The effect of the use of commercial tempeh starter on the diversity of Rhizopus tempeh in Indonesia.

At present, only a single Rhizopus species, R. microsporus, can be found in fresh tempeh produced in Java, Indonesia. The loss of diversity of Rhizopus in tempeh has been associated with the widespread use of commercial tempeh starter in Indonesia since the 2000s. However, the identities of the previous Rhizopus strains associated with tempeh, which have been preserved in a culture collection in Indonesia, have not been verified. The present study aimed to verify the identities of 22 Rhizopus strains isolated from tempeh produced using the traditional tempeh starters from the 1960s to the 2000s. Phylogenetic analysis based on the ITS regions in the rRNA gene sequence data, revealed that the Rhizopus strains belonged to the species R. arrhizus (five strains); R. delemar (14 strains); and R. microsporus (three strains). Verification of the identities of these Rhizopus strains in the present study confirmed the loss of diversity of Rhizopus species in tempeh produced in Indonesia, particularly in Java. Our findings confirmed that the morphological changes in Rhizopus species isolated from tempeh as a result of centuries of domestication.

Molecular epidemiology of zygomycosis and their related factors in tertiary referral centers in southern Iran.

INTRODUCTION: For the best management of the zygomycosis in immunocompromised patients, the present study aims to detect and identify the etiologic agents by DNA sequencing method and their related factors in clinical samples of patients. METHODOLOGY: Clinical samples from 1,058 patients admitted in 11 university hospitals in Shiraz, Southern Iran were collected between July 2015 and July 2018. All samples (bronchoalveolar lavage, sputum, blood, tissue) were examined by routine microscopic and culture tests for zygomycetes. The etiologic agents were identified by the molecular method and sequencing. RESULTS: Direct microscopic examinations or pathology smear, culture, and PCR were positive in 61 (5.8%), 15 (1.4%), and 103 (9.7%) patients, respectively. According to EORTC/MSG criteria, the rates of proven, probable, and possible zygomycosis were 59.2% (61/103), 14.6% (15/103), and 26.2% (27/103 patients), respectively. The most prevalent etiologic agents according to sequencing were Rhizopus oryzae (44 cases), Rhizopus microsporus (31 cases), Rhizopus stolonifer (15 cases). Twenty-two patients (21.4%) with positive PCR died. There were significant relations between zygomycosis and the underlying disease (p = 0.043) and prior antifungal therapy (p = 0.023). White blood cell count was in the normal range in 14.1% of patients, and the means of erythrocyte sedimentation rate (ESR) and C reactive protein (CRP) were 65 mm/hour and 57 mg/L, respectively. CONCLUSIONS: Molecular methods and sequencing may have considered as suitable tools to diagnose zygomycosis. Identification of the etiologic agents may be considered as the future antifungal therapy and management of the respective patients.

Microsatellite analysis of the population structure in Rhizopus arrhizus.

OBJECTIVES: Rhizopus arrhizus is recognized as an emergent agent of superficial and invasive mucormycosis. Despite an increasing number of these infections, the molecular epidemiology of Rhizopus species has not been well studied. MATERIALS AND METHODS: In this study, 43 R. arrhizus strains (25 environmental and 18 clinical isolates) were genotyped using six novel panels of microsatellite markers. RESULTS: Upon the analysis of 43 isolates, 4-8 distinct alleles were detected for each marker. The discriminatory power for the individual markers ranged from 0·522 to 0·830. The combination of all six markers yielded 33 different haplotypes with a high degree of discrimination (0·989 D value). A four-marker combination were selected as the most parsimonious panel achieving D > 0·95. One clinical isolate and one environmental isolate shared the same genotype suggesting the possible nosocomial outbreak of mucormycosis in hospitalized patients. We have noted that the strains isolated from cutaneous mucormycosis were different from the strains isolated from rhino-orbito-cerebral mucormycosis. Then, the hypothesis of particular tropism of infectious strains for a given site is not excluded. The standardized indices of association IA and rBarD were significantly different from zero (P < 0·01), suggesting a prevailing clonal reproduction. The environmental population was significantly differentiated from clinical populations (Fst = 0·2249). CONCLUSIONS: Microsatellite typing method described in our study showed an excellent degree of discriminatory power. It is a promising tool for illuminating the molecular epidemiology of R. arrhizus species, including strain relatedness and transmission pathways.

Alterations in gut bacterial and fungal microbiomes are associated with bacterial Keratitis, an inflammatory disease of the human eye.

