Esta pesquisa foi suportada por concessões de Saúde Pública dos Estados Unidos Serviço AI69233 e AI073843 do Instituto Nacional de Alergia e Doenças Infecciosas. EPS é um Burroughs Wellcome Fellow na patogênese de doenças infecciosas. KPH foi financiado pelo Celular e Molecular Microbiology concessão de Treinamento Programa 5 T32 A107611-10.

1. Purificação da hemoglobina a partir de sangue fresco <ol><li> Adquirir sangue humano fresco suplementado com um anticoagulante. Manter o sangue em gelo ou a 4 ° C durante a purificação. </li><li> Centrifugar o sangue durante 20 minutos a 1500 x g. As células vermelhas do sangue (RBC) irá ficar na parte inferior do tubo. Aspirar cuidadosamente o sobrenadante e ressuspender o pellet cuidadosamente em solução gelada de NaCl a 0,9% (w / v). Voltar a centrifugar e lavar 3 vezes. </li><li> Ressuspender o sedimento em 1 volume de mM arrefecido com gelo 10 Tris-HCl (pH 8,0). Isto irá induzir a lise dos glóbulos vermelhos devido à pressão osmótica. Adicionar tolueno a ~ 20% do volume final. </li><li> Incubar a 4 ° C durante a noite em um espeto. </li><li> Centrifuga-se o lisado a 20.000 xg durante 1 hora. Recolher a fracção hemolisado meio deixam os tolueno (flutuante no topo) e pellet. Usar uma pipeta de gargalo longo de recolher a fracção do meio. </li><li> Passar através de filtro de seringa de 0,44 | im. Se a solução contiver partículasmatéria e não pode ser passada através do filtro, repetir o passo 1.5. </li><li> Purificar a hemoglobina (Hb) com uma cromatografia líquida de alta performance (HPLC) em coluna de permuta aniónica (Varian, PL-SAX 1000 por 8 um, 150 mm x 4,6 mm). A fase móvel A é 10 mM Tris-HCl (pH 8,0) e a fase móvel B é 10 mM Tris-HCl (pH 8,0) + 0,5 M de NaCl. Um gradiente de 0% -100% de solvente B é administrado ao longo de 2 min a taxa de fluxo de 2,0 ml / min. A eluição é controlada com base na absorção (λ: 410 nm e 280 nm). Recolher apenas a fracção caracterizada por uma cor vermelho brilhante e um pico de absorção proeminente (Figura 1). </li><li> Dialisar a eluição contra salino tamponado com fosfato (PBS) durante a noite e, em seguida, por algumas horas, de novo. Esterilizar por passagem através de um filtro de seringa de 0,22 um. </li><li> Para medir a concentração de Hb, preparar soluções-padrão de concentrações conhecidas de hemoglobina em PBS. Determinar a concentração de Hb no exemplo por mistura das soluções-padrão (ver a tabela com reagentes nosed) ou da solução de amostra com o reagente de Drabkin 2x (preparada a partir de pó), a uma proporção de 1:1. Por exemplo, misturar 100 ul de solução de Hb com o reagente de Drabkin 100 ul de 2x, em placas de 96 poços. Incubar durante 15 min e a absorvância medida a 540 nm. Desenhar uma curva padrão e determinar a concentração de Hb na amostra. Cinco a 15 mg / ml rendimentos são típicos. </li><li> Executar 15-20 ug purificado hemoglobina em 15% SDS-PAGE em duplicado. Stain um dos géis; transferir as proteínas a partir de um outro gel para uma membrana de nitrocelulose e imunotransf erência para a hemoglobina (Figura 2). </li><li> Congelar e armazenar alíquotas de 1 ml de hemoglobina em azoto líquido. </li></ol> 2. Preparação de Ferro-empobrecem meios de crescimento <ol><li> Prepare Roswell Park Memorial Institute (RPMI) caldo por dissolução do pó de RPMI em água, adicionar bicarbonato de sódio, tal como recomendado pelo fabricante, e 1% de ácidos cassamino (CA) (w / v). Esterilizar por passagem através de um filtro de 0,2 um e refrig lojarado. </li><li> Prepare-metal empobrecido RPMI (NRPMI) pela adição de 7% (w / v) Chelex 100 e misturando durante a noite numa placa de agitação. Remover Chelex 100, passando através de um filtro de 0,2 um e armazenamento refrigerado. Suplemento meios essenciais com metais não-ferrosos: 25 fiM de ZnCl2, 25 mM de MnCl2, 100 mM de CaCl2 e 1 mM de MgCl2 preparado antecipadamente como uma solução