Nous sommes reconnaissants à Michael Feiss et Jean Sippy pour l&#39;orientation sur la création et la purification de phages. Nous remercions Michael Elowitz pour fournir le logiciel de reconnaissance cellulaire, Schnitzcell. Travail dans le laboratoire Golding est pris en charge par des subventions du National Institutes of Health (R01GM082837), la National Science Foundation (082.265, PFC: Centre de physique des cellules vivantes), la Fondation Welch (Grant Q-1759) et Human Frontier Science Programme (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>

Aucun conflit d&#39;intérêt déclaré.

Souches bactériennes, plasmides et de phages: Souche LE392 est supF. Il a été choisi de supprimer la mutation Sam7 dans le génome du phage (voir le tableau 1 pour plus de détails). Ainsi, lysogènes induits finira lyser et libérer des particules de phages, comme les cellules infectées volonté qui ont choisi la voie lytique. Lysogènes cellules sont cultivées à 30 ° C en raison de la présence de la température cI 857 sensible allèle dans le génome du phage. Après l&#39;induction de chaleur, Gpd-EYFP et de type sauvage Gpd sont co-exprimés à partir du génome de λ LZ1 et le plasmide pPlate * D respectivement. En conséquence, la capside du phage nouvellement créé LZ2 λ contient un mélange de Gpd-EYFP et protéines GPD. Ce phage mosaïque est structurellement stable et suffisamment pour permettre la détection fluorescente de phages individuels 5. pP RE - mCherry est un plasmide rapporteur utilisé pour détecter choix de la pathwa lysogèney. Le promoteur P RE est activée par CII lors de l&#39;établissement de la lysogénie 1,11. pP RE - mCherry 5 a été dérivé de pE-gfp 11 par le remplacement gfp avec mCherry 12. Pour plus de détails, voir nos travaux antérieurs 5. Paramètres Condition de croissance: Pendant l&#39;induction lysogène (section 1), tremblements légers à 180 rpm donne un rendement bon virus 13. L&#39;utilisation de glucose dans le milieu de croissance doit être évitée car le métabolisme du glucose génère acides produits métaboliques, et les particules lambda matures sont instables à pH acide 13. L&#39;addition de MgSO 4 vise à stabiliser la capside de phage 3. Pour phages porteurs de type sauvage cI (au lieu de cI 857), le lysogène peut être induite en utilisant le C altération de l&#39;ADN mitomycine agent 3. À l&#39;étape 1.3, l&#39;incubation à 37 ° C ne devrait normalement pas dépasser 90 minutes. Il est usef<html> ul pour vérifier la densité cellulaire par DO 600 toutes les 30 min. Pour un bon lysat, DO 600 tombe à environ 0,2 ou moins, et le reste de OD 600 est le résultat de débris cellulaires. Incubation trop long peut entraîner une baisse du rendement depuis le phage phage nouvellement créée peut commencer à injecter leur ADN dans les débris cellulaires. Pour obtenir une bande de phage visible (au moins 1 x 10 11 particules de phages) aux étapes 2.11 et 2.13, croître d&#39;au moins 500 ml de culture à l&#39;étape 1.2. L&#39;ajout de 0,2% de maltose dans le milieu de croissance dans les étapes 5.1 et 5.2 vise à induire l&#39;expression de l&#39;agneau, le récepteur de phage lambda adsorption 3,14. La dilution de 1000 fois au lieu de 100 fois dans l&#39;étape 5.2 vise à réduire le niveau de fond mCherry du journaliste pP RE plasmide - mCherry. À l&#39;étape d&#39;injection de 5,5 pour déclencher l&#39;ADN du phage, 35 ° C est choisie pour éviter l&#39;induction de l&#39;allèle sensible à la température cI857. Purification phage: jove_content &quot;&gt; Les étapes de purification de phages (étapes 2.1 à 2.11) peut être remplacé par d&#39;autres protocoles de purification 5, mais l&#39;ultracentrifugation finale par gradient de CsCl équilibre (étapes 2.12 et 2.13) est inévitable. rotors à godets oscillants sont nécessaires aux étapes 2.10 et 2.12 à assurer pointus bandes de phages visibles. Obtention d&#39;un stock de phages pur peut facilement prendre jusqu&#39;à une semaine, il est donc nécessaire de vérifier le titre du phage en cours de route pour s&#39;assurer que rien se passe mal pendant les étapes intermédiaires. Manipulation phage: Pendant toutes les procédures de purification dans la section 2, il est essentiel de traiter le lysat de phage doucement pour éviter le cisaillement des queues des têtes de phages phages. Au cours de l&#39;infection des cellules dans la section 5 (par exemple, les étapes 5.5 à 5.7), il est également essentiel pour éviter le cisaillement des particules de phage de la cellule infectée. Notez que si le phage est cisaillé à partir de la cellule infectée après l&#39;injection de l&#39;ADN, le résultat est un &quot;sombre&quot; infection, à savoir l&#39;enfection résultat sera observé dans l&#39;expérience, mais le phage infectant le seront pas. Pour minimiser ces problèmes, nous utilisons un embout de pipette large lors de la manipulation ou le mélange des phages phages / cellules. DAPI essai: La coloration du stock de phages avec du DAPI (section 4) est une méthode rapide et efficace pour vérifier la pureté du stock de phage. Il peut également être utilisé pour tester une possible dégradation d&#39;un stock de phages existant au fil du temps. Pour une pure souche, la co-localisation des signaux YFP et DAPI au microscope à fluorescence devrait être proche de 100%. On observe généralement que moins de 1% des spots YFP ne contiennent DAPI (représentant capsides, sans le génome viral), ce qui indique que ces particules n&#39;ont pas réussi à emballer l&#39;ADN viral, ou déjà injecté leur ADN ailleurs. Moins de 1% des spots ne contiennent DAPI YFP (correspondant à la non-fluorescents phages). Si ce n&#39;est pas le cas, les étapes 2,12 par 2,14 besoin d&#39;être répété dans oOMMANDE à purifier à nouveau. En ce qui concerne les paramètres d&#39;imagerie, la configuration de microscope à l&#39;étape 4.3 est pas aussi critique que dans la section 5, car aucun long terme imagerie des cellules vivantes est nécessaire ici. Toutefois, en gardant les paramètres de microscopie mêmes que dans la section 5 est utile si l&#39;on souhaite calibrer l&#39;intensité de fluorescence d&#39;une seule particule de phage. Si la dalle de PBS-agarose n&#39;est pas très propre, ou trop DAPI colorant est utilisé, quelques taches DAPI correspondant à l&#39;ADN de phage peuvent être entourés d&#39;un «halo». Si trop peu de colorant DAPI est utilisé, le signal provenant du canal DAPI peut être très faible. Système de microscope: Pour l&#39;imagerie dans la section 6, nous utilisons un microscope à épifluorescence commerciale inversée (Eclipse TE2000-E, Nikon) avec un objectif 100x (Plan Fluo, ouverture numérique de 1,40, immersion dans l&#39;huile) et standards des jeux de filtres (Nikon). La source lumineuse est une lampe à fluorescence avec Arc de commande de l&#39;intensité lumineuse. Les fonctions suivantes sont contrôlés par ordinateur: x, y et z potion; champ clair et volets fluorescence et de fluorescence choix de filtre. Une fonction d&#39;auto-focus est nécessaire. Dans le cas contraire, l&#39;accent peut facilement dériver pendant le film time-lapse (normalement 4 heures). La capacité d&#39;acquérir de multiples positions (x, y) à chaque point est utile, car elle permet de suivre l&#39;actualité des infections multiples en parallèle. En général, nous acquérir 8 positions de scène dans chaque film, à la suite jusqu&#39;à 100 événements d&#39;infection. La caméra que nous utilisons est une