Dysbiosis, or imbalance in the gut microbiome, has been implicated in auto-immune, inflammatory, neurological diseases as well as in cancers. More recently it has also been shown to be associated with ocular diseases. In the present study, the association of gut microbiome dysbiosis with bacterial Keratitis, an inflammatory eye disease which significantly contributes to corneal blindness, was investigated. Bacterial and fungal gut microbiomes were analysed using fecal samples of healthy controls (HC, n = 21) and bacterial Keratitis patients (BK, n = 19). An increase in abundance of several antiinflammatory organisms including Dialister, Megasphaera, Faecalibacterium, Lachnospira, Ruminococcus and Mitsuokella and members of Firmicutes, Veillonellaceae, Ruminococcaceae and Lachnospiraceae was observed in HC compared to BK patients in the bacterial microbiome. In the fungal microbiome, a decrease in the abundance of Mortierella, Rhizopus, Kluyveromyces, Embellisia and Haematonectria and an increase in the abundance of pathogenic fungi Aspergillus and Malassezia were observed in BK patients compared to HC. In addition, heatmaps, PCoA plots and inferred functional profiles also indicated significant variations between the HC and BK microbiomes, which strongly suggest dysbiosis in the gut microbiome of BK patients. This is the first study demonstrating the association of gut microbiome with the pathophysiology of BK and thus supports the gut-eye axis hypothesis. Considering that Keratitis affects about 1 million people annually across the globe, the data could be the basis for developing alternate strategies for treatment like use of probiotics or fecal transplantation to restore the healthy microbiome as a treatment protocol for Keratitis.

Beneficial Effects of Dietary Tempeh Prepared with Rhizopus stolonifer on Liver Function in Rats Fed with a High-Fat Diet.

The regional standard for tempeh established by the Codex Alimentarius defines the use of Rhizopus oligosporus, R. oryzae, and/or R. stolonifer as soybean tempeh starters. However, comparative studies on the functions of tempeh prepared with these Rhizopus species are scarce. In the present study, we examined the effects of dietary tempeh prepared with these three Rhizopus species using rats fed with a high-fat diet. Compared to the control diet, consumption of tempeh prepared with R. stolonifer significantly suppressed serum levels of aspartate transaminase, total bilirubin, and ammonium (indices of liver function). However, less or no suppression was observed with tempeh prepared with R. oligosporus or R. oryzae. Serum levels of triglyceride, total cholesterol, HDL cholesterol, and glucose were unaffected. Liver levels of free cholesterol, a parameter relating to liver injury, were significantly decreased by the three types of the tempeh examined; however, there was no difference in the free cholesterol levels among the tempeh groups. We conclude that the ingestion of tempeh prepared with R. stolonifer might have beneficial effects pertaining to the liver function in rats fed with high-fat diets.

Mucormycosis outbreak due to Rhizopus microsporus after arthroscopic anterior cruciate ligament reconstruction surgery evaluated by RAPD and MALDI-TOF Mass spectrometry.

BACKGROUND: Rhizopus microsporus is one of the main causative agents of mucormycosis. These mycoses are mostly described as isolated cases involving uncontrolled diabetes mellitus or immunosuppressed patients. In this work we report a nosocomial outbreak of mucormycosis due to R. microsporum involving three young immunocompetent patients whom underwent arthroscopic anterior cruciate ligament reconstruction surgery in a seven-month time span. PROCEDURES: During the outbreak period, a total of 32 surgeries of this type were performed in the clinic (mucormycosis prevalence of 9.375%). The three patients presented healthcare-associated Mucormycosis comprising the bone surrounding one of the fixation screws (femoral or tibial). In addition to these three strains, another three R. microsporus strains isolated in the medical center during the same period of time were included in the study. One of these fungi was isolated from a skin lesion of a kidney transplant patient while the other two strains were isolated from environmental sources. Classical, mass spectrometry-based (MALDI-TOFF) and molecular identification were performed. Genetic relatedness was established by Rep-PCR (RAPD variant) and by single-linkage cluster analysis mass spectra. Cluster analysis was performed by unweighed pair group method with arithmetic mean (UPGMA). MAIN FINDINGS: All the strains were identified as R. microsporum by the used phenotypic and genetic tools. Clinical strains fell into 2 different clusters separating the renal transplant recipient strain from the three strains isolated post ACLR surgery, which clustered together. CONCLUSIONS: The established genetic/mass spectra relatedness between the three post-surgery isolates suggests that these cases may be considered a healthcare-associated mucormycosis outbreak.

Identification of Mucorales in patients with proven invasive mucormycosis by polymerase chain reaction in tissue samples.

