Bu çalışma, Burroughs-Wellcome Kariyer Ödülü ve GY Liu Sağlık hibe AI074832 Ulusal Sağlık Enstitüleri tarafından desteklenmiştir.

1. Enfeksiyon MRSA (Enfeksiyon önce iki Gün) Hazırlama <ol><li> Bir hisse senedi kültürünün bir kan agar (Triptikaz Soya Agar (TSA)) plaka MRSA loopful İnokülasyon. </li><li> Kanlı agar plaka üzerinde hemoliz fenotip (her koloni etrafında açık bir bölge) kontrol edin. </li><li> Diğer deneylerin tutarlı bir hemolitik fenotip bir koloni seçin. </li><li> 15 ml snap-kapaklı tüp, tahsis antibiyotik gerektiğinde, 3 ml Todd Hewitt Broth (THB) koloni İnokülasyon. </li><li> 37 ° ° C&#39;de 220 rpm&#39;de sallayarak ile geceleme. </li></ol> 2. Enfeksiyon Fare hazırlanması (Hastalığın bir Gün önce) <ol><li> Farelerin büyüklüğüne bağlı olarak, 3 x 4 cm alan farelerin arka kapalı kürk tıraş. </li><li> ~ 5 mm 3 tüy dökücü krem (yerel bir uyuşturucu deposu satın) traş bölgeye uygulayın. </li><li> Tüy dökücü krem, cilt yüzeyinde yaklaşık 1 dakika inkübe izin verin. </li><li> GKD 2 O ıslak kağıt havlu </li><li> Tüy dökücü krem, ıslak kağıt havlu ile silin. </li></ol> 3. (Day Enfeksiyon) Enfeksiyon MRSA hazırlanması <ol><li> 50 ml vidalı kapaklı tüp içinde, önceden ısıtılmış THB 10 ml 1:500 1:1000 için gecelik bakteri kültürü sulandırınız. </li><li> 37 ° C yaklaşık 2,5 saat için bir 540 2.5 ulaşıncaya kadar, 220 rpm&#39;de titreme ile inkübe. </li><li> 4, 10 dakika süreyle 3225 xg&#39;de santrifüj yoluyla MRSA toplayın ° C </li><li> Süpernatantı atın. </li><li> Bakteriyel pelletini tekrar 10 ml Dulbecco&#39;nun fosfat tamponlu salin (DPBS). </li><li> 3. ve 4. adımları tekrarlayın. </li><li> DPBS içinde istenilen konsantrasyonda bakteri pelletini tekrar. </li><li> 8 Ekim - 1 Ekim seri bakteriyel süspansiyon sulandırmak. </li><li> Plaka kanlı agar plakaları üzerine seyreltilmiş bakteriyel süspansiyon. </li><li> ° C gecede 37 plakalar inkübe edin. </li><li> Gözlemleyin ve inocula hemolitik fenotip kayıt. </li><li> Plaka üzerinde Birimleri oluşturulması Colony (CFU) sayısını saymak. </li><li> Inocula plaka CFU sayısına göre hesaplayın. </li></ol> 4. Subkutan Enfeksiyon MRSA (Day Enfeksiyon ve enfeksiyon sonrası 3 Gün) <ol><li> Traş alanda subkutan 100 mcL bakteriyel süspansiyon enjekte edilir. </li><li> Farelerin hayatta emin olmak için enjeksiyon sonrası 3 ila 5 saat hayvanları gözlemleyin. </li><li> Günlük hayvanların geri lezyon gözlemleyin ve gerektiğinde günlük lezyon alanı kaydı. Lezyon gözlem, lezyon büyüklüğü ve morfolojisi kayıt içermelidir. Her iki kas ve deri lezyonlarının lezyonun uzunluğu ve genişliği çarpılarak quantitated. Düzensiz şekilli lezyonlar, daha küçük simetrik parçalar halinde parçalanır ve her bir parçanın aynı yöntemi ile ölçülür olması gerekir. Lezyon ölçümler bilgisayar destekli histomorfometrik değerlendirme programı (NIH kullanılabilir açık kaynak ImageJ kullanabilirsiniz <a href="http://rsb.info.nih.gov/ij">http://rsb.info.nih.gov/ij/</a> 2). </li><li> Servikal dislokasyon izofluran inhalasyonu gün 3 sonrası enfeksiyon hayvanların Kurban. </li><li> Gözlemleyin ve cilt üzerinde lezyon kayıt. </li><li> Steril makas bir çift Nick cilt. </li><li> Cildin soyulmasına dikkatli ve gözlemlemek ve kas lezyon kaydetmek. </li><li> Vergi lezyon ve yaklaşık 2 - 5 mm çevresi. </li><li> Dalak ve böbrekler toplayın. </li><li> Bir doku Homojenizatör kullanarak dokular homojenize veya doku ve DPBS 100 mcL içeren bir mikrofuge&#39;de tüpüne 1 ml şırınga pistonu tekrarlanan uygulama tarafından. </li><li> Her bir tüp DPBS 900 mcL ekleyin. </li><li> 5 dakika boyunca vorteksleyin örnekleri. </li><li> Seri DPBS 10 1 10 6 süspansiyon sulandırmak. </li><li> Plaka TKP plakalar üzerinde seyreltilmiş süspansiyonlar. </li></ol> 5. Temsilcisi Sonuçlar: <ol><li> Hemen her subkutan enjeksiyondan sonra, bir bleb enfeksiyonu (Şekil 1A) doğru bir şekilde yapılırsa, deri yüzeyinde görülen olacaktır. Bleb deri yüzeyinde görülürse, enjeksiyon çok derin olabilir ve bu enfeksiyon (lezyon boyutu, CFU) sonucu etkileyebilir. </li><li> Bakteriyel süspansiyon seri seyreltilerek gereken ve CFU her bir deney için kan agar plaklarına incelenmesi gerekir. 1) inocula homojen fenotip olup olmadığını; 2) enfeksiyon için tam bir canlı bakteri sayısı: Bu adım önemli bilgiler verecektir. </li><li> Lezyonlar ve bakteriyel hayatta kalma, çeşitli zaman noktalarında sonrası enfeksiyon değerlendirilebilir. Burada, 3. gün sonrası enfeksiyon, enfeksiyon inceledi. Lezyon boyutları cilt yüzeyi (: 1x 10 9 Şekil 1D: Şekil 1B 1 x 10 7 CFU) analiz edilir ve kas yüzeyi (Şekil 1C: 1x 10 9 </s&gt; Şekil 1E: 1 x 10 7 CFU). Lezyon alanı (mm) x genişliği (mm) (Şekil 2 A ve B) doku hasarı değerlendirmek için ölçülen olabilir süresini eşittir. Bu sonuç, belirli bir virülans faktörü doku hasarı ölçüde belirlemenize yardımcı olabilir. Ayrıca, lezyonlar eksize ve DPBS homojenize ve enfeksiyon yerinde bakterilerin canlılığını gösterir CFU numarası (Şekil 2C), ölçmek için kaplama. </li></ol><img src="/files/ftp_upload/2528/2528fig1.jpg" alt="Şekil 1" /> Şekil 1. Cilt ve kas lezyonları CD1 fareler MRSA (LAC) ile bir kanadında subkutan ekildi. Resimleri zaman 0 ve 3. gün sonrası enfeksiyon alınmıştır. (A) Zaman 0 sonrası enfeksiyon (pi), (B) - (G) 3. Gün pi; B, D ve F: deri lezyonları, C, E ve G: kas lezyonları, B ve C: 1x 10 9 CFU bulaşmış, D ve E: 1x 10 7 CFU enfekte; F ve G: alay. <img src="/files/ftp_upload/2528/2528fig2.jpg" alt="Şekil 2" /> Şekil 2. Cilt lezyon büyüklüğü ve canlı bakteri CD1 fareleri enfekte MRSA (LAC) 3. gün sonrası enfeksiyon Deri ve kas lezyonları enfeksiyon yerinde saymak. (A) Cilt lezyon büyüklüğü. (B) Kas lezyon boyutu. (C) Toplam doku CFU. H: 1 x 10 9 CFU inocula L: 1 x 10 7 CFU inocula. *: P &lt;0.05, Mann-Whitney testi.

