We thank Saran Kupul for technical assistance. This work was supported in part by NIH grant R01-AI104952 to FW. Further support was provided by the University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR), an NIH funded program (P30 AI027767) that was made possible by the following NIH Institutes: NIAID, NIMH, NIDA, NICHD, NHLDI, NIA.

1. biofilm Initiation <ol><li> Cultiver une culture de biofilm organisme produisant dans un milieu riche en nutriments. Inoculer Staphylococcus aureus souche Newman dans 10 ml de milieu Mueller-Hinton à partir d&#39; un stock de glycérol. Effectuer tous les travaux impliquant la manipulation de S. aureus avec des gants et dans une enceinte de biosécurité. </li><li> Incuber pendant 16 heures à 37 ° C sur un agitateur rotatif (100-200 rpm). </li><li> Déterminer la DO 600 de la culture en utilisant un spectrophotomètre et d&#39; une cuvette standard (longueur de trajet: 1 cm). </li><li> Calculer le volume (V) de la culture nécessaire pour préparer 20 ml d&#39;inoculum avec OD 600 (inoculum) de 0,1 à chelexed médias Roswell Park Memorial Institute (cRPMI) 9 en utilisant la formule: [V = 20 ml * 0,1 / OD 600 (culture )] Remarque: La référence 9 fournit des détails sur la préparation cRPMI. <ol><li> Ajouter le volume calculé de culture (ci-dessus) dans un tube de centrifugeuse et un culot de cellules par centrifugation pendant 1 min à 10 000 x g. Pour prepare biofilm inoculum, aspirer le milieu de culture et remettre en suspension le culot cellulaire dans 20 ml passé cRPMI. </li></ol></li><li> Verser inoculum dans un réservoir de 25 ml. </li><li> Retirez délicatement le couvercle d&#39;une plaque de cheville 96 puits stérile en le tirant vers le haut. Remarque: Il faut prendre soin de ne pas contaminer les chevilles qui pendent du couvercle. Le couvercle peut être placé à l&#39;envers sur une surface propre au sein d&#39;une enceinte de sécurité biologique. </li><li> En utilisant une pipette multi-canal de 200 pi, ajouter 125 inoculum ul à chaque puits de lignes A à G de la plaque inférieure. </li><li> Remplissez moyen cRPMI 125 ul stérile dans chaque puits de la rangée H comme un contrôle de stérilité. </li><li> Prenez le peg-couvercle et le maintenir au-dessus du fond de la plaque avec les chevilles vers le bas. Alignez soigneusement les piquets individuels avec leurs puits respectifs. Éviter tout contact physique entre la plaque et les chevilles. Une fois aligné, abaissez soigneusement le couvercle vers le bas. Évitez les désalignements comme réajustements effectués après les chevilles ont touché la plaque de fond peut contcontrôles de stérilité aminer dans la rangée H. </li><li> Sceller le couvercle à la plaque avec un film de paraffine pour éviter l&#39;évaporation et le placer dans un sac en plastique refermable, scellant hermétiquement. Gardez le volume d&#39;air dans le sac aussi bas que possible pour minimiser l&#39;évaporation. </li><li> Incuber sans agitation à 37 ° C pendant 48 heures dans un incubateur à CO 2 de 5%. Note: 48 h de croissance a été optimale pour S. aureus, mais varieront en fonction de l&#39;organisme utilisé. Remarque: Bien que l&#39; incubation statique est un bon point de départ pour optimiser le dosage, en secouant doucement à 150 rpm sur un agitateur de microplaques est une variante possible qui peut améliorer la formation de biofilm de certains S. aureus. </li></ol> 2. Préparation de la plaque biofilm Défi <ol><li> Le jour du défi de biofilm prendre une plaque de 96 puits fraîche et ajouter 100 pi de cRPMI, équilibrées à la température ambiante, à chaque puits de lignes B à H. Pour éviter des résultats incohérents, utiliser une plaque de 96 puits qui peut contenir 200 pides supports lorsque les pions sont à l&#39;intérieur, et comporte des puits ayant des dimensions identiques à celles de la plaque biofilm d&#39;initiation. </li><li> Diluer jusqu&#39;à 4 composés testés séparément dans 1,0 ml de cRPMI à 2 x la concentration finale désirée, afin de tenir compte de la dilution finale dans l&#39;étape 2.7. Note: Un bon point de départ est 8 fois supérieure à la concentration inhibitrice des cellules planctoniques. </li><li> Ajouter 200 pi de composé 1 dans le puits A1 à A3, le composé 2 dans les puits A4 à A6, composé 3 dans les puits A7 à A9 et composé 4 dans les puits A10 à A12. </li><li> En série diluer les composés d&#39;essai à l&#39;aide d&#39;une pipette multicanaux et transférer 100 pi de la ligne A à la ligne B et mélanger. </li><li> De cette manière, continuer à transférer 100 ul puits à puits. Mélanger après chaque transfert et arrêter la ligne F. </li><li> Après le mélange, expulser 100 pi de la ligne F dans un conteneur de