<html> 1. Hémoculture bouillons Signaler comme «Positive» <ol><li> Retirez la bouteille d&#39;hémoculture signalé du cabinet continue d&#39;incubation de surveillance et le placer dans une enceinte de sécurité biologique. Remarque: Les bouteilles peuvent contenir des micro-organismes dangereux et les précautions universelles doivent être suivies. En raison du risque d&#39;aérosols infectieux dans l&#39;échantillonnage, toutes les procédures d&#39;échantillonnage doivent être effectuées dans une hotte à flux laminaire de biosécurité de classe II. </li></ol> 2. Coloration de Gram est préparé <ol><li> Préparer une coloration de Gram à partir du bouillon de culture de sang signalé conformément aux protocoles institutionnels locaux. Remarque: Lorsque des organismes Gram négatifs sont identifiés sur la microscopie du bouillon de culture de sang est traité selon la méthode suivante. Lorsque des organismes Gram positifs sont identifiés, une méthode moléculaire alternatif visant l&#39;identification génétique et les marqueurs de résistance est appliquée au bouillon (pas abordé dans cet article) 13. </li></ol>e &quot;&gt; 3. Transfert de sang Marqué Bouillon de culture à un tube de séparation de sérum <ol><li> Mélanger doucement le flacon d&#39;hémoculture en inversant 2-3x. </li><li> Au sein de l&#39;enceinte de sécurité biologique fixer une seringue de 10 ml à un dispositif de transfert du sang de sécurité. </li><li> Attacher le dispositif de transfert du sang vers le flacon de culture de sang et de retirer 5 ml du bouillon dans la seringue. Transférer le bouillon de la Colombie-Britannique aspiré dans un tube de séparation de sérum. </li></ol> 4. Concentration de sang Bouillon de culture <ol><li> Centrifuger le tube de séparation de sérum à 1 250 xg pendant 15 min, ce qui supprime un grand volume de globules rouges. </li><li> Aspirer et jeter le surnageant à l&#39;aide d&#39;une pipette de transfert stérile en prenant soin de laisser environ 1 ml de la couche leuco-plaquettaire immédiatement au-dessus de l&#39;interface gel / fluide. Remarque: Les bouteilles d&#39;aérobie montrent une nette séparation des globules rouges au fond du tube avec l&#39;interface gel / fluide apparaissant une couleur blanc opaque. En revanche, lorsqueappliqué à la anaérobie bouillon de culture de sang lytique l&#39;interface gel / liquide apparaît comme une couleur rouge foncé en raison des composants de cellules lysées de sang rouge restant en suspension dans le surnageant. </li></ol> 5. Répétez les étapes de lavage centrifugation <ol><li> Mélanger délicatement les dernières 1 ml de buffy manteau fluide au-dessus de l&#39;interface de gel avec une pipette stérile, puis transférer la totalité du volume dans un tube de 1,5 ml. Centrifuger le nouveau tube à 288 g pendant 30 sec. </li><li> Transférer le surnageant en utilisant un jetable de 1 ml pipette de transfert en plastique dans un nouveau tube de 1,5 ml et jeter le tube avec le culot. Remarque: Il est essentiel lors de cette étape que l&#39;on prend soin d&#39;éviter le transfert de la pastille. Ceci est particulièrement important pour un échantillon en cours de traitement à partir de la bouteille de BC anaérobie comme le surnageant reste pigmenté et le culot est pas toujours clairement visible. </li></ol> 6. Lyse des cellules résiduelles </p&gt; <ol><li> Centrifugeuse l&#39;échantillon à 18 407 xg pendant 1 min, puis aspirer (et conserver) le surnageant à l&#39;aide d&#39;une amende transfert à bout pipette pour laisser le moins de liquide résiduel que possible sans perturber le culot. </li><li> Reprendre le culot résiduel dans 1 ml de DNAse et RNAse stérile eau libre par aspiration et refoulement. Remarque: Certains auteurs ont décrit l&#39;utilisation de solutions d&#39;alcool à la place de l&#39;eau stérile pour remettre le culot qui rend les bactéries non viables. </li><li> Centrifuger la solution remises en suspension à 18 407 g pendant 1 min. </li></ol> 7. Extraction des protéines bactériennes <ol><li> Aspirer et jeter le surnageant, à nouveau par aspiration avec une pointe pipette bien veiller à ce que le plus de liquide possible est retiré. </li><li> Reprendre le culot dans 10 ul d&#39;acide formique (70% v / v). Remarque: L&#39;acide formique peut affecter le corps si elle est inhalée ou si elle vient en contact avec la peau. Lors de la préparation ou la manipulation de solutions, il est rRecommandés que le personnel utilise une hotte de chimiste en plus de l&#39;équipement de protection individuelle. </li><li> Bien mélanger à l&#39;aide de la pipette afin d&#39;assurer une remise en suspension solution homogène. Utilisation de l&#39;embout de pipette pour perturber physiquement la pastille peut être nécessaire pour assurer une solution plus homogène. </li></ol> 8. Préparation de MALDI-TOF Plaque de mire <ol><li> Inoculer une assiette propre cible MALDI-TOF avec 2 pi de la suspension préparée par point cible. Préparer trois sites cibles pour chacun des échantillons de surmonter les problèmes