Phenotypic and genotypic characterization of Staphylococcus spp. isolated from mastitis milk and cheese processing: Study of adherence and biofilm formation.

The aim of this study was to identify the phenotypic and genotypic profiles of Staphylococcus spp. isolated from mastitis milk and cheese processing plant.To evaluate the biofilm production of wild-type strains on contact surfaces by testing different factors through adhered cells and biofilm quantifications, finally, these biofilms were observed by Scanning Electron Microscopy (SEM). Congo red agar (CRA) plate method was used to identify slime production by strains. Screening of genes encoding adhesion factors and biofilm formation was carried out using PCR. After strains selection, adhesion and biofilm assays were designed testing different times (12, 48, 96 h), strains (n = 13), contact surfaces (stainless steel and polypropylene), and temperatures (5 °C and 25 °C); and then, bacterial count and crystal violet staining were conducted. Relative frequencies of positive on CRA and genes presence were determined, and Friedman test was applied for bacterial counts and OD values. Additionally, significant factors (P ≤ .05) were subjected to multiple comparisons using the Nemenyi test. The slime production in CRA was observed by visual inspection in 38.7% of strains. A large distribution of genes was described among strains, implying a high variability of genotypic profiles. Moreover, relative frequencies of CRA positive and gene presence were described. The developed assay showed that the strain, temperature, contact surface, were significant for both variables. The SEM corroborated the findings, showing greater biofilm formation on stainless steel at 25 °C. Thus, it is essential to highlight the importance of temperature control and material with low superficial energy to avoid biofilm formation by staphylococci.

Molecular characterization and antibiotic resistance of Staphylococcus spp. isolated from cheese processing plants.

The aim of this research paper was to characterize coagulase-positive and coagulase-negative staphylococci from raw milk, Minas cheese, and production lines of Minas cheese processing. One hundred isolates from 3 different cheese producers were characterized using molecular approaches, such as PCR, molecular typing, and DNA sequencing. Staphylococcus aureus (88% of the isolates) was the most abundant followed by Staphylococcus epidermidis, Staphylococcus hyicus, and Staphylococcus warneri. Among the 22 enterotoxin genes tested, the most frequent was seh (62% of the isolates), followed by selx and ser. Hemolysin genes were widely distributed across isolates, and Panton-Valentine leukocidin and toxic shock syndrome toxin genes were also identified. Methicillin-resistant S. aureus were staphylococcal cassette chromosome mec III, IVa, IVd, and others nontypeable. In the phenotypic antibiotic resistance, multiresistant isolates were detected and resistance to penicillin was the most observed. Using spa typing, we identified several types and described a new one, t14969, isolated from cheese. These findings suggest that antibiotic resistance and potentially virulent strains from different sources can be found in the Brazilian dairy processing environment. Further research should be conducted with collaboration from regulatory agencies to develop programs of prevention of virulent and resistant strain dissemination in dairy products and the processing environment.

Efficiency of a cleaning protocol for the removal of enterotoxigenic Staphylococcus aureus strains in dairy plants.

Staphylococci are considered a major concern in dairy plants mainly due to the intensive production flow, automation of processing plants and increased demand in the microbiological quality of dairy products. This study aimed to identify S. aureus strains isolated from three Brazilian dairy plants, evaluate the influence of time, temperature and contact surface on the bacterial adhesion process, as well as the efficiency of simulated hygiene and sanitation protocol in removing adhered cells. For genotypic analyses, the presence of icaA and icaD in strains was evaluated. Adherence assays were performed in biofilm reactor, comparing the influence of 2 temperatures (5°C and 35°C), 2 surfaces (stainless steel and polypropylene) and 4 contact times (3, 6, 12h and post-sanitization). To evaluate the process effectiveness in removing adhered cells, neutral detergent and sanitizing agent based on sodium hypochlorite were used in order to simulate the situation observed in one of the dairy plants analyzed. The presence of icaA and icaD genes was determined in 75.3% and 77.6% of strains, respectively; 70.6% of isolates showed both genes, whereas 17.6% showed no genes. Genes for enterotoxin production were found in all samples, relating to SEG and SEH toxins. The number of cells adhered on both surfaces was about 3 and 6 log10 CFU/cm2 at temperatures of 5°C and 35°C, respectively, for most situations evaluated, with significant increase over the evaluation period. In general, the temperature of 35°C favored greater adherence of S. aureus. At 5°C, there was a considerable number of adhered cells, but in populations significantly lower than those observed at 35°C. The cleaning and sanitizing protocol was ineffective in removing adhered cells; better performance of sodium hypochlorite was observed at 5°C, which should be related to lower adherence observed at this temperature. Thus, the process was not able to reduce the number of S. aureus bacteria adhered on both surfaces to safe levels under the conditions evaluated.