BACKGROUND: Accurate diagnosis of mucormycosis, a life-threatening fungal infection, remains a challenge for physicians. OBJECTIVES: To identify the causative Mucorales in fresh clinical samples and formalin-fixed paraffin-embedded (FFPE) samples of patients with proven mucormycosis by molecular method. PATIENTS/METHODS: Fresh clinical samples of patients with proven mucormycosis according to the EORTC/MSG criteria admitted between 2015 and 2017 and histopathologically proven FFPE archives collected during 2004-2007 and 2015-2017 from Mazandaran University-affiliated hospitals of northern Iran were included. Seminested PCR targeting the 18S rDNA of Mucorales and ITS region was performed, and PCR products were then sequenced. RESULTS: While culture was positive only in 5 of 9 (56%) of fresh specimen cases, PCR was positive in all 9 (100%) histologically proven mucormycosis. Ten of 18 (56%) FFPE samples were PCR-positive. Overall, Mucorales PCR was positive in 19 of 27 (70%) samples. Mucorales species were Rhizopus arrhizus in 16 (84%) cases, R. arrhizus/Amylomyces rouxii in 2 (10.5%) cases and Rhizopus stolonifer in one case (5.5%). Among 27 mucormycosis cases, 25 (93%) cases were rhinocerebral, and 2 (7%) cases were disseminated. Diabetes mellitus (74%) and neutropaenia (63%) were the main risk factors. CONCLUSIONS: Seminested PCR targeting 18S rDNA region of Mucorales is useful for identification of the causative agents of mucormycosis.

Molecular Confirmation of the Linkage between the Rhizopus oryzae CYP51A Gene Coding Region and Its Intrinsic Voriconazole and Fluconazole Resistance.

Rhizopus oryzae is the most prevalent causative agent of mucormycosis, an increasingly reported opportunistic fungal infection. These Mucorales are intrinsically resistant to Candida- and Aspergillus-active antifungal azole drugs, such as fluconazole (FLC) and voriconazole, respectively. Despite its importance, the molecular mechanisms of its intrinsic azole resistance have not been elucidated yet. The aim of this work was to establish if the Rhizopus oryzaeCYP51 genes are uniquely responsible for intrinsic voriconazole and fluconazole resistance in these fungal pathogens. Two CYP51 genes were identified in the R. oryzae genome. We classified them as CYP51A and CYP51B based on their sequence similarity with other known fungal CYP51 genes. Later, we obtained a chimeric Aspergillus fumigatus strain harboring a functional R. oryzae CYP51A gene expressed under the regulation of the wild-type A. fumigatusCYP51A promoter and terminator. The mutant was selected after transformation by using a novel procedure taking advantage of the FLC hypersusceptibility of the A. fumigatusCYP51A deletion mutant used as the recipient strain. The azole susceptibility patterns of the A. fumigatus transformants harboring R. oryzae CYP51A mimicked exactly the azole susceptibility patterns of this mucormycete. The data presented in this work demonstrate that the R. oryzae CYP51A coding sequence is uniquely responsible for the R. oryzae azole susceptibility patterns.

Phylogenetic and Phylogenomic Definition of Rhizopus Species.

Phylogenomic approaches have the potential to improve confidence about the inter-relationships of species in the order Mucorales within the fungal tree of life. Rhizopus species are especially important as plant and animal pathogens and bioindustrial fermenters for food and metabolite production. A dataset of 192 orthologous genes was used to construct a phylogenetic tree of 21 Rhizopus strains, classified into four species isolated from habitats of industrial, medical and environmental importance. The phylogeny indicates that the genus Rhizopus consists of three major clades, with R. microsporus as the basal species and the sister lineage to R. stolonifer and two closely related species R. arrhizus and R. delemar A comparative analysis of the mating type locus across Rhizopus reveals that its structure is flexible even between different species in the same genus, but shows similarities between Rhizopus and other mucoralean fungi. The topology of single-gene phylogenies built for two genes involved in mating is similar to the phylogenomic tree. Comparison of the total length of the genome assemblies showed that genome size varies by as much as threefold within a species and is driven by changes in transposable element copy numbers and genome duplications.

Rhizopus arrhizus Invasive Infection due to Self-Inflicted Scratch Injuries in a Diabetic Patient with Non-ketotic Acidosis.

Mucormycosis is a rare infection caused by members included in the subphylum Mucoromycotina. Characterized by the histopathological hallmark of angioinvasion, these infections affect most often patients with certain underlying conditions carrying immunosuppression (haematological neoplasias, diabetic ketoacidosis and other forms of acidosis, and iron overload) or immunocompetent patients with traumatic mucocutaneous barriers breakdown and direct inoculation of the mould. A case is presented in which a rare underlying condition (non-ketotic acidosis) and a rare cause of cutaneous injuries collide. Prognosis, treatment options and management decisions are described thoroughly.

