Bu çalışma Sağlık ABD Ulusal Sağlık Enstitüleri tarafından desteklenen [hibe ASB R01AI-39.108] ve Amerikan Kalp Derneği [hibe SDG 0630219N ve YQX için YARDIM 09GRNT2180065].

1. Enfeksiyon için MRSA suşları hazırlanması <ol><li> Soya Agar (TSA) plaka Triptikaz bir koyun kanı (özel reaktifler ve ekipman Tablo bakınız) -80 bir stok tüp MRSA kültür loopful ° C inoküle ve 37 ° C&#39;de bir gece inkübe edin. </li><li> Hiçbir kir olmadığından emin olmak için kan agar (benzer koloni fenotipleri) üzerinde kültürün saflığı kontrol edin. </li><li> Koyun kanlı TSA plakadan bir koloni seçin ve 15 ml ek kapaklı tüp içinde 5 ml Triptikaz Soya Broth (TSB) içine koloni aşılamak. </li><li> 37 inkübe ° C&#39;de 200 rpm&#39;de sallayarak bir gecede. </li></ol> 2. Cerrahi Kateterler hazırlanması 10 cm uzunluğa ve steril bir forseps ile ucu basarak bir ucunu eritmek; polietilen boru (Becton Dickinson, sipariş no 427.401. PE10) kesin. Kateterin bir ucunu sızdırmazlık amacı kateterizasyon sırasında kanama kaçınmaktır. 3. Pre-surgery Hazırlık ve Anestezi <ol><li> Isofluorane-oksijen gaz karışımı (% 50:% 50) içeren bir odaya koyun Sprague-Dawley sıçan (Harlan, Indianapolis, Ind kadın, 250-300 gram) anestezik kadar (etkisi sürer örneğin kaslar gevşer ve pedal refleksleri vardır edilir ), ve gaz karışımı ile anesteziye cerrahisi sırasında bir durumda muhafaza hayvanlar. </li><li> Çeneden sadece Betadine ve% 70 etanol ile sternum altındaki boyun bölgesi temizleyin. </li></ol> 4. Cerrahi Prosedür <ol><li> Ameliyat boyunca steril tekniği kullanın. Dikey olarak sternum yukarıda boyun sadece cilt katmanı üzerinden bir insizyon (1-15 cm) olun. </li><li> Künt diseksiyon kullanarak, eğri dişli forseps 2 çift kullanılarak sağ karotis arter açığa fasya ayırın. </li><li> Yavaşça boyun kavite arter dışarı çekin ve ipek sütür yerine iki adet 10 cm uzunlukta arter altında ve maruz sefalik sonunda arter kapalı kravat, arte bir klip yerleştirinkanamayı önlemek için ry. </li><li> Bir kateter introducer (Becton Dickinson, sipariş no. 406.999) kullanarak arter üst küçük bir delik açın, arter delikten bir forseps ile bir kateter takmak, klibini kaldırmak ve kalp doğru kateter aşağı itmek kadar direniş karşılanmaktadır. </li><li> Arterin kaudal ucu çevresinde gevşek dikiş bağlayın ve kateter tarafından belirlenen yerde olduğunda ipek sütür ile yerde kateter sabitleyin: i) kateteri uzunluğu (4-5 cm); daha fazla ilerlemesini ii) direnç ve iii) kalp atışı ile kateter pulsasyon. Deney kalanı için bir yerde kateter bırakın. </li><li> Aşırı sütür ve kateter uçları kesilmiş ve kateter hiçbir kanama olmadığından emin olun. Boyun deri altında gevşek biter Tuck ve cilt klipler ile cilt kapatın. </li><li> Anestezi kurtarıldı kadar ılık bir yerde kafeste sıçan yerleştirin ve yiyecek ve su sağlıyor. Sırasında sık sık kontrol edin ve sıçansonra anestezi kurtarma. </li><li> Bu ameliyat bir &quot;Kategori E&quot; işlemdir. Ağrı üretilen prosedürleri hayvanlara sıkıntı eşlik MRSA enfeksiyonlarının beri analjezikler tarafından hafiflememiş olduğunu. </li></ol> 5.. MRSA enfeksiyonu <ol><li> Enfeksiyon 1 ile 7 gün ameliyat sonrası arasında yapılan, ancak deneyler içinde tutarlı devam edilebilir. </li><li> Temiz sıçan% 70 etanol ile kuyruk ve istenilen hücre sayımı (10 4 - En S. aureus için 10 6 kob / hayvan) 0.5 ml MRSA enjekte. Kuyruk damarından intravenöz G 1/2 inç iğne 27 ile Dikkat : MRSA kültür biyogüvenlik düzeyi 2 (BSL2) ait personel ve çevre için potansiyel tehlike ılımlı vardır. </li><li> Hemostaz kafesine sıçan dönmeden önce oluşana kadar yerinde basınç tutun. </li></ol> 6. Kurban Sıçanlar ve Kültür Hedef Dokular <ol><li> Sodyum pentobarbital (200 mg ip enjeksiyonu ile sıçan Kurban/ Kg) 1-6 gün post-enfeksiyonu sonrası. </li><li> Sırtında sıçan yerleştirin ve% 70 etanol ile göğüs silin. </li><li> Sternum altındaki göğüs V şeklinde bir kesi yapın ve kalp maruz sternumun her iki tarafındaki kaburga kıkırdağı kesti. </li><li> Kadar yavaşça kalp çekin ve klip aorta yakın kalp dokusu ile ve kalp kurtarmak kadar incelemek. </li><li> 4x4 inç gazlı bez içinde bir steril Petri tabağına yerleştirin kalp, sol ventrikül iç duvarından bir kesim yapmak ve sol taraflı odası açın. </li><li> Kateter yerleştirme için görsel olarak kontrol edin. Inceleyin ve makas ve forseps ile valfinden vejetasyonlar kaldırın. </li><li> Tartılır ve homojenize vejetasyon ve kantitatif kültür için PBS ile seri dilüsyonlar olun. </li></ol> 7. Temsilcisi Sonuçlar Hemen artere kateter takılması ve kalp yönüne doğru 4-5 cm kateter aşağı iterek sonra, bir direnç experien olacakdüşürebilmiştir. Herhangi bir direnç hissedilen edilirse, kateter başarıyla kateterin yerleştirilmesi etkileyebilecek, kalp odasının sol tarafında içine yerleştirilir edilmemiş olabilir. Daha önce yayınlanmış 9 doğru bir kateter Şekil 1 &#39;de gösterilmiştir. MRSA örnekleri parçası enfeksiyonu numunenin enfeksiyonu ile saflık için tam canlı bakteri sayısı emin niceliksel kültürlenebilir gerekir. Buna ek olarak, vejetasyonlar kurtarıldı organizmalar inokulum kullanılanlar ile aynı olmalıdır. Tablo 1, bir S. nin ölümcüllüğünün bir örnek sunar Daha önce 9 yayımlandı sıçan endokardit modelinde aureus suşu. Tüm hayvanlar 10 5 ve 10 6 cfu inokulum ile meydan ve 3 ile 6 gün yüksek S. ile enfeksiyonların geliştirilen endocardits sonra sakrifiye kardiyak vejetasyon yanı sıra böbrek ve dalak aureus yoğunlukları ( <strong&gt; Tablo 1). Steril kapak kültürlerinde Ratlarda bulaşmamış kabul edilir. <img alt="Şekil 1" src="/files/ftp_upload/3863/3863fig1.jpg" /> Şekil 1. Kateter doğru bir yerde (kalp odasının sol tarafında) olduğunu, ve çok sayıda vejetasyon aort kapakları 9 civarında görülebilir. <table border="1"><tbody><tr><td> Ekim (Animas sayısı) </td><td colspan="3"> Doku 10 CFU / g ± SD bir oturum Ortalama: </td></tr><tr><td></td><td> Bitki örtüsü </td><td> Böbrek </td><td> Dalak </td></tr><tr><td> 10 6 CFU / hayvan (9) </td><td> 10.36 ± 0.85 </td><td> 7.30 ± 0.64 </td><td> 6.70 ± 0.57 </td></tr><tr><td> 10 5 CFU / hayvan (8) </td><td> 9.93 ± 0.53 </td><td> 7.14 ± 0.53 </td><td> 6.44 ± 0.63</td></tr><tr><td> 10 4 CFU / hayvan (7) </td><td> 3.46 ± 0.50 * </td><td> 1.81 ± 0.74 * </td><td> 1.58 ± 0.59 * </td></tr></tbody></table> Bir SD, standart sapma. P 10 5 veya 10 6 CFU-meydan hayvanlar ile ilgili &lt;0.001. Tablo 1. S. rat endokardit model 9 farklı inokulum ile kardiyak vejetasyonların aureus yoğunluğu.

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K., Rivera, J. J., Smeds, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structural+model+of+the+Staphylococcus+aureus+ClfA-fibrinogen+interaction+opens+new+avenues+for+the+design+of+anti-staphylococcal+therapeutics.">A structural model of the Staphylococcus aureus ClfA-fibrinogen interaction opens new avenues for the design of anti-staphylococcal therapeutics.</a> PLoS Pathog. 4, e1000226 (2008).</li><li>Boles, B. R., Horswill, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+biofilm+disassembly.">Staphylococcal biofilm disassembly.</a> Trends Microbiol. , (2011).</li><li>Tseng, C. W., Sanchez-Martinez, M., Arruda, A., Liu, G. 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Çıkar çatışması ilan etti.

Sıçan endokarditi bakteriyel enfeksiyonların 9-11 tedavisinde patogenezine ve antimikrobiyal ajanlar in vivo çalışmalar için önemli ve iyi karakterize hayvan modeli. Ayrıca, sıçan endokardit modelinde akut ve sub-akut enfeksiyonların bir kompozit temsil eder ve yakından insan meslektaşı ve insan doğal kapak endokarditi taklit eder. Ayrıca, bir de konut kateter eşliğinde, klasik bir biyofilm ilişkili enfeksiyon, klinik ortamlarda 12 ortak ve zor bir mesele temsil eder. E...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Triptikaz Soya Agar Kan agar,% 5 koyun kanı (TSA) </td><td> Hardy Diagnostics </td><td> A10BX </td><td></td></tr><tr><td> Triptikaz Soya Broth (TSB) </td><td> Becton Dickinson </td><td> 211825 </td><td></td></tr><tr><td> Çalkalamalı inkübatör </td><td> Labnet </td><td> I5311-DS </td><td></td></tr><tr><td> Polietilen boru </td><td> Becton Dickinson </td><td> 427401 </td><td></td></tr><tr><td> Kateter introducer </td><td> Becton Dickinson </td><td> 6999 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Isofluorance </td><td> Batı Medikal Desty, Inc </td><td> 2147 </td><td> Güvenlik ve kullanım bilgileri izleyin </td></tr><tr><td> Cerrahi Aletler </td><td> Güzel Bilim Araçlar ve Biyomedikal Araştırma Instruments, Inc </td><td></td><td> Ihtiyaçlarınız için iki şirketlerden aletleri bul </td></tr></tbody></table>

experimental endocarditis model of methicillin resistant staphylococcus aureus (mrsa) in rat

Endokardit dahil Endovasküler enfeksiyonları, yaşamı tehdit eden enfeksiyon sendromdur 1-3. Staphylococcus aureus kabul edilemeyecek kadar yüksek morbidite ve hatta uygun antimikrobiyal ajan tedavileri 4-6 ile mortalite ile bu sendromun en yaygın dünya çapında nedenidir. Nedeniyle Metisiline dirençli S. için enfeksiyonlarda artış aureus (MRSA), vankomisin klinik tedavi başarısızlıkları ve linezolid ve daptomisin direnci artan sorunların yüksek oranda bu enfeksiyonu olan tüm hastalar daha karmaşık bir yönetim var ve yüksek sağlık masrafları 7, 8 yol açtı. Ayrıca, antibiyotik tedavisi sonuçları ile en son çalışmalarda klinik ortamlarda kurulmuştu ve bu nedenle de hasta-hasta arasında değişen ana faktörler tarafından etkilenir olabileceği vurgulanmalıdır. Bu nedenle, ana faktör hayvan-to-hayvana benzer olan endovasküler enfeksiyon ilgili bir hayvan modeli daha fazladırmikrobiyal patogenez yanı sıra, yeni antimikrobiyal ajanların etkinliğini araştırmak için çok önemlidir. Rat Endokardit yakın insan doğal kapak endokarditi yaklaşan köklü bir deneysel hayvan modelidir. Bu model özellikle stafilokok virulans faktörlerinin rolü ve stafilokok endokardit için antibiyotik tedavisi rejimlerinin etkinliğini incelemek için kullanılmıştır. Bu yazıda, bakteriyel patogenezi ve antibiyotik tedavisine yanıt araştırmak için kullanılan olabilir MRSA nedeniyle deneysel endokardit modeli açıklamak.

Watch this Scientific Journal Video about Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat at JoVE.com

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Metisiline dirençli nedeniyle deneysel rat endokardit modeli<em&gt; S. aureus</em&gt;.

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 ...

experimental-endocarditis-model-methicillin-resistant-staphylococcus

Research

JoVE Journal

Immunology and Infection

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

15.0K Görüntüleme.  Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. Metisiline dirençli nedeniyle deneysel rat endokardit modeli S. aureus.

Video: Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

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

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 ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Kabul edilmiş Transfer ve antijenik Challenge Deneysel Otoimmün Encephalomyelitis (EAE) Dayanıklı Fare Suşlarında yanıtsızlık Aşmak

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

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 ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Bir fare modeli için değerlendirmek doğuştan gelen bağışıklık yanıtı Staphylococcus aureus enfeksiyon

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...