Daniela Jakšić Despot

A year-round investigation of indoor airborne fungi in Croatia

This study assessed the composition of aeromycota at a grain mill and four dwellings (two apartments and two basements) as well as in outdoor air duriAbstract This study assessed the composition of aeromycota at a grain mill and four dwellings (two apartments and two basements) as well as in outdoor air during one year in Zagreb, Croatia. The incidence of Aspergilli from sections Flavi, Nigri, and Versicolores was also assessed. Airborne fungi were collected using an air-sampler and DG-18 agar plates. The average concentrations of airborne fungi in the grain mill ranged from 14,310 to 40,000 cfu m-3, which was above the hazardous level (104 cfu m-3), whereas the values statistically estimated using Feller’s correction were up to six times higher. Concentrations in the apartment (163-1244 cfu m-3) were lower than in outdoor air (286-2090 cfu m-3) and lower than in the basement (697-1203 cfu m-3), except in the warmer period of the year when they were similar. The most abundant species throughout the year were Cladosporium spp. (90-100 %), Penicillium spp. (40-100 %), and Alternaria spp. (10-100 %), which are common for temperate climates. Aspergilli from the Flavi (50- 100 %) and Nigri (15-40 %) sections as well as A. ochraceus (15-60 %) and Eurotium spp. (85-100 %) were the most abundant at the grain mill and were rarely found in outdoor air. In the basement, Aspergilli (Versicolores) were more abundant than in the apartment. The excess of aeromycoparticles in the grain mill throughout the year may have represented a serious health risk to mill workers. This is the first Croatian one-year study of indoor airborne fungi in a grain mill and dwellings; however monitoring should continue over a longer period Sažetak Cilj rada bio je ispitati varijacije učestalosti (%) i koncentracija (cfu m-3) plijesni u zraku mlina žitarica i četiriju stambenih prostora (dva stana i dva podruma) u odnosu na vanjski zrak tijekom jedne godine u Zagrebu (Hrvatska). Učestalost Aspergillus vrsta iz sekcija Flavi, Nigri i Versicolores također je ispitana. Plijesni iz zraka su uzorkovane pomoću uređaja Air-sampler MAS 100 Eco u kojem su postavljene DG- 18-agarske ploče. Prosječna koncentracija aerogenih plijesni u mlinu bila je između 14.310 i 40.000 cfu m-3, što je veće od koncentracije (104 cfu m-3) koja se smatra opasnom za zdravlje. Procijenjene vrijednosti koncentracija plijesni u zraku mlina, dobivene Felerovom korekcijom, čak su šest puta veće od izmjerenih koncentracija. U stanovima (163-1244 cfu m-3) koncentracije aerogenih plijesni bile su manje nego u vanjskom zraku (286-2090 cfu m-3) i podrumima (697-1203 cfu m-3), izuzev u toplijim mjesecima kada su izmjerene vrijednosti bile slične. Tijekom godine dominirale su vrste iz rodova Cladosporium spp. (90-100 %), Penicillium spp. (40-100 %), i Alternaria spp. (10-100 %), koje su uobičajene u područjima s umjerenom klimom. Aspergile iz sekcija Flavi (50-100 %) i Nigri (15-40 %), A. ochraceus (15-60 %) i Eurotium spp. (85-100 %) dominirali su u zraku mlina, a u uzorcima vanjskog zraka rijetko su detektirane. Vrste Aspergillus iz sekcije Versicolores s većom učestalošću nađene u podrumima nego u stanovima. Veliko opterećenje mlina aerogenim plijesnima tijekom cijele godine može biti opasno za zdravlje radnika. Ujedno, ovo je prvo jednogodišnje ispitivanje sezonske varijabilnosti u sastavu plijesni u zraku mlina žitarica i stambenih prostora u Hrvatskoj. Međutim, monitoring treba nastaviti tijekom dužeg razdoblja.

