Microbiological status of donor human milk - A single center study from Poland.

Human milk is considered the most suitable source of nutrition for infants. Donor human milk from human milk banks (HMB) is recommended as the best alternative for infants whose mothers' own milk is unavailable. Microbiological screening of milk donated to HMB is important to ensure the quality and safety of the pasteurised human milk. This article describes the microbiological status of human milk donated to the Regional Human Milk Bank in Torun, Poland. Statistical data regarding the microbiological analysis of milk from 292 donors were collected in the years 2013-2021. Total of 538 milk samples were tested. Only in 6% of human milk samples the bacteria level was above the required standard and/or the milk had potentially pathogenic bacteria. The main core of donors' breastmilk bacteria represents the skin microbiota, and the composition of the microbiota is strictly related to the surrounding environment. The most abundant genera detected in milk samples were the Staphylococcus group. Prolonged hospitalisation of infants' mothers and/or offsprings is associated with potentially pathogenic bacteria colonization in milk. The use of the modern identification method MALDI-TOF resulted in more accurate results compared to the biochemical methods. Our analysis indicates that most of the tested milk samples (94%), both expressing at home and in hospital environments, meet the criteria for admission to the human milk bank. Effective techniques for identifying microorganisms ensure that donor milk from human milk banks meets the guidelines set for these units.

Microbial Assessment in A Rare Norwegian Book Collection: A One Health Approach to Cultural Heritage.

Microbial contamination poses a threat to both the preservation of library and archival collections and the health of staff and users. This study investigated the microbial communities and potential health risks associated with the UNESCO-classified Norwegian Sea Trade Archive (NST Archive) collection exhibiting visible microbial colonization and staff health concerns. Dust samples from book surfaces and the storage environment were analysed using culturing methods, qPCR, Next Generation Sequencing, and mycotoxin, cytotoxicity, and azole resistance assays. Penicillium sp., Aspergillus sp., and Cladosporium sp. were the most common fungi identified, with some potentially toxic species like Stachybotrys sp., Toxicladosporium sp., and Aspergillus section Fumigati. Fungal resistance to azoles was not detected. Only one mycotoxin, sterigmatocystin, was found in a heavily contaminated book. Dust extracts from books exhibited moderate to high cytotoxicity on human lung cells, suggesting a potential respiratory risk. The collection had higher contamination levels compared to the storage environment, likely due to improved storage conditions. Even though overall low contamination levels were obtained, these might be underestimated due to the presence of salt (from cod preservation) that could have interfered with the analyses. This study underlines the importance of monitoring microbial communities and implementing proper storage measures to safeguard cultural heritage and staff well-being.

Assessment of Portuguese fitness centers: Bridging the knowledge gap on harmful microbial contamination with focus on fungi.

The lack of knowledge regarding the extent of microbial contamination in Portuguese fitness centers (FC) puts attendees and athletes at risk for bioaerosol exposure. This study intends to characterize microbial contamination in Portuguese FC by passive sampling methods: electrostatic dust collectors (EDC) (N = 39), settled dust (N = 8), vacuum filters (N = 8), and used cleaning mops (N = 12). The obtained extracts were plated in selective culture media for fungi and bacteria. Filters, EDC, and mop samples' extracts were also screened for antifungal resistance and used for the molecular detection of the selected Aspergillus sections. The detection of mycotoxins was conducted using a high-performance liquid chromatograph (HPLC) system and to determine the cytotoxicity of microbial contaminants recovered by passive sampling, HepG2 (human liver carcinoma) and A549 (human alveolar epithelial) cells were employed. The results reinforce the use of passive sampling methods to identify the most critical areas and identify environmental factors that influence microbial contamination, namely having a swimming pool. The cardio fitness area presented the highest median value of total bacteria (TSA: 9.69 × 102 CFU m-2.day-1) and Gram-negative bacteria (VRBA: 1.23 CFU m-2.day-1), while for fungi it was the open space area, with 1.86 × 101 CFU m-2.day-1. Aspergillus sp. was present in EDC and in filters used to collect settled dust. Reduced azole susceptibility was observed in filters and EDC (on ICZ and VCZ), and in mops (on ICZ). Fumonisin B2 was the only mycotoxin detected and it was present in all sampling matrixes except settled dust. High and moderate cytotoxicity was obtained, suggesting that A549 cells were more sensitive to samples' contaminants. The observed widespread of critical toxigenic fungal species with clinical relevance, such as Aspergillus section Fumigati, as well as Fumonisin B2 emphasizes the importance of frequent and effective cleaning procedures while using shared mops appeared as a vehicle of cross-contamination.