occasionnels de lit échoué. </li><li> Laisser la plaque cible MALDI-TOF à sécher. La vitesse de séchage peut être augmentée en plaçant la plaque sur le bord de la hotte afin d&#39;optimiser le débit d&#39;air à travers la plaque. </li><li> Superposer chaque spot séché avec 1 pl de la solution de matrice (10 mg / ml α-cyano-4-hydroxycinnamique (HCCA), 50% d&#39;acétonitrile, l&#39;acide trifluoroacétique 2,5%). Remarque: solution Matrix peut affecter le corps si elle est inhalée ou si elle vient en contact avec la peau. Lors de la préparation ou la manipulation de solutions, il est recommandé que le personnel utilise une hotte de chimiste en plus de l&#39;équipement de protection individuelle. </li><li> Laisser la plaque cible MALDI-TOF à sécher sur le bord de la hotte de laboratoire, comme décrit ci-dessus. Assurez-vous une préparation homogène remplit chacun des points cibles sur la plaque. </li></ol> 9. Lieu MALDI-TOF plaque cible dans le spectromètre de masse <ol><li> Insérer la plaque cible MALDI-TOF dans le spectromètre de masse MALDI-TOF. S&#39;assurer que la plaque est assis au ras des plaques de ressort. Remarque: ne pas insérer la plaque cible jusqu&#39;à ce que tous les points cibles sont secs. </li><li> S&#39;assurer que le joint en caoutchouc est propre de peluches, poussières et des cheveux pour permettre les conditions de vide nécessaires peuvent être créés. </li><li> Fermez le couvercle du stade MALDI-TOF et puis appuyez sur le bouton &quot;in / out&quot; sur le devant de l&#39;appareil. Le couvercle sera maintenant verrouiller et permettre le vide nécessaire à créer. </li></ol> 10. MALDI-TOF MS Spectra acquisition <ol><li> Ouvrez le MALDI Biotypage et logiciels FlexControl. </li><li> Dans le logiciel de dactylographie sélectionnez &quot;nouvelle classification&quot; dans le menu déroulant intitulé &quot;fichier&quot;. Nommez le projet et puis sélectionnez «nouveau». Sélectionnez &quot;Suivant&quot; pour terminer l&#39;installation du logiciel de dactylographie. </li><li> Dans la fenêtre du logiciel de contrôle d&#39;identifier le graphique sur l&#39;écran de la plaque cible. Mettre en évidence les points cibles MALDI-TOF qui sont en usage en déplaçant la souris sur les points inoculés tout en maintenant le bouton gauche de la souris. </li><li> Utilisez le bouton droit de la souris pour cliquer n&#39;importe où sur les postes cibles sélectionnés, puis sélectionnez &quot;ajouter analytes&quot; dans le menu déroulant. </li><li> Ajouter sang culture détails d&#39;identification de la colonne &quot;ID&quot; pour chacun des points cibles et cliquez sur &quot;Suivant&quot; lorsque vous avez terminé. </li><li> Sélectionnez la base de données de spectres &quot;de la taxonomie&quot; commercial et sélectionnez &quot;Suivant&quot;. Remarque: Labotoires peuvent utiliser supplémentaires en interne ou bases de données commerciales pour augmenter le nombre de spectres de référence. </li><li> Cliquez sur &quot;Terminer&quot; et le MALDI-TOF MS commencera à générer des spectres. Remarque: Les paramètres MALDI-TOF ont été selon les paramètres de fabrication (en mode linéaire positif, 60 Hz fréquence de laser, 20 kV tension d&#39;accélération, 16,7 kV tension de IS2, 170 ns retard d&#39;extraction, et 2,000-20,137 m / z gamme). </li><li> Si le logiciel de contrôle MALDI échoue à générer des pics, saisie manuelle des spectres peut être effectuée (méthode non décrite). </li></ol> 11. Analyse de post interprétation de MALDI-TOF MS Partitions <ol><li> Une fois les spectres sont acquis et l&#39;analyse est terminée, cliquez sur le bouton &quot;+&quot; à côté de l&#39;identification des espèces sur le logiciel de dactylographie pour développer la liste d&#39;identification de chaque cible. </li><li> Étudier le top 5 des identifications, notant si le top 5 sont similaires. Discordants top 5 des genres avec des scores ≥ 1.7 de la même cibleplace a évoqué la possibilité de bouillons mixtes. Remarque: la technologie MALDI-TOF est peu sensible pour la détection de bouillons mixtes. Par exemple, quand un bouillon contient des espèces mixte un score élevé de confiance peut être identifié pour que l&#39;une des espèces mixtes. </li><li> Quand un score ≥ 2.0 est noté pour le plus grand match sur un point cible, signaler les espèces. </li><li> Lorsque le score le plus élevé sur un point cible est ≥ 1,7, mais &lt;2,0, rapport que les genres. Si les critères supplémentaires des 5 meilleurs identifications sont concordantes, ensuite rapport au niveau de l&#39;espèce. </li></ol> 12. Enlèvement de la plaque cible MALDI-TOF <ol><li> Lorsque tous les spectres sont presse le bouton &quot;in / out&quot; sur la machine MALDI-TOF. </li><li> Ouvrez le MALDI-TOF plaque cible récipient et retirer la plaque cible MALDI-TOF. </li><li> Si vous utilisez une plaque cible MALDI-TOF réutilisables, nettoyer toutes les taches de cibles à l&#39;aide d&#39;alcool et de stocker la plaque MALDI-TOF à température ambiante une fois qu&#39;elle est propre et sèche. </li></ol>

