Resistant enterococci isolated from raw sheep’s milk and cheeses from South region of Brazil

Enterococci have been used as sentinel organisms for monitoring antimicrobial resistance in food, humans, and other animals. In this sense, the present study evaluated the antimicrobial susceptibility profile and the presence of genes associated with resistance to erythromycin (msrC and ermB) and tetracycline [tet(M) and/or tet(L)] in enterococci isolated from raw sheep’s milk and cheeses (colonial, feta-, and pecorino-type) from South region of Brazil. A total of 156 enterococci were isolated from milk (n=80) and cheese (n=76) samples, identified by MALDI-TOF. Enterococcus faecalis (50.6%; n=79) was the most frequent species isolated from both samples. According to in vitro susceptibility tests, enterococci strains were not susceptible to the most commonly antimicrobial agents used in human and veterinary medicine. The frequency of MDR strains in enterococci isolated from milk (53.7%) was higher than those from cheese (24.2%). The tet(M) gene was the most commonly detected among tetracycline not-susceptible strains. The present study provided the first evidence of antimicrobial not-susceptible enterococci in raw sheep’s milk and cheeses in South Brazil. Drug-resistant strains, particularly those that are MDR, constitute a One Health issue.Os enterococos têm sido usados como organismos sentinela para monitorar o padrão de suscetibilidade a antimicrobianos em alimentos, humanos e outros animais. Neste sentido, o presente estudo objetivou avaliar o perfil de susceptibilidade a antimicrobianos e os genes associados com a resistência a eritromicina (msrC and ermB) e à tetraciclina [tet(M) and/or tet(L)] em enterococos isolados de leite cru de ovelha e queijos (colonial, tipo-feta e tipo-pecorino) do Sul do Brasil. Um total de 156 enterococos foram isolados de leite (n=80) e queijo (n=76), identificados por MALDI-TOF. Enterococcus faecalis (50,6%; n=79) foi a espécie mais frequentemente isolada de ambas as amostras. De acordo com o teste de suscetibilidade in vitro, as cepas de enterococos não foram susceptíveis aos agentes antimicrobianos mais comumente utilizados na clínica humana e veterinária. A frequência de cepas de enterococos MDR isoladas do leite (53,7%) foi superior à do queijo (24,2%). O gene tet(M) foi o mais comumente detectado entre as cepas não susceptíveis à tetraciclina. O presente estudo fornece as primeiras evidências de enterococos não susceptíveis aos antimicrobianos em leite cru de ovelha e queijos no Sul do Brasil. Cepas resistentes a drogas, particularmente as que são MDR, representam uma preocupação de Saúde Única

Evaluation of enterotoxins and antimicrobial resistance in microorganisms isolated from raw sheep milk and cheese : ensuring the microbiological safety of these products in southern Brazil

This study emphasizes the importance of monitoring the microbiological quality of animal products, such as raw sheep’s milk and cheese, to ensure food safety. In Brazil, there is currently no legislation governing the quality of sheep’s milk and its derivatives. Therefore, this study aimed to evaluate: (i) the hygienic-sanitary quality of raw sheep’s milk and cheese produced in southern Brazil; (ii) the presence of enterotoxins and Staphylococcus spp. in these products; and (iii) the susceptibility of the isolated Staphylococcus spp. to antimicrobial drugs and the presence of resistance genes. A total of 35 samples of sheep’s milk and cheese were examined. The microbiological quality and presence of enterotoxins were accessed using Petrifilm and VIDAS SET2 methods, respectively. Antimicrobial susceptibility tests were conducted using VITEK 2 equipment and the disc diffusion method. The presence of resistance genes tet(L), sul1, sul2, ermB, tetM, AAC(6)’, tetW, and strA were evaluated through PCR. In total, 39 Staphylococcus spp. were obtained. The resistance genes tetM, ermB, strA, tetL, sul1, AAC(6)’, and sul2 were detected in 82%, 59%, 36%, 28%, 23%, 3%, and 3% of isolates, respectively. The findings revealed that both raw sheep’s milk and cheese contained Staphylococcus spp. that exhibited resistance to antimicrobial drugs and harbored resistance genes. These results underscore the immediate need for specific legislation in Brazil to regulate the production and sale of these product

