Abstract Real-time PCR (qPCR) has been used for rapid identification of Salmonella Typhimurium and Staphylococcus aureus in dairy foods, but is unable to differentiate viable and unviable pathogens. Ethidium bromide monoazide (EMA), a DNA-intercalating agent, can detect only viable cells because selectively enter cells considered unviable and bind to their DNA, inhibiting its amplification during qPCR. The objective was to establish a protocol for detection of viable Salmonella Typhimurium and S. aureus, experimentally inoculated in coalho cheese, by the use of EMA combined with qPCR. The protocol was effective for the identification of viable Salmonella Typhimurium in coalho cheese but not for the S. aureus cells. Concentrations of viable Salmonella Typhimurium cells of 1 CFU/10 g of coalho cheese could be detected. The monoplex protocol enables the rapid and specific identification of viable Salmonella Typhimurium in coalho cheese, making it an alternative method for the quality and safety control of cheeses.

Resumo: O objetivo deste trabalho foi caracterizar bacteriófagos autóctones e determinar sua atividade lítica em Staphylococcus aureus. Seis fagos foram isolados de água de lavagem de pisos de estábulos por meio do enriquecimento de cultura com três estirpes de S. aureus. Todos os fagos foram caracterizados pela digestão do DNA com enzimas de restrição e pelo sequenciamento do fragmento de DNA que codifica a endolisina. Cada fago foi testado contra 100 estirpes de S. aureus isoladas de casos de mastite bovina e de produtos lácteos pelo método de lise em placa. Sequências do gene de endolisina apresentaram alta conservação, com mais de 99% de similaridade a nível do nucleotídeo entre os fagos isolados. Foram identificados três domínios envolvidos no reconhecimento e na lise da parede celular bacteriana. Dois bacteriófagos isolados de estábulos apresentam alta atividade lítica em S. aureus, em ampla gama de estirpes, o que indica seu potencial para estudos de fagoterapia em gado leiteiro ou como agente de controle biológico para produtos lácteos.

Abstract: The objective of this work was to characterize autochthonous bacteriophages and to determine their lytic activity on Staphylococcus aureus. Six phages were isolated from dairy barn flush water through enrichment cultures with three S. aureus strains. All phages were characterized by DNA digestion by restriction enzymes and sequencing of the DNA fragment encoding endolysin. Each phage was tested against 100 S. aureus strains isolated from bovine mastitis and from dairy products using the lysis-plate method. The sequences of the endolysin gene were highly conserved, with nucleotide similarity higher than 99% among the isolated phages. Three domains involved in the recognition and lysis of the bacterial cell wall were identified. Two bacteriophages isolated from the dairy barns present high lytic activity on S. aureus, on a wide range of host strains, indicating their potential for studies on phage therapy in dairy cattle or as a biological control agent for dairy products.

This study aimed to evaluate the presence of enterotoxigenic S. aureus in the endogenous starter and in Artisan Minas cheeses from the Serra da Canastra. Sixteen samples of endogenous starters and cheese were collected during the rainy and dry seasons. The isolation and enumeration of S. aureus were performed using the PetrifilmTM-Rapid S. aureus Plate Count method. The presence of enterotoxin in the cheese samples was analyzed by the Optimal Sensitivity Plate (OSP) method and the ELFA-VIDAS®-Staph enterotoxin-II assay. S. aureus strains were tested for their ability to produce enterotoxins using the Optimal Sensitivity Plate (OSP) method and the polymerase chain reaction (PCR) assay for the classical enterotoxin genes. The Optimal Sensitivity Plate (OSP) method data showed that staphylococcal enterotoxin A (SEA) was detected in 75% of the cheese samples, but no toxin was detected with the ELFA-VIDAS method. It was found that 12.5% of the isolated strains produced staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin C (SEC). When using the the polymerase chain reaction (PCR) assay, only one isolate was found to harbor an enterotoxin gene, contrary our expectations. However, discrepancies between the immunological and molecular assays are not uncommon. Despite the fact that most isolates did not produce classical enterotoxins, high S. aureus counts in the cheese samples causes concern since there is a risk of the presence of non-classical enterotoxins.

