Aspergillus Species Research Articles

Galactomannan Antigen Assay for the Diagnosis of Invasive Aspergillosis: A Study from a Tertiary Care Hospital of Central India

Invasive Aspergillosis (IA) is a severe and fatal infection, especially in immunocompromised patients. Galactomannan is a polysaccharide antigen present in the cell wall of Aspergillus species, which is secreted into the blood and other body fluids during hyphal growth. Therefore, detecting galactomannan antigen is very useful in diagnosing IA, along with clinical features and radiological findings. The study period was one year (January 2022 to December 2022). The data was collected retrospectively from the medical records and case sheets of all clinically suspected invasive aspergillosis patients. Galactomannan antigen assay was performed using an FDA-approved Platelia Aspergillus EIA test kit, and results were interpreted according to the manufacturer’s instructions (Cut off > 0.5). A total of 236 clinically suspected Invasive Aspergillosis cases were enrolled in the study. Galactomannan positivity was predominantly seen in patients aged 40 – 60 years, with male preponderance. Of 236 patients, 14.40% were immunocompromised, and 85.59% were immunocompetent. According to EORTC/MSG definitions, we got one proven IA case, 21 probable cases (8.89%), and nine (3.81%) possible cases. In immunocompetent individuals also, we observed 48.72% (115/236) galactomannan positivity, especially in old Pulmonary Tuberculosis (PTB) patients. Galactomannan positivity was higher in Broncho alveolar lavage (BAL) samples (n=70, 85.36%) than in serum samples (n=77, 46.67%). We found culture positivity of 14.06%, with Aspergillus fumigatus being the commonest isolate, followed by Aspergillus flavus. There is increased positivity of galactomannan in BAL samples compared to serum; hence BAL is a better specimen for diagnosis of Invasive Pulmonary Aspergillosis (IPA).

Urinary Tract Infection by Aspergillus Species in Post-Menopausal Woman

Background:: Urinary tract infection by Aspergillus species is an uncommon but potentially serious complication in post-menopausal women, often associated with recurrent UTIs or immunocompromise. Case Presentation:: We present a case of a deceased 67-year-old female with complaints of fever, abdominal pain, diabetes, hypertension, pain and burning during urination admitted to hospital. Microscopic examination confirmed the Aspergillus infection. The patient could not be followed up due to COVID. Discussion:: Urinary Tract infection with Aspergillus species is few and far occurrence. Especially combined with a medical history of non-immune compromises like diabetes, kidney dysfunction and hypertension. Voriconazole anti-fungal medicine worked well on patients with improved conditions. In spite of diabetes and other medical conditions, neutrophils and total lymphocyte count showed elevated levels, which need further studies. Conclusion:: Prompt diagnosis and targeted antifungal therapy are essential in managing urinary tract infections caused by Aspergillus species in postmenopausal women to prevent complications.

Interplay of Cytokines and Chemokines in Aspergillosis.

Aspergillosis is a fungal infection caused by various species of Aspergillus, most notably A. fumigatus. This fungus causes a spectrum of diseases, including allergic bronchopulmonary aspergillosis, aspergilloma, chronic pulmonary aspergillosis, and invasive aspergillosis. The clinical manifestations and severity of aspergillosis can vary depending on individual immune status and the specific species of Aspergillus involved. The recognition of Aspergillus involves pathogen-associated molecular patterns (PAMPs) such as glucan, galactomannan, mannose, and conidial surface proteins. These are recognized by the pathogen recognition receptors present on immune cells such as Toll-like receptors (TLR-1,2,3,4, etc.) and C-type lectins (Dectin-1 and Dectin-2). We discuss the roles of cytokines and pathogen recognition in aspergillosis from both the perspective of human and experimental infection. Several cytokines and chemokines have been implicated in the immune response to Aspergillus infection, including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), CCR4, CCR17, and other interleukins. For example, allergic bronchopulmonary aspergillosis (ABPA) is characterized by Th2 and Th9 cell-type immunity and involves interleukin (IL)-4, IL-5, IL-13, and IL-10. In contrast, it has been observed that invasive aspergillosis involves Th1 and Th17 cell-type immunity via IFN-γ, IL-1, IL-6, and IL-17. These cytokines activate various immune cells and stimulate the production of other immune molecules, such as antimicrobial peptides and reactive oxygen species, which aid in the clearance of the fungal pathogen. Moreover, they help to initiate and coordinate the immune response, recruit immune cells to the site of infection, and promote clearance of the fungus. Insight into the host response from both human and animal studies may aid in understanding the immune response in aspergillosis, possibly leading to harnessing the power of cytokines or cytokine (receptor) antagonists and transforming them into precise immunotherapeutic strategies. This could advance personalized medicine.

