Siamo grati a Michael Feiss e Jean Sippy per la guida sulla creazione di fago e purificazione. Ringraziamo Michael Elowitz per la fornitura del software di riconoscimento delle cellule, Schnitzcell. Il lavoro in laboratorio Golding è sostenuta da sovvenzioni dal National Institutes of Health (R01GM082837), la National Science Foundation (082.265, PFC: Centro per la Fisica delle cellule viventi), la Fondazione Welch (Grant Q-1759) e Human Frontier Science Programma (RGY 70/2008).

<ol>
	<li>Oppenheim, A. B., Kobiler, O., Stavans, J., Court, D. L., Adhya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switches+in+bacteriophage+lambda+development.">Switches in bacteriophage lambda development.</a> Annu. Rev. Genet. 39, 409-429 (2005).</li><li>Ptashne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A genetic switch : phage lambda revisited. , (2004).</li><li>Hendrix, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lambda II. , (1983).</li><li>Hershey, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Bacteriophage lambda. , (1971).</li><li>Zeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decision+making+at+a+subcellular+level+determines+the+outcome+of+bacteriophage+infection.">Decision making at a subcellular level determines the outcome of bacteriophage infection.</a> Cell. 141, 682-691 (2010).</li><li>Edgar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+infection+is+targeted+to+cellular+poles.">Bacteriophage infection is targeted to cellular poles.</a> Mol. Microbiol. , (2008).</li><li>Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current protocols in molecular biology. , (1994).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a laboratory manual. , (2001).</li><li>Fasman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical handbook of biochemistry and molecular biology. , (1989).</li><li>Kaiser, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+internal+structure+of+bacteriophage+lambda.">On the internal structure of bacteriophage lambda.</a> J. Gen. Physiol. 49, 171-178 (1966).</li><li>Kobiler, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+kinetic+analysis+of+the+bacteriophage+lambda+genetic+network.">Quantitative kinetic analysis of the bacteriophage lambda genetic network.</a> Proc Natl Acad Sci. 102, 4470-4475 (2005).</li><li>Shaner, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nat. Biotechnol. 22, 1567-1572 (2004).</li><li>Zeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Personal communication with M. Feiss. , .</li><li>Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adsorption+of+coliphage+lambda+to+its+host:+effect+of+variations+in+the+surface+density+of+receptor+and+in+phage-receptor+affinity.">The adsorption of coliphage lambda to its host: effect of variations in the surface density of receptor and in phage-receptor affinity.</a> J. Mol. Biol. 103, 521-536 (1976).</li></ol>

Nessun conflitto di interessi dichiarati.