Fatal Pulmonary Mucormycosis due to Rhizopus homothallicus.

We report here a case of cavitary pneumonia due to Rhizopus homothallicus in a diabetic patient. This is the first proven case of R. homothallicus infection in Western countries and the third case described worldwide. The organism was isolated from lung biopsy and identified after amplification and sequencing of the internal transcribed spacer region.

Primary Cutaneous Mucormycosis Caused by Rhizopus oryzae: A Case Report and Review of Literature.

Mucormycosis is an invasive infection caused by opportunistic fungi. Rhizopus, Lichtheimia, Mucor and Rhizomucor are the most common isolated genera. Primary cutaneous mucormycosis is usually related to traumatic injuries, but immunocompromised cases are associated with underlying conditions such as diabetes mellitus and malignancies. The treatment of choice is surgical debridement and liposomal amphotericin B. We present a 40-year-old male with fever and a painful necrotic lesion on the middle back and history of poorly controlled diabetes mellitus. Rhizopus oryzae was isolated and identified using an internal transcribed spacer regions ITS1 and ITS2. An initial good response to treatment was observed; however, 7 days later a diabetic ketoacidosis due to poor adherence to treatment caused a lethal outcome.

Fermentation Optimization and Unstructured Kinetic Model for Cellulase Production by Rhizopus stolonifer var. reflexus TP-02 on Agriculture By-Products.

Agricultural by-products, rice straw, wheat bran juice, and soybean residue, were used as substrates for cellulase production using Rhizopus stolonifer var. reflexus TP-02. The culture medium was optimized though uniform design experimentation during shaking flask fermentation, and the ideal formulation obtained for filter paper enzyme (FPase) production was 10 % bran diffusion juice, 1 % rice straw, 0.17 % urea, 0.17 % soybean residue, 0.11 % KH2PO4, and 0.027 % Tween 80, and the maximal FPase activity in the culture supernatant was 13.16 U/mL at an incubation time of 3 days. A kinetic model for cellulase production in batch fermentation was subsequently developed. The unstructured kinetic model considered three responses, namely biomass, cellulase, and sugar. Models for the production of three types of cellulase components (i.e., endoglucanases, cellobiohydrolases, and ß-glucosidases) were established to adequately describe the cellulase production pattern. It was found that the models fitted the experimental data well under pH 5.0 and 6.0, but only the avicelase production model predicted the experimental data under pH-uncontrolled conditions.

Differentiation of clinically relevant Mucorales Rhizopus microsporus and R. arrhizus by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

This study addresses the usefulness of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS for reliable identification of the two most frequently occurring clinical species of Rhizopus, namely Rhizopus arrhizus with its two varieties, arrhizus and delemar, and Rhizopus microsporus. The test-set comprised 38 isolates of clinical and environmental origin previously identified by internal transcribed spacer (ITS) sequencing of rDNA. Multi-locus sequence data targeting three gene markers (ITS, ACT, TEF ) showed two monophylic clades for Rhizopus arrhizus and Rhizopus microsporus (bootstrap values of 99 %). Cluster analysis confirmed the presence of two distinct clades within Rhizopus arrhizus representing its varieties arrhizus and delemar. The MALDI Biotyper 3.0 Microflex LT platform (Bruker Daltonics) was used to confirm the distinction between Rhizopus arrhizus and Rhizopus microsporus and the presence of two varieties within the species Rhizopus arrhizus. An in-house database of 30 reference main spectra (MSPs) was initially tested for correctness using commercially available databases of Bruker Daltonics. By challenging the database with the same strains of which an in-house database was created, automatic identification runs confirmed that MALDI-TOF MS is able to recognize the strains at the variety level. Based on principal component analysis, two MSP dendrograms were created and showed concordance with the multi-locus tree; thus, MALDI-TOF MS is a useful tool for diagnostics of mucoralean species.

Deciphering the uniqueness of Mucoromycotina cell walls by combining biochemical and phylogenomic approaches.

