Este trabalho foi financiado pelo National Institutes of Health EUA [concessão R01AI-39108 para ASB] ea American Heart Association [subsídios SDG 0630219N e AID 09GRNT2180065 para YQX].

1. Preparando o MRSA de Infecção <ol><li> Inocule uma ansa cheia de cultura MRSA de um tubo de estoque a -80 ° C a uma sangue de ovelha Trypticase Soy placa de agar (TSA) (ver Tabela de reagentes específicos e equipamentos), e incubar a 37 ° C durante a noite. </li><li> Verifique a pureza da cultura sobre a placa de ágar sangue (fenótipos semelhantes colônia) para ter certeza não há contaminação. </li><li> Escolha uma colónia da placa de sangue de ovelha TSA e inocular a colónia em 5 ml Trypticase Soy Broth (TSB) em um tubo de 15 ml snap-tapado. </li><li> Incubar a 37 ° C durante a noite com agitação a 200 rpm. </li></ol> 2. Preparando Cateteres Cirurgia Corte o tubo de polietileno (PI10,. Becton Dickinson, nenhuma ordem 427401) a 10 cm comprimento, e derreter uma extremidade pressionando a ponta com uma pinça estéreis. O objectivo da selagem uma extremidade do cateter é evitar hemorragia durante a cateterização. 3. Pré-sPreparação urgery e Anestesia <ol><li> Rato lugar Sprague-Dawley (Harlan, Indianapolis, Ind. fêmea, 250-300 gramas) em uma câmara contendo isofluorano em oxigénio da mistura de gases (50%: 50%) até que o anestésico tem efeito (por exemplo, os músculos estão relaxados e reflexos de pedais estão ausentes ), e manter os animais em um estado de anestesia durante a cirurgia com a mistura de gás. </li><li> Limpe a área do pescoço do queixo até um pouco abaixo do esterno com Betadine e etanol 70%. </li></ol> 4. Procedimento Cirúrgico <ol><li> Use uma técnica asséptica durante toda a cirurgia. Fazer uma incisão (1-1,5 cm) verticalmente através da pele só camada do pescoço acima do esterno. </li><li> Usando dissecção romba, separar a fáscia para expor a artéria carótida direita com 2 pares de pinças curvas dentadas. </li><li> Puxe delicadamente a artéria para fora da cavidade do pescoço, e coloque dois comprimentos de 10 cm de fio de seda sob a artéria e amarrar a artéria na extremidade exposta cefálica, coloque um clipe sobre a artery para evitar sangramento. </li><li> Fazer um pequeno furo no topo da artéria utilizando um introdutor de cateter (Becton Dickinson, ordem n. 406.999), inserir um cateter com um fórceps através do orifício na artéria, remover o grampo e empurrar para baixo do cateter para o coração até que a resistência seja cumprido. </li><li> Amarrá a sutura solto em torno da extremidade caudal da artéria e fixar o cateter no lugar com a sutura de seda quando o cateter está no lugar, tal como determinado por: i) o comprimento do cateter inserido (4-5 cm); resistência ii) um maior avanço e iii) pulsação do cateter com o batimento cardíaco. Deixar o cateter no lugar durante o resto da experiência. </li><li> Cortar sutura em excesso e as extremidades do cateter, e se certificar de que não há sangramento a partir do cateter. Dobre as pontas soltas sob a pele do pescoço e feche a pele com grampos de pele. </li><li> Coloque o rato na gaiola em um lugar morno até que recuperou da anestesia, e fornecer comida e água. Verifique o rato freqüentemente durante eapós a recuperação da anestesia. </li><li> Esta cirurgia é um &quot;Categoria E&quot; procedimento. Os procedimentos produzidos a dor é sem alívio com analgésicos desde infecções por MRSA que acompanham angústia para os animais. </li></ol> 5. Infecção por MRSA <ol><li> A infecção pode ser realizada entre 1 e 7 dias pós-cirurgia, mas manter consistente dentro experimentos. </li><li> Limpeza da cauda de rato com etanol a 70%, e injectar 0,5 ml MRSA com uma contagem de célula desejado (10 abril-10 junho ufc / animal para a maioria das cepas de S. aureus). Com 27 G agulha 1/2 polegada por via intravenosa a partir da veia da cauda Cuidado : cultura MRSA pertence ao nível de biossegurança 2 (BSL2) e tem potencial perigo moderado para o pessoal e do ambiente. </li><li> Manter a pressão no local até que a hemostase ocorre antes de retornar o rato para o compartimento. </li></ol> 6. Ratos sacrifício e tecidos-alvo Cultura <ol><li> Sacrificar ratos por injecção ip de pentobarbital de sódio (200 mg/ Kg) após 1 a 6 dias após a infecção. </li><li> Coloque o rato sobre as costas e limpe o peito com etanol 70%. </li><li> Fazer uma incisão em forma de V no peito abaixo esterno, e cortado a cartilagem de nervuras de cada lado do esterno para expor o coração. </li><li> Puxe o coração se suavemente, e clipe através do tecido do coração perto da aorta e dissecar até libertar o coração. </li><li> Coloque o coração em uma placa de Petri esterilizada com 4x4 polegadas dentro de gaze, faça um corte através da parede do ventrículo esquerdo interior e abrir a câmara do lado esquerdo. </li><li> Verifique visualmente para a colocação do cateter. Examinar e remover vegetações da válvula com uma tesoura e pinças. </li><li> Pesar e homogeneizar as vegetações, e fazer diluições seriadas com PBS para a cultura quantitativa. </li></ol> 7. Os resultados representativos Imediatamente após a inserção do cateter na artéria e empurrando para baixo a cerca de 4-5 cm do cateter para o coração, uma resistência será experienCED. Se nenhuma resistência é detectado, o cateter pode não ter sucesso, foi inserido no lado esquerdo da câmara do coração, que podem afectar a colocação do cateter. Um correcto posicionamento do cateter publicado anteriormente 9 é mostrada na Figura 1. Parte das amostras de infecção por MRSA precisam de ser cultivadas quantitativa a certificar-se a contagem viável exacta bacteriana para a infecção e pureza da amostra infecção. Além disso, os organismos recuperados a partir de vegetações deve ser os mesmos que os utilizados no inoculo. A Tabela 1 apresenta um exemplo de virulência de um S. aureus estirpe no modelo de rato endocardite, que foi publicado anteriormente 9. Todos os animais desafiados com inóculos de 10 5 e 10 6 ufc e sacrificados entre 3 a 6 dias após infecção endocardite desenvolveram com S. alta densidades aureus em vegetações cardíacas, bem como os rins e do baço ( <strong&gt; Tabela 1). Ratos com culturas de válvulas estéreis são considerados não infectados. <img alt="A Figura 1" src="/files/ftp_upload/3863/3863fig1.jpg" /> Figura 1. O cateter está em um lugar correto (lado esquerdo da câmara do coração), e vegetações diversas são visíveis em torno da válvula aórtica 9. <table border="1"><tbody><tr><td> Inóculo (n º de animas) </td><td colspan="3"> A média de log 10 CFU / g de tecido ± DP um em: </td></tr><tr><td></td><td> Vegetação </td><td> Rim </td><td> Baço </td></tr><tr><td> 10 6 UFC / animal (9) </td><td> 10,36 ± 0,85 </td><td> 7,30 ± 0,64 </td><td> 6,70 ± 0,57 </td></tr><tr><td> 10 5 UFC / animal (8) </td><td> 9,93 ± 0,53 </td><td> 7,14 ± 0,53 </td><td> 6,44 ± 0,63</td></tr><tr><td> 10 4 UFC / animal (7) </td><td> 3,46 ± 0,50 * </td><td> 1,81 ± 0,74 * </td><td> 1,58 ± 0,59 * </td></tr></tbody></table> um desvio, SD padrão. P &lt;0,001 em relação ao 10 5 ou 10 6 CFU-desafiados animais. Tabela 1. S. densidade aureus em vegetações cardíacas com diferentes inóculos na endocardite ratos 9.

