NIAID hibe K22AI081748.

1. Damla Akış Biyofilm Reaktörü <ol><li> Damla akış biyofilm reaktörü monte edilmiş ve otoklava (Biosurface Technologies ya da özel olarak tasarlanmış sürümleri mevcuttur genellikle üniversite makine atölyeleri tarafından yapılabilir, bkz. Şekil 1). Meclis odaları ve güvence odasına kapakları iliştirilmesi kupon içerir. Biyofilm orta (triptik soy broth 2 gram / L ve glukoz 2 gram / L) ve akma besin boru ile birlikte odası, otoklav ile sterilize edilir. </li><li> Damla akış reaktör, reaktör, düz bir yüzey üzerine yerleştirerek, atık su boru hatları sıkma, 10 ml triptik soy broth her ​​odasını doldurmak ve S. 10 mcL ekleyerek Aşılama preformed. aureus kültürü triptik soy broth gecede büyüdü. Inoküle reaktör daha sonra 18 saat boyunca 37 ° C inkübatör yerdir. </li><li> 18 saat inkübasyondan sonra, atık su boru unclamped ve reaktör, 10 derecelik açıyla kesilmiş ahşap bir blok üzerine yerleştirilir. </li><li> Aseptik sürekli akış besin suyu şişesi gibi geleneksel besin hortumu bağlayın. Maksimum hız (pompa modeline göre değişir) pompa çalıştırarak, pompa ve başbakan tüpler aracılığıyla boru hattı besleyin. </li><li> Akma boru, pompa durdurmak ve her tüp sonuna kadar bağlantı iğne (22 gauge, 1 inç) eklemek astarlanmalıdır. Etanol silin ve aseptik giriş stoper üzerinden iğne eklemek odasına giriş stoper silin. </li><li> Pompa açın ve kuponlar üzerinde (akış hızı ~ 125 mcL / dakika) medya yavaş yavaş damla izin. Medya giriş stoper limandan çıkış portuna kupon boyunca aşağıya doğru akmalıdır. Bazen uygun drenaj için reaktör kontrol, 2-5 gün (uygulamaya bağlı olarak) sürekli akış reaktör çalıştırın. </li><li> Damla akış reaktör biyofilm hasat için, pompayı durdurun ve reaktör dikkatle iğneleri çıkarın. Reaktör daha sonra düz bir yüzeye yerleştirilir ve kuponlar aseptik olarak steril forseps kullanılarak silinebilir. Mikroskopi isteniyorsa, kuponlarını buna göre işlenmiş olabilir (Şekil 2B bir damla biyofilm reaktörde yetişen bir S. aureus biyofilm bir tarama elektron mikrografı). Biyofilm biyokütle veya fizyoloji çalışmaları ölçümü çalışmanın amacı ise, biyofilm bir hücre kazıyıcı kullanarak kupon silinebilir. Forseps ile kupon tutarken, fosfat içeren konik bir tüp içine yavaşça kupon kapalı biyofilm kazımak bir hücre kazıyıcı kullanarak tamponlu salin. Not: biyofilm koloni oluşturan birim ölçmek için, kümeleri parçalara ayırma ve homojen bir süspansiyon formu Homojenizatör bir doku ile hasat biyofilm homojenize gereklidir. Çeşitli modeller doku homojenizatörler bu uygulama için uygundur. Biyofilm örnekleri homojenize 1 dakika boyunca tam hızda bir Fisher Scientific Tissuemiser Homojenizatör (ürün # 15-338-420) kullanmaktadır. Biyofilm homojenize yapılmaması örnek mevcut birimleri oluşturan koloni küçümsenmesi neden olacaktır. </li></ol> 2. Döner Disk Biyofilm Reaktörü <ol><li> Dönen disk biyofilm reaktörü (yapılan bkz. Şekil 3 Biosurface Teknolojileri temin edinilebilir veya özel olabilir) toplandı ve otoklava. Ilk etapta reaktör birleştirin yuvalarına dönen diskin disk kupon iplik ve bir taşma bağlantı noktası ile 1 litrelik cam beher içine yerleştirerek. Medya akış ve havalandırma sağlamak için delikli bir dizi 15-lastik tıpa reaktör kap olarak kullanılır. Reaktör, biyofilm medya (triptik soy broth 2 g / L ve glukoz 2 g / L), ve giriş boruları, daha sonra otoklav ile sterilize edilir. </li><li> Dönen disk biyofilm reaktör, reaktör içine 250 mL steril ortamda koyarak inoküle ve bir gecede S. 0,5 mL ekleyerek aureus kültürü triptik soy broth yetiştirilen. Reaktör sonra, 250 rpm bir heyecan plaka üzerine yerleştirilir ve istenilen sıcaklıkta gece inkübe edilir. </li><li> 16 saat inkübasyondan orta rezervuar giriş boru bağlantı noktasına bağlamak ve peristaltik pompa bağlamak sonra. Pompa daha sonra açmak ve akış ~ 0.25 ml / dak (akış istenilen büyüme hızına bağlı olarak değiştirilebilir). </li><li> 24 saat sonra reaktör durdurmak ve aseptik biyofilm kupon dokunmadan diski çıkarın. Kuponları steril forseps kullanarak çıkarın ve fosfat içine her bir dip gevşek bağlı herhangi bir bakteri kaldırmak için tamponlu salin. </li><li> Antimikrobiyal bileşik testi için, cips ilgi bileşikleri içeren bir 96 plaka ayrı ayrı kuyu içine yerleştirilir. Inkübasyondan sonra, cips, 1 ml fosfat tamponu tuzlu su içeren 1.5 ml mikrosantrifüj tüp içine aktarılır ve bir doku bozulmadan biyofilm dağıtmak için homojenizatör ile homojenize. </li><li> Hücreler daha sonra seri seyreltilmiş ve canlı koloni formi belirlemek için besin agar üzerine kaplamang birimleri. </li><li> Not: Yukarıdaki protokol çok varyasyon yapılabilir. Örneğin, dönen disk kuponlar ile kaplı olabilir ya da etkinliğini test etmek için potansiyel anti-biyofilm bileşikler yapılmış. Biyofilm dispersant dağıtıcı bileşikler ve ekli karşı müstakil bakteri miktarının belirlenmesi kupon kuluçka olarak değerlendirilebilir. </li></ol> 3. Temsilcisi Sonuçlar Şekil 1 ise damla akış reaktör kadar bir dizi bir örnek gösterilmiştir. Biyofilm bol miktarda akışı üç gün sonra, kupon yüzeyi, Şekil 2A üzerinde birikir. Toplam biyokütle bakteri suşu ve hassas bir büyüme şartlarına bağlı olarak değişir. S. taramalı elektron mikrografı Şekil 2B damla akış reaktör yetiştirilen aureus biyofilm gösterilmiştir. Dönen bir disk reaktör rakam 3A gösterilmiştir. Açıklanan protokol, bir biyofilm oluşturma yeteneğine sahip, genellikle herhangi bir mikroorganizmanın özel gereksinimlerine adapte edilebilir. Şekil 3B 18 plastik diskler yapıştırılmış iplik disk gösterir. Bu disk yetiştirilen biyofilm, özellikle de antibiyotik testi için uygun ve yüksek tekrarlanabilir sonuçlar 11 verim . <img src="/files/ftp_upload/2470/2470fig1.jpg" alt="Şekil 1" /> Şekil 1 damla akım reaktöründe kurulum. Önemli bileşenleri etiketlenir. <img src="/files/ftp_upload/2470/2470fig2.jpg" alt="Şekil 2" /> Şekil 2: Örnek bir damla akışı S. aureus biyofilm. A) Bu biyofilm açıklanan protokol izleyen üç gün için yetiştirilir. Kapaklar reaktörün ilk iki odalarından sarı S. göstermek için kaldırılır aureus biyofilm biyokütle. S. B) Sacnning elektron mikrografı aureus biyofilm damla akış reaktöründe büyüdü. <img src="/files/ftp_upload/2470/2470fig3.jpg" alt="Şekil 3" /> Şekil 3 Dönen disk reaktör. A) Örnek dönen disk çalışan bir reaktör. Anahtar bileşeni olarak etiketlenir. B) dönen bir disk kapatın.

