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Ochratoxin A (OTA) is a toxin produced by several Aspergillus species, mainly those belonging to section Circumdati and section Nigri. The presence of OTA in cheese has been reported recently in cave cheese in Italy. As artisanal cheese production in Brazil has increased, the aim of this study was to investigate the presence of ochratoxin A and related fungi in artisanal cheese consumed in Brazil. A total of 130 samples of artisanal cheeses with natural moldy rind at different periods of maturation were collected. Of this total, 79 samples were collected from 6 producers from Canastra region in the state of Minas Gerais, since this is the largest artisanal cheese producer region; 13 samples from one producer in the Amparo region in the state of São Paulo and 36 samples from markets located in these 2 states. Aspergillus section Circumdati occurred in samples of three producers and some samples from the markets. A. section Circumdati colony counts varied from 102 to 106 CFU/g. Molecular analysis revealed Aspergillus westerdijkiae (67 %) as the most frequent species, followed by Aspergillus ostianus (22 %), and Aspergillus steynii (11 %). All of these isolates of A. section Circumdati were able to produce OTA in Yeast Extract Sucrose Agar (YESA) at 25 °C/7 days. OTA was found in 22 % of the artisanal cheese samples, ranging from 1.0 to above 1000 µg/kg, but only five samples had OTA higher than 1000 µg/kg. These findings emphasize the significance of ongoing monitoring and quality control in the artisanal cheese production process to minimize potential health risks linked to OTA contamination.

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Riverbed sediments are dynamic freshwater environments colonized by a great diversity of microorganisms which play important roles in supporting freshwater ecosystem by performing a vast array of metabolic functions. Recent evidence generated by HTS approaches has revealed that the structure of sediment microbial communities is influenced by natural seasonal variations in water such as temperature or streamflow as well by disturbances caused by local human activities. Here, a spatiotemporal analysis of sediment microbial distribution from São Francisco River headwaters section was conducted using Illumina 16S rRNA-V4 region amplicon sequencing in order to accomplish three major goals: (i) to investigate whether the diversity and composition of bacterial communities accessed in riverbed sediments vary in response to distinct land-use activities; (ii) to estimate whether the diversity patterns vary between the dry and wet seasons; and (iii) to evaluate whether the diversity of bacterial metabolic functions, predicted by PICRUSt2 approach, varies similarly to the estimated taxonomic diversity. Our findings revealed that bacterial communities in the sediment show differences in diversity and taxonomic composition according to the anthropic activities performed in the local environment. However, the patterns in which this taxonomic diversity is spatially structured show differences between the dry and wet seasons. On the other hand, the most changes in predicted bacterial metabolic functions were verified between sediment samples accessed in portions of the river located in protected and unprotected areas. Our findings contributed with new evidence about the impact of typical land-use practices conducted in countryside landscapes from developing countries on riverbed bacterial communities, both in their taxonomic and functional structure.

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Canastra Minas Artisanal Cheese is produced in the Brazilian State of Minas Gerais using raw milk, rennet, and pingo, a natural endogenous starter culture (fermented whey) collected from the previous day's production. Due to the use of raw milk, the product can carry microorganisms that may cause foodborne diseases (FBD), including Staphylococcus aureus. Genomic characterization of S. aureus is an important tool to assess diversity, virulence, antimicrobial resistance, and the potential for causing food poisoning due to enterotoxin production. This study is aimed at exploring the genomic features of S. aureus strains isolated from Canastra Minas Artisanal Cheeses. Multilocus sequence typing (MLST) classified these strains as ST1, ST5, and a new profile ST7849 (assigned to the clonal complex CC97). These strains belonged to four spa types: t008, t127, t359, and t992. We identified antimicrobial resistance genes with phenotypic correlation against methicillin (MRSA) and tetracycline. Virulome analysis revealed genes associated with iron uptake, immune evasion, and potential capacity for adherence and biofilm formation. The toxigenic potential included cyto- and exotoxins genes, and all strains presented the genes that encode for Panton-Valentine toxin and hemolysin, and two strains encoded 4 and 8 Staphylococcal enterotoxin (SE) genes. The results revealed the pathogenic potential of the evaluated S. aureus strains circulating in the Canastra region, representing a potential risk to public health. This study also provides useful information to monitor and guide the application of control measures to the artisanal dairy food production chain.

