Nous remercions les techniciens du laboratoire de bactériologie du CHU de Lausanne pour leur aide à la mise en œuvre des techniques de laboratoire.

Ce protocole a été développé et validé en suivant les processus de recherche et développement et les règles éthiques de notre institution avant d&#39;être mis en œuvre comme un outil de routine. 1 Préparation d&#39;une culture bactérienne du sang Pellet par ammonium procédure chlorure érythrocytes-lyse <ol><li> Préparation d&#39;une hémoculture positive pour la centrifugation ultérieure. <ol><li> Stériliser le bouchon de culture de sang. Ajouter 70% d&#39;éthanol sur le bouchon de la bouteille et de le brûler. NOTE: Ne pas effectuer cette étape sous une hotte à flux laminaire. </li><li> Déplacer la bouteille à une hotte à flux laminaire. Recueillir 5 ml de la culture de sang avec une seringue de 20 G. </li><li> Ajouter 5 ml de la culture de sang dans un tube à centrifuger de 50 ml contenant 45 ml d&#39;eau stérile (H 2 O) et de mélanger l&#39;échantillon. </li></ol></li><li> Préparation d&#39;une culture de sang par lyse culot bactérien centrifugation. <ol><li> Centrifuger les échantillons à 1000 g pendant 10 min à température ambiante. </li><li> Remove du surnageant (qui contient principalement du H 2 O et de cellules sanguines) avec une pompe à vide de laboratoire. </li><li> Remettre en suspension le culot dans 1 ml de chlorure d&#39;ammonium artisanale solution (0,15 M de NH 4 Cl, 1 mM KHCO 3) de lyse. Décomposer le culot par grattage et au vortex et centrifuger le tube de l&#39;échantillon à 140 xg pendant 10 min à température ambiante. </li><li> Jeter le surnageant qui contient principalement les globules rouges débris. <ol><li> Si la pastille est restée hémorragique, remettre en suspension le culot dans 2 ml d&#39;eau stérile distillée et 2 0 en outre à lyser les globules rouges résiduels et laver le culot. Centrifuger l&#39;échantillon à 140 g pendant 10 min à température ambiante et jeter le surnageant. </li></ol></li><li> Remettre en suspension le culot dans 200 pl de H 2 O </li></ol></li><li> Subculture de culture de sang sur divers milieux de gélose sélective et non sélective. <ol><li> Prélever 1 ml de la culture de sang avec une seringue de 20 G. </li><li> Inoculer une goutte de culture de sang into quatre quadrants sur gélose au sang, une gélose au chocolat, de l&#39;agar agar et Schaedler McConkey. </li><li> Incuber à 37 ° C, la gélose au sang et des plaques d&#39;agar au chocolat dans une atmosphère de CO2 à 5%, la gélose de McConkey dans une atmosphère normale, et la plaque de gélose Schaedler dans des conditions anaérobies. </li><li> Suivez la croissance bactérienne sur les différents médias. </li><li> Vérifier la pureté bactérienne. </li></ol></li></ol> 2 Identification par MALDI-TOF MS <ol><li> L&#39;identification directe à partir du culot de sang. <ol><li> Transfert 1 pl de la pastille de sang sur une plaque cible MALDI et laisser sécher sur une plate-forme de chauffage à 42 ° C. </li><li> Superposer l&#39;échantillon avec de l&#39;acide formique 1 ul de 70% et laisser sécher sur une plate-forme de chauffage à 42 ° C. </li><li> Superposer l&#39;échantillon avec 1 pl de matrice MALDI et laisser sécher sur une plate-forme C 42 ° chauffage (solution de α-cyano-4-hydroxycinnamique dans 50% d&#39;acétonitrile-2.5% d&#39;acide trifluoroacétique saturé). </li><li> Passez à la MALDI-TOF MS avec un système MALDI-TOF MS comme décrit par les fabricants. </li><li> Si le score d&#39;identification est valide au niveau de l&#39;espèce (par exemple, avec un score &lt;2), effectuer une extraction de protéines de l&#39;échantillon de granulés dans le sang. </li></ol></li><li> Identification de la protéine après extraction du culot de la culture de sang. <ol><li> Mélanger 20 ul de la pastille avec 1 ml d&#39;éthanol à 70% et centrifugation à 13 000 xg pendant 2 minutes à température ambiante. </li><li> Jeter le surnageant et ajouter 25 ul d&#39;acide formique à 70% et 25 ul de 100% d&#39;acétonitrile à la pastille. Vortex vigoureusement pour remettre en suspension les culots. Remettre en suspension granulés difficiles en les décomposant avec des embouts de pipette avant vortex. </li><li> Centrifuger à 13 000 g pendant 2 min à