Questo lavoro è stato finanziato dal National Institutes of Health concede AI62983 (ayk), AI22535 (GPB).

1. Modello murino di P. aeruginosa GI colonizzazione e diffusione <ol><li> Topi C3H/HeN (6-8 settimane vecchio, femmina, Harlan) sono trattati con antibiotici per via orale ad esaurire flora commensale e poi mono-colonizzati da PA come descritto in precedenza 4. </li></ol> 2. La raccolta murini contenuto del cieco Luminal <ol><li> Immergere un mortaio pulito in acciaio inox in una vasca di azoto liquido. </li><li> Euthanize topi per asfissia di anidride carbonica. </li><li> Topo carcassa sicuro su una tavola di polistirolo e doccia con etanolo al 95%. Fai una incisione mediana longitudinale attraverso la pelle dallo sterno al perineo. Riflettono la pelle e peritoneo per esporre la cavità addominale. </li><li> Resecare il cieco intero. Tenere il cieco con una pinza sopra la malta in acciaio inox. Snip entrambe le estremità del cieco con le forbici dissezione. </li><li> Inserire una punta P1000 pipetta riempita con 1 ml di tampone flushate del cieco (10 mM TrisHCl, 1 mM EDTA, 200 mM NaCl) 5 nelle estremità prossimale del cieco. Svuotare il buffer del cieco flushate e del cieco contenuto luminale nel mortaio in acciaio inox. 1 ml di tampone flushate del cieco sarà sufficiente per il lavaggio di tutto il contenuto del cieco. Cecale contenuti flushate si blocca subito dopo entrare in contatto con la malta. </li><li> Macinare il contenuto flushate del cieco con un pestello sterile. </li><li> Terreno posto ghiacciato del cieco contenuto luminale in una provetta 50 ml conica in polipropilene immerso in un ghiaccio secco / etanolo bagno. </li><li> Ripetere i punti 2,2 per 2,7 per ogni mouse aggiuntivo. </li><li> Conservare a -80 ° C. </li></ol> 3. Batteriche RNA Isolation <ol><li> Caldo 1 volume (in base al volume finale di due contenuti del cieco del lume, circa 3 ml) di acido fenolo / cloroformio (5:1, v / v) contenuta in un tubo 50 ml per centrifuga Oak Ridge con il tappo fissato in parafilm in 65 ° C acqua sporca. </li><li> Fate bollire 0,5 volume di tampone di lisi (2% SDS, 16 mM EDTA, 200 mM NaCl) 6 contenuta in un tubo in polipropilene da 50 ml conica per 5 minuti. </li><li> Aggiungi tampone bollente lisi del cieco a contenuto luminale. Omogeneizzare. Fate bollire per 5 minuti con vortex periodica. </li><li> Aggiungere i 100 ° C il contenuto del cieco / campione lisi ai 65 ° C acido fenolo / cloroformio nella provetta da centrifuga Oak Ridge. Con tappo a tenuta con parafilm. </li><li> Incubare a 65 ° C per 10 minuti con vortex periodico (ogni 2 minuti). </li><li> Centrifugare campione a 2.500 g a 4 ° per 15 minuti. </li><li> Con attenzione trasferimento fase acquosa (evitando qualsiasi dell&#39;interfaccia bianco) per un nuovo 50 ml centrifuga Oak Ridge. Il volume della fase acquosa sarà circa il 50% del volume totale dei contenuti del cieco, tampone di lisi, e acido fenolo / cloroformio. Aggiungere un volume equivalente di acido fenolo / cloroformio. </li><li> Con tappo a tenuta con parafilm e mescolare bene nel vortex ad alta velocità. </li><li> Centrifugare campione a 2.500 g a 4 ° per 15 minuti. </li><li> Ripetere i passaggi 3,7-3,9 finché non ci sarà alcuna interfaccia visibile bianco tra le fasi acquosa e organica. </li><li> Con attenzione trasferimento fase acquosa in una nuova centrifuga 50 ml Oak Ridge. Aggiungere un uguale volume di cloroformio / alcool isoamilico (24:1, v / v). </li><li> Guarnizione tappo e mescolare bene nel vortex. </li><li> Centrifugare campione a 2.500 g a 4 ° per 15 minuti. Rimuovere fase acquosa (volume sarà circa il 50% del volume totale di reazione) a 15 tubo di polipropilene ml e aggiungere un uguale volume di isopropanolo. </li><li> Incubare a -20 ° per almeno 2 ore o durante la notte. </li><li> Centrifugare