Publications by authors named "Jos Houbraken"

80 Publications

The Environmental Spread of in Tyrol, Austria.

Microorganisms 2021 Mar 5;9(3). Epub 2021 Mar 5.

Institute of Hygiene & Medical Microbiology, Medical University of Innsbruck, 6020 Innsbruck, Austria.

Fungal infections due to species have become a major cause of morbidity and mortality among immunocompromised patients. At the Medical University of Innsbruck, and related species are the second most common causative agents of aspergillosis. In this one-year study we collected environmental samples to investigate (i) the environmental distribution, (ii) the ecological niche of in Tyrol, (iii) the genetic relatedness of environmental and clinical isolates and the correlation between those two groups of isolates, and (iv) the antifungal susceptibility patterns. was present in 5.4% of 3845 environmental samples, with a significantly higher frequency during winter (6.8%) than summer (3.9%). An increased abundance in Tyrol's Eastern part was detected which is in agreement with the proof of clinical cases. In total, 92% of environmental and 98% of clinical isolates were amphotericin B resistant; 22.6% and 9.8% were resistant against posaconazole. Overall, 3.9% of clinical isolates were resistant against voriconazole. Short tandem repeat analysis identified three major genotypes persisting in Tyrol. Soil from agricultural cornfields seems to be an important source; the environmental frequency of correlates with the high incidence of infections in certain geographical areas.
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http://dx.doi.org/10.3390/microorganisms9030539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998223PMC
March 2021

Preservation stress resistance of melanin deficient conidia from Paecilomyces variotii and Penicillium roqueforti mutants generated via CRISPR/Cas9 genome editing.

Fungal Biol Biotechnol 2021 Apr 2;8(1). Epub 2021 Apr 2.

TIFN, Agro Business Park 82, 6708 PW, Wageningen, The Netherlands.

Background: The filamentous fungi Paecilomyces variotii and Penicillium roqueforti are prevalent food spoilers and are of interest as potential future cell factories. A functional CRISPR/Cas9 genome editing system would be beneficial for biotechnological advances as well as future (genetic) research in P. variotii and P. roqueforti.

Results: Here we describe the successful implementation of an efficient AMA1-based CRISPR/Cas9 genome editing system developed for Aspergillus niger in P. variotii and P. roqueforti in order to create melanin deficient strains. Additionally, kusA mutant strains with a disrupted non-homologous end-joining repair mechanism were created to further optimize and facilitate efficient genome editing in these species. The effect of melanin on the resistance of conidia against the food preservation stressors heat and UV-C radiation was assessed by comparing wild-type and melanin deficient mutant conidia.

Conclusions: Our findings show the successful use of CRISPR/Cas9 genome editing and its high efficiency in P. variotii and P. roqueforti in both wild-type strains as well as kusA mutant background strains. Additionally, we observed that melanin deficient conidia of three food spoiling fungi were not altered in their heat resistance. However, melanin deficient conidia had increased sensitivity towards UV-C radiation.
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http://dx.doi.org/10.1186/s40694-021-00111-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8017634PMC
April 2021

Identification and in vitro antifungal susceptibility of causative agents of onychomycosis due to Aspergillus species in Mashhad, Iran.

Sci Rep 2021 Mar 24;11(1):6808. Epub 2021 Mar 24.

Department of Medical Microbiology, The People's Hospital of Suzhou New District, Suzhou, 215000, Jiangsu, People's Republic of China.

Aspergillus species are emerging causative agents of non-dermatophyte mold onychomycosis. In this study, 48 Aspergillus isolates were obtained from patients with onychomycosis in Mashhad, Iran, during 2015-2018. The aim is to identify the Aspergillus isolates to the species level by using partial calmodulin and beta-tubulin gene sequencing and MALDI-TOF MS, and to evaluate their in vitro susceptibility to ten antifungal drugs: terbinafine, itraconazole, voriconazole, posaconazole, ravuconazole, isavuconazole, caspofungin, micafungin, anidulafungin and amphotericin B according to CLSI M38-A3. Our results indicate that A.flavus (n = 38, 79%) is the most common Aspergillus species causing onychomycosis in Mashhad, Iran. Other detected species were A. terreus (n = 3), A. tubingensis (n = 2), A. niger (n = 1), A. welwitschiae (n = 1), A. minisclerotigenes (n = 1), A. citrinoterreus (n = 1) and A. ochraceus (n = 1). Aspergillus flavus, A. terreus and A. niger isolates were correctly identified at the species level by MALDI-TOF MS, while all cryptic species were misidentified. In conclusion, A. flavus is the predominant Aspergillus species causing onychomycosis due to Aspergillus spp. in Mashhad, Iran. MALDI-TOF MS holds promise as a fast and accurate identification tool, particularly for common Aspergillus species. It is important that the current database of reference spectra, representing different Aspergillus species is expanded to increase the precision of the species-level identification. Terbinafine, posaconazole and echinocandins were in vitro most active against the studies Aspergillus isolates and terbinafine could be the first choice for treatment of onychomycosis due to Aspergillus.
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http://dx.doi.org/10.1038/s41598-021-86038-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7991633PMC
March 2021

Genetic and Phenotypic Characterization of in-Host Developed Azole-Resistant Isolates.

J Fungi (Basel) 2021 Feb 25;7(3). Epub 2021 Feb 25.