Species diversity and cytotoxic potency of airborne sterigmatocystin-producing Aspergilli from the section Versicolores

This study presents the distribution and species diversity of sterigmatocystin-producing Aspergilli from the section Versicolores in the indoor air of apartment-AP, basements-BS and grain mill-GM in Croatia, as well as the cytotoxic potency of isolates. The species comprised 0.7-20% of total airborne fungi detected in the AP, 11-55% in the BS, and 0-2% in the GM. Based on CaM sequences, seven species were identified; dominant were Aspergillus jensenii and Aspergillus creber, followed by Aspergillus protuberus, Aspergillus venenatus, Aspergillus tennesseensis, Aspergillus amoenus, Aspergillus griseoaurantiacus and three undescribed species. All of the identified species produced sterigmatocystin-STC (HPLC/UV-VIS); A. griseoaurantiacus (208.29μg/mL) and A. jensenii (1.192-133.63μg/mL) produced the highest levels, the lowest were detected in A. protuberus and A. tennesseensis (0.117-2.749μg/mL). Lower species diversity was obtained in the GM due to overgrowth with more propulsive fungi. Relatively high STC levels (0.06-2.35μg/g) detected in 52% of GM dust samples confirmed the presence of STC-producers, although this STC cannot be exclusively attributed to Aspergilli (Versicolores). STC and the majority of STC-producing Aspergilli were cytotoxic to human lung A549 cells (IC50 0.9-2.3μg/mL) and THP-1 macrophage-like cells (IC50 0.3-0.6μg/mL) in relatively low concentrations suggesting that humans can be at high risk during chronic exposure.

Cytotoxic and genotoxic potencies of single and combined spore extracts of airborne OTA-producing and OTA-non-producing Aspergilli in Human lung A549 cells

Aspergillus sclerotiorum (AS) is a well-known producer of ochratoxin A (OTA) while Aspergillus pseudoglaucus (AP) produces a wide range of extrolites with poorly investigated toxicity. These species are frequently co-occur in grain mill aeromycota. The aim of this study was to determine OTA levels in spore extracts using HPLC and immunoaffinity columns, and to examine the cytotoxicity of pure OTA, OTA-positive (AS-OTA(+)) and OTA-negative (AS-OTA(-)) spore extracts, as well as of AP spore extract, on human lung adenocarcinoma cells A549, individually and in combination, using a colorimetric MTT test (540nm). To establish which type of cell death predominated after treatments, a quantitative fluorescent assay with ethidium bromide and acridine orange was used, and the level of primary DNA damage in A549 cells was evaluated using the alkaline comet assay. OTA was detected in spore extracts (0.3-28µg/mL) of 3/6 of the AS strains, while none of the tested AP strains were able to produce OTA. Taking into account the maximum detected concentration of OTA in the spores, the daily intake of OTA by inhalation was calculated to be 1ng/kg body weight (b.w.), which is below the tolerable daily intake for OTA (17ng/kg b.w.). Using the MTT test, the following IC50 values were obtained: single OTA (53μg/mL); AS-OTA(+) (mass concentration 934μg/mL corresponds to 10.5μg/mL of OTA in spore extract); and 2126μg/mL for AP. The highest applied concentration of AS-OTA(-) spore extract (4940μg/mL) decreased cell viability by 30% and IC50 for the extract could not be determined. Single OTA and AS-OTA(+) and combinations (AP+AS-OTA(+) and AP+AS-OTA(-)) in subtoxic concentrations provoked significant primary DNA damage, apoptosis, and to a lesser extent, necrosis in A549 cells. Mixture of AP+AS-OTA(+) and AP+AS-OTA(-) in subtoxic concentrations showed dominant additive interactions. Despite the low calculated daily intake of OTA by inhalation, our results suggest that chronic exposure to high levels of OTA-producing airborne fungi in combination with other more or less toxic moulds pose a significant threat to human health due to their possible additive and/or synergistic interactions.

Identification of Aspergillus species in Central Europe able to produce G-type aflatoxins

The occurrence of potential aflatoxin producing fungi was examined in various agricultural products and indoor air in Central European countries including Hungary, Serbia and Croatia. For species identification, both morphological and sequence based methods were applied. Aspergillus flavus was detected in several samples including maize, cheese, nuts, spices and indoor air, and several isolates were able to produce aflatoxins. Besides, three other species of Aspergillus section Flavi, A. nomius, A. pseudonomius and A. parasiticus were also isolated from cheese, maize and indoor air, respectively. This is the first report on the occurrence of A. nomius and A. pseudonomius in Central Europe. All A. nomius, A. pseudonomius and A. parasiticus isolates were able to produce aflatoxins B1, B2, G1 and G2. The A. nomius isolate came from cheese produced very high amounts of aflatoxins (above 1 mg ml⁻¹). All A. nomius, A. pseudonomius and A. parasiticus isolates produced much higher amounts of aflatoxin G1 then aflatoxin B1. Further studies are in progress to examine the occurrence of producers of these highly carcinogenic mycotoxins in agricultural products and indoor air in Central Europe.

