Ce travail a été soutenu par une bourse de carrière Burroughs-Wellcome et par les Instituts nationaux de la santé accorde AI074832 au GY Liu.

1. Préparation de la SDRM de l&#39;infection (deux jours avant l&#39;infection) <ol><li> Inoculer une anse de SARM à partir d&#39;une culture d&#39;actions à une gélose au sang (gélose trypticase-soja (TSA)) la plaque. </li><li> Vérifiez le phénotype hémolyse (une zone claire autour de chaque colonie) sur la plaque de gélose au sang. </li><li> Choisissez une colonie qui a un phénotype hémolytique qui est compatible avec d&#39;autres expériences. </li><li> Inoculer la colonie dans 3 ml de bouillon Todd Hewitt (THB), avec l&#39;antibiotique approprié, si nécessaire, dans un composant logiciel enfichable 15 ml tube à bouchon. </li><li> Incuber à 37 ° C pendant une nuit sous agitation à 220 rpm. </li></ol> 2. Préparation de la souris de l&#39;infection (un jour avant l&#39;infection) <ol><li> Selon la taille de la souris, se raser la fourrure au large une zone de 3 x 4 cm sur le dos de souris. </li><li> Appliquer ~ 5 mm 3 de crème dépilatoire (acheté dans un magasin local de drogue) pour la zone rasée. </li><li> Laissez la crème dépilatoire à incuber sur la surface de la peau pendant environ 1 min. </li><li> Serviettes de papier humide avec ddH 2 O. </li><li> Essuyez la crème dépilatoire avec la serviette en papier humide. </li></ol> 3. Préparation du SARM d&#39;infection (le jour de l&#39;infection) <ol><li> Diluer la culture bactérienne à la nuit de 1:500 à 1:1000 avec 10 ml de pré-chauffé THB, dans un 50 ml à bouchon à vis tube. </li><li> Incuber à 37 ° C pendant environ 2,5 heures avec agitation à 220 rpm, jusqu&#39;à 540 A atteint 2,5. </li><li> Recueillir SARM par centrifugation à 3225 xg pendant 10 min à 4 ° C. </li><li> Jeter le surnageant. </li><li> Reprendre le culot bactérien dans 10 ml de phosphate de Dulbecco saline tamponnée (DPBS). </li><li> Répétez les étapes 3 et 4. </li><li> Reprendre le culot bactérien dans du DPBS à une concentration désirée. </li><li> Série diluer la suspension bactérienne à partir 10 janvier à 10 août. </li><li> Assiette de la suspension diluée bactérienne sur gélose au sang. </li><li> Incuber les plaques à 37 ° C pendant la nuit. </li><li> Observer et enregistrer le phénotype hémolytique de l&#39;inoculum. </li><li> Compter les unités formant colonie (UFC) Numéro de la plaque. </li><li> Calculer les inoculums basé sur le nombre UFC sur la plaque. </li></ol> 4. Infection sous-cutanée de SARM (sur le jour de l&#39;infection et 3 jours post-infection) <ol><li> Injecter par voie sous cutanée 100 uL de suspension bactérienne dans la zone rasée. </li><li> Observer les animaux de 3 à 5 heures après l&#39;injection pour s&#39;assurer que les souris sont vivantes. </li><li> Observez la lésion sur le dos de l&#39;animal tous les jours et enregistrer la zone de lésion quotidienne quand elle est nécessaire. L&#39;observation des lésions devrait inclure l&#39;enregistrement de la taille de la lésion et la morphologie. Les deux lésions cutanées et musculaires sont quantifiés en multipliant la longueur et la largeur de la lésion. Lésions de forme irrégulière doivent être décomposés en petits morceaux symétriques, et chaque pièce mesurée par la même méthode. Mesures lésion peut également utiliser un programme assisté par ordinateur évaluation histomorphométrique (ImageJ, open-source disponible à partir du NIH