déchets. Ne pas transférer tout liquide aux lignes G et H. </li><li> Ajouter 100 ul de cRPMI à chaque puits de la plaque à l&#39;aide d&#39;une pipette multicanaux pouramener le volume à 200 ul. Voir la Figure 1 pour mise en page finale de la plaque. </li></ol> 3. biofilm Défi <ol><li> Ajouter 200 pi de tampon phosphate salin (PBS) à une plaque de 96 puits stérile fraîche qui comporte des puits ayant des dimensions identiques à celles de la plaque biofilm d&#39;initiation. </li><li> Retirez le sac en plastique et un film de paraffine de incubé plaque biofilm d&#39;initiation. </li><li> Soulevez le couvercle vers le haut. Éviter tout contact entre les piquets et le fond de la plaque, car cela pourrait endommager le biofilm. Gardez partie inférieure pour la suite OD 600 analyse (voir 3.8). </li><li> Rincez les cellules planctoniques en plaçant le peg-couvercle sur la plaque contenant du PBS (de l&#39;étape 3.1). Immerger chevilles pendant 5 secondes, sortir de la PBS, et submergez une fois de plus, en faisant attention de ne pas frapper les chevilles contre les côtés du puits. </li><li> Placez le peg-couvercle rincés dans la plaque de défi. Évitez tout contact inutile entre les piquets et plaque de fond, car cela endommagerait le biofilm conduisant àdes résultats incohérents. </li><li> plaque d&#39;étanchéité avec un film de paraffine et placer dans un sac en plastique refermable comme décrit à l&#39;étape 1.10. </li><li> Incuber la plaque sans agitation pendant 24 heures à 37 ° C dans un incubateur à CO2 à 5%. </li><li> Prendre en bas de la plaque biofilm d&#39;initiation de 3,3. Resuspendre les bactéries dans chaque puits à l&#39; aide d&#39; une pipette multicanaux et mesurer la DO 600 avec un lecteur de microplaque approprié pour confirmer la croissance se produisant dans tous les puits , mais les contrôles de stérilité dans la rangée H. </li></ol> 4. biofilm Détermination <ol><li> Utiliser un lecteur de plaque muni d&#39;une chambre d&#39;incubation et capable d&#39;effectuer des mesures de fluorescence pour détecter la cinétique de conversion résazurine. Mettre en place une cinétique mesure de fluorescence 24 heures en utilisant une excitation de 530 nm et 590 nm émission. </li><li> Réglez la température de la chambre à 37 ° C et la plaque ont lu toutes les 20 min. Configurer le lecteur de plaque à mesurer à partir du fond de la plaque selon le constructeur instructions. </li><li> Préparer la plaque de lavage en ajoutant 200 pi de PBS avec une pipette multicanaux à une plaque de 96 puits stérile. </li><li> Préparer le milieu de récupération de biofilm par addition de 400 ul de 0,8 mg / ml de résazurine solution mère à 20 ml de milieu cRPMI à une concentration finale de 16 pg / ml de résazurine. Note: résazurine est un irritant connu de la peau. Utilisez un sarrau de laboratoire, des lunettes de protection et des gants lors de la manipulation. CRPMI que les médias de récupération de biofilm a été utilisé car il fournit le signal de fond le plus bas, mais il peut être remplacé par les médias Mueller Hinton, le cas échéant. </li><li> Ajouter 150 ul de milieu de récupération de biofilm avec une pipette multicanaux à chaque puits d&#39;une plaque à 96 puits fraîche. </li><li> Transfert de plaque de défi de l&#39;incubateur dans une enceinte de sécurité biologique et retirez soigneusement le sac en plastique et le film d&#39;étanchéité. </li><li> Retirez le peg-couvercle de la plaque de défi et rincer les cellules planctoniques dans du PBS comme décrit dans 3.4. Gardez le fond de la plaque de défi pour OD ultérieure 600 analyse (voir 4.10). </li><li> Après rinçage, placer la cheville couvercle dans la partie inférieure de la plaque contenant le milieu de récupération du biofilm. </li><li> Envelopper le côté de la plaque avec un film de paraffine, en faisant attention à ne pas obstruer le fond des puits de périmètre. Immédiatement après avoir enveloppé la plaque, placez-le sur le lecteur de plaque et de commencer une lecture cinétique sur une période de 24 heures en lançant précédemment fait résazurine protocole cinétique de l&#39;étape 4.1. </li><li> Pour quantifier la croissance des cellules planctoniques qui peuvent ou peuvent ne pas se produire pendant défi, lisez la DO 600 de l&#39;ensemble du fond de la plaque de défi en utilisant un lecteur de plaque micro. Suivez les instructions données à l&#39;étape 3.8. Remarque: mélanger soigneusement le contenu de chaque puits comme cellules excrétion hors du biofilm ont tendance à croître dans les touffes au fond du puits. Les bulles dans le puits vont fortement interférer avec OD 600 lectures et doivent être évités ou supprimés avant la lecture. </li></ol>