Department of Medical Microbiology, Radboud University Medical Center, 6525 GANijmegen, The Netherlands.

is a pathogenic fungal species that can cause pulmonary aspergillosis, and triazole compounds are used for the treatment of these infections. Prolonged exposure to azoles may select for compensatory mutations in the genome, resulting in azole resistance. Here, we characterize a series of 11 isogenic strains isolated from a patient with pulmonary aspergillosis. Over a period of three months, the initially azole-susceptible strain developed itraconazole and voriconazole resistance. Short tandem repeat analysis and whole-genome sequencing revealed the high genetic relatedness of all isolates, indicating an infection with one single isolate. In contrast, the isolates were macroscopically highly diverse, suggesting an adaptation to the environment due to (epi)genetic changes. The whole-genome sequencing of susceptible and azole-resistant strains showed a number of mutations that might be associated with azole resistance. The majority of resistant strains contain a Y119F mutation in the gene, which corresponds to the Y121F mutation found in . One azole-resistant strain demonstrated a divergent set of mutations, including a V99A mutation in a major facilitator superfamily (MSF) multidrug transporter (AFLA 083950).
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http://dx.doi.org/10.3390/jof7030164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996152PMC
February 2021

Fatal Rhinofacial Mycosis Due to : Case Report and Review of Published Literature.

Front Microbiol 2020 22;11:595375. Epub 2020 Dec 22.

Center of Expertise in Mycology, Radboud University Medical Center/Canisius Wilhelmina Hospital, Nijmegen, Netherlands.

Background: A 73-year-old female suffering from acute myeloid leukemia presented with progressive rhinofacial mycosis. Suspecting it to be mucormycosis, the antifungal amphotericin B (AMB) was administered empirically, but the patient did not respond as planned. The fungus was then isolated from the biopsied tissue and morphologically identified as a species of . Necrosis progressed and she died of cerebral hemorrhage. Since is susceptible to AMB, and several other species can be misidentified as , the observed resistance necessitated a re-examination of the fungal isolate.

Methods: The fungal strain was re-isolated and re-examined morphologically. Additionally, genomic DNA was extracted from the fungus and sequences were obtained from three genomic regions [the rDNA internal transcribed spacer (ITS) region, and portions of the β-tubulin and calmodulin genes] to more accurately identify this strain. Its antifungal susceptibility was assessed using multiple compounds and our findings were compared with literature data.

Results: The fungal culture again yielded an isolate morphologically identical to . Molecular analyses, however, revealed the strain to be , a close relative of in section , and it exhibited resistance to AMB. Reviewing the literature, only five other cases of infection in humans have been reported worldwide.

Conclusion And Clinical Importance: The rhinofacial mycosis of the patient was actually due to . The initial misidentification of the fungus, coupled with its resistance to AMB, could be the reason treatment did not help the patient. We postulate that clinical infections may be underreported and that accurate and speedy pathogen identification is important so that an effective antifungal regimen can be administered.
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http://dx.doi.org/10.3389/fmicb.2020.595375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782315PMC
December 2020

Conidial heat resistance of various strains of the food spoilage fungus Paecilomyces variotii correlates with mean spore size, spore shape and size distribution.

Food Res Int 2020 11 6;137:109514. Epub 2020 Jul 6.

TiFN, P.O. Box 557, 6700 AN Wageningen, the Netherlands; Westerdijk Fungal Biodiversity Institute, Applied & Industrial Mycology, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands. Electronic address:

Contamination by spores is often the cause of fungal food spoilage. Some distinct strains of the food spoilage fungus Paecilomyces variotii are able to produce airborne conidia that are more heat-resistant than similar species. These ellipsoid asexual spores can vary in size between strains, but also within strains. Here, we compared four measurement techniques to measure conidia size and distribution of five heat-sensitive and five heat-resistant P. variotii strains. Light microscopy (LM), Scanning Electron Microscopy (SEM) and Coulter Counter (CC) were used to measure and compare the spherical equivalent diameter, while CC and flow cytometry were used to study spore size distributions. The flow cytometry data was useful to study spore size distributions, but only relative spore sizes were obtained. There was no statistic difference between the method used of spore size measurement between LM, SEM and CC, but spore size was significantly different between strains with a 2.4-fold volume difference between the extremes. Various size distribution and shape parameters were correlated with conidial heat resistance. We found significant correlations in mean spore size, aspect ratio, roundness and skewness in relation to heat resistance, which suggests that these parameters are indicative for the conidial heat resistance of a P. variotii strain.
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http://dx.doi.org/10.1016/j.foodres.2020.109514DOI Listing
November 2020

Revisiting an Strain Isolated from an Egyptian Sugarcane Field in 1930.

Microorganisms 2020 Oct 22;8(11). Epub 2020 Oct 22.

Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands.

The aflatoxin type B and G producer was described in 2012 and was firstly reported from sputum, hospital air (Brazil), and soil (Colombia). Later, several survey studies reported the occurrence of this species in different foods and other agricultural commodities from several countries worldwide. This short communication reports on an old fungal strain (CBS 108.30), isolated from (grey sugarcane mealybug) from an Egyptian sugarcane field in (or before) 1930. This strain was initially identified as ; however, using the latest taxonomy schemes, the strain is, in fact, . These data and previous reports indicate that is strongly associated with sugarcane, and pre-harvest biocontrol approaches with non-toxigenic strains are likely to be more successful than those using non-toxigenic strains. Further studies on the association between and might shed light on the distribution (and aflatoxin contamination) of this species in sugarcane. Additionally, the interaction between , and sugarcane crop under different scenarios of climate change will be critical in order to get more insight into the host-pathogen interaction and host resistance and propose appropriate prevention strategies to decrease mycotoxin contamination and crop loss due to attack.
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http://dx.doi.org/10.3390/microorganisms8111633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7690594PMC
October 2020

New species in section and an overview of section .

Int J Syst Evol Microbiol 2020 Oct 4;70(10):5401-5416. Epub 2020 Sep 4.

Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.

sections and are accommodated in the subgenus . In the present study, a polyphasic approach using morphology and multi-gene phylogeny was applied to investigate the taxonomy of these two sections. Based on the phylogenetic analysis, section includes 25 species, which can be assigned to four series: , , and is newly described in this section and this species was isolated from a cigarette from PR China and belongs to series . It is clearly distinct from other members in this series based on ITS, , and sequences. section members like and are important opportunistic pathogens, it is speculative that more pathogenetic species will be found by using polyphasic taxonomy approaches. section includes five species, the growth rates on agar media and size and ornamentation of conidia are important characters for differentiating species in section .
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http://dx.doi.org/10.1099/ijsem.0.004425DOI Listing
October 2020

Contact lens-related fungal keratitis.

Lancet Infect Dis 2020 09;20(9):1100

Department of Medical Microbiology, Leiden University Medical Center, Leiden, Netherlands.

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http://dx.doi.org/10.1016/S1473-3099(20)30126-2DOI Listing
September 2020

Variation Among Biosynthetic Gene Clusters, Secondary Metabolite Profiles, and Cards of Virulence Across Species.

Genetics 2020 10 17;216(2):481-497. Epub 2020 Aug 17.

Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235

is a major human pathogen. In contrast, and the recently described , the two species most closely related to , are not known to be pathogenic. Some of the genetic determinants of virulence (or "cards of virulence") that A. possesses are secondary metabolites that impair the host immune system, protect from host immune cell attacks, or acquire key nutrients. To examine whether secondary metabolism-associated cards of virulence vary between these species, we conducted extensive genomic and secondary metabolite profiling analyses of multiple , one , and multiple strains. We identified two cards of virulence (gliotoxin and fumitremorgin) shared by all three species and three cards of virulence (trypacidin, pseurotin, and fumagillin) that are variable. For example, we found that all species and strains examined biosynthesized gliotoxin, which is known to contribute to virulence, consistent with the conservation of the gliotoxin biosynthetic gene cluster (BGC) across genomes. For other secondary metabolites, such as fumitremorgin, a modulator of host biology, we found that all species produced the metabolite but that there was strain heterogeneity in its production within species. Finally, species differed in their biosynthesis of fumagillin and pseurotin, both contributors to host tissue damage during invasive aspergillosis. biosynthesized fumagillin and pseurotin, while biosynthesized fumagillin and biosynthesized neither. These biochemical differences were reflected in sequence divergence of the intertwined fumagillin/pseurotin BGCs across genomes. These results delineate the similarities and differences in secondary metabolism-associated cards of virulence between a major fungal pathogen and its nonpathogenic closest relatives, shedding light onto the genetic and phenotypic changes associated with the evolution of fungal pathogenicity.
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http://dx.doi.org/10.1534/genetics.120.303549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536862PMC
October 2020

New section and species in .

MycoKeys 2020 7;68:75-113. Epub 2020 Jul 7.

Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands Westerdijk Fungal Biodiversity Institute Utrecht Netherlands.

is a monophyletic genus containing seven sections. The number of species in grows rapidly due to reliable and complete sequence data contributed from all over the world. In this study agricultural soil samples from Fujiang, Guangdong, Jiangxi, Shandong, Tibet and Zhejiang provinces of China were collected and analyzed for fungal diversity. Based on a polyphasic approach including phylogenetic analysis of partial ITS, , and gene sequences, macro- and micro-morphological analyses, six of them could not be assigned to any described species, and one cannot be assigned to any known sections. Morphological characters as well as their phylogenetic relationship with other species are presented for these putative new species. is combined in Talaromyces section Subinflati as .
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http://dx.doi.org/10.3897/mycokeys.68.52092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360636PMC
July 2020

Diversity and toxigenicity of fungi and description of sp. nov. from cereals, legumes and soils in north-central Nigeria.

MycoKeys 2020 8;67:95-124. Epub 2020 Jun 8.

Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands Westerdijk Fungal Biodiversity Institute Utrecht Netherlands.

Mycological investigation of various foods (mainly cowpea, groundnut, maize, rice, sorghum) and agricultural soils from two states in north-central Nigeria (Nasarawa and Niger), was conducted in order to understand the role of filamentous fungi in food contamination and public health. A total of 839 fungal isolates were recovered from 84% of the 250 food and all 30 soil samples. Preliminary identifications were made, based on macro- and micromorphological characters. Representative strains (n = 121) were studied in detail using morphology and DNA sequencing, involving genera/species-specific markers, while extrolite profiles using LC-MS/MS were obtained for a selection of strains. The representative strains grouped in seven genera (, , , , , and ). Amongst the 21 species that were isolated during this study was one novel species belonging to the species complex, , obtained from groundnut and sorghum in Nasarawa state. The examined strains produced diverse extrolites, including several uncommon compounds: averantinmethylether in ; aspergillimide in ; heptelidic acid in ; desoxypaxillin, kotanin A and paspalitrems (A and B) in , and ; aurasperon C, dimethylsulochrin, fellutanine A, methylorsellinic acid, nigragillin and pyrophen in ; cyclosporins (A, B, C and H) in ; methylorsellinic acid, pyrophen and secalonic acid in ; aspulvinone E, fonsecin, kojic acid, kotanin A, malformin C, pyranonigrin and pyrophen in ; and all compounds in sp. nov., , and . This study provides snapshot data for prediction of food contamination and fungal biodiversity exploitation.
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http://dx.doi.org/10.3897/mycokeys.67.52716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295817PMC
June 2020

Thermotolerant and Thermophilic Mycobiota in Different Steps of Compost Maturation.

Microorganisms 2020 Jun 11;8(6). Epub 2020 Jun 11.

