Feed production, swine and slaughterhouse: where is the highest occupational exposure to Aspergillus spp. in this production line?

Purpose - This study intended to characterize fungal contamination in two swine farms, in one feed production unit, and also in one swine slaughterhouse. We aimed to identify where the highest occupational exposure to Aspergillus spp. was detected during the production line.

Prevalence of Aspergillus section Fumigati in Portuguese slaughterhouses: a fungal and mycotoxin concern

Introduction - Within the Aspergillus genus, Aspergillus fumigatus species is one of the most ubiquitous saprophytic fungi and is considered the species with higher clinical relevance. The fungi belonging to the Fumigati section are the most common cause of invasive aspergillosis and a major source of infection related mortality in immunocompromised patients. One of the most abundant metabolites produced by Aspergillus fumigatus is the metabolite gliotoxin, which exhibits a diverse array of biologic effects on the immune system. Further, environments contaminated with A. fumigatus may be the cause or enhance respiratory problems in the workers of those specific settings. These species produce specific allergens and mycotoxins that could cause respiratory disorders. Aim of the study - The aim of the present work was to determine the prevalence of Aspergillus section Fumigati by cultural and molecular methods in poultry; swine and bovine; and large animal (bovine and horses) slaughterhouses.

Occupational exposure to Aspergillus spp. in poultry and swine feed production

Workers from feed production often develop allergic respiratory symptoms and fungi are likely to be a significant contributing factor to these symptoms. This study intended to characterize fungal contamination in two feed production units, one for poultry and other for swine consumption. We aimed at identifying which unit presented the highest risk of occupational exposure to Aspergillus spp.

Prevalence of Aspergillus fumigatus complex in waste sorting and incineration plants: an occupational threat

Aspergillus fumigatus is one of the major ubiquitous saprophytic fungi and it is considered one of the fungal species with higher clinical relevance. This study aimed at characterising the prevalence of A. fumigatus complex in one waste-sorting plant and also in one incineration plant. Conventional and molecular methodologies were applied in order to detect its presence. Aspergillus fumigatus complex was the second most frequently found in the air from the waste-sorting plant (16.0%) and from the incineration plant (18.0%). Regarding surfaces, it ranked the third species most frequently found in the waste-sorting plant (13.8%) and the second in the incineration plant (22.3%). In the waste-sorting plant, it was possible to amplify by qPCR DNA from the A. fumigatus complex in all culture-positive sampling sites plus one other sampling site that was negative by culture analysis. Considering the observed fungal load, it is recommended to apply preventive and protective measures in order to avoid or minimise worker's exposure.

Biodegradação de deoxinivalenol por aspergillus oryzae e rhizopus sp.: um estudo bioquímico de degradação e toxicidade

A contaminação aleatória de alimentos por micotoxinas afeta as condições de sanidade das dietas de humanos e animais. Dentre as toxinas fúngicas, deoxinivalenol (DON) se destaca pela freqüente contaminação de produtos agrícolas e alimentos e pela sua resistência a degradação pelo emprego de métodos tradicionais de processamento, o que motiva políticas de controle e a busca por técnicas de descontaminação. A descontaminação biológica empregando processsos fermentativos tem sido apontada como uma alternativa promissora, pois permite degradar micotoxinas através do sistema enzimático microbiano e melhorar características funcionais e sensoriais de matérias-primas e insumos alimentícios. Este trabalho teve por objetivo estudar condições e mecanismos de biodegradação de deoxinivalenol empregando Aspergillus oryzae e Rhizopus sp. em sistemas fermentativos submersos. Para tanto, foi necessário adequar metodologia para reação de derivação na determinação cromatográfica de DON; estudar o potencial e condições de degradação via fermentação submersa por Aspergillus oryzae e Rhizopus sp.; e avaliar a atividade de oxidoredutases e a citotoxicidade dos extratos fementados. A otimização da metodologia estabeleceu a melhor condição para a reação de derivação com 200 µL de anidrido trifluoroacético e 18 mg de bicarbonato de sódio, durante 6 minutos a 74 °C na faixa entre 7 e 21 µg de DON. A quantificação de DON residual no meio fermentado mostrou que as espécies fúngicas Rhizopus sp. e Aspergillus oryzae possuem a capacidade de degradar DON demonstrando índices médios de 87,4 e 62,4% respectivamente, principalmente quando o meio submerso foi água estéril e fermentação realizada durante 48 horas. A velocidade máxima de degradação neste intervalo foi de 10,8 e 12,4 ppb/h, observando também um aumento na atividade específica da enzima peroxidase. Os extratos dos fermentados com A. oryzae e Rhizopus sp. apresentaram efeito de inibição de proliferação celular (IC50) quando concentrados 10 vezes em 48 e 72 horas respectivamente. Os meios fermentados com Rhizopus sp. apresentaram menor efeito (1,5 vezes) quando comparado com Aspergillus oryzae.

In vitro experimental environments lacking or containing soil disparately affect competition experiments of Aspergillus flavus and co-occurring fungi in maize grains

In vitro experimental environments are used to study interactions between microorganisms, and predict dynamics in natural ecosystems. This study highlights that experimental in vitro environments should be selected to closely match the natural environment of interest during in vitro studies to strengthen extrapolations about aflatoxin production by Aspergillus and competing organisms. Fungal competition and aflatoxin accumulation was studied in soil, cotton wool or tube (water-only) environments, for Aspergillus flavus competition with Penicillium purpurogenum, Fusarium oxysporum or Sarocladium zeae within maize grains. Inoculated grains were incubated in each environment at two temperature regimes (25oC and 30oC). Competition experiments showed interaction between main effects of aflatoxin accumulation and environment at 25oC, but not so at 30oC. However, competition experiments showed fungal populations were always interacting with their environments. Fungal survival differed after the 72-hour incubation in different experimental environments. Whereas, all fungi incubated within the soil environment survived; in the cotton-wool environment, none of the competitors of A. flavus survived at 30 oC. With aflatoxin accumulation, F. oxysporum was the only fungus able to interdict aflatoxin production at both temperatures. This occurred only in the soil environment and fumonisins accumulated instead. Smallholder farmers in developing countries face serious mycotoxin contamination of their grains, and soil is a natural reservoir for the associated fungal propagules, and a drying and storage surface for grains on these farms. Studying fungal dynamics in the soil environment and other environments in vitro can provide insights into aflatoxin accumulation post harvest.

A survey on prevalence rate of aflatoxigenic species of Aspergillus and residues of aflatoxins by ELISA method in cold-water cultural fish (rainbow trout) feeds in Tehran and West Azarbayjan provinces-Iran

Aflatoxins are one kind of fungal toxins produced by species of toxigenic Aspergillus (A. flavus and A. parasiticus) and in other words they are secondary metabolites which are considered as one of the threatening factors of food consumer's health. In this research 96 samples of cold-water cultural fish feed, rainbow trout, during the seasons of spring and summer of 2007 (every fifteenth of the month) were randomized (by simple and stratified random) to determine: 1. The prevalence rate of aflatoxigenic species of Aspergillus in stored feed of cold-water cultural fish in West Azarbayjan cultural fish farms in both seasons (spring and summer); 2. The residues of total aflatoxin in stored feed of fish in cultural fish farms of West Azarbayjan in both seasons by ELISA method; and 3. The residues of that toxin in feed produced in aquatic feed factories in Tehran and West Azarbyjan provinces with the same method. In order to study prevalence rate of toxigenic species of Aspergillus, pour-plate culture method by general medium such as Malt Extract Agar (M.E.A.) and Sabouraud-Dextrose Agar (S.D.A.) and by standard No.997 of Iranian Standard Institute were used. The produced colonies were examined microscopically. To determine the aflatoxins residues, ELISA method using Agra-Quant kit of Romer Lab company, were applied. The results of this survey indicated that only 8.3% of the samples were infected by A. flavus. A. parasiticus was not observed. There were no significant differences between the prevalence rate of AFT and seasons/months, either (P<0.05). Evaluating mean of aflatoxin rate showed that the rates of this variable are lower than the tolerance levels designated by the joint FAO/WHO expert committee (The mean of AFT in all data was lower than 11 ppb). Furthermore, mean of total AFT residues rates of stored feed of various cultural center of West Azarbayjan and Tehran factories were comparable in spring and summer, and no significant differences were observed (P<0.05). But there were significant differences between the total aflatoxin rates in the feed of West Azarbayjan factory and spring and summer (P<0.05), and AFT residues in spring (8.6 ppb) were higher than summer (6.1 ppb). Prevalence rates of AFT in Tehran feed factories (9.2 ppb) are higher than W. Azarbayjan (7.4 ppb). In other words, location was considered as a decisive factor in total AFT rates of samples. Moreover, the results indicated that there was significant difference between total aflatoxin rates of feed and cultural centers (P<0.05). The mean of AFT rates in embankment dam cultural fish farms (6.75 ppb) and multi-functions cultural fish farms (6.25 ppb) was higher than individual cultural pond (4.67 ppb). In conclusion, the finally results of this survey indicated that the lower rates of Aspergillus is not effective on the presence of total aflatoxin rates in trout feed. Due to low levels of aflatoxin rates (lower than 20 ppb), the produced feed of cold-cultural fishes, Rainbow Trout, in Tehran and West Azarbayjan provinces, in spring and summer of 2007, were safe and healthy both for fish and their consumers.

Effect of commercial starter cultures and native yeasts on ochratoxin a production by aspergillus westerdijkiae and penicillium nordicum in meat products

Processed meat products are of worldwide importance and, because of their intrinsic factors as well as the processing methods, they are highly prone to fungal and mycotoxin contamination. Ochratoxin A (OTA) is the most significant mycotoxin in processed meat products. Penicillium nordicum is considered to be responsible for OTA contamination of meat products, as it is highly adapted to salt and protein-rich matrices and is moderately psycrotrophic. However, another OTA-producing fungus, Aspergillus westerdijkiae, adapted to carbon-rich matrices such as cereals and coffee beans, has been recently associated with high levels of OTA in meat products. Several Lactic Acid Bacteria (LAB) and yeasts have been tested as biocontrol agents against P. nordicum growth and OTA production in meat products, with promising results, but none of the studies have considered A. westerdijkiae. The aim of this work was to evaluate in vitro the effect of a commercial starter culture used in sausage fermentation and four yeasts isolated from dry-cured sausage on these two OTA-producing fungi, both in terms of fungal growth and of OTA production, using different meat-based culture media as model systems. The mechanisms underlying the observed effect were also studied. For this purpose, C. krusei, C. zeylanoides, R. mucilaginosa, R. glutinis, a mix of these yeasts and the starter culture were co-inoculated with P. nordicum and A. westerdijkiae in industrial sausage, traditional sausage, and ham-based media, under conditions of water activity, salt concentration and temperature that mimic real conditions at beginning and end of sausage curing process. Fungal growth was determined by measuring colony diameter, and OTA production was quantified by HPLC-FLD after extraction with methanol. Yeasts where found to inhibit significantly the growth of both fungi. P. nordicum was unable to produce detectable OTA in both sausage-based media under any condition. In ham, yeasts reduced OTA production, while the starter culture significantly increased it. Unexpectedly, OTA production by A. westerdijkiae was significantly stimulated in all media tested by all microorganisms. Matrix has a significant effect on OTA production by P. nordicum, but not by A. westerdijkiae, for which only temperature showed to have effect. By testing the mechanisms of action by which starter culture and C. zeylanoides influenced fungal responses, we were able to determine that direct contact and simultaneous growth of test organisms were the mechanisms more significantly involved in the responses. In conclusion, ochratoxigenic fungi do not all respond to antagonistic microorganisms in the same way. The use of biocontrol agents with the intent of reducing fungal growth and mycotoxin production by one fungus can have unexpected effects on others, thus leading to unforeseen safety problems. Further experiments are recommended to properly understand the reasons behind the different effects of microorganisms, to ensure their safe as biocontrol agents.

PCR-Internal Transcribed Spacer (ITS) genes sequencing and phylogenetic analysis of clinical and environmental Aspergillus species associated with HIV-TB co infected patients in a hospital in Abeokuta, southwestern Nigeria.

Background: Aspergillosis has been identified as one of the hospital acquired infections but the contribution of water and inhouse air as possible sources of Aspergillus infection in immunocompromised individuals like HIV-TB patients have not been studied in any hospital setting in Nigeria. Objective: To identify and investigate genetic relationship between clinical and environmental Aspergillus species associated with HIV-TB co infected patients. Methods: DNA extraction, purification, amplification and sequencing of Internal Transcribed Spacer (ITS) genes were performed using standard protocols. Similarity search using BLAST on NCBI was used for species identification and MEGA 5.0 was used for phylogenetic analysis. Results: Analyses of sequenced ITS genes of selected fourteen (14) Aspergillus isolates identified in the GenBank database revealed Aspergillus niger (28.57%), Aspergillus tubingensis (7.14%), Aspergillus flavus (7.14%) and Aspergillus fumigatus (57.14%). Aspergillus in sputum of HIV patients were Aspergillus niger, A. fumigatus, A. tubingensis and A. flavus. Also, A. niger and A. fumigatus were identified from water and open-air. Phylogenetic analysis of sequences yielded genetic relatedness between clinical and environmental isolates. Conclusion: Water and air in health care settings in Nigeria are important sources of Aspergillus sp. for HIV-TB patients.

Antifungal activity of extracts from Atacama Desert fungi against Paracoccidioides brasiliensis and identification of Aspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values ≤ 125.0 μg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 μg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 μg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

Beiträge zur kenntniss der pilzgattung Aspergillus ...

Inaug.-diss.--Strassburg.

Harnessing bacterial signals for suppression of biofilm formation in the nosocomial fungal pathogen Aspergillus fumigatus

Faced with the continued emergence of antibiotic resistance to all known classes of antibiotics, a paradigm shift in approaches toward antifungal therapeutics is required. Well characterized in a broad spectrum of bacterial and fungal pathogens, biofilms are a key factor in limiting the effectiveness of conventional antibiotics. Therefore, therapeutics such as small molecules that prevent or disrupt biofilm formation would render pathogens susceptible to clearance by existing drugs. This is the first report describing the effect of the Pseudomonas aeruginosa alkylhydroxyquinolone interkingdom signal molecules 2-heptyl-3-hydroxy-4-quinolone and 2-heptyl-4-quinolone on biofilm formation in the important fungal pathogen Aspergillus fumigatus. Decoration of the anthranilate ring on the quinolone framework resulted in significant changes in the capacity of these chemical messages to suppress biofilm formation. Addition of methoxy or methyl groups at the C5–C7 positions led to retention of anti-biofilm activity, in some cases dependent on the alkyl chain length at position C2. In contrast, halogenation at either the C3 or C6 positions led to loss of activity, with one notable exception. Microscopic staining provided key insights into the structural impact of the parent and modified molecules, identifying lead compounds for further development.

Comparação da produção de enzimas celulolíticas e hemicelulolíticas por linhagens mutantes e parental do Aspergillus niger 3T5B8.

Diversos trabalhos têm procurado aumentar a eficiência da hidrólise enzimática da biomassa lignocelulósica. Nesse contexto, o melhoramento de cepas produtoras de enzimas celulolíticas e hemicelulolíticas pode resultar em misturas enzimáticas mais eficientes. A linhagem parental de Aspergillus niger 3T5B8, referenciada como produtora de poligalacturonase, foi utilizada para o melhoramento genético visando aumentar a produção de celulases e hemicelulases. A produção das enzimas CMCase, xilanase, beta-glicosidase e poligalacturonase por fermentação submersa usando duas linhagens mutantes P49 e P83 foi avaliada e comparada com a linhagem parental. Os resultados mostraram um destaque para a cepa P83 com um aumento na produção de 56% de CMCase, 76% de beta-glicosidase, 23% de xilanase e 216% na poligalacturonase.

Caracterização das lipases obtidas por duas cepas de Aspergillus niger.

As lipases são enzimas que catalisam a hidrólise parcial ou total de triacilglicerois produzindo ácidos graxos livres, diacilglicerol, monoacilglicerol e glicerol. Este trabalho caracterizou a especificidade, a temperatura ótima e o pH ótimo das lipases obtidas por duas cepas de Aspergillus niger, sendo uma selvagem C e outra mutante 11T53A14. Os resultados mostram que os extratos enzimáticos apresentam especificidade diferentes, sendo a cepa selvagem mais específica para ácidos graxos de 8 carbonos e a cepa mutante inespecífica em relação ao tamanho do ácido graxo. As duas cepas apresentaram atividade em uma ampla faixa de pH, porém observou-se uma redução da atividade de mais de 50% em pH acima de 9,0. Em relação à temperatura, as lipases das duas cepas se mostraram mais ativas a 35°C.

Fungal spore fragmentation as a function of airflow rates and fungal generation methods

The aim of this study was to characterise and quantify the fungal fragment propagules derived and released from several fungal species (Penicillium, Aspergillus niger and Cladosporium cladosporioides) using different generation methods and different air velocities over the colonies. Real time fungal spore fragmentation was investigated using an Ultraviolet Aerodynamic Particle Sizer (UVASP) and a Scanning Mobility Particle Sizer (SMPS). The study showed that there were significant differences (p < 0.01) in the fragmentation percentage between different air velocities for the three generation methods, namely the direct, the fan and the fungal spore source strength tester (FSSST) methods. The percentage of fragmentation also proved to be dependant on fungal species. The study found that there was no fragmentation for any of the fungal species at an air velocity ≤ 0.4 m/s for any method of generation. Fluorescent signals, as well as mathematical determination also showed that the fungal fragments were derived from spores. Correlation analysis showed that the number of released fragments measured by the UVAPS under controlled conditions can be predicted on the basis of the number of spores, for Penicillium and Aspergillus niger, but not for Cladosporium cladosporioides. The fluorescence percentage of fragment samples was found to be significantly different to that of non-fragment samples (p < 0.0001) and the fragment sample fluorescence was always less than that of the non-fragment samples. Size distribution and concentration of fungal fragment particles were investigated qualitatively and quantitatively, by both UVAPS and SMPS, and it was found that the UVAPS was more sensitive than the SMPS for measuring small sample concentrations, and the results obtained from the UVAPS and SMAS were not identical for the same samples.