Os autores gostariam de agradecer à Cystic Fibrosis Foundation que forneceu financiamento para esta pesquisa e à MedImmune por sua generosa doação de anticorpos anti-Psl0096. Para este estudo, as imagens foram realizadas no centro de imagens CAMiLoD da Universidade de Toronto.

A aprovação do Conselho de Ética em Pesquisa (CEP) é necessária para coletar e armazenar amostras de escarro de seres humanos. Os estudos aqui apresentados foram aprovados pelo Hospital for Sick Children REB#1000058579.
1. Coleta de Escarro
<ol><li>Armazenar o escarro expectorado em um copo de coleta estéril e armazenar imediatamente a 4 °C por um máximo de 24 h antes da fixação. NOTA: Deixar o escarro demasiado tempo a 4 °C sem fixação pode levar à degradação celular, particularmente degradação dos glóbulos brancos. A fixação o mais rápido possível é preferível.</li>
	<li>Transferir as amostras de escarro para um tubo estéril de 15 mL.</li>
	<li>Adicionar um volume igual de paraformaldeído (PFA) a 4% às amostras de escarro. Por exemplo, se a amostra de escarro for de 0,5 mL, adicionar 0,5 mL de PFA a 4%. Misture por inversão suave.</li>
	<li>Incubar o escarro durante a noite a 4 °C.</li>
	<li>Lavar a amostra adicionando 5 mL de solução salina tamponada com fosfato (PBS) a cada 2 mL de escarro fixo. NOTA: Não é necessária centrifugação para esta etapa de lavagem.</li>
	<li>Retire cuidadosamente o sobrenadante com uma pipeta. NOTA: Evite sugar o escarro com a pipeta, apontando a ponta para longe do escarro e aspirar muito lentamente o líquido da superfície. A etapa de lavagem não envolve centrifugação para não perturbar a integridade estrutural dos tampões de escarro.</li>
	<li>Repita as etapas 1.6-1.7 mais duas vezes.</li>
	<li>Ressuspender o pellet em 2x o volume de PBS com azida sódica a 0,01 % (p/v).</li>
	<li>Conservar o escarro a 4 °C.</li>
</ol>2. MiPACT (técnica de limpeza de tecidos) processamento de escarro
<ol><li>Desgaseifique os componentes do hidrogel (30% 29:1 acrilamida:bis-acrilamida, hardner e PBS) em um recipiente selado contendo uma embalagem anaeróbia antes por 72 h. NOTA: A embalagem anaeróbia e o recipiente selado são necessários porque o oxigênio inibe a polimerização da acrilamida. Alternativamente, o oxigênio pode ser removido realizando esta etapa em um exaustor anaeróbico, aplicando um vácuo em um recipiente selado ou borbulhando gás N2 através da mistura de acrilamida.<ol><li>Adicionar 2 ml de uma solução de acrilamida:bis-acrilamida a 30% 29:1 a um tubo de 15 ml com a tampa desligada no interior do recipiente anaeróbio selado.</li>
		<li>Faça um estoque concentrado de 10% (p/v) de endurecedor adicionando 0,5 g de endurecedor a 5 mL de PBS em um tubo de 15 mL. Deixe a tampa fora do tubo e guarde no recipiente anaeróbio selado.</li>
		<li>Deixe alguns mL de PBS estéril em um tubo, com a tampa desligada, dentro do recipiente anaeróbio.</li>
	</ol></li>
	<li>Completar 5 mL de solução de hidrogel com concentração final de endurecedor a 0,2% e acrilamida:bis-acrilamida a 29:1 a 4% em PBS em tubo de 15 mL. Misture por inversão e filtre esterilizar.</li>