Microbiologia do leite de ovelha e queijos produzidos no sul do Brasil : um estudo sobre a microbiota, qualidade microbiológica e resistência a antimicrobianos de Staphylococcus spp. isolados

O leite de ovelha e o queijo, vêm se destacando como uma importante atividade agropecuária no Brasil. Estes apresentam valor agregado e benefícios à saúde. O queijo apresenta uma microbiota própria, relacionada a qualidade do leite. A presença de microrganismos pode ser responsável pela deterioração e causa de infecções alimentares. O presente estudo teve por objetivo avaliar a diversidade microbiana presente em amostras de leite cru de ovelha e dos queijos produzidos na região sul do Brasil, assim como a qualidade microbiológica e presença de microrganismos resistentes a antimicrobianos beta-lactâmicos, rifanpicinas, oxazolidinonas, sulfonamidas, macrolídeos, fluoroquinolonas, tetraciclinas, nitrofuranos, anfenicois e lincosamidas. As amostras de leite cru foram coletadas em três fazendas distintas (n = 5 cada fazenda) e amostras de quatro tipos de queijo – tipo-feta, frescal, colonial e tipo pecorino, (n = 5 cada) - foram obtidas em fazendas e/ou adquiridas no comércio local. Para a análise da microbiota, o DNA das amostras foi extraído e a região V4 do gene 16S rRNA foi amplificada por PCR. Após, as amostras foram submetidas ao sequenciamento de alto desempenho no equipamento MiSeq da Illumina. A qualidade microbiológica e a análise de enterotoxinas foram determinadas pelos métodos petrifilm e VIDAS SET2, respectivamente. Foi realizado o isolamento de Staphylococcus spp., que foram avaliados quando à suscetibilidade antimicrobiana e a presença de genes de resistência tet(L), sul1, sul2, ermB, tetM, GN, tetW e strA. Os resultados do sequenciamento mostraram 501 Variantes de Sequências de Amplicons (ASV), sendo 429 ASV correspondentes a amostras de leite e 72 ASV para as amostras de queijo. Os índices de Chao1 e Shannon mostraram que a diversidade bacteriana nas amostras de queijos foi inferior às amostras de leites. As amostras de leites mostraram a presença de 87 gêneros, incluindo Phyllobacterium spp. (42,59%), Staphylococcus spp. (29,20%), Pseudomonas spp. (10,96%), Lactococcus spp. (5,10%) e Acinetobacter spp. (1,38%). Nas amostras de queijos, os gêneros mais abundantes foram os Streptococcus spp. (73,15%), Lactobacillus spp. (19,91%), Lactococcus spp. (2,46%) e Staphylococcus spp. (1,74%). A análise de beta diversidade mostrou que houve diferenças significativas na estrutura das comunidades microbianas entre as amostras de leite e queijos. Com relação ao estudo de segurança dos alimentos, foi possível observar uma maior contagem de microrganismos nas amostras de leite de ovelha cru do que no queijo. Em relação a ocorrência de enterotoxinas foi observada em uma amostra de leite. Foram obtidos 39 isolados de Staphylococcus spp. (15 em queijo e 24 em leite de ovelha cru). Em 12,5% de leite de ovelha cru foi identificado S. aureus e outras espécies foram identificadas: S. sciuri, S. simulans, S. lentus, S. pseudintermedius, S. chromogenes e S. warneri. Com relação ao perfil fenotípico de resistência a antimicrobianos, 90% dos isolados apresentaram resistência a pelo menos um antimicrobiano avaliado, e a multirresistência foi detectada em cepas de S. aureus, S. pseudintermedius e Staphylococcus coagulase negativa. Quanto aos genes de resistência, 82% dos isolados carregavam o gene tetM, 59% o ermB, 36% o strA, 28% o tetL, 23% o gene sul1 e 3% GN e sul2. O gene tetW não foi detectado em nenhum isolado. Esses achados impactam a indústria de laticínios, representando um risco à saúde pública. Os resultados podem auxiliar na estruturação de estratégias que garantam