<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> Fare deri ve yumuşak doku enfeksiyonu modeli bir patojenin in vivo virülans değerlendirmesi için güçlü bir araçtır. S. patojenite aureus, deri ve yumuşak doku enfeksiyonu, bir takım parametreler bağlı olarak değişebilir . Bunlar inokulum büyüklüğü, bakteriyel büyüme aşamasında, aşılama derinliği, fareler yaş, ve farenin genetik arka plan 7, 8. Bu model kullanılarak virülans işlevini incelerken, sonuçları tutarlı olacağını sağlamak için bu parametreler...

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subcutaneous infection of methicillin resistant staphylococcus aureus (mrsa)

MRSA, halk sağlığı için dünya çapında bir bir tehdit ve MRSA deri ve yumuşak doku enfeksiyonları, şimdi Amerika Birleşik Devletleri&#39;nde tüm yumuşak doku enfeksiyonları yarısından fazlasının hesabı. Yumuşak doku enfeksiyonları arasında, miyozit, pyomyositis ve nekrotizan fasiit giderek MRSA toplum kaynaklanan ilişkili olarak rapor edilmiştir. MRSA ve daha şiddetli enfeksiyonlara yol açan ev sahibi bağışıklık arasındaki etkileşimi anlamak için, host ve bakteriyel faktörler çalışma izin, hayvan modellerinde durumu kritik öneme sahiptir. S. patogenezinde değerlendirmek için birçok enfeksiyon modelleri tanıtıldı yüzeyel cilt enfeksiyonu sırasında aureus. Burada, deri, derialtı ve kas patolojileri inceleyen bir subkutan enfeksiyon modeli açıklanmaktadır.

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

Murin deri ve yumuşak doku enfeksiyonu modeli metisiline dirençli Staphylococcus aureus (MRSA) ve ev sahibi immünolojik yanıtlar virülans fonksiyonu değerlendirmek için kullanılmaktadır. Burada, biz, deri ve yumuşak doku enfeksiyonu için bir deri altı enfeksiyon modeli sundu.

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 ...

subcutaneous-infection-methicillin-resistant-staphylococcus-aureus

Research

JoVE Journal

Immunology and Infection

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

22.9K Görüntüleme.  Cedars-Sinai Medical Center. Murin deri ve yumuşak doku enfeksiyonu modeli metisiline dirençli Staphylococcus aureus (MRSA) ve ev sahibi immünolojik yanıtlar virülans fonksiyonu değerlendirmek için kullanılmaktadır. Burada, biz, deri ve yumuşak doku enfeksiyonu için bir deri altı enfeksiyon modeli sundu.

Video: 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.

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 ...

İmmünoloji ve Enfeksiyon

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. ...

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.

Çok ilaca dirençli Demonstrating<em&gt; Mycobacterium tuberculosis</em&gt; Amplifikasyon Microarray

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.

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 ...

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.

Yalıtım, genişleme ve Adipojenik indüksiyon CD34 + CD31 + insan Omental ve deri altı yağ dokusu endotel hücrelerden

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

Subkutan Enfeksiyon Metisiline Dirençli Stafilokok Aureus (MRSA)

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Etkileşimi incelenmesi

Çok ilaca dirençli Demonstrating

Biyofilm İlişkili Hedefleme

bir Genotiplendirme

Yatay Gen Transferi Çalışmasında Metodolojisi

Yalıtım, genişleme ve Adipojenik indüksiyon CD34 + CD31 + insan Omental ve deri altı yağ dokusu endotel hücrelerden