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	<li>Petti, C. A., Fowler, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+bacteremia+and+endocarditis.">Staphylococcus aureus bacteremia and endocarditis.</a> Cardiol. Clin. 21, 219-233 (2003).</li><li>Bashore, T. M., Cabell, C., Fowler, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+infective+endocarditis.">Update on infective endocarditis.</a> Curr. Probl. Cardiol. 31, 274-352 (2006).</li><li>Fowler, V. G., Justice, A., Moore, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+hematogenous+complications+of+intravascular+catheter-associated+Staphylococcus+aureus+bacteremia.">Risk factors for hematogenous complications of intravascular catheter-associated Staphylococcus aureus bacteremia.</a> Clin. Infect. Dis. 17, 313-320 (1993).</li><li>Wisplinghoff, H., Bischoff, T., Tallent, S. M., Seifert, H., Wenzel, R. P., Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+bloodstream+infections+in+US+hospitals:+analysis+of+24,179+cases+from+a+prospective+nationwide+surveillance+study.">Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study.</a> Clin. Infect. Dis. 39, 309-317 (2004).</li><li>Hoen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Special+issues+in+the+management+of+infective+endocarditis+caused+by+gram-positive+cocci.">Special issues in the management of infective endocarditis caused by gram-positive cocci.</a> Infect Dis. Clin. North Am. 16, 437-452 (2002).</li><li>Moise, P. A., Hershberger, E., Amodio-Groton, M. I., Lamp, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+clinical+outcomes+when+utilizing+high-dose+(&gt;+or+=8+mg/kg)+daptomycin+therapy.">Safety and clinical outcomes when utilizing high-dose (&gt; or =8 mg/kg) daptomycin therapy.</a> Ann. Pharmacother. 43, 1211-1219 (2009).</li><li>Sakoulas, G., Brown, J., Lamp, K. C., Friedrich, L. V., Lindfield, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+outcomes+of+patients+receiving+daptomycin+for+the+treatment+of+Staphylococcus+aureus+infections+and+assessment+of+clinical+factors+for+daptomycin+failure:+a+retrospective+cohort+study+utilizing+the+Cubicin+Outcomes+Registry+and+Experience.">Clinical outcomes of patients receiving daptomycin for the treatment of Staphylococcus aureus infections and assessment of clinical factors for daptomycin failure: a retrospective cohort study utilizing the Cubicin Outcomes Registry and Experience.</a> Clin. Ther. 31, 1936-1945 (2009).</li><li>Xiong, Y. Q., Willard, J., Kadurugamuwa, J. L., Yu, J., Francis, K. P., Bayer, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+in+vivo+bioluminescent+imaging+for+evaluating+the+efficacy+of+antibiotics+in+a+rat+Staphylococcus+aureus+endocarditis+model.">Real-time in vivo bioluminescent imaging for evaluating the efficacy of antibiotics in a rat Staphylococcus aureus endocarditis model.</a> Antimicrob. Agents Chemother. 49, 380-387 (2005).</li><li>Peerschke, E. I., Bayer, A. S., Ghebrehiwet, B., Xiong, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gC1qR/p33+blockade+reduces+Staphylococcus+aureus+colonization+of+target+tissues+in+an+animal+model+of+infective+endocarditis.">gC1qR/p33 blockade reduces Staphylococcus aureus colonization of target tissues in an animal model of infective endocarditis.</a> Infect. Immun. 74, 4418-4423 (2006).</li><li>Ganesh, V. K., Rivera, J. J., Smeds, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structural+model+of+the+Staphylococcus+aureus+ClfA-fibrinogen+interaction+opens+new+avenues+for+the+design+of+anti-staphylococcal+therapeutics.">A structural model of the Staphylococcus aureus ClfA-fibrinogen interaction opens new avenues for the design of anti-staphylococcal therapeutics.</a> PLoS Pathog. 4, e1000226 (2008).</li><li>Boles, B. R., Horswill, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+biofilm+disassembly.">Staphylococcal biofilm disassembly.</a> Trends Microbiol. , (2011).</li><li>Tseng, C. W., Sanchez-Martinez, M., Arruda, A., Liu, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+Infection+of+Methicillin+Resistant+Staphylococcus+Aureus+(MRSA).">Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA).</a> J. Vis. Exp. (48), e2528 (2011).</li></ol>