Analysis of Mycotoxins and Cytotoxicity of Airborne Molds Isolated from the Zoological Garden-Screening Research.

OBJECTIVE: The objective of this paper was to assess the airborne mold contamination, secondary metabolite profiles, and cytotoxicity of the dominant fungal species isolated from the air in selected rooms at a Zoological Garden. MATERIALS AND METHODS: Fungal concentrations were measured with MAS-100 air samplers. The collected airborne fungi were identified using a combination of morphological and molecular methods. The cytotoxicity of 84 strains belonging to two Penicillium and Aspergillus genera was determined using the quantitative colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt) assay. The mycotoxins were detected using high-performance liquid chromatography (HPLC) with a mass spectrometry detector. RESULTS: The ITS gene was amplified and sequenced to identify the 132 species. For mycotoxicological and cytotoxicity analyses, 52 Penicillium isolates and 32 Aspergillus representatives were selected. Cytotoxicity was confirmed in 97.6% of cases analyzed. Using the LC-MS/MS method, 42 out of 84 strains produced at least one of the following toxins: ochratoxin A, ochratoxin B, patulin, gliotoxin, roquefortine C, griseofulvin, sterigmatocystin, fumonisin B2, moniliformin, and mycophenolic acid. CONCLUSIONS: Analytical methods for assessing the presence of mycotoxins in fungal isolates collected directly from the air have proven to be an effective tool. Our research provides new information on the occurrence of potentially toxin-producing molds within a zoo.

Antagonistic properties against Fusarium sporotrichioides and glycosylation of HT-2 and T-2 toxins by selected Trichoderma strains.

The present study assessed the ability of Trichoderma to combat F. sporotrichioides, focusing on their antagonistic properties. Tests showed that Trichoderma effectively inhibited F. sporotrichioides mycelial growth, particularly with T. atroviride strains. In co-cultures on rice grains, Trichoderma almost completely reduced the biosynthesis of T-2 and HT-2 toxins by Fusarium. T-2 toxin-α-glucoside (T-2-3α-G), HT-2 toxin-α-glucoside (HT-2-3α-G), and HT-2 toxin-ß-glucoside (HT-2-3ß-G) were observed in the common culture medium, while these substances were not present in the control medium. The study also revealed unique metabolites and varying metabolomic profiles in joint cultures of Trichoderma and Fusarium, suggesting complex interactions. This research offers insights into the processes of biocontrol by Trichoderma, highlighting its potential as a sustainable solution for managing cereal plant pathogens and ensuring food safety.

Assessment of waste workers occupational risk to microbial agents and cytotoxic effects of mixed contaminants present in the air of waste truck cabin and ventilation filters.

Workers in the waste-processing industry are potentially exposed to high concentrations of biological contaminants, leading to respiratory and digestive problems and skin irritations. However, few data on the exposure of waste collection truck (WCT) drivers are available. The goal was to document the microbial risk of the waste collection truck (WCT) workers while in the vehicle cab. Long-period sampling using the truck air filters (CAF) and short time ambient air sampling in the cab were used. The potential release of microbial particles from CAFs was also investigated since it could contribute to the microbial load of the cabin air. A combination of analytical methods also helped assess the complex mixture of the biological agents. Aspergillus sections Fumigati and Flavi, E. coli, Enterobacter spp. and Legionella spp. were detected in the CAF of trucks collecting three types of waste. The highest levels of bacteria and fungi were found in the CAF from organic WCT. The highest endotoxin concentrations in CAF were 300 EU/cm2. Most of the CAF showed cytotoxic effects on both lung cells and hepatocytes. Only one mycotoxin was detected in a CAF. The maximal concentrations in the ambient WCT air varied according to the type of waste collected. The highest proportion (84%) of the air samples without cytotoxic effects on the lungs cells was for the recyclable material WCTs. The results revealed the potential microbial risk to workers from a complex mixture of bio-contaminants in the cabs of vehicles collecting all types of waste. The sustained cytotoxic effect indicates the potential adverse health-related impact of mixed contaminants (biological and non-biological) for the workers. Overall, this study highlights the benefits of using complementary sampling strategy and combined analytical methods for a the assessment of the microbial risk in work environments and the need to implement protective measures for the workers.Implications: Exposure to microbial agents is a well-known occupational hazard in the waste management sector. No previous study had evaluated the cytotoxicity of ambient air and ventilation filters to document worker exposure to a combination of contaminants during waste collection. This research confirms the usefulness of ventilation filters for long-term characterization of exposure to infectious agents, azole-resistant fungi, coliform bacteria and mycotoxin. Overall, this study highlights the importance of using several sampling and analysis methods for a comprehensive assessment of microbial risk in work environments, as well as the need to implement appropriate protective measures for collection workers.