O objetivo deste trabalho foi isolar e caracterizar bactérias ruminais tolerantes a taninos obtidas de vacas mestiças Holandês x Zebu alimentadas com dieta composta por capim-elefante (Pennisetum purpureum) picado com ramos novos de angico-vermelho (Parapiptadenia rigida) e folhas de bananeira (Musa sp.). Um total de 117 cepas bacterianas foram isoladas a partir de cultivos de enriquecimento da microbiota ruminal em meio contendo extrato de taninos. Destas, 11 foram capazes de tolerar até 3 g L-1 de taninos. Procedimentos clássicos de caracterização indicaram que diferentes grupos, morfológicos e fisiológicos, estavam representados. Perfis dos fragmentos de restrição com Alu1 e Taq1 dos produtos de PCR de 1.450 bp do gene 16S rRNA agruparam os 11 isolados nos tipos I a VI. O sequenciamento dos produtos PCR 16S rRNA foi utilizado para identificação. Das 11 estirpes estudadas, sete não foram identificáveis pelos métodos utilizados neste trabalho, duas eram estirpes de Butyrivibrio fibrisolvens e duas de Streptococcus bovis.

The objective of this work was to isolate and characterize tannin-tolerant ruminal bacteria from crossbred Holstein x Zebu cows fed a chopped mixture of elephant grass (Pennisetum purpureum), young stems of "angico-vermelho" (Parapiptadenia rigida), and banana tree (Musa sp.) leaves. A total of 117 bacteria strains were isolated from enrichment cultures of rumen microflora in medium containing tannin extracts. Of these, 11 isolates were able to tolerate up to 3 g L-1 of tannins. Classical characterization procedures indicated that different morphological and physiological groups were represented. Restriction fragments profiles using Alu1 and Taq1 of 1,450 bp PCR products from the 16S rRNA gene grouped the 11 isolates into types I to VI. Sequencing of 16S rRNA PCR products was used for identification. From the 11 strains studied, seven were not identifiable by the methods used in this work, two were strains of Butyrivibrio fibrisolvens, and two of Streptococcus bovis.

Com os objetivos de quantificar, isolar e caracterizar bactérias psicrotróficas contaminantes de leite cru refrigerado, produzido na região da Zona da Mata de Minas Gerais e Sudeste do Rio de Janeiro, foram analisadas amostras de leite coletadas de 20 tanques coletivos e 23 tanques individuais. As contagens de bactérias psicrotróficas nas amostras de leite para os dois tipos de tanques de refrigeração variaram entre 10² e 10(7) Unidades Formadoras de Colônias (UFC) ml-1, porém, um maior número de tanques coletivos apresentou contagens acima de 1 x 10(5) UFC ml-1. Foi verificada a predominância de bactérias psicrotróficas gram-negativas (81,2%), que foram identificadas pelos sistemas API 20E e API 20NE nos gêneros: Aeromonas, Alcaligenes, Acinetobacter, Burkholderia,Chryseomonas, Enterobacter, Ewingella, Klebsiella, Hafnia, Methylobacterium, Moraxella, Pantoea, Pseudomonas, Serratia, Sphingomonas e Yersinia. As bactérias gram-positivas (18,8%) foram identificadas com API 50 CH, API Coryne e API Staph, nos gêneros: Bacillus, Brevibacterium, Cellum/Microbacterium, Kurthia e Staphylococcus. Os sistemas API utilizados não identificaram todos os isolados bacterianos. Pseudomonas foi o gênero mais isolado e P. fluorescens foi a espécie predominante. A maioria dos isolados bacterianos apresentou atividade proteolítica e/ou lipolítica a temperaturas de refrigeração de 4°C, 7°C e 10°C, evidenciando seu alto potencial de deterioração do leite e dos produtos lácteos. Os resultados ressaltam que maior atenção deve ser dada aos procedimentos que impeçam a contaminação do leite por esses microrganismos.

