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Fundação de Amparo a Pesquisa do Estado de Minas Gerais The presence of microbial biofilm attached milking equipment, formed by inadequate cleaning, are a constant source of contamination of the milking animals and which goes to the milk industry, compromising both the animal health and the quality of the milk produced. The objective of this study was to identify strains of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli producing biofilm in cows with mastitis milk samples, milk samples from the expansion tank and swabs of the set of liners for milking, and evaluate training biofilm coatings on four different stainless steel, comprising polypropylene, polypropylene with zeolite to 3% polypropylene with 6% zeolite and polypropylene with glass and silver. For phenotypic characterization of the strains were used for tests with congo red agar (CRA) microplate assay with crystal violet dye, counting colony forming units of biofilm cells, extracting the matrix by sonication, quantification of extracellular proteins and polysaccharides, confirmed by analysis of images generated by laser confocal microscopy. Of the 294 samples analyzed were identified eight strains of Staphylococcus aureus, Staphylococcus epidermidis and 22 three Escherichia coli, those eight, 1:11 were producing biofilm, respectively. Escherichia coli bacteria that has been formed over biofilm in all materials, and for coating stainless steel comprising polypropylene with 6% zeolite had a higher bond biofilm when compared to polypropylene and glass with silver (p < 0,05). The material consists of silver, glass and polypropylene had microbial biofilms of grip, but at a lower concentration than the other materials tested, representing themselves in this way as an alternative to coat surfaces of utensils and equipment related to milking. A presença de biofilme microbiano aderido em equipamentos de ordenha, formado por limpezas inadequadas, constituem uma constante fonte de contaminação para os animais ordenhados e do leite que vai para a indústria, comprometendo tanto a saúde do animal quanto a qualidade do leite produzido. O objetivo deste estudo foi identificar cepas de Staphylococcus aureus, Staphylococcus epidermidis, e Escherichia coli produtoras de biofilme em amostras de leite de vacas com mastite, amostras de leite do tanque de expansão e suabes do conjunto de teteiras para ordenhadeira, e avaliar a formação de biofilme em quatro revestimentos para aço inox diferentes, composto por polipropileno, polipropileno com zeolita a 3%, polipropileno com zeolita a 6% e polipropileno com vidro e prata. Para caracterização fenotípica das cepas foram utilizados os testes com ágar vermelho congo (CRA), teste de microplacas com o corante cristal violeta, contagem de unidades formadoras de colônias das células em biofilme, extração da matriz por sonicação, quantificação de proteínas extracelulares e polissacarídeos, confirmados por análise de imagens geradas pela microscopia confocal a laser. Das 294 amostras analisadas foram identificadas oito cepas de Staphylococcus aureus, três Staphylococcus epidermidis e 22 Escherichia coli, dessas oito, uma e 11 foram produtoras de biofilme, respectivamente. A Escherichia coli foi a bactéria que mais formou biofilme em todos os materiais, e o revestimento para aço inóx composto por polipropileno com zeolita a 6% teve uma maior adesão de biofilme microbiano quando comparado ao polipropileno com prata e vidro (pvalor < 0,05). O material composto por prata, vidro e polipropileno teve aderência de biofilmes microbianos, porém em menor concentração que os outros materiais testados, representando-se desta forma como alternativa para revestir superfícies de utensílios e equipamentos relacionados a ordenha. Mestre em Ciências Veterinárias

The presence of biofilm-forming bacteria from the mammary gland of dairy cows adhered to equipment in the milking environment represents one of the major causes of bacterial resistance during mastitis treatment. The aim of this study was to identify strains of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli in milk samples from cows with mastitis, as well as in the expansion tank and milking set liners. We aimed to quantify the extracellular proteins and polysaccharides in the biofilm produced by each strain. A total of 294 samples were collected from a dairy farm in the municipality of Uberlândia, Minas Gerais. To identify the S. aureus, S. epidermidis and E. coli isolates responsible for biofilm production, we tested the phenotype using the Congo red agar (CRA) and microplate adhesion tests. Protein quantification was performed with a Bicinchoninic Acid Protein Assay Kit (BCA kit), and polysaccharides were quantified by the phenol sulfuric acid method. We identified eight strains of S. aureus, one strain of S. epidermidis and 11 strains of E. coli responsible for biofilm production, all of which showed a higher concentration of polysaccharides than proteins in the matrix. Escherichia coli was considered the most prevalent bacterium among the samples, and S. aureus was determined to be the largest biofilm producer. The results of the CRA and microplate adhesion tests were similar in regard to identification of the biofilm-producing strains according to their phenotype and matrix composition. The classification of S. aureus strains as major biofilm producers is of great concern for producers, as such bacteria are considered one of the predominant contagious etiological agents that cause bovine mastitis. In addition, our observation that E. coli and S. epidermidis can produce biofilms highlights the need to reassess prophylactic measures to avoid the adhesion of biofilm-producing bacteria.

This study evaluated the microbiological quality of the water of 33 poultry sheds seeking to identify the presence of biofilm-producing Escherichia coli. The swab samples of the surface of the drinkers were analyzed by the inhibition test to detect the most effective disinfectant for removing these bacterial communities. The multiple-tube and mesophilic counts techniques were used to analyze the water samples. The swabs were plated on EMB agar followed by Rugai medium to identify E. coli . The biofilm was detected by the optical density readings and the Congo red agar method. Overall, 15 strains of Escherichia coli were detected in the swabs of the drinkers, of which 8 were biofilm producers. Regarding the water quality, 15 (45.45%) of the 33 evaluated water samples had bacterial growth while only two (13.33%) produced gas. The test with disinfectants showed that chlorine and chlorhexidine were not effective to control the E. coli biofilm production. Therefore, a strict control is essential to ensure the safety of the water supplied to the animals in broiler sheds and to eliminate the biofilm-forming E. coli that have shown to be potentially resistant to the sanitizers commonly used in the cleaning processes.

A presença de biofilme microbiano nos equipamentos de ordenha, ocasionada pela limpeza inadequada, é uma fonte constante de contaminação dos animais ordenhados e do leite que é enviado para a indústria. O biofilme também compromete a saúde dos animais e a qualidade do leite produzido. O objetivo deste estudo foi avaliar a formação de biofilme em quatro diferentes revestimentos para aço inoxidável, compostos por polipropileno virgem, polipropileno com 3% de zeólita, polipropileno com 6% de zeólita e polipropileno com vidro e prata. A formação de biofilme nos quatro revestimentos testados foi avaliada pela contagem de unidades formadoras de colônias de células no biofilme, extração da matriz por sonicação e quantificação de proteínas extracelulares e polissacarídeos, conforme confirmado pela análise de imagens geradas por microscopia confocal de varredura a laser. Cinco amostras de produção de biofilmeForam selecionados Staphylococcus aureus , um de Staphylococcus epidermidis e cinco de Escherichia coli , cuja produção de biofilme foi avaliada em um estudo anterior para avaliar a adesão aos diferentes revestimentos. Escherichia coli foi a bactéria com maior produção de biofilme em todos os revestimentos testados, sendo que a maior adesão do biofilme microbiano ocorreu no polipropileno com 6% de zeólita quando comparado ao polipropileno com prata e vidro. Esperava-se que o material composto de prata, vidro e polipropileno apresentasse menor aderência aos biofilmes microbianos, mas neste estudo não foi observada diferença entre os revestimentos com polipropileno virgem, com 3% de zeólita, e com vidro e prata. La presencia de biofilm microbiano adherido a los equipos de ordeño, formado por una limpieza inadecuada, es una fuente constante de contaminación para los animales ordeñados y para la leche que va a la industria, comprometiendo tanto la salud del animal como la calidad de la leche producida. El objetivo de este estudio fue evaluar la formación de biopelículas en cuatro recubrimientos diferentes de acero inoxidable, compuestos de polipropileno virgen, polipropileno con 3% de zeolita, polipropileno con 6% de zeolita y polipropileno con vidrio y plata. Para la determinación de la formación de biofilm en los cuatro recubrimientos ensayados, se contaron las unidades formadoras de colonias de las células en biofilm, extracción de matriz por sonicación, cuantificación de proteínas extracelulares y polisacáridos, confirmada por análisis de imágenes generadas por microscopía confocal láser. . Se seleccionaron cinco muestras de Staphylococcus aureus, una de Staphylococcus epidermidis y cinco de Escherichia coli productoras de biofilms, cuya producción de biofilm fue evaluada en un estudio previo realizado para evaluar la adherencia a diferentes recubrimientos. Escherichia coli fue la bacteria con mayor producción de biopelícula en todos los recubrimientos probados y el polipropileno con 6% de zeolita mostró una mayor adhesión de biopelícula microbiana en comparación con el polipropileno con plata y vidrio. Se esperaba que el material compuesto por plata, vidrio y polipropileno tuviera menor adherencia a las biopelículas microbianas, pero en este estudio se observó que no hubo diferencia entre los recubrimientos de polipropileno virgen, con 3% de zeolita y con vidrio y plata. A presença de biofilme microbiano aderido em equipamentos de ordenha, formado por limpezas inadequadas, constituem uma constante fonte de contaminação para os animais ordenhados e para o leite que vai para a indústria, comprometendo tanto a saúde do animal quanto a qualidade do leite produzido. O objetivo desse estudo foi avaliar