I ceppi batterici, fagi e plasmidi: Strain LE392 è supF. E &#39;stato scelto per sopprimere la SAM7 mutazione nel genoma del fago (vedi Tabella 1 per i dettagli). Così, alla fine lysogens indotte lisi e rilasciare particelle fagiche, le cellule infettate volontà che hanno scelto la via litico. Lisogeni cellule vengono coltivate a 30 ° C a causa della presenza del termosensibile cI 857 allele nel genoma fagico. Dopo l&#39;induzione di calore, GPD-EYFP e di tipo selvatico GPD sono co-espressi dal genoma di λ LZ1 e il plasmide pPlate * D rispettivamente. Come risultato, il capside del fago λ appena creato LZ2 contiene una miscela di GPD-EYFP e proteine ​​GPD. Questo fago mosaico è strutturalmente stabile e sufficientemente fluorescente per consentire l&#39;individuazione di fagi individuali 5. pP RE - mCherry è un plasmide reporter utilizzato per rilevare scelta del pathwa lisogenay. Il RE promotore P viene attivato dal CII durante la creazione di lisogenia 1,11. pP RE - mCherry 5 è stato derivato da pE-GFP 11 sostituendo GFP con mCherry 12. Per maggiori dettagli si veda il nostro precedente lavoro 5. Condizioni di crescita Parametri: Durante l&#39;induzione lysogen (sezione 1), lieve agitazione a 180 rpm dà una resa virus buono 13. Uso di glucosio nel mezzo di crescita deve essere evitato il metabolismo del glucosio genera acidi prodotti metabolici, e particelle lambda mature sono instabili a pH acido 13. L&#39;aggiunta di MgSO4 mira a stabilizzare il fago capside 3. Per i fagi che trasportano wild type CI (invece di CI 857), il lysogen può essere indotta con il DNA-danneggiamento agente Mitomicina C 3. Nella fase 1.3, l&#39;incubazione a 37 ° C non dovrebbe di norma superare i 90 minuti. È Usef<html> ul per verificare la densità cellulare da OD 600 ogni 30 min. Per un buon lisato, OD 600 scende a circa 0,2 o meno, e il restante OD 600 è un risultato di detriti cellulari. Incubazione troppo lungo può causare una resa inferiore, poiché il fago fago appena creato può iniziare a iniettare loro DNA in detriti cellulari. Per ottenere una banda visibile fago (almeno 1 x 10 11 particelle fagiche) nei passi 2,11 e 2,13, crescono almeno 500 ml cultura nel passaggio 1,2. L&#39;aggiunta di 0,2% maltosio nel mezzo di crescita nelle fasi 5.1 e 5.2 mira ad indurre l&#39;espressione di agnello, il recettore per il fago lambda adsorbimento 3,14. Il 1000 volte diluizione invece di 100 volte al punto 5.2 è volta a ridurre il livello di fondo mCherry dal RE plasmide giornalista pP - mCherry. Nel Passaggio 5,5 iniettabile DNA fagico triggering, 35 ° C viene scelta per evitare l&#39;induzione della temperatura sensibile allele cI857. Phage Purificazione: jove_content &quot;&gt; Le fasi di depurazione dei fagi (passaggi da 2.1 a 2.11) può essere sostituito con altri 5 protocolli di purificazione, ma il finale attraverso ultracentrifugazione gradiente di equilibrio CsCl (Piazza di 2.12 e 2.13), è inevitabile. Rotori oscillanti sono necessari passi 2,10 e 2.12 per garantire taglienti bande fagiche visibili. Ottenere un titolo puro fago può facilmente richiedere fino a una settimana, per cui è necessario controllare il titolo fago lungo la strada per assicurarsi che nulla vada storto durante le fasi intermedie. Phage Manipolazione: Durante tutte le procedure di purificazione di cui alla sezione 2, è fondamentale per gestire fago lisato con delicatezza per evitare di taglio code fago da teste fagiche. Durante l&#39;infezione delle cellule nella sezione 5 (ad esempio, attraverso i passaggi 5,5 5,7), è anche fondamentale per evitare il taglio di particelle fagiche dalla cellula infettata. Si noti che se il fago viene tagliato dalla cellula infetta dopo iniezione suo DNA, il risultato è una infezione &quot;scuro&quot;, cioè inrisultato perfezione sarà osservato nell&#39;esperimento, ma il fago infettante non lo farà. Per ridurre al minimo tali problemi, si usa una punta larga pipetta ogni volta che la gestione fagi o il fago / cella miscela. DAPI di prova: La colorazione dello stock del fago con DAPI (sezione 4) è un metodo rapido ed efficace per verificare la purezza dello stock fago. Può anche essere usato per testare possibile degradazione di un magazzino fago esistente nel tempo. Per un archivio pura, la co-localizzazione di segnali YFP e DAPI al microscopio a fluorescenza dovrebbe essere prossimo al 100%. Abbiamo tipicamente osservare che meno dell&#39;1% dei punti YFP non contengono DAPI (che rappresenta capsidi senza genoma virale), che indica che queste particelle non correttamente impacchettare il DNA virale o già iniettato loro DNA altrove. Meno dell&#39;1% dei punti DAPI non contengono YFP (corrispondente a non fluorescenti fagi). Se questo non è il caso, attraverso passaggi 2,12 2,14 necessità di essere ripetuto in order per purificare nuovamente. Per quanto riguarda i parametri di imaging, la configurazione microscopio al punto 4.3 non è così critica come al punto 5 perché non a lungo termine live-cell imaging è necessario qui. Tuttavia, mantenendo le stesse impostazioni microscopia