Ce travail a été financé par les National Institutes of Health subventions AI62983 (AYK), AI22535 (GPB).

1. Modèle murin de P. Colonisation IG aeruginosa et de diffusion <ol><li> Souris C3H/HeN (6-8 semaines vieux, femmes, Harlan) sont traités avec des antibiotiques oraux pour épuiser la flore commensale et mono-colonisés par PA tel que décrit précédemment 4. </li></ol> 2. Récolte murin caecum contenu luminal <ol><li> Plongez un mortier d&#39;acier inoxydable, nettoyé dans un bain d&#39;azote liquide. </li><li> Euthanasier les souris par asphyxie de dioxyde de carbone. </li><li> La carcasse de souris sécurisé sur une planche en mousse de polystyrène et d&#39;une douche avec de l&#39;éthanol à 95%. Faire une incision médiane longitudinale à travers la peau du sternum au périnée. Refléter la peau et le péritoine pour exposer la cavité abdominale. </li><li> Réséquer le caecum entier. Tenez le cæcum avec une pince sur le mortier en acier inoxydable. Snip les deux extrémités du cæcum avec des ciseaux de dissection. </li><li> Insérez une extrémité de la pipette P1000 rempli avec 1 ml de tampon flushate caecal (10 mM Tris-HCl, 1 mM EDTA et 200 mM de NaCl) 5 dans l&#39;extrémité proximale du caecum. Vide le tampon flushate caecale et caecal contenu luminal dans le mortier en acier inoxydable. 1 ml de tampon flushate caecale sera suffisant pour rincer la totalité du contenu caecal. Caecale contenus flushate gèlera immédiatement après l&#39;entrée en contact avec le mortier. </li><li> Broyer le contenu caecal flushate avec un pilon stériles. </li><li> Placez au sol congelé caecale contenu luminal dans un tube de 50 ml en polypropylène coniques immergés dans une glace sèche / éthanol bain. </li><li> Répétez les étapes 2.2 à 2.7 pour chaque souris supplémentaire. </li><li> Conserver à -80 ° C. </li></ol> 3. Isolement ARN bactérien <ol><li> Chaud 1 volume (basée sur le volume final de deux caecale contenu luminal, environ 3 ml) d&#39;acide phénol / chloroforme (5:1, v / v) contenus dans un tube de 50 ml Oak Ridge centrifugeuse avec le bouchon fixé dans parafilm dans un 65 · Bain C eau. </li><li> Faites bouillir 0,5 volume de tampon de lyse (2% SDS, 16 mM EDTA et 200 mM de NaCl) 6 contenue dans un tube en polypropylène de 50 ml conique pour 5 minutes. </li><li> Ajouter un tampon de lyse bouillant pour caecale contenu luminal. Homogénéiser. Faire bouillir pendant 5 minutes avec vortex périodiques. </li><li> Ajoutez les 100 ° C contenu caecal / échantillon lyse de la 65 ° C l&#39;acide phénol / chloroforme dans le tube Oak Ridge centrifugeuse. Bouchon d&#39;étanchéité avec du parafilm. </li><li> Incuber à 65 ° C pendant 10 minutes avec vortex périodiques (toutes les 2 minutes). </li><li> Centrifuger l&#39;échantillon à 2500 g à 4 ° pendant 15 minutes. </li><li> Soigneusement transfert de phase aqueuse (en évitant tout de l&#39;interface blanc) pour une nouvelle centrifugeuse 50 ml d&#39;Oak Ridge. Le volume de la phase aqueuse sera d&#39;environ 50% du volume total des matières fécales, un tampon de lyse, et l&#39;acide phénol / chloroforme. Ajouter un volume égal d&#39;acide phénol / chloroforme. </li><li> Bouchon d&#39;étanchéité avec du parafilm et bien mélanger au vortex à grande vitesse. </li><li> Centrifuger l&#39;échantillon à 2500 g à 4 ° pendant 15 minutes. </li><li> Répétez les étapes 3.7 à 3.9 jusqu&#39;à ce qu&#39;il n&#39;y a pas d&#39;interface entre les blancs visibles phases aqueuses et organiques. </li><li> Soigneusement transfert de phase aqueuse à une nouvelle centrifugeuse 50 ml d&#39;Oak Ridge. Ajouter un volume égal de chloroforme / alcool isoamylique (24:1, v / v). </li><li> Bouchon d&#39;étanchéité et bien mélanger au vortex. </li><li> Centrifuger l&#39;échantillon à 2500 g à 4 ° pendant 15 minutes. Retirer phase aqueuse (volume sera d&#39;environ 50% du volume total de réaction) à 15 ml tube en polypropylène coniques et ajouter un volume égal d&#39;isopropanol. </li><li> Incuber à -20 ° C pendant au moins 2 heures ou jusqu&#39;au lendemain. </li><li> Centrifuger l&#39;échantillon à 2500 g à 4 ° pendant 45 minutes. Un gel-like granules formera au fond du tube. Retirer le surnageant. </li><li> Laver le culot avec 1 ml d&#39;éthanol glacé à 70%. Vortex. Centrifuger l&#39;échantillon à moins 10.000 g à 4 ° C pendant 5 minutes. </li><li> Enlever le surnageant et inverser le tube de laisser le culot sec à température ambiante pendant 10 minutes. </li><li> Reprendre le culot d&#39;ARN dans 200 pl de l&#39;eau sans RNase. </li></ol> 4. DNase Traitement (Turbo DNA-Free, Applied Biosystems) <ol><li> Ajouter 20 ul de tampon 10X DNase Turbo et Turbo 2 ul de DNase et mélanger doucement. </li><li> Incuber à 37 ° pendant 20 minutes. </li><li> Ajouter 20 ul de réactif d&#39;inactivation resuspendues DNase et bien mélanger. </li><li> Incuber 5 minutes à température ambiante, en mélangeant de temps en temps. </li><li> Centrifuger à 10000 g à température ambiante pendant 1,5 minutes et transférer le surnageant dans un tube de centrifugeuse de nouvelles. </li><li> Ajouter un volume de froid (4 °) l&#39;acide phénol / chloroforme et bien mélanger au vortex pendant 1 minute. </li><li> Centrifuger à 12500 g à température ambiante pendant 2 minutes. </li><li> Transférer la phase aqueuse dans un nouveau tube et ajouter 1 volume de chloroforme / alcool isoamylique (49:1 v / v), vortex. </li><li> Centrifuger à 12500 g à température ambiante pendant 2 minutes. </li><li> Transférer la phase aqueuse (environ 50% du volume total de réaction) à un nouveau tube. </li><li> Ajouter 0,1 volume d&#39;acétate de sodium 3M, pH 5,5 et2,5 volumes d&#39;éthanol glacé de 100%. Vortex. </li><li> Incuber à -20 ° C pendant au moins 2 heures ou à -80 ° pendant 30 minutes. </li><li> Centrifuger à 12000 g pendant 30 minutes à 4 ° </li><li> Retirer le surnageant. </li><li> Laver le culot avec 1 ml d&#39;éthanol glacé à 70% par centrifugation pendant 5 minutes à 10000 g à 4 °. </li><li> Retirer le surnageant et sécher l&#39;air pendant 10 minutes. </li><li> Reprendre le culot dans 100 ul d&#39;eau sans RNase. Il peut être nécessaire de placer dans le bain d&#39;eau 65 ° à resuspendre et dissoudre le culot. </li><li> Pour tester l&#39;efficacité du traitement à la DNase, une norme RT-PCR pour l&#39;amplification d&#39;une protéine ribosomique (ou gène de référence) pourrait être effectuée pour les échantillons traités et non traités, sans utiliser de commandes RT. </li></ol> 5. Étape de nettoyage ARN (Qiagen, RNeasy Kit) <ol><li> Se reporter aux pages 56-57 de Qiagen RNeasy Mini Handbook (4 e édition, avril 2006). Suivre les indications. </li></ol> 6. Les résultats représentatifs Le montant de l&#39;ARN total de bactéries récupérées en utilisant ce protocole est d&#39;environ 2-3 mg à partir de deux cecums. L&#39;ARN récupéré est en quantité suffisante et de qualité pour qPCR subséquentes, le profilage de transcription, l&#39;ARN et des expériences Seq. La pureté ARN est systématiquement évaluée en mesurant le rapport 260nm/280nm 7 de l&#39;échantillon, mais cette méthode ne donne aucune information sur l&#39;intégrité ARN. Le bioanalyseur Agilent est une plateforme microfluidique à base de dimensionnement, de quantification et de contrôle de la qualité de l&#39;ADN, ARN, protéines et des cellules, et utilise une métrique intégrité de l&#39;ARN connue sous le nom Nombre intégrité de l&#39;ARN (RIN) 8. L&#39;ARN extrait avec ce protocole produit des ratios 260/280 allant de 1,7 à 2,0 et des valeurs du RIN ≥ 7,0. Un exemple d&#39;analyse Agilent Bioanalyzer de l&#39;ARN bactérien récupéré par ce protocole est montré dans la figure 1. <img src="/files/ftp_upload/3293/3293fig1.jpg" alt="Figure 1"/> Figure 1. Agilent Bioanalyzer électrophérogramme et gel-like image de Pseudomonas aeruginosa échantillon total ARN isolés et récupérés à partir murin contenu caecal. 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>

Aucun conflit d&#39;intérêt déclaré.

La méthode d&#39;extraction d&#39;ARN décrite ici permet la récupération de quantités suffisantes d&#39;ARN Pseudomonas haute qualité totale aeruginosa récoltés par le tractus gastro-intestinal murin. Cette méthode n&#39;est pas limitée à P. aeruginosa et peut potentiellement être appliqué à d&#39;autres bactéries. La récupération des organismes microbiens suffisante de l&#39;intestin varient considérablement d&#39;un organisme à. Dans notre modèle murin, P. aeruginosa colonise généralement les voies gastro-intestinal murin à des niveaux compris entre 5 x 10 7 à 5 x 10 8 ufc / g fèces 4. Comme le contenu récupéré caecale sont à peu près 0,5 gramme, le nombre estimé de P. aeruginosa récupéré à partir de deux cecums se situe entre 5 x 10 7 à 5 x 10 8 ufc. Si d&#39;autres microorganismes sont utilisés, il serait prudent de vérifier les niveaux de colonisation IG et ensuite calculer le nombre de cecums nécessaires pour récupérer le nombre ciblé de ufc. Il est également important de noter que lorsque vous utilisez ce modèle particulier murins, les souris traitées aux antibiotiques n&#39;est pas infecté par le PA n&#39;ont pas des quantités mesurables de l&#39;ARN isolé à partir de leur contenu caecal. Notre modèle murin de la colonisation à Pseudomonas aeruginosa gastro-intestinaux et les tentatives d&#39;imiter la diffusion de la pathogenèse de P. aeruginosa bactériémie chez les patients du cancer et greffe de cellules souches. Dans cette population de patients, la flore commensale est souvent appauvri secondaire à un traitement antibiotique ou chimiothérapeutique (par exemple l&#39;épuisement des antibiotiques IG flore commensale) résultant de la prolifération des microbes pathogènes (par exemple les mono-association avec P. aeruginosa), puis diffusion ultérieure, après la suppression immunitaire. Les avantages et les limites de ce modèle murin ont déjà été abordées précédemment 4. Le but de cette étude est de fournir une méthodologie pour isoler microbienne ARN total à partir du tractus gastro-intestinal. Ce protocole peut être facilement adaptée à d&#39;autres modèles murins de cette étude d&#39;autres aspects de la pathogenèse microbienne dans le tractus gastro-intestinal (c&#39;est à dire des interactions commensales de la flore, les effets des bactéries sur les maladies inflammatoires de l&#39;intestin, etc.) Un avantage de cette méthode est l&#39;incorporation de plusieurs étapes de lyse, y compris de gel / dégel, une rupture mécanique (broyage au mortier / pilon, homogénéisation), l&#39;ébullition, et la lyse chimique (p. ex FDS). Malgré la multitude d&#39;étapes de lyse, certains micro-organismes (notamment les bactéries Gram positif et les champignons / levures) peuvent exiger d&#39;autres perturbations mécaniques. Après la lyse chaude / acide phénol-chloroforme incubation (étape 3.5), l&#39;addition de billes (0,1 mm pour les bactéries et / ou 0,5 à 0,7 mm pour la levure) et une suite de billes battre étape devrait être suffisante pour lyser ces 9 organismes, 10. Étant donné la nature complexe des matériaux récupérés de l&#39;murin cæcum, le froid, les extractions répétées acid-phenol/chloroform (étape 3.7 à 3.9) sont absolument nécessaires pour obtenir une qualité acceptable de l&#39;ARN et l&#39;intégrité des autres réactions en aval. Entre 3-5 extractions froide acid-phenol/chloroform peuvent être nécessaires avant que l&#39;interface blanche entre les phases aqueuse et organique est éliminée. Enfin, la combinaison des deux dans le traitement DNase (étape 4) et le protocole RNeasy Cleanup (étape 5) sont indispensables pour éliminer l&#39;ADN contaminant et petits non ARNm (5s et ARNt). Comme indiqué précédemment, l&#39;ARN récupéré en utilisant ce protocole est de quantité et qualité suffisantes pour le profilage de transcription ultérieure 3, qPCR (inédit), et de l&#39;ARN Seq (inédit) des expériences.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> Mortier et pilon </td><td> Fisher </td><td> 12-947-1 </td></tr><tr><td> Homogénéisateur </td><td> Omni </td><td> TM-125 </td></tr><tr><td> Oak Ridge tubes de centrifugation (50 ml) </td><td> Nalgene </td><td> 3119-0050 </td></tr><tr><td> Acide phénol / chloroforme </td><td> Ambion </td><td> AM9720 </td></tr><tr><td> Chloroforme </td><td> Sigma-Aldrich </td><td> C2432 </td></tr><tr><td> Alcool isoamylique </td><td> Sigma-Aldrich </td><td> W205702 </td></tr><tr><td> Isopropanol </td><td> Sigma-Aldrich </td><td> 190764 </td></tr><tr><td> Diéthyl pyrocarbonate (DEPC) </td><td> Sigma-Aldrich </td><td> 40718 </td></tr><tr><td> DNase </td><td> Ambion </td><td> AM2238 </td></tr><tr><td> Kit RNeasy </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> Acétate de sodium 3M </td><td> Ambion </td><td> AM9740 </td></tr><tr><td> L&#39;éthanol à 100% </td><td> Sigma-Aldrich </td><td> E7023 </td></tr></tbody></table>

rna isolation of pseudomonas aeruginosa colonizing the murine gastrointestinal tract

Pseudomonas aeruginosa (PA) des infections entraîner une morbidité et une mortalité importantes chez les hôtes dont le système immunitaire, comme les patients atteints de leucémie, de graves brûlures ou une transplantation d&#39;organe. Chez les patients à haut risque de développer des infections sanguines PA, la gastro-intestinal (GI) est le principal réservoir de la colonisation 2, mais les mécanismes par lesquels les transitions d&#39;un microbe PA asymptomatiques coloniser d&#39;une espèce envahissante, et souvent mortelles, des agents pathogènes ne sont pas claires. Auparavant, nous avons effectué des expériences in vivo de profilage de transcription en récupérant l&#39;ARNm PA partir de cellules bactériennes résidant dans la cecums du colonisé souris 3 afin d&#39;identifier les changements dans l&#39;expression des gènes bactériens lors de modifications au statut immunitaire de l&#39;hôte. Comme avec n&#39;importe quelle expérience de profilage de transcription, l&#39;étape limitante est l&#39;isolement des quantités suffisantes de l&#39;ARNm de haute qualité. Étant donné l&#39;abondance des enzymes, des débris, des résidus alimentaires et les matières particulaires dans le tractus gastro-intestinal, le défi de l&#39;isolement de l&#39;ARN est intimidante. Ici, nous présentons une méthode pour l&#39;isolement fiable et reproductible de l&#39;ARN bactérien isolé du tractus gastro-intestinal murin. Cette méthode utilise un modèle bien établi murin de PA GI colonisation et la neutropénie induite par la diffusion 4. Une fois que la colonisation IG avec PA est confirmé, les souris sont euthanasiées et le contenu caecal sont récupérés et surgelés. L&#39;ARN est ensuite extrait en utilisant une combinaison d&#39;une rupture mécanique, l&#39;ébullition, le phénol / chloroforme, extractions, traitement à la DNase, et la chromatographie d&#39;affinité. Quantité et la pureté sont confirmées par spectrophotométrie (Nanodrop Technologies) et bioanalyseur (Agilent Technologies) (figure 1). Cette méthode de GI microbienne isolement d&#39;ARN peut être facilement adaptée à d&#39;autres bactéries et champignons ainsi.

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

Une méthode fiable pour l&#39;isolement des ARN<em&gt; Pseudomonas aeruginosa</em&gt; Récupéré cecums murin est décrite. L&#39;ARN récupéré est en quantité suffisante et de qualité pour qPCR subséquentes, le profilage de transcription, l&#39;ARN et des expériences Seq. Cette technique peut être adaptée à l&#39;isolement d&#39;ARN des autres microbes intestinaux.

Isolement des ARN Pseudomonas aeruginosa La colonisation du tractus gastro-intestinal murin

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 vues.  University of Texas Southwestern Medical Center. Une méthode fiable pour l'isolement des ARN Pseudomonas aeruginosa Récupéré cecums murin est décrite. L'ARN récupéré est en quantité suffisante et de qualité pour qPCR subséquentes, le profilage de transcription, l'ARN et des expériences Seq. Cette technique peut être adaptée à l'isolement d'ARN des autres microbes intestinaux.

Vidéo: 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-culture des modèles Pseudomonas aeruginosa Les biofilms Cultivé sur le Live cellules des voies respiratoires de l&#39;homme

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

Recherche

Immunologie et infection

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 et le biofilm Saccharomyces cerevisiae en cellules à flux

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.

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 ...

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

Isolement des lymphocytes de souris muqueuses génitales Tract

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.

Chronique à long terme<em&gt; Pseudomonas aeruginosa</em&gt; Infection des voies respiratoires chez les souris

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 lésion pulmonaire induite par modèle

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.

Venez sur le côté de la lumière: In Vivo surveillance des Infections à Pseudomonas aeruginosa Biofilm dans les plaies chroniques dans un modèle murin glabres diabétique

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.

Réplication de l’ordonné, bibliothèque non redondante de la souche Pseudomonas aeruginosa PA14 Transposon Mutants d’Insertion

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.

Méthodes pour détecter les cytotoxiques amyloïdes suivant Infection de pulmonaire cellules endothéliales par 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.

Visualisation de Candida albicans dans le tractus gastro-intestinal murine utilisant l'hybridation 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 ...