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	<li>Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+Biofilms.">Microbial Biofilms.</a> Annu. Rev. Microbiol. 49, 711-745 (1995).</li><li>Costerton, J. W., Cheng, K. J., Gessey, G. G., Ladd, T. I., Nickel, J. C., Dasgupta, M., Marrie, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms+in+nature+and+disease.">Bacterial biofilms in nature and disease.</a> Ann. Rev. Microbiol. 41, 435-464 (1987).</li><li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+biofilm+formation+in+Pseudomonas+fluorescens+WCS365+proceeds+via+multiple,+convergent+signaling+pathways:+a+genetic+analysis.">Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis.</a> Mol. Micro. 28, 449-461 (2002).</li><li>Boles, B. R., Horswill, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agr-mediated+dispersal+of+Staphylococcus+aureus+biofilms.">Agr-mediated dispersal of Staphylococcus aureus biofilms.</a> PLoS Pathog. 4, e1000052-e1000052 (2008).</li><li>Goeres, D. M., Haamilton, M. A., Beck, N. A., Buckingham-Meyer, K., Hilyard, J., Loetterle, L. A., Walker, D. 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Farklı reaktörler yetiştirilen Biyofilmler genellikle farklı özelliklere sahip olacak ve her reaktör farklı uygulamalar vardır. Bir damla akış biyofilm reaktörü ve dönen bir disk reaktör: Bu çalışmada, iki biyofilm reaktörlerin kullanımı açıklanmaktadır. Damla akış reaktörleri düşük bir hava-sıvı arayüzü kesme biyofilm yetiştirmek için yararlı olan ve çeşitli koşullara uyarlanabilir. Biz büyük bir miktarda biyofilm biyokütle arzu edilir çalışmaları için son derece kullanı?...