	<li>Retire as amostras de escarro do frigorífico e, em seguida, corte em pequenas secções (cerca de 5 mm de diâmetro) em condições estéreis com um bisturi. NOTA: Se o escarro é bastante fluido, então esta etapa ainda pode ser realizada, no entanto, é preciso mais paciência e prática para remover o escarro da solução de armazenamento com pinça antes de usar o bisturi para separar as frações desejadas.</li>
	<li>Coloque as amostras de escarro cortadas dentro de um poço de uma lâmina de vidro de cobertura de 8 câmaras.</li>
	<li>Adicionar 300 μL de solução de hidrogel esterilizada por filtro a partir do passo 2.2 a cada um dos orifícios que contêm escarro.</li>
	<li>Colocar o vidro de cobertura de 8 câmaras dentro de um recipiente selado contendo uma embalagem anaeróbia durante 3 h a 37 °C. OBS: Uma vez polimerizado o hidrogel com escarro embutido deve ser a consistência de gel firme.</li>
	<li>Transferir as amostras de escarro de hidrogel solidificado para um tubo de cultura de 15 mL contendo 5 mL de dodecil sulfato de sódio (SDS) a 8%, pH 8, e deixar as amostras limparem por 3-14 dias a 37 °C (com ou sem agitação), até que o escarro fique transparente. NOTA: Os tempos de clareamento dependem da composição do escarro e geralmente podem ser diminuídos com a agitação. No entanto, tome cuidado para não aumentar a velocidade de agitação a ponto de interromper a integridade da amostra. Mais amostras ricas em DNA levam mais tempo para limpar em comparação com amostras ricas em muco.</li>
	<li>Decantar a solução de SDS a 8% para um recipiente de recolha de resíduos. Usando pinças estéreis, transfira cada amostra embebida em hidrogel para um tubo cônico estéril de 50 mL. Adicionar 10 mL de PBS a cada um dos tubos cônicos de 50 mL para lavar os hidrogéis e deixar a solução descansar por 30 min a 1 h antes da decantação. Repita isso 2x mais vezes. NOTA: Esta etapa de lavagem não envolve centrifugação. O excesso de líquido e sobrenadante são cuidadosamente removidos com uma pipeta.</li>
	<li>Armazenar as amostras lavadas em PBS com azida sódica a 0,01% (p/v) e inibidor de RNase 1x a 4 °C.</li>
</ol>3. Protocolo de hibridização in situ fluorescente de hidrogel (FISH)
<ol><li>Retire as amostras de hidrogel de sua solução de armazenamento usando pinças estéreis e coloque as amostras em uma superfície estéril (como uma lâmina de vidro ou placa de Petri).</li>
	<li>Usando um bisturi estéril corte os hidrogéis em fatias de ~ 1 mm de espessura.</li>
	<li>Coloque as secções de 1 mm de hidrogel dentro de tubos estéreis de 1,5 ml.</li>
	<li>Preparar 1 mL de tampão de hibridização (formamida 25%, NaCl 0,9M, Tris-HCl 20 mM [pH 7,6], SDS 0,01%, H2Opurificado e deionizado) em tubo de 1,5 mL.</li>
	<li>Adicione a sonda PseaerA fluorescentemente marcada (150,7 nM12) ao tampão de hibridização e misture por inversão. NOTA: A solução de formalina deve ser mantida longe de uma chama aberta. O tampão de hibridização pode ser preparado em avançado e armazenado em alíquotas a -20 °C13.</li>