in sezione 5 è utile se si vuole calibrare l&#39;intensità di fluorescenza di una singola particella fagica. Se il PBS-agarosio lastra non è molto pulito, o troppo colorante DAPI viene utilizzato, alcuni punti DAPI corrispondenti a DNA del fago può essere circondato da un &quot;alone&quot;. Se troppo poco colorante DAPI viene utilizzato, il segnale dal canale DAPI può essere molto debole. Microscopio di sistema: Per l&#39;imaging nella sezione 6, si usa un microscopio a epifluorescenza invertito commerciale (Eclipse TE2000-E, Nikon), con un obiettivo 100x (Piano di Fluo, apertura numerica 1,40, immersione in olio) e standard di set di filtri (Nikon). La sorgente di luce di fluorescenza è una lampada ad arco con controllo di intensità luminosa. Le seguenti caratteristiche sono controllati dal computer: x, y, z Pozione, campo chiaro e fluorescenza persiane, e la scelta del filtro di fluorescenza. Una funzione di messa a fuoco automatica è necessaria. In caso contrario, la messa a fuoco può facilmente allontanarsi durante il time-lapse film (normalmente 4 ore). La capacità di acquisire più posizioni (x, y) in ogni punto è utile, in quanto permette di seguire eventi di infezione multipli in parallelo. Noi di solito acquisire 8 posizioni stadio in ogni film, in seguito fino a 100 eventi di infezione. La fotocamera che usiamo è una soluzione raffreddata 512x512 CCD da 16x16 pixel fotocamera micron con una gamma dinamica di 16 bit (Cascade512, fotometrici). Acquisizione viene eseguita utilizzando MetaMorph software (Molecular Devices). Il microscopio deve essere posto in un ambiente a temperatura controllata, in alternativa, la fase microscopio dovrebbe essere circondato da una camera a temperatura controllata. Acquisizione di immagini: Per live-cell imaging, è fondamentale per evitare inutile esposizione del campione, che potrebbe portare a candeggio e phototoxicity. Pertanto, è meglio caratterizzare il vostro primo sistema per trovare una esposizione ottimale la luce che consenta di rilevare la fluorescenza pur non portando ad una crescita cellulare eccessiva sbiancamento o inibizione. Per ottenere una buona immagine di fluorescenza, giocare con l&#39;intensità della luce eccitante, tempo di esposizione e guadagno della telecamera. Nei passaggi 6,2-6,3, l&#39;intervallo di 10 min cornice è scelta allo scopo di minimizzare l&#39;esposizione della luce. In ogni frame, un solo in-fuoco dell&#39;immagine è necessaria in contrasto di fase (per riconoscimento cellulare) e canali fluorescenti (per determinare il destino della cellula). Nel primo punto temporale, tuttavia, più z-posizione immagini attraverso il canale YFP devono catturare tutti fagi infettano sulla superficie cellulare. Il tempo di esposizione YFP nel frame iniziale può anche essere superiore a quella utilizzata per il film accelerato nei tempi successivi. Image Analysis: Attentamente contare le particelle fagiche intorno alla superficie delle cellule nella fase 7.1. Comeosservato in precedenza, si prende una serie di z-stack attraverso il canale YFP al punto 6.2. Tuttavia, questo può ancora lasciare alcune particelle fagiche fluorescenti out-of-focus, che sfida il conteggio. La lunghezza della cella nel lasso di tempo iniziale è misurata utilizzando il software Metamorph. La lunghezza della cella può essere misurata ImageJ o altri strumenti software. Inoltre, un programma automatizzato casa costruita Matlab può essere molto utile per ottenere informazioni quali il cambiamento di fluorescenza nel tempo lungo linee cellulari.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Cloroformio </td><td> Fisher Scientific </td><td> C298-500 </td></tr><tr><td> NaCl </td><td> Fisher Scientific </td><td> S271-3 </td></tr><tr><td> DNasi I </td><td> Sigma </td><td> D4527-10KU </td></tr><tr><td> RNasi </td><td> Sigma </td><td> R4642-10MG </td></tr><tr><td> PEG8000 </td><td> Fisher Scientific </td><td> BP233-1 </td></tr><tr><td> SM tampone </td><td> Teknova </td><td> S0249 </td></tr><tr><td> NZYM </td><td> Teknova </td><td> N2062 </td></tr><tr><td> CsCl </td><td> Sigma </td><td> C3011-250G </td></tr><tr><td> Siringa </td><td> Becton Dickinson </td><td> 309585 </td></tr><tr><td> Ago </td><td> Becton Dickinson </td><td> 305176 </td></tr><tr><td> Dialisi cassetta </td><td> Thermo Scientific </td><td> 66333 </td></tr><tr><td> Vetrino da microscopio </td><td> Corning </td><td> 2947-75x50 </td></tr><tr><td> Agarosio </td><td> Fisher Scientific </td><td> BP160-100 </td></tr><tr><td> SW40Ti ultra-trasparente tubo </td><td> Beckman Coulter </td><td> 344060 </td></tr><tr><td> SW60Ti ultra-trasparente tubo </td><td> Beckman Coulter </td><td> 344062 </td></tr><tr><td> SW40Ti rotore </td><td> Beckman Coulter </td><td> 331302 </td></tr><tr><td> SW60Ti rotore </td><td> Beckman Coulter </td><td> 335649 </td></tr><tr><td> Rifrattometro </td><td> Fisher Scientific </td><td> 13-947 </td></tr><tr><td> Microscopio a epifluorescenza </td><td> Nikon </td><td> Eclipse TE2000-E </td></tr></tbody></table> Tabella 2. Reagenti e apparecchiature.