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

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<td>Drip Flow Reactors</td>
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the use of drip flow and rotating disk reactors for staphylococcus aureus biofilm analysis

Doğada en çok mikrop 1 Bakteriyel biyofilm bir matriks içinde kaplı. Biyofilm yüzey ile ilişkili topluluklar gibi mevcut düşünce ve bir yüzey 2 Biyofilm oluşumu ve gelişimi bağlı yaygın microtiter plakalar veya akış gibi toplu sistemlerini kullanarak laboratuvarda incelenir sistemleri, akış hücreleri gibi. Bu metodolojileri mutant ve kimyasal kütüphaneleri (microtiter plakalar) görselleştirme (akış hücreleri) 4 için 3 ya da büyüyen biyofilm tarama için yararlıdır. Damla akışı biyofilm reaktörü ve dönen disk biyofilm reaktörü: Burada iki ek tip akış sistemi biyofilm büyüyen Staphylococcus aureus için ayrıntılı protokolleri mevcut. Damla akış biyofilm reaktörleri düşük kesme koşulları altında yetiştirilen biyofilm çalışma için tasarlanmış dört paralel test kanalları, her bir standart cam mikroskop lamı büyüklüğünde kupon tutma kapasitesine sahip, ya da bir kateter ya da çalıştıktan uzunluğu 5 damla akış reaktör oluşur . Damla akış reaktör mikrosensor izleme, genel biyofilm çalışmaları, biyofilm cryosectioning örnekleri, yüksek biyokütle üretimi, tıbbi malzeme değerlendirmeler ve kalıcı bir tıbbi cihazın denenmesi için idealdir. 6,7,8,9 Dönen disk reaktör, çıkarılabilir kuponlar için girinti içeren bir teflon disk oluşur. Çıkarılabilir 10 kupon yazan işlenebilir herhangi bir malzemeden yapılmış olabilir. Dönen disk alt diskin dönme yüzey yıkama kuponlar arasında sıvı yüzey kesme oluşturmak için izin veren bir çubuk mıknatıs içerir. 18 kupon içeren tüm disk, 1000 ml&#39;lik cam yan kol reaktör damar yerleştirilir. Disk, manyetik karıştırıcı ile döndürülmüş ise sıvı bir büyüme ortamı gemi dolaşıyor. Kupon reaktör damarı çıkarılmış ve ardından daha fazla çalışma veya mikroskopi görüntüleme biyofilm örnek toplamak için kazınır. Döner disk reaktörler biyosit etkinlik, biyofilm kaldırılması ve anti-kirlenme malzemeleri performans laboratuvar değerlendirmeler için tasarlanmıştır. 9,11,12,13

Watch this Scientific Journal Video about The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis at JoVE.com

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

Damla akış reaktörleri, açık sistem akış biyofilm kullanan ve diski reaktörler dönen için Protokoller ayrıntılı olarak sunulmuştur.

Damla Akış ve Döner Disk Reaktörler kullanın Staphylococcus aureus Biyofilm Analizi

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

the-use-drip-flow-rotating-disk-reactors-for-staphylococcus-aureus

Research

JoVE Journal

Immunology and Infection

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

17.0K Görüntüleme.  University of Michigan. Damla akış reaktörleri, açık sistem akış biyofilm kullanan ve diski reaktörler dönen için Protokoller ayrıntılı olarak sunulmuştur.

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa ve Saccharomyces cerevisiae Biyofilm Akış Hücreler

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

İmmünoloji ve Enfeksiyon

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.

Subkutan Enfeksiyon Metisiline Dirençli Stafilokok 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 ...

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.

Metisiline Dirençli Deneysel Endokardit Modeli<em&gt; Staphylococcus aureus</em&gt; (MRSA) Sıçan

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.

Demir Kaynağı Olarak İnsan Hemoglobin kullanarak Staphylococcus aureus Büyüme

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.

Metisiline diren

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.

Etkileşimi incelenmesi<em&gt; Staphylococcus aureus</em&gt; Akım Sitometri ve Time Lapse Mikroskopi tarafından Nötrofiller ile

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

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.

Biyofilm İlişkili Hedefleme<em&gt; Staphylococcus aureus</emKullanma&gt; Resazurin Tabanlı İlaç-duyarlılık Deneyi

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.

bir Genotiplendirme<em&gt; Staphylococcus aureus</em&gt; Tarafından Ribozomal 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.

Yatay Gen Transferi Çalışmasında Metodolojisi<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 ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

İnsan monosit alt kümeleri tam kan tarafından karakterizasyonu Akış Sitometresi Analizi

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...