The author thanks R. Boss, A. Cosandey, C. Fournier, I. Ivanovic, and J. Naskova for their excellent work.

1. Staphylococcus aureus izolatları <ol><li> S. 10 ul yayıldı aureus hazır bir kültür veya bir koloni,% 5 koyun kanı (BA) içeren Kolumbiya agar plakaları üzerinde seçici bir ortam üzerinde büyütülür. </li><li> 24 saat için 18 saat, 37 ° C &#39;de aerobik inkübe edin. </li></ol> 2. DNA Ekstraksiyon <ol><li> 10 mM Tris-HCl ve 10 mM Na2EDTA, pH 8.5 ihtiva eden TEl tamponu hazırlayın. 15 dakika boyunca 121 ° C&#39;de alikotları ve otoklav hazırlayın. </li><li> 100 ul TEL steril plastik döngü veya steril ahşap kürdan ve tekrar süspansiyon kullanılarak BA 1 koloni toplayın. vidalı kapakları ile 1.5 ml tüpler kullanın. </li><li> 10 dakika boyunca 95 ° C&#39;de inkübe edin. Daha sonra, ıslak buz üzerinde hemen örnekleri yerleştirin. </li><li> PCR (PCR için = şablon DNA) için uygun H 2 O 100: numuneleri 1 seyreltin. olmayan seyreltildi ve seyreltilmiş numune, 1 ay boyunca -20 ° C&#39;de muhafaza edilebilir. </li></ol> 3. RS-PCR <ol><lPCR tüpü 1.5 ul H içine her bir numune pipet için i&gt; 2 O, Taq Master Mix ul 12.5, 2 ul G1 astar 10 uM, 2 ul L1 astar 10 uM ve saf yaklaşık 30 ng karşılık gelen şablon DNA 7 ul DNA, 2. </li><li> Standart bir PCR döngü aşağıdaki döngü programı çalıştırmak: 15 dakika, 7 dakika boyunca 55 ° C &#39;de 2 dakika rampa ve tavlama takip 1 dakika için 94 ° C kapsayan 27 çevrim, ardından 95 ° C. Bir başka 2 dakika rampa sonra 2 dakika süreyle 72 ° C&#39;de uzatma. 4 ° C&#39;ye kadar soğutulduktan takiben 10 dakika boyunca 72 ° C&#39;de son uzatma PCR sonlandırma. Not: PCR ürünleri, 5 gün boyunca -20 ° C&#39;de saklanabilir. </li><li> Her çalışma için, RS-PCR, yani pozitif kontrol, bilinen bir genotip (normalde GTP) bir örneği, ve bir negatif kontrolü (H2O). </li></ol> 4. Elektroforez <ol><li> Birlikte DNA (MED kiti) için bir ticari minyatür elektroforez kiti kullanınPC ve yüklü yazılım bağlı bir Bioanalyzer ile. dikkatle üreticinin gelen kılavuzları inceleyin. </li><li> çip astar istasyonu kurmak ve üreticinin kılavuzuna göre şırınga klibi ayarlayın. </li><li> üreticinin kılavuzuna göre jel boya karışımı hazırlayın. </li><li> (Üreticinin kılavuzuna göre) jel boya karışımı yükleniyor. <ol><li> kullanımdan önce, 30 dakika boyunca oda sıcaklığında jel boya karışımı dengelenmesi. </li><li> Çip astar istasyonunda yeni bir DNA çip koyun. iyi işaretlenmiş G. jel boya Pipet 9 ul </li><li> piston 1 ml konumlandırılmış olduğundan emin olun ve sonra çip hazırlama istasyonu kapatın. Basın piston o şırınga klibi tarafından tutulan kadar. 30 sn sonra şırınga klibi yayınlayacak için tam bekleyin. </li><li> 5 saniye bekleyin. Yavaşça 1 ml konumuna pistonu geri çekin. </li><li> Her iki kuyularda Açık çip hazırlama istasyonu ve jel boya pipet 9 ul &#39;G&#39; olarak işaretlenmiş. </li></ol></li><li> M Yüklemearker <ol><li> 12 örnek kuyularda ve iyi merdivende marker Pipet 5 ul. Boş olan bu 13 kuyu herhangi bırakmayın. </li></ol></li><li> Merdiven ve Örnekleri yükleniyor. <ol><li> Pipet merdiven işareti ile iyi işaretlenmiş DNA merdiveninin 1 ul. </li><li> Numunenin Pipet 1 ul (kullanılan kuyular) ya da deiyonize su (kullanılmayan kuyuların) 1 ul. </li><li> 2400 rpm&#39;de 1 dakika için adaptör ve vorteks yatay çip koyun. </li><li> 5 dakika içinde Bioanalyzer çipi çalıştırın. </li></ol></li><li> Analizi Koşu <ol><li> Bioanalyzer ve bağlı bilgisayarınızı başlatın. yazılımını açın. </li><li> Bioanalyzer kapağını açın ve (çip yalnızca bir şekilde oturabilir) yonga tutucu içine çip yerleştirin. dikkatlice kapağını kapatın. </li><li> Yazılımın Enstrüman bağlamında &quot;Elektroforez&quot;&gt; &quot;dsDNA&quot;&gt; &quot;DNA 7500&quot; i seçin. Yazılımın güncel Dosya Öneki kabul edin veya değiştirin.