température ambiante. Transfert 1 pi du surnageant (qui contiennent des protéines extraites) sur une plaque cible MALDI et laisser sécher sur une plate-forme de chauffage à 42 ° C. Procédez comme décrit dans le paragraphe 2.1.2. </li></ol></li></ol> 3. bactérienne Identification et les tests de sensibilité aux antibiotiques avec un système automatisé microbienne <ol><li> Préparation d&#39;une suspension bactérienne d&#39;identification bactérienne et AST avec un système automatisé microbienne. <ol><li> Préparer un tube en matière plastique contenant 3 ml de solution stérile de NaCl à 0,45% compatible avec le système automatisé microbienne. </li><li> Ajouter une quantité suffisante de la culture de culot de sang dans la solution de NaCl à 0,45% pour obtenir une suspension bactérienne correspondant à une turbidité McFarland de 0,6 à 0,8. Mesure de la turbidité en utilisant un appareil à densitomètre. NOTE: Le volume du culot bactérien de la culture de sang varie de 50 à 200 pl pour obtenir une turbidité McFarland de 0,6 à 0,8. </li></ol></li><li> Inoculer les bactéries Gram-positif (GP) ou Gram-négatif (GN) cartes automatisés d&#39;identification et les cartes AST automatisés pour les tests de sensibilité aux antimicrobiens des Cocci Gram-positives et Gram-négatives bactéries comme décrit par le fabricant. REMARQUE: identification avec GP ou cartes GN n&#39;est pas appliquée si MALDI-TOF MS valeur du score d&#39;identification est&gt; 2. Vérifier la pureté de la suspension bactérienne par repiquage la suspension bactérienne sur gélose au sang (GP et GN) et la gélose Mac Conkey (GN) plaques. </li></ol> 4 essais de sensibilité aux antibiotiques par disque Diffusion Assay Le test de diffusion sur disque est décrit par le Comité européen sur les tests de sensibilité aux antimicrobiens (EUCAST, Version 3.0, Avril 2013, www.eucast.org). <ol><li> Préparation d&#39;une suspension bactérienne à effectuer des tests de sensibilité aux antimicrobiens par un essai de diffusion de disques. <ol><li> Préparer un tube en verre contenant 3 ml de solution stérile de NaCl à 0,9%. </li><li> Ajouter une quantité suffisante de la culture de culot de sang dans la solution de NaCl à 0,9% pour obtenir une suspension bactérienne correspondant à une turbidité de 0,5 McFarland. Mesure de la turbidité en utilisant un appareil à densitomètre. REMARQUE: Le volume de la culture de sangculot bactérien varie de 50 à 200 pl pour obtenir une turbidité McFarland de 0,6 à 0,8. </li><li> Vortex la suspension bactérienne. </li></ol></li><li> L&#39;inoculation de la suspension bactérienne sur de la gélose de Mueller-Hinton (MH) ou Mueller-Hinton agar agar organismes-fastidieuse (MHF) (MH supplémenté avec 5% de sang de cheval défibriné et 20 mg / L de β-nicotinamide adénine dinucléotide). <ol><li> Trempez un coton-tige stérile dans la suspension bactérienne et retirez délicatement l&#39;excès de liquide en tournant la tige sur la paroi interne du tube de verre. </li><li> Etaler la suspension bactérienne de façon égale sur toute la surface de la plaque de gélose MH / MHF par écouvillonnage ou dans trois directions à l&#39;aide d&#39;un rotateur de plaque automatisé. </li></ol></li><li> Application des disques antimicrobiens sur des plaques de gélose ensemencées. <ol><li> Appliquer près les disques sur la surface des plaques de gélose ensemencées séchées manuellement ou à l&#39;aide d&#39;un distributeur de sensibilité sur disque. </li><li> Incuber la plaque à la tempe appropriérature et de l&#39;atmosphère, comme décrit dans la méthode de diffusion EUCAST disque de test de sensibilité aux antimicrobiens (Version 3.0, Avril 2013, www.eucast.org). </li></ol></li></ol> 5. automatisé PCR à base de test de diagnostic pour la détection de SARM <ol><li> Utilisation d&#39;une micro-pipette, transférer 50 pi de la pastille de culture de sang dans le flacon de réactif d&#39;échantillon fourni avec le kit SARM automatisé de test de diagnostic basé sur la PCR et vortex pendant 20 s. </li><li> L&#39;utilisation d&#39;une micropipette de 1 ml, distribuer l&#39;échantillon dans l&#39;orifice d&#39;échantillon de la cartouche. Insérez la cartouche dans le système PCR automatisé et commencer le dosage comme décrit par le fabricant. </li></ol>

