Nous reconnaissons l&#39;appui de l&#39;Institut national du Royaume-Uni pour la recherche en santé, le Dr Hadwen Trust pour la recherche humaine, le Royaume-Uni leader de financement de la recherche médicale charité exclusivement des techniques de recherche non-animale pour remplacer l&#39;expérimentation animale, et le Wellcome Trust (Grant 089 215). Nous reconnaissons également Novartis Pharmaceuticals UK Ltd (l&#39;éducation sans restriction de subvention).

<ol>
	<li>Andrews, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BSAC+standardized+disc+susceptibility+testing+method+(version+8).">BSAC standardized disc susceptibility testing method (version 8).</a> J. Antimicrob. Chemother. 64, 454-489 (2009).</li><li>Worlitzsch, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+reduced+mucus+oxygen+concentration+in+airway+Pseudomonas+infections+of+cystic+fibrosis+patients.">Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients.</a> J. Clin. Invest. 109, 317-325 (2002).</li><li>Smith, A. L., Fiel, S. B., Mayer-Hamblett, N., Ramsey, B., Burns, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+testing+of+Pseudomonas+aeruginosa+isolates+and+clinical+response+to+parenteral+antibiotic+administration:+lack+of+association+in+cystic+fibrosis.">Susceptibility testing of Pseudomonas aeruginosa isolates and clinical response to parenteral antibiotic administration: lack of association in cystic fibrosis.</a> Chest. 123, 1495-1502 (2003).</li><li>Sriramulu, D. D., Lunsdorf, H., Lam, J. S., Romling, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcolony+formation:+a+novel+biofilm+model+of+Pseudomonas+aeruginosa+for+the+cystic+fibrosis+lung.">Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung.</a> J. Med. Microbiol. 54, 667-676 (2005).</li><li>Garbe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+JG024,+a+pseudomonas+aeruginosa+PB1-like+broad+host+range+phage+under+simulated+infection+conditions.">Characterization of JG024, a pseudomonas aeruginosa PB1-like broad host range phage under simulated infection conditions.</a> BMC Microbiol. 10, 301 (2010).</li><li>Naughton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+AES-1+exhibits+increased+virulence+gene+expression+during+chronic+infection+of+cystic+fibrosis+lung.">Pseudomonas aeruginosa AES-1 exhibits increased virulence gene expression during chronic infection of cystic fibrosis lung.</a> PLoS One. 6, e24526 (2011).</li><li>Moskowitz, S. M., Foster, J. M., Emerson, J. C., Gibson, R. L., Burns, J. 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+Pseudomonas+biofilm+susceptibilities+to+assign+simulated+antibiotic+regimens+for+cystic+fibrosis+airway+infection.">Use of Pseudomonas biofilm susceptibilities to assign simulated antibiotic regimens for cystic fibrosis airway infection.</a> J. Antimicrob. Chemother. 56, 879-886 (2005).</li><li>Field, T. R., White, A., Elborn, J. S., Tunney, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+oxygen+limitation+on+the+in+vitro+antimicrobial+susceptibility+of+clinical+isolates+of+Pseudomonas+aeruginosa+grown+planktonically+and+as+biofilms.">Effect of oxygen limitation on the in vitro antimicrobial susceptibility of clinical isolates of Pseudomonas aeruginosa grown planktonically and as biofilms.</a> Eur. J. Clin. Microbiol. Infect. Dis. 24, 677-687 (2005).</li><li>Bjarnsholt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+biofilms+in+the+respiratory+tract+of+cystic+fibrosis+patients.">Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.</a> Pediatr. Pulmonol. 44, 547-558 (2009).</li><li>Hill, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+susceptibilities+of+Pseudomonas+aeruginosa+isolates+derived+from+patients+with+cystic+fibrosis+under+aerobic,+anaerobic,+and+biofilm+conditions.">Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.</a> J. Clin. Microbiol. 43, 5085-5090 (2005).</li><li>Fung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+of+Pseudomonas+aeruginosa+in+a+mucin-containing+synthetic+growth+medium+mimicking+cystic+fibrosis+lung+sputum.">Gene expression of Pseudomonas aeruginosa in a mucin-containing synthetic growth medium mimicking cystic fibrosis lung sputum.