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

Il est important lors de l&#39;application MALDI-TOF MS de bouillon de culture de sang que les étapes de post de centrifugation sont effectuées avec suffisamment de soin à ne pas remixer les composants séparés. Il est particulièrement important de supprimer les constituants de culture de sang et de protéines cellulaires humaines, y compris l&#39;hémoglobine, qui peuvent produire des pointes interférant avec les spectres MALDI-TOF. Bien que MS fabrique recommander couper du score de ...

Les patients atteints d&#39;une infection sanguine (BSI) en raison de bactéries continuent d&#39;avoir une forte mortalité à l&#39;hôpital, allant de 6 à 48% 1. La livraison de l&#39;antibiotique empirique approprié favorise la survie et dans le sous-ensemble de patients atteints de sepsis sévère, chaque heure de retard à un traitement approprié est corrélée à une diminution de la survie de 2,3. En conséquence, un objectif clé du laboratoire clinique est de détecter rapidement, identifier et communiquer la présence de bactéries dans les cultures de sang pour éclairer les décisions cliniques. Il a été démontré que le laboratoire de microbiologie a la plus grande influence sur la thérapie antimicrobienne au moment de la notification de la coloration de Gram 4 et récemment, une étude d&#39;observation a démontré que le temps de désorption laser assistée par matrice / ionisation de spectrométrie de masse de vol (MALDI-TOF MS) réalisée directement sur ​​des bouillons de culture de sang influencer la prescription dans plus d&#39;un tiers de BSI causée par des bactéries Gram négatif 5. 6,7. La technologie est maintenant bien établie et a été intégrée dans de nombreux laboratoires pour l&#39;identification rapide et précise des micro-organismes isolés sur solide 6,8 médiatique. L&#39;application directe de MALDI-TOF MS à la culture de sang (BC) de bouillon qui ont signalé «positif» pour les micro-organismes recours à des cliniciens et des gestionnaires de laboratoire en raison de la possibilité d&#39;obtenir une identification plus rapide des micro-organismes à faible coût. L&#39;utilité clinique de l&#39;application directe de MALDI-TOF MS de bouillon de culture de sang a été limitée par le large éventail de sensibilités observées par rapport aux méthodes basées sur la culture de phénotypique norme d&#39;identification, avec les rapports de l&#39;identification réussie des bactéries Gram négatives allant de 47 à 98,9 9-11%. La variation de la sensibilité probableconcerne la composition BC bouillon, la concentration bactérienne initiale, la variation dans les méthodes de préparation des échantillons ainsi que le réseau des organismes Gram négatif rencontrées dans les populations d&#39;étude 9. Par rapport à ces autres protocoles publiés la méthode présentée ici permet d&#39;éviter l&#39;utilisation de l&#39;éthanol, le chlorure d&#39;ammonium ou supplémentaire (non-matrice) acétonitrile. Par conséquent, le culot bactérien demeure viable (jusqu&#39;à ce que le point d&#39;extraction de la protéine) permettant méthodes potentielles de test phénotypique de susceptibilité à être appliqués directement sur ces micro-organismes dans le bouillon. En outre, la méthode présentée a été montré pour être peu coûteux, fiable et rapide de l&#39;identification bactérienne disponible dans 25 min de la culture de sang Gram résultats de la coloration, avec un minimum de «pratique» du temps 12. Cette méthode est un protocole simple en interne spin-lyse en utilisant l&#39;extraction de l&#39;acide formique est appliqué directement sur des bouillons de culture de sang positifs pour identifier Gram négatif bacteria avec la technologie MALDI-TOF MS.

