Mucor thermorhizoides-A New Species from Post-mining Site in Sudety Mountains (Poland).

Mucor representatives are mostly rapidly growing cosmopolitan soil saprotrophs of early diverged Mucoromycotina subphylum. Although this is the most speciose genus within the group, some lineages are still understudied. In this study, new species of Mucor was isolated from the post-mining area in southwestern Poland, where soil chemical composition analysis revealed high concentration of hydrocarbons and heavy metals. Phylogenetic analysis based on multigene phylogeny showed that the new isolate clusters distinctly from other Mucor species as a sister group to Mucor microsporus. New species Mucor thermorhizoides Abramczyk (Mucorales, Mucoromycota) is characterized by the extensive rhizoid production in elevated temperatures and formation of two layers of sporangiophores. It also significantly differs from M. microsporus in the shape of spores and the size of sporangia. M. thermorhizoides was shown to be able to grow in oligotrophic conditions at low temperatures. Together with M. microsporus they represent understudied and highly variable lineage of the Mucor genus.

Penicillin G acylase production by Mucor griseocyanus and the partial genetic analysis of its pga gene.

Penicillin acylases (penicillin amidohydrolase, EC 3.5.1.11) are a group of enzymes with many applications within the pharmaceutical industry, and one of them is the production of semi-synthetic beta-lactam antibiotics. This enzyme is mainly produced by bacteria but also by some fungi. In the present study, the filamentous fungus Mucor griseocyanus was used to produce penicillin acylase enzyme (PGA). Its ability to express PGA enzyme in submerged fermentation process was assessed, finding that this fungal strain produces the biocatalyst of interest in an extracellular way at a level of 570 IU/L at 72 h of fermentation; in this case, a saline media using lactose as carbon source and penicillin G as inducer was employed. In addition, a DNA fragment (859 bp) of the pga from a pure Mucor griseocyanus strain was amplified, sequenced, and analyzed in silico. The partial sequence of pga identified in the fungi showed high identity percentage with penicillin G acylase sequences deposited in NCBI through BLAST, especially with the ß subunit of PGA from the Alcaligenes faecalis bacterium¸ which is a region involved in the catalytic function of this protein. Besides, the identification of domains in the penicillin G acylase sequence of Mucor griseocyanus showed three conserved regions of this protein. The bioinformatic results support the identity of the gen as penicillin G acylase. This is the first report that involves sequencing and in silico analysis of Mucor griseocyanus strain gene encoding PGA.

Mitochondrial Citrate Transport System in the Fungus Mucor circinelloides: Identification, Phylogenetic Analysis, and Expression Profiling During Growth and Lipid Accumulation.

The mitochondrial citrate transport system, composed of citrate and malate transporters (MTs), can regulate the citrate efflux from mitochondria to cytosol, and then citrate is cleaved into OAA and acetyl-CoA which can be used for fatty acid (FA) biosynthesis. However, in the fungus Mucor circinelloides the molecular mechanism of citrate efflux from the mitochondria by this system and its role in FA synthesis is unclear. In the present study, we have analyzed the genome of high lipid-producing strain WJ11 and the low lipid-producing strain CBS 277.49 to find the potential genes involving in this system. Five potential genes are present in the genome of WJ11. These genes encode one citrate transport protein (CT), one tricarboxylate carrier (TCT), one MT, and two 2-oxoglutarate:malate antiporters (SoDIT-a and SoDIT-b). However, the genome of CBS 277.49 contains the same set of genes, except for the presence of just one SoDIT. The proteins from WJ11 had similar properties as their counterparts in CBS 277.49. Moreover, phylogenetic analyses revealed the evolutionary relationship of these proteins and illuminated their typical motifs related to potential functions. Additionally, the expression of these genes was analyzed to predict the possible functions in lipid metabolism in M. circinelloides. This is the first study to report the in silico analysis of structures and functions of the mitochondrial citrate transport system in M. circinelloides. This work showed a new strategy for research for the selection of candidate genes for further detailed functional investigation of the mitochondrial citrate transport system in lipid accumulation.

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.

Mucor chuxiongensis sp. nov., a novel fungal species isolated from rotten wood.