</a> J. Med. Microbiol. 59, 1089-1100 (2010).</li><li>Andrews, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BSAC+standardized+disc+susceptibility+testing+method+(version+8).">BSAC standardized disc susceptibility testing method (version 8).</a> The Journal of antimicrobial chemotherapy. 64, 454-489 (2009).</li><li>Rybtke, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+implication+of+Pseudomonas+aeruginosa+biofilms+in+infections.">The implication of Pseudomonas aeruginosa biofilms in infections.</a> Inflamm Allergy Drug Targets. 10, 141-157 (2011).</li><li>Moreau-Marquis, S., Stanton, B. A., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+biofilm+formation+in+the+cystic+fibrosis+airway.">Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway.</a> Pulm. Pharmacol. Ther. 21, 595-599 (2008).</li><li>Ahmed, S. A., Gogal, R. M., Walsh, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+rapid+and+simple+non-radioactive+assay+to+monitor+and+determine+the+proliferation+of+lymphocytes:+an+alternative+to+[3H]thymidine+incorporation+assay.">A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay.</a> J. Immunol. Methods. 170, 211-224 (1994).</li><li>Fothergill, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+antibiotic+treatment+on+bacteriophage+production+by+a+cystic+fibrosis+epidemic+strain+of+Pseudomonas+aeruginosa.">Effect of antibiotic treatment on bacteriophage production by a cystic fibrosis epidemic strain of Pseudomonas aeruginosa.</a> Antimicrob. Agents Chemother. 55, 426-428 (2011).</li><li>Singh, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quorum-sensing+signals+indicate+that+cystic+fibrosis+lungs+are+infected+with+bacterial+biofilms.">Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms.</a> Nature. 407, 762-764 (2000).</li><li>Mowat, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+Aeruginosa+Population+Diversity+and+Turnover+in+Cystic+Fibrosis+Chronic+Infections.">Pseudomonas Aeruginosa Population Diversity and Turnover in Cystic Fibrosis Chronic Infections.</a> Am. J. Respir. Crit. Care. Med. , (2011).</li><li>Fothergill, J. L., Mowat, E., Ledson, M. J., Walshaw, M. J., Winstanley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+in+phenotypes+and+genotypes+within+populations+of+Pseudomonas+aeruginosa+in+the+cystic+fibrosis+lung+during+pulmonary+exacerbations.">Fluctuations in phenotypes and genotypes within populations of Pseudomonas aeruginosa in the cystic fibrosis lung during pulmonary exacerbations.</a> J. Med. Microbiol. 59, 472-481 (2010).</li><li>Fouhy, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusible+signal+factor-dependent+cell-cell+signaling+and+virulence+in+the+nosocomial+pathogen+Stenotrophomonas+maltophilia.">Diffusible signal factor-dependent cell-cell signaling and virulence in the nosocomial pathogen Stenotrophomonas maltophilia.</a> J. Bacteriol. 189, 4964-4968 (2007).</li><li>Winstanley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+introduced+genomic+prophage+islands+are+critical+determinants+of+in+vivo+competitiveness+in+the+Liverpool+Epidemic+Strain+of+Pseudomonas+aeruginosa.">Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa.</a> Genome Res. 19, 12-23 (2009).</li><li>Carter, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subtype+of+a+Pseudomonas+aeruginosa+cystic+fibrosis+epidemic+strain+exhibits+enhanced+virulence+in+a+murine+model+of+acute+respiratory+infection.">A subtype of a Pseudomonas aeruginosa cystic fibrosis epidemic strain exhibits enhanced virulence in a murine model of acute respiratory infection.</a> J. Infect. Dis. 202, 935-942 (2010).