<ol>
	<li>Alberti, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+sepsis+and+infection+in+ICU+patients+from+an+international+multicentre+cohort+study.">Epidemiology of sepsis and infection in ICU patients from an international multicentre cohort study.</a> Intensive care medicine. 28, 108-121 (2002).</li><li>Angus, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+severe+sepsis+in+the+United+States:+analysis+of+incidence,+outcome,+and+associated+costs+of+care.">Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care.</a> Critical care medicine. 29, 1303-1310 (2001).</li><li>Vincent, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis+in+European+intensive+care+units+results+of+the+SOAP+study.">Sepsis in European intensive care units results of the SOAP study.</a> Critical care medicine. 34, 344-353 (2006).</li><li>Micek, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Empiric+combination+antibiotic+therapy+is+associated+with+improved+outcome+against+sepsis+due+to+Gram-negative+bacteria+a+retrospective+analysis.">Empiric combination antibiotic therapy is associated with improved outcome against sepsis due to Gram-negative bacteria a retrospective analysis.</a> Antimicrob Agents Chemother. 54, 1742-1748 (2010).</li><li>Seifert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+importance+of+microbiological+findings+in+the+diagnosis+and+management+of+bloodstream+infections.">The clinical importance of microbiological findings in the diagnosis and management of bloodstream infections.</a> Clin Infect Dis. 48, S238-S245 (2009).</li><li>Barenfanger, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+mortality+associated+with+prompt+Gram+staining+of+blood+cultures.">Decreased mortality associated with prompt Gram staining of blood cultures.</a> American journal of clinical pathology. 130, 870-876 (2008).</li><li>Munson, E. L., Diekema, D. J., Beekmann, S. E., Chapin, K. C., Doern, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+treatment+of+bloodstream+infection:+laboratory+reporting+and+antimicrobial+management.">Detection and treatment of bloodstream infection: laboratory reporting and antimicrobial management.</a> J Clin Microbiol. 41, 495-497 (2003).</li><li>Clerc, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry+on+the+clinical+management+of+patients+with+Gram-negative+bacteremia+a+prospective+observational+study.">Impact of matrix-assisted laser desorption ionization time-of-flight mass spectrometry on the clinical management of patients with Gram-negative bacteremia a prospective observational study.</a> Clin Infect Dis. 56, 1101-1107 (2013).</li><li>Meex, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+identification+of+bacteria+from+BacT/ALERT+anaerobic+positive+blood+cultures+by+MALDI-TOF+MS+MALDI+Sepsityper+kit+versus+an+in-house+saponin+method+for+bacterial+extraction.">Direct identification of bacteria from BacT/ALERT anaerobic positive blood cultures by MALDI-TOF MS MALDI Sepsityper kit versus an in-house saponin method for bacterial extraction.</a> Journal of medical microbiology. 61, 1511-1516 (2012).</li><li>Moussaoui, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry+identifies+90%+of+bacteria+directly+from+blood+culture+vials.">Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directly from blood culture vials.</a> Clin Microbiol Infect. 16, 1631-1638 (2010).