Sensory quality of Camembert-type cheese: Relationship between starter cultures and ripening molds.

Starter cultures and ripening molds used in the manufacture of moldy cheese aimed at obtaining characteristic flavors and textures considerably differ among dairy industries. Thus, the study of variables inherent to the process and their influence on sensory patterns in cheese can improve the standardization and control of the production process. The aim of this work was to study the influence of three different variables on the sensory quality of Camembert-type cheese: type of lactic bacteria, type of ripener molds and inoculation method. Batches of Camembert-type cheese were produced using O or DL-type mesophilic starter culture, ripened with Penicillium camemberti or Penicillium candidum and mold inoculation was made directly into the milk or by spraying. All batches were sensorially evaluated using Quantitative Descriptive Analysis (QDA) with panelists trained for various attributes. Among the combinations analyzed, those resulting in more typical Camembert-type cheese were those using O-type mesophilic starter culture and P. candidum maturation mold directly applied into the milk or sprayed and those using DL-type mesophilic starter and P. camemberti ripener mold applied by surface spraying. These results demonstrate, therefore, that the combination of different ripener molds, inoculation methods and starter cultures directly influences the sensory quality of Camembert-type cheese, modifying significantly its texture, appearance, aroma and taste.

Pulsed-Field Gel Electrophoresis characterization of Listeria monocytogenes isolates from cheese manufacturing plants in São Paulo, Brazil.

This study aimed to evaluate the occurrence of Listeria monocytogenes in cheese and in the environment of three small-scale dairy plants (A, B, C) located in the Northern region state of São Paulo, Brazil, and to characterize the isolates using conventional serotyping and PFGE. A total of 393 samples were collected and analyzed from October 2008 to September 2009. From these, 136 came from dairy plant A, where only L. seeligeri was isolated. In dairy plant B, 136 samples were analyzed, and L. innocua, L. seeligeri and L. welshimeri were isolated together with L. monocytogenes. In dairy plant C, 121 samples were analyzed, and L. monocytogenes and L. innocua were isolated. Cheese from dairy plants B and C were contaminated with Listeria spp, with L. innocua being found in Minas frescal cheese from both dairy plants, and L. innocua and L. monocytogenes in Prato cheese from dairy plant C. A total of 85 L. monocytogenes isolates were classified in 3 serotypes: 1/2b, 1/2c, and 4b, with predominance of serotype 4b in both dairy plants. The 85 isolates found in the dairy plants were characterized by genomic macrorestriction using ApaI and AscI with Pulsed Field Gel Electrophoresis (PFGE). Macrorestriction yielded 30 different pulsotypes. The presence of indistinguishable profiles repeatedly isolated during a 12-month period indicated the persistence of L. monocytogenes in dairy plants B and C, which were more than 100 km away from each other. Brine used in dairy plant C contained more than one L. monocytogenes lineage. The routes of contamination were identified in plants B and C, and highlighted the importance of using molecular techniques and serotyping to track L. monocytogenes sources of contamination, distribution, and routes of contamination in dairy plants, and to develop improved control strategies for L. monocytogenes in dairy plants and dairy products.

Antimicrobial activity of yerba mate (Ilex paraguariensis St. Hil.) against food pathogens.

Yerba mate (Ilex paraguariensis St. Hil.) has been studied for its important biological activities mainly attributed to phenolic compounds. This study evaluated the antimicrobial activity of methanolic and ethanolic extracts of yerba mate against food pathogens, such as Staphylococcus aureus, Listeria monocytogenes, Salmonella Enteritidis and Escherichia coli through minimum inhibitory (MIC) and bactericidal (MBC) concentrations, in addition to the determination of chemical composition by gas chromatography with mass spectrometry (GC-MS) and phenolic content. The most effective extract had its activity evaluated under different pH conditions by growth curve analysis. All microorganisms except E. coli were inhibited. The ethanolic extract showed the lowest MIC/MBC (0.78/0.78 mg/ml), the highest phenolic content (193.9 g.GAE/kg) and the presence of chlorogenic acid derivatives, especially 3-O-caffeoylquinic and caffeic acid. This extract was able to inhibit microbial growth at pH 7 and 8.

Antimicrobial activity of yerba mate (Ilex paraguariensis St. Hil.) against food pathogens / Antimicrobial activity of yerba mate (Ilex paraguariensis St. Hil.) against food pathogens.

Yerba mate (Ilex paraguariensis St. Hil.) has been studied for its important biological activities mainly attributed to phenolic compounds. This study evaluated the antimicrobial activity of methanolic and ethanolic extracts of yerba mate against food pathogens, such as Staphylococcus aureus, Listeria monocytogenes, Salmonella Enteritidis and Escherichia coli through minimum inhibitory (MIC) and bactericidal (MBC) concentrations, in addition to the determination of chemical composition by gas chromatography with mass spectrometry (GC-MS) and phenolic content. The most effective extract had its activity evaluated under different pH conditions by growth curve analysis. All microorganisms except E. coli were inhibited. The ethanolic extract showed the lowest MIC/MBC (0.78/0.78 mg/ml), the highest phenolic content (193.9 g.GAE/kg) and the presence of chlorogenic acid derivatives, especially 3-O-caffeoylquinic and caffeic acid. This extract was able to inhibit microbial growth at pH 7 and 8.

Incidence of Listeria monocytogenes in cheese manufacturing plants from the northeast region of São Paulo, Brazil.

The incidence of Listeria monocytogenes in three cheese manufacturing plants from the northeastern region of São Paulo, Brazil, was evaluated from October 2008 to September 2009. L. monocytogenes was found in samples from two plants, at percentages of 13.3% (n = 128) and 9.6% (n = 114). Samples of raw and pasteurized milk, water, and Minas Frescal cheese were negative for L. monocytogenes, although the pathogen was isolated from the surface of Prato cheese and in brine from one of the plants evaluated. L. monocytogenes was also isolated from different sites of the facilities, mainly in non-food contact surfaces such as drains, floors, and platforms. Serotype 4b was the most predominant in the plants studied. The results of this study indicate the need for control strategies to prevent the dispersion of L. monocytogenes in the environment of cheese manufacturing plants.

Avaliação do nível de adequação às boas praticas de fabricação em lactário hospitalar / Evaluation the level of compliance with good manufacturing practices in hospital lactary

O presente trabalho teve por objetivo avaliar as condições higiênico-sanitárias de um lactário, visando subsidiar a implementação do sistema APPCC na produção de fórmulas infantis. Em consideração às Boas Práticas na produção das fórmulas, como pré-requisito na implementação do sistema, realizou-se o levantamento das condições operacionais e higiênico-sanitárias com a aplicação de uma Lista de Verificação adaptada ao lactário. Realizaram-se também, medições de tempo de exposição e da temperatura do ambiente, de equipamentos e das fórmulas infantis nas etapas de manipulação e produção. Os resultados obtidos demonstraram que, as condições operacionais e higiênico-sanitarias atenderam 66 por cento dos procedimentos preconizados pela legislação sanitária. As não-conformidades mais relevantes referiram-se aos manipuladores de alimentos.