	<li>Adicione 200-500 μL de tampão de hibridização a cada seção de ~1 mm de hidrogel e certifique-se de que toda a amostra de hidrogel esteja submersa.</li>
	<li>Permitir que a sonda PseaerA hibridize com as amostras de hidrogel, colocando-as no escuro por ~ 18-24 h, a 46 °C, sem agitar.</li>
	<li>Decantar o tampão de hibridização em um recipiente de coleta de resíduos.</li>
	<li>Enxaguar as amostras uma vez com tampão de lavagem esterilizado por filtro (NaCl 337,5 mM, Tris-HCl 20 mM, EDTA (ácido etilenodiaminotetracético) 5 mM [pH 7,2], SDS 0,01% e H2O purificado e deionizado) adicionando 1 mL de tampão de lavagem a cada um dos tubos de 1,5 mL e, em seguida, removê-lo. NOTA: O tampão de lavagem pode ser feito com antecedência e armazenado à temperatura ambiente (RT).</li>
	<li>Adicionar 1 ml de tampão de lavagem fresco aos tubos e, em seguida, incubar as amostras no escuro durante 6 h a 48 °C, sem agitar.</li>
</ol>4. Ligação de anticorpos Hydrogel e Psl0096
<ol><li>Remova estérilmente o tampão de lavagem usando um pipetador de 1 mL.</li>
	<li>Enxaguar as amostras com uma solução de BSA a 2% (p/v) em PBS, adicionando e removendo 1 ml da solução de BSA a 2%.</li>
	<li>Adicionar 500 μL da solução de BSA/PBS a 2% às amostras de hidrogel para bloquear a ligação proteica não específica. Em seguida, incube as amostras durante a noite, no escuro, em RT, sem agitar.</li>
	<li>Remover estérilmente a solução de bloqueio utilizando uma pipeta de 1 ml.</li>
	<li>Preparar a solução de anticorpos Psl0096-Texas Red diluindo o anticorpo para uma concentração final de 0,112 μg/mL em 500 μL de BSA/PBS fresco a 2%.</li>
	<li>Adicionar a solução de 500 μL de anticorpos às amostras de hidrogel e incubá-las em RT por 6 h, protegidas da luz, sem agitar.</li>
</ol>5. Coloração DAPI (4′,6′-diamidino-2-fenilindol)
<ol><li>Preparar a solução de correspondência do índice de refração (RIMS) adicionando 40 g de meio de gradiente de densidade não iónico, 30 μL de Tween20, 3 μg de azida sódica e 30 ml de PBS a um balão contendo uma barra de agitação magnética. Agite a solução durante 15 minutos num agitador magnético ou até dissolver completamente.</li>
	<li>Filtrar esterilizar a solução num tubo cónico de 50 ml utilizando uma seringa de 10 ml e um filtro estéril de 0,2 μm. NOTA: A solução pode ser armazenada a 4 °C durante vários meses.</li>
	<li>Remova a solução de anticorpos Psl0096-Texas Red com uma pipeta estéril de 1 mL.</li>
	<li>Enxaguar as amostras de hidrogel adicionando e removendo 1 mL de PBS.</li>
	<li>Incubar as amostras de hidrogel com 250 μL de solução de RIMS e 10 μg/mL de DAPI em TR com agitação suave, no escuro, durante a noite.</li>
	<li>Antes da aquisição de imagens confocais, monte as amostras em câmaras de perfusão de 0,9 mm ou 1,7 mm e sele com uma lamínula de vidro. OBS: Após FISH e/ou imunohistoquímica e antes da imersão em RIMS, a coloração fluorescente de lectinas pode ser aplicada se a visualização da mucosa do escarro for desejada12.</li>
</ol>6. Exames por imagem
<ol><li>Realize imagens confocais de microscopia de varredura a laser usando técnicas padrão em aumentos de 25x, 40x, 63x ou 100x.</li>
</ol>

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O objetivo deste protocolo é permitir vislumbrar a organização in situ das células de P. aeruginosa no escarro de pacientes com FC. As amostras de escarro devem ser armazenadas a 4 °C até serem processadas, se não puderem ser imediatamente fixadas. Foi demonstrado que o número de células de P. aeruginosa no escarro não muda significativamente se processado em 1 h, 24 h ou 48 h, quando armazenado a 4 °C, embora se deixado a 25 °C por 24 ou 48 h, a contagem de células bacterianas aumentará s...

Neste estudo, experimentos foram delineados para visualizar a estrutura in vivo de Pseudomonas aeruginosa no escarro de pacientes pediátricos com fibrose cística (FC). As infecções por P. aeruginosa tornam-se crônicas em 30-40% da população pediátrica com FC; Uma vez estabelecidas, as infecções crônicas são quase impossíveis de serem eliminadas1. Isolados de P. aeruginosa de pacientes com infecção precoce são geralmente mais suscetíveis aos antimicrobianos, portanto, estes são tratados com antibióticos anti-pseudomonas para prevenir o estabelecimento de infecção crônica2. Infelizmente, nem todos os isolados de P. aeruginosa são efetivamente eliminados do pulmão após a antibioticoterapia. Os mecanismos precisos associados à falha antibiótica ainda não foram totalmente elucidados. Estudos prévios demonstraram que variações na densidade celular, agregação e produção de polissacarídeos podem afetar a eficácia dos antibióticos3. P. aeruginosa produz três polissacarídeos extracelulares: Pel, Psl ealginato 4. A maioria das cepas de P. aeruginosa tem a capacidade genética de expressar cada um dos exopolissacarídeos, embora frequentemente um tipo de polissacarídeo seja expresso predominantemente5. O alginato de exopolissacarídeo está associado a infecções crônicas no pulmão da FC, resultando em um fenótipo mucoide 6,7. Os polissacarídeos Pel e Psl têm múltiplas funções, incluindo auxiliar a fixação inicial e a manutenção da estrutura do biofilme, conferindo resistência aos antibióticos8.
Métodos com o objetivo de visualizar in vivo estruturas de tecidos têm sido desenvolvidos para uma variedade de tipos de amostras9,10,11. Mais recentemente, eles foram adaptados para visualizar comunidades microbianas in vivo dentro do escarro de pacientes com FC12. A otimização de um protocolo de limpeza tecidual específico para a identificação de comunidades microbianas dentro do escarro foi desenvolvida por DePas et al., 201612. O termo MiPACT, que significa microbial identification after Passive CLARITY technique foi cunhado para a depuração do escarro de FC11,12. Para as técnicas de limpeza de tecidos, os espécimes são primeiramente fixados, depois tornados transparentes, deixando intacta sua arquitetura inerente para coloração e visualização microscópica11. A fixação e a limpeza de amostras de escarro de FC permitem que os pesquisadores respondam a perguntas relacionadas à estrutura do biofilme, densidade celular bacteriana, associações polimicrobianas e associações entre patógenos e células hospedeiras. A vantagem de examinar diretamente as bactérias que foram preservadas no escarro é que elas podem ser analisadas e visualizadas em um contexto específico do hospedeiro. Embora o crescimento in vitro de isolados clínicos em laboratório para experimentação possa ser muito informativo, tais métodos são incapazes de recriar completamente o ambiente pulmonar da FC, resultando em uma desconexão entre os resultados laboratoriais e os desfechos dos pacientes.
Os métodos aqui apresentados podem ser usados para fixar e limpar o escarro para visualizar bactérias, seja de pacientes com FC ou de pacientes com outras infecções respiratórias. O tipo específico de coloração e análise microscópica aqui descrita é a hibridização in situ fluorescente (FISH), seguida pela ligação de anticorpos anti-PSL dentro do hidrogel e subsequente análise via microscopia confocal de varredura a laser (CLSM). Após a limpeza tecidual, outros métodos de imunohistoquímica e microscopia também podem ser aplicados.

<table>
	<tbody>
		<tr>
			<td>29:1 acrylamide bisacrylamide, 30 % solution</td>
			<td>BioRad</td>
			<td>161-0146</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Chambered Coverglass Nunc Lab-Tek</td>
			<td>ThermoFischer Scientific</td>
			<td>155411</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaerogen2.5L</td>
			<td>Oxid Inc.</td>
			<td>35108</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverwell perfusion chambers</td>
			<td>Electron Microscopry Sciences</td>
			<td>70326 -12/-14</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoDenz</td>
			<td>Sigma</td>
			<td>D2158</td>
			<td></td>
		</tr>
		<tr>
			<td>Protect RNA Rnase Inhibitor</td>
			<td>Sigma</td>
			<td>R7387</td>
			<td></td>
		</tr>
		<tr>
			<td>PseaerA - GGTAACCGTCCCCCTTGC</td>
			<td>Eurofins</td>
			<td></td>
			<td>Order Details: Product: Modified DNA Oligo; Name: PseaerA; Sequence: [Alexa488]GGTAACCGTCCCCCTTGC; Synthesis: 50 nmol; Purification: HPLC; Ship state: Full yield (dry)</td>
		</tr>
		<tr>
			<td>Psl0096-Texas Red</td>
			<td>Medimmune</td>
			<td></td>
			<td>The Psl0096-Texas red antibodies were a gift kindly provided by Medimmune and the company should be contacted for order inquiries.</td>
		</tr>
		<tr>
			<td>VA-044 Hardener</td>
			<td>Wako</td>
			<td>27776-21-21</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of pseudomonas aeruginosa within the sputum of cystic fibrosis patients