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Dans cette étude nous avons utilisé un modèle in vitro roman basé sur l&#39;ASM à reproduire P. conditions biofilm aeruginosa dans les 4 poumon FC. Le modèle a été modifié avec succès à petite échelle, à haut débit des tests des agents antimicrobiens. Les étapes critiques de ce test sont les suivants: <ol><li> Conformément préparation des médias ASM et la stérilité maintien. Nous avons consacré de longues heures à l&#39;optimisation de la manière dont chaque composant est ajouté à obtenir des résultats reproductibles à chaque fois. Filtration de l&#39;ASM est lent mais il est préférable d&#39;autoclavage, ce qui peut endommager le composant mucine. Nous ne conseillons pas l&#39;addition d&#39;antibiotiques, comme suggéré par d&#39;autres 11, car cela pourrait imposer considérables pressions sélectives, des mutations d&#39;entraînement, induire la lyse prophage 16 et modifier de manière significative l&#39;expression de plusieurs gènes bactériens. </li><li> Le test doit être optimisée en fonction de l&#39;o du volumef ASM utilisé. Shaking vitesses sont augmentées pour les petits volumes et une plus courte biofilm du cycle de vie est observée. </li></ol> Une application évidente du modèle à petite échelle biofilm ASM est la détermination plus réaliste de susceptibilités biofilm antimicrobiens (BSMIC 90). Niches anaérobies et microaérophiles sont présents dans le poumon des FC et il est évident que l&#39;oxygène est limité au plus profond de maturité biofilms 2, 17. Nous démontrons ici que 10/14 P. clinique isolats aeruginosa de patient atteint de mucoviscidose présentent une considérable crachats (4 - ≥ 128 fois) diminution de la sensibilité à la tobramycine dans des conditions microaérophiles en ASM. Les résultats de cette étude suggèrent que les antibiotiques, tels que la tobramycine, pourrait être moins efficace contre P. infections aeruginosa dans les poumons CF que ceux indiqués par les méthodes classiques de test de sensibilité. Ces résultats reflètent les études antérieures sur la sensibilité aux antimicrobiens des biofilms 10. Petit-échelle des dosages ASM ainsi fournir une plate-forme simple, le débit élevé pour générer des données significatives de sensibilité aux antibiotiques afin de mieux éclairer les décisions thérapeutiques. Le test est limité dans la même manière que les tests de sensibilité aux antibiotiques classiques par le fait que des colonies isolées sont cueillies pour le dépistage qui peuvent ne pas être représentatif de la population dans son ensemble. Cependant, nous croyons qu&#39;une approche (i) à l&#39;aide non-surface de croissance biofilm fixé et (ii) applicable à des conditions microaérophiles, représente une alternative claire et un potentiel d&#39;amélioration des méthodes existantes. Nous concluons que cet essai est un modèle approprié pour étudier P. populations biofilm aeruginosa. D&#39;autres tests dans des contextes cliniques serait de vérifier si la sensibilité aux antibiotiques sur la base de biofilm développé P. aeruginosa pourrait conduire à des choix différents antibiotiques avec potentiellement de meilleurs résultats cliniques et microbiologiques. Des enquêtes similaires en utilisant des modèles classiques de biofilm ont montré que les valeurs BSMIC conduiredes recommandations différentes pour le traitement antibiotique 5,17. En plus des tests de l&#39;efficacité des agents anti-infectieux, le système ASM représente un pas cher, alternative simple et reproductible pour des modèles animaux pour des études telles que celles visant à comprendre la diversification de P. populations aeruginosa. Nous avons observé une hétérogénéité importante dans les populations naturelles de P. aeruginosa récupéré de patient atteint de mucoviscidose crachats 18, 19. Comme la diversification phénotypique et génotypique peut être observée au cours de la croissance à l&#39;ASM 4 (et nos données non publiées), ce qui en fait un joli modèle in vitro des maladies pulmonaires FC. La relative simplicité du modèle ASM, il est facile de concevoir des expériences d&#39;adaptation à long terme visant, par exemple, à surveiller les effets des antibiotiques ou autres facteurs de stress sur P. Population de divergence aeruginosa. En outre, d&#39;autres bactéries pathogènes peuvent être cultivées dansASM. Par exemple, Fouhy et al. 2007 ont utilisé l&#39;ASM pour étudier la formation de biofilm par S. maltophillia 20.