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Microbial starter cultures are used in the production of many cheeses around the world, such as Parmigiano-Reggiano, in Italy, Époisses, in France, and Canastra, in Brazil, providing many of the unique features of these cheeses. Bacteriophages (phages) are ubiquitous and well known to modulate the structure of bacterial communities, and recent data indicate that cheeses contain a high abundance of naturally occurring phages. Here, we analyze the viral and bacterial metagenomes of Canastra cheese: a traditional artisanal Brazilian cheese produced using an endogenous starter culture and raw milk. Over 1,200 viral operational taxonomic units were recovered using both isolated viral-like particles and complete metagenomic DNA. Common viral families identified included Siphoviridae and Myoviridae, with 40% of putative phage genomes unidentified at the family level of classification. We observed very high phage diversity, which varied greatly across different cheese producers, with 28% of phage genomes detected in only one producer. Several metagenome-assembled genomes were recovered for lactic acid-producing bacteria, as well as nonstarter bacterial species, and we identified several phage-bacterium interactions, at the strain level of resolution, varying across distinct cheese producers. We postulate that at least one bacterial strain detected could be endogenous and unique to the Canastra cheese-producing region in Brazil and that its growth seems to be modulated by autochthonous phages present in this artisanal production system. This phage-host relationship is likely to influence the fermentation dynamics and ultimately the sensorial profile of these cheeses, with implications for other similar cheese production systems around the world. IMPORTANCE Our work demonstrated a dynamic yet stable microbial ecosystem during cheese production using an endogenous starter culture. This was observed across several distinct producers and was marked by genomic evidence of continued phage-bacterium interactions, such as the presence of bacterial defense mechanisms. Furthermore, we provide evidence of unique microbial signatures for each individual cheese producer studied in the region, a fact that may have profound consequences on product traceability. This was the first effort to describe and understand the bacteriophage composition and ecological dynamics within the Brazilian Canastra cheese production system. The study of this prototypical backslopping production system provides a solid background for further mechanistic studies of the production of many cheeses around the world.

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Introdução: No Brasil, a legislação exige maturação mínima de 60 dias para queijos produzidos com leite cru, salvo se houver comprovação científica de que a redução do prazo não compromete a segurança do produto, como ocorreu com alguns queijos artesanais tradicionais de diferentes regiões do estado de Minas Gerais. No estado de São Paulo, onde os produtores seguem formulações e processos específicos individuais, a falta de um padrão de pesquisa validado dificulta a avaliação da segurança microbiológica deste tipo de queijo, pois essas pesquisas são dispendiosas e não há garantia de que os parâmetros estabelecidos pelos pesquisadores seriam aceitos pelos serviços de inspeção. Objetivo: Para abordar este desafio, esta proposta visa estabelecer um modelo de pesquisa científica padronizado para avaliar a segurança de queijos de leite cru maturados por menos de 60 dias. Métodos: Com base na proposta do DIPOA para classificação de Risco de Estabelecimentos, foi criado um checklist para classificação dos estabelecimentos/produtos, considerando risco do produto, volume processado e características de desempenho do estabelecimento em relação às Boas Práticas de Fabricação.  Resultados: A pontuação obtida pela aplicação do checklist na produção do queijo em questão determina o risco do produto como baixo, médio, alto ou muito alto e auxilia no estabelecimento do número mínimo de amostras a serem avaliadas mensalmente. Discussão: Espera-se que essa pesquisa auxilie produtores e órgãos de registro, fiscalização e inspeção no processo de legalização dos queijos artesanais de leite cru, possibilitando o desenvolvimento rural e a fixação de mão de obra no campo. (AU)