Serological Cross-Reactivity of Various Aspergillus spp. with Aspergillus fumigatus: A Diagnostic Blind Spot

Introduction: Aspergillus fumigatus is the most common airborne allergen of the Aspergillus family. However, allergies to Aspergillus spp. are increasing, and subsequently, allergies to Aspergillus species other than fumigatus are also on the rise. Commercial diagnostic tools are still limited to Aspergillus fumigatus. Hence, there is a need for improved tests. We decided to investigate the correlation between serological sensitization to A. fumigatus and other Aspergillus species. Methods: Hundred and seven patients with positive skin prick tests to A. fumigatus were included in this study. Immunoglobulin E (IgE) concentrations against A. fumigatus, A. terreus, A. niger, A. flavus, and A. versicolor were measured from specimens by fluorescent enzyme-linked immunoassays. Results: Patients showed considerably higher IgE concentrations against A. fumigatus (6.00 ± 15.05 kUA/L) than A. versicolor (0.30 ± 1.01 kUA/L), A. niger (0.62 ± 1.59 kUA/L), A. terreus (0.45 ± 1.12 kUA/L), or A. flavus (0.41 ± 0.97 kUA/L). Regression analysis yielded weak positive correlations for all Aspergillus spp., but low r2 values and heteroscedastic distribution indicate an overall poor fit of the calculated models. Conclusion: Serological sensitization against A. fumigatus does not correlate with sensitization against other Aspergillus spp. To detect sensitization against these, other diagnostic tools like a skin prick test solution of different Aspergillus spp. are needed.

Fungal and bacterial species richness in biodeteriorated seventeenth century Venetian manuscripts

Historical paper documents are susceptible to complex degradation processes, including biodeterioration, which can progressively compromise their aesthetic and structural integrity. This study analyses seventeenth century handwritten historical letters stored at the Correr Museum Library in Venice, Italy, exhibiting pronounced signs of biodeterioration. The techniques used encompassed traditional colony isolation on agar plates and proteomics analyses, employing nanoscale liquid chromatography coupled with high-resolution mass spectrometry (nano-LC–MS). Fluorescence microscopy was used for the first time in the historical paper biodeterioration context to supplement the conventional stereoscopic, optical, and scanning electron microscopic imaging techniques. This method enables the visualisation of microorganisms beyond and beneath the paper’s surface through their natural intrinsic autofluorescence in a non-invasive and non-destructive way. The results demonstrate a diverse, complex, and abundant microbiota composed of coexisting fungal and bacterial species (Ascomycota, Mucoromycota, Basidiomycota, Proteobacteria, and Actinobacteria), along with mite carcasses, insects, parasites, and possibly protists. Furthermore, this study reveals certain species that were not previously documented in the biodeterioration of historical paper, including human pathogens, such as Histoplasma capsulatum, Brucella, Candida albicans, and species of Aspergillus (A. flavus, A. fumigatus, A. oryzae, A. terreus, A. niger) known to cause infections or produce mycotoxins, posing substantial risk to both artefacts and humans.