Three strains of a novel mucoralean fungus were isolated from samples of decayed wood, which were collected from three locations near the city of Chuxiong, Yunnan province, China. These isolates were identified as a novel species through comparison of sequences in the ITS sequence, the D1/D2 domains of the LSU rRNA gene, and other taxonomic characteristics. The results demonstrated that these isolates represent a novel mucoralean fungus species belonging to the genus Mucor. The ITS sequence of Mucor chuxiongensissp. nov. differed from its closest relative, Mucor guilliermondii CBS 174.27T, by 13.1 % sequence divergence (39 substitutions and 38 gaps), and the D1/D2 sequences of the novel strains differed by 13 nt substitutions and one gap (1.9 %) from the ex-type strain. On potato dextrose agar, malt extract agar and synthetic mucor agar, the isolates grew slowly below 10 °C, rapidly at 25-30 °C, and could not grow well at 35 °C. The holotype strain of Mucor chuxiongensis sp. nov. is NYNU 174111 (CICC 41666T=CBS 143707T).

Outbreak of Invasive Wound Mucormycosis in a Burn Unit Due to Multiple Strains of Mucor circinelloides f. circinelloides Resolved by Whole-Genome Sequencing.

Mucorales are ubiquitous environmental molds responsible for mucormycosis in diabetic, immunocompromised, and severely burned patients. Small outbreaks of invasive wound mucormycosis (IWM) have already been reported in burn units without extensive microbiological investigations. We faced an outbreak of IWM in our center and investigated the clinical isolates with whole-genome sequencing (WGS) analysis. We analyzed M. circinelloides isolates from patients in our burn unit (BU1, Hôpital Saint-Louis, Paris, France) together with nonoutbreak isolates from Burn Unit 2 (BU2, Paris area) and from France over a 2-year period (2013 to 2015). A total of 21 isolates, including 14 isolates from six BU1 patients, were analyzed by whole-genome sequencing (WGS). Phylogenetic classification based on de novo assembly and assembly free approaches showed that the clinical isolates clustered in four highly divergent clades. Clade 1 contained at least one of the strains from the six epidemiologically linked BU1 patients. The clinical isolates were specific to each patient. Two patients were infected with more than two strains from different clades, suggesting that an environmental reservoir of clonally unrelated isolates was the source of contamination. Only two patients from BU1 shared one strain, which could correspond to direct transmission or contamination with the same environmental source. In conclusion, WGS of several isolates per patients coupled with precise epidemiological data revealed a complex situation combining potential cross-transmission between patients and multiple contaminations with a heterogeneous pool of strains from a cryptic environmental reservoir.IMPORTANCE Invasive wound mucormycosis (IWM) is a severe infection due to environmental molds belonging to the order Mucorales. Severely burned patients are particularly at risk for IWM. Here, we used whole-genome sequencing (WGS) analysis to resolve an outbreak of IWM due to Mucor circinelloides that occurred in our hospital (BU1). We sequenced 21 clinical isolates, including 14 from BU1 and 7 unrelated isolates, and compared them to the reference genome (1006PhL). This analysis revealed that the outbreak was mainly due to multiple strains that seemed patient specific, suggesting that the patients were more likely infected from a pool of diverse strains from the environment rather than from direct transmission among them. This study revealed the complexity of a Mucorales outbreak in the settings of IWM in burn patients, which has been highlighted based on WGS combined with careful sampling.

The gut mycobiome composition is linked to carotid atherosclerosis.

The mycobiotic component of the microbiota comprises an integral, yet under-researched, part of the gastrointestinal tract. Here, we present a preliminary study of the possible contribution of gut mycobiota to sub-clinical atherosclerosis in a well-characterised group of obese and non-obese subjects in association with the Framingham Risk Score (FRS) and carotid intima-media thickness (cIMT). From all taxa identified, the relative abundance of the phylum Zygomycota, comprising the family Mucoraceae and genus Mucor, was negatively associated with cIMT and this association remained significant after controlling for false discovery rate. Obese subjects with detectable Mucor spp. had a similar cardiovascular risk profile as non-obese subjects. Interestingly, the relative abundance of Mucor racemosus was negatively associated both with FRS and cIMT. Partial least square discriminant analyses modelling, evaluating the potential relevance of gut mycobiota in patients stratified by mean values of cIMT, showed that even a 1 component model had a high accuracy (0.789), with a high R2 value (0.51). Variable importance in projection scores showed that M. racemosus abundance had the same impact in the model as waist-to-hip ratio, high-density lipoprotein-cholesterol, fasting triglycerides or fasting glucose, suggesting that M. racemosus relative abundance in the gut may be a relevant biomarker for cardiovascular risk.

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.

Antioxidants Potential of the Filamentous Fungi (Mucor circinelloides).