campione a 2.500 g a 4 ° per 45 minuti. Un gel-like pellet si forma sul fondo della provetta. Rimuovere il surnatante. </li><li> Lavare il pellet con 1 ml di ghiacciata etanolo al 70%. Vortex. Centrifugare a campione a 10.000 g a 4 ° per 5 minuti. </li><li> Rimuovere il supernatante e invertire il tubo per far asciugare il pellet a temperatura ambiente per 10 minuti. </li><li> Risospendere il pellet di RNA in 200 ml di acqua RNase-free. </li></ol> 4. DNasi Trattamento (Turbo DNA-Free, Applied Biosystems) <ol><li> Aggiungere 20 ml di tampone 10X Turbo DNasi e 2 microlitri Turbo DNasi e mescolare delicatamente. </li><li> Incubare a 37 ° per 20 minuti. </li><li> Aggiungere 20 ml di reagente risospeso inattivazione DNasi e mescolare bene. </li><li> Incubare 5 minuti a temperatura ambiente, mescolando di tanto in tanto. </li><li> Centrifugare a 10.000 g a temperatura ambiente per 1,5 minuti e trasferire il surnatante in una provetta da microcentrifuga nuovo. </li><li> Aggiungere un volume di freddo (4 °) L&#39;acido fenolo / cloroformio e mescolare accuratamente vortexando per 1 minuto. </li><li> Centrifugare a 12.500 g a temperatura ambiente per 2 minuti. </li><li> Trasferire la fase acquosa in una nuova provetta e aggiungere 1 volume di cloroformio / alcool isoamilico (49:1 v / v), vortice. </li><li> Centrifugare a 12.500 g a temperatura ambiente per 2 minuti. </li><li> Trasferire la fase acquosa (circa il 50% del volume totale di reazione) in una nuova provetta. </li><li> Aggiungere 0,1 volumi di acetato di sodio 3M pH 5.5 e2,5 volumi di ghiaccio freddo etanolo al 100%. Vortex. </li><li> Incubare a -20 ° per almeno 2 ore oa -80 ° per 30 minuti. </li><li> Centrifugare a 12.000 g per 30 minuti a 4 ° </li><li> Rimuovere il surnatante. </li><li> Lavare il pellet con 1 ml di ghiacciata etanolo al 70% per centrifugazione per 5 minuti a 10.000 g a 4 °. </li><li> Rimuovere il surnatante e l&#39;aria secca per 10 minuti. </li><li> Risospendere il pellet in 100 ml di acqua RNase-free. Potrebbe essere necessario posto nel 65 ° di durezza bagno per risospendere e sciogliere il precipitato. </li><li> Per testare l&#39;efficacia del trattamento DNasi, una RT-PCR, reazione standard per l&#39;amplificazione di una proteina ribosomiale (o gene altro riferimento) potrebbe essere eseguito per campioni trattati e non trattati, con nessun controllo RT. </li></ol> 5. RNA sottofase di cancellazione (Qiagen, RNeasy Kit) <ol><li> Fare riferimento alle pagine 56-57 della Qiagen RNeasy Mini Handbook (4 ª edizione, aprile 2006). Seguite le indicazioni fornite. </li></ol> 6. Rappresentante Risultati La quantità di RNA batterico totale recuperati utilizzando questo protocollo è di circa 2-3 mg da due cecums. L&#39;RNA recuperato è di quantità e qualità sufficienti per qPCR successivi, profili di trascrizione, e l&#39;RNA esperimenti Seq. Purezza RNA è normalmente valutata misurando il rapporto 260nm/280nm 7 del campione, ma questo metodo non fornisce alcuna informazione circa l&#39;integrità di RNA. Il Bioanalyzer Agilent è una piattaforma basata su microfluidica per il dimensionamento, la quantificazione e il controllo della qualità del DNA, RNA, proteine ​​e cellule, e utilizza una metrica integrità RNA conosciuta come numero di integrità dell&#39;RNA (RIN) 8. RNA estratto con questo protocollo produce 260/280 rapporti che vanno dal 1,7-2,0 e valori RIN ≥ 7.0. Un esempio di analisi Agilent Bioanalyzer dell&#39;RNA batterico recuperato da questo protocollo è illustrato nella figura 1. <img src="/files/ftp_upload/3293/3293fig1.jpg" alt="Figura 1"/> Figura 1. Agilent Bioanalyzer elettroferogramma e gel-come immagine di Pseudomonas aeruginosa totale del campione di RNA isolati e recuperati da topo contenuti del cieco. 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>

