Este trabalho foi financiado pelos Institutos Nacionais de Saúde concede AI62983 (AYK), AI22535 (GPB).

1. Modelo murino de P. Colonização aeruginosa GI e Difusão <ol><li> Camundongos C3H/HeN (6-8 semanas de idade, do sexo feminino, Harlan) são tratadas com antibióticos orais para esgotar flora comensal e então mono-colonizados com PA como descrito anteriormente 4. </li></ol> 2. Colheita murino Cecal Conteúdo Luminal <ol><li> Mergulhe uma argamassa de aço inoxidável limpa em um banho de nitrogênio líquido. </li><li> Euthanize ratos por asfixia dióxido de carbono. </li><li> Carcaça de camundongo segura em uma placa de isopor e chuveiro com etanol a 95%. Faça uma incisão mediana longitudinal através da pele do esterno para o períneo. Refletir a pele e peritônio para expor a cavidade abdominal. </li><li> Ressecção do ceco inteiro. Segure o ceco com uma pinça sobre a argamassa de aço inoxidável. Snip ambas as extremidades do ceco com tesouras de dissecação. </li><li> Inserir uma ponteira P1000 cheia com 1 ml de tampão flushate cecal (10 mM TrisHCl, 1 mM EDTA e 200 mM NaCl) 5 na extremidade proximal do ceco. Liberar o buffer flushate cecal e conteúdo cecal luminal na argamassa de aço inoxidável. 1 ml de tampão flushate cecal será suficiente para a lavagem de todo o conteúdo cecal. Conteúdo cecal flushate irá congelar imediatamente após entrar em contato com a argamassa. </li><li> Moer o conteúdo flushate cecal com um pilão estéril. </li><li> Solo congelado lugar cecal conteúdo luminal em um tubo de polipropileno 50 ml submersa em um banho de gelo / etanol seco. </li><li> Repita os passos 2.2 a 2.7 para cada rato adicionais. </li><li> Armazenar a -80 ° C. </li></ol> 3. Isolamento de bactérias RNA <ol><li> Volume 1 quente (com base no volume final de dois cecal conteúdo luminal, cerca de 3 ml) de ácido fenol / clorofórmio (5:1, v / v) contido em um carvalho de 50 ml tubo de centrifugação cume com a tampa garantidos em parafilme em 65 · Banho de água C. </li><li> Ferva 0,5 volume de tampão de lise (2% SDS, 16 mM EDTA e 200 mM NaCl) 6 contida em um tubo de polipropileno 50 ml por 5 minutos. </li><li> Adicionar tampão de lise de ebulição para cecal conteúdo luminal. Homogeneizar. Ferva por 5 minutos com vortex periódica. </li><li> Adicione a 100 ° C conteúdo cecal / amostra de lise para a 65 ° C ácido fenol / clorofórmio no tubo de ensaio de Oak Ridge. Seal tampa com parafilme. </li><li> Incubar a 65 ° C por 10 minutos com vortex periódicas (a cada 2 minutos). </li><li> Centrifugar a 2.500 g de amostra a 4 ° por 15 minutos. </li><li> Transferir cuidadosamente fase aquosa (evitando qualquer da interface branco) para uma centrífuga de 50 ml fresco Oak Ridge. O volume da fase aquosa será de aproximadamente 50% do volume total do conteúdo cecal, tampão de lise e ácido fenol / clorofórmio. Adicionar um volume igual de ácido fenol / clorofórmio. </li><li> Seal tampa com parafilme e misturar bem em vórtice em alta velocidade. </li><li> Centrifugar a 2.500 g de amostra a 4 ° por 15 minutos. </li><li> Repita as etapas 3,7-3,9 até que não haja interface visível em branco entre as fases aquosa e orgânica. </li><li> Cuidado de transferência de fase aquosa a uma nova centrífuga 50 ml de Oak Ridge. Adicionar um volume igual de clorofórmio / álcool isoamílico (24:1, v / v). </li><li> Seal tampa e misture bem em vórtice. </li><li> Centrifugar a 2.500 g de amostra a 4 ° por 15 minutos. Remover fase aquosa (volume será de aproximadamente 50% do volume total da reacção) a 15 tubo de polipropileno ml cônico e adicionar um volume igual de isopropanol. </li><li> Incubar a -20 ° por pelo