Occurrence of multi-resistant Staphylococcus aureus in artisan goat coalho cheese in northeastern Brazil

The artisanal goat coalho cheese is one of the products obtained that stand out in the dairy goat farming of the Northeast of Brazil. Despite its importance, goat cheese is often made under inadequate hygienic-sanitary conditions and usually uses raw goat's milk, increasing the risk of product contamination. Among the pathogens carried by goat coalho cheese, Staphylococcus aureus stands out, being responsible for cases of food poisoning and persistent infections that are difficult to treat. This study aimed to evaluate the contamination, genotypic and phenotypic resistance of Staphylococcus aureus isolated from artisanal coalho cheese made with goat milk produced in the Northeast region of Brazil. This study analyzed only artisanal coalho cheeses made with raw goat's milk and purchased directly from farms. Twelve samples of artisanal coalho cheeses made with raw goat's milk were collected (1 sample per property) in 8 municipalities in the state of Pernambuco, Northeast region of Brazil. For microbiological analysis of enumeration of Colony Forming Units (CFU/g) of Staphylococcus spp. the methodology recommended by the International Organization for Standardization (2019) and recognized by the Ministry of Agriculture, Livestock and Supply was used. After enumeration, 5 colonies were selected per enumerated plate, a total of 180 Staphylococcus spp. was obtained. These were subjected to thermal extraction of genetic material to search for the nuc gene by Polymerase Chain Reaction, the isolates carrying the nuc gene were subjected to genotypic and phenotypic evaluation of antimicrobial resistance. After the phenotypic analysis, the Multiple Antimicrobial Resistance Index was evaluated. In all samples, Staphylococcus spp. and were considered unfit for consumption, with the lowest count being 9.4x103 CFU/g and the highest 6.4x106 CFU /g. Of the 180 isolates, 28.34% (51/180) were positive for the detection of the nuc gene. All resistance genes except mecA, mecC, and norB were detected. Of the 51 S. aureus isolates, 31.37% (16/51) were considered multi-resistant and presented a Multiple Antimicrobial Resistance Index above 0.2. After microbiological analysis it was found that all samples of coalho cheese were out of standards and unfit for human consumption in accordance with Ordinance no 146/1996 of the Ministry of Agriculture, Livestock and Supply of Brazil. Furthermore, the contamination of goat coalho cheeses is a risk to public health. During sample collection were found inadequate hygiene conditions in the environment used for cheese production. The presence of Staphylococcus aureus can be attributed to hygienic-sanitary failures in cheese production. From a health point of view, it is even more alarming when it comes to S. aureus carrying resistance genes. Although the 51 S. aureus isolates did not carry the mecA, mecC, norB genes and did not show phenotypic resistance to cefoxitin and oxacillin, all other genes were detected, indicating the circulation of S. aureus carrying the tet(L) genes, tet(M), tet-38, msrA, norA, and norC, which so far had not been reported in the production chain of goat coalho cheese in Brazil. Furthermore, the evaluation of the Multiple Antimicrobial Resistance Index identified the occurrence of multiple resistance to antimicrobials in 31.37% (16/51) of S. aureus at high risk to human health. The results obtained are quite worrying and serve as a warning to the scientific community and the Food Safety and Hygiene Inspection Services.(AU)

Methicillin-resistant Staphylococcus epidermidis in handler of cheese made with goat's milk in Brazil / Staphylococcus epidermidis resistente à meticilina em manipulador de queijo elaborado com leite de cabra no Brasil

ABSTRACT: In the Northeastern Brazil, artisanal cheese production is an important local economic activity for small producers. Methicillin-resistant Staphylococcus is responsible for causing infection in animals and humans. This study described the first detection of methicillin-resistant S. epidermidis isolated in the nasal cavity of a handler of coalho cheese made with goat's milk in Northeastern Brazil. This brief communication highlighted the importance of adopting biosafety measures by cheese handlers, in order to reduce possible contamination and the spread of pathogens in the production chain of this type of artisanal cheese in Brazil.