Introduction: In Brazil, the legislation requires a minimum ripening period of 60 days for raw milk cheeses, unless scientific evidence shows that the reduction in the ripening time does not compromise food safety, as seen with traditional artisanal cheeses from the state of Minas Gerais. In the state of Sao Paulo, where many producers have developed their own specific formulations and processes, the lack of a standardized validated research protocol for these types of cheese hinders their microbiological safety evaluation, since this type of research is costly and there are no guarantees that the parameters stablished by the researchers would be accepted by the inspection services. Objectives: To address this challenge, this proposal aims to stablish a standardized scientific research protocol for assessing the safety of raw milk cheeses ripened for less than 60 days. Methods: Based on the DIPOA (Department of Inspection of Products of Animal Origin) risk classification model for establishments, a checklist was created to categorize establishments/products, taking into consideration the product's risk, processed volume, and the establishment's performance assessed on the basis of good manufacturing practices. Results: The score obtained through the checklist application determines the final risk level of the product, categorized as low, medium, high, or very high, and helps determining the minimum number of samples to be evaluated monthly. Discussion: This research protocol may help producers, regulatory, inspection, and registration bodies in the legalization process of raw milk artisanal cheeses, enabling rural development and fixation of workforce in rural settings. (AU)

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Raw milk samples were collected from 200 dairy cows belonging to Girolando 1/2, Gyr, Guzera, and Holstein breeds, and the bacterial diversity was explored using 16S rRNA amplicon sequencing. SCC analysis showed that 69 animals were classified as affected with subclinical mastitis. The milk bacterial microbiome was dominated by Firmicutes, Proteobacteria, and Actinobacteria, with an increase of Firmicutes in animals with subclinical mastitis and Proteobacteria in healthy animals. At the family and genus level, the milk bacterial microbiome was dominated by Staphylococcus, Acinetobacter, Pseudomonas, members of the family Enterobacteriaceae, Lactococcus, Aerococcus, members of the family Rhizobiaceae, Anaerobacillus, Streptococcus, members of the family Intrasporangiaceae, members of the family Planococcaceae, Corynebacterium, Nocardioides, and Chryseobacterium. Significant differences in alpha and beta diversity analysis suggest an effect of udder health status and breed on the composition of raw bovine milk microbiota. LEfSe analysis showed 45 and 51 discriminative taxonomic biomarkers associated with udder health status and with one of the four breeds respectively, suggesting an effect of subclinical mastitis and breed on the microbiota of milk in cattle.

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Boza is a traditional low-alcohol fermented beverage from the Balkan Peninsula, frequently explored as a functional food product. The product is rich in Lactic Acid Bacteria (LAB) and some of them can produce bacteriocins. In this study, a sample of Boza from Belogratchik, Bulgaria, was analyzed for the presence of bacteriocinogenic LAB, and after analyses by RAPD-PCR, three representative isolates were characterized by genomic analyses, using whole genome sequencing. Isolates identified as Pediococcus pentosaceus ST75BZ and Pediococcus pentosaceus ST87BZ contained operons encoding for bacteriocins pediocin PA-1 and penocin A, while isolate identified as Pediococcus acidilactici ST31BZ contained only the operon for pediocin PA-1 and a CRISPR/Cas system for protection against bacteriophage infection. The antimicrobial activity of bacteriocins produced by the three isolates was inhibited by treatment of the cell-free supernatants with proteolytic enzymes. The produced bacteriocins inhibited the growth of Listeria monocytogenes, Enterococcus spp. and some Lactobacillus spp., among other tested species. The levels of bacteriocin production varied from 3200 to 12,800 AU/ml recorded against L. monocytogenes 104, 637 and 711, measured at 24 h of incubation at 37 °C. All bacteriocins remained active after incubation at pH 2.0-10.0. The activity mode of the studied bacteriocins was bactericidal, as determined against L. monocytogenes 104, 637 and 711. In addition, bactericidal activity was demonstrated using a cell leakage ß-galactosidase assay, indicating a pore formation mechanism as a mode of action. The present study highlights the importance of combining genomic analyses and traditional microbiological approaches as way of characterizing microbial interactions in fermented foods.