</li><li>Seng, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ongoing+revolution+in+bacteriology+routine+identification+of+bacteria+by+matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry.">Ongoing revolution in bacteriology routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.</a> Clin Infect Dis. 49, 543-551 (2009).</li><li>Stevenson, L. G., Drake, S. K., Murray, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+bacteria+in+positive+blood+culture+broths+by+matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry.">Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry.</a> J Clin Microbiol. 48, 444-447 (2010).</li><li>Croxatto, A., Prod&amp;34hom, G., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+MALDI-TOF+mass+spectrometry+in+clinical+diagnostic+microbiology.">Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology.</a> FEMS Microbiol Rev. 36, 380-407 (2012).</li><li>Chen, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+bacterial+identification+in+positive+blood+cultures+by+use+of+two+commercial+matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry+systems.">Direct bacterial identification in positive blood cultures by use of two commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry systems.</a> J Clin Microbiol. 51, 1733-1739 (2013).</li><li>Prod&amp;34hom, G., Bizzini, A., Durussel, C., Bille, J., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry+for+direct+bacterial+identification+from+positive+blood+culture+pellets.">Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets.</a> J Clin Microbiol. 48, 1481-1483 (2010).</li><li>Prod&amp;34hom, G., Durussel, C., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+blood-culture+bacterial+pellet+preparation+for+faster+accurate+direct+bacterial+identification+and+antibiotic+susceptibility+testing+with+the+VITEK+2+system.">A simple blood-culture bacterial pellet preparation for faster accurate direct bacterial identification and antibiotic susceptibility testing with the VITEK 2 system.</a> Journal of medical microbiology. 62, 773-777 (2013).</li><li>Clerc, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry+and+PCR-based+rapid+diagnosis+of+Staphylococcus+aureus+bacteraemia.">Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR-based rapid diagnosis of Staphylococcus aureus bacteraemia.</a> Clin Microbiol Infect. , (2013).</li><li>Martiny, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+rapid+microbial+identification+directly+from+positive+blood+cultures+using+matrix-assisted+laser+desorption/ionization+time-of-flight+mass+spectrometry+on+patient+management.">Impact of rapid microbial identification directly from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on patient management.</a> Clin Microbiol Infect. 19, E568-E581 (2013).</li><li>Stoneking, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Would+earlier+microbe+identification+alter+antibiotic+therapy+in+bacteremic+emergency+department+patients.">Would earlier microbe identification alter antibiotic therapy in bacteremic emergency department patients.</a> The Journal of emergency medicine. 44, 1-8 (2013).</li><li>Nordmann, P., Dortet, L., Poirel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+extended-spectrum-beta-lactamase-producing+Enterobacteriaceae.">Rapid detection of extended-spectrum-beta-lactamase-producing Enterobacteriaceae.</a> J Clin Microbiol. 50, 3016-3022 (2012).</li></ol>

Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Par rapport aux approches diagnostiques d&#39;hémoculture positifs classiques, l&#39;acquisition rapide d&#39;un culot bactérien en utilisant le chlorure lyse approche de centrifugation d&#39;ammonium réduire le temps de signaler l&#39;identification de 16 à 24 heures et le temps de signaler AST de 24 à 48 h (figures 1 et 3). Introduction rapide d&#39;une antibiothérapie appropriée est essentielle pour améliorer les résultats des patients souffrant d&#39;infections ...

Les bactériémies et septicémie chez les patients hospitalisés sont une cause majeure de morbidité et de mortalité. Ainsi, la mortalité liée à la circulation sanguine infections est observée chez environ 14% à 37% des patients hospitalisés et peut augmenter de 35% en unités de soins intensifs des patients 1-3. L&#39;identification rapide de l&#39;agent infectieux est essentiel pour guider le traitement optimal des antimicrobiens et à accroître le succès de la thérapie antimicrobienne 4,5. L&#39;analyse rapide de la coloration de Gram de hémoculture positive a déjà un impact significatif sur l&#39;adaptation de la thérapie antimicrobienne 6,7 mais l&#39;identification précise de l&#39;agent infectieux est nécessaire pour fournir le traitement antibiotique le mieux adapté aux patients. Par exemple, les différents régimes de traitement antibiotique doivent être mis en œuvre suivant une bactériémie avec les entérocoques et les streptocoques qui sont difficiles à distinguer par la coloration de Gram. De même, l&#39;identification à l&#39;espèce Level est nécessaire pour détecter les entérobactéries Gram négatif ampC codant pour un gène chromosomique qui confère une résistance accrue aux β-lactames 8. Avec une hémoculture positive, l&#39;approche diagnostique classique consiste à repiquer l&#39;agent infectieux sur différentes plaques d&#39;agar, qui nécessite plusieurs heures d&#39;incubation supplémentaire identification préalable avec diverses approches, y compris des tests biochimiques, la croissance sur différents milieux sélectifs et les systèmes d&#39;identification microbienne automatisés. Le temps les résultats d&#39;une approche diagnostique classique est d&#39;environ 1 à 3 jours. L&#39;émergence de la technologie désorption laser assistée par matrice / ionisation temps de vol spectrométrie de masse (MALDI-TOF) pour l&#39;identification rapide des micro-organismes a fourni un nouvel outil pour identifier rapidement les micro-organismes de colonies cultivées sur des plaques d&#39;agar-agar, mais aussi directement à partir de sang positif cultures (Figure 1) 9-12. Thl&#39;utilisation de l&#39;e de MALDI-TOF d&#39;identifier un agent infectieux à partir d&#39;hémocultures a considérablement réduit le temps aux résultats de quelques minutes au lieu des heures et des jours requis par les méthodes traditionnelles. Comme on le verra par Croxatto et al. 13, l&#39;efficacité de l&#39;identification MALDI-TOF repose sur différents paramètres, y compris la pureté et la quantité du micro-organisme. Ces deux critères sont facilement obtenus à partir des colonies distinctes cultivées sur des plaques d&#39;agar mais nécessaire un traitement pré-analytique pour l&#39;enrichissement bactérienne et purification à partir d&#39;échantillons complexes tels que la culture de sang, qui contiennent plusieurs composants cellulaires et des protéines qui peuvent interférer avec identification MALDI-TOF. Les méthodes d&#39;isolement de divers micro-organismes de culture de sang ont été utilisés dans un certain nombre d&#39;études, y compris la saponine ou une autre méthode de détergents doux pour l&#39;extraction bactérienne 9,14, méthode de séparation de sérum 10, la lyse des méthodes de centrifugation 12 </shaut&gt; et solutions commercialement tels que le kit de sepsityper. Notre laboratoire de diagnostic bactériologique a mis au point un sang-culture simple préparation de culot bactérien à base de chlorure d&#39;ammonium érythrocytes-lyse qui permettent une identification rapide de bactéries et de levures par MALDI-TOF et des systèmes d&#39;identification automatisés (figure 2) 15. Cette préparation de pastilles hémoculture également fournir un échantillon pour d&#39;autres applications directes en aval tels que la coloration de Gram, tests de diagnostic basés sur la PCR automatisées telles que POCT-PCR pour la détection rapide de résistant à la méthicilline Staphylococcus aureus (SARM) et les tests de sensibilité aux antibiotiques des Les systèmes automatisés d&#39;AST et / ou par des essais de diffusion de disque sur plaques d&#39;agar-agar (figure 3). Dans ce travail, nous décrivons les différentes étapes de la préparation du culot bactérien sang-culture, comme expliqué par Prod&#39;hom et al. 15 (figure 4). Nous allons également déscribe les protocoles pour trois des principales applications qui peuvent être exécutées sur le culot de la culture de sang: Identification par MALDI-TOF 15, l&#39;identification (ID) et les tests de sensibilité aux antibiotiques (AST) avec les systèmes automatisés 16 pour les entérobactéries et les staphylocoques et PCR automatisé test diagnostique pour la détection du SARM 17.