estéril x 1000. Use recipientes de plástico descartáveis ​​para esta etapa para evitar a contaminação de ferro a partir de re-utilizáveis ​​suprimentos. </li></ol> 3. Crescimento Staphylococcus aureus utilizando hemoglobina como uma fonte de ferro Sole <ol><li> Streak S. aureus para isolamento em agar de soja tríptica (TSA) a partir de um estoque congelado. Incubar a 37 ° C durante 20-24 hr. </li><li> Ressuspender o ácido etilenodiamino-N, N&#39;-bis (ácido 2-hidroxifenilacético) (EDDHA) em etanol anidro a 100 mM. EDDHA não entra em solução, mas é esterilizada por etanol. </li><li> Adicionar EDDHA de RPMI a uma concentração final de 0,5 mM. PermitirEDDHA para dissolver, pelo menos, 30 minutos antes de prosseguir para o passo seguinte. Devido ao lote-a-lote de variação, a concentração final de EDDHA pode precisar de ser reduzidos para 0,25 mM para permitir o crescimento bacteriano. </li><li> Inocular colónias isoladas de S. aureus em 5 ml de meio RPMI contendo EDDHA em 15 ml com tampa de rosca tubos cónicos. Incubar a 37 ° C com agitação a 180 rotações por minuto (rpm) durante 16-20 horas. </li><li> Centrifugar as culturas durante a noite para 5 min a 7500 xg e ressuspender o sedimento em NRPMI contendo 0,5 mM de EDDHA. Normalizar a OD 600 ~ 3. </li><li> Prepare NRPMI contendo 2,5 ug por ml de Hb e 0,1-1,0 mM EDDHA. Devido a variação entre os lotes, a concentração necessária EDDHA de quelação de ferro livre no NRPMI pode variar. </li><li> Ul subcultura 10 de suspensão bacteriana a partir do passo 3,5 em 1 ml NRPMI + + EDDHA Hb num tubo de 15 ml com tampa de rosca cónica. </li><li> Incubar as culturas a 37 ° C durante até 48 horas com agitação a 180 rpm ou num tambor giratório. </li><li&gt; Cada hr 6-12 tomar leituras de DO 600, removendo 50 ul da cultura e da sua mistura com 150 ul de PBS, em placas de 96 poços. </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+weak+protein-protein+complexes+transfer+heme+across+the+cell+wall+of+Staphylococcus+aureus.">Transient weak protein-protein complexes transfer heme across the cell wall of Staphylococcus aureus.</a> Journal of the American Chemical Society. 133, 14176-14179 (2011).</li><li>Muryoi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+the+iron-regulated+surface+determinant+(Isd)+heme+transfer+pathway+in+Staphylococcus+aureus.">Demonstration of the iron-regulated surface determinant (Isd) heme transfer pathway in Staphylococcus aureus.</a> J. Biol. Chem. 283, 28125-28136 (2008).</li><li>Reniere, M. L., Skaar, E. P. <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+haem+oxygenases+are+differentially+regulated+by+iron+and+haem.">Staphylococcus aureus haem oxygenases are differentially regulated by iron and haem.</a> Mol. Microbiol. 69, 1304-1315 (2008).</li><li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+hemin+transfer+from+IsdA+to+IsdC+in+the+iron-regulated+surface+determinant+(Isd)+heme+acquisition+system+of+Staphylococcus+aureus.">Direct hemin transfer from IsdA to IsdC in the iron-regulated surface determinant (Isd) heme acquisition system of Staphylococcus aureus.</a> J. Biol. Chem. 283, 6668-6676 (2008).</li><li>Pishchany, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+for+human+hemoglobin+enhances+Staphylococcus+aureus+infection.">Specificity for human hemoglobin enhances Staphylococcus aureus infection.</a> Cell Host Microbe. 8, 544-550 (2010).</li><li>Pishchany, G., Dickey, S. E., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+the+Staphylococcus+aureus+heme+iron+transport+components+IsdA+and+IsdB.">Subcellular localization of the Staphylococcus aureus heme iron transport components IsdA and IsdB.</a> Infect. Immun. 77, 2624-2634 (2009).</li><li>Torres, V. J., Pishchany, G., Humayun, M., Schneewind, O., Skaar, E. P. <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+IsdB+is+a+hemoglobin+receptor+required+for+heme+iron+utilization.">Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization.</a> J. Bacteriol. 188, 8421-8429 (2006).</li><li>Kim, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IsdA+and+IsdB+antibodies+protect+mice+against+Staphylococcus+aureus+abscess+formation+and+lethal+challenge.">IsdA and IsdB antibodies protect mice against Staphylococcus aureus abscess formation and lethal challenge.</a> Vaccine. 28, 6382-6392 (2010).</li><li>Cheng, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+requirements+for+Staphylococcus+aureus+abscess+formation+and+persistence+in+host+tissues.">Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues.</a> Faseb J. 23, 3393-3404 (2009).</li><li>Andreini, C., Bertini, I., Cavallaro, G., Holliday, G. L., Thornton, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+ions+in+biological+catalysis:+from+enzyme+databases+to+general+principles.">Metal ions in biological catalysis: from enzyme databases to general principles.</a> J. Biol. Inorg. Chem. 13, 1205-1218 (2008).</li><li>Weinberg, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+availability+and+infection.">Iron availability and infection.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1790, 600-605 (2009).</li><li>Drabkin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+the+Hemin+Chromoproteins.">Metabolism of the Hemin Chromoproteins.</a> Physiological Reviews. 31, 345-431 (1951).</li><li>Graversen, J. H., Madsen, M., Moestrup, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD163:+a+signal+receptor+scavenging+haptoglobin-hemoglobin+complexes+from+plasma.">CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma.</a> The international journal of biochemistry &amp; cell biology. 34, 309-314 (2002).</li><li>Torres, V. J., et al. <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+Fur+regulates+the+expression+of+virulence+factors+that+contribute+to+the+pathogenesis+of+pneumonia.">Staphylococcus aureus Fur regulates the expression of virulence factors that contribute to the pathogenesis of pneumonia.</a> Infect. Immun. 78, 1618-1628 (2010).</li><li>Hammer, N. D., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Staphylococcus+aureus+Iron+Acquisition.">Molecular Mechanisms of Staphylococcus aureus Iron Acquisition.</a> Annu. Rev. Microbiol. , (2011).</li><li>Hurd, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iron-regulated+surface+proteins+IsdA,+IsdB,+and+IsdH+are+not+required+for+heme+iron+utilization+in+Staphylococcus+aureus.">The iron-regulated surface proteins IsdA, IsdB, and IsdH are not required for heme iron utilization in Staphylococcus aureus.</a> Fems. Microbiology Letters. 329, 93-100 (2012).</li><li>Boys, B. L., Kuprowski, M. C., Konermann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetric+behavior+of+hemoglobin+alpha-+and+beta-+subunits+during+acid-induced+denaturation+observed+by+electrospray+mass+spectrometry.">Symmetric behavior of hemoglobin alpha- and beta- subunits during acid-induced denaturation observed by electrospray mass spectrometry.</a> Biochemistry. 46, 10675-10684 (2007).</li><li>Williams, R. C., Tsay, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+convenient+chromatographic+method+for+the+preparation+of+human+hemoglobin.">A convenient chromatographic method for the preparation of human hemoglobin.</a> Analytical Biochemistry. 54, 137-145 (1973).</li><li>Shen, T. 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O ferro é um nutriente essencial necessário por organismos de todos os reinos da vida 15. Em vertebrados, o ferro é sequestrado para evitar a toxicidade causada por este elemento. Este seqüestro também esconde ferro de micróbios invasores em um processo conhecido como imunidade nutricional 16. Em resposta, os patógenos evoluíram estratégias que burlam a imunidade nutricional. Um tal mecanismo depende de hemoglobina, que é o mais abundante fonte de ferro no interior do hospedeiro 17.<...