menos duas horas ou durante a noite. </li><li> Centrifugar a 2.500 g de amostra a 4 ° por 45 minutos. Um gel-like pellet formarão no fundo do tubo. Remover o sobrenadante. </li><li> Lave o pellet com 1 ml de etanol gelado 70%. Vortex. Centrífuga de amostra em menos 10.000 g a 4 ° por 5 minutos. </li><li> Remover o sobrenadante e inverter o tubo para deixar o pellet secar em temperatura ambiente por 10 minutos. </li><li> Ressuspender o sedimento em 200 mL de RNA de RNase água. </li></ol> 4. DNase Tratamento (Turbo DNA-Livre, a Applied Biosystems) <ol><li> Adicionar 20 mL de tampão 10X DNase Turbo e 2 l Turbo DNase e misture delicadamente. </li><li> Incubar a 37 ° por 20 minutos. </li><li> Adicionar 20 mL de reagente ressuspenso inativação DNase e misture bem. </li><li> Incubar 5 minutos à temperatura ambiente, mexendo ocasionalmente. </li><li> Centrifugar a 10.000 g à temperatura ambiente durante 1,5 minutos e transferir o sobrenadante para um tubo de microcentrífuga novo. </li><li> Adicionar um volume de ácido (4 º) frio de fenol / clorofórmio e misture bem em vortex por 1 minuto. </li><li> Centrifugar a 12.500 g à temperatura ambiente por 2 minutos. </li><li> Transferir a fase aquosa para um novo tubo e adicionar 1 volume de clorofórmio / álcool isoamílico (49:1 v / v), vórtice. </li><li> Centrifugar a 12.500 g à temperatura ambiente por 2 minutos. </li><li> Transferir a fase aquosa (aproximadamente 50% do volume total de reacção) para um novo tubo. </li><li> Adicionar 0,1 volume de acetato de sódio 3M, pH 5,5 e2,5 volumes de etanol gelado de 100%. Vortex. </li><li> Incubar a -20 ° por pelo menos 2 horas ou a -80 ° por 30 minutos. </li><li> Centrifugar a 12.000 g por 30 minutos a 4 ° </li><li> Remover o sobrenadante. </li><li> Lave o pellet com 1 ml de etanol gelado 70% por centrifugação por 5 minutos a 10.000 g a 4 °. </li><li> Remover o sobrenadante e secar o ar por 10 minutos. </li><li> Ressuspender o sedimento em 100 ml de RNase água. Pode precisar para colocar em banho-maria 65 ° a ressuspender e dissolver o sedimento. </li><li> Para testar a eficácia do tratamento DNase, um padrão de reação RT-PCR para a amplificação de uma proteína ribossomal (ou gene outra referência) poderia ser realizado para as amostras tratadas e não tratadas, utilizando qualquer controles RT. </li></ol> 5. RNA etapa de limpeza (Qiagen, RNeasy Kit) <ol><li> Consulte as páginas 56-57 da Qiagen RNeasy Mini Manual (4 ª Edição, Abril de 2006). Siga o caminho. </li></ol> 6. Resultados representante A quantidade de bactérias RNA total recuperado usando esse protocolo é de aproximadamente 2-3 mg de dois cecums. O RNA recuperado é de quantidade e qualidade suficientes para qPCR subseqüentes, perfis de transcrição, RNA e experimentos Seq. Pureza RNA é avaliada rotineiramente através da medição da relação de 260nm/280nm 7 da amostra, mas este método não fornece nenhuma informação sobre a integridade do RNA. O Bioanalyzer Agilent é uma plataforma microfluídica baseado para o dimensionamento, quantificação e controle de qualidade de DNA, RNA, proteínas e células, e utiliza uma métrica integridade RNA conhecido como RNA Número Integrity (RIN) 8. RNA extraído com este protocolo produz 260/280 proporções que variam 1,7-2,0 e valores RIN ≥ 7.0. Um exemplo de uma análise Bioanalyzer Agilent do RNA bacteriano recuperado por este protocolo é mostrado na Figura 1. <img src="/files/ftp_upload/3293/3293fig1.jpg" alt="Figura 1"/> Figura 1. Agilent Bioanalyzer electroferograma e gel-como a imagem de Pseudomonas aeruginosa amostra de RNA total isolado e recuperado de murino conteúdo cecal. RIN 8.0.