Complementary sampling strategy and combined analytical methods are helpful in risk assessment.Air filter analysis (long-term sampling) assesses the presence of airborne biological contaminants over a long period.The type of waste collected influences the microbiological hazard of the workers.Waste collection workers are potentially exposed to infectious and mycotoxin-producing fungi.Cytotoxic assays revealed that waste collection workers are potentially.

Co-occurrence of T-2 and HT-2 Mycotoxins and α and ß Anomers of Their Glucosides in Wheat and Oat Grains.

The aim of this study was to simultaneously determine T-2 and HT-2 toxins and the α and ß anomers of their glucosides to assess their content in wheat and oat grains harvested in Poland (2020-2022). Of 298 wheat samples, only 14 (5%) contained the sum of the T-2 and HT-2 toxins (average 34.2 µg/kg; 10.6-67.7 µg/kg). In oat (n = 129), these compounds were detected much more frequently (70% of samples) at an average level of 107.5 µg/kg (6.9-949.1 µg/kg). The sum of T-2 and HT-2 glucosides was detectable in 3% of the wheat (average 16.3 µg/kg; 7.1-39.4 µg/kg) and 65% of the oat samples (average 35.1 µg/kg; 4.0-624.1 µg/kg). Following the study, T-2-3-α-glucoside was identified as the only naturally occurring anomer, while both anomers of HT-2-3-glucosides were detected with higher contents and occurrence rates of HT-2-3-ß-glucoside than the α anomer of this compound.

Effect of Selected Entomopathogenic Fungal Species on Embryonic Development of Ascaris suum (Nematoda).

The aim of the study was to evaluate the potential of using five selected species of entomopathogenic fungi (Beauveria bassiana, B. brongniartii, Conidiobolus coronatus, Isaria fumosorosea, and Metarhizium robertsii) in the bioregulation of the dispersive stages of the parasitic nematode-Ascaris suum. Experimental cultures of each of the selected entomopathogenic fungi, as well as a control culture without fungi, were incubated with A. suum eggs at 26 °C for 28 days. Development of the A. suum eggs was observed using a light microscope on the 7th, 14th, 21st, and 28th days of incubation. The API-ZYM® test was used to determine, semiquantitatively, the activity of 19 hydrolytic enzymes from the entomopathogenic fungi. The cytotoxicity of the fungi was determined using tetrazole salt MTT. It was found that none of the five tested strains of entomopathogenic fungi showed an ovicidal effect, and none of them colonized the A. suum egg shells. However, ovistatic activity was observed mainly until the 14th day of incubation by I. fumosorosea, M. robertsii, and B. bassiana. In the MTT test, M. robertsii showed moderate cytotoxicity, while the other species showed low cytotoxicity. Among the strains tested, I. fumosorosea showed the highest spectrum of hydrolase production (13 out of 19 enzymes gave a positive reaction from 3 to 5; 20-40 nM or more). The absence of morphological changes in the A. suum egg shells suggests that the antagonistic effect of the studied entomopathogenic fungi may be due to their cytotoxicity, associated with the production of secondary metabolites-toxins (M. robertsii) and enzymatic activity (I. fumosorosea).

A Chemically Defined Medium That Supports Mycotoxin Production by Stachybotrys chartarum Enabled Analysis of the Impact of Nitrogen and Carbon Sources on the Biosynthesis of Macrocyclic Trichothecenes and Stachybotrylactam.

Stachybotrys chartarum (Hypocreales, Ascomycota) is a toxigenic fungus that is frequently isolated from water-damaged buildings or improperly stored feed. The secondary metabolites formed by this mold have been associated with health problems in humans and animals. Several authors have studied the influence of environmental conditions on the production of mycotoxins, but these studies focused on undefined or complex substrates, such as building materials and media that impeded investigations of the influence of specific nutrients. In this study, a chemically defined cultivation medium was used to investigate the impact of several nitrogen and carbon sources on growth of S. chartarum and its production of macrocyclic trichothecenes (MTs) and stachybotrylactam (STLAC). Increasing concentrations of sodium nitrate were found to positively affect mycelial growth, the level of sporulation, and MT production, while ammonium nitrate and ammonium chloride had an inhibitory effect. Potato starch was the superior and most reliable carbon source tested. Additionally, we observed that the level of sporulation was correlated with the production of MTs but not with that of STLAC. In this study, we provide a chemically well-defined cultivation medium suitable for standardized in vitro testing of the capacity of S. chartarum isolates to produce macrocyclic trichothecenes. IMPORTANCE Macrocyclic trichothecenes (MTs) are highly toxic secondary metabolites that are produced by certain Stachybotrys chartarum strains, which consequently pose a risk for animals and humans. To identify hazardous, toxin-producing strains by analytical means, it is important to grow them under conditions that support MT production. Nutrients determine growth and development and thus the synthesis of secondary metabolites. Complex rich media are commonly used for diagnostics, but batch differences of supplements pose a risk for inconsistent data. We have established a chemically defined medium for S. chartarum and used it to analyze the impact of nitrogen and carbon sources. A key finding is that nitrate stimulates MT production, whereas ammonium suppresses it. Defining nutrients that support MT production will enable a more reliable identification of hazardous S. chartarum isolates. The new medium will also be instrumental in analyzing the biosynthetic pathways and regulatory mechanisms that control mycotoxin production in S. chartarum.

Microbial contamination in grocery stores from Portugal and Spain - The neglected indoor environment to be tackled in the scope of the One Health approach.

Microbial contamination in grocery shops (GS) should be evaluated since food commodities are commonly handled by workers and customers increasing the risk of food contamination and disease transmission. The aim of this study was to evaluate the microbial contamination in Portuguese and Spanish GS with a multi-approach protocol using passive (electrostatic dust cloths and surface swabs) sampling methods. The molecular detection of Aspergillus sections, mycotoxin analysis, screening of azole resistance as well as cytotoxicity measurement were conducted to better estimate the potential health risks of exposure and to identify possible relations between the risk factors studied. Fruits/vegetables sampling location was the one identified has being the most contaminated (bacteria and fungi) area in GS from both countries. Aspergillus section Fumigati and Fusarium species were observed in samples from Portuguese groceries with reduced susceptibilities to azoles commonly used in the clinical treatment of fungal infections. Fumonisin B2 was detected in Portuguese GS possible unveiling this emergent threat concerning occupational exposure and food safety. Overall, the results obtained raise concerns regarding human health and food safety and must be surveilled applying a One Health approach.

Toxico-pathological effects of ochratoxin A and its diastereoisomer under in ovo conditions and in vitro evaluation of the toxicity of these toxins against the embryo Gallus gallus fibroblast cell line.

Herein, we conducted a comparative study on the embryotoxicity of ochratoxin A (OTA) and its diastereomer 2'R-ochratoxin A (2'R-OTA) under in ovo conditions, as well as assess the in vitro embryotoxicity of these substances together with ochratoxin B and α-ochratoxin, using chicken (Gallus gallus domesticus) embryo cell lines. In ovo tests involved egg incubation of 8 different groups (i.e., control "0"-no puncture or injection (standard incubation); "00"-punctured eggs without injection; "OTA 0.25," "OTA 0.50," "OTA 0.75," "2'R-OTA 0.25," "2'R-OTA 0.50," "2'R-OTA 0.75"-eggs containing OTA or 2'R-OTA at 0.25, 0.50, and 0.75 µg/egg concentration, respectively). The results confirmed OTA's impact on early and late embryo mortality, where chick hatchability decreased with increasing toxin dosage. Both OTA and 2'R-OTA demonstrated embryotoxicity, however, in the case of the highest OTA diastereomer dose, nearly 11% higher chick hatchability was observed compared with the group that received OTA. 2'R-OTA dosage did not reduce parameters chick quality compared to chicks hatched from control group eggs. OTA concentrations were higher than 2'R-OTA detected in chicken organs such as liver and kidney, whereas 2'R-OTA concentrations were higher in blood serum and heart. The presented studies highlighted the differences in the ability to accumulate toxins in certain organs, which, to a certain extent, may affect the potential toxicity on individual organs. Additionally, during in vitro tests, when assessing the cytotoxic effects of OTA and its analogues toward the chicken embryonic cell line in an MTT assay, the cell metabolic activity was inhibited to a comparable extent at 27-times higher concentration of 2'R-OTA than OTA (0.24 µM). Also, comparably lower toxicity was attributed to the remaining OTA derivatives.

Tea contamination by mycotoxins and azole-resistant mycobiota - The need of a One Health approach to tackle exposures.

Despite tea beneficial health effects, there is a substantial risk of tea contamination by harmful pathogens and mycotoxins. A total of 40 tea samples (17 green (raw) tea; 13 black (fermented) tea; 10 herbal infusions or white tea) were purchased from different markets located in Lisbon district during 2020. All products were directly available to consumers either in bulk (13) and or in individual packages (27). Bacterial analysis was performed by inoculating 150 µL of samples extracts in tryptic soy agar (TSA) supplemented with 0.2 % nystatin medium for mesophilic bacteria, and in Violet Red bile agar (VRBA) medium for coliforms (Gram-negative bacteria). Fungal research was performed by spreading 150 µL of samples in malt extract agar (MEA) supplemented with 0.05 % chloramphenicol and in dichloran-glycerol agar (DG18) media. The molecular detection of the Aspergillus sections Fumigati, Nidulantes, Circumdati and Flavi was carried out by Real Time PCR (qPCR). Detection of mycotoxins was performed using high performance liquid chromatograph (HPLC) with a mass spectrometry detector. Azole resistance screening was achieved following the EUCAST guidelines. The highest counts of total bacteria (TSA) were obtained in green raw tea (81.6 %), while for coliform counts (VRBA) were found in samples from black raw tea (96.2 %). The highest fungal counts were obtained in green raw tea (87.7 % MEA; 69.6 % DG18). Aspergillus sp. was the most prevalent genus in all samples on MEA (54.3 %) and on DG18 (56.2 %). In the raw tea 23 of the samples (57.5 %) presented contamination by one to five mycotoxins in the same sample. One Aspergillus section Fumigati isolate from green tea beverage recovered form itraconazole-Sabouraud dextrose agar (SDA) medium, presented itraconazole and posaconazole E-test MICs above MIC90 values. Our findings open further discussion regarding the One-Health approach and the necessary investment in researching biological hazards and azole-resistance associated with the production and consumption of tea (in particular green tea).

Comprehensive assessment of occupational exposure to microbial contamination in waste sorting facilities from Norway.

Introduction: It is of upmost importance to contribute to fill the knowledge gap concerning the characterization of the occupational exposure to microbial agents in the waste sorting setting (automated and manual sorting). Methods: This study intends to apply a comprehensive field sampling and laboratory protocol (culture based-methods and molecular tools), assess fungal azole resistance, as well as to elucidate on potential exposure related health effects (cytotoxicity analyses). Skin-biota samples (eSwabs) were performed on workers and controls to identify other exposure routes. Results: In personal filter samples the guidelines in one automated industry surpassed the guidelines for fungi. Seasonal influence on viable microbial contamination including fungi with reduced susceptibility to the tested azoles was observed, besides the observed reduced susceptibility of pathogens of critical priority (Mucorales and Fusarium sp.). Aspergillus sections with potential toxigenic effect and with clinical relevance were also detected in all the sampling methods. Discussion: The results regarding skin-biota in both controls´ and workers´ hands claim attention for the possible exposure due to hand to face/mouth contact. This study allowed concluding that working in automated and manual waste sorting plants imply high exposure to microbial agents.

The Presence of Molds and Their Secondary Metabolites in Purple Coneflower-Based Dietary Supplements (Echinacea purpurea (L.) Moench).

Purple coneflower (Echinacea purpurea (L.) Moench) is a plant in the family Asteraceae, mainly grown in North America. Echinacea purpurea has been used in conventional medicine. The plant has immuno-stimulating and antibacterial properties, but neither mold contamination nor a mycotoxin presence have been evaluated. Our goal is to determine the degree to which molds and mycotoxins contaminate dietary supplements based on purple coneflower distributed on the Polish market. We analyzed 21 samples divided into four groups: sachets (n = 5), dry raw material (n = 3), capsules (n = 9), and tablets (n = 4). The mycological analysis of dietary supplements shows that the average number of molds is 1012 cfu/g, and the most common molds are Aspergillus spp., Phoma spp. and Eurotium spp. The mycotoxins most common in the samples are ZEN (18/21), DON (5/21) and T-2 toxin (3/21).