Nessun conflitto di interessi dichiarati.

Il metodo di estrazione di RNA qui descritto consente il recupero di una quantità sufficiente di alta qualità RNA totale Pseudomonas aeruginosa raccolte dal tratto GI murino. Questo metodo non si limita a P. aeruginosa e possono potenzialmente essere applicato ad altri batteri. Il recupero di organismi microbici sufficiente da parte dell&#39;intestino varia significativamente da un organismo all&#39;altro. Nel nostro modello murino, P. aeruginosa tipicamente colonizza il tratto gastrointestinale murino a livelli tra 5 x 10 7 a 5 x 10 8 CFU / g feci 4. Dal momento che il contenuto recuperato del cieco sono circa 0,5 grammi, il numero stimato di P. aeruginosa recuperato da due cecums è compresa tra 5 x 10 7 a 5 x 10 8 cfu. Se altri microrganismi vengono utilizzati, sarebbe prudente per verificare i livelli di colonizzazione GI e quindi calcolare il numero di cecums necessari per recuperare il numero mirato di cfu. E &#39;anche importante notare che quando si utilizza questo particolare modello murino, topi trattati con antibiotico non infettati con PA non quantificabile quantità di RNA isolati dal loro contenuto cecale. Il nostro modello murino di colonizzazione Pseudomonas aeruginosa gastrointestinale e tentativi di diffusione di emulare la patogenesi di P. aeruginosa batteriemia nel cancro e pazienti sottoposti a trapianto di cellule staminali. In questa popolazione di pazienti, la flora commensale è spesso impoverito secondaria al trattamento con antibiotici o chemioterapici (ad esempio l&#39;esaurimento antibiotico di GI flora commensale) con conseguente proliferazione di microbi patogeni (ad esempio mono-associazione con P. aeruginosa) e successiva diffusione, dopo la soppressione immunitaria. I vantaggi ei limiti di questo modello murino sono già stati affrontati in precedenza 4. Lo scopo di questo studio è di fornire una metodologia per isolare microbica RNA totale dal tratto GI. Questo protocollo può essere facilmente adattato ad altri modelli murini che lo studio di altri aspetti della patogenesi microbica nel tratto gastrointestinale (per esempio la flora commensale interazioni, effetti batterica sulle malattie infiammatorie intestinali, ecc.) Un vantaggio di questo metodo è l&#39;incorporazione di passi lisi multiple compreso gelo / disgelo, distruzione meccanica (polverizzando con malta / pestello, omogeneizzazione), bollitura, e lisi chimica (per esempio, SDS). Nonostante la moltitudine di passi lisi, alcuni microrganismi (in particolare batteri Gram-positivi e funghi / lievito) possono richiedere ulteriori rottura meccanica. Dopo il caldo lisi / acido fenolo-cloroformio incubazione (Step 3.5), l&#39;aggiunta di perline (0,1 mm per i batteri e / o di 0,5-0,7 mm per lievito) e una successiva perline battendo passo dovrebbe essere sufficiente per lisare questi organismi 9, 10. Data la natura complessa del materiale recuperato dal topo cieco, le ripetute freddo estrazioni acid-phenol/chloroform (Passo 3,7-3,9) sono assolutamente necessari per ottenere qualità accettabile RNA e l&#39;integrità per ulteriori reazioni a valle. Tra i 3-5 freddo estrazioni acid-phenol/chloroform può essere richiesto prima che l&#39;interfaccia bianca tra le fasi acquosa e organica è eliminata. Infine, la combinazione di entrambi i trattamenti DNasi (punto 4) e il protocollo di pulizia RNeasy (Fase 5) sono essenziali per la rimozione del DNA contaminante e piccoli non-mRNA (5s e tRNA). Come affermato in precedenza, l&#39;RNA recuperato utilizzando questo protocollo è di quantità e qualità sufficienti per il profiling di trascrizione successivi 3, qPCR (inedito), e RNA Seq (inedito) esperimenti.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Mortaio e pestello </td><td> Pescatore </td><td> 12-947-1 </td></tr><tr><td> Omogeneizzatore </td><td> Omni </td><td> TM-125 </td></tr><tr><td> Oak Ridge provette (50 ml) </td><td> Nalgene </td><td> 3119-0050 </td></tr><tr><td> Acido fenolo / cloroformio </td><td> Ambion </td><td> AM9720 </td></tr><tr><td> Cloroformio </td><td> Sigma-Aldrich </td><td> C2432 </td></tr><tr><td> L&#39;alcol isoamilico </td><td> Sigma-Aldrich </td><td> W205702 </td></tr><tr><td> Isopropanolo </td><td> Sigma-Aldrich </td><td> 190764 </td></tr><tr><td> Dietil pirocarbonato (DEPC) </td><td> Sigma-Aldrich </td><td> 40718 </td></tr><tr><td> DNasi </td><td> Ambion </td><td> AM2238 </td></tr><tr><td> RNeasy Kit </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> 3M sodio acetato </td><td> Ambion </td><td> AM9740 </td></tr><tr><td> 100% di etanolo </td><td> Sigma-Aldrich </td><td> E7023 </td></tr></tbody></table>

rna isolation of pseudomonas aeruginosa colonizing the murine gastrointestinal tract