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The authors have nothing to disclose.

Ici, nous avons décrit un dosage de biofilm modifié pour déterminer l&#39; activité d&#39;inhibiteurs testés sur S. biofilms aureus mettant l&#39; accent sur ​​l&#39;état métabolique des cellules de biofilm associé. Alors que l&#39;initiation de biofilm décrit et procédures de contestation des recommandations du fabricant principalement simulées, l&#39;utilisation de résazurine colorant pour détecter et quantifier les cellules associées à un biofilm qui survivent un défi inhibiteur ...

Microbes pathogènes peuvent former des biofilms in vivo conduisant à des infections chroniques non actifs 1. L&#39; infection par le biofilm associé est un facteur de risque grave de procédures médicales qui impliquent l&#39; implantation d&#39;objets étrangers (par exemple, le remplacement des os artificiels, des implants mammaires) ou l&#39;installation des tubes trachéaux ou cathéters urinaires 2. Dans ces contextes, le traitement anti-infectieux est presque toujours nécessaire que les infections à base de biofilm sont rarement effacés sur leur propre, même chez les personnes immunocompétentes. Staphylococcus aureus est l&#39; un des agents pathogènes les plus fréquemment observés impliqués dans les complications liées à biofilm qui se produisent lors de l&#39;utilisation de dispositifs médicaux invasifs 3. Malheureusement, la nature même de biofilms comme une barrière protectrice qui les rend plus résistantes au traitement que les cellules planctoniques 1,4, et l&#39; évaluation de l&#39; efficacité clinique prédit est un élément essentiel à la fois initialele développement de médicaments, ainsi que la surveillance de la résistance aux médicaments. Il est de plus en plus reconnu que les conditions de laboratoire, axées sur les cultures planctoniques, peuvent ne pas représenter fidèlement la maladie dans le monde réel 5. Aggravant encore le problème, la réplication des phénotypes biofilm est difficile, et les modèles de biofilm existants sont fastidieux et souffrent de haute inter et intra-essai variabilité 6. Ainsi, de nombreux chercheurs, par nécessité, par défaut aux dosages cellulaires planctoniques de sensibilité aux médicaments, ce qui pourrait négliger un aspect important de la virulence bactérienne et la maladie. Nous décrivons ici le protocole de dosage de bactéries, en particulier S. aureus, dans les biofilms pré-cultivés en utilisant une base de 7,8 à 96 puits de système de biofilm. Bien que le protocole pour la formation de biofilm et de défi suit essentiellement la recommandation du fabricant, nous présentons une méthodologie riche en information alternative pour quantifier la viabilité du bioFilm après la provocation. En bref, les bactéries sont mises en culture dans la plaque de cheville, où les biofilms se forment sur les pions en saillie fixés au couvercle de la plaque. Après la formation du biofilm, les ergots sont légèrement trempées dans le puits d&#39;une plaque fraîche rempli avec du PBS pour éliminer les cellules planctoniques. Le peg-couvercle, avec biofilms attachés, est transféré à une nouvelle plaque de défi, contenant diverses concentrations d&#39;antibiotiques à doser. Après une seconde incubation, les couvercles sont de nouveau retirées, lavées et transférées à une plaque de recouvrement contenant un colorant résazurine, où ils sont soumis à une incubation finale. la conversion résazurine peut être enregistré cinétiquement ou pris comme une lecture du point final après une période de récupération définie. Cette méthode de quantification de la viabilité des biofilms à base de colorant diffère considérablement des UFC (unités formant colonies) fastidieuses méthodologie basée-count décrit dans le protocole original 7. OD 600 mesures de la plaque de défi de la drogue et la résazurine cinétique de conversion servent de viabilité de lectureouts de planctoniques et biofilm cellules, respectivement, offrant une information riche et techniquement simple test rapide, fiable, pour la survie de biofilm.