<table border="1"><tbody><tr><td> Nom de réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> L&#39;ADN de sperme de poisson </td><td> Sigma-Aldrich </td><td> 74782 </td></tr><tr><td> Mucine de porc estomac, de type II </td><td> Sigma-Aldrich </td><td> M2378 </td></tr><tr><td> L-alanine </td><td> Acros Organics </td><td> 102830250 </td></tr><tr><td> L-Arginine </td><td> Sigma-Aldrich </td><td> A5006 </td></tr><tr><td> L (+)-asparagine monohydraté </td><td> Acros Organics </td><td> 175271000 </td></tr><tr><td> L (+)-acide aspartique </td><td> Acros Organics </td><td> 105041000 </td></tr><tr><td> L-cystéine </td><td> Sigma-Adrich </td><td> 168149 </td></tr&gt; <tr><td> L (+)-acide glutamique </td><td> Acros Organics </td><td> 156211000 </td></tr><tr><td> L-glutamine </td><td> Sigma-Aldrich </td><td> G3126 </td></tr><tr><td> Glycine </td><td> Acros Organics </td><td> 220911000 </td></tr><tr><td> L-histidine </td><td> Sigma-Adrich </td><td> H8000 </td></tr><tr><td> L-isoleucine </td><td> Sigma-Aldrich </td><td> I2752 </td></tr><tr><td> L-leucine </td><td> Sigma-Aldrich </td><td> L8000 </td></tr><tr><td> L (+)-lysine monochlorhydrate </td><td> Acros Organics </td><td> 125222500 </td></tr><tr><td> L-Méthionine </td><td> Sigma-Aldrich </td><td> M9625 </td></tr><tr><td> L-phénylalanine </td><td> Acros Organics </td><td> 130310250 </td></tr><tr><td> L-proline </td><td> Sigma-Aldrich </td><td> P0380 </td></tr><tr><td> L-sérine </td><td> Acros Organics </td><td> 132660250 </td></tr><tr><td> L-Thréonine </td><td> Acros Organics </td><td> 138930250 </td></tr><tr><td> L (-)-tryptophane </td><td> Acros Organics </td><td> 140590250 </td></tr><tr><td> L-Tyrosine </td><td> Acros Organics </td><td> 140641000 </td></tr><tr><td> L-valine </td><td> Sigma-Aldrich </td><td> V0500 </td></tr><tr><td> L&#39;acide diéthylènetriaminepentaacétique </td><td> Sigma-Aldrich </td><td> 32318 </td></tr><tr><td> NaCl </td><td> Fisher Scientific </td><td> S/3160/60 </td></tr><tr><td> KCl </td><td> BDH </td><td> BDH0258 </td></tr><tr><td&gt; KOH </td><td> BDH </td><td> BDH0262 </td></tr><tr><td> Émulsion de jaune d&#39;oeuf </td><td> Sigma-Aldrich </td><td> 17148 </td></tr><tr><td> ME 2 Pompe à vide à membrane </td><td> Vacuubrand </td><td> 696126 </td></tr><tr><td> Filtres Steritop (taille des pores: 0,22 um, la taille du cou: 45 mm) </td><td> Millipore </td><td> SCGPT10RE </td></tr><tr><td> Luria-Bertani </td><td> Sigma </td><td> L2897 </td></tr><tr><td> Microplaques de 96 puits </td><td> Sarstedt </td><td> 82.1581 </td></tr><tr><td> Tissus de 24 puits culture traités plaques </td><td> Iwaki </td><td> 3820-024 </td></tr><tr><td> Packs CampyGen génération de gaz </td><td> Oxoid </td><td> CN0025 </td></tr><tr><td> Chambre microaérophile </td><td> Oxoid </td><td> HP0011 </td></tr><tr><td> Tobramycine sulfate </td><td> Sigma-Aldrich </td><td> T1783 </td></tr><tr><td> Cellulase, à partir d&#39;Aspergillus Niger </td><td> Sigma-Aldrich </td><td> 22178 </td></tr><tr><td> Résazurine </td><td> Sigma-Aldrich </td><td> 199303 </td></tr><tr><td> Citrate.H 2 0 </td><td> BDH </td><td> BDH0288 </td></tr><tr><td> Fluostar oméga lecteur de microplaques </td><td> BMG-Labtech </td><td> SPECTROstar Omega </td></tr></tbody></table>