ASPERGILLOMA PADA TUBERKULOSIS PARU: LITERATURE REVIEW

Indonesia ranking among the top six countries with the highest TB burden. In the aftermath of pulmonary TB, fungal infections, particularly those caused by Aspergillus species, have emerged as a notable complication. The predominant species, Aspergillus fumigatus, is responsible for a spectrum of conditions ranging from invasive to noninvasive aspergillosis. This literature review aims to provide an exhaustive analysis of post-TB pulmonary aspergilloma, a clinical condition characterized by the growth of Aspergillus fungal balls within pre-existing lung cavities. Utilizing databases such as PubMed, Science Direct, EBSCO, Emerald, Scopus, and Google Scholar, and employing keywords such as "tuberculosis," "aspergillus," "aspergilloma," and "pulmonary," we meticulously applied the PICO framework and the CRAAP (Currency, Relevance, Authority, Accuracy, and Purpose) criteria to select 20 pertinent articles. The findings indicate that pulmonary aspergilloma manifests in approximately 11% to 17% of individuals post-TB treatment, presenting a clinical spectrum from asymptomatic to severe, life-threatening hemoptysis. Classification into simple and complex aspergilloma is based on specific clinical and radiological criteria. Diagnosis is contingent upon the identification of clinical symptoms, assessment of risk factors, and the corroboration of Aspergillus infection through radiological (chest X-ray and CT scan) and serological or microbiological evidence. Management of pulmonary aspergilloma entails non-operative and operative strategies, with surgical intervention being the definitive treatment, especially in cases of recurrent or massive hemoptysis. The review underscores the persistent challenge of TB in Indonesia, exacerbated by secondary fungal infections such as aspergillosis among pulmonary TB survivors. Aspergillus fumigatus is identified as the primary fungal pathogen, leading to pulmonary aspergilloma.

A review of recently introduced Aspergillus, Penicillium, Talaromyces and other Eurotiales species.

The order Eurotiales is diverse and includes species that impact our daily lives in many ways. In the past, its taxonomy was difficult due to morphological similarities, which made accurate identification of species difficult. This situation improved and stabilised with recent taxonomic and nomenclatural revisions that modernised Aspergillus, Penicillium and Talaromyces. This was mainly due to the availability of curated accepted species lists and the publication of comprehensive DNA sequence reference datasets. This has also led to a sharp increase in the number of new species described each year with the accepted species lists in turn also needing regular updates. The focus of this study was to review the 160 species described between the last list of accepted species published in 2020 until 31 December 2022. To review these species, single-gene phylogenies were constructed and GCPSR (Genealogical Concordance Phylogenetic Species Recognition) was applied. Multi-gene phylogenetic analyses were performed to further determine the relationships of the newly introduced species. As a result, we accepted 133 species (37 Aspergillus, two Paecilomyces, 59 Penicillium, two Rasamsonia, 32 Talaromyces and one Xerochrysium), synonymised 22, classified four as doubtful and created a new combination for Paraxerochrysium coryli, which is classified in Xerochrysium. This brings the number of accepted species to 453 for Aspergillus, 12 for Paecilomyces, 535 for Penicillium, 14 for Rasamsonia, 203 for Talaromyces and four for Xerochrysium. We accept the newly introduced section Tenues (in Talaromyces), and series Hainanici (in Aspergillus sect. Cavernicolarum) and Vascosobrinhoana (in Penicillium sect. Citrina). In addition, we validate the invalidly described species Aspergillus annui and A. saccharicola, and series Annuorum (in Aspergillus sect. Flavi), introduce a new combination for Dichlaena lentisci (type of the genus) and place it in a new section in Aspergillus subgenus Circumdati, provide an updated description for Rasamsonia oblata, and list excluded and recently synonymised species that were previously accepted. This study represents an important update of the accepted species lists in Eurotiales. Taxonomic novelties: New sections: Aspergillus section Dichlaena Visagie, Kocsubé & Houbraken. New series: Aspergillus series Annuorum J.J. Silva, B.T. Iamanaka, Frisvad. New species: Aspergillus annui J.J. Silva, M.H.P. Fungaro, Frisvad, M.H. Taniwaki & B.T. Iamanaka; Aspergillus saccharicola J.J. Silva, Frisvad, M.H.P. Fungaro, M.H. Taniwaki & B.T. Iamanaka. New combinations: Aspergillus lentisci (Durieu & Mont.) Visagie, Malloch, L. Kriegsteiner, Samson & Houbraken; Xerochrysium coryli (Crous & Decock) Visagie & Houbraken. Citation: Visagie CM, Yilmaz N, Kocsubé S, Frisvad JC, Hubka V, Samson RA, Houbraken J (2024). A review of recently introduced Aspergillus, Penicillium, Talaromyces and other Eurotiales species. Studies in Mycology 107: 1-66. doi: 10.3114/sim.2024.107.01.