Laboratory of Mycology, DISTAV Department of Earth, Environmental and Life Science, University of Genoa, Corso Europa 26, 16132 Genoa, Italy.

Composting is a complex process in which various micro-organisms, mainly fungi and bacteria, are involved. The process depends on a large number of factors (biological, chemical, and physical) among which microbial populations play a fundamental role. The high temperatures that occur during the composting process indicate the presence of thermotolerant and thermophilic micro-organisms that are key for the optimization of the process. However, the same micro-organisms can be harmful (allergenic, pathogenic) for workers that handle large quantities of material in the plant, and for end users, for example, in the indoor environment (e.g., pots in houses and offices). Accurate knowledge of thermotolerant and thermophilic organisms present during the composting stages is required to find key organisms to improve the process and estimate potential health risks. The objective of the present work was to study thermotolerant and thermophilic mycobiota at different time points of compost maturation. Fungi were isolated at four temperatures (25, 37, 45, and 50 °C) from compost samples collected at five different steps during a 21-day compost-maturation period in an active composting plant in Liguria (northwestern Italy). The samples were subsequently plated on three different media. Our results showed a high presence of fungi with an order of magnitude ranging from 1 × 10 to 3 × 10 colony-forming units (CFU) g. The isolated strains, identified by means of specific molecular tools (ITS, beta-tubulin, calmodulin, elongation factor 1-alpha, and LSU sequencing), belonged to 45 different species. Several thermophilic species belonging to genera and were detected, which could be key during composting. Moreover, the presence of several potentially harmful fungal species, such as , and were found during the whole process, including the final product. Results highlighted the importance of surveying the mycobiota involved in the composting process in order to: (i) find solutions to improve efficiency and (ii) reduce health risks.
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http://dx.doi.org/10.3390/microorganisms8060880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355412PMC
June 2020

Molecular Identification and In Vitro Antifungal Susceptibility of Aspergillus Isolates Recovered from Otomycosis Patients in Western China.

Mycopathologia 2020 Jun 28;185(3):527-535. Epub 2020 Apr 28.

Microbiology Department, People's Hospital of Suzhou National New & Hi-Tech Industrial Development Zone, Suzhou, Jiangsu, China.

Aspergillus species are the most common causative agents involved in otomycosis. In this study, 45 Aspergillus isolates were obtained from patients with otomycosis in western China during 2013-2016. The aim of this study is to identify the Aspergillus isolates to the species level by using β-tubulin gene sequencing and to evaluate their in vitro susceptibility to nine antifungal drugs: amphotericin B, itraconazole, voriconazole, posaconazole, ravuconazole, isavuconazole, caspofungin, micafungin and anidulafungin according to CLSI M38-A2. Our results indicate that A. tubingensis (18/45) is the predominant Aspergillus species causing ear infections in western China, which is three times more than its sibling species A. niger (6/45) and A. welwitschiae (2/45). Other detected species were A. fumigatus (n = 8), A. terreus (n = 7) and A. flavus (n = 4). Antifungal susceptibility data indicate that triazoles and echinocandins are active against the most Aspergillus isolates. There are no significant differences in the susceptibility among A. niger, A. tubingensis and A. welwitschiae to each of the antifungals tested. One azole-resistant A. fumigatus isolate with a TR34/L98H mutation in the CYP51A gene and one posaconazole-resistant A. terreus isolate presented among the studied isolates. In conclusion, A. tubingensis is the most prevalent Aspergillus species causing otomycosis in western China. Posaconazole and echinocandins are potential drugs for treatment of otomycosis due to Aspergillus; however, in vivo efficacy remains to be determined.
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http://dx.doi.org/10.1007/s11046-020-00448-7DOI Listing
June 2020

Fungal Diversity and Mycotoxins in Low Moisture Content Ready-To-Eat Foods in Nigeria.

Front Microbiol 2020 9;11:615. Epub 2020 Apr 9.

Department of Agrobiotechnology (IFA-Tulln), Institute of Bioanalytics and Agro-Metabolomics, University of Natural Resources and Life Sciences Vienna (BOKU), Tulln, Austria.

Low moisture content ready-to-eat foods vended in Nigerian markets could be pre-packaged or packaged at point of sale. These foods are widely and frequently consumed across Nigeria as quick foods. Despite their importance in the daily diets of Nigerians, a comprehensive study on the diversity of fungi, fungal metabolite production potential, and mycotoxin contamination in the foods has not yet been reported. Therefore, this study assessed the diversity of fungi in 70 samples of low moisture content ready-to-eat foods [cheese balls, (cassava-based), (a mix of cereals and nuts) and popcorn] in Nigeria by applying a polyphasic approach including morphological examination, genera/species-specific gene marker sequencing and secondary metabolite profiling of fungal cultures. Additionally, mycotoxin levels in the foods were determined by LC-MS/MS. Fungal strains ( = 148) were recovered only from . Molecular analysis of 107 representative isolates revealed 27 species belonging to 12 genera: , , , , , , , , , and in the Ascomycota, and and in the Basidiomycota. To the best of our knowledge , , , , and are reported in African food for the first time. A total of 21 uncommon metabolites were found in cultures of the following species: andrastin A and sporogen AO1 in ; paspalin in ; lecanoic acid and rugulusovin in ; sclerotin A in and ; barceloneic acid, festuclavine, fumigaclavine, isochromophilons (IV, VI, and IX), ochrephilone, sclerotioramin, and sclerotiorin in ; epoxyagroclavine, infectopyron, methylorsellinic acid and trichodermamide C in ; moniliformin and sporogen AO1 in ; and aminodimethyloctadecanol in . Twenty-four mycotoxins in addition to other 73 fungal and plant toxins were quantified in the foods. In , cheeseballs, popcorn and were 1, 6, 12, and 23 mycotoxins detected, respectively. Deoxynivalenol, fumonisins, moniliformin, aflatoxins and citrinin contaminated 37, 31, 31, 20, and 14% of all food samples, respectively. Overall, citrinin had the highest mean concentration of 1481 μg/kg in the foods, suggesting high citrinin exposures in the Nigerian populace. Fungal and mycotoxin contamination of the foods depend on pre-food and post-food processing practices.
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http://dx.doi.org/10.3389/fmicb.2020.00615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161469PMC
April 2020

Moulds and their secondary metabolites associated with the fermentation and storage of two cocoa bean hybrids in Nigeria.