use of artificial sputum medium to test antibiotic efficacy against pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung

Il est de plus en plus préoccupés par la pertinence des tests in vitro de sensibilité aux antimicrobiens lorsqu&#39;ils sont appliqués à des isolats de P. aeruginosa de fibrose kystique (FK) patients. Les méthodes existantes assurée par un seul ou de quelques isolats cultivés en aérobiose et en plancton. Prédéterminés seuils sont utilisés pour définir si les bactéries sont sensibles ou résistantes à un antibiotique donné 1. Toutefois, au cours des infections pulmonaires chroniques dans les FC, P. populations aeruginosa existent dans des biofilms et il est évident que l&#39;environnement est en grande partie microaérophile 2. La différence marquée dans des conditions entre les bactéries dans les poumons et ceux lors de tests a remis en question la pertinence de fiabilité et même de ces 3 tests. Moyennes expectoration artificielle (ASM) est un milieu de culture contenant les composants de l&#39;expectoration patient atteint de mucoviscidose, y compris les acides aminés, et l&#39;ADN de mucine libre. P aeruginosa &lt;/ Em&gt; La croissance de la croissance au cours des infections imite ASM FC, avec la formation de biofilm structures d&#39;auto-agrégation et 4,5,6 divergence de la population. L&#39;objectif de cette étude était de développer un test de microtitrage plaque d&#39;étudier la sensibilité aux antimicrobiens de P. aeruginosa basé sur la croissance à l&#39;ASM, qui est applicable aux deux conditions microaérophiles et aérobie. Un test a été développé ASM dans un format microplaque. P. biofilms aeruginosa ont été autorisés à se développer pour 3 jours avant l&#39;incubation avec des agents antimicrobiens à des concentrations différentes pour 24 heures. Après la perturbation du biofilm, la viabilité cellulaire a été mesurée par coloration avec la résazurine. Ce test a été utilisé pour déterminer la concentration minimale inhibitrice des cellules sessiles (SMIC) de la tobramycine pour 15 différents P. isolats aeruginosa dans des conditions aérobies et microaérophiles et les valeurs du SMIC ont été comparés à ceux obtenus avec la croissance de bouillon standard. Bien qu&#39;il n&#39;y aitcertains éléments de preuve pour les valeurs de CMI accrues pour les isolats cultivés dans ASM par rapport à leurs homologues planctoniques, les plus grandes différences ont été trouvées avec des bactéries testées dans des conditions microaérophiles, qui ont montré une résistance beaucoup plus porté à un&gt; 128 fois, vers la tobramycine dans le système ASM lors par rapport à des tests réalisés dans des conditions aérobies. L&#39;absence d&#39;association entre les méthodes actuelles de tests de sensibilité et le résultat clinique a mis en doute la validité des méthodes actuelles 3. Plusieurs modèles in vitro ont déjà été utilisés pour étudier P. biofilms aeruginosa 7, 8. Cependant, ces méthodes reposent sur ​​des biofilms de surface ci-jointes, tandis que les biofilms ASM ressemblent à ceux observés dans le poumon FC 9. En outre, la concentration en oxygène réduite dans le mucus a été montré pour modifier le comportement de P. aeruginosa 2 et affectent la sensibilité aux antibiotiques 10. Par conséquent using ASM dans des conditions microaérophiles peuvent fournir un environnement plus réaliste dans lequel d&#39;étudier la sensibilité aux antimicrobiens.