solution refroidie 512x512 CCD 16x16 pixel appareil photo um avec une plage dynamique de 16 bits (Cascade512, Photometrics). L&#39;acquisition est réalisée à l&#39;aide du logiciel MetaMorph (Molecular Devices). Le microscope doit être placé dans une chambre à température contrôlée, en alternative, la platine du microscope doit être entourée par une chambre à température contrôlée. Acquisition d&#39;image: Pour imagerie des cellules vivantes, il est essentiel d&#39;éviter toute exposition inutile de l&#39;échantillon, ce qui pourrait conduire à la décoloration et phototoxicity. Par conséquent, il est préférable de commencer par caractériser le système pour trouver une exposition optimale de la lumière qui permet la détection de fluorescence tout en ne conduisant pas à une croissance cellulaire excessive de blanchiment ou d&#39;inhibition. Pour obtenir une bonne image de fluorescence, jouer avec l&#39;intensité lumineuse d&#39;excitation, le temps d&#39;exposition et de gain de la caméra. Dans les étapes 6.2 à 6.3, l&#39;intervalle de 10 min est choisie de cadre dans le but de réduire l&#39;exposition de la lumière. Dans chaque image, un seul de mise au point d&#39;image est nécessaire à contraste de phase (pour la reconnaissance des cellules) et les canaux fluorescents (pour déterminer le devenir des cellules). Dans le premier point de temps, cependant, de multiples z-position des images à travers le canal YFP sont nécessaires pour capturer tous les phages infectant sur la surface cellulaire. Le temps d&#39;exposition YFP dans le cadre initial peut également besoin d&#39;être supérieure à celle utilisée pour le film time-lapse dans les délais ultérieurs. Analyse d&#39;images: Très soigneusement compter les particules de phage dans la surface de la cellule à l&#39;étape 7,1. Commeindiqué ci-dessus, nous prenons une série de z-piles à travers le canal YFP à l&#39;étape 6.2. Toutefois, cela peut encore laisser des particules de phages fluorescentes out-of-focus, qui contestent le décompte. La longueur de la cellule dans le délai initial est mesuré à l&#39;aide du logiciel Metamorph. La longueur de la cellule peut également être mesurée par ImageJ ou d&#39;autres outils logiciels. En outre, une maison automatisée construite programme Matlab peut être très utile pour obtenir des informations telles que le changement de fluorescence au cours du temps le long lignées cellulaires.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Chloroforme </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> DNase I </td><td> Sigma </td><td> D4527-10KU </td></tr><tr><td> RNase </td><td> Sigma </td><td> R4642-10MG </td></tr><tr><td> PEG8000 </td><td> Fisher Scientific </td><td> BP233-1 </td></tr><tr><td> Tampon SM </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> Seringue </td><td> Becton Dickinson </td><td> 309585 </td></tr><tr><td> Aiguille </td><td> Becton Dickinson </td><td> 305176 </td></tr><tr><td> Cassette de dialyse </td><td> Thermo Scientific </td><td> 66333 </td></tr><tr><td> Lame de microscope </td><td> Corning </td><td> 2947-75x50 </td></tr><tr><td> Agarose </td><td> Fisher Scientific </td><td> BP160-100 </td></tr><tr><td> SW40Ti ultra-clair, tube de </td><td> Beckman Coulter </td><td> 344060 </td></tr><tr><td> SW60Ti ultra-clair, tube de </td><td> Beckman Coulter </td><td> 344062 </td></tr><tr><td> SW40Ti rotor </td><td> Beckman Coulter </td><td> 331302 </td></tr><tr><td> SW60Ti rotor </td><td> Beckman Coulter </td><td> 335649 </td></tr><tr><td> Réfractomètre </td><td> Fisher Scientific </td><td> 13-947 </td></tr><tr><td> Microscope à épifluorescence </td><td> Nikon </td><td> Eclipse TE2000-E </td></tr></tbody></table> Tableau 2. Réactifs et matériel.