Most fungi from the Mucoromycotina lineage occur in ecosystems as saprobes, although some species are phytopathogens or may induce human mycosis. Mucoromycotina represent early diverging models that are most valuable for understanding fungal evolution. Here we reveal the uniqueness of the cell wall structure of the Mucoromycotina Rhizopus oryzae and Phycomyces blakesleeanus compared with the better characterized cell wall of the ascomycete Neurospora crassa. We have analysed the corresponding polysaccharide biosynthetic and modifying pathways, and highlight their evolutionary features and higher complexity in terms of gene copy numbers compared with species from other lineages. This work uncovers the presence in Mucoromycotina of abundant fucose-based polysaccharides similar to algal fucoidans. These unexpected polymers are associated with unusually low amounts of glucans and a higher proportion of chitin compared with N. crassa. The specific structural features are supported by the identification of genes potentially involved in the corresponding metabolic pathways. Phylogenomic analyses of genes encoding carbohydrate synthases, polysaccharide modifying enzymes and enzymes involved in nucleotide-sugar formation provide evidence for duplication events during evolution of cell wall metabolism in fungi. Altogether, the data highlight the specificity of Mucoromycotina cell walls and pave the way for a finer understanding of their metabolism.

Species boundaries and nomenclature of Rhizopus arrhizus (syn. R. oryzae).

Rhizopus arrhizus (Mucorales, Mucoromycotina) is the prevalent opportunist worldwide among the mucoralean species causing human infections. On the other hand the species has been used since ancient times to ferment African and Asian traditional foods and condiments based on ground soybeans. As producer of organic acids and hydrolytic enzymes it is widely applied in food industry and biotechnology. Using a set of 82 strains we studied phylogenetic and biological species boundaries within Rhizopus arrhizus s.l. to test the taxonomic status of R. delemar that was recently separated from R. arrhizus. Sequence analyses based on the internal transcribed spacer region, the gene of the largest subunit of the RNA polymerase II, a part of the actin gene, and the translation elongation factor 1-α as well as amplified fragment length polymorphism analysis were performed. Phenotypic characters such as enzyme profiles and growth kinetics were examined and the mating behavior was tested. Molecular analyses supported the existence of two phylogenetic species. However, the results of the mating test suggest that the mating barrier is still not complete. No physiological, ecological or epidemiological distinction could be found beside the difference in the production of organic acids. Consequently the status of varieties is proposed for the two phylogenetic species. Because the description of the first described R. arrhizus is considered to be conclusive we recommend the use of Rhizopus arrhizus var. arrhizus and var. delemar.

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.

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.

Mucormycosis in haematological patients: case report and results of prospective study in Saint Petersburg, Russia.

We prospectively observed 36 haematological patients with mucormycosis from nine hospitals of St. Petersburg during 2004-2013. The most frequent underlying diseases were acute leukaemia (64%), and main risk factors were prolonged neutropenia (92%) and lymphocytopenia (86%). In 50% of the patients, mucormycosis was diagnosed 1-65 days after invasive aspergillosis. Main clinical form of mucormycosis was pulmonary (64%), while two or more organ involvement was noted in 50% of the cases. The most frequent aetiological agents of mucormycosis were Rhizopus spp. (48%). Twelve-week survival rate was 50%. Combination therapy (echinocandins + amphotericin B forms) and recovery from the underlying disease significantly improved the survival rate.

Mucormycosis outbreak associated with hospital linens.

BACKGROUND: Mucormycosis is an invasive fungal infection with a high fatality rate. We investigated an outbreak of mucormycosis in a pediatric hospital to determine routes of pathogen transmission from the environment and prevent additional infections. METHODS: A case was defined as a hospital-onset illness consistent with mucormycosis, confirmed by culture or histopathology. Case-patient medical records were reviewed for clinical course and exposure to items and locations within the hospital. Environmental samples were collected from air and surfaces. Fungal isolates collected from case-patients and the environmental samples were identified using DNA sequencing. RESULTS: Five case-patients had hospital-associated cutaneous mucormycosis over an 11-month period; all subsequently died. Three case-patients had conditions known to be associated with susceptibility to mucormycosis, while 2 had cardiac conditions with persistent acidosis. The cases occurred on several different wards throughout the hospital, and hospital linens were the only exposure identified as common to the case-patients. Rhizopus species were recovered from 26 (42%) of 62 environmental samples from clean linens and associated areas and from 1 (4%) of 25 samples from nonlinen-related items. Case-patients were infected with Rhizopus delemar, which was also isolated from cultures of clean linens and clean linen delivery bins from the off-site laundry facility. CONCLUSIONS: Hospital linens were identified as a vehicle that carried R. delemar into contact with susceptible patients. Fungal species identification using DNA-based methods is useful for corroborating epidemiologic links in hospital outbreak investigations. Hospital linens should be laundered, packaged, shipped and stored in a manner that minimizes exposure to environmental contaminants.