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A endocardite rato é um modelo animal importante e bem caracterizado para estudos in vivo na patogénese e agentes antimicrobianos para o tratamento de infecções bacterianas 9-11. Além disso, o modelo de rato endocardite representa um composto de infecções agudas e sub-aguda, e imita homólogo humano e endocardite válvula humana nativa. Além disso, acompanhados por um cateter na habitação, que representa uma infecção clássica associada ao biofilme, um problema comum e difícil n...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Placa de ágar sangue, sangue de carneiro 5% em Trypticase Soy ágar (TSA) </td><td> Diagnóstico Hardy </td><td> A10BX </td><td></td></tr><tr><td> Trypticase Soy Broth (TSB) </td><td> Becton Dickinson </td><td> 211825 </td><td></td></tr><tr><td> Agitando incubadora </td><td> Labnet </td><td> I5311-DS </td><td></td></tr><tr><td> Tubagem de polietileno </td><td> Becton Dickinson </td><td> 427401 </td><td></td></tr><tr><td> Introdutor de cateter </td><td> Becton Dickinson </td><td> 6999 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Isofluorance </td><td> Western Medical Suppy, Inc </td><td> 2147 </td><td> Siga as informações de segurança e manuseio </td></tr><tr><td> Instrumentos Cirúrgicos </td><td> Belas Ferramentas e Instrumentos de Pesquisa de Ciências Biomédicas, Inc. </td><td></td><td> Encontre os instrumentos das duas empresas para as suas necessidades </td></tr></tbody></table>