à <a href="http://rsb.info.nih.gov/ij">http://rsb.info.nih.gov/ij/</a> ) 2. </li><li> Sacrifice des animaux au jour 3 après l&#39;infection par inhalation d&#39;isoflurane suivi par dislocation cervicale. </li><li> Observer et enregistrer la lésion sur la peau. </li><li> Nick la peau avec une paire de ciseaux stériles. </li><li> Peler la peau soigneusement et observer et d&#39;enregistrer la lésion sur le muscle. </li><li> Accise de la lésion et d&#39;environ 2 - 5 mm de la zone environnante. </li><li> Recueillir la rate et les reins. </li><li> Homogénéiser les tissus à l&#39;aide d&#39;un homogénéiseur de tissus ou par l&#39;application répétée d&#39;un piston d&#39;une seringue de 1 mL dans un tube à centrifuger contenant le tissu et 100 uL de DPBS. </li><li> Ajouter 900 uL de DPBS à chaque tube. </li><li> Vortex les échantillons pendant 5 min. </li><li> Série diluer la suspension avec du DPBS janvier 10 à juin 10. </li><li> Assiette des suspensions diluées sur des plaques THA. </li></ol> 5. Les résultats représentatifs: <ol><li> Immédiatement après chaque injection sous-cutanée, une cloque sera observée sur la surface de la peau lorsque l&#39;infection est fait correctement (figure 1A). Si aucune bulle est observé sur la surface de la peau, l&#39;injection peut être trop profond, et cela peut affecter le résultat de l&#39;infection (taille de la lésion, UFC). </li><li> La suspension bactérienne doit être dilué en série et l&#39;UFC doivent être examinés sur gélose au sang pour chaque expérience. Cette étape donnera des informations importantes: 1) si l&#39;inoculum ont phénotype homogène; 2) l&#39;exacte comptent viables bactérienne de l&#39;infection. </li><li> Les lésions et la survie bactérienne peut être évaluée à différents temps post-infection. Ici, nous avons examiné l&#39;infection sur trois jours post-infection. La taille des lésions sont analysés sur la surface de la peau (Figure 1B: 1x 10 9; Figure 1D: 1 x 10 7 UFC) et sur ​​la surface du muscle (figure 1C: 1x 10 9 </sjusqu&#39;à&gt;; figure 1E: 1 x 10 7 UFC). La zone de la lésion est égale à la longueur (mm) x largeur (mm) peuvent être mesurés pour évaluer les dommages aux tissus (fig. 2 A et B). Ce résultat peut aider à déterminer l&#39;étendue des dommages aux tissus causés par un facteur de virulence donné. De plus, les lésions sont excisées et homogénéisé dans DPBS, et plaqué pour quantifier le nombre UFC (figure 2C), qui indique la viabilité des bactéries sur le site de l&#39;infection. </li></ol><img src="/files/ftp_upload/2528/2528fig1.jpg" alt="Figure 1" /> Figure 1. La peau et les lésions musculaires. CD1 souris ont été inoculées sous-cutanée sur un flanc par le SARM (BAC). Les photos ont été prises à 0 heure et le jour 3 après l&#39;infection. (A) 0 Temps de post-infection (pi), (B) - (G) Jour 3 pi; B, D. et F: lésions cutanées, C, E et G: lésions musculaires; B et C: 1x 10 9 UFC infectés; D et E: 1x 10 7 UFC infectés; F et G: maquette. <img src="/files/ftp_upload/2528/2528fig2.jpg" alt="Figure 2" /> Figure 2. Taille de la lésion de la peau et les bactéries viables comptent sur ​​le site de l&#39;infection sur trois jours post-infection. Lésions cutanées et musculaires sur le SARM (BAC) infectés souris CD1. (A) la taille des lésions cutanées. (B) la taille des lésions musculaires. (C) Total des tissus UFC. H: 1 x 10 9 UFC inoculums; L: 1 x 10 7 UFC inoculums. *: P &lt;0,05, test de Mann-Whitney.

<ol>
	<li>Ahn, J. Y., Song, J. Y., Yun, Y. S., Jeong, G., Choi, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+Staphylococcus+aureus-infected+septic+mice+by+suppression+of+early+acute+inflammation+and+enhanced+antimicrobial+activity+by+ginsan.">Protection of Staphylococcus aureus-infected septic mice by suppression of early acute inflammation and enhanced antimicrobial activity by ginsan.</a> FEMS Immunol Med Microbiol. 46, 187-197 (2006).</li><li>Girish, V., Vijayalakshmi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affordable+image+analysis+using+NIH+Image/ImageJ.">Affordable image analysis using NIH Image/ImageJ.</a> Indian J Cancer. 41, 47-47 (2004).</li><li>Hahn, B. L., Onunkwo, C. C., Watts, C. J., Sohnle, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+dissemination+and+cutaneous+damage+in+a+mouse+model+of+staphylococcal+skin+infections.">Systemic dissemination and cutaneous damage in a mouse model of staphylococcal skin infections.</a> Microb Pathog. 47, 16-23 (2009).</li><li>Hruz, P., Zinkernagel, A. S., Jenikova, G., Botwin, G. J., Hugot, J. P., Karin, M., Nizet, V., Eckmann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOD2+contributes+to+cutaneous+defense+against+Staphylococcus+aureus+through+alpha-toxin-dependent+innate+immune+activation.">NOD2 contributes to cutaneous defense against Staphylococcus aureus through alpha-toxin-dependent innate immune activation.</a> Proc Natl Acad Sci U S A. 106, 12873-12878 (2009).</li><li>Ji, Y., Zhang, B., Van, S. F., Horn, P. W. a. r. r. e. n., Woodnutt, G., Burnham, M. K., Rosenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+critical+staphylococcal+genes+using+conditional+phenotypes+generated+by+antisense+RNA.">Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA.</a> Science. 293, 2266-2269 (2001).</li><li>Somerville, G. A., Beres, S. B., Fitzgerald, J. R., DeLeo, F. R., Cole, R. L., Hoff, J. S., Musser, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+serial+passage+of+Staphylococcus+aureus:+changes+in+physiology,+virulence+factor+production,+and+agr+nucleotide+sequence.">In vitro serial passage of Staphylococcus aureus: changes in physiology, virulence factor production, and agr nucleotide sequence.</a> J Bacteriol. 184, 1430-1437 (2002).</li><li>Tseng, C. W., Kyme, P., Low, J., Rocha, M. A., Alsabeh, R., Miller, L. G., Otto, M., Arditi, M., Diep, B. A., Nizet, V., Doherty, T. M., Beenhouwer, D. O., Liu, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+Panton-Valentine+leukocidin+contributes+to+inflammation+and+muscle+tissue+injury.">Staphylococcus aureus Panton-Valentine leukocidin contributes to inflammation and muscle tissue injury.</a> PLoS One. 4, e6387-e6387 (2009).</li><li>von Kockritz-Blickwede, M., Rohde, M., Oehmcke, S., Miller, L. S., Cheung, A. L., Herwald, H., Foster, S., Medina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+mechanisms+underlying+the+genetic+predisposition+to+severe+Staphylococcus+aureus+infection+in+the+mouse+model.">Immunological mechanisms underlying the genetic predisposition to severe Staphylococcus aureus infection in the mouse model.</a> Am J Pathol. 173, 1657-1668 (2008).</li></ol>

<ol><li> La peau douce et murin modèle d&#39;infection des tissus est un outil puissant pour l&#39;évaluation in vivo de virulence d&#39;un pathogène. La pathogénicité de S. aureus dans les infections cutanées et des tissus mous peuvent varier en fonction d&#39;un certain nombre de paramètres. Il s&#39;agit notamment de taille de l&#39;inoculum, la phase de croissance bactérienne, la profondeur de l&#39;inoculation, l&#39;âge des souris, et le fond génétique de la souris 7, 8. Lorsque l&#39;on examine la ...