Int J Food Microbiol 2020 Mar 14;316:108490. Epub 2019 Dec 14.

Institute of Bioanalytics and Agro-Metabolomics, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU), Konrad Lorenzstr. 20, A-3430 Tulln, Austria; Department of Microbiology, Babcock University, Ilishan Remo, Ogun State, Nigeria. Electronic address:

Fungi and mycotoxin contamination of cocoa beans during fermentation and storage may constitute a hazard in the cocoa value chain and risk to consumers of its products. In this study, fungal profile and secondary metabolite patterns in two cocoa bean hybrids, F and T series, during fermentation and storage were determined. Additionally, secondary metabolite production by the recovered fungi in the beans was examined in culture media. Fungal isolates spanned six genera and eight species: Aspergillus niger, A. tamarii, Paecilomyces variotii, Penicillium citrinum, Pseudopithomyces palmicola, Simplicillium sp., Talaromyces atroroseus and Talaromyces sp.. In both hybrids, Aspergilli (38%) dominated the other fungi while more than one half of all the fungal isolates were from the beans in storage. Among the diverse secondary metabolites produced in media by the isolates were uncommon compounds, e.g. aspulvinone E produced by A. niger, aspterric acid by P. variotii, scalusamid A and sydowinin A by P. citrinum, norlichexanthone and siccanol by Simplicillium, and fallacinol and orsellinic acid by Talaromyces. The strains of P. citrinum produced up to 372 mg/kg citrinin. Forty-four fungal metabolites were quantified in both bean hybrids across the various processing stages, with about 86% occurring in the fermented beans stored for 30 days. The nephrotoxic citrinin, which was not previously reported in cocoa beans worldwide, was the only mycotoxin found in the fermented beans at overall mean concentration of 368 μg/kg. Additionally, its metabolite, dihydrocitrinone, was detected in fermented and stored beans. Consumption of freshly fermented cocoa beans may result in citrinin exposure. Appropriate fungal and mycotoxin control measures are proposed.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2019.108490DOI Listing
March 2020

Partial characteristics of hemolytic factors secreted from airborne Aspergillus and Penicillium, and an enhancement of hemolysis by Aspergillus micronesiensis CAMP-like factor via Staphylococcus aureus-sphingomyelinase.

J Microbiol 2019 Dec 4;57(12):1086-1094. Epub 2019 Nov 4.

Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.

One of the advantages for initial survival of inhaled fungal spores in the respiratory tract is the ability for iron acquisition via hemolytic factor-production. To examine the ability of indoor Aspergillus and Penicillium affecting hemolysis, the secreted factors during the growth of thirteen strains from eight species were characterized in vitro for their hemolytic activity (HA) and CAMP-like reaction. The hemolytic index of HA on human blood agar of Aspergillus micronesiensis, Aspergillus wentii, Aspergillus westerdijkiae, Penicillium citrinum, Penicillium copticola, Penicillium paxilli, Penicillium steckii, and Penicillium sumatrense were 1.72 ± 0.34, 1.61 ± 0.41, 1.69 ± 0.16, 1.58 ± 0.46, 3.10 ± 0.51, 1.22 ± 0.19, 2.55 ± 0.22, and 1.90 ± 0.14, respectively. The secreted factors of an Aspergillus wentii showed high HA when grown in undernourished broth at 25°C at an exponential phase and were heat sensitive. Its secreted proteins have an estimated relative molecular weight over 50 kDa. Whereas, the factors of Penicillium steckii were secreted in a similar condition at a late exponential phase but showed low HA and heat tolerance. In a CAMP-like test with sheep blood, the synergistic hemolytic reactions between most tested mold strains and Staphylococcus aureus were identified. Moreover, the enhancement of α-hemolysis of Staphylococcus aureus could occur through the interaction of Staphylococcus aureus-sphingomyelinase and CAMP-like factors secreted from Aspergillus micronesiensis. Further studies on the characterization of purified hemolytic- and CAMP-like-factors secreted from Aspergillus wentii and Aspergillus micronesiensis may lead to more understanding of their involvement of hemolysis and cytolysis for fungal survival prior to pathogenesis.
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http://dx.doi.org/10.1007/s12275-019-9133-4DOI Listing
December 2019

The diversity and ecological roles of Penicillium in intertidal zones.

Sci Rep 2019 09 19;9(1):13540. Epub 2019 Sep 19.

School of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul, 08826, South Korea.

Members of the genus Penicillium are commonly isolated from various terrestrial and marine environments, and play an important ecological role as a decomposer. To gain insight into the ecological role of Penicillium in intertidal zones, we investigated the Penicillium diversity and community structure using a culture-dependent technique and a culture independent metagenomic approach using ITS (ITS-NGS) and partial β-tubulin (BenA-NGS) as targets. The obtained isolates were tested for halotolerance, enzyme activity, and polycyclic aromatic hydrocarbons (PAHs) degradation. A total of 96 Penicillium species were identified from the investigated intertidal zones. Although the BenA-NGS method was efficient for detecting Penicillium, some species were only detected using conventional isolation and/or the ITS-NGS method. The Penicillium community displayed a significant degree of variation relative to season (summer and winter) and seaside (western and southern coast). Many Penicillium species isolated in this study exhibited cellulase and protease activity, and/or degradation of PAHs. These findings support the important role of Penicillium in the intertidal zone for nutrient recycling and pollutant degradation.
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http://dx.doi.org/10.1038/s41598-019-49966-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753150PMC
September 2019

Talaromyces atroroseus in HIV and non-HIV patient: A first report from Indonesia.