a qualidade e segurança dos produtos, servindo de base para a criação de legislação específica para leites de ovelha e queijos produzidos no Brasil.Sheep's milk and cheese have been highlighted as an important agricultural activity in Brazil. These have added value and health benefits. Since cheese has its own microbiota, which is related to milk quality. Nonetheless, the presence of microorganisms responsible for spoilage and food infections must be investigated. Thus, the present study aimed to evaluate the microbial diversity present in samples of raw sheep milk and cheese produced in southern Brazil, as well as the microbiological quality. Presence of microorganisms resistant to antimicrobials as beta-lactam, rifanpicins, oxazolidinones, sulfonamides, macrolides, fluoroquinolones, tetracyclines, nitrofurans, amphenicols and lincosamides. In addition, the isolation of species of Staphylococcus spp. Raw milk samples were collected from three different farms (n = 5 each farm) and samples of four types of cheese - feta-type, fresh, colonial and pecorino-type, (n = 5 each) - were obtained from farms and/or or purchased from local businesses. For the analysis of the microbiota, the DNA of the samples was extracted and the V4 region of the 16S rRNA gene was amplified by PCR. Afterwards, the samples were submitted to highperformance sequencing in the MiSeq equipment from Illumina. Microbiological quality and enterotoxin analysis were determined by the petrifilm and VIDAS SET2 methods, respectively. Staphylococcus spp. was isolated and evaluated for antimicrobial susceptibility and the presence of resistance genes tet(L), sul1, sul2, ermB, tetM, GN, tetW and strA. The sequencing results showed 501 Amplicon Sequence Variants (ASV), with 429 ASV corresponding to milk samples and 72 ASV to cheese samples. The Chao1 and Shannon indices showed that bacterial diversity in cheese samples was lower than in milk samples. Milk samples showed the presence of 87 genera, including Phyllobacterium spp. (42.59%), Staphylococcus spp. (29.20%), Pseudomonas spp. (10.96%), Lactococcus spp. (5.10%) and Acinetobacter spp. (1.38%). In cheese samples, the most abundant genera were Streptococcus spp. (73.15%), Lactobacillus spp. (19.91%), Lactococcus spp. (2.46%) and Staphylococcus spp. (1.74%). Beta diversity analysis showed that there were significant differences in the structure of microbial communities between milk and cheese samples. With regard to the food safety study, it was possible to observe a higher count of microorganisms in samples of raw sheep's milk than in cheese. Regarding the occurrence of enterotoxins, it was observed in a milk sample. 39 isolates of Staphylococcus spp. (15 in cheese and 24 in raw sheep's milk). In 12.5% of raw sheep milk, S. aureus was identified and other species were identified: S. sciuri, S. simulans, S. lentus, S. pseudintermedius, S. chromogenes and S. warneri. Regarding the phenotypic profile of antimicrobial resistance, 90% of the isolates showed resistance to at least one antimicrobial agent evaluated, and multidrug resistance was detected in strains of S. aureus, S. pseudintermedius and Staphylococcus coagulase-negative. For the resistance genes, 82% of the isolates carried out the tetM gene, 59% the ermB, 36% the strA, 28% the tetL, 23% the sul1 gene and 3% GN and sul2. The tetW gene was not detected in any isolate. These findings impact the dairy industry, which poses a risk to public health. The results contribute to the structuring of strategies that guarantee product quality and safety. Furthermore, these data can serve as a basis for the elaboration of specific legislation for both sheep's milk and cheese produced in Brazil