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Events of soil contamination by heavy metals are mostly related to human activities that release these metals into the environment as emissions or effluents. Among the industrial activities related to heavy metal pollution, cement production plants are considered one of the most common sources. In this work we applied the High-throughput sequencing approach called 16 S rDNA metabarcoding to perform the taxonomic characterization of the prokaryotic communities of the soil surrounding three cement plants as well as two areas outside the influence of the cement plants that represented agricultural production environments free of heavy metal contamination (control areas). We applied the environmental genomics approaches known as "structural community metrics" (α- and ß-diversity metrics) and "functional community metrics" (PICRUSt2 approach) to verify whether or not the effects of heavy metal contamination in the study area generated impacts on soil bacterial communities. We found that the impact related to the elevation of heavy metal concentration due to the operation of cement plants in the surrounding soil can be considered smooth according to globally recognized indices such as Igeo. However, we identified that both the taxonomic and functional structures of the communities surrounding cement plants were different from those found in the control areas. We consider that our findings contribute significantly to the general understanding of the effects of heavy metals on the soil ecosystem by showing that light contamination can disturb the dynamics of ecosystem services provided by soil, specifically those associated with microbial metabolism.

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Information concerning the factors affecting the circulation and distribution of free-roaming dogs is crucial in developing control actions and limiting the spread of zoonoses. The present study analyzes the influence of gender, sterilization, and environment on the spatial distribution of free-roaming dogs in urban settings. Animals were captured/recaptured in seven consecutive morning sampling efforts conducted at 2-monthly intervals in control and intervention areas in a medium-size town in southeastern Brazil. Capture locations were georeferenced and captured animals were microchipped before being released at their original capture sites. Dogs captured in the intervention area were subjected additionally to surgical sterilization prior to release. Home range (HR) areas were calculated by applying the minimum convex polygon method to dogs that had been captured at least three times. Land coverage zones were determined from satellite images and overlaid on maps of the study areas along with the locations of 22 commercial food outlets. HR areas showed a global mean of 448 m2 and a median of 28 m2, values that were smaller than those reported previously for dogs in rural regions. The median HR of females (64.m2) was higher than that of males (15 m2), while median HRs of animals in the control and intervention areas were similar (27 and 28.5 m2, respectively). Variability of HR was high, although animals with small HRs predominated. Free-roaming dogs grouped primarily in urbanized and transitional regions, and their spatial distribution was positively correlated with locations of commercial food outlets. While sterilization did not influence HR size, the search for food was a key factor in determining mobility and spatial aggregation of free-roaming dogs. Our findings are pertinent in understanding the ecology of free-roaming dogs in urban environments and will be applicable to strategies aimed at promoting animal welfare and preventing the dissemination of zoonoses.

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Brazil is the second largest ethanol producer in the world and largest using sugarcane feedstock. Bacteria contamination is one of the most important issues faced by ethanol producers that seek to increase production efficiency. Each step of production is a selection event due to the environmental and biological changes that occur. Therefore, we evaluated the influence of the selection arising from the ethanol production process on diversity and composition of bacteria. Our objectives were to test two hypotheses, (1) that species richness will decrease during the production process and (2) that lactic acid bacteria will become dominant with the advance of ethanol production. Bacterial community assemblage was accessed using 16S rRNA gene sequencing from 19 sequential samples. Temperature is of great importance in shaping microbial communities. Species richness increased between the decanter and must steps of the process. Low Simpson index values were recorded at the fermentation step, indicating a high dominance of Lactobacillus. Interactions between Lactobacillus and yeast may be impairing the efficiency of industrial ethanol production.

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