Contagem de microrganismos aeróbios mesófilos, coliformes totais, Escherichia Coli e Staphylococcus coagulase positiva, em leite humano cru de banco de leite humano / Count of aerobic microorganisms, coliforms, Escherichia coli and Staphylococcus coagulase positive, in human milk from human milk bank

O leite materno é o alimento mais adequado ao recém-nascido, devido ao seu valor nutricional e fisiológico. Possui propriedades imunológicas para proteção ao trato gastrintestinal e do sistema respiratório da criança. Como muitas vezes a criança está impossibilitada de receber o leite materno pelo aleitamento natural, os bancos de leite humano (BLH`s) suprem essa necessidade. O leite humano, por ser manipulado nos BLH`s (bancos de leite humano), necessita de tecnologias adequadas para o seu manuseio, devido aos microrganismos presentes no ambiente comum à mãe, à criança e aos utensílios usados no seu armazenamento. O objetivo deste trabalho foi quantificar os microrganismos aeróbios mesófilos, Staphylococcus coagulase positivo (ECP), coliformes totais e Escherichia coli, do leite cru recebido no BLH (banco de leite humano) do Hospital Santa Casa de Misericórdia em Limeira, SP.

Avaliação microbiológica e sensorial de queijo minas frescal embalados sob atmosfera modificada / Microbiological and sensory evaluation of cheese frescal under modified atmosphere

Foram preparadas amostras para três tratamentos: controle, vácuo e atmosfera modificada de 70 por cento CO2 e 30 por cento N2 (ATM). Para os tratamentos com Vácuo e ATM, as amostras foram acondicionadas em embalagens PEAD (poliestireno de alta densidade) e armazenadas a 4ºC. Após a preparação dos tratamentos foram retiradas amostras para as análises microbiológicas no 1º dia de armazenamento do produto e para análises sensoriais, nos 1º, 6º, 13º, 17º, 21º, 24º, 28º, 31º,35º e 40º dias de armazenamento. Segundo os aspectos microbiológicos, o queijo Minas Frescal não estava em condições para o consumo. Foi realizado o teste de aceitabilidade com 30 provadores não treinados, sendo medidos os atributos de cor, aparência geral e odor. O controle foi rejeitado no 17º dia de armazenamento, enquanto os queijos sob ATM e vácuo mantiveram aceitação por 40 dias para os atributos de cor, odor e aparência.

Influência do congelamento com nitrogênio líquido na sobrevivência de Vibrio cholerae em caudas de lagosta / Influence of the freezing with liquid nitrogen on the survival of the Vibrio cholerae in lobster tails

Caudas de lagosta foram artificialmente inoculadas por imersäo numa suspensäo de V. cholerae, drenadas, pesadas e, após uma noite de estocagem sob refrigeraçäo para favorecer a aderência da bactéria, congeladas a -20ºC, -60ºC e -120ºC, durante 5, 40 e 120 minutos em cada temperatura. Imediatamente após, as caudas foram descongeladas sob refrigeraçäo e lavadas por agitaçäo com soluçäo salina fisiológica. Na água de lavagem foi feita contagem de V. cholerae. A temperatura de -20ºC teve ligeiro efeito na reduçäo da populaçäo da bactéria. Temperaturas mais baixas de congelamento tiveram efeito progressivo na reduçäo das contagens. O congelamento a -120ºC/120 minutos reduziu a populaçäo da bactéria em 3,5 ciclos logarítmicos. Houve em todas as temperaturas uma correlaçäo direta entre o aumento do tempo de congelamento e a inativaçäo de V. cholerae. Caudas artificialmente contaminadas também foram congeladas a -20ºC e -120ºC por 40 minutos e a seguir estocadas a -18ºC por 28 dias. Semanalmente foi realizada contagem de V. cholerae. O congelamento a -120ºC reduziu em 3,3 ciclos logarítmicos a populaçäo do microrganismo na primeira semana, permanecendo esta praticamente constante até o final da estocagem. No congelamento a -20ºC constatou-se que durante a estocagem houve uma diminuiçäo lenta e contínua das contagens de V. cholerae. O congelamento foi capaz de diminuir, mas näo de eliminar a populaçäo da bactéria presente nas caudas de lagosta. O tratamento mais eficiente foi o congelamento a -120ºC por 40 minutos