Chronic Suppurative Otitis Media: A Prospective Descriptive Study of the Microbiology and Antimicrobial Susceptibility Patterns.

This study aims to identify dominant causative pathogens of chronic suppurative otitis media (CSOM) and their antimicrobial susceptibility patterns. The secondary objective is to assess the cholesteatoma concurrence among this population. A prospective descriptive study. Port Elizabeth Provincial Hospital, Eastern Cape, South Africa. Aural swabs of purulent otorrhoea were collected from patients with CSOM attending the otorhinolaryngology outpatient department between April 2022 and June 2023. Samples were analyzed to determine the microbiological spectrum and evaluate antimicrobial sensitivities. Patient files were reviewed to identify cases with cholesteatoma. The study population size of 169 enabled representative results for a confidence interval of 90%. Patients were divided into 2 age categories: children (0-12 years) and adults (>12 years). Adults represented the majority of cases (76.9%) and females (58.6%) were affected more than males. Fungal isolates, Candida species and Aspergillus species, were the most common (20.8%). This was followed by Pseudomonas aeruginosa (12.4%) and Staphylococcus aureus (11.8%). 3.6% of cases cultured Mycobacterial tuberculosis which was high compared to global estimates. Susceptibility patterns showed 94.3% sensitivity of fungal cultures to fluconazole. Pseudomonas aeruginosa was 100% susceptible to piperacillin-tazobactam and cefepime, but only 76.2% sensitive to ciprofloxacin. Cholesteatoma concurrence was identified in 17.8%. CSOM remains a public health challenge. This study highlights the need to consider fungal etiology in instances where standard antibiotic treatments prove ineffective. Given the variety of cultured organisms and their susceptibility patterns, incorporating culture-directed therapy should be considered standard practice for chronic otorrhoea patients.

Unveiling ochratoxin A and ochratoxigenic fungi in Brazilian artisanal Cheeses: Insights from production to consumption

Ochratoxin A (OTA) is a toxin produced by several Aspergillus species, mainly those belonging to section Circumdati and section Nigri. The presence of OTA in cheese has been reported recently in cave cheese in Italy. As artisanal cheese production in Brazil has increased, the aim of this study was to investigate the presence of ochratoxin A and related fungi in artisanal cheese consumed in Brazil.A total of 130 samples of artisanal cheeses with natural moldy rind at different periods of maturation were collected. Of this total, 79 samples were collected from 6 producers from Canastra region in the state of Minas Gerais, since this is the largest artisanal cheese producer region; 13 samples from one producer in the Amparo region in the state of São Paulo and 36 samples from markets located in these 2 states. Aspergillus section Circumdati occurred in samples of three producers and some samples from the markets. A. section Circumdati colony counts varied from 102 to 106 CFU/g. Molecular analysis revealed Aspergillus westerdijkiae (67 %) as the most frequent species, followed by Aspergillus ostianus (22 %), and Aspergillus steynii (11 %). All of these isolates of A. section Circumdati were able to produce OTA in Yeast Extract Sucrose Agar (YESA) at 25 °C/7 days. OTA was found in 22 % of the artisanal cheese samples, ranging from 1.0 to above 1000 µg/kg, but only five samples had OTA higher than 1000 µg/kg. These findings emphasize the significance of ongoing monitoring and quality control in the artisanal cheese production process to minimize potential health risks linked to OTA contamination.

Clinicomycological Study of the Spectrum of Pulmonary Aspergillosis at a Tertiary Care Hospital in Central India.

