essential in thermal adaptation and virulence of M. irregularis. CONCLUSION: We found some common genes expressed at high temperature, while some others specifically related to M. irregularis isolates with different virulence and thermal adaptation. Further research of genes involved in the pathogenic process is needed for the development of potential targeted antifungal.

Characterization of mitogenomes from four Mucorales species and insights into pathogenicity.

BACKGROUND: Mucorales, as one major order of Zygomycetes fungi, can infect human beings and cause serious consequence. We have noticed the pathogenicity of Mucorales is closely related to energy metabolism, while mitochondria play the role of energy factories in almost all biological activities. METHODS: Virulence of M irregularis, M hiemalis, L corymbifera and R arrhizus were verified in Galleria mellonella larvae, as well as mitochondrial gene copies analysed with RT-qPCR. Mitogenomes of the four Mucorales species were sequenced based on illumina NovaSeq technology to study their characteristic features and functional regions. RESULTS: Variant virulence of M irregularis, M hiemalis, L corymbifera and R arrhizu were verified by clinical retrospective data and our G mellonella infection models, also copies of mitochondrial genes indicated the significant associations with pathogenicity. A total of 274.18 clean reads were generated to be assembled; the complete mitogenomes of the four Mucorales species were obtained with totally different length. After the genomes annotated and compared, M irregularis was found more similar with M hiemalis than those of L corymbifera and R arrhizus, especially the small (rrns) and large (rrnl) subunits of mitochondrial ribosomal RNA (rRNA) genes. The GC content, ncRNAs and the distribution of the SNPs and InDels were also compared, and the GC content rate of fungi seems to be related to the fungal thermal adaptability. In addition, linear mitogenomes of these four Mucorales showed diverse arrangements of orf genes and directionality of some conserved gene elements. CONCLUSION: This study uncovered the pathogenicity variances among the four Mucorales species and the relationship between their mitogenomic features and clinical pathogenicity. Further studies like spatial structure of mitochondrial genomes and the comprehensive analysis of transcription regulation are needed.

Vacuum-Assisted Closure and Skin Grafting Combined with Amphotericin B for Successful Treatment of an Immunocompromised Patient with Cutaneous Mucormycosis Caused by Mucor irregularis: A Case Report and Literature Review.

Cutaneous mucormycosis caused by Mucor irregularis (M. irregularis) is a rare condition that typically occurs in immunocompetent patients. Herein, we describe an immunocompromised patient with cutaneous M. irregularis infection who was successfully treated with debridement combined with vacuum assisted closure (VAC) negative pressure technique and split-thickness skin grafting. We present this case owing to its complexity and rarity and the successful treatment with surgical therapy. A 58-year-old man presented to our hospital with a history of skin ulcers and eschar on the right lower leg since two months. He had been receiving methylprednisolone therapy for bullous pemphigoid that occurred five months prior to the present lesions. Histopathological examination of a right leg lesion showed broad, branching hyphae in the dermis. Fungal culture and subsequent molecular cytogenetic analysis identified the pathogen as M. irregularis. After admission, methylprednisolone was gradually tapered and systemic treatment with amphotericin B (total dose 615 mg) initiated along with others supportive therapies. However, the ulcers showed no improvement, and amphotericin B had to be discontinued owing to development of renal dysfunction. After extensive surgical debridement combined with VAC and skin grafting, his skin ulcers were healed; subsequent fungal cultures of the lesions were negative. The patient exhibited no signs of recurrence at 36-month follow-up. Twenty-six cases with M. irregularis-associated cutaneous mucormycosis in literature were reviewed.

In Vivo and In Vitro Impairments in T Helper Cell and Neutrophil Responses against Mucor irregularis in Card9 Knockout Mice.

Mucor irregularis is a frequently found fungus in Asia, especially China, and it causes primary cutaneous mucormycosis with a high rate of disfigurement. Caspase recruitment domain-containing protein 9 (Card9) is an essential adaptor molecule downstream of C-type lectin receptors. It mediates the activation of nuclear factor kappa B (NF-κB), regulates T helper 1 (Th1) and Th17 differentiation, and plays an important role in fungal immune surveillance. CARD9 deficiency correlates with the increased susceptibility to many fungal infections, including cutaneous mucormycosis caused by M. irregularis However, the underlying immunological mechanisms were not elucidated. Our study established a murine model of subcutaneous M. irregularis infection, and we isolated immune cells, including bone marrow-derived macrophages, bone marrow-derived dendritic cells, naive T cells, and neutrophils, from wild-type (WT) and Card9 knockout (Card9-/- ) mice to examine the antifungal effect of Card9 on M. irregularis in vivo and in vitroCard9-/- mice exhibited increased susceptibility to M. irregularis infection. Impaired local cytokine and chemokine production, NF-κB (p65) activation, and Th1/17 cell differentiation and partially impaired neutrophil-dependent antifungal immunity were observed in Card9-/- mice. This work enriches our knowledge of the relationship between CARD9 deficiency and mucormycosis.

Biodegradation of fluorene by the newly isolated marine-derived fungus, Mucor irregularis strain bpo1 using response surface methodology.