Unveiling the Occupational Exposure to Microbial Contamination in Conservation-Restoration Settings.

Assuring a proper environment for the fulfillment of professional activities is one of the Sustainable Development Goals and is contemplated in the One Health approach assumed by the World Health Organization. This particular study is applied to an often neglected sector of our society-the conservators/restorers-despite the many health issues reported by these professionals. Three different specialties (textiles, paintings and wood sculpture) and locations were selected for evaluation by placement of electrostatic dust cloths. After treatment of the samples, bacterial and fungal contamination were assessed, as well as mycotoxin determination, the presence of azole-resistant strains and cytotoxicity of the microorganisms encountered. Bacteria were only present in one of medias used and showed relatively low numbers. The highest level of contamination by fungi was identified in one of the textiles settings. The textile area also showed the highest variability for fungi. Aspergillus sp. are one indicator of possible environmental issues, and A. sections Fumigati and Circumdati were particularly relevant in two of the settings and identified in all of them. No mycotoxins were detected and the large majority of the fungi identified were non-cytotoxic. Overall, these can be considered low-contaminated environments but attention should be given to the Aspergillus sp. contamination. Additional studies are needed not only to make these results more robust, but also to test if the environmental sampling alone is the best approach in a setting where there is very little movement and dust displacement and where professionals are in very close proximity to the artefacts being treated, which may suggest the existence of a micro-atmosphere worth evaluating and comparing to the obtained results.

Production of Satratoxin G and H Is Tightly Linked to Sporulation in Stachybotrys chartarum.

Stachybotrys chartarum is a toxigenic fungus that is frequently isolated from damp building materials or improperly stored forage. Macrocyclic trichothecenes and in particular satratoxins are the most potent mycotoxins known to be produced by this fungus. Exposure of humans or animals to these secondary metabolites can be associated with severe health problems. To assess the pathogenic potential of S. chartarum isolates, it is essential to cultivate them under conditions that reliably promote toxin production. Potato dextrose agar (PDA) was reported to be the optimal nutrition medium for satratoxin production. In this study, the growth of S. chartarum genotype S strains on PDA from two manufacturers led to divergent results, namely, well-grown and sporulating cultures with high satratoxin concentrations (20.8 ± 0.4 µg/cm2) versus cultures with sparse sporulation and low satratoxin production (0.3 ± 0.1 µg/cm2). This finding is important for any attempt to identify toxigenic S. chartarum isolates. Further experiments performed with the two media provided strong evidence for a link between satratoxin production and sporulation. A comparison of three-point and one-point cultures grown on the two types of PDA, furthermore, demonstrated an inter-colony communication that influences both sporulation and mycotoxin production of S. chartarum genotype S strains.

Leptin in Human Milk-One of the Key Regulators of Nutritional Programming.

Breast milk is the optimal food for infants and toddlers, providing basic nutrients. It is also a source of many biologically active substances. Among them are hormones responsible for metabolic balance. One of the hormones taken in with breast milk by a breastfed baby is leptin. This hormone is involved in the regulation of appetite, informing the brain about the body's energy resources. Having the correct mechanisms related to the action of leptin is a factor reducing the risk of obesity. The natural presence of leptin in the composition of breast milk suggests that it has a specific role in shaping the health of a breastfed child. Obesity as a disease of civilization affects more and more people, including children. The development of this disease is multifaceted and determined by many factors, including genetic and environmental factors such as eating habits and low physical activity. Behind obesity, there are complex mechanisms in which many elements of the human body are involved. Understanding the effects of breastfeeding as a natural source of leptin can help prevent childhood obesity and development of this disease in future life.

Microbial contamination and metabolite exposure assessment during waste and recyclable material collection.