Knowing the spectrum, prevalence, and modes of diagnosis of pulmonary aspergillosis (PA) will be beneficial to clinicians for its early diagnosis and management. This study aims to estimate the prevalence, spectrum, and role of serological tests and radiological findings in the diagnosis of PA. A total of 150 patients were suspected of having PA after obtaining relevant clinical history and radiological imaging. The patients were grouped into each spectrum of PA as invasive PA (IPA), chronic necrotizing PA (CNPA), aspergilloma, allergic bronchopulmonary aspergillosis (ABPA) based on predisposing factors, clinical and radiological findings, and the guidelines of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG). Samples (bronchoalveolar lavage (BAL), sputum, blood) were collected from these patients and processed in a microbiology lab. BAL and sputum were subjected to microscopy by potassium hydroxide mount, calcofluor white mount, and culture. The serum was separated from blood by centrifugation and subjected to specific serological tests based on the spectrum of PA that the patient was suspected to have. For IPA, serum and BAL galactomannan antigen enzyme-linked immunosorbent assay (ELISA) was performed. For CNPA and aspergilloma, the anti-Aspergillus IgG antibody ELISA was performed. For ABPA, the tests performed were total immunoglobulin E (IgE) ELISA, Aspergillus fumigatus-specific IgE ELISA, and anti-Aspergillus immunoglobulin G (IgG) antibody ELISA. After compiling the clinical, radiological, culture, and serological findings, patients were diagnosed to have a particular spectrum of PA. The prevalence of IPA was 1.4%, CNPA was 4%, ABPA was 3.2%, and aspergilloma was 2.9%. CNPA was the predominant spectrum of PA in our study. Culture positivity for Aspergillus species was seen the highest in aspergilloma patients, followed by IPA, ABPA, and CNPA patients. A. fumigatus was the most common causative agent of PA, except for IPA for which Aspergillus flavus was the most common causative. Aspergillus niger and Aspergillus terreus were less the frequent causes of PA. A combination of radiological, microbiological, and serological tests along with clinical correlation is needed to confirm the diagnosis of PA.

Cyp51A mutations, protein modeling, and efflux pump gene expression reveals multifactorial complexity towards understanding Aspergillus section Nigri azole resistance mechanism

Black Aspergillus species are the most common etiological agents of otomycosis, and pulmonary aspergillosis. However, limited data is available on their antifungal susceptibility profiles and associated resistance mechanisms. Here, we determined the azole susceptibility profiles of black Aspergillus species isolated from the Indian environment and explored the potential resistance mechanisms through cyp51A gene sequencing, protein homology modeling, and expression analysis of selected genes cyp51A, cyp51B, mdr1, and mfs based on their role in imparting resistance against antifungal drugs. In this study, we have isolated a total of 161 black aspergilli isolates from 174 agricultural soil samples. Isolates had variable resistance towards medical azoles; approximately 11.80%, 3.10%, and 1.24% of isolates were resistant to itraconazole (ITC), posaconazole (POS), and voriconazole (VRC), respectively. Further, cyp51A sequence analysis showed that non-synonymous mutations were present in 20 azole-resistant Aspergillus section Nigri and 10 susceptible isolates. However, Cyp51A homology modeling indicated insignificant protein structural variations because of these mutations. Most of the isolates showed the overexpression of mdr1, and mfs genes. Hence, the study concluded that azole-resistance in section Nigri cannot be attributed exclusively to the cyp51A gene mutation or its overexpression. However, overexpression of mdr1 and mfs genes may have a potential role in drug resistance.

Detection and molecular characterization of aflatoxin and ochratoxin produce aspergillus species in capsicum spices in Saudi Arabia

Capsicum is a common spice used in food flavouring. However, they are prone to mycotoxin contamination. Mycotoxins are natural toxins produced by filamentous fungi and can pose serious risks to human health. Fungal contamination was assessed in 130 randomly collected samples following ISO 21527–2:2008 standards. Results revealed that 84.6% of the samples exceeded the acceptable fungal count limit (102 CFU/g) according to Gulf Cooperation Council (GCC) standards (GSO1016:2015). The predominant fungal isolates were Aspergillus (51.1%), notably Aspergillus flavus (38.8%) and Aspergillus niger (37.7%). Molecular characterization focused on crucial genes associated with aflatoxin (AF) and ochratoxin (OT) biosynthesis, 14.4% of the isolates exhibited all targeted AF genes. The mycotoxin analysis, conducted on 34.6% of samples via liquid chromatography-mass spectrometry (LC-MS), detected AFB1 in 28.8% (0.2–13.8 μg/kg) and OTA in 35.5% (6.87–59.00 μg/kg) of the tested samples. This study demonstrates the need of implementing rules governing the methods of storing, shipping, and packing spices in Saudi Arabia, which may help to minimizes the prevalence of toxigenic fungus and mycotoxins. This was the first study in KSA that focused on Aspergillus in Capsicum products.