Med Mycol 2020 Jun;58(4):560-563

Department of Parasitology, Faculty of Medicine, Universitas Indonesia.

We performed morphology, molecular study and antifungal susceptibility test on 10 Talaromyces sp. isolates: eight clinical isolates (human immunodeficiency virus (HIV) and non-HIV-patient) and two isolates from rats. All strains produced red soluble pigment and microscopically showed Penicillium-like structure in room temperature and yeast-like structure in 37°C. Based on molecular analysis, nine isolates were identified as Talaromyces atroroseus (including the isolates from rats) and one as T. marneffei. Our susceptibility result of T. marneffei supports the use of amphotericin B, itraconazole for talaromycosis marneffei management. Talaromyces atroroseus showed variable MIC to echinocandin, azole derivatives, 5-flucytosine and amphotericin B.
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http://dx.doi.org/10.1093/mmy/myz090DOI Listing
June 2020

Mutations, Extrolite Profiles, and Antifungal Susceptibility in Clinical and Environmental Isolates of the Aspergillus viridinutans Species Complex.

Antimicrob Agents Chemother 2019 11 22;63(11). Epub 2019 Oct 22.

Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands.

The past decade has seen an increase in aspergillosis in humans and animals due to species complex members. Azole resistance is common to these infections, carrying a poor prognosis. gene mutations are the main cause of acquired azole resistance in This study aimed to determine if the azole-resistant phenotype in complex members is associated with mutations or extrolite profiles. The gene of clinical and environmental isolates was amplified using novel primers, antifungal susceptibility was tested using the Clinical and Laboratory Standards Institute methodology, and extrolite profiling was performed using agar plug extraction. Very high azole MICs were detected in 84% of the isolates (31/37). The MICs of the newer antifungals luliconazole and olorofim (F901318) were low for all isolates. sequences revealed 113 nonsynonymous mutations compared to the sequence of wild-type M172A/V and D255G, previously associated with azole resistance, were common among all isolates but were not correlated with azole MICs. Two environmental isolates with nonsusceptibility to itraconazole and high MICs of voriconazole and isavuconazole harbored G138C, previously associated with azole-resistant Some novel mutations were identified only among isolates with high azole MICs. However, homology modeling did not cause a significant protein structure change for these mutations. There was no correlation between extrolite patterns and susceptibility. For complex isolates, mutations and the extrolites that they produced were not major causes of antifungal resistance. Luliconazole and olorofim show promise for treating azole-resistant infections caused by these cryptic species.
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http://dx.doi.org/10.1128/AAC.00632-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6811395PMC
November 2019

The Emergence of Rare Clinical Species in Qatar: Molecular Characterization and Antifungal Susceptibility Profiles.

Front Microbiol 2019 30;10:1677. Epub 2019 Jul 30.

Division of Microbiology, Department of Laboratory Medicine and Pathology, Hamad Medical Corporation, Doha, Qatar.

are ubiquitous mold species that infect immunocompetent and immunocompromised patients. The symptoms are diverse and range from allergic reactions, bronchopulmonary infection, and bronchitis, to invasive aspergillosis. The aim of this study was to characterize 70 isolates recovered from clinical specimens of patients with various clinical conditions presented at Hamad general hospital in Doha, Qatar, by using molecular methods and to determine their antifungal susceptibility patterns using the Clinical and Laboratory Standards Institute (CLSI) M38-A2 reference method. Fourteen species were identified by sequencing β-tubulin and calmodulin genes, including 10 rare and cryptic species not commonly recovered from human clinical specimens. is reported in this study for the first time in patients with fungal rhinosinusitis ( = 6) and one patient with a lower respiratory infection. Moreover, is reported in a patient with fungal rhinosinusitis which is considered as the first report ever from clinical specimens. In addition, is reported for the first time in a patient with cystic fibrosis. In general, our strains exhibited low MIC values for most of the antifungal drugs tested. One strain of showed high MECs for echinocandins and low MICs for the rest of the drugs tested. Another strain of exhibited high MIC for itraconazole and categorized as non-wild type. These findings require further analysis of their molecular basis of resistance. In conclusion, reliable identification of species is achieved by using molecular sequencing, especially for the emerging rare and cryptic species. They are mostly indistinguishable by conventional methods and might exhibit variable antifungal susceptibility profiles. Moreover, investigation of the antifungal susceptibility patterns is necessary for improved antifungal therapy against aspergillosis.
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http://dx.doi.org/10.3389/fmicb.2019.01677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697061PMC
July 2019

The most heat-resistant conidia observed to date are formed by distinct strains of Paecilomyces variotii.

Environ Microbiol 2020 03 24;22(3):986-999. Epub 2019 Oct 24.

TiFN, P.O. Box 557, 6700 AN, Wageningen, The Netherlands.