RESUMO: Na região Nordeste do Brasil, a cadeia de produção de queijo artesanal é uma atividade local importante para pequenos produtores. Staphylococcus resistentes à meticilina são responsáveis por causar infecções em animais e seres humanos. Neste estudo descreve-se a primeira detecção de S. epidermidis resistente à meticilina isolado da cavidade nasal de um manipulador de queijo coalho elaborado com leite de cabra no Nordeste do Brasil. Este relato destaca a importância da adoção de medidas de biossegurança por manipuladores de queijo, a fim de reduzir possíveis contaminações e a disseminação de patógenos na cadeia produtiva deste tipo de queijo artesanal no Brasil.

Avaliação da contaminação por Staphylococcus aureus em queijo coalho artesanal elaborado com leite de cabra produzido no estado de Pernambuco / Evaluation of contamination by Staphylococcus aureus in artisanal curd cheese made from goat's milk produced in the state of Pernambuco

A fabricação de queijo coalho artesanal elaborado com leite de cabra é composta pelas etapas de obtenção do leite, refrigeração, manipulação e armazenamento, que aumentam o risco de contaminação do produto. Objetivou-se neste estudo avaliar o nível de contaminação por Staphylococcus aureus em amostras de queijo coalho artesanal produzido com leite de cabra cru no estado de Pernambuco, Brasil, bem como avaliar a concordância entre a técnica oficial da Instrução Normativa nº62/2003 (Mapa) e a técnica molecular (gene nuc) para identificar S. aureus no queijo. Houve crescimento de colônias típicas de Staphylococcus aureus em 100% das amostras, e a contagem variou de 7,0×103 a 8,6×106 UFC/g. Das 30 amostras analisadas, 18 (60,0%) apresentaram valores superiores ou iguais a 105UFC/g, e 21 (70,0%) estavam contaminadas por S. aureus. A concordância entre os métodos de diagnóstico de S. aureus em queijo coalho caprino foi moderada. O nível de contaminação dos queijos revela a necessidade de ações de melhoria das condições de elaboração do produto, a fim de garantir um produto seguro aos consumidores.(AU)

The manufacture of artisanal Coalho cheese made from goat's milk is composed of the steps of obtaining milk, refrigeration, handling and storage that increase the risk of product contamination. The objective of this study was to evaluate the level of contamination by Staphylococcus aureus in samples of artisanal Coalho cheese produced with raw goat's milk in the state of Pernambuco, Brazil. In addition to evaluating the agreement between the official technique of Normative Instruction nº62/2003 (MAPA) and the molecular technique (nuc gene) to identify S. aureus in cheese. There was growth of typical Staphylococcus aureus colonies in 100% of the samples and the count ranged from 7.0×103 to 8.6×106 CFU/g. Of the 30 analyzed samples, 18 (60.0%) presented values greater than or equal to 105CFU/g and 21 (70.0%) were contaminated by S. aureus. The agreement between the diagnostic methods of S. aureus in goat cheese was moderate. The level of contamination of cheeses reveals the need for actions to improve the preparation conditions of the product in order to guarantee a safe product to consumers.(AU)

Bactérias láticas produtoras de folato e riboflavina: Isolamento e avaliação do seu potencial de aplicação na produção de produtos lácteos caprinos com alto teor de vitaminas / Folate and riboflavin producing lactic acid bacteria: isolation and evaluation of their potential application for production of goat dairy products with higher vitamin content