<table border="0" cellpadding="0" cellspacing="0" width="612">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">&#160;Mueller-Hinton Medium</td>
			<td style="width:89px;">Oxoid</td>
			<td style="width:103px;">CM0405</td>
			<td style="width:195px;">Follow recommendations of manufacturer</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">RPMI-medium</td>
			<td style="width:89px;">Corning&#160;</td>
			<td style="width:103px;">17-105-CV</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:227px;">CRPMI</td>
			<td style="width:89px;">Ref 9</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">RPMI-1640 medium chelexed for 1h with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Chelex 100 Resin</td>
			<td style="width:89px;">Bio Rad</td>
			<td style="width:103px;">142-2822</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">MBEC-plates</td>
			<td style="width:89px;">Innovotech</td>
			<td style="width:103px;">19111</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Resazurin Sodium Salt</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">R7017</td>
			<td style="width:195px;">800&#181;g/ml in DI water&#160;&#160;&#160;&#160; Filter sterile</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Micro Plate Shaking Platform&#160;</td>
			<td style="width:89px;">Heidolph Titramax 1000</td>
			<td style="width:103px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Cytation 3 Plate Reader</td>
			<td style="width:89px;">Biotek</td>
			<td style="width:103px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:58px;width:227px;">Gen5 software</td>
			<td style="width:89px;">Biotek</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">Recording and analysis of resazurin conversion</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Neocuproine</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">N1501&#160;</td>
			<td style="width:195px;">prepare 10 mM stock in 100% Ethanol, store at -80&#186;C</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Copper sulfate</td>
			<td style="width:89px;">Acros Organics</td>
			<td style="width:103px;">7758-99-8</td>
			<td style="width:195px;">prepare a 100 mM stock solution in water, store at 4&#186;C</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:227px;">Cu-Neocuproine</td>
			<td style="width:89px;">Self-Made</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Gentamicin</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">G3632-1G</td>
			<td style="width:195px;"></td>
		</tr>
	</tbody>
</table>

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.

Le dosage de la résazurine est suffisamment sensible pour détecter de manière fiable très peu de cellules viables capables de se répliquer dans un milieu exempt de drogue après la provocation. Dans cette méthodologie, nous définissons le biofilm l&#39;éradication de concentration minimale que la plus faible concentration à laquelle aucune conversion de résazurine est vu dans les 24 heures. Le dosage de la résazurine repose sur des molécules oxydantes présentes dans des cell...

Watch this Scientific Journal Video about Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay at JoVE.com

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

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

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.

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

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Immunology and Infection

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

12.9K vues.  University of Alabama at Birmingham. 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. 

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

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Plaque de microtitrage biofilm essai de formation

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

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Immunologie et infection

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 ...

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

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 United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

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 ...

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. ...

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, ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

Visualisation de l&#39;Agar Charcoal résazurine Essai pour semi-quantitative, Moyen-débit Enumération des mycobactéries

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

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 ...