Prevalence and Concentration of Airborne Microorganisms Isolated from Domestic Toilets in Port Harcourt, Rivers State, Nigeria

Introduction: Microorganisms are ubiquitous in nature and transient airborne microorganisms have been shown to constitute major health hazards.
 Aim: This study was carried out to assess the microbial air quality in ten (10) domestic toilets located in mile-3, Alakahia, Choba, Ada-George and Rumuokwuta, Port Harcourt, Rivers state, Nigeria.
 Methodology: Microbial air quality was sampled using sedimentation techniques. Freshly prepared Nutrient Agar, MacConkey and Sabouraud Dextrose Agar (SDA) plates were placed one meter above the floor of the toilets for 1 hours at different sections of each toilet exposed to an open air. The agar plates were closed and transported to the Laboratory were incubation took place at 37°C for 24 hours and 25°C for 3-5 days for growth of bacteria and fungi respectively. Isolates were characterized and identified by standard microbiological methods.
 Results: The bacteria isolated were Staphylococcus species 22(22.45%), Bacillus species 20 (20.41%), Enterococcus species 20(20.41%), Escherichia coli 16(16.32%), Micrococcus species 15 (15.31%), Klebsiella species 3(3.06%), and Proteus species 2(2.04%) while Aspergillus species 14(27.45%), Penicillium species 16(23.53%), Fusarium species 10(19.60%), Mucor species 8(15.69%), and Rhizopus species 7(13.73%) were the fungi identified. The highest bacteria colony count before and after flushing were 52.380×103CFU/m3/hr & 67.261× 103CFU/m3/ hrrespectively while the lowest bacteria counts observed before and after flushing 19.047× 103CFU/m3/hr & 39.286×103CFU/m3/hr while the highest and lowest fungal count before and after flushing were 10.119×103CFU/m3/hr & 13.690×103CFU/m3/hr and 5.952×103CFU/m3/hr & 5.953×103CFU/m3/hr respectively.
 Conclusion: This study shows that considerable numbers of both pathogenic bacteria and fungi particles were released into the air in higher quantity after flushing domestic toilets when compared to air quality before flushing. These organisms have been implicated in major and minor infectious diseases. Inhalation or contact may easily lead to infection especially in immunocompromised individuals and the older adults. To maintain the health of users, it is necessary to carefully manage the environmental factors that promote the growth and multiplication of microorganisms in domestic toilet environment.

The forced activation of asexual conidiation in Aspergillus niger simplifies bioproduction

Aspergillus niger is an efficient cell factory for organic acids production, particularly l-malic acid, through genetic manipulation. However, the traditional method of collecting A. niger spores for inoculation is labor-intensive and resource-consuming. In our study, we used the CRISPR-Cas9 system to replace the promoter of brlA, a key gene in Aspergillus conidiation, with a xylose-inducible promoter xylP in l-malic acid-producing A. niger strain RG0095, generating strain brlAxylP. When induced with xylose in submerged liquid culture, brlAxylP exhibited significant upregulation of conidiation-related genes. This induction allowed us to easily collect an abundance of brlAxylP spores (>7.1 × 106/mL) in liquid xylose medium. Significantly, the submerged conidiation approach preserves the substantial potential of A. niger as a foundational cellular platform for the biosynthesis of organic acids, including but not limited to l-malic acid. In summary, our study offers a simplified submerged conidiation strategy to streamline the preparation stage and reduce labor and material costs for industrial organic acid production using Aspergillus species.