A detecção precoce e a erradicação de Pseudomonas aeruginosa nos pulmões de pacientes com fibrose cística podem reduzir a chance de desenvolver infecção crônica. O desenvolvimento de infecções crônicas por P. aeruginosa está associado a um declínio na função pulmonar e aumento da morbidade. Portanto, há um grande interesse em elucidar as razões para a falha na erradicação da P. aeruginosa com antibioticoterapia, que ocorre em aproximadamente 10-40% dos pacientes pediátricos. Um dos muitos fatores que podem afetar a depuração de P. aeruginosa e a suscetibilidade a antibióticos pelo hospedeiro são variações na organização espacial (como agregação ou formação de biofilme) e produção de polissacarídeos. Portanto, estávamos interessados em visualizar as características in situ da P. aeruginosa no escarro de pacientes com FC. Uma técnica de limpeza tecidual foi aplicada às amostras de escarro após a incorporação das amostras em uma matriz de hidrogel para reter as estruturas 3D em relação às células hospedeiras. Após a limpeza do tecido, etiquetas fluorescentes e corantes foram adicionados para permitir a visualização. Hibridização in situ fluorescente foi realizada para a visualização das células bacterianas, ligação de anticorpos anti-Psl marcados fluorescentemente para a visualização do exopolissacarídeo e coloração DAPI para corar células hospedeiras para obter insight estrutural. Esses métodos permitiram a obtenção de imagens de alta resolução da P. aeruginosa no escarro de pacientes com FC por meio de microscopia confocal de varredura a laser.

O planejamento geral do experimento está resumido na Figura 1 e na Figura 2. A Figura 1 apresenta um resumo dos protocolos de processamento e desobstrução de escarro. O processamento e a depuração do escarro podem levar até 17 dias. No entanto, o protocolo pode ser interrompido, e as amostras podem ser armazenadas após a fixação com PFA (dia 2) ou após a limpeza do tecido (dias 5-17, dependendo do tempo de clareamento). Na...

Watch this Scientific Journal Video about Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients at JoVE.com

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Este protocolo fornece métodos para visualização de células bacterianas e polissacarídeos do lócus de síntese de polissacarídeos (Psl) no escarro de pacientes com fibrose cística.

Visualização de Pseudomonas aeruginosa no Escarro de Pacientes com Fibrose Cística

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

visualization-pseudomonas-aeruginosa-within-sputum-cystic-fibrosis

Research

JoVE Journal

Immunology and Infection

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

2.9K visualizações.  Hospital for Sick Children. Este protocolo fornece métodos para visualização de células bacterianas e polissacarídeos do lócus de síntese de polissacarídeos (Psl) no escarro de pacientes com fibrose cística.

Vídeo: Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

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 ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

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 ...

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 ...

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. ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Um modelo de mouse transiente Transientized IL-8 para o<em&gt; In Vivo</em&gt; Monitoramento de Longo Prazo de Respostas Inflamatórias

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

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 ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Cultura de Variante de Pequena Colônia de Pseudomonas aeruginosa e Quantitação de seu Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

Visualização de Pseudomonas aeruginosa no Escarro de Pacientes com Fibrose Cística

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