New sterigmatocystin-producing species of Aspergillus section Versicolores from indoor air in Croatia

Multilocus DNA sequence-based identification methods raised the number of known species assigned to the Aspergillus section Versicolores. Currently, there are 16 species accepted in the section, including A. amoenus, A. austroafricanus, A. creber, A. cvjetkovicii, A. fructus, A. griseoaurantiacus, A. hongkongensis, A. jensenii, A. protuberus, A. puulaauensis, A. subversicolor, A. sydowii, A. tabacinus, A. tennesseensis, A. venenatus, and A. versicolor. Based on morphological identifications, most of these species were identified as either A. sydowii or A. versicolor, with the latter reported to have a world-wide distribution, growing in many habitats. Aspergillus versicolor has been implicated in health hazards including sick building syndrome, human and animal mycoses, and contamination of food and feed were assigned primarily to this species. A. versicolor is still commonly isolated from indoor surveys, even though species such as A. jensenii and A. creber seem more common. From indoor air samples collected at a grain mill in Croatia, we isolated an undescribed species assigned to the Aspergillus section Versicolores. A polyphasic approach, including sequence-based methods, morphological and physiological studies, was used for species characterization and in this paper is described as Aspergillus pepii. Additionally, sterigmatocystin producing abilities have been confirmed. Based on a combined phylogenetic tree, morphological features and sterigmatocystin producing abilities, A. pepii is closely related to A. versicolor. Further studies should explore the frequency of the species in indoor environments and its medical, industrial, and environmental significance.

Occurrence of black Aspergilli in indoor environments of six countries.

Abstract Black Aspergilli (Aspergillus section Nigri) are widely distributed in various habitats. They act as food spoilage organisms, human pathogens, and mycotoxin producers and are frequently encountered in indoor environments. Black Aspergilli, specifically A. niger, A. welwitschiae, and A. carbonarius, produce different ochratoxins and fumonisins. Ochratoxins are known to induce renal disorders following inhalation, which necessitates the determination of potential mycotoxin-producing species in our environment. This paper aimed to compare the diversity and species distribution of black Aspergilli in the indoor environments of six different countries using morphological and molecular methods. A total of 178 black Aspergillus isolates were identified from six countries. In contrast with results from previous studies, A. niger was not the only black Aspergillus detected in indoor air. Species distribution differed among countries, although the distribution in European countries (Croatia, Hungary, the Netherlands, and Turkey) with a temperate climate was considerably similar. The highest species diversity was observed in indoor samples from Thailand, while the lowest was found in Algeria. Potentially ochratoxin- and fumonisin-producing fungi were detected in the indoor air of all six countries. Further studies need to clarify the effect of these fungi and their mycotoxins on human and animal health. Sažetak Crne plijesni roda Aspergillus široko su rasprostranjene u različitim životnim prostorima. Razgrađivači su hrane, ljudski patogeni i proizvođači mikotoksina. Također ih se često može pronaći u unutarnjim prostorima, a pojedine vrste poput A. niger, A. welwitschiae i A. carbonarius proizvode ohratoksine i fumonizine. Udisanje ohratoksina uzrokuje poremećaje bubrežne funkcije, pa je iznimno važno odrediti plijesni u našem okolišu koje bi ih mogle proizvoditi. U ovom se radu uspoređuje raznolikost i rasprostranjenost vrsta crnih plijesni roda Aspergillus u unutarnjem prostoru šest različitih zemalja morfološkim i molekularnim metodama. Analizirano je ukupno 178 uzoraka. Za razliku od prethodnih studija, u zraku unutarnjih prostora nije pronađena samo A. niger. Rasprostranjenost vrsta u europskim zemljama (Hrvatska, Mađarska, Nizozemska i Turska), gdje je klima umjerena, bila je umnogome slična. Najveća raznolikost uočena je u uzorcima iz Tajlanda, a najmanja u Alžiru. Da bi se razjasnili štetni učinci navedenih plijesni i mikrotoksina na zdravlje ljudi i životinja, potrebna su daljnja istraživanja.