Exposure to resistant fungi across working environments and time

Antifungal resistance has emerged as a significant health concern with increasing reports of resistant variants in previously susceptible species. At present, little is known about occupational exposure to antifungal-resistant fungi. This study aimed to investigate Danish workers' occupational exposure to airborne fungi resistant to first-line treatment drugs. A retrospective study was performed on a unique collection of personal exposure samples gathered over a twenty-year period from Danish working environments, in sectors including agriculture, animal handling, waste management, and healthcare. A total of 669 samples were cultivated at 37 °C and fungal colonies were identified using MALDI-TOF MS. Subsequently, identification was confirmed by amplicon sequencing the genes of calmodulin and beta-tubulin to unveil potential cryptic species.Infectious fungi (495 isolates from 23 species) were tested for resistance against Itraconazole, Voriconazole, Posaconazole, and Amphotericin B. Working environments were highly variable in the overall fungal exposure, and showed vastly different species compositions. Resistance was found in 30 isolates of the species Aspergillus fumigatus (4 of 251 isolates), A. nidulans (2 of 13), A. niger complex (19 of 131), A. versicolor (3 of 18), and A. lentulus (2 of 2). Sequence analysis revealed several cryptic species within the A. niger complex including A. tubingensis, A. luchuensis, and A. phoenicis. Among the resistant A. fumigatus isolates, two contained the well-described TR34/L98H mutation in the cyp51A gene and promoter region, while the remainder harbored silent mutations.The results indicate that the working environment significantly contributes to exposure to resistant fungi, with particularly biofuel plant workers experiencing high exposure. Differences in the prevalence of resistance across working environments may be linked to the underlying species composition.

Enhancement of cellulolytic enzyme production from intrageneric protoplast fusion of Aspergillus species and evaluating the hydrolysate scavenging activity

BackgroundLignocellulosic biomass provides a great starting point for the production of energy, chemicals, and fuels. The major component of lignocellulosic biomass is cellulose, the employment of highly effective enzymatic cocktails, which can be produced by a variety of microorganisms including species of the genus Aspergillus, is necessary for its utilization in a more productive manner. In this regard, molecular biology techniques should be utilized to promote the economics of enzyme production, whereas strategies like protoplast fusion could be employed to improve the efficacy of the hydrolytic process.ResultsThe current study focuses on cellulase production in Aspergillus species using intrageneric protoplast fusion, statistical optimization of growth parameters, and determination of antioxidant activity of fermentation hydrolysate. Protoplast fusion was conducted between A. flavus X A. terreus (PFFT), A. nidulans X A. tamarii (PFNT) and A. oryzae X A. tubingensis (PFOT), and the resultant fusant PFNT revealed higher activity level compared with the other fusants. Thus, this study aimed to optimize lignocellulosic wastes-based medium for cellulase production by Aspergillus spp. fusant (PFNT) and studying the antioxidant effect of fermentation hydrolysate. The experimental strategy Plackett-Burman (PBD) was used to assess how culture conditions affected cellulase output, the best level of the three major variables namely, SCB, pH, and incubation temperature were then determined using Box-Behnken design (BBD). Consequently, by utilizing an optimized medium instead of a basal medium, cellulase activity increased from 3.11 U/ml to 7.689 U/ml CMCase. The following medium composition was thought to be ideal based on this optimization: sugarcane bagasse (SCB), 6.82 gm; wheat bran (WB), 4; Moisture, 80%; pH, 4; inoculum size, (3 × 106 spores/ml); and incubation Temp. 31.8 °C for 4 days and the fermentation hydrolysate has 28.13% scavenging activities.ConclusionThe results obtained in this study demonstrated the significant activity of the selected fusant and the higher sugar yield from cellulose hydrolysis over its parental strains, suggesting the possibility of enhancing cellulase activity by protoplast fusion using an experimental strategy and the fermentation hydrolysate showed antioxidant activity.

Carvacrol nanocapsules as a new antifungal strategy: Characterization and evaluation against fungi important for grape quality and to control the synthesis of ochratoxins