a formação de biofilme em quatro revestimentos para aço inox diferentes, compostos por polipropileno virgem, polipropileno com zeolita a 3%, polipropileno com zeolita a 6% e polipropileno com vidro e prata. Para a determinação da formação de biofilme nos quatro revestimentos testados, foi realizada a contagem de unidades formadoras de colônias das células em biofilme, extração da matriz por sonicação, quantificação de proteínas extracelulares e polissacarídeos, confirmados por análise de imagens geradas pela microscopia confocal a laser. Foram selecionadas cinco amostras de Staphylococcus aureus, uma de Staphylococcus epidermidis e cinco de Escherichia coli produtoras de biofilme cuja produção de biofilme foi avaliada em estudo anterior realizado para avaliar a adesão nos diferentes revestimentos. A Escherichia coli foi a bactéria com maior produção de biofilme em todos os revestimentos testados e o polipropileno com zeolita a 6% apresentou uma maior adesão de biofilme microbiano quando comparado ao polipropileno com prata e vidro. Esperava-se que o material composto por prata, vidro e polipropileno tivesse menor aderência de biofilmes microbianos, mas nesse estudo observou-se que não houve diferença entre os revestimentos de polipropileno virgem, com zeolita a 3% e com vidro e prata.

Background: Leptospirosis is a zoonosis that affects many species of mammals and occurs endemically in Brazil. The biofilm matrix provides structure and protection to the biofilm cells working as a physical barrier to antibiotic agents, which are attached or consumed by the matrix components. However, this attribute varies according to the matrix, antimicrobial agent and biofilm age. Leptospira may change morphologically according to environmental conditions, including cell aggregation and biofilm formation. Leptospira can colonize the ducts of kidney from hosts for a long time, forming a biofilm, which is believed to be an important factor for their maintenance in animals and in the environment. Thus, the objective of this research was to determine the biofilm formation capacity of four strains of Leptospira interrogans.Materials, Methods & Results: The strains were typified by WHO/FAO/OIE and National Collaborating Center for Reference and Research on Leptospirosis (Kit Biomedical Research, Amsterdam, Netherlands). Leptospira interrogans strains, two isolated from cattle and two isolated from dogs were biofilms tested for adhesion on polystyrene plates, extracellular matrix composition and confocal microscopy. In the plating adhesion test, the suspension was inoculated into 96-well sterile polystyrene microplates with flat bottom at a ratio of 1:200 in EMJH medium, followed by 24 h incubation at 28°C, with medium renewal after 12 h. After this period the wells were washed three times with sterile PBS and following incubation; the plates were dried in the oven at 60°C for 30 min and added 200 μL of 1% violet crystal for five min. Subsequently, the plates were washed with distilled water, after complete removal, 200 μL of acetic acid 33% was added and the readings were performed at 570 nm in the ELISA reader. The proteins and polysaccharides were quantified in a scraped pooled sample diluted in 0.85% sterile saline solution to achieve an optimal amount for testing used reagents of the BCA kit. The polysaccharide content was determined by adding into a tube, an aliquot of 0.5 mL from the pooled sample, 0.5 mL of phenol and then immediately 2.5 mL of sulfuric acid. The solution was homogenized and left to react for 15 min at room temperature. The reading was performed at 490 nm in ELISA reader. The strains were compared regarding polysaccharides and protein matrices using analysis of variance (ANOVA) and Tukey test. At confocal microscopy the strains were incubated with the tested polypropylene material for 24 h. The materials were washed with sterile phosphate buffer and stained with propidium iodide. The reading was performed using a Laser Scanning Confocal Microscope (Zeiss 710) with laser excitation (488 nm) and 580-680 nm emission filters for propidium iodide (red marking). All strains displayed strong adherence on microplate and the amount of polysaccharides in biofilm was not statistically different among the studied strains, but the amount of protein was significantly different in strain 4 (P > 0.5). The confocal microscopy showed the adherence of the Leptospira spp. strains to polypropylene material after washing.Discussion: Biofilm production plays an important role in the maintenance of a chronic infection by Leptospira interrogans with renal colonization. The exopolysaccharide (EPS) has various functions, such as checking insolubility in water; giving the three-dimensional conformation of the biofilm; protecting cells from physical (mechanical action, irradiation and temperature variations), chemical.