Combined genotyping strategy reveals structural differences between Aspergillus flavus lineages from different habitats impacting human health

Aspergillus flavus is a filamentous fungus which is widespread on agricultural products and also able to cause various human diseases. This species is frequently isolated from indoor air as well, furthermore, it is known as a common causal agent of keratomycosis, particularly in subtropical and tropical areas. It is also able to produce aflatoxins, one of the most carcinogenic mycotoxins which are harmful to animals and humans. In this study, 59 A. flavus isolates from four different habitats and 1 A. minisclerotigenes isolate were investigated. The isolates were identified and confirmed at the species level by the sequence analysis of a part of their calmodulin gene. Applying a combined analysis of UP‐PCR, microsatellite, and calmodulin sequence data, the four group of isolates formed separate clusters on the phylogenetic tree. Examining the distribution of mating type genes MAT1‐1 and MAT1‐2, a ratio of approximately 3:1 was determined, and no correlation was found between the carried mating type gene and the aflatoxin production capability. HPLC analysis revealed that none of the examined isolates collected from indoor air or maize in Central Europe were able to produce aflatoxins, while about half of the isolates from India produced these mycotoxins under the test conditions.

Jednogodišnje istraživanje razine plijesni u zraku unutarnjih prostora

This study assessed the composition of aeromycota at a grain mill and four dwellings (two apartments and two basements) as well as in outdoor air duriAbstract This study assessed the composition of aeromycota at a grain mill and four dwellings (two apartments and two basements) as well as in outdoor air during one year in Zagreb, Croatia. The incidence of Aspergilli from sections Flavi, Nigri, and Versicolores was also assessed. Airborne fungi were collected using an air-sampler and DG-18 agar plates. The average concentrations of airborne fungi in the grain mill ranged from 14,310 to 40,000 cfu m-3, which was above the hazardous level (104 cfu m-3), whereas the values statistically estimated using Feller’s correction were up to six times higher. Concentrations in the apartment (163-1244 cfu m-3) were lower than in outdoor air (286-2090 cfu m-3) and lower than in the basement (697-1203 cfu m-3), except in the warmer period of the year when they were similar. The most abundant species throughout the year were Cladosporium spp. (90-100 %), Penicillium spp. (40-100 %), and Alternaria spp. (10-100 %), which are common for temperate climates. Aspergilli from the Flavi (50- 100 %) and Nigri (15-40 %) sections as well as A. ochraceus (15-60 %) and Eurotium spp. (85-100 %) were the most abundant at the grain mill and were rarely found in outdoor air. In the basement, Aspergilli (Versicolores) were more abundant than in the apartment. The excess of aeromycoparticles in the grain mill throughout the year may have represented a serious health risk to mill workers. This is the first Croatian one-year study of indoor airborne fungi in a grain mill and dwellings; however monitoring should continue over a longer period Sažetak Cilj rada bio je ispitati varijacije učestalosti (%) i koncentracija (cfu m-3) plijesni u zraku mlina žitarica i četiriju stambenih prostora (dva stana i dva podruma) u odnosu na vanjski zrak tijekom jedne godine u Zagrebu (Hrvatska). Učestalost Aspergillus vrsta iz sekcija Flavi, Nigri i Versicolores također je ispitana. Plijesni iz zraka su uzorkovane pomoću uređaja Air-sampler MAS 100 Eco u kojem su postavljene DG- 18-agarske ploče. Prosječna koncentracija aerogenih plijesni u mlinu bila je između 14.310 i 40.000 cfu m-3, što je veće od koncentracije (104 cfu m-3) koja se smatra opasnom za zdravlje. Procijenjene vrijednosti koncentracija plijesni u zraku mlina, dobivene Felerovom korekcijom, čak su šest puta veće od izmjerenih koncentracija. U stanovima (163-1244 cfu m-3) koncentracije aerogenih plijesni bile su manje nego u vanjskom zraku (286-2090 cfu m-3) i podrumima (697-1203 cfu m-3), izuzev u toplijim mjesecima kada su izmjerene vrijednosti bile slične. Tijekom godine dominirale su vrste iz rodova Cladosporium spp. (90-100 %), Penicillium spp. (40-100 %), i Alternaria spp. (10-100 %), koje su uobičajene u područjima s umjerenom klimom. Aspergile iz sekcija Flavi (50-100 %) i Nigri (15-40 %), A. ochraceus (15-60 %) i Eurotium spp. (85-100 %) dominirali su u zraku mlina, a u uzorcima vanjskog zraka rijetko su detektirane. Vrste Aspergillus iz sekcije Versicolores s većom učestalošću nađene u podrumima nego u stanovima. Veliko opterećenje mlina aerogenim plijesnima tijekom cijele godine može biti opasno za zdravlje radnika. Ujedno, ovo je prvo jednogodišnje ispitivanje sezonske varijabilnosti u sastavu plijesni u zraku mlina žitarica i stambenih prostora u Hrvatskoj. Međutim, monitoring treba nastaviti tijekom dužeg razdoblja.