Fluorene, a low molecular weight polycyclic aromatic hydrocarbon (PAH), is of immense environmental interest because of its carcinogenicity, teratogenicity, mutagenicity, toxicity and persistence to microbial degradation. Existentially, there is paucity of information on PAH degradation by fungi isolated from marine environment. Therefore, this study investigated fluorene degradation efficiency of marine derived filamentous fungus, Mucor irregularis strain bpo1 (GenBank Accession Number: MK373020). Response Surface Methodology (RSM) using Box-Behnken Design (BBD) was successfully deployed in the optimization of process parameters (pH-7, temperature-32.5 °C, substrate concentration-100 mg L-1 and dry weight-2 g) resulting in 81.50% fluorene degradation on 5th day. The design and regression model were found to be statistically significant, adequate and appropriate with p < 0.0001, F value= 202.39, and predicted coefficient of determination (R2 =0.9991). Optimization of the vital constituents of the mineral salt medium (MSM) used for the study using RSM-Central Composite Design (CCD) resulted in 79.80% fluorene degradation rate. Enhanced fluorene degradation efficiency (82.50%) was recorded when the optimized process variables were subjected to growth-linked validation experiments. The enzyme activities revealed 87%, 59% and 31% induction of laccase, manganese peroxidase and lignin peroxidase respectively. Four metabolites; 9H-fluoren-9-one, benzene-1,2-dicarboxylic acid, 2-hydroxybenzoic acid and phenol obtained after the experiment were characterized and confirmed with GC-MS analysis. The findings revealed the promising potentials of M. irregularis in PAH degradation and by extension green remediation technology.

Disfiguring Mucor irregularis Infection Cured by Amphotericin B and Itraconazole: A Case Report and Treatment Experience.

We report a case of primary cutaneous mucormycosis caused by Mucor irregularis. A 52-year-old male farmer was presented to our hospital with a history of progressive nodule and plaque with ulceration on the face for two and a half years. Broad, aseptate hyphae were seen in direct KOH examination and biopsy. Fungal culture showed light yellow filamentous colonies. The rRNA sequencing revealed M. irregularis was the responsible fungus. Amphotericin B in gradually increasing dose and itraconazole were administered. When the cumulative dose of amphotericin B was 1500 mg, the skin lesion improved significantly with remaining scars on the face. Then, the patient received sequential oral itraconazole treatment for 8 months. There was no recurrence up to now through follow-ups.

Successful Treatment by Surgery of a Primary Cutaneous Mucormycosis Caused by Mucor irregularis.

We report a case of primary cutaneous mucormycosis caused by Mucor irregularis. A 66-year-old man was presented to our hospital with a history of gradually enlarging plaque on the right leg for about a year. The identification of pathogen based on the fungus morphology and DNA sequencing revealed M. irregularis as the responsible fungus for skin lesion. The lesion was removed incidentally by a surgery procedure, and no recrudescence was seen during a follow-up of 24-month observation.

Fast technique for the identification of Gilbertella persicaria via optical microscopy.

Gilbertella persicaria is an important phytopathogen that is confused with Mucor spp. and Rhizopus spp. The main distinguishing characteristic of G. persicaria is the presence of appendages in sporangiospores, and their observation by conventional staining techniques generally fails. A technique is described using light microscopy for fast and reliable diagnosis.

Characterization of Two New Records of Mucoralean Species Isolated from Gut of Soldier Fly Larva in Korea.

While surveying the diversity of fungi of the order Mucorales, two isolates, EML-PUKI12-1 and EML-PUKI06-1, were obtained from the gut of soldier fly larvae inhabiting the bulrush at a pond located in the Chonnam National University Arboretum, Gwangju, Korea. The isolates were confirmed as Mucor irregularis and Mucor fragilis species, respectively, based on the morphological characteristics and phylogenetic analysis of rDNA internal transcribed spacer region. Such mucoralean species belonging to undiscovered taxa has not previously been described in Korea.

Primary cutaneous mucormycosis caused by Mucor irregularis in an elderly person.

Primary cutaneous mucormycosis is a rare but often lethal severe fungal infection, which usually occurs in immunocompromised patients. We report a case of primary cutaneous mucormycosis caused by Mucor irregularis in an elderly patient. Seven months after the surgical dissection of the involved skin, cutaneous mucormycosis recurred at the peripheral edge of the skin graft. Shortly subsequent to the administration of liposomal amphotericin B, the remaining skin lesion was excised again. M. irregularis is rarely but increasingly reported as a cause of mucormycosis in immunocompetent individuals, especially in Asian farmers. M. irregularis may be largely disseminated in the soils of Asia and thus the trivial trauma at the time of farm work may be a trigger for the onset. These cases tend to leave severe cosmetic damage even in healthy individuals, although the vital prognosis is not affected.

Taxonomy and epidemiology of Mucor irregularis, agent of chronic cutaneous mucormycosis.

Mucormycosis usually presents as a progressive infection with significant angio-invasion. Mucormycosis due to Mucor irregularis (formerly Rhizomucor variabilis var. variabilis), however, is exceptional in causing chronic cutaneous infection in immunocompetent humans, ultimately leading to severe morbidity if left untreated. More than 90 % of the cases known to date were reported from Asia, mainly from China. The nearest neighbour of M. irregularis is the saprobic species M. hiemalis. The aim of this study was to evaluate the taxonomic position, epidemiology, and intra- and inter-species diversity of M. irregularis based on 21 strains (clinical n = 17) by multilocus analysis using ITS, LSU, RPB1 and RPB2 genes, compared to results of cluster analysis with amplified fragment length polymorphism (AFLP) data. By combining MLST and AFLP analyses, M. irregularis was found to be monophyletic with high bootstrap support, and consisted of five subgroups, which were not concordant in all partitions. It was thus confirmed that M. irregularis is a single species at 96.1-100 % ITS similarity and low recombination rates between populations. Some geographic structuring was noted with some localised populations, which may be explained by limited air-dispersal. The natural habitat of the species is likely to be in soil and decomposing plant material.