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subcutaneous infection of methicillin resistant staphylococcus aureus (mrsa)

Le SARM est une menace mondiale pour la santé publique, et la peau SARM et des tissus mous représentent désormais plus de la moitié de toutes les infections des tissus mous dans les États-Unis. Parmi les infections des tissus mous, la myosite, pyomyosite, et la fasciite nécrosante ont été de plus en plus signalés en association avec le SARM provenant de la communauté. Pour comprendre l&#39;interaction entre le SARM et immunité de l&#39;hôte conduisant à l&#39;infection plus sévère, la disponibilité de modèles animaux est critique, permettant l&#39;étude de l&#39;hôte et facteurs bactériens. Plusieurs modèles d&#39;infection ont été introduites afin d&#39;évaluer la pathogenèse de S. aureus lors d&#39;une infection cutanée superficielle. Ici, nous décrivons un modèle d&#39;infection cutanée qui examine la peau, sous-cutanée, et de pathologies musculaires.

Watch this Scientific Journal Video about Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA at JoVE.com

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

La peau douce et murin modèle d&#39;infection des tissus est utilisé pour évaluer la fonction de virulence de Staphylococcus aureus résistant à la méthicilline (SARM) et les réponses immunologiques hôte. Ici, nous avons présenté un modèle d&#39;infection sous-cutanée de la peau et les infections des tissus mous.

Infection sous-cutanée du Staphylococcus aureus résistant à la méthicilline (SARM)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

subcutaneous-infection-methicillin-resistant-staphylococcus-aureus

Research

JoVE Journal

Immunology and Infection

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

22.9K vues.  Cedars-Sinai Medical Center. La peau douce et murin modèle d'infection des tissus est utilisé pour évaluer la fonction de virulence de Staphylococcus aureus résistant à la méthicilline (SARM) et les réponses immunologiques hôte. Ici, nous avons présenté un modèle d'infection sous-cutanée de la peau et les infections des tissus mous.

Vidéo: Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

L&#39;utilisation des flux de goutte et des réacteurs de disque rotatif pour Staphylococcus aureus Analyse du biofilm

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Recherche

Immunologie et infection

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

Modèle endocardite expérimentale de résistant à la méthicilline<em&gt; Staphylococcus aureus</em&gt; (SARM) chez le rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Croissance Staphylococcus aureus en utilisant l&#39;hémoglobine humaine comme source de fer

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

PCR multiplex pour le typage des staphylocoques Cassette Chromosome Mec types I 

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

L&#39;étude des interactions de<em&gt; Staphylococcus aureus</em&gt; Avec des neutrophiles par cytométrie en flux et microscopie Time Lapse

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Faisant preuve d&#39;une multi-résistante<em&gt; Mycobacterium tuberculosis</em&gt; Amplification de puces à ADN

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Ciblage biofilm Associated<em&gt; Staphylococcus aureus</em&gt; Utilisation de résazurine Basé Essai de sensibilité aux médicaments

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

génotypage des<em&gt; Staphylococcus aureus</em&gt; par Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Méthodologie de l&#39;étude du transfert horizontal de gènes dans<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Isolement, Expansion et adipocytaire Induction de CD34 + CD31 + des cellules endothéliales du tissu adipeux humain épiploïques et sous-cutanée

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Infection sous-cutanée du Staphylococcus aureus résistant à la méthicilline (SARM)

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L'utilisation des flux de goutte et des réacteurs de disque rotatif pour Staphylococcus aureus Analyse du biofilm

Modèle endocardite expérimentale de résistant à la méthicilline

Croissance Staphylococcus aureus en utilisant l'hémoglobine humaine comme source de fer

PCR multiplex pour le typage des staphylocoques Cassette Chromosome Mec types I

L'étude des interactions de

Faisant preuve d'une multi-résistante

Ciblage biofilm Associated

génotypage des

Méthodologie de l'étude du transfert horizontal de gènes dans

Isolement, Expansion et adipocytaire Induction de CD34 + CD31 + des cellules endothéliales du tissu adipeux humain épiploïques et sous-cutanée