Pseudomonas aeruginosa (PA) causa infezioni in significativa morbidità e mortalità in host con sistema immunitario compromesso, come i pazienti affetti da leucemia, gravi ustioni, o trapianti di organi 1. Nei pazienti ad alto rischio di sviluppare infezioni del sangue PA, il tratto gastrointestinale (GI) è il principale serbatoio per la colonizzazione 2, ma i meccanismi con cui transizioni PA da un microbo ad una colonizzazione asintomatica invasiva, e spesso mortale, patogeno non sono chiare. In precedenza, abbiamo effettuato esperimenti in vivo profilazione trascrizione mRNA PA attraverso il recupero di cellule batteriche residenti nel cecums di topi colonizzati 3, per identificare i cambiamenti nell&#39;espressione genica batterica durante alterazioni dello stato immunitario dell&#39;ospite. Come con qualsiasi esperimento profilazione trascrizione, il fattore limitante è l&#39;isolamento di una sufficiente quantità di mRNA di alta qualità. Data l&#39;abbondanza di enzimi, detriti, residui di cibo, e particolato nel tratto gastrointestinale, la sfida di isolamento dell&#39;RNA è scoraggiante. Qui, vi presentiamo un metodo per l&#39;isolamento affidabile e riproducibile di RNA batterico recuperato dal tratto GI murino. Questo metodo utilizza un consolidato modello murino di colonizzazione PA GI e neutropenia indotta dalla diffusione 4. Colonizzazione volta GI con PA è confermata, i topi sono contenuti eutanasia e del cieco vengono recuperati e flash congelati. L&#39;RNA viene poi estratta usando una combinazione di distruzione meccanica, bollitura, fenolo / cloroformio estrazioni, trattamento DNase, e cromatografia di affinità. Quantità e la purezza sono confermati da spettrofotometria (Tecnologie NanoDrop) e Bioanalyzer (Agilent Technologies) (Fig. 1). Questo metodo di isolamento microbico GI RNA può essere facilmente adattato ad altri batteri e funghi, come pure.

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

Un metodo affidabile per l&#39;isolamento di RNA<em&gt; Pseudomonas aeruginosa</em&gt; Recuperato da cecums murino è descritto. L&#39;RNA recuperato è di quantità e qualità sufficienti per qPCR successivi, profili di trascrizione, e l&#39;RNA esperimenti Seq. Questa tecnica può essere adattata per l&#39;isolamento di RNA di altri microbi intestinali.

Isolamento del RNA Pseudomonas aeruginosa Colonizzare la Murine tratto gastrointestinale

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 Views.  University of Texas Southwestern Medical Center. Un metodo affidabile per l'isolamento di RNA Pseudomonas aeruginosa Recuperato da cecums murino è descritto. L'RNA recuperato è di quantità e qualità sufficienti per qPCR successivi, profili di trascrizione, e l'RNA esperimenti Seq. Questa tecnica può essere adattata per l'isolamento di RNA di altri microbi intestinali.

Video: 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-coltura Modelli di Pseudomonas aeruginosa Biofilm Cresciuto in diretta cellule delle vie aeree umane

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

Ricerca

Immunologia e infezioni

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 biofilm Saccharomyces cerevisiae in celle di flusso

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 di un terreno dell&#39;espettorato artificiale per testare l&#39;efficacia degli antibiotici contro<em&gt; Pseudomonas aeruginosa</em&gt; In condizioni più rilevanti al polmone Fibrosi Cistica

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

Isolamento dei linfociti da mouse mucosa del tratto genitale

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.

Long Term cronica<em&gt; Pseudomonas aeruginosa</em&gt; Infezione delle vie aeree nei topi

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 Induced Lung Injury Modello

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.

Vieni nel lato di luce: In Vivo monitoraggio di Pseudomonas aeruginosa Biofilm infezioni nelle ferite croniche in un modello murino Hairless diabetico

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.

Replica di ordinato, Nonredundant biblioteca del ceppo di Pseudomonas aeruginosa PA14 trasposone inserimento mutanti

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.

Metodi per rilevare amiloidi seguente infezione di polmonare endoteliali cellule citotossiche da 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.

Visualizzazione degli albicani Candida nel tratto gastrointestinale Murine utilizzando l'ibridazione fluorescente in Situ

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 ...