<table border="0" cellpadding="0" cellspacing="0" width="1319">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">BACTEC Plus Aerobic/F Medium</td>
			<td style="width:307px;">Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right" style="width:188px;">442192</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Lytic/10 Anaerobic /F Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442265</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Peds Plus Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442194</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer - Blood transfer device</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">364880</td>
			<td>Single use sampling device reducing the risk of needlestick injury</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer SST 5.0mL, Advance plus</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">367954</td>
			<td>Serum separating tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe 10 mL</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">302143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Samco, USA</td>
			<td>222-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette (fine tipped)</td>
			<td>Samco, USA</td>
			<td>232-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge tube (2.0 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030.120.094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile water (DNAse and RNAse free)</td>
			<td>Life Technologies, Carlsbad, California, USA</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>F0507</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix solution&#160;</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td align="right">285074</td>
			<td>10 mg/ml &#945;-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Benchtop microflex LT MALDI-TOF MS</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td></td>
			<td>Utilizing BioTyper 3.1 (Build 65) and FlexControl 3.3 (Build 99) software</td>
		</tr>
	</tbody>
</table>

rapid identification of gram negative bacteria from blood culture broth using maldi-tof mass spectrometry

Un rôle important du laboratoire de microbiologie clinique est de fournir une identification rapide de bactéries causant l&#39;infection sanguine. Identification traditionnelle nécessite la sous-culture de signalé bouillon de culture de sang avec identification disponible uniquement après que les colonies sur gélose solide ont mûri. MALDI-TOF MS est une méthode fiable pour l&#39;identification rapide de la majorité des bactéries cliniquement pertinentes lorsqu&#39;il est appliqué à des colonies sur un milieu solide. L&#39;application de MALDI-TOF MS directement bouillon de culture de sang est une approche intéressante car elle a le potentiel d&#39;accélérer l&#39;identification des espèces de bactéries et d&#39;améliorer la gestion clinique. Cependant, un problème important à surmonter est la pré-analyse suppression de brouilleurs résines, des protéines et de l&#39;hémoglobine contenue dans le sang culture spécimens qui, s&#39;il n&#39;est pas retiré, interfèrent avec les spectres MS et peuvent entraîner des scores d&#39;identification discrimination insuffisantes ou faibles. En outre, il est nécessaire de concentrer bacteria pour développer les spectres d&#39;une qualité suffisante. La méthode présentée décrit la concentration, la purification et l&#39;extraction des bactéries Gram négatives permettant l&#39;identification précoce des bactéries à partir d&#39;un bouillon de culture de sang signalé.

Le MS spectres MALDI-TOF généré sont comparés à la base de données de référence intégrée des spectres. Un score logarithmique est attribué pour la confiance du match entre l&#39;isolat de test et la base de données de référence isolats, avec la recommandation d&#39;un score ≥ 1,7 nécessaires à l&#39;identification probable au niveau de la génération (figure 1) et ≥ 2,0 pour une identification probable des espèces (Figure 2). Rapport au niveau de l&#39;espèce lors...

Watch this Scientific Journal Video about Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry at JoVE.com

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

L&#39;application de laser désorption / ionisation de temps assistée par matrice de vol (MALDI-TOF), la spectrométrie de masse (MS) directement à un bouillon de culture de sang accélère l&#39;identification de bactéries. La méthode présentée est une méthode rapide et fiable pour l&#39;identification des bactéries à Gram négatif directement à partir de bouillon de culture de sang.

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 ...

rapid-identification-gram-negative-bacteria-from-blood-culture-broth

Research

JoVE Journal

Immunology and Infection

23.8K vues.  Westmead Hospital.  L'application de laser désorption / ionisation de temps assistée par matrice de vol (MALDI-TOF), la spectrométrie de masse (MS) directement à un bouillon de culture de sang accélère l'identification de bactéries. La méthode présentée est une méthode rapide et fiable pour l'identification des bactéries à Gram négatif directement à partir de bouillon de culture de sang. 

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

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 ...

Recherche

Immunologie et infection

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Purification et visualisation de lipopolysaccharide de bactéries à Gram négatif par Hot-phénol aqueux d&#39;extraction

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Détection des bactéries à l&#39;aide ADNzymes fluorogènes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

Isolation de Double Negative αβ cellules T du rein

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

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

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

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 ...

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, ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

Identification des bactéries pathogènes rares par 16S ARNr Gene Séquençage et MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

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 ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextrane augmente l’efficacité de la Transduction des gènes des cellules NK primaires murines et humaines

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...