Development of cottage cheese lactose free with fiber addition and reduction of sodium and fat

The objective of this work was to develop, characterize and determine the shelf life of lactose-free cottage cheese with fiber addition and sodium and fat reduction. Experimental tests were performed to determine the final cheese formulation, which was evaluated at different storage times according to microbiological tests recommended by the current legislation and physicochemical parameters. The product developed met the physical-chemical standards when compared to the control cheese, presenting  41 and 30 % reduction of sodium and fat, respectively, and an increase of approximately 5,31 % of inulin, without altering the sensorial characteristics. The cheese presented stability during the 42 days evaluated, not suffering any physicalchemical alterations (acidity) and microbiological significant that compromised the safety and quality of the product. The addition of fiber and sodium reduction also did not negatively influence the sensoriality of the product, being an alternative of products lactose free and with functional properties for the dairy market

DESENVOLVIMENTO DE REQUEIJÃO CREMOSO SEM LACTOSE

Lactose intolerance is the name given to a dysfunction of the body, which has a deficiency or lack of production of enzyme lactase (β-galactosidase), responsible for the conversion of lactose into glucose and galactose. It is common in all age groups and ethnicities, its main consequence is the impossibility of ingesting products containing lactose, such as milk and its derivatives. Anticipating this, the food industry hydrolyzes lactose in order to obtain a product compatible with the disease, without compromising the characteristics already found in products without this treatment. Therefore, the objective of this work was to develop a “requeijão cremoso” without lactose. Skim milk were used as the main ingredient and β-galactosidase was added in concentrations of 0.07%, 0.1% and 0.2%, the hydrolysis time was 90 minutes at a pH of 6.55 and a temperature of 40 ºC. Analyses mode by high performance liquid chromatography showed that the degree of hydrolysis of the formulations was satisfactory. The developed product was adequate to the standards established by Brazilian legislation regarding to microbiological and physico-chemical standards. The sensorial analysis was performed with a hedonic scale of 9 points with a panel of 100 untrained judges, showing no statistically significant difference between samples of lactose-free and traditional curd. Thus, the formulation containing 0.07% of β-galactosidase enzyme can be considered a low-lactose curd, it is a good alternative for intolerants, since it has a similar taste and cost to the traditional one.A intolerância a lactose é o nome dado a uma disfunção do organismo que apresenta uma deficiência ou ausência da produção da enzima lactase (β-galactosidase), responsável pela conversão da lactose em glicose e galactose. É comum em todas as faixas etárias e etnias, sua principal consequência é a impossibilidade de ingestão de produtos contendo lactose, como leite e seus derivados. Antecipando-se a isso, a indústria alimentícia hidrolisa a lactose a fim de obter um produto compatível aos portadores dessa doença, sem comprometer as características já encontradas em produtos sem esse tratamento. Sendo assim, o objetivo deste trabalho foi desenvolver um requeijão cremoso sem lactose. Utilizou-se leite desnatado como matéria-prima e β-galactosidase em concentrações de 0,07%, 0,1% e 0,2%. O tempo de hidrólise foi de 90 minutos a um pH de 6,55 e temperatura de 40 ºC. A cromatografia líquida de alta eficiência demonstrou que o grau de hidrólise das formulações foi satisfatório. O produto desenvolvido adequou-se aos padrões estabelecidos pela legislação brasileira quanto aos limites microbiológicos e físico-químicos. A análise sensorial foi realizada com escala hedônica de 9 pontos com painel de 100 julgadores não treinados, não mostrando diferença estatística significativa entre amostras de requeijão sem lactose e tradicional. Sendo assim, a formulação contendo 0,07% de enzima β-galactosidase pode ser considerada um requeijão com baixo teor de lactose e é uma ótima alternativa para intolerantes, pois possui sabor e custo similar ao tradicional