Influência de diferentes tipos de vinagre e do hipoclorito de sódio na sobrevivência de Vibrio cholerae em folhas de alface (Lactucca sativa) artificialmente contaminadas e sobre a microbiota natural / Influence of different types of vinegar and sodium hypochlorite on the survival of Vibrio cholerae in artificially contaminated lettuce leaves (Lactucca sativa) and on the natural microflora

Folhas de alface foram artificialmente contaminadas com cultura de Vibrio cholerae 01, de maneira de obter uma contagem de 6-7 log NMP/g. Após uma noite sob refrigeraçäo as folhas contaminadas foram imergidas em diferentes soluçöes desinfetantes durante 15 minutos. Avaliaram-se soluçöes de diferentes tipos e marcas de vinagre a 6 por cento, soluçäo de hipoclorito de sódio (NaOCl) a 100mg/l de cloro ativo em pH 7,0, soluçäo de ácido acético a 0,2 por cento e 0,3 por cento e tratamento combinado de NaOCl a 100mg/l por 15 minutos seguido de tratamento com soluçäo de vinagre a 6 por cento por 15 minutos. Após o tratamento os resíduos das diferentes soluçöes nas folhas de alface foram neutralizados e determinada a populaçäo de Vibrio cholerae. Folhas naturalmente contaminadas foram tratadas com soluçäo de vinagre de vinho tinto a 6 por cento e a seguir, após neutralizaçäo dos resíduos de ácido acético, foi determinada a populaçäo de bactérias do grupo coliforme pelo método do Número Mais Provável e de microrganismos mesófilos aeróbios totais por inoculaçäo em profundidade. Em todos os ensaios com folhas artificialmente contaminadas com V. cholerae, a reduçäo das contagens deste foi acentuada, reduzindo a populaçäo inicial de 6-7 log NMP/g para menos de 0,5 log MNP/g, inativando praticamente a populaçäo da bactéria. O ajuste do pH da soluçäo de NaOCl para 7,0, melhorou sensivelmente o desempenho do produto, o qual foi ainda inferior ao vinagre. O tratamento com NaOCl (100mg/l) seguido de vinagre (por cento) näo superou os resultados obtidos pelo tratamento apenas com vinagre. O uso de ácido acético puro nas concentraçöes aproximadas da soluçäo de vinagre apresentou resultado similar a este. O efeito do vinagre sobre a microbiota natural da alface, incluindo coliformes totais e fecais näo foi täo acentuado quanto sobre o V. cholerae

Evaluation of different chlorine based disinfectantes and vinegar agaisnt Vibrio cholerae present in lettuce

Folhas de alface foram contaminadas com uma suspensäo de V. cholerae 01 contendo aproximadamente 10(6) UFC/ml. Amostras de 25g foram imersas durante 15 minutos em soluçöes de hipoclorito de sódio (NaOCl) e 3 diferentes produtos comerciais à base de di-cloro-isocianurato de sódio, todas elas contendo 100mg/l de cloro residual livre. Amostras de 25g também foram imersas por 15 e 30 minutos em soluçäo de NaOCl contendo 200mg/l de cloro ativo. Amostras similares foram imersas em soluçöes de vinagre comercial a 2 p/cento, 4 p/cento e 6 p/cento (v/v) e avaliadas após 15 minutos de tratamento. Após desinfecçäo, os resíduos das diferentes substâncias químicas foram neutralizados e enumerada a populaçäo de V. cholerae, determinando-se também a populaçäo da bactéria em folhas näo-tratadas. O vinagre foi o melhor dos produtos avaliados, seguido em ordem decrescente de eficiência pelos compostos à base de di-cloro-isocianurato de sódio e NaOCl. Em geral a reutilizaçäo das soluçöes de cloro näo affetou sua eficiência