O valor nutricional e as características físico-químicas fazem com que os produtos lácteos caprinos sejam muito apreciados e tenham um alto valor agregado. Muitas bactérias láticas (BAL) possuem a capacidade de produzir compostos com propriedades benéficas à saúde, inclusive vitaminas do grupo B, como a riboflavina (B2) e o folato (B9), que participam de importantes funções metabólicas. O presente estudo objetivou isolar e identificar BAL capazes de produzir folato e riboflavina, utilizando leite de cabra cru e de queijos de cabra como fonte, para em seguida, avaliar a produção destas vitaminas em leite de cabra UHT, para estimar uma possível aplicação na produção de derivados lácteos de cabra com teores mais altos destas vitaminas. Um total de 179 isolados de BAL, sendo 87 provenientes de leite e 92 de queijos, foram obtidas e analisados quanto à produção de folato e de riboflavina extracelular (EC) e intracelular (IC), empregando-se ensaios microbiológicos apropriados. A produção de folato em meio de cultura a 37ºC foi observada em 151 isolados (84,4%) e de riboflavina em 15 isolados (8,4%), sendo que 14 produziram as duas vitaminas concomitantemente. A média da produção folato total (EC + IC) foi 138,8 µg/L, sendo que em 77 isolados (51%) a produção estava acima da média. Houve diferença significativa entre as médias de produção de folato total pelos isolados de leite e de queijo. A média da produção de riboflavina total (EC + IC) foi 363,7 µg/L, sendo que em 9 isolados (60%) a produção foi acima da média. Com base no RAPD-PCR e sequenciamento da porção 16S do rDNA, foram obtidos 19 perfis genéticos diferentes e identificadas 7 espécies, com predominância de Streptococcus thermophilus (7 isolados), Weissella paramensenteroides (6 isolados) e Lactococcus lactis subsp. lactis (4 isolados). Oito isolados produtores de folato acima da média foram selecionados e submetidos à avaliação da produção de folato em leite de cabra UHT a 37°C, sendo 7 positivas, com média da concentração de folato total de 120,55 µg/L, variando de 12,97 a 261,91 µg/L. Os melhores produtores de folato em leite de cabra UHT foram Lc. lactis subsp. lactis FP368 e St. thermophilus FP34v, FP170v e FP268v. A concentração de folato produzido por estes isolados foi acima da média, evidenciando seu interessante potencial de aplicação na produção de novos derivados lácteos caprinos com teores aumentados desta vitamina. Por outro lado, não foi possível detectar a produção de riboflavina em leite de cabra por nenhum dos isolados testados

Due to the high nutritional value and physicochemical characteristics, goat milk and cheeses are highly appreciated. Many strains of lactic acid bacteria (LAB) are able to produce vitamins from the B complex, such as riboflavin (B2) and folate (B9). These vitamins have important metabolic roles. The present study aimed to isolate riboflavin- and folate-producing LAB strains from goat milk and cheeses, and evaluate the potential application of those strains in the production of goat dairy products with higher content of these vitamins. A total of 179 LAB isolates were obtained from milk (87) and cheese (92) samples. The isolates were evaluated for production of extra (EC) and intracellular (IC) riboflavin and folate, applying appropriate microbiological methods. Among these isolates, 151 (84.4%) were able to produce folate, while 15 (8.4%) displayed the ability to produce riboflavin, and 14 produced both vitamins. The average production of total folate (EC + IC) was 138.8 µg/L, and the amount of folates produced by 77 isolates (51%) were above this average. The differences observed for the average production of total folate from milk and cheese isolates were statistically significant. For total riboflavin (EC+ IC), the average rate was 363.7 µg/L. Nine isolates (60%) presented production rates above the average. No significant difference was observed between the average production of total riboflafin from milk or cheese isolates (319.3 µg/L and 379.8 µg/L, respectively). Based on RAPD-PCR and 16S rDNA sequencing, 19 different genetic profiles were obtained and 7 species were identified, with predominance of Streptococcus thermophilus (7 isolates), Weissella paramensenteroides (6 isolates), and Lactococcus lactis subsp. lactis (4 isolates). Eight isolates that produced folate above the average were selected and tested for vitamins production in UHT goat milk at 37°C. Seven isolates produced an average of 120.55 µg/L of folate in the milk and concentration varied from 12.97 to 261,91 µg/L. The best folate producers were Lactococcus lactis subsp. lactis FP368, Streptococcus thermophilus FP34v, Streptococcus thermophilus FP170v and Streptococcus thermophilus FP268v. The amount of folate produced by these isolates surpassed the average, and was above the amounts described in other studies, evidencing their potential application in the production of goat dairy products with higher content of folate. None of the tested isolates was able to produce riboflavin in UHT goat milk

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese

Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain (Lc. lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products.

Maduración de queso de cabra con cepa autóctona de Leuconostoc mesenteroides aislada de queso artesanal / Ripening of Goat Cheese With a Native Strain of Leuconostoc mesenteroides Isolated from Artisan Cheese

Se elaboró queso de cabra madurado empleando una cepa liofilizada de Leuconostoc mesenteroides como cultivo iniciador, aislada a partir de la flora natural de quesos de cabra, elaborados artesanalmente con leche cruda de cabra del municipio Torres, estado Lara, Venezuela. El proceso de maduración fue de 4 sem, a 14°C y 70% de humedad relativa. Durante el tiempo de maduración, se evaluaron semanalmente los cambios en pH, sólidos totales y parámetros de textura; mientras que el contenido de proteínas, se determinó al inicio y al final del estudio. Al finalizar la maduración, se realizó un estudio con consumidores para evaluar la aceptabilidad de los quesos obtenidos en comparación con un queso control inoculado con una cepa comercial. El contenido de sólidos totales disminuyó entre 23 y 31% a lo largo del estudio (p<0,05), el pH en 6%, mientras que el contenido proteico no registró cambios al final de la maduración. La dureza y gomosidad en los quesos se incrementaron entre 1,64 y 2N; y 0,72 y 0,83N, respectivamente, con la maduración; correlacionándose positivamente con la variación en sólidos totales (R²>0,90) para ambos quesos mientras que la adhesividad y cohesividad no registraron cambios importantes. En la evaluación sensorial, el queso elaborado con L. mesenteroides obtuvo una aceptabilidad promedio del 70%, al compararlo con un queso de cabra obtenido a partir de una cepa comercial de Lactococcus lactis.

A ripened goat cheese was developed using a lyophilized strain of Leuconostoc mesenteroides as starter culture. This strain was isolated from the natural flora of artisan goat cheese made with raw milk in the municipality of Torres, the State of Lara, Venezuela. The maturation process lasted 4 weeks, at 14°C and 70% relative humidity. During the ripening period, changes in pH, total solids, and texture parameters where registered every week, while protein content was determined at baseline and at the end of the study. After maturation was completed, its acceptability was assessed through a consumer test against a control cheese inoculated with a commercial strain. The total solids content fell between 23 and 31% throughout the study (p<0.05), the pH at 6%, while the protein content was unchanged at the end of ripening. The hardness and gumminess in cheese increased between 1,64 and 2N; and 0,72 and 0,83N, respectively, with maturation; their were positively correlated with the variation in total solids (R²>0,90) for both cheeses while adhesiveness and cohesiveness showed no major changes. In the sensory evaluation, the cheese made from L. mesenteroides obtained an average acceptability of 70%, when compared with a goat cheese obtained from a commercial strain of Lactococcus lactis.

Producción y Composición de la Leche y Queso Fresco Pasteurizado de Cabras Mestizas Canarias Suplementadas con Grasa Sobrepasante / Production and Composition of Milk and Fresh Pasteurized Cheese in Crossbred Canarias Goats, Supplemented with Protected Fat

Para determinar el efecto de la grasa sobrepasante (GSP) con alto contenido de ácidos grasos poli-insaturados sobre la producción y composición de la leche y queso fresco pasteurizado, se utilizaron 22 cabras mestizas (entre 3/4 y 5/8 Canarias) de primer parto, de las cuales 11 se asignaron al grupo tratamiento y 11 al grupo control, de la Unidad Experimental de Producción Caprina (UEPC), de la Facultad de Ciencias Veterinarias de la Universidad Central de Venezuela, Maracay, Venezuela. Los análisis químicos se realizaron tanto a leche como a los quesos para determinación de acidez titulable, pH, Wisconsin Mastitis Test, cloruros, grasa, caseína, proteínas totales, sólidos totales, humedad, cenizas (Cen), sólidos no grasos y lactosa. Asimismo, se determinó el contenido de ácidos grasos C18:1, C18:2, C18:3 presentes en los quesos. Los resultados revelan que la incorporación de 3% de GSP en la dieta de los animales aumentó significativamente la producción de leche en 43% (P = 0,0019) y presentó una tendencia (P= 0,0821) a aumentar los días en lactancia. No se afectaron las características, ni la composición de la leche y quesos, pero por el hecho de producir más leche sin afectar sus componentes, resultó en una mayor concentración de todos los componentes (kg/lactancia). Hubo diferencias en la concentración de ácidos grasos C18:1 (P=0,0064), C18:2 (P=0,4632), C18:3 (P=0,0322), precursores de ácidos ω-3 y ω-6 en los quesos elaborados. No se afectó el rendimiento quesero, pero al aumentar la producción de leche, aumentó la producción de queso en 47,09% en el grupo tratamiento. No se alteraron las características físico-químicas y propiedades organolépticas de los quesos frescos. La suplementación con 3% de GSP no afectó las características físico-químicas ni organolépticas de los quesos. De igual manera, tampoco afectó el consumo de alimento y la condición corporal de las cabras.

The effect of protected fat (PF) with high levels of polyunsaturated fatty acids on the production and composition of milk and fresh cheese in crossbred Canarias goats was evaluated. Experiments were conducted at the Goat Production Experimental Unit of the College of Veterinary Sciences of the Central University of Venezuela, Maracay, the State of Aragua, Venezuela . Twenty-two crossbred (3/4 and 5/8) Canarias goats from first calving were used. Animals were allotted into two groups: Group 1 (n =11): Treatment group; Group 2 (n = 11): Control group. The following chemical analyses were performed for milk and cheese: pH, titratable acidity; Wisconsin Mastitis Test; chloride; fat; casein; total proteins; total solids; humidity; ash; non-fatty solids; lactose; and fatty acids (C18:1, C18:2, and C18:3) content present in cheese. Results show that addition of 3% PF caused a significant (P=0.0019) increase in milk production (43%), with a tendency (P=0.0821) to increase lactation days. Neither the characteristics nor the composition of milk and cheese were affected by PF inclusion, but the production of more milk without affecting its composition resulted in a greater concentration (kg/lactation) of all its components. There were differences in the content of the following fatty acids: C18:1 (P=0,0064), C18:2 (P=0,4632), C18:3 (P=0,0322), precursors of ω-3 and ω-6 in processed cheese. Cheese performance was not affected, but the increase in milk production, increased cheese production (47.09%) in treatment group. Treatment did not significantly affect physicochemical and organoleptic properties of fresh cheese, voluntary consumption of food, and body condition.

Evaluation of Petrifilm™ method for enumerating aerobic bacteria in Crottin goat's cheese / Evaluación de PetrifilmTM para la enumeración de bacterias aerobias en queso de cabra Crottin

The PetrifilmTM Aerobic Count Plate (ACP) developed by 3M laboratories, is a ready-to-use culture medium system, useful for the enumeration of aerobic bacteria in food. PetrifilmTMwas compared with a standard method in several different food products with satisfactory results. However, many studies showed that bacterial counts in PetrifilmTM were significantly lower than those obtained with conventional methods in fermented food. The purpose of this study was to compare the PetrifilmTM method for enumerating aerobic bacteria with a conventional method (PCA) in Crottin goat's cheese. Thirty samples were used for the colony count. The mean count and standard deviation were 7.18 ± 1.17 log CFU g-1 on PCA and 7.11 ± 1.05 log CFU g-1 on PetrifilmTM. Analysis of variance revealed no significant differences between both methods (t = 1.33, P = 0.193). The Pearson correlation coefficient (0.971, P=0.0001) indicated a strong linear relationship between the PetrifilmTM and the standard method. The results showed that PetrifilmTM is suitable and a convenient alternative to this standard method for the enumeration of aerobic flora in goat soft cheese.

PetrifilmTM Aerobic Count Plate (ACP) desarrollado por 3M es un sistema listo para usar, empleado para el recuento de bacterias aerobias en alimentos. PetrifilmTMfue comparado con los métodos estándar en diferentes productos alimenticios con resultados satisfactorios. Sin embargo, en alimentos fermentados, algunos estudios mostraron que el recuento de bacterias aerobias en PetrifilmTM fue significativamente menor que aquellos obtenidos con los métodos convencionales (PCA). El propósito de este estudio fue comparar el método PetrifilmTM para el recuento de bacterias aerobias con un método convencional en queso de cabra Crottin. Se usaron 30 muestras para el recuento de colonias. Las medias y desviaciones estándar fueron 7,18 ± 1,17 log UFC g-1 en PCA y 7,11 ± 1,05 log UFC g-1 en PetrifilmTM. El análisis de varianza mostró que no había diferencia significativa entre ambos métodos (t = 1,33, P = 0,193). El coeficiente de correlación fue 0,971 ( P = 0,0001) indicando una fuerte correlación lineal. Los resultados muestran a PetrifilmTM como un método apropiado y una alternativa conveniente a los métodos estándar para la cuantificación de flora aeróbica en queso blando de cabra.