This study aimed to quantify, isolate and characterize psychrotrophic bacteria from refrigerated raw milk produced at the ‘Mata’ Region of Minas Gerais State and Southeast of Rio de Janeiro State, Brazil. Raw milk samples, were collected at the farms, from 20 collective refrigerated tanks and 23 individual refrigerated tanks. The psychrotrophic bacteria counting ranged from 10² to 10(7) Colony Forming Units (CFU) ml-1 for both types of refrigerated tanks, but most of the collective tanks showed counts higher than 1 x 10(5) CFU ml-1. Predominance of psychrotrophic gram-negative bacteria (81.2%), that were identified by API 20E and API 20NE as belonging to genera: Aeromonas, Alcaligenes, Acinetobacter, Burkholderia, Chryseomonas, Enterobacter, Ewingella, Klebsiella, Hafnia, Methylobacterium, Moraxella, Pantoea, Pseudomonas, Serratia, Sphingomonas e Yersinia were oserved. The gram-positive bacteria (18.8%), were identified by API 50 CH, API Coryne and API Staph, to genera: Bacillus, Brevibacterium, Cellum/Microbacterium, Kurthia e Staphylococcus. The API systems utilized could not identify all the bacterial isolates. Pseudomonas was the genus most isolated with P. fluorescens as the predominant species. Most of the isolates presented proteolytic and/or lipolytic activity at 4°C, 7°C and 10°C showing high potential for milk and milk products spoilage. The results indicated that more attention must be taken to the procedures necessaries to reduce milk contamination with psychrotrophic bacteria.

Neste estudo, foi comparada a contagem de células somáticas (CCS) de amostras pareadas de leite de 86 cabras pelo método eletrônico (Somacount 300) calibrado com padrão de vaca, com a técnica padrão de contagem microscópica direta utilizando corante pyronina Y - verde de metila. As cabras eram das raças Saanen e Toggenburg de um criatório localizado na Zona da Mata de Minas Gerais. Em complementação, foi avaliado o efeito do conservante Bronopol® no leite de cabras sobre a CCS. Para as 86 amostras de leite sem conservante, a média das leituras de CCS determinadas com o microscópio foi menor e diferiu (p <FONT FACE=Symbol>£</FONT> 0,05) da média de CCS lidas pelo Somacount 300 padronizado com leite de vaca. Porém, a média de leituras de CCS das 86 amostras com Bronopol® lidas no microscópio não apresentou diferença significativa (p>0,05) em relação à obtida com o Somacount 300; e a curva de estimação de CCS no microscópio em função do Somacount foi significativa em nível de 95% com alta correlação, demonstrando a possibilidade de se utilizar o Somacount 300 padronizado com leite de vaca para leituras de CCS de leite de cabras conservado com Bronopol® dentro dos limites de CCS analisados neste estudo.

In this study, there was a comparison of SCC from paired milk samples of 86 goats by the electronic method (Somacount 300) calibrated with cow milk standard, with the pyronin Y-methyl green stain direct microscopic method. The goats were of Saanen and Toggenburg breeds from a farm located at the Zona da Mata of the Minas Gerais State. In addition, was evaluated the effect of Bronopol® on the SCC in goat milk was evaluated. For the unpreserved 86 milk samples, the SCC mean determined with the microscopic method was smaller and differed (p <FONT FACE=Symbol>£</FONT> 0.05) from that obtained with the Somacount 300 calibrated with cow milk. However, the SCC mean of the 86 milk samples with Bronopol® did not differ between the two methods (p>0.05). The estimated SCC curve for the microscopic in function to the Somacount significant with high correlation, demonstrating the possibility of using the Somacount 300 calibrated with cow milk for counting somatic cells in goat milk preserved with Bronopol® within the range of SCC analyzed in this study.