Three important strains of Mucorcircinelloides grown in complete and minimal media for specified period (72 h, 120 h and 168 h) under submerged fermentation conditions were investigated for their potential antioxidants/secondary metabolite production. All mycelial extracts demonstrated effective antioxidant activities in terms of ß-carotene/linoleic acid bleaching, radical scavenging, reduction of metal ions and chelating abilities against ferrous ions. Different extraction methods and solvent systems affected the recovery yield and antioxidant activities of the extracts significantly (p ≤ 0.05). Ethanolic extracts were found to be rich source of antioxidant components and subsequently more effective in antioxidant properties. Fermentation period and media used also significantly affected (p ≤ 0.05) the antioxidant production and the resulting antioxidant properties. The (ethanolic) extracts of all the strains from late exponential growth phase (120 h) showed highest antioxidant production with topmost reducing, chelating and radical scavenging capabilities. Strain MC277.49 was found to be the highest producer of antioxidants followed by MC108.16 and WJ11. Phenolic compounds were detected significantly in higher (p ≤ 0.05) amount succeeded by the condensed tannins and flavonoids. Total phenol content of each extract was attributed to overall antioxidant capacity. Submerged fermentation with nutritional stress conditions were found to be excellent way of producing surplus amount of natural antioxidants/secondary metabolites with their vast potential commercial application in food and pharmaceutical industries.

Phylogenetic analysis reveals two genotypes of the emerging fungus Mucor indicus, an opportunistic human pathogen in immunocompromised patients.

Mucormycosis is a rare fungal infection caused by Mucor indicus. Phylogenetic analysis of many M. indicus isolates, mainly sampled from different clinical and environmental specimens collected worldwide, revealed two genotypes, I and II, based on ITS and D1/D2 LSU rDNA sequences. A retrospective review of the literature revealed 13 cases. Eight (76.9%) patients had disseminated infections, and the overall mortality rate was 30.7%. A pulmonary infection caused by M. indicus genotype I in a liver transplant recipient was disseminated to include the skin and was successfully treated with liposomal amphotericin B and aggressive surgery. M. indicus can infect a wide variety of patients with no real preference for the site of infection. We concluded that M. indicus has emerged as a significant cause of invasive mycosis in severely immunocompromised patients worldwide. Early diagnosis and initiation of appropriate therapy could enhance survival in these immunocompromised patient populations.

Comparative Proteome Analysis between High Lipid-Producing Strain Mucor circinelloides WJ11 and Low Lipid-Producing Strain CBS 277.49.

Mucor circinelloides is one of few oleaginous fungi that produces a useful oil rich in γ-linolenic acid, but it usually only produces <25% total lipid. Nevertheless, we isolated a new strain WJ11 that can produce up to 36% lipid of cell dry weight. In this study, we have systematically analyzed the global changes in protein levels between the high lipid-producing strain WJ11 and the low lipid-producing strain CBS 277.49 (15%, lipid/cell dry weight) at lipid accumulation phase through comparative proteome analysis. Proteome analysis demonstrated that the branched-chain amino acid and lysine metabolism, glycolytic pathway, and pentose phosphate pathway in WJ11 were up-regulated, while the activities of tricarboxylic acid cycle and branch point enzyme for synthesis of isoprenoids were retarded compared with CBS 277.49. The coordinated regulation at proteome level indicate that more acetyl-CoA and NADPH are provided for fatty acid biosynthesis in WJ11 compared with CBS 277.49.

A new mercury-accumulating Mucor hiemalis strain EH8 from cold sulfidic spring water biofilms.

Here, we report about a unique aquatic fungus Mucor hiemalisEH8 that can remove toxic ionic mercury from water by intracellular accumulation and reduction into elemental mercury (Hg0 ). EH8 was isolated from a microbial biofilm grown in sulfidic-reducing spring water sourced at a Marching's site located downhill from hop cultivation areas with a history of mercury use. A thorough biodiversity survey and mercury-removal function analyses were undertaken in an area of about 200 km2 in Bavaria (Germany) to find the key biofilm and microbe for mercury removal. After a systematic search using metal removal assays we identified Marching spring's biofilm out of 18 different sulfidic springs' biofilms as the only one that was capable of removing ionic Hg from water. EH8 was selected, due to its molecular biological identification as the key microorganism of this biofilm with the capability of mercury removal, and cultivated as a pure culture on solid and in liquid media to produce germinating sporangiospores. They removed 99% of mercury from water within 10-48 h after initial exposure to Hg(II). Scanning electron microscopy demonstrated occurrence of intracellular mercury in germinating sporangiospores exposed to mercury. Not only associated with intracellular components, but mercury was also found to be released and deposited as metallic-shiny nanospheres. Electron-dispersive x-ray analysis of such a nanosphere confirmed presence of mercury by the HgMα peak at 2.195 keV. Thus, a first aquatic eukaryotic microbe has been found that is able to grow even at low temperature under sulfur-reducing conditions with promising performance in mercury removal to safeguard our environment from mercury pollution.