Fungi are a problem for viticulture as they can lead to deterioration of grapes and mycotoxins production. Despite the widespread use of synthetic fungicides to control fungi, their impact on the agricultural ecosystem and human health demand safer and eco-friendly alternatives. This study aimed to produce, characterize and assess the antifungal activity of carvacrol loaded in nanocapsules of Eudragit® and chia mucilage as strategy for controlling Botrytis cinerea, Aspergillus flavus, Aspergillus carbonarius, and Aspergillus niger. Eudragit® and chia mucilage were suitable wall materials, as both favored the encapsulation of carvacrol into nanometric diameter particles. Fourier Transform Infrared Spectroscopy (FTIR) analysis suggested a successful incorporation of carvacrol into both nanocapsules, which was confirmed by presenting a good encapsulation efficiency and loading capacity. Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) analyses revealed adequate thermal resistance. All fungi were sensible to carvacrol treatments and B. cinerea was the most sensitive compared to the Aspergillus species. Lower concentrations of encapsulated carvacrol than the unencapsulated form were required to inhibit fungi in the in vitro and grape assays. Additionally, lower levels of carvacrol (unencapsulated or encapsulated) were used to inhibit fungal growth and ochratoxin synthesis on undamaged grapes in comparison to those superficially damaged, highlighting the importance of management practices designed to preserve berry integrity during cultivation, storage or commercialization. When sublethal doses of carvacrol were used, the growth of A. niger and A. carbonarius was suppressed by at least 45 %, and ochratoxins were not found. The nanoencapsulation of carvacrol using Eudragit® and chia mucilage has proven to be an alternative to mitigate the problems with fungi and mycotoxins faced by the grape and wine sector.

Real-time monitoring of mycelial growth in liquid culture using hyphal dispersion mutant of Aspergillus fumigatus.

Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti-Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δags1Δgtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δags1Δgtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δags1Δags2Δags3Δgtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti-Aspergillus agents.

Study of cytotoxicity in neuroblastoma cell line exposed to patulin and citrinin

Mycotoxins can be found in food and feed storage as well as in several kinds of foodstuff and are capable of harming mammals and some of them even in small doses. This study investigated on the undifferentiated neuronal cell line SH-SY5Y the effects of two mycotoxins: patulin (PAT) and citrinin (CTN), which are predominantly produced by fungi species Penicillium and Aspergillus. Here, the individual and combined cytotoxicity of PAT and CTN was investigated using the cytotoxic assay MTT. Our findings indicate that after 24 h of treatment, the IC50 value for PAT is 2.01 μM, which decreases at 1.5 μM after 48 h. In contrast, CTN did not attain an IC50 value at the tested concentration. Therefore, we found PAT to be the more toxic compared to CTN. However, the combined treatment suggests an additive toxic effect. With 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) DCFH-DA assay, ROS generation was demonstrated after CTN treatment, but PAT showed only small changes. The mixture presented a very constant behavior over time. Finally, the median-effect/combination index (CI-) isobologram equation demonstrated an additive effect after 24 h, but an antagonistic effect after 48 h for the interaction of the two mycotoxins.

Evaluation of Tolerance and Uptake of Cd and Mn for Microfungi Aspergillus flavus, Aspergillus oryzae, and Aspergillus terreus Isolated from Landfill Soil Collected from Bangar, La Union Philippines

Excessive deposition of heavy metals into the environment due to anthropogenic activities necessitates an eco-friendly clean-up strategy. Among microorganisms, limited studies have been made on the mycoremediation potential of microfungi. This paper evaluated three landfill microfungal isolates of Aspergillus species for tolerance and uptake to Cd and Mn. Culture media optimization was also performed for the evaluation of the tolerance index and heavy metal analysis of soil samples from the landfill site. Among the nine heavy metals analyzed, Mn and Fe were detected in relatively high amounts, while Cd, Ni, and Cu were detected in a moderate range. Luxuriant mycelial growth of A. oryzae (MK120548.1) and A. flavus (MH864264.1) was observed in potato dextrose agar while A. terreus (MH047280.1) grew best in potato sucrose agar. In terms of tolerance index, A. oryzae (MK120548.1) and A. flavus (MH864264.1) demonstrated high tolerance to Cd up to 10 mg/kg. A. oryzae (MK120548.1) showed high tolerance to Mn up to 1,000 mg/kg while A. flavus (MH864264.1) exhibited a very high 10,000 mg/kg tolerance. In terms of metal uptake, A. oryzae (MK120548.1) showed the highest metal uptake of up to 654 mg/kg of Cd, while A. terreus (MH047280.1) exhibited the highest metal uptake of 997 mg/kg ofMn. With these findings, A. oryzae (MK120548.1), A. flavus (MH864264.1), and A. terreus (MH047280.1) have considerable mycoremediation potential. Bioremediation studies in conjunction with plants can be explored to further assess the potential of these Aspergillus species.