Made available in DSpace on 2022-04-28T19:01:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-01-01 Staphylococcus is an important agent of mastitis, especially when biofilm producers are related to a number of mechanisms of resistance to different types of infections. The objective of this research was to evaluate the susceptibility and resistance of Staphylococcus aureus strains isolates from cases of bovine mastitis and milking environment under conditions of biofilm, compared to three different concentrations in different antimicrobials. Thirty-two strains used were S. aureus that, due to the inhibition tests, minimal biofilm eradication concentration were tested using cephalexin, amoxicillin and rifampin at concentrations of 30 mg/mL, and 50 mg/mL 100 mg/mL for 12 hours, and efficiency of tests evaluated by counting the colony forming units and the microplate test. The results revealed that among the tested antibiotics cephalexin showed the best efficiency at the three concentrations tested, and rifampin and amoxicillin were more efficient at concentrations of 50 and 100 mg/mL. These data demonstrate the importance of targeted antibiotic therapy associated with the correct cleaning of milking equipment, thus, preventing the formation of biofilm, avoiding persistent microbial adhesion in equipment, and the spread of microorganisms by ascending channel teats among herd animals. Universidade Federal de Uberlandia Universidade Estadual de Santa Cruz Universidade Federal de Uberlandia, Instituto de Ciencias Biomedicas, Area de Imunologia Microbiologia e Parasitologia Universidade Estadual Paulista “Julio de Mesquita Filho”, Departamento de Medicina Veterinaria Preventiva e Reproducao Animal Universidade Estadual Paulista “Julio de Mesquita Filho”, Departamento de Medicina Veterinaria Preventiva e Reproducao Animal

A IgY e assim classificada porque os anticorpos maternos sao transferidos do soro sanguineo para a gema do ovo, desta forma, para adquirir anticorpos reativos para determinado tipo de antigeno, o sacrificio de animais seria evitado, uma vez que os mesmos podem ser extraidos da gema em grande quantidade, o que nao ocorre na utilizacao de mamiferos. Objetiva-se avaliar a reatividade de imunoglobulinas Y de galinhas imunizadas com vacina B19 atraves producao de anticorpos policlonais especificos, detectaveis em testes oficiais para brucelose, tais como, Antigeno Acidificado Tamponado (AAT) e 2-Mercaptoetanol (2-ME), e no ELISA indireto. Foram utilizadas quatro galinhas, divididas aleatoriamente em dois grupos experimentais, sendo um grupo controle (Grupo 1) e um grupo imunizado com vacina B19 de Brucela abortus (Grupo 2). No Grupo 1 as galinhas foram imunizadas com 250 µL de PBS, e no Grupo 2 com 250 µL da Vacina B19 diluida em PBS, ambos adicionados de adjuvante. Os dois grupos foram imunizados seis vezes durante 13 semanas, quinzenalmente. Para avaliar a producao e reatividade da IgY foram realizadas sete coletas de sangue quinzenais, sendo a primeira, uma semana antes da primeira imunizacao e as demais uma semana apos cada imunizacao; e coletas diarias de ovos a partir de uma semana antes da primeira imunizacao, sendo estes separados por grupo e por semana. A IgY proveniente da gema do ovo foi purificada a partir de um pool semanal gema de ovos de cada grupo, utilizando-se os metodos de delipidacao atraves da diluicao em agua acida e a precipitacao com sulfato de amonio. As galinhas do Grupo 1, nao foram reagentes aos testes, enquanto do Grupo 2 produziram anticorpos reativos a este antigeno detectaveis em todos os testes realizados para o diagnostico de brucelose bovina. Conclui-se que as galinhas produziram anticorpos IgY reagentes nos testes sorologicos realizados, sendo a imunoglobulina Y um potencial antigeno para producao de anticorpos especificos, a fim de serem utilizados em testes diagnosticos. SUMMARY The IgY is classified as such because maternal antibodies are transferred from the serum to the egg yolk. Thereby to acquire antibodies reactive to a particular type of antigen, animal killing could be avoided since the antibodies can now be extracted from the yolk in large quantity, something that does not occur with mammals. This study aims to evaluate the reactivity of immunoglobulin Y of chickens immunized with B19 through the production of specific polyclonal antibodies, detectable in official tests for brucellosis such as Buffered Acidified Antigen (AAT) and 2-mercaptoethanol (2-ME), and ELISA. Four hens were randomly divided into two experimental groups, one of which the control group (Group 1) and the second, a group immunized with Brucella abortus B19 vaccine (Group 2). The chickens of Group 1 were immunized with 250 μ L of PBS while in Group 2 with 250 μ L of B19 vaccine diluted in PBS; adjuvant was added in both. The two groups were immunized six times during 13 weeks, fortnightly. To evaluate the production and reactivity of IgY seven blood samples were collected biweekly, the first, a week before the first immunization and the others a week after each immunization; and eggs were harvested daily starting from a week before the first immunization, and separated by group per week. The IgY from the egg yolk was purged from an egg yolk pool of each group prepared weekly, using the delipidation by dilution with water and acid precipitation with ammonium sulfate method. The chickens of Group 1 were not reactive to the tests, whereas the chickens in Group 2 produced antibodies reactive to this antigen detectable in all brucellosis diagnosis tests. It is concluded that the chickens produced IgY antibody reagents in serological tests performed, and the immunoglobulin Y is a potential antigen for production of specific antibodies that can be used in diagnostic tests.

A prevalencia da mastite por Staphylococcus aureus em rebanhos leiteiros ocorre devido a sua alta infectividade associada a fatores de virulencia que conferem ao micro-organismo a capacidade de se instalar no parenquima mamario. Objetivou-se avaliar o perfil de sensibilidade e resistencia de estirpes de Staphylococcus aureus produtoras de biofilmes isoladas de casos de mastite bovina e ambiente de ordenha. Foram avaliadas 32 estirpes, sendo 28 isoladas de leite de vacas com mastite, duas isoladas de insufladores, uma isolada de mangueira condutora de leite e uma do leite do tanque de expansao. Apos o isolamento e caracterizacao das estirpes como Staphylococcus aureus foram realizados testes para avaliar a capacidade de producao de biofilmes pelo teste de microplacas, e o perfil de sensibilidade e resistencia de estirpes em modelo de adesao em biofilmes frente ao antimicrobiano cefalexina nas concentracoes de 30mg/mL e 100mg/mL no tempo de 12 horas de contato das bacterias com o antimicrobiano. Os resultados revelaram que apos a producao de biofilmes de 32 estirpes, 2 (6,25%) foram resistentes a cefalexina na concentracao de 30mg e 8 (25,00%) foram resistentes na concentracao de 100mg. A alta concentracao de cefalexina tornou ineficaz a penetracao do antimicrobiano pela matriz do biofilme dificultando sua atuacao, sabendo-se que as bacterias quando em biofilmes estao mais resistentes do que na forma livre. Por isso, e importante realizar uma correta avaliacao do uso e da concentracao dos antimicrobianos para o adequado tratamento das infeccoes, principalmente das mastites estafilococicas. SUMMARY The prevalence of mastitis transmitted by Staphylococcus aureus in dairy herds is due to its high infectivity rate associated with virulence factors that give the micro-organism the ability to settle in the breast. This study aimed to evaluate the sensitivity and resistance of Staphylococcus aureus strains capable of producing biofilms in isolates from cases of bovine mastitis and milking environment. We evaluated 32 strains, 28 strains isolated from milk of cows with mastitis, two isolated from vacuum tubes, one isolated from the milk hose and another from the milk of the expansion tank. After isolation and characterization of strains such as Staphylococcus aureus, tests were performed to evaluate biofilms production capacity using the microplate test, and the susceptibility profile and resistance of strains using the biofilm adhesion model against the antimicrobial cephalexin at concentrations of 30 and 100 mg/mL for 12 hours of contact time of the bacteria with the antimicrobial. The results showed that after production of biofilms of 32 strains, 2 (6.25%) were resistant to cephalexin concentration of 30 mg while 8 (25.00%) were resistant at the concentration of 100 mg. The high concentration of cephalexin rendered ineffective the antimicrobial penetration by the biofilm matrix hindering its performance; it is known that when the bacteria are in biofilms they become more resistant than in the free form. It is concluded that it is important to make a correct assessment of the use and concentration of antimicrobials for the appropriate treatment of infections, particularly of staphylococcal mastitis.