Effect of temperature, pH, and water activity on Mucor spp. growth on synthetic medium, cheese analog and cheese.

The Mucor genus includes a large number of ubiquitous fungal species. In the dairy environment, some of them play a technological role providing typical organoleptic qualities to some cheeses while others can cause spoilage. In this study, we compared the effect of relevant abiotic factors for cheese production on the growth of six strains representative of dairy technological and contaminant species as well as of a non cheese related strain (plant endophyte). Growth kinetics were determined for each strain in function of temperature, water activity and pH on synthetic Potato Dextrose Agar (PDA), and secondary models were fitted to calculate the corresponding specific cardinal values. Using these values and growth kinetics acquired at 15 °C on cheese agar medium (CA) along with three different cheese types, optimal growth rates (µopt) were estimated and consequently used to establish a predictive model. Contrarily to contaminant strains, technological strains showed higher µopt on cheese matrices than on PDA. Interestingly, lag times of the endophyte strain were strongly extended on cheese related matrices. This study offers a relevant predictive model of growth that may be used for better cheese production control but also raises the question of adaptation of some Mucor strains to the cheese.

Lysine and Glutamic Acids as the End Products of Multi-response of Optimized Fermented Medium by Mucor mucedo KP736529.

BACKGROUND: Amino acids are important for living organisms, they acting as crucial for metabolic activities and energy generation, wherein the deficiency in these amino acids cause various physiological defects. OBJECTIVE: The aim of this study is to investigate the effect of some nutritional factors on the amino acids production by Mucor mucedo KP736529 during fermentation intervals. METHODOLOGY: Mucor mucedo KP736529 was selected according to proteolytic activity. Corn steep liquor and olive cake were used in the fermented medium during Placket-Burman and central composite design to maximize the production of lysine and glutamic acids. RESULTS: During the screening by Plackett-Burman design, olive cake and Corn Steep Liquor (CSL) had potential importance for the higher production of amino acids. The individual fractionation of total amino acids showed both lysine and glutamic as the major amino acids associated with the fermentation process. Moreover, the Central Composite Design (CCD) has been adopted to explain the interaction between olive cake and CSL on the production of lysine and glutamic acids. The model recorded significant F-value, with high values of R 2, adjusted R 2 and predicted R 2 for both lysine and glutamic, indicating the validity of the data. Solving equation for maximum production of lysine recorded theoretical levels of olive cake and CSL, being 2.58 and 1.83 g L -1, respectively, with predicting value of lysine at 1.470 µg mL -1, whereas the predicting value of glutamic acid reached 0.805 mg mL -1 at levels of 2.49 and 1.93 g L -1 from olive cake and CSL, respectively. The desirability function (D) showed the actual responses being 1.473±0.009 and 0.801±0.004 µg mL -1 for lysine and glutamic acids, respectively. CONCLUSION: The model showed adequate validity to be applied in a large-scale production of both lysine and glutamic acids.

Obesity changes the human gut mycobiome.

The human intestine is home to a diverse range of bacterial and fungal species, forming an ecological community that contributes to normal physiology and disease susceptibility. Here, the fungal microbiota (mycobiome) in obese and non-obese subjects was characterized using Internal Transcribed Spacer (ITS)-based sequencing. The results demonstrate that obese patients could be discriminated by their specific fungal composition, which also distinguished metabolically "healthy" from "unhealthy" obesity. Clusters according to genus abundance co-segregated with body fatness, fasting triglycerides and HDL-cholesterol. A preliminary link to metabolites such as hexadecanedioic acid, caproic acid and N-acetyl-L-glutamic acid was also found. Mucor racemosus and M. fuscus were the species more represented in non-obese subjects compared to obese counterparts. Interestingly, the decreased relative abundance of the Mucor genus in obese subjects was reversible upon weight loss. Collectively, these findings suggest that manipulation of gut mycobiome communities might be a novel target in the treatment of obesity.

Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides.

Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates ß-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of ß-carotene (i.e. zeaxanthin and ß-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of ß-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than ß-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and ß-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the ß-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene.

Rapid screening for human-pathogenic Mucorales using rolling circle amplification.

Mucormycosis has emerged as a relatively common severe mycosis in patients with haematological and allogeneic stem cell transplantation. Source of transmission is from unidentified sources in the environment. Early diagnosis of infection and its source of contamination are paramount for rapid and appropriate therapy. In this study, rolling circle amplification (RCA) is introduced as a sensitive, specific and reproducible isothermal DNA amplification technique for rapid molecular identification of six of the most virulent species (Rhizopus microsporus, R. arrhizus var. arrhizus, R. arrhizus var. delemar, Mucor irregularis, Mucor circinelloides, Lichtheimia ramosa, Lichtheimia corymbifera). DNAs of target species were successfully amplified, with no cross reactivity between species. RCA can be considered as a rapid detection method with high specificity and sensitivity, suitable for large screening.

Molecular characterization and in vitro antifungal susceptibility of 80 clinical isolates of mucormycetes in Delhi, India.

Mucormycosis is a highly aggressive disease which is usually fatal in immunocompromised patients. The species of mucormycetes show significant differences in susceptibility to amphotericin B, azoles and terbinafine. The precise species level identification for this fungal group could be achieved by internal transcribed-spacer (ITS) region sequencing. Herein, we present the largest series of antifungal susceptibility data of molecularly characterised isolates of mucormycetes reported so far from India. Eighty isolates originating from 71 patients comprised 50 (62.5%) from pulmonary cases, 15 (19%) from rhino-orbital-cerebral, 13 (16.2%) from cutaneous and 2 (2.5%) from disseminated mucormycosis. ITS and D1/D2 regions sequencing of the isolates identified, Rhizopus arrhizus var. delemar (n = 25), R. arrhizus var. arrhizus (n = 15), R. microsporus (n = 17), R. stolonifer (n = 3), Syncephalastrum racemosum (n = 11), Apophysomyces elegans (n = 2), A. variabilis (n = 2), Lichtheimia ramosa (n = 3) and Mucor circinelloides f. lusitanicus (n = 2). Amplified fragment length polymorphism analysis was done to genotype Rhizopus isolates and revealed 5 clusters of R. arrhizus, which were well separated from R. microsporus. Amphotericin B was the most potent antifungal followed by posaconazole, itraconazole and isavuconazole. Etest and CLSI MICs of amphotericin B showed 87% agreement. Overall, the commonest underlying condition was uncontrolled diabetes mellitus. Records of 54 patients revealed fatalities in 28 cases.

Mucor irregularis infection around the inner canthus cured by amphotericin B: a case report and review of published literatures.

We report a case of primary cutaneous mucormycosis caused by Mucor irregularis. A 47-year-old farmer was presented to our clinic with the history of progressive red plaque around the inner canthus following dacryocystectomy about a year earlier. Linear, aseptate hyphae were seen by direct KOH examination and in biopsy. Fungal culture revealed light yellow filamentous colonies that were identified as Mucor irregularis by nucleotide sequencing of rRNA gene. Amphotericin B and dexamethasone were used in gradually increasing dosage. The treatment lasted 43 days, and the patient received 760 mg total amphotericin B. The patient was discharged after 2 months of treatment. The plaque became smooth, and fungal culture was negative. There was no recurrence for half a year through telephone follow-ups. A review of published studies revealed 23 cases of Mucor irregularis infection. Most cases resulted following injuries or surgical complications. Farmers and manual laborers were most at risk with males outnumbering females among patients. Amphotericin B and its liposomal preparations remain most effective treatment choices.

Primary cutaneous mucormycosis presenting as a giant plaque: uncommon presentation of a rare mycosis.

Mucormycosis is an uncommon systemic mycosis affecting the immunocompromised individuals. It is usually caused by organisms of the genera Rhizopus and Mucor, although rarely other organisms have also been implicated. Mycoses due to these angioinvasive fungi have an acute onset, rapidly progressive course with high mortality rate. A rare and less well known is the chronic subtype of primary cutaneous mucormycosis (PCM). Herein, we report a case of PCM clinically presenting as a chronic, giant destructive plaque in a young immunocompetent male and coin the term chronic granulomatous mucormycosis. A clinicopathological classification for cutaneous mucormycosis is also proposed.