<table border="0" cellpadding="0" cellspacing="0" width="1130">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">20 needle gauge&#160;</td>
			<td style="width:268px;">Terumo, Leuven, Belgium</td>
			<td style="width:239px;">NN-2038R</td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">50 ml Falcon tube</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>352070</td>
			<td>50 ml centrifuge tubes</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ammonium chlorure</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>101145</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Potassium hydrogen carbonate</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>60340</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>F0507</td>
			<td>Flammable, corrosive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-Cyano-4-hydroxycinnamic acid</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>70990</td>
			<td>Acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Acetonitrile</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>271004</td>
			<td>Flammable, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Trifluoroacetic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>T6508</td>
			<td>Corrosive, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 60 instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27202</td>
			<td>automated microbial system instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-positive (GP) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21342</td>
			<td>automated GP&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-P580 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>22233</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-negative (GN) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21341</td>
			<td>automated GN&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-N242 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>413391</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Xpert MRSA</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>&#160;GXMRSA-100N-10</td>
			<td>nucleic acid amplification technology MRSA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">GeneXpert IV instrument</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>GXIV-4-D</td>
			<td>nucleic acid amplification technology instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Microflex LT MALDI-TOF MS instrument</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>BDAL microflex LT/SH</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MSP 96 target&#160; steel BC</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>280799</td>
			<td>MALDI target plate</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Densitometer Densicheck instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27208</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MALDI Sepsityper kit 50</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>8270170</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mac Conkey agar</td>
			<td>Biolife, Milano, Italy</td>
			<td>4016702</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mueller-Hinton agar</td>
			<td>Oxoid, Hampshire, England</td>
			<td>CM0337</td>
			<td>Mueller-Hinton agar (MH)&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MHF agar</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>43901</td>
			<td>Mueller-Hinton agar-fastidious organisms agar (MHF)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD columbia III agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254071</td>
			<td>blood agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD chocolate agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254089</td>
			<td>chocolate agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD schaedler agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254084</td>
			<td>Schaedler agar</td>
		</tr>
	</tbody>
</table>

preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing

Les bactériémies et septicémie sont une cause majeure de morbidité et de mortalité. Le succès des patients atteints de bactériémie dépend de l&#39;identification rapide de l&#39;agent infectieux pour guider le traitement antibiotique optimal. L&#39;analyse de la coloration de Gram de la culture de sang positive peut être réalisée rapidement et déjà d&#39;impact significatif sur le traitement antibiotique. Cependant, l&#39;identification précise de l&#39;agent infectieux est toujours nécessaire pour établir le traitement optimal ciblée. Nous présentons ici une préparation de culot bactérien simple et rapide à partir d&#39;une hémoculture positive qui peut être utilisé comme un échantillon de plusieurs applications essentielles en aval telles que l&#39;identification par MALDI-TOF MS, les tests de sensibilité aux antibiotiques (AST) par méthode de diffusion sur disque ou systèmes AST automatisés et par PCR automatisé à base de tests de diagnostic. La performance de ces systèmes d&#39;identification et AST différentes appliquées directement sur les culots bactériens d&#39;hémoculture est très similar à la performance s&#39;obtient normalement à partir des colonies isolées cultivées sur des plaques d&#39;agar-agar. Par rapport aux approches conventionnelles, l&#39;acquisition rapide d&#39;un culot bactérien réduit considérablement le temps de signaler la fois l&#39;identification et AST. Ainsi, suite à sang positivité de la culture, de l&#39;identification par MALDI-TOF peut être signalée en moins de 1 heure alors que les résultats d&#39;AST par des systèmes AST automatisés ou des essais de diffusion de disque dans les 8 à 18 heures, respectivement. De même, les résultats d&#39;un test rapide basé sur la PCR peuvent être communiqués aux cliniciens moins de 2 heures suivant le rapport d&#39;une bactériémie. Ensemble, ces résultats démontrent que la préparation rapide d&#39;un culot bactérien de la culture du sang a un impact significatif sur le temps d&#39;identification et AST redressement et donc sur le succès de patients souffrant d&#39;infections de la circulation sanguine.