Watch this Scientific Journal Video about Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung at JoVE.com

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Actuelle des tests de diagnostic de sensibilité aux antimicrobiens repose sur la croissance planctonique des isolats en nutriments riches, des conditions aérobies. Ici, nous employons un moyen alternatif d&#39;expectoration artificielle pour étudier la sensibilité aux antimicrobiens des biofilms de Pseudomonas aeruginosa dans des conditions aérobies et microaérophiles plus représentatives de la fibrose pulmonaire kystique.

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 ...

use-artificial-sputum-medium-to-test-antibiotic-efficacy-against

Research

JoVE Journal

Immunology and Infection

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

32.5K vues.  University of Liverpool. Actuelle des tests de diagnostic de sensibilité aux antimicrobiens repose sur la croissance planctonique des isolats en nutriments riches, des conditions aérobies. Ici, nous employons un moyen alternatif d'expectoration artificielle pour étudier la sensibilité aux antimicrobiens des biofilms de Pseudomonas aeruginosa dans des conditions aérobies et microaérophiles plus représentatives de la fibrose pulmonaire kystique.

Vidéo: Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

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 ...

Recherche

Immunologie et infection

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

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

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

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

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 ...

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. ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Un modèle de pneumonie robuste en immunocompétents Rongeurs pour évaluer Efficacité antibactérienne contre<i&gt; S. pneumoniae</i&gt;,<i&gt; H. influenzae</i&gt;,<i&gt; K. pneumoniae</i&gt;,<em&gt; P. aeruginosa</em&gt; ou<em&gt; A. baumannii</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

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

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

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

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

Un modèle de souris transmissible transitoirement IL-8 pour le<em&gt; In vivo</em&gt; Suivi à long terme des réponses inflammatoires

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

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing methods in general. Additionally, little evidence exists comparing handwashing methods to determine which are most efficacious at removing infectious pathogens. Research is needed to provide evidence for the different approaches to handwashing that may be employed during infectious disease outbreaks. Here, a laboratory method to assess the efficacy of handwashing methods at removing microorganisms from hands and their persistence in rinse water is described. Volunteers' hands are first spiked with the test organism and then washed with each handwashing method of interest. Generally, surrogate microorganisms are used to protect human subjects from disease. The number of organisms remaining on volunteers' hands after washing is tested using a modified "glove juice" method: the hands are placed in gloves with an eluent and are scrubbed to suspend the microorganisms and make them available for analysis by membrane filtration (bacteria) or plaque assay (viruses/bacteriophages). Rinse water produced from the handwashing is directly collected for analysis. Handwashing efficacy is quantified by comparing the log reduction value between samples taken after handwashing to samples with no handwashing. Rinse water persistence is quantified by comparing rinse water samples from various handwashing methods to samples collected after handwashing with just water. While this method is limited by the need to use surrogate organisms to preserve the safety of human volunteers, it captures aspects of handwashing that are difficult to replicate in an in vitro study and fills research gaps on handwashing efficacy and the persistence of infectious organisms in rinse water.

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

Une méthode pour tester l&#39;efficacité du lavage des mains pour l&#39;élimination des pathogènes infectieux émergents

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

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 ...

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe infections, complications, and mortality worldwide with a huge impact on our society and on the health system. In particular, patients with compromised immune systems or pre-existing and chronic pathologies, such as cystic fibrosis (CF), are subjected to frequent antibiotic treatments to control the infections with the appearance and diffusion of multidrug resistant isolates. Therefore, there is an urgent need to address alternative therapies to counteract bacterial infections. Use of bacteriophages, the natural enemies of bacteria, can be a possible solution. The protocol detailed in this work describes the application of phage therapy against Pseudomonas aeruginosa infection in CF zebrafish embryos. Zebrafish embryos were infected with P. aeruginosa to demonstrate that phage therapy is effective against P. aeruginosa infections as it reduces lethality, bacterial burden and pro-inflammatory immune response in CF embryos.

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Presented here is a protocol for Pseudomonas aeruginosa infection and phage therapy application in cystic fibrosis (CF) zebrafish embryos.

Application de thérapie de phage pour contrecarrer l’infection de Pseudomonas aeruginosa dans les embryons de poisson zèbre de fibrose kystique

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.