Fungi colonize habitats by means of spores. These cells are stress-resistant compared with growing fungal cells. Fungal conidia, asexual spores, formed by cosmopolitan fungal genera like Penicillium, Aspergillus and Peacilomyces are dispersed by air. They are present in places where food products are stored and as a result, they cause food spoilage. Here, we determined the heterogeneity of heat resistance of conidia between and within strains of Paecilomyces variotii, a spoiler of foods such as margarine, fruit juices, canned fruits and non-carbonized sodas. Out of 108 strains, 31 isolates showed a conidial survival >10% after a 10-min-heat treatment at 59°C. Three strains with different conidial heat resistance were selected for further phenotyping. Conidia of DTO 212-C5 and DTO 032-I3 showed 0.3% and 2.6% survival in the screening respectively, while survival of DTO 217-A2 conidia was >10%. The decimal reduction times of these strains at 60°C (D value) were 3.7 ± 0.08, 5.5 ± 0.35 and 22.9 ± 2.00 min respectively. Further in-depth analysis revealed that the three strains showed differences in morphology, spore size distributions, compatible solute compositions and growth under salt stress. Conidia of DTO 217-A2 are the most heat-resistant reported so far. The ecological consequences of this heterogeneity of resistance, including food spoilage, are discussed.
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http://dx.doi.org/10.1111/1462-2920.14791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065192PMC
March 2020

The preservative propionic acid differentially affects survival of conidia and germ tubes of feed spoilage fungi.

Int J Food Microbiol 2019 Oct 8;306:108258. Epub 2019 Jul 8.

Trouw Nutrition, Stationsstraat 77, 3811 MH Amersfoort, the Netherlands.

Propionic acid is widely used as a preservative in (poultry) feed. In this study we have isolated and identified fungal strains from nine samples poultry feed originating from different countries. The majority of the strains were Aspergilli with a eurotium-morph, such as Aspergillus proliferans and A. chevalieri. These and three other species were selected and tested for their sensitivity towards the feed preservative propionic acid, among them Penicillium lanosocoeruleum. The determined MIC values of 6.1-31 mM of these poultry feed specific fungi were well in the range as described in literature. Propionic acid (at 31 mM) damages conidia (spores) in a species dependent fashion after a 24-hour-treatment. The majority of the conidia (over 70%) of P. lanosocoeruleum germinated within 60 h on agar medium, while 50 and 80% of the A. chevalieri and A. proliferans conidia did not, respectively. Dependent on the species, cell damage was visible after incubation with propionic acid. Germ tubes of P. lanosocoeruleum in a biofilm showed extensive (85%) cell death after a 30 min treatment with propionic acid and slightly lower sensitivity was observed with A. proliferans (62% cell death). Microscopic analysis of these fungal biofilms revealed extensive damage to the cell membrane and showed distorted intracellular structures. Fluorescent life-dead staining of the germ tubes showed a clear dose response of propionic acid indicating a fungicidal effect on these growing cells. These results show that conidia can be inactivated by propionic acid, but that germ tubes show a much higher sensitivity. These observations shed new light on the mode of action of this important preservative to prevent fungal contamination of feed.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2019.108258DOI Listing
October 2019

First case of fungal keratitis due to in Iran.

Curr Med Mycol 2019 Jun;5(2):45-48

Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands.

Background And Purpose: Herein, we report the first case of fungal keratitis due to in a 68-year-old rural woman admitted to the Ophthalmology Center of Khatam-Al-Anbia Hospital in Mashhad, northeast of Iran.

Case Report: The patient presented with severe pain, burning, foreign body sensation, and reduced vision in her right eye. She had long-term uncontrolled diabetes and was not able to close her eye due to an anatomical problem with the eyelid. The cornea smear sample was cultured, and the fungus was initially identified as . The isolated strain was further identified by sequencing a part of the calmodulin gene as . The patient did not respond to any antifungal treatments (e.g., amphotericin B and voriconazole drops, and fluconazole 300 mg/day); therefore, she was eventually subjected to corneal transplantation surgery.

Conclusion: Fungal keratitis can be caused by the less common species. The reliable identification of the causative agents can be accomplished by the implementation of molecular methods.
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http://dx.doi.org/10.18502/cmm.5.2.1162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626709PMC
June 2019

Mould spoilage of foods and beverages: Using the right methodology.

Food Microbiol 2019 Aug 30;81:51-62. Epub 2018 Mar 30.

Westerdijk Fungal Biodiversity Institute, Dept. Applied and Industrial Mycology, Uppsalalaan 8, Utrecht, CT 3584, The Netherlands.

Fungal spoilage of products manufactured by the food and beverage industry imposes significant annual global revenue losses. Mould spoilage can also be a food safety issue due to the production of mycotoxins by these moulds. To prevent mould spoilage, it is essential that the associated mycobiota be adequately isolated and accurately identified. The main fungal groups associated with spoilage are the xerophilic, heat-resistant, preservative-resistant, anaerobic and psychrophilic fungi. To assess mould spoilage, the appropriate methodology and media must be used. While classic mycological detection methods can detect a broad range of fungi using well validated protocols, they are time consuming and results can take days or even weeks. New molecular detection methods are faster but require good DNA isolation techniques, expensive equipment and may detect viable and non-viable fungi that probably will not spoil a specific product. Although there is no complete and easy method for the detection of fungi in food it is important to be aware of the limitation of the methodology. More research is needed on the development of methods of detection and identification that are both faster and highly sensitive.
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http://dx.doi.org/10.1016/j.fm.2018.03.016DOI Listing
August 2019

, a new species from (L.) Dunal.

Mycology 2019 Mar 12;10(1):49-60. Epub 2018 Dec 12.

School of Biosciences, Mahatma Gandhi University, Kottayam, India.