Dans l&#39;étude effectuée par Prod&#39;hom et al. 15, culots bactériens obtenus par lyse de chlorure d&#39;ammonium centrifugation de 122 hémoculture positive à partir de 78 patients ont été analysés par MALDI-TOF MS. Sur 122 hémoculture positive, 95 (77,9%) ont été correctement identifiés au niveau de l&#39;espèce et un (0,8%) au niveau du genre. Les 26 (21,3%) des pastilles d&#39;hémoculture restants n&#39;ont donné aucune identification fiable par MALDI-TOF. Parmi ceux-ci, 21 sont ...

Watch this Scientific Journal Video about Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing at JoVE.com

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Une préparation de culot bactérien rapide à partir d&#39;une culture de sang positif peut être utilisé comme échantillon pour des applications telles que l&#39;identification par MALDI-TOF, la coloration de Gram, les tests de sensibilité aux antibiotiques et le test basé sur la PCR. Les résultats peuvent être transmis rapidement aux cliniciens pour améliorer les résultats des patients souffrant d&#39;infections du courant sanguin.

Préparation d&#39;une hémoculture Pellet pour Rapid bactérienne identification et les tests de sensibilité aux antibiotiques

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

preparation-blood-culture-pellet-for-rapid-bacterial-identification

Research

JoVE Journal

Immunology and Infection

35.4K vues.  University Hospital Center and University of Lausanne.  Une préparation de culot bactérien rapide à partir d'une culture de sang positif peut être utilisé comme échantillon pour des applications telles que l'identification par MALDI-TOF, la coloration de Gram, les tests de sensibilité aux antibiotiques et le test basé sur la PCR. Les résultats peuvent être transmis rapidement aux cliniciens pour améliorer les résultats des patients souffrant d'infections du courant sanguin. 

Vidéo: Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Une plaque de microtitrage à 96 puits à base Méthode de formation suivi et les tests de sensibilité antifongique des Candida albicans Les biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Recherche

Immunologie et infection

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Protocole pour vaccins recombinants contre le SRAS RBD-base: préparation de protéines, vaccination des animaux et de détection de neutralisation

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Une procédure de 1,5 heure pour l&#39;identification des Enterococcus Espèces directement à partir d&#39;hémocultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Test de sensibilité aux antimicrobiens du complexe Mycobacterium tuberculosis des drogues de première et deuxième par dilution en bouillon dans un format microplaque

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Présentation de contrainte de cisaillement dans l&#39;étude de l&#39;adhésion bactérienne

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Schéma de flux de travail d&#39;une journée pour la détection de pathogènes et des bactéries tests de résistance antimicrobienne à partir d&#39;hémocultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

Identification rapide des bactéries à Gram négatif de sang Bouillon de culture à l&#39;aide MALDI-TOF spectrométrie de masse

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Une méthode de référence de microdilution de bouillon pour dalbavancine<em&gt; In Vitro</em&gt; Tests de sensibilité des bactéries qui se développent Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Une méthode rapide pour le traitement du sang pour augmenter le rendement des niveaux peptidiques plasma dans le sang humain

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Culture de Macrophages Factor macrophage colony-stimulating Differentiated humaine monocytes dérivés

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...