<ol>
	<li>Bodey, G. P., Bolivar, R., Fainstein, V., Jadeja, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infections+caused+by+Pseudomonas+aeruginosa.">Infections caused by Pseudomonas aeruginosa.</a> Rev Infect Dis. 5, 279-279 (1983).</li><li>Bertrand, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endemicity,+molecular+diversity+and+colonisation+routes+of+Pseudomonas+aeruginosa+in+intensive+care+units.">Endemicity, molecular diversity and colonisation routes of Pseudomonas aeruginosa in intensive care units.</a> Intensive Care Med. 27, 1263-1268 (2001).</li><li>Koh, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+in+vivo+transcription+profiling+for+identifying+Pseudomonas+aeruginosa+genes+needed+for+gastrointestinal+colonization+and+dissemination.">Utility of in vivo transcription profiling for identifying Pseudomonas aeruginosa genes needed for gastrointestinal colonization and dissemination.</a> PLoS One. 5, e15131-e15131 (2010).</li><li>Koh, A. Y., Priebe, G. P., Pier, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+Pseudomonas+aeruginosa+in+a+murine+model+of+gastrointestinal+colonization+and+dissemination+in+neutropenia.">Virulence of Pseudomonas aeruginosa in a murine model of gastrointestinal colonization and dissemination in neutropenia.</a> Infect Immun. 73, 2262-2272 (2005).</li><li>Alexander, R. J., Raicht, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+total+RNA+from+human+stool+samples.">Purification of total RNA from human stool samples.</a> Dig Dis Sci. 43, 2652-2658 (1998).</li><li>Fitzsimons, N. A., Akkermans, A. D., de Vos, W. M., Vaughan, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+gene+expression+detected+in+human+faeces+by+reverse+transcription-PCR.">Bacterial gene expression detected in human faeces by reverse transcription-PCR.</a> J Microbiol Methods. 55, 133-140 (2003).</li><li>Manchester, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+UV+methods+for+measurement+of+protein+and+nucleic+acid+concentrations.">Use of UV methods for measurement of protein and nucleic acid concentrations.</a> Biotechniques. 20, 968-970 (1996).</li><li>Schroeder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Mol Biol. 7, 3-3 (2006).</li><li>Turnbaugh, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+core+gut+microbiome+in+obese+and+lean+twins.">A core gut microbiome in obese and lean twins.</a> Nature. 457, 480-484 (2009).</li></ol>