</li><li> yazılımda mevcut start düğmesine tıklayın. Yazılımın örnek adlarını girin. Çip çalışma bittiğinde (sonra yaklaşık 30 dakika) Run mesajın sonu Bioanalyzer yazılımında görünür. </li><li> üreticinin talimatlarına göre Bioanalyzer elektrotları çipini çıkarın ve temizleyin. </li></ol></li></ol> 5. Elektroforez Profilinden genotip çıkarımlar yapma <ol><li> (Yazılım yazarlardan serbestçe kullanılabilir) Mahal yazılımını yükleyin. </li><li> &gt; &quot;Import Referans Veri&quot; &quot;Dosya&quot;: Mahal yazılımı içine alma referans verileri </li><li> Bioanalyzer yazılımı Veri bağlamında çalışır elektroforez açın. Bir numunede bir elektroforez profili seçin. </li><li> İlgili zirveleri için kontrol edin. <ol><li> &quot;Araçlar&quot;&gt; &quot;Mean Floresans&quot;: İlgili zirveleri kontrol etmek için Mahal yazılımını kullanın. &quot;Floresan&quot; kutusuna yerleştirinfloresans değerleri, Bioanalyzer yazılımından okunabilir şekilde, floresans (FU) olarak gösterilir. artan düzende en yüksek floresan 4 (3) zirveleri takın ve virgülle ayrılmış. Örnek: 126.130.132.137. NOT: tüm bu zirveleri FU Bioanalyzer yazılımı ile gösterilir, bunlar komplo onları okuyarak tahmin edilmesi gerekmektedir. </li></ol></li><li> &quot;Hesapla&quot; tuşuna basınız. gözlenen floresan kutusuna &quot;Minimal pik yüksekliği&quot; verilen değeri aştığı takdirde bir zirve geçerlidir. </li><li> genotip anlaması. <ol><li> Mahal metin kutusu &quot;Giriş Peaks (bp)&quot; artan sırayla ilgili tüm piklerin bp ölçü ve virgülle ayrılmış içine yerleştirin. Örnek: 440.462.519.579.637. NOT: Bu en yüksek boyutlarda tüm Bioanalyzer yazılımı ile gösterilir, bunlar grafiğinden onları okuyarak tahmin edilmesi gerekmektedir. </li></ol></li><li> &quot;Hesapla&quot; tuşuna basınız. </li><li> listesindekutu, asgari kare Mahalanobis mesafe &quot;d2m&quot; arayın. Bu ≥0.05 sağ karşılık gelen p değeri daha sonra olasılık sıfır hipotezi H 0 reddedilir ≥5% ise (H 0: gözlenen ve referans profili aynıdır) gözlenen profilini özdeş olduğu kabul edilmektedir, yani belirtilen genotip referans profili. </li><li> D2m az ancak karşılık gelen P değerinin alan boş ise, bir olasılık &lt;% 5 ile H 0 reddediyoruz. Not: Bu d2m çok az olsa da profilleri eşit olduğu anlamına gelir. Bu tür bir sonuç, sigara ortalama elektroforez koşulları kaynaklanabilir. <ol><li> Bu sapmaların düzeltmek için, değeri 395 &quot;Tepe (bp) Gözlenen&quot; metin kutusuna takın ve &quot;Hesapla&quot; tuşuna basınız. Minimal d2m ve karşılık gelen P değeri ≥0.05 için tekrar kontrol edin. Başarılı değilse, değerleri 405, 415, ve 425 deneyin. NOT: Bazen küçük adımlar bile vardırbelirtilen. Gözlenen profil Referans veri mevcut değildir başarılı de, bu yeni genotip veya varyant olabilir. </li></ol></li><li> çeşitli Profiller <ol><li> Yineleyin ayrı ayrı 5.6 her elektroforez profili için 5.9 için yineleyin. </li></ol></li></ol>

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The authors have nothing to disclose.

RS-PCR (&gt; 30 saf DNA / tahlil ng) &#39;de DNA&#39; sının aşırı olduğunda bütün prosedürün en kritik adımdır 2. Bunu tüm tepe başlangıçta başlangıç ​​ve bitiş Genel olarak, bir profilin çözünürlüğü en uygunudur. iki tepe birbirine çok yakın ise, çözünürlük eksik. kılavuzu tanımlama gerekli olduğu şekilde, bu koşullar altında, Bioanalyzer yazılımı olmayan pik tanımlanmasına yol açabilir. bