Aspergillus Section Circumdati Research Articles

Assessment of mycobiota in Thai pigmented rice: Insights into ochratoxin A and citrinin production by Aspergillus and Penicillium species

Pigmented rice has gained interest for its nutritional and health benefits attributed to its bioactive compounds. Despite these advantages, the vulnerability of pigmented rice to mycotoxin contamination poses a major concern. This study examined the mycobiota of seven Thai pigmented rice varieties, with a focus on fungi capable of producing ochratoxin A (OTA) and citrinin. The study analyzed 110 pigmented rice samples and found that the Aspergillus section Aspergillus was predominant (50–82.35%), followed by Penicillium species (12.50–70.59%). One isolate of A. carbonarius from Aspergillus section Nigri (1/112) and two isolates from Aspergillus section Circumdati (2/61)—A. steynii and A. flocculosus—produced OTA. However, none of the Penicillium species produced OTA, though citrinin production was notable in P. citrinum (128/280). The study found that the Black Jasmine rice and Red Jasmine rice varieties were more susceptible to mycotoxin production, while the Luem Pua glutinous rice and Hom Nil rice varieties displayed more resistance to the accumulation of both mycotoxins. These findings should guide targeted strategies to mitigate mycotoxin risks and ensure the safety of pigmented rice in the food chain.

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.

Simultaneous detection of mycotoxigenic Aspergillus species of sections Circumdati and Flavi using multiplex digital PCR.

Populations of ochratoxin-producing Aspergillus section Circumdati species and aflatoxin-producing Aspergillus section Flavi species frequently coexist in soil and are the main sources of mycotoxin contamination of tree nuts. Identification of mycotoxigenic Aspergillus species in these sections is difficult using traditional isolation and culture methods. We developed a multiplex digital PCR (dPCR) assay to detect and quantify Aspergillus ochraceus, Aspergillus westerdijkiae, and Aspergillus steynii (section Circumdati), as well as Aspergillus flavus and Aspergillus parasiticus (section Flavi), in environmental samples based on species-specific calmodulin gene sequences. Relative quantification of each species by dPCR of mixed-species templates correlated with corresponding DNA input ratios. Target species could be detected in soil inoculated with conidia from each species. Non-target species of sections Circumdati, Flavi, and Nigri were generally not detectable using this dPCR method. Detected non-target species (Aspergillus fresenii, Aspergillus melleus, Aspergillus sclerotiorum, and Aspergillus subramanianii) were discernible from A. ochraceus in dual-template dPCR reactions based on differential fluorescence intensity.

87 Portuguese Groceries: A Potential Indoor Environment to Be Used as Public Health Sentinel for Fungal and Mycotoxins Contamination

Abstract This study aimed to characterize microbial contamination in Portuguese grocery stores. The research was carried out in 15 grocery stores in Cascais, Portugal. Electrostatic dust cloths (EDC) and surface swabs were used on 3 sampling sites: checkout, fruits/vegetable, and warehouse/dispenser. Fungal contamination was characterized by culture-based methods, through the inoculation onto two different culture media: malt extract agar (MEA) supplemented with chloramphenicol (0.05 %) and dichloran-glycerol agar (DG18). Screening of azole resistance was performed by inoculation of EDC extracts´ in azole-supplemented Sabouraud dextrose agar (SDA) media, according to EUCAST guideline. A total of 39 samples were screened for mycotoxins contamination. The highest fungal contamination was obtained from fruits/vegetables in swabs (76 % MEA; 71 % DG18). The most prevalent genera in swabs was Cladosporium sp. (54 % MEA). Regarding EDC, besides Penicillium sp. (63 %) the most prevalent were Aspergillus sections Circumdati (25 %) and Aspergilli (35 %). Penicillium sp. was highly prevalent in the checkout (61 % SDA; 87 % voriconazole), and warehouse/dispenser sectors (73% voriconazole; 52% posaconazole). Circumdati and Fumigati were the most prevalent Aspergillus sections in fruits/vegetables (73 % and 64 % in voriconazole, respectively). Four mycotoxins (fumonisin B1, B2, B3) were detected simultaneously in most of the 36 samples. Identifying the most critical workplaces in groceries concerning fungal and mycotoxins contamination is of upmost importance to human health (workers and consumers) when using a One Health approach.

The Secondary Metabolites and Biosynthetic Diversity From Aspergillus ochraceus.

Aspergillus ochraceus, generally known as a food spoilage fungus, is the representative species in Aspergillus section Circumdati. A. ochraceus strains are widely distributed in nature, and usually isolated from cereal, coffee, fruit, and beverage. Increasing cases suggest A. ochraceus acts as human and animal pathogens due to producing the mycotoxins. However, in terms of benefits to mankind, A. ochraceus is the potential source of industrial enzymes, and has excellent capability to produce diverse structural products, including polyketides, nonribosomal peptides, diketopiperazine alkaloids, benzodiazepine alkaloids, pyrazines, bis-indolyl benzenoids, nitrobenzoyl sesquiterpenoids, and steroids. This review outlines recent discovery, chemical structure, biosynthetic pathway, and bio-activity of the natural compounds from A. ochraceus.

Genome and Metabolome MS-Based Mining of a Marine Strain of Aspergillus affinis.

Aspergillus section Circumdati encompasses several species that express both beneficial (e.g., biochemical transformation of steroids and alkaloids, enzymes and metabolites) and harmful compounds (e.g., production of ochratoxin A (OTA)). Given their relevance, it is important to analyze the genetic and metabolic diversity of the species of this section. We sequenced the genome of Aspergillus affinis CMG 70, isolated from sea water, and compared it with the genomes of species from section Circumdati, including A. affinis’s strain type. The A. affinis genome was characterized considering secondary metabolites biosynthetic gene clusters (BGCs), carbohydrate-active enzymes (CAZymes), and transporters. To uncover the biosynthetic potential of A. affinis CMG 70, an untargeted metabolomics (LC-MS/MS) approach was used. Cultivating the fungus in the presence and absence of sea salt showed that A. affinis CMG 70 metabolite profiles are salt dependent. Analyses of the methanolic crude extract revealed the presence of both unknown and well-known Aspergillus compounds, such as ochratoxin A, anti-viral (e.g., 3,5-Di-tert-butyl-4-hydroxybenzoic acid and epigallocatechin), anti-bacterial (e.g., 3-Hydroxybenzyl alcohol, l-pyroglutamic acid, lecanoric acid), antifungal (e.g., lpyroglutamic acid, 9,12,13-Trihydroxyoctadec-10-enoic acid, hydroxyferulic acid), and chemotherapeutic (e.g., daunomycinone, mitoxantrone) related metabolites. Comparative analysis of 17 genomes from 16 Aspergillus species revealed abundant CAZymes (568 per species), secondary metabolite BGCs (73 per species), and transporters (1359 per species). Some BGCs are highly conserved in this section (e.g., pyranonigrin E and UNII-YC2Q1O94PT (ACR toxin I)), while others are incomplete or completely lost among species (e.g., bikaverin and chaetoglobosins were found exclusively in series Sclerotiorum, while asperlactone seemed completely lost). The results of this study, including genome analysis and metabolome characterization, emphasize the molecular diversity of A. affinis CMG 70, as well as of other species in the section Circumdati.

Storage fungi and ochratoxin A associated with arabica coffee bean in postharvest processes in Northern Thailand

Arabica coffee is one of the most popular varieties globally; however, during postharvest and storage, it can be contaminated by fungi which produce mycotoxins such as ochratoxin A (OTA). The goal of this study was to determine fungal contamination and OTA levels of arabica coffee produced from Chiang Rai and Chiang Mai provinces in Northern Thailand. Aspergillus and Penicillium were mainly isolated and evaluated from coffee samples in the postharvest process. Coffee cherries, parchment coffee, and green coffee beans were commonly found contaminated with fungi. Coffee beans were contaminated with Penicillium, Aspergillus section Flavi, Aspergillus section Nigri, and Aspergillus section Circumdati at 1.2–10%. Fungal contamination in parchment coffee was studied using three drying methods. The results showed that coffee dried in a greenhouse had higher fungal contamination than samples dried on concrete ground or on a bamboo table. Whether basic storage packaging of green coffee beans affects fungal contamination was also investigated. Green coffee beans in three packaging types showed similar levels of Penicillium, while of the three packaging types, coffee in a polypropylene woven bag showed significantly less Aspergillus than those in a ramie sack or a plastic mesh bag. Fourteen strains of Aspergillus section Circumdati were isolated from coffee samples and 5 isolates showed green-blue fluorescence under ultraviolet light suggesting a potential of OTA production. The morphology of Aspergillus section Circumdati isolates were studied and five isolates with similar morphological characters were further identified to the species level based on ITS and the β-tubulin gene regions. Sequencing and phylogenetic analysis suggested that four isolates were grouped with A. westerdijkiae and one isolate with A. melleus. Analysis of OTA in parchment coffee, green coffee beans, and roasted coffee was performed using HPLC. The results showed that OTA was detected in only one sample of roasted coffee at 0.39 μg kg−1.

Diversity of Mycobiota in Spanish Grape Berries and Selection of Hanseniaspora uvarum U1 to Prevent Mycotoxin Contamination.

The occurrence of mycotoxins on grapes poses a high risk for food safety; thus, it is necessary to implement effective prevention methods. In this work, a metagenomic approach revealed the presence of important mycotoxigenic fungi in grape berries, including Aspergillus flavus, Aspergillus niger aggregate species, or Aspergillus section Circumdati. However, A. carbonarius was not detected in any sample. One of the samples was not contaminated by any mycotoxigenic species, and, therefore, it was selected for the isolation of potential biocontrol agents. In this context, Hanseniaspora uvarum U1 was selected for biocontrol in vitro assays. The results showed that this yeast is able to reduce the growth rate of the main ochratoxigenic and aflatoxigenic Aspergillus spp. occurring on grapes. Moreover, H. uvarum U1 seems to be an effective detoxifying agent for aflatoxin B1 and ochratoxin A, probably mediated by the mechanisms of adsorption to the cell wall and other active mechanisms. Therefore, H. uvarum U1 should be considered in an integrated approach to preventing AFB1 and OTA in grapes due to its potential as a biocontrol and detoxifying agent.

Melinacidin-Producing Acrostalagmus luteoalbus, a Major Constituent of Mixed Mycobiota Contaminating Insulation Material in an Outdoor Wall.

Occupants may complain about indoor air quality in closed spaces where the officially approved standard methods for indoor air quality risk assessment fail to reveal the cause of the problem. This study describes a rare genus not previously detected in Finnish buildings, Acrostalagmus, and its species A. luteoalbus as the major constituents of the mixed microbiota in the wet cork liner from an outdoor wall. Representatives of the genus were also present in the settled dust in offices where occupants suffered from symptoms related to the indoor air. One strain, POB8, was identified as A. luteoalbus by ITS sequencing. The strain produced the immunosuppressive and cytotoxic melinacidins II, III, and IV, as evidenced by mass spectrometry analysis. In addition, the classical toxigenic species indicating water damage, mycoparasitic Trichoderma, Aspergillus section Versicolores, Aspergillus section Circumdati, Aspergillus section Nigri, and Chaetomium spp., were detected in the wet outdoor wall and settled dust from the problematic rooms. The offices exhibited no visible signs of microbial growth, and the airborne load of microbial conidia was too low to explain the reported symptoms. In conclusion, we suggest the possible migration of microbial bioactive metabolites from the wet outdoor wall into indoor spaces as a plausible explanation for the reported complaints.

The Genomic Regions That Contain Ochratoxin A Biosynthetic Genes Widely Differ in Aspergillus Section Circumdati Species.

Aspergillus section Circumdati includes 27 species, some of which are considered ochratoxin A (OTA) producers. However, there is considerable controversy about their potential OTA synthesis ability. In this work, the complete genomes of 13 species of Aspergillus section Circumdati were analyzed in order to study the cluster of OTA biosynthetic genes and the region was compared to those previously reported in A. steynii and A. westerdijkiae. The results obtained reveal that the genomes of some species in this section, including A. affinis, A. cretensis, A. elegans, A. muricatus, A. pulvericola, A. roseoglobulosus, and A. subramanianii, contain a potentially functional OTA biosynthetic cluster. Therefore, they might be able to synthesize the toxin. On the contrary, A. melleus, A. ochraceus, A. ostianus, A. persii, A. sclerotiorum, A. sesamicola, and A. westlandensis contain a truncated version of the cluster that lacks many of the genes involved in OTA biosynthesis, which might be related to their inability to produce OTA. The gain/loss pattern is different in all species, which suggests that the genetic evolution of this region might be due to independent events.

Settleable Dust and Bioburden in Portuguese Dwellings.

Monitoring campaigns in several buildings have shown that occupants exposed to contaminated indoor air generally exhibit diverse health symptoms. This study intends to assess settleable dust loading rates and bioburden in Portuguese dwellings by passive sampling onto quartz fiber filters and electrostatic dust cloths (EDCs), respectively. Settled dust collected by EDCs was analyzed by culture-based methods (including azole-resistance screening) and qPCR, targeting four different toxigenic Aspergillus sections (Flavi, Fumigati, Circumdati, and Nidulantes). Dust loading rates and bioburden showed higher variability in the summer season. In both seasons, Penicillium sp. was the one with the highest prevalence (59.1% winter; 58.1% summer), followed by Aspergillus sp. in winter (13.0%). Fungal contamination increased in the winter period, while bacterial counts decreased. Aspergillus sections Circumdati and Nidulantes, detected in voriconazole supplemented media, and Aspergillus sections Fumigati and Nidulantes, detected by molecular tools, were found in the winter samples. This study reinforces the importance of applying: (a) Passive sampling methods in campaigns in dwellings; (b) two different culture media (MEA and DG18) to assess fungi; (c) in parallel, molecular tools targeting the most suitable indicators of fungal contamination; and (d) azole resistance screening to unveil azole resistance detection in fungal species.

Occurrence of aflatoxin- and ochratoxin A-producing Aspergillus species in Thai dried chilli

The present study aimed to examine the presence of aflatoxin- and ochratoxin A (OTA)-producing fungi in dried chilli ( Capsicum spp.). A total of 120 chilli samples, including 60 samples each of chilli powder and dried chilli pods, were examined for fungal contamination. Results showed that 86.7% and 96.7% of chilli powder and dried chilli pods, respectively, were contaminated by fungi at a concentration of <10 3 CFU/g. Overall, 500 fungal isolates were recovered and identified using a morphological method. The most frequent strain was Aspergillus section Flavi (46.6%), followed by Aspergillus section Nigri (34%), Penicillium (7.6%), Aspergillus section Aspergillus (7.6%), Aspergillus section Circumdati (2.2%) and Rhizopus (2%). In total, 37.5% and 43.8% of Aspergillus section Flavi isolates obtained from dried chilli powder and dried chilli pods, respectively, produced aflatoxin. Of the 96 aflatoxigenic isolates, 96.9% of the strains produced aflatoxin B and 3.1% produced both aflatoxin B and G. However, β-tubulin sequence analysis revealed that all aflatoxin-producing isolates were identical to A. flavus . Moreover, 170 isolates of Aspergillus section Nigri , 11 of Aspergillus section Circumdati , 2 of Aspergillus section Flavi producing yellow-to-cinnamon colonies and 38 of Penicillium spp. were examined for their ability to produce OTA. In total, 1 isolate of Aspergillus section Nigri and 2 isolates of Aspergillus section Flavi produced OTA. The first isolate was identified as A. carbonarius , whereas the latter two isolates were both A. alliaceus . The highest OTA production by A. carbonarius (622.4 ng/g) was observed following incubation on MEA at 25 °C for 21 days. Conversely, the two A. alliaceus isolates produced OTA at levels of 583.7 and 743.7 ng/g, respectively, after cultivation on YES at 25 °C for 21 days. To the best of our knowledge, this is the first report regarding the natural occurrence of aflatoxin- and OTA-producing fungi in dried chilli from Thailand. • More than 95% of dried chilli powder and dried chilli pods collected in Thailand was contaminated with fungi. • Aspergillus section Flavi is the most common species in dried chilli. • Aspergillus section Flavi in dried chili samples is frequently aflatoxigenic strain. • Ochratoxin A producing species, A. carbonarius and A. alliaceus , were detected in dried chilli powder.

Occurrence and diversity of Aspergillus in commercial yerba mate elaborated for the Brazilian beverage ‘chimarrão’

Dried leaves and stems of Ilex paraguariensis St. Hil. (yerba mate) are used to make a popular beverage in some countries of South America, commonly known as “chimarrão”. The present study was designed to evaluate the occurrence of toxigenic Aspergillus in yerba mate in order to define the mycotoxin risk associated with this foodstuff. All samples tested were positive for fungal contamination, and the fungal load per sample ranged from 2.0 × 102 to 1.6 × 104 CFU/g. Aspergillus section Nigri was found in all samples and represented 76.5% of the total fungi isolated. Aspergillus section Circumdati, Aspergillus section Flavi and Aspergillus section Cremei were found at low frequencies. Thirteen different Aspergillus species were identified. The most common species found was A. luchuensis, which does not produce any harmful toxin for humans. A. niger, A. welwitschiae, A. flavus and A. novoparasiticus, all potentially toxigenic species, were found only in small quantities. The A. niger and A. welwitschiae strains were cultured to test for ochratoxin A and fumonisin B2 production. Only one strain producing ochratoxin A was found, but approximately 29% of the strains were positive for fumonisin B2. The A. flavus and A. novoparasiticus strains were tested for aflatoxins production, and 63% were positive. A. pallidofulvus, recently assigned to A. section Circumdati, was reported for the first time in herbs. All A. pallidofulvus strains analyzed in this study were negative for ochratoxin A production. In conclusion, A. section Nigri occurs with high frequency in yerba mate, and A. luchuensis is the predominant species. Although toxigenic species were found in this herb, the incidence was low.

Detection of Ochratoxigenic Potential in some Aspergillus and Penicillium Isolates from Vineyard Soil, Fresh and Dried Grapes by ELISA

Several species of Aspergillus and Penicillium were isolated and identified from samples of vineyard soil, fresh grape berries and dried vine fruits collected from grapevine nursery and shops in Duhok province. The ochratoxigenic potential of some of their strains was evaluated by ELISA technique. Ochratoxin A (OA) was detected in cultures of two species of section Nigri (A.carbonarius and A.niger aggreg.), two species of Aspergillus section Circumdati (A.ochraceus and A.westerdijkia) and one species of OA was found at levels from 0.64 to 0.72 ng ml in A.carbonarius isolates, from 0.64 to 0.66 ng ml in A.niger aggreg. isolates, from 0.23 to 0.65 ng ml in A.ochraceus isolates and 0.40 ngml in A.westterdijkia isolate, whereas the level was from 0.61 to 0.64 ngml in Penicillium verrucosum.تم عزل وتشخیص العدید من الأنواع الفطریة العائدة للجنسین اسبرجلس و بنیسیلیوم من عینات ترب حقول الأعناب فضلا عن ثمار العنب الطریة و الجافة والتی جمعت من محافظه دهوک. تم الکشف عن قابلیه إنتاج هذه العزلات لإنتاج السم Aspergillus section Nigri تم الکشف عن إنتاج السم من قبل عزلتان من ELISA. الفطری اوکراتوکسین باستخدام تقنیه Aspergillus section Circumdati (A.ochraceus وعزلتان من (A.carbonarius A.niger aggreg.), .Penicillium (P.verrucosum و عزله من A.westerdijkiae A.niger 0.64 نانوغرام / ملغم وفى عزلات - ما بین 0.72 A.carbonarius تراوح مستوى إنتاج السم من قبل عزلات 0.23 و فی عزله - مابین 0.65 A.ochraceus 0.64 نانوغرام/ ملغرام وفى عزلات - مابین 0.66 aggreg. 0.61 نانوغرام/ ملغم فى عزلات - 0.40 نانوغرام/ ملغرام بینما تراوح الإنتاج مابین - 0.64 A.westerdijkiaie .Penicillium verrucosum

Risk management of ochratoxigenic fungi and ochratoxin A in maize grains by bioactive EVOH films containing individual components of some essential oils

Aspergillus steynii and Aspergillus tubingensis are possibly the main ochratoxin A (OTA) producing species in Aspergillus section Circumdati and section Nigri, respectively. OTA is a potent nephrotoxic, teratogenic, embryotoxic, genotoxic, neurotoxic, carcinogenic and immunosuppressive compound being cereals the first source of OTA in the diet. In this study bioactive ethylene-vinyl alcohol copolymer (EVOH) films containing cinnamaldehyde (CINHO), linalool (LIN), isoeugenol (IEG) or citral (CIT) which are major components of some plant essential oils (EOs) were produced and tested against A. steynii and A. tubingensis growth and OTA production in partly milled maize grains. Due to the favourable safety profile, these bioactive compounds are considered in the category “GRAS”. The study was carried out under different water activity (0.96 and 0.99 aw), and temperature (24 and 32 °C) conditions. ANOVA showed that class of film, fungal species, aw and temperature and their interactions significantly affected growth rates (GR), ED50 and ED90 and the doses for total fungal growth inhibition and OTA production. The most effective EVOH films against both species were those containing CINHO. ED50, ED90 and doses for total growth and OTA inhibition were 165–405, 297–614, 333–666 μg of EVOH-CINHO/plate (25 g of maize grains), respectively, depending on environmental conditions. The least efficient were EVOH-LIN films. ED50, ED90 and doses for total growth and OTA inhibition were 2800–>3330, >3330 and >3330 μg of EVOH-LIN/plate (25 g of maize grains), respectively. The effectiveness of the bioactive films increased with increasing doses. Overall, A. tubingensis was less sensitive to treatments than A. steynii. Depending on the species, aw and temperature affected GR and OTA production in a different way. In A. steynii cultures, optimal growth occurred at 0.96 aw and 32 °C while optimal OTA production happened at 0.99 aw and 32 °C. In A. tubingensis cultures optimal growth happened at 0.99 aw and 32 °C, although the best conditions for OTA production were 0.99 aw and 24 °C. Thus, these species can be very competitive in warm climates and storage conditions. The EVOH-CINHO films followed by EVOH-IEG and EVOH-CIT films, designed in this study and applied in vapour phase, can be potent antifungal agents against A. steynii and A. tubingensis and strong inhibitors of OTA biosynthesis in maize grains at very low doses. This is the first study on the impact that interacting environmental conditions and bioactive films containing individual components of EOs have on the growth of these ochratoxigenic fungi and on OTA production in maize grains.

Aspergillus spp. prevalence in different Portuguese occupational environments: What is the real scenario in high load settings?

ABSTRACTThe genus Aspergillus is one of the most prevalent regarding fungi in several highly contaminated occupational environments. The goal of the current study was to assess the prevalence of Aspergillus spp. in different settings, focusing on those where a higher load of fungal contamination is expected according to the European Agency for Safety and Health at Work. A specific protocol to ensure a more accurate assessment of the exposure to Aspergillus spp. is proposed aimed at allowing a detailed risk characterization and management. Two wastewater treatment plants, one wastewater elevation plant, four waste treatment plants, three cork industries, five slaughter houses, four feed industries, one poultry pavilion, and two swineries, all located in the outskirts of Lisbon, were assessed. In total, 125 air samples and 125 surface samples were collected and analysed by culture-based methods. Real-time polymerase chain reaction was performed to detect fungal presence in 100 samples, targeting the Aspergillus sections Circumdati, Flavi, and Fumigati. The highest prevalence of Aspergillus spp. was found in wastewater treatment plants (69.3%; 31.1%), waste treatment plants (34.8%; 73.6%), and poultry feed industry (6.3%; 26.1%), in air and surfaces, respectively. Aspergillus spp. was also prevalent in cork industry (0.9%; 23.4%), slaughter houses (1.6%; 17.7%), and swineries (7.4%; 9.5%), in air and surfaces, respectively. The Aspergillus sections more prevalent in the air and surfaces of all the assessed settings were the Nigri section (47.46%; 44.71%, respectively), followed by Fumigati (22.28%; 27.97%, respectively) and Flavi (10.78%; 11.45%, respectively) sections. Aspergillus section Fumigati was successfully amplified by qPCR in 18 sampling sites where the presence of this fungal species had not been identified by conventional methods. It should be highlighted that the occupational exposure burden is due not only to the Aspergillus load, but also to the toxigenic potential of this genus. Based on our results, a protocol relied in the application of conventional and molecular methods in parallel is herein suggested aimed at allowing a better risk characterization and management.

Fungal levels in houses in the Fukushima Daiichi Nuclear Power Plant evacuation zone after the Great East Japan Earthquake

ABSTRACTResidences located within 20 km of the damaged Fukushima Daiichi Nuclear Power Plant were evacuated shortly after the Great East Japan Earthquake. The levels of airborne and surface fungi were measured in six houses in the evacuation zone in August 2012 and February 2013. Airborne fungal levels in all of the houses in the summer were higher than the environmental standard levels for residential houses published in Architectural Institute of Japan (>1000 colony-forming units [CFU]/m3). In two houses whose residents rarely returned to visit, fungal levels were extremely high (>52,000 CFU/m3). Although fungal levels in the winter were much lower than those in the summer, they were still higher than environmental standard levels in several houses. Indoor fungal levels were significantly inversely related to the frequency with which residents returned, but they were not correlated with the air exchange rates, temperature, humidity, or radiation levels. Cladosporium spp. and Penicillium spp. were detected in every house. Aspergillus section Circumdati (Aspergillus ochraceus group) was also detected in several houses. These fungi produced ochratoxin A and ochratoxin B, which have nephrotoxic and carcinogenic potential. The present study suggests that further monitoring of fungal levels is necessary in houses in the Fukushima Daiichi Nuclear Power Plant evacuation zone, and that some houses may require fungal disinfection.Implications: The results suggest that residents’ health could be at risk owing to the high levels of airborne fungi and toxic fungi Aspergillus section Circumdati. Therefore, monitoring and decontamination/disinfection of fungi are strongly recommended before residents are allowed to return permanently to their homes. In addition, returning to home with a certain frequency and adequate ventilation are necessary during similar situations, e.g., when residents cannot stay in their homes for a long period, because fungal levels in houses in the Fukushima Daiichi Nuclear Power Plant evacuation zone were inversely correlated with the frequency with which residents returned to visit their houses.

A new approach to assess occupational exposure to airborne fungal contamination and mycotoxins of forklift drivers in waste sorting facilities

The waste management industry is an important employer, and exposure of waste-handling workers to microorganisms is considered an occupational health problem. Besides fungal contamination, it is important to consider the co-occurrence of mycotoxins in this setting. Forklifts with closed cabinet and air conditioner are commonly used in waste industry to transport waste and other products within the facilities, possibly increasing the risk of exposure under certain conditions. The aim of this study was to assess the fungal contamination and mycotoxin levels in filters from the air conditioning system of forklift cabinets, as an indicator to assess occupational exposure of the drivers working in a waste sorting facility. Cytotoxicity was also assessed to understand and characterize the toxicity of the complex mixtures as present in the forklift filters. Aqueous extracts of filters from 11 vehicles were streaked onto 2% malt extract agar (MEA) with chloramphenicol (0.05g/L) media, and in dichloran glycerol (DG18) agar-based media for morphological identification of the mycobiota. Real-time quantitative PCR amplification of genes from Aspergillus sections Fumigati, Flavi, Circumdati, and Versicolores was also performed. Mycotoxins were analyzed using LC-MS/MS system. Cytotoxicity of filter extracts was analyzed by using a MTT cell culture test. Aspergillus species were found most frequently, namely Aspergillus sections Circumdati (MEA 48%; DG18 41%) and Nigri (MEA 32%; DG18 17.3%). By qPCR, only Aspergillus section Fumigati species were found, but positive results were obtained for all assessed filters. No mycotoxins were detected in aqueous filter extracts, but most extracts were highly cytotoxic (n=6) or medium cytotoxic (n=4). Although filter service life and cytotoxicity were not clearly correlated, the results suggest that observing air conditioner filter replacement frequency may be a critical aspect to avoid worker's exposure. Further research is required to check if the environmental conditions as present in the filters could allow the production of mycotoxins and their dissemination in the cabinet during the normal use of the vehicles.

Fungal contamination in green coffee beans samples: A public health concern

ABSTRACTStudies on the microbiology of coffee cherries and beans have shown that the predominant toxigenic fungal genera (Aspergillus and Penicillium) are natural coffee contaminants. The aim of this study was to investigate the distribution of fungi in Coffea arabica L. (Arabica coffee) and Coffea canephora L. var. robusta (Robusta coffee) green coffee samples obtained from different sources at the pre-roasting stage. Twenty-eight green coffee samples from different countries of origin (Brazil, Timor, Honduras, Angola, Vietnam, Costa Rica, Colombia, Guatemala, Nicaragua, India, and Uganda) were evaluated. The fungal load in the contaminated samples ranged from 0 to 12330 colony forming units (CFU)/g, of which approximately 67% presented contamination levels below 1500 CFU/g, while 11% exhibited intermediate contamination levels between 1500 and 3000 CFU/g. Contamination levels higher than 3000 CFU/g were found in 22% of contaminated coffee samples. Fifteen different fungi were isolated by culture-based methods and Aspergillus species belonging to different sections (complexes). The predominant Aspergillus section detected was Nigri (39%), followed by Aspergillus section Circumdati (29%). Molecular analysis detected the presence of Aspergillus sections Fumigati and Circumdati. The% coffee samples where Aspergillus species were identified by culture-based methods were 96%. Data demonstrated that green coffee beans samples were contaminated with toxigenic fungal species. Since mycotoxins may be resistant to the roasting process, this suggests possible exposure to mycotoxins through consumption of coffee. Further studies need to be conducted to provide information on critical points of coffee processing, such that fungal contamination may be reduced or eliminated and thus exposure to fungi and mycotoxins through coffee handling and consumption be prevented.

Multilocus Phylogeny and Antifungal Susceptibility of Aspergillus Section Circumdati from Clinical Samples and Description of A. pseudosclerotiorum sp. nov.

A multilocus phylogenetic study was carried out to assess species identity of a set of 34 clinical isolates from Aspergillus section Circumdati from the United States and to determine their in vitro antifungal susceptibility against eight antifungal drugs. The genetic markers used were the internal transcribed spacer (ITS) region, and fragments of the beta-tubulin (BenA), calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) genes. The drugs tested were amphotericin B, itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, micafungin, and terbinafine. The most common species sampled was A. westerdijkiae (29.4%), followed by a novel species, which was described here as A. pseudosclerotiorum (23.5%). Other species identified were A. sclerotiorum (17.6%), A. ochraceus (8.8%), A. subramanianii (8.8%), and A. insulicola and A. ochraceopetaliformis, with two isolates (5.9%) of each. The drugs that showed the most potent activity were caspofungin, micafungin, and terbinafine, while amphotericin B showed the least activity.