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> HPLC em coluna de permuta aniónica </td><td> Varian </td><td> PL1551-3802 </td><td></td></tr><tr><td> Reagente de Drabkin </td><td> Sigma </td><td> D5941-6VL </td><td></td></tr><tr><td> Padrão de hemoglobina </td><td> Pointe Científico </td><td> H7506-STD </td><td></td></tr><tr><td> RPMI </td><td> HyClone </td><td> SH30011.02 </td><td></td></tr><tr><td> Chelex forma sódica 100 </td><td> Sigma </td><td> C7901 </td><td></td></tr><tr><td> EDDHA </td><td> LGC Standards GmbH </td><td> ANC 001 </td><td></td></tr><tr><td> Anticorpo hemoglobina um </td><td> Santa Cruz Biotechnology, Inc </td><td> SC-21005 </td><td> &lt;/ Td&gt; </tr><tr><td> Agar de soja tríptica </td><td> BD </td><td> 236920 </td><td></td></tr></tbody></table>

staphylococcus aureus growth using human hemoglobin as an iron source

S. aureus é uma bactéria patogênica que requer ferro para realizar funções vitais metabólicas e causam doenças. O reservatório mais abundante de ferro no interior do hospedeiro humano é heme, que é o cofactor da hemoglobina. Para adquirir ferro da hemoglobina, S. aureus utiliza um elaborado sistema conhecido como o determinante superfície de ferro-regulado (DSI) do sistema 1. Componentes da hemoglobina Isd sistema host primeiro ligar, em seguida, extrair e importar heme, e, finalmente, libertar ferro de heme no citoplasma bacteriano 2,3. Este caminho tem sido dissecado através de numerosos estudos in vitro 4-9. Além disso, a contribuição do sistema Isd a infecção tem sido repetidamente demonstrado em modelos de ratos 8,10-14. Que institui a contribuição do sistema Isd à hemoglobina derivada de aquisição de ferro e de crescimento tem se mostrado mais desafiador. Ensaios de crescimento utilizando hemoglobina como fonte única de ferro é complicada by a instabilidade da hemoglobina disponível comercialmente, contaminando ferro livre no meio de crescimento, e a toxicidade associada com quelantes de ferro. Aqui apresentamos um método que supera estas limitações. Hemoglobina de alta qualidade é preparado a partir de sangue fresco e armazenado em azoto líquido. Hemoglobina purificada é complementada em ferro-esgotar meio imitando o ambiente pobre em ferro encontrado por agentes patogénicos no interior do hospedeiro vertebrado. Pela fome S. aureus de ferro livre e completando com uma forma minimamente manipuladas de hemoglobina que induzem o crescimento de uma maneira que é totalmente dependente da capacidade de se ligar a hemoglobina, extrair heme, heme passar através do envelope celular bacteriana e degradar heme no citoplasma. Este ensaio será útil para pesquisadores que procuram elucidar os mecanismos de aquisição de ferro em S. hemoglobin-/heme-derived aureus e, eventualmente, outros agentes patogénicos bacterianos.

Temos hemoglobina humana purificada a partir de hemolisado com HPLC (Protocolo passo 1.7). Figura 1 mostra registada a absorvância do eluato a 280 e 410 nm, comprimentos de onda. Fracção 5 foi recolhido e outras fracções foram descartados. Os rendimentos de 5-15 miligramas de hemoglobina por mililitro de fluido são normalmente adquiridas. Hemoglobina purificada foi analisada por SDS-PAGE em duplicado e os géis foram corados quer para as proteínas ou transferidas para nitrocelulose e imunotrans (...

Watch this Scientific Journal Video about Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source at JoVE.com

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Descrevemos aqui um ensaio de crescimento de<em&gt; Staphylococcus aureus</em&gt; Usando hemoglobina como a única fonte de ferro nutrientes disponíveis. Este ensaio estabelece o papel dos fatores bacterianos envolvidos na hemoglobina derivada de aquisição de ferro.

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 ...

staphylococcus-aureus-growth-using-human-hemoglobin-as-an-iron-source

Research

JoVE Journal

Immunology and Infection

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

16.4K visualizações.  Vanderbilt University Medical School. Descrevemos aqui um ensaio de crescimento de Staphylococcus aureus Usando hemoglobina como a única fonte de ferro nutrientes disponíveis. Este ensaio estabelece o papel dos fatores bacterianos envolvidos na hemoglobina derivada de aquisição de ferro.

Vídeo: Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

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 ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Relação custo-benefício Método de Rastreamento Fonte microbiana, utilizando específicas Vírus Humano e Animal

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

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 ...

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 ...

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

This protocol describes a method for the purification of polymorphonuclear leukocytes from whole human blood and two distinct assays that quantify the cytotoxicity of Staphylococcus aureus against these important innate immune cells.

Quantificando a citotoxicidade de Staphyloccus aureus contra os leukócitos polimorfóides humanos

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes ...