Mycobiota and mycotoxins in various kinds of vegetables and fruits as potential health risk factors for consumers - summary of a multiyear study.

INTRODUCTION: Between 2015-2020, the concentrations and diversity of filamentous fungi as well as the levels of mycotoxins in 4 classes of vegetables and fruits were studied in samples from farms located in eastern Poland. The short communication summarizes the results with the use of statistical analysis and indicates the potential health hazards associated with the consumption of the produce contaminated with fungi and/or mycotoxins. The concentrations of filamentous fungi in all examined samples were moderate (2.813 - 4.146 log10 CFU g -1). The highest values were noted in root vegetables, whereas the lowest values in fruit vegetables. The mycobiota revealed a marked biodiversity (67 species and 33 genera). Penicillium and Fusarium prevailed in the mycobiota of root vegetables, whereas Cladosporium and Alternaria in the remaining classes. Most of identified species (88.1%) were described as potentially pathogenic. The importance of mycotoxins as a potential health risk factor for vegetable consumers, was confirmed by the detection of a high prevalence (>55%) of total aflatoxin (AFT). The level of AFT in berry fruits was significantly higher than in other classes. The high prevalence (58.5%) of deoxynivalenol was noted in raspberries. CONCLUSIONS: Filamentous fungi present in the examined vegetables and fruits may pose a potential health risk for consumers due to a high prevalence of potentially pathogenic species, mostly those producing mycotoxins.

Mycobiota of berry fruits - levels of filamentous fungi and mycotoxins, composition of fungi, and analysis of potential health risk for consumers.

INTRODUCTION AND OBJECTIVE: The aim of the study was to determine the presence, concentration and generic composition of filamentous fungi. Selected mycotoxins were also determined: total aflatoxins and deoxynivalenol. MATERIAL AND METHODS: In 2017-2018, 40 samples of strawberry fruits and 40 samples of red raspberry fruits were collected. In 2019-2020, 37 samples of fresh strawberry fruits and 41 samples of fresh red raspberry fruits were collected on conventional farms located in eastern Poland and were subjected to mycological examination. The concentration and species composition of filamentous fungi were determined by the method of plate dilutions on malt agar. The isolated strains were identified using macroscopic and microscopic methods. Samples were also analysed for the presence of aflatoxin B1, total aflatoxin and deoxynivalenol using ELISA tests. RESULTS: The median concentrations of fungi were moderate or low: 3.079 and 3.491 log10 CFU g-1 for strawberries and raspberries, respectively. Fungi of the genus Cladosporium prevailed in the mycobiota of berry fruits, accounting for 84.3% of total isolates in raspberries and 34.8% in strawberries. The occurrence of total aflatoxins was detected in the majority of tested samples (73.2% in raspberries and 70.3% in strawberries) but never exceeded the level of 4.0 µg kg-1 assumed as safe. Deoxynivalenol has been detected only in raspberries with the prevalence of 58.5%. Its median concentration was 242.0 µg kg-1 and in 7 out of 41 samples (17.0%) exceeded the level of 750.0 µg kg-1, assumed as safe. CONCLUSIONS: Filamentous fungi and mycotoxins occurred in the examined berries at levels that mostly do not represent a health risk for immunocompetent people, but might pose such risk for immuno-compromised and/or atopic consumers.

Levels of filamentous fungi and selected mycotoxins in leafy and fruit vegetables and analysis of their potential health risk for consumers.

INTRODUCTION AND OBJECTIVE: The aim of the study was to determine the presence, concentration and generic composition of filamentous fungi. Considering the significant role of mycotoxins in the pathogenicity of fungal contaminants of vegetables, the scope of the study was extended by determination of aflatoxins and deoxynivalenol. MATERIAL AND METHODS: In the years 2019-2020, samples of vegetables (lettuce, spinach, tomato, red pepper) collected on conventional farms located in eastern Poland were subjected to mycological examination. The concentration and species composition of filamentous fungi were determined by the method of plate dilutions on malt agar. The isolated strains were identified with the use of macroscopic and microscopic methods. Samples were also analyzed for the presence of aflatoxin B1 (AFB1), total aflatoxin (AFT) and deoxynivalenol (DON) using the immunoenzymatic ELISA method. RESULTS: The median concentrations of filamentous fungi ranged from 2.778-3.204 log10 CFU g -1. Overall, 40 fungal species were identified in the examined vegetables, of which 38 are classified as potentially pathogenic for humans. The mean prevalence values for AFB1 and AFT were moderate or high (16.0-60.0% and 57.8-75.6%, respectively) and very low for DON (0-2.2%). The median concentrations of filamentous fungi, AFB1 and AFT were distinctly greater in leafy vegetables than on non-leafy tomato and pepper fruits, and the differences were highly significant (P<0.001). CONCLUSIONS: The levels of filamentous fungi and mycotoxins in Polish vegetables could be classified as moderate or low. The abundant presence of species with various pathogenic abilities may pose a risk for some categories of people consuming raw vegetables, mostly for immuno-compromised persons or atopics susceptible to food allergy caused by ingested moulds.

The effect of fruit on the extracellular enzyme profiles of fungi.

INTRODUCTION: In recent years, the number of diseases caused by fungal pathogens has increased significantly. Many species of fungi are pathogenic for plants, causing a threat to food production and to humans, and are among the causes of chronic diseases. OBJECTIVE: The aim of the study is to determine the enzyme profiles of fungi, depending on the different types of fruit with which they have contact, and to determine the differences in these profiles in relation to the substrate on which they are grown. MATERIAL AND METHODS: Six strains of fungi identified as Cladosporium sphaerospermum, Fusarium poae, Alternaria alternata, Penicillium expansum, Penicillium verucosum and Acremonium strictum, isolated from fruits, were selected and analyzed for enzymatic profiles. The enzymatic activity was assessed using the API ZYM test (bioMerieux, France). RESULTS: In the majority of the 6 fungal strains isolated from fruits, enzymes belonging to glycol-hydrolases were the most active. The exception was Acremonium strictum, where phosphatases dominated. Among most fungal isolates, the enzymes ß- glucosidase and N-acetyl-ß-glucosaminidase showed the highest activity. The highest ß-glucosidase activities were found in Cladosporium sphaerospermum and Penicillium expansum. On the other hand, lipase, α-fucosidase and α-chymotrypsin showed the least activity. The least activity of these enzymes or their complete absence was observed in Fusarium poae, Alternaria alternata, Penicillium expansum and Acremonium strictum. CONCLUSIONS: The activity of hydrolytic enzymes in the isolated fungi depended on the addition of fruit and the type of medium. Individual fruits can increase or decrease the activity of the enzymes. Fungi present in fruit have pathogenic properties and can be possible risk factors for fungal infections.

Acute hypersensitivity pneumonitis in woodworkers caused by inhalation of birch dust contaminated with Pantoea agglomerans and Microbacterium barkeri.

CASE DESCRIPTION: Five workers (2 males and 3 females) employed in a furniture factory located in eastern Poland developed hypersensitivity pneumonitis (HP) after the pine wood used for furniture production was replaced by birch wood. All of them reported onset of respiratory and general symptoms (cough, shortness of breath, general malaise) after inhalation exposure to birch dust, showed crackles at auscultation, ground-glass attenuations in HRCT examination, and lymphocytosis in the BAL examination. The diagnosis of acute HP was set in 4 persons and the diagnosis of subacute HP in one. IDENTIFICATION OF SPECIFIC ALLERGEN: Samples of birch wood associated with evoking disease symptoms were subjected to microbiological analysis with the conventional and molecular methods. Two bacterial isolates were found to occur in large quantities (of the order 108 CFU/g) in examined samples: Gram-negative bacterium of the species Pantoea agglomerans and a non-filamentous Gram-positive actinobacterium of the species Microbacterium barkeri. In the test for inhibition of leukocyte migration, 4 out of 5 examined patients showed a positive reaction in the presence of P. agglomerans and 2 in the presence of M. barkeri. Only one person showed the presence of precipitins to P. agglomerans and none to M. barkeri. In the inhalation challenge, which is the most relevant allergological test in the HP diagnostics, all patients reacted positively to P. agglomerans and only one to M. barkeri. The results indicate that P. agglomerans developing in birch wood was the main agent causing HP in the workers exposed to the inhalation of dust from this wood, while the etiologic role of M. barkeri is probably secondary. CONCLUSION: The results demonstrate that apart from fungi and filamentous actinobacteria, regarded until recently as causative agents of HP in woodworkers, Gram-negative bacteria and non-filamentous actinobacteria may also elicit disease symptoms in the workers processing wood infected with large amounts of these microorganisms. The results obtained also seem to indicate that cellular-mediated reactions are more significant for causing disease symptoms compared to those that are precipitin-mediated.

Fungal diversity of root vegetables and soil rhizosphere collected from organic and conventional farms in Eastern Poland.

OBJECTIVE: Determination of the concentration and species composition of filamentous fungi in root vegetables (carrots and red beetroots), and the adjacent rhizosphere soil, collected on organic and conventional farms in Eastern Poland. MATERIAL AND METHODS: During the period 2015-2016, a total number of 80 samples of vegetables and 40 samples of soil were examined. From each type of farm, 20 samples of vegetables and 20 samples of the adjacent soil were examined. In addition, the study included 20 samples of vegetables from organic farms and 20 samples of vegetables from conventional farms purchased on the markets in the city of Lublin in Eastern Poland. In order to determine the concentration and species composition of filamentous fungi, both in vegetables and soil, the method of plate dilutions on Malt Agar medium (Difco) with chloramphenicol was used. The study was conducted in two parallel repetitions. Inoculated media were incubated at the temperature of 30° C for 72 hours, then at room temperature for 72 hours. The species composition of fungal flora was determined using macroscopic and microscopic methods, with the help of keys and atlases. RESULTS: It was found that the mean concentration of fungi was higher in vegetables and soil from conventional farms than in those from organic farms. In the case of carrots and soil from conventional farms, this concentration was 4.93 and 5.10 log10 CFU g-1, respectively, whereas from organic farms - respectively, 3.81 and 4.20 log10 CFU g-1. In the case of beetroots and soil from conventional farms, the mean concentrations were also higher compared to organic farms - 5.09 vs. 3.93 and 4.95 vs. 4.23 log10 CFU g-1. In the examined vegetables and rhizosphere soil, 61 species of filamentous fungi were found, of which 12 belonged to the genus Penicillium, 4 to the genus Fusarium, and 2 species each to the genera: Aspergillus, Cladosporium and Trichoderma. As many as 28 (45.9%) fungal species that occurred in vegetables and soil are regarded as pathogenic for humans.

Variations in the Rate of Infestations of Dogs with Zoonotic Nematodes and the Contamination of Soil in Different Environments.

Companion animals are an important aspect in human life. However, they may also be considered a source of pathogens. An example of zoonotic parasitoses is toxocarosis or cutaneous larva migrans (CLM). The aim of the study was to detect zoonotic nematodes of dogs living in different areas and the intensity of contamination in parasite polluted environments that are hazardous to human health. The fecal samples were examined using standard flotation and decantation methods as well as McMaster's quantitative technique. The soil samples in urban and rural areas were examined using a modified flotation method as described by Quinn et al. Statistical analyses were performed by IBM SPSS Statistics Version 23. The overall prevalence of parasites in dogs was 38%, 17.02% and 56.60% from urban and rural areas, respectively. The percentage values of nematodes important for human health (Toxocaracanis, Ancylostomatidae, Trichurisvulpis) remained at the same level (16%). The infected dogs were dominated by a single parasite species, the main was T.canis (28.95%). In total, 54.30% of the soil samples were contaminated with parasite eggs. The contamination of urban and rural sandpits was 40% and 60%, respectively. The molecular examinations of soil samples using LAMP (loop-mediated isothermal amplification) confirmed the presence of nematode eggs of the species T.canis in all samples previously classified as positive.

Microbiological characterization of vegetables and their rhizosphere soil in Eastern Poland.

The aim of this study was to investigate the bacteriological quality of 5 kinds of vegetables (lettuce, dill, radish, beetroot, carrot) and their rhizosphere soil, originating from conventional farms located in the Lublin Province of Eastern Poland. A total number of 35 samples of fresh vegetables (FV) taken immediately from soil, 35 samples of soil from rhizosphere of these vegetables (SR) and 35 samples of vegetables sold at retail in the markets in Lublin (VR) were examined. The samples were analysed for the content of: aerobic mesophilic bacteria (AMB) grown at 30°C and 37°C, Gram-negative bacteria of Enterobacteriaceae family, faecal coliform (FC) bacteria, Salmonella spp., and Clostridium perfringens. Median AMB values determined at 30°C for FV, SR and VR were 5.27, 5.00, and 5.00 log10 CFU g-1 , respectively, being significantly greater compared to those recorded at 37°C. The exceeding of the threshold value of 6.0 log10 CFU g-1 proposed by Gelosa (1998) was noted only in 5 FV samples grown at 30°C (14.3%), and in 3 FV samples grown at 37°C (8.6%). The threshold value was never exceeded in SR and VR samples. Median concentrations of Enterobacteriaceae determined for FV, SR and VR were 4.03, 3.87, and 3.04 log10 CFU g-1, respectively. Eleven species of Enterobacteriaceae were identified in the FV, SR and VR samples. The percent of samples containing Escherichia coli was greatest for VR (22.9%), smaller for FV (17.1%) and smallest for SR (5.7%). The median concentrations of the faecal coliform bacteria (FC), determined by culture at 44°C, were low, amounting to 1.000 log10 CFU g-1 for FV and SR and 0.00 for VR. All examined vegetable and soil samples tested negative for the presence of Salmonella. The median concentrations of Clostridium perfringens were low, amounting to 0.00 log10 CFU g-1 for all categories of samples. This bacterium was relatively common in soil samples with the prevalence of 40.0%, but very rare in vegetable samples (occurring in 5.7% of FV and in none of VR samples). In conclusion, the results of the present study generally indicate that the microbiological quality of Polish vegetables grown on conventional farms is satisfactory and safe for consumers.