following cell-fate in e. coli after infection by phage lambda

Il sistema comprendendo batteriofago (fago) lambda e del batterio E. coli si è servito a lungo come paradigma per la cella-destino 1,2 determinazione. Seguendo l&#39;infezione simultanea della cella da un numero di fagi, uno dei due percorsi è scelto: litico (virulenta) o lisogena (dormiente) 3,4. Abbiamo recentemente sviluppato un metodo per l&#39;etichettatura fluorescente fagi singoli, e sono stati in grado di esaminare la post-infezione decisionale in tempo reale al microscopio, a livello di fagi singoli e cellule 5. Qui, descriviamo la procedura completa per eseguire gli esperimenti di infezione descritti nel nostro precedente lavoro 5. Questo include la creazione di fagi fluorescenti, infezione delle cellule, l&#39;imaging al microscopio e l&#39;analisi dei dati. Il fago fluorescente è un &quot;ibrido&quot;, co-esprimono versioni wild-type e YFP-fusione della proteina del capside GPD. Un lisato fago grezzo viene prima ottenuto inducendo una lysogen del GPD-EYFP (Enhbilanciato giallo fluorescente delle proteine) fago, ospitare un plasmide che esprime di tipo selvatico GPD. Una serie di fasi di purificazione vengono poi effettuate, seguito da DAPI-etichettatura e immagini al microscopio. Questo viene fatto al fine di verificare l&#39;uniformità, confezionamento efficienza DNA, segnale di fluorescenza e la stabilità strutturale del magazzino fago. L&#39;adsorbimento iniziale di fagi a batteri viene eseguita su ghiaccio, seguito da una breve incubazione a 35 ° C per attivare l&#39;iniezione del DNA virale 6. Il fago / miscela di batteri viene poi spostato alla superficie di una lastra sottile agar nutriente, coperto con un vetrino coprioggetto e ripreso sotto un microscopio a epifluorescenza. Il post-infezione processo è seguito per 4 ore, a intervallo di 10 min. Posizioni a più stadi sono tracciati in modo tale che ~ 100 infezioni delle cellule può essere rintracciata in un singolo esperimento. Ad ogni punto di posizione e tempo, le immagini vengono acquisite in contrasto di fase e fluorescenti canali rosso e verde. Il contrasto di fase immagine è utilizzato in seguito per l&#39;assemblaggio cell riconoscimento mentre i canali fluorescenti sono usate per caratterizzare il risultato dell&#39;infezione: produzione di nuovi fagi fluorescenti (verde) seguita da lisi cellulare, o l&#39;espressione di fattori lisogenia (rosso), seguito da crescita cellulare ripresa e divisione. Gli acquisiti filmati time-lapse sono trattati con una combinazione di metodi manuali e automatizzati. I risultati delle analisi dei dati di identificazione dei parametri di infezione per ogni evento infezione (ad esempio il numero e le posizioni di infettare fagi), così come risultato l&#39;infezione (lisi / lisogenia). Ulteriori parametri possono essere estratti se desiderato.

Watch this Scientific Journal Video about Following Cell-fate in E. coli After Infection by Phage Lambda at JoVE.com

Following Cell-fate in E. coli After Infection by Phage Lambda

In questo articolo viene descritta la procedura per la preparazione di una versione fluorescente marcato del batteriofago lambda, infezione di<em&gt; E. coli</em&gt; Batteri, dopo l&#39;esito dell&#39;infezione sotto il microscopio, e l&#39;analisi dei risultati di infezione.

A seguito di Cell-destino<em&gt; E. coli</em&gt; Dopo l&#39;infezione da fago lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

following-cell-fate-in-e-coli-after-infection-by-phage-lambda

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Immunology and Infection

Following Cell-fate in E. coli After Infection by Phage Lambda

23.2K Views.  University of Illinois at Urbana-Champaign. In questo articolo viene descritta la procedura per la preparazione di una versione fluorescente marcato del batteriofago lambda, infezione di E. coli Batteri, dopo l'esito dell'infezione sotto il microscopio, e l'analisi dei risultati di infezione.

Video: Following Cell-fate in E. coli After Infection by Phage Lambda

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

Monitoraggio Traffico di cellule immunitarie fluorescente Rods prioni ore dopo l&#39;infezione intraperitoneale

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

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Immunologia e infezioni

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 ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Totale Estrazione di proteine ​​e 2-D elettroforesi su gel Metodi per<em&gt; Burkholderia</em&gt; Specie

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Scalabile High Throughput Selection Da sintetici Biblioteche Antibody fagi-visualizzato

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Non invasiva Modello di neuropatogeno<em&gt; Escherichia coli</em&gt; L&#39;infezione neonatale nel ratto

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Fenomica fagi: Approcci fisiologici per caratterizzare Novel virali Proteine

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Trasformazione di lievito probiotico e il loro recupero da gastrointestinali immunitario tessuti dopo sonda gastrica nei topi

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

Agenti patogeni di origine alimentare Screening utilizzando Magneto-fluorescente nanosensore: Rilevazione rapida di e. Coli O157: H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Rilevamento della colonizzazione di Escherichia Coli enteroemorragica in murino Host di sistema Non invasivo In Vivo bioluminescenza

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Creazione di una densa trasposone inserimento Biblioteca mediante coniugazione batterica Enterobacterial ceppi come Escherichia Coli e Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...