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

Le système comprend des bactériophages (phages) lambda et de la bactérie E. coli a longtemps servi de paradigme pour 1,2 détermination cellulaire destin. Suite à l&#39;infection simultanée de la cellule par un certain nombre de phages, l&#39;une des deux voies est choisi: lytique (virulente) ou lysogène (en sommeil) 3,4. Nous avons récemment développé une méthode de fluorescence étiquetage phages individuels, et ont été en mesure d&#39;examiner la décision après l&#39;infection en temps réel sous le microscope, au niveau des phages et les cellules individuelles 5. Nous décrivons ici la procédure complète pour réaliser les expériences d&#39;infection décrits dans nos travaux antérieurs 5. Cela comprend la création de phages fluorescentes, l&#39;infection des cellules, imagerie sous le microscope et l&#39;analyse des données. Le phage fluorescent est un «hybrides», co-exprimant les versions de type sauvage et YFP-fusion de la protéine de capside GPD. Un lysat de phage brut est d&#39;abord obtenue en induisant un lysogène de l&#39;(GPD-EYFP Enhancées Yellow Fluorescent Protein) phage, hébergeant un plasmide de type sauvage exprimant GPD. Une série d&#39;étapes de purification sont ensuite réalisée, suivie par DAPI-étiquetage et l&#39;imagerie au microscope. Ceci est fait afin de vérifier l&#39;uniformité, l&#39;efficacité empaquetage de l&#39;ADN, signal de fluorescence et de la stabilité structurelle du stock de phages. L&#39;adsorption initiale de phages à des bactéries est effectuée sur la glace, puis suivie par une courte incubation à 35 ° C pour déclencher l&#39;injection d&#39;ADN viral 6. Le phage / bactérie mélange est ensuite déplacé vers la surface d&#39;une plaque de gélose nutritive mince, recouverte d&#39;une lamelle couvre-objet imagé et sous un microscope à épifluorescence. Le processus post-infection est suivie pendant 4 heures, à 10 min d&#39;intervalle. Positions à plusieurs étages sont suivis tels que ~ 100 infections cellulaires peuvent être retrouvées dans une seule expérience. A chaque point de position et de temps, les images sont acquises dans le contraste de phase et les canaux rouge et vert fluo. L&#39;image en contraste de phase est utilisé ultérieurement pour automatiser les celreconnaissance l tandis que les canaux fluorescents sont utilisés pour caractériser l&#39;issue infection: la production de nouveaux phages fluorescent (vert), suivie par la lyse cellulaire, ou l&#39;expression de facteurs lysogénie (rouge), suivie par une croissance cellulaire a repris et la division. Les acquis time-lapse films sont traités en utilisant une combinaison de méthodes manuelles et automatisées. Les résultats d&#39;analyse de données pour l&#39;identification des paramètres des infections pour chaque événement infection (par exemple le nombre et les positions d&#39;infecter les phages) ainsi que les résultats infection (lyse / lysogénie). D&#39;autres paramètres peuvent être extraites si désiré.

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

Cet article décrit la procédure à suivre pour la préparation d&#39;une version marquée par fluorescence du bactériophage lambda, l&#39;infection des<em&gt; E. coli</em&gt; Les bactéries, à l&#39;issue infection sous le microscope et l&#39;analyse des résultats d&#39;infection.

Après Cell-sort<em&gt; E. coli</em&gt; Après infection par le phage 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

Research

JoVE Journal

Immunology and Infection

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

23.2K vues.  University of Illinois at Urbana-Champaign. Cet article décrit la procédure à suivre pour la préparation d'une version marquée par fluorescence du bactériophage lambda, l'infection des E. coli Les bactéries, à l'issue infection sous le microscope et l'analyse des résultats d'infection.

Vidéo: 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.

Surveillance des cellules immunitaires trafic fluorescent Rods Prion heures après l&#39;infection par voie intrapéritonéale

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

Recherche

Immunologie et infection

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.

L&#39;utilisation de moyen d&#39;expectoration artificielle pour tester l&#39;efficacité des antibiotiques contre<em&gt; Pseudomonas aeruginosa</em&gt; Dans des conditions plus pertinents à la fibrose kystique du poumon

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.

Extraction des protéines totales et 2-D Méthodes électrophorèse sur gel pour<em&gt; Burkholderia</em&gt; Espèces

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.

Scalable High Throughput sélection à partir de bibliothèques d&#39;anticorps synthétiques phage

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.

Modèle non-invasive de neuropathogènes<em&gt; Escherichia coli</em&gt; Maladies infectieuses chez le rat nouveau-né

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.

Phenomics de phages: approches physiologiques à caractériser de nouvelles protéines virales

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.

Transformation de la levure probiotique et leur récupération à partir gastro immunitaire tissus suivants gavage chez des souris

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.

Pathogènes d’origine alimentaire préalable à l’aide de magnéto-fluorescent NANOCAPTEUR : Détection rapide d’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.

Détection de la colonisation de Escherichia Coli entéro-hémorragique en hôte murin par système de Bioluminescence Non invasif In Vivo

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.

Création d’un Dense Transposon Insertion bibliothèque à l’aide de la conjugaison bactérienne dans les entérobactéries souches telles que Escherichia Coli et 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 ...