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.

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.

Extracellular Hsp70 induces inflammation and modulates LPS/LTA-stimulated inflammatory response in THP-1 cells.

Extracellular Hsp70 (eHsp70) can act as damage-associated molecular pattern (DAMP) via Toll-like receptors TLR2 and TLR4, and stimulate immune and inflammatory responses leading to sterile inflammation and propagation of already existing inflammation. It was found elevated in the blood of patients with chronic obstructive pulmonary disease (COPD), who might suffer occasional bacterial colonizations and infections. We used a monocytic THP-1 cell line as a cellular model of systemic compartment of COPD to assess inflammatory effects of eHsp70 when present alone or together with bacterial products lypopolysaccharide (LPS) and lypoteichoic acid (LTA). THP-1 cells were differentiated into macrophage-like cells and treated with various concentrations of recombinant human Hsp70 protein (rhHsp70), LPS (TLR4 agonist), LTA (TLR2 agonist), and their combinations for 4, 12, 24, and 48 h. Concentrations of IL-1α, IL-6, IL-8, and TNF-α were determined by ELISA. Cell viability was assessed by MTS assay, and mode of cell death by luminometric measurements of caspases-3/7, -8, and -9 activities. rhHsp70 showed cell protecting effect by suppressing caspases-3/7 activation, while LPS provoked cytotoxicity through caspases-8 and -3/7 pathway. Regarding inflammatory processes, rhHsp70 alone induced secretion of IL-1α and IL-8, but had modulatory effects on release of all four cytokines when applied together with LPS or LTA. Combined effect with LPS was mainly synergistic, and with LTA mainly antagonistic, although it was cytokine- and time-dependent. Our results confirmed pro-inflammatory function of extracellular Hsp70, and suggest its possible implication in COPD exacerbations caused by bacterial infection through desensitization or inappropriate activation of TLR2 and TLR4 receptors.

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 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.

Occurrence of black Aspergilli in indoor environments of six countries.

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.