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O método de extração de RNA descrita aqui permite a recuperação de quantidades suficientes de alta qualidade Pseudomonas aeruginosa RNA total colhido a partir do trato GI murino. Este método não é restrito a P. aeruginosa e pode potencialmente ser aplicada a outras bactérias. A recuperação de microrganismos suficientes a partir do intestino irá variar significativamente de organismo para organismo. Em nosso modelo murino, P. aeruginosa tipicamente coloniza o trato GI murino em níveis entre 5 x 10 7 a 5 x 10 8 ufc / g fezes 4. Uma vez que o conteúdo recuperado cecal são aproximadamente 0,5 grama, o número estimado de P. aeruginosa recuperou de duas cecums está entre 5 x 7-5 x 10 10 8 ufc. Se outros microorganismos são utilizados, seria prudente para verificar os níveis de colonização GI e depois calcular o número de cecums necessário para recuperar o número alvo de ufc. Também é importante notar que, ao usar este modelo em particular murino, tratado com antibióticos camundongos não infectados com o PA tem nenhum valor quantificável de RNA isolado de seu conteúdo cecal. Nosso modelo murino de Pseudomonas aeruginosa gastrointestinal e as tentativas de divulgação para emular a patogênese de P. aeruginosa bacteremia em pacientes de câncer e transplante de células-tronco. Nesta população de pacientes, comensais da flora é muitas vezes esgotadas secundárias ao tratamento com antibióticos ou quimioterápicos (por exemplo, o esgotamento antibiótico de GI comensais flora), resultando em crescimento de micróbios patogênicos (por exemplo, mono-associação com P. aeruginosa) e posterior divulgação após a supressão imunológica. As vantagens e limitações deste modelo murino, já foram abordados anteriormente 4. O objetivo deste estudo é fornecer uma metodologia para isolar RNA total microbiana do trato GI. Este protocolo pode ser facilmente adaptado para outros modelos murinos que estudam outros aspectos da patogênese microbiana no trato GI (ou seja, interações comensais flora, efeitos bacteriana na doença inflamatória intestinal, etc.) Uma vantagem deste método é a incorporação das etapas de lise múltiplos, incluindo congelamento / descongelamento, a perturbação mecânica (pulverização com argamassa / pilão homogeneização), fervendo, e lise química (por exemplo SDS). Apesar da multiplicidade de etapas de lise, alguns microorganismos (principalmente bactérias gram-positivas e fungos / leveduras) podem exigir a interrupção mecânica adicional. Após a lise quente / ácido fenol-clorofórmio de incubação (Passo 3.5), a adição de contas (0,1 mm para as bactérias e / ou 0,5-0,7 mm para levedura) e um passo-a bater talão subseqüentes deve ser suficiente para lisar estes 9 organismos, 10. Dada a natureza complexa de materiais recuperados a partir do ceco murino, o frio acid-phenol/chloroform repetidas extrações (Passo 3,7-3,9) são absolutamente necessários para atingir a qualidade aceitável RNA e integridade para outras reações a jusante. Entre 3-5 frio acid-phenol/chloroform extrações podem ser necessários antes que a interface branco entre as fases aquosa e orgânica é eliminada. Finalmente, a combinação de ambos o tratamento DNase (Etapa 4) e Protocolo de Limpeza RNeasy (Passo 5) são essenciais para a remoção de DNA contaminante e pequenas não mRNA (5s e tRNA). Como dito anteriormente, o RNA recuperado através da utilização deste protocolo é de quantidade e qualidade suficientes para posterior transcrição perfil 3, qPCR (não publicado), e Seq RNA (não publicado) experimentos.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> Almofariz e pilão </td><td> Pescador </td><td> 12-947-1 </td></tr><tr><td> Homogeneizador </td><td> Omni </td><td> TM-125 </td></tr><tr><td> Oak Ridge tubos de centrífuga (50 ml) </td><td> Nalgene </td><td> 3119-0050 </td></tr><tr><td> Ácido fenol / clorofórmio </td><td> Ambion </td><td> AM9720 </td></tr><tr><td> Clorofórmio </td><td> Sigma-Aldrich </td><td> C2432 </td></tr><tr><td> Álcool isoamílico </td><td> Sigma-Aldrich </td><td> W205702 </td></tr><tr><td> Isopropanol </td><td> Sigma-Aldrich </td><td> 190764 </td></tr><tr><td> Dietil pyrocarbonate (DEPC) </td><td> Sigma-Aldrich </td><td> 40718 </td></tr><tr><td> DNase </td><td> Ambion </td><td> AM2238 </td></tr><tr><td> RNeasy Kit </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> 3M acetato de sódio </td><td> Ambion </td><td> AM9740 </td></tr><tr><td> O etanol 100% </td><td> Sigma-Aldrich </td><td> E7023 </td></tr></tbody></table>

rna isolation of pseudomonas aeruginosa colonizing the murine gastrointestinal tract