Waste workers are exposed to bioaerosols when handling, lifting and dumping garbage. Bioaerosol exposure has been linked to health problems such as asthma, airway irritant symptoms, infectious, gastrointestinal and skin diseases, and cancer. Our objective was to characterize the exposure of urban collectors and drivers to inhalable bioaerosols and to measured the cytotoxic effect of air samples in order to evaluate their health risk. Personal and ambient air sampling were conducted during the summer of 2019. Workers from 12 waste trucks collecting recyclables, organic waste or compost were evaluated. Bacteria and fungi were cultured, molecular biology methods were used to detect microbial indicators, cytotoxic assays were performed and endotoxins and mycotoxins were quantified. Domestic waste collectors were exposed to concentrations of bacteria and endotoxins above the recommended limits, and Aspergillus section Fumigati was detected at critical concentrations in their breathing zones. Cytotoxic effects were observed in many samples, demonstrating the potential health risk for these workers. This study establishes evidence that waste workers are exposed to microbial health risks during collection. It also demonstrates the relevance of cytotoxic assays in documenting the general toxic risk found in air samples. Our results also suggest that exposures differ depending on the type of waste, job title and discharge/unloading locations.

Six Feet under Microbiota: Microbiologic Contamination and Toxicity Profile in Three Urban Cemeteries from Lisbon, Portugal.

Cemeteries are potential environmental reservoirs of pathogenic microorganisms from organic matter decomposition. This study aimed to characterize the microbial contamination in three cemeteries, and more specifically in grave diggers' facilities. One active sampling method (impingement method) and several passive sampling methods (swabs, settled dust, settled dust filters and electrostatic dust cloths-EDC) were employed. The molecular detection of Aspergillus sections and SARS-CoV-2, as well as mycotoxin analysis, screening of azole resistance, and cytotoxicity measurement were also conducted. Total bacteria contamination was 80 CFU·m-2 in settled dust samples, reached 849 CFU·m-2 in EDC and 20,000 CFU·m-2 in swabs, and ranged from 5000 to 10,000 CFU·m-2 in filters. Gram-negative bacteria (VRBA) were only observed in in settled dust samples (2.00 × 105 CFU·m-2). Regarding Aspergillus sp., the highest counts were obtained in DG18 (18.38%) and it was not observed in azole-supplemented SDA media. SARS-CoV-2 and the targeted Aspergillus sections were not detected. Mycophenolic acid was detected in one settled dust sample. Cytotoxic effects were observed for 94.4% filters and 5.6% EDC in A549 lung epithelial cells, and for 50.0% filters and 5.6% EDC in HepG2 cells. Future studies are needed in this occupational setting to implement more focused risk management measures.

Virulence Factor Genes and Cytotoxicity of Streptococcus agalactiae Isolated from Bovine Mastitis in Poland.

Streptococcus agalactiae can produce a wide variety of virulence factors, including toxins and proteins which facilitate adhesion to and colonization and invasion of the host cells. There are few reports on the characteristics of field isolates from bovine mastitis in Poland. Thus, the aim of this study was to determine the occurrence of types of hemolysis on blood agar, virulence factor genes, and cytotoxicity of S. agalactiae isolates derived from cows with mastitis across Poland. The study included 68 isolates. Virulence genes were tested using standard PCR, and cytotoxicity was determined using methylthiazol tetrazolium (MTT) and lactate dehydrogenase (LDH) tests. Among the tested isolates, 89.7% were ß-hemolytic, 8.8% γ-hemolytic, and 1.5% alpha-hemolytic. The only genes detected in all isolates were the cfb, cspA, hylB, and sip genes. Cytotoxicity assessment based on the LDH test revealed that isolates were cytotoxic only to Vero cells. However, according to the results obtained from the MTT test, more than half of the isolates exhibited low cytotoxicity to both SK and Vero cells, whereas the other isolates showed moderate or no cytotoxicity to both cell lines. Our research confirms the prevalence of various virulence genes in S. agalactiae isolated from Polish dairy herds, which have previously been found in isolates recovered from human and animal infections. For the first time, the presence of bac- and scpB-positive isolates of S. agalactiae was determined in Polish dairy cattle, and the cytotoxicity of bovine isolates was assessed. IMPORTANCE We believe that this manuscript is one of the few reports on the characteristics of field S. agalactiae isolates derived from cases of bovine mastitis in cows in Poland in terms of the occurrence of virulence genes and cytotoxicity. For the first time, the presence of bac- and scpB-positive isolates of S. agalactiae was determined in Polish dairy cattle, and the cytotoxicity of bovine isolates was assessed.