experimental endocarditis model of methicillin resistant staphylococcus aureus (mrsa) in rat

Infecções endovasculares, incluindo endocardite, são fatais síndromes infecciosas 1-3. Staphylococcus aureus é a causa mais comum em todo o mundo dessas síndromes com morbidade e mortalidade inaceitavelmente elevada, mesmo com tratamentos adequados agente antimicrobiano 4-6. O aumento em infecções devidas a resistente à meticilina S. aureus (MRSA), as altas taxas de falhas vancomicina clínicas de tratamento e os problemas crescentes de linezolida e resistência à daptomicina tem tudo ainda mais complicada a gestão de pacientes com essas infecções, e levou a custos de saúde de elevada 7, 8. Além disso, deve salientar-se que os estudos mais recentes com os resultados do tratamento com antibióticos têm sido baseados em cenários clínicos e, portanto, pode muito bem ser influenciada por factores do hospedeiro variando de paciente para paciente. Portanto, um modelo animal de infecção relevante endovascular em que factores do hospedeiro são semelhantes a partir de animal para animal é maiscrucial para investigar patogénese microbiana, bem como a eficácia de novos agentes antimicrobianos. Endocardite em rato é um bem estabelecida modelo experimental animal que se aproxima endocardite humana nativa. Este modelo tem sido utilizado para examinar o papel de determinados factores de virulência de estafilococos e da eficácia dos regimes de tratamento com antibióticos para endocardite estafilocócica. Neste relatório, nós descrevemos o modelo de endocardite experimental devido à MRSA que poderia ser usado para investigar patogênese bacteriana e da resposta ao tratamento com antibióticos.

Watch this Scientific Journal Video about Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat at JoVE.com

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Endocardite modelo experimental de ratos devido à meticilina-resistente<em&gt; S. aureus</em&gt;.

Modelo Experimental de Endocardite resistente à meticilina<em&gt; Staphylococcus aureus</em&gt; (MRSA) em Rato

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

experimental-endocarditis-model-methicillin-resistant-staphylococcus

Research

JoVE Journal

Immunology and Infection

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

15.0K visualizações.  Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. Endocardite modelo experimental de ratos devido à meticilina-resistente S. aureus.

Vídeo: Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

O uso do fluxo de gotejamento e Reatores de disco rotativo para Staphylococcus aureus Análise de biofilme

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Imunologia e Infecção

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

Infecção subcutânea de Staphylococcus aureus resistente (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Superando indiferença em Encefalomielite autoimune experimental (EAE) linhagens mais resistentes por transferência adotiva e desafio antigênico

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Staphylococcus aureus usando hemoglobina humana como uma fonte de ferro

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Multiplex PCR Ensaio para a digita

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

Estudo das interacções de<em&gt; Staphylococcus aureus</em&gt; Com neutrófilos por citometria de fluxo e Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Segmentação de biofilme Associated<em&gt; Staphylococcus aureus</em&gt; Usando resazurina Baseado Ensaio Drogas susceptibilidade

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

genotipagem de<em&gt; Staphylococcus aureus</em&gt; por Ribossômico Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Metodologia para o Estudo da transferência horizontal de genes em<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Um modelo do rato para avaliar a resposta imune inata à infecção de Staphylococcus aureus

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...