Medicinal plants are considered as sources of novel and unexplored groups of endophytic microorganisms. A study on endophytic fungal species from the medicinal plant (L.) Dunal resulted in the isolation of a isolate (WSR 62) with antibiotic activity. Phylogenetic analysis showed that the isolate belongs to section , and it is most closely related to . Subsequent detailed phylogenetic analyses using partial β-tubulin (), calmodulin () and DNA-dependent RNA polymerase II () gene sequences of a larger number of related strains revealed the distinctiveness of the isolate in the clade. The isolate grows fast on Czapek yeast autolysate agar (CYA) and malt extract agar (MEA) incubated at 25°C, 30°C and 37°C. The obverse colony colour is dominated by the conspicuous production of cleistothecia and is greyish yellow on CYA and yellowish brown on MEA. Production of cleistothecia containing prominent spinose ascospores was present on all tested agar media. Based on the phylogenetic analysis and the phenotypic characterisation, strain WSR 62 from is described here as a novel species named
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http://dx.doi.org/10.1080/21501203.2018.1555868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6394320PMC
March 2019

Comparative genotyping and phenotyping of Aspergillus fumigatus isolates from humans, dogs and the environment.

BMC Microbiol 2018 09 17;18(1):118. Epub 2018 Sep 17.

Microbiology, Department of Biology, Utrecht University, Utrecht, The Netherlands.

Background: Aspergillus fumigatus is a ubiquitous saprotrophic fungus and an opportunistic pathogen of humans and animals. Humans and animals can inhale hundreds of A. fumigatus spores daily. Normally this is harmless for humans, but in case of immunodeficiency, invasive pulmonary aspergillosis (IPA) can develop with a high mortality rate. A. fumigatus also causes non-invasive mycoses like sino-nasal aspergillosis (SNA) in dogs.

Results: In this study we compared A. fumigatus isolates from humans with suspected IPA, dogs with SNA, and a set of environmental isolates. Phylogenetic inference based on calmodulin (CaM) and beta-tubulin (benA) sequences did not reveal A. fumigatus sub-groups linked to the origin of the isolates. Genotyping and microsatellite analysis showed that each dog was infected by one A. fumigatus genotype, whereas human patients had mixed infections. Azole resistance was determined by antifungal susceptibility testing and sequencing of the cyp51A gene. A total of 12 out of 29 human isolates and 1 out of 27 environmental isolates were azole resistant. Of the azole resistant strains, 11 human isolates showed TR/L98H (n = 6) or TR46/Y121F/T289A (n = 5). Phenotypically, isolates from dogs were more variable in growth speed and morphology when compared to those isolated from human and the environment.

Conclusions: 1. A. fumigatus from dogs with SNA are phenotypically very diverse in contrast to their environmental and human counterparts. 2. Phenotypic variability can be induced during the chronic infection process in the sinus of the dogs. The basis of this heterogeneity might be due to genomic differences and/or epigenetic variations. 3. Differences in dogs is a could be a result of within-host adaption and might be triggered by environmental factors in the sinus, however this hypothesis still needs to be tested.
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http://dx.doi.org/10.1186/s12866-018-1244-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142626PMC
September 2018

Talaromyces borbonicus, sp. nov., a novel fungus from biodegraded Arundo donax with potential abilities in lignocellulose conversion.

Mycologia 2018 Mar-Apr;110(2):316-324. Epub 2018 May 29.

a Department of Chemical Sciences , University of Naples Federico II, Complesso Universitario Monte S. Angelo , via Cintia, 4 80126 Naples , Italy.

A novel fungal species able to synthesize enzymes with potential synergistic actions in lignocellulose conversion was isolated from the biomass of Arundo donax during biodegradation under natural conditions in the Gussone Park of the Royal Palace of Portici (Naples, Italy). In this work, this species was subjected to morphological and phylogenetic analyses. Sequencing of its genome was performed, resulting in 28 scaffolds that were assembled into 27.05 Mb containing 9744 predicted genes, among which 396 belong to carbohydrate-active enzyme (CAZyme)-encoding genes. Here we describe and illustrate this previously unknown species, which was named Talaromyces borbonicus, by a polyphasic approach combining phenotypic, physiological, and sequence data.
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http://dx.doi.org/10.1080/00275514.2018.1456835DOI Listing
January 2019

Identification of fungal causative agents of rhinosinusitis from Mashhad, Iran.

Curr Med Mycol 2017 Sep;3(3):5-9

Sinus and Surgical Endoscopic Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Background And Purpose: Rhinosinusitis is a common disorder, influencing approximately 20% of the population at some time of their lives. It was recognized and reported with expanding recurrence over the past two decades worldwide. Undoubtedly, correct diagnosis of fungi in patients with fungal rhinosinusitis affects the treatment planning and prognosis of the patients. Identification of the causative agents using the standard mycological procedures remains difficult and time-consuming.

Materials And Methods: Based on clinical and radiological parameters, 106 patients suspected of fungal rhinosinusitis were investigated in this cross-sectional prospective study from April 2012 to March 2016 at an otorhinolaryngology department. In this study, internal transcribed spacer (ITS) and calmodulin () sequencing were respectively validated as reliable techniques for the identification of Mucorales and to species level (both agents of fungal rhinosinusitis).

Results: Of these, 63 (59.4%) patients were suspected of allergic fungal rhinosinusitis (AFRS), 40 (37.7%) patients suspected of acute invasive fungal rhinosinusitis (AIFRS), and 3 (2.8%) patients suspected of mycetoma. In patients suspected of AFRS, AIFRS, and mycetoma only 7, 29, and 1 had positive fungal culture, respectively. After ITS and sequencing, was the most common species isolated from non-invasive forms, and and were more frequently isolated from invasive forms.

Conclusion: is the most common agent of fungal rhinosinusitis in Iran, unlike most other reports from throughout the world stating that is the most frequent causative agent of this disease.
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http://dx.doi.org/10.29252/cmm.3.3.5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5914920PMC
September 2017