Pseudomonas aeruginosa (PA) resultado infecções em significativa morbidade e mortalidade em hosts com sistemas imunológicos comprometidos, tais como doentes com leucemia, queimaduras graves, ou transplantes de órgãos 1. Em pacientes de alto risco para o desenvolvimento de infecções da corrente sanguínea PA, do trato gastrointestinal (TGI) é o principal reservatório para a colonização 2, mas os mecanismos pelos quais as transições de um micróbio PA colonização assintomática a uma invasivo, e muitas vezes mortal, patógeno não são claras. Anteriormente, foi realizada em experimentos in vivo perfis de transcrição, recuperando mRNA PA a partir de células bacterianas residentes no cecums de ratos colonizados 3, a fim de identificar mudanças na expressão gênica de bactérias durante a alterações do estado imunológico do hospedeiro. Como acontece com qualquer experiência de perfis de transcrição, o passo limitante da velocidade está no isolamento de quantidades suficientes de mRNA de alta qualidade. Dada a abundância de enzimas, detritos, resíduos de alimentos, e as partículas no trato gastrintestinal, o desafio do isolamento do RNA é assustador. Aqui, apresentamos um método para o isolamento fiável e reprodutível de RNA bacterianos recuperados do trato gastrointestinal de camundongos. Este método utiliza um modelo bem estabelecido murino de PA colonização GI e neutropenia induzida por difusão 4. Colonização, uma vez GI com PA for confirmada, os ratos são sacrificados e conteúdo cecal são recuperados e flash congelado. RNA é então extraído usando uma combinação de perturbação mecânica, ferver, fenol / clorofórmio extrações, tratamento de DNase, e cromatografia de afinidade. Quantidade e pureza são confirmadas por espectrofotometria (Nanodrop Technologies) e Bioanalyzer (Agilent Technologies) (Fig. 1). Este método de GI isolamento microbiano RNA pode ser facilmente adaptado para outras bactérias e fungos.

Watch this Scientific Journal Video about RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract at JoVE.com

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Um método confiável para o isolamento do RNA<em&gt; Pseudomonas aeruginosa</em&gt; Recuperou de cecums murino é descrito. O RNA recuperado é de quantidade e qualidade suficientes para qPCR subseqüentes, perfis de transcrição, RNA e experimentos Seq. Esta técnica pode ser adaptada para o isolamento de RNA outros micróbios intestinais.

Isolamento de RNA Pseudomonas aeruginosa Colonizar o trato gastrointestinal Murino

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

rna-isolation-pseudomonas-aeruginosa-colonizing-murine

Research

JoVE Journal

Immunology and Infection

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

19.0K visualizações.  University of Texas Southwestern Medical Center. Um método confiável para o isolamento do RNA Pseudomonas aeruginosa Recuperou de cecums murino é descrito. O RNA recuperado é de quantidade e qualidade suficientes para qPCR subseqüentes, perfis de transcrição, RNA e experimentos Seq. Esta técnica pode ser adaptada para o isolamento de RNA outros micróbios intestinais.

Vídeo: RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-cultura de Modelos Pseudomonas aeruginosa Biofilmes Grown no Live Células Airway Humanos

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Imunologia e Infecção

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa e Saccharomyces cerevisiae Biofilme em Células de Fluxo

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso de meio de escarro Artificial para testar a eficácia antibiótica contra<em&gt; Pseudomonas aeruginosa</em&gt; Em condições mais relevantes para o Pulmão Fibrose Cística

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

O isolamento de linfócitos de rato Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Crônica de Longo Prazo<em&gt; Pseudomonas aeruginosa</em&gt; Infecção das vias aéreas em ratos

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

Pseudomonas aeruginosa Lesão Pulmonar Induzida Modelo

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

Venha para o lado da luz: In Vivo monitoramento de infecções por Pseudomonas aeruginosa biofilme em feridas crônicas em um modelo murino Hairless diabética

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Replicação do requisitado, biblioteca não redundante de estirpe Pseudomonas aeruginosa PA14 Transposon mutantes de inserção

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Métodos para detecção de citotóxicos amiloides seguir infecção de pulmonar células endoteliais por Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Visualização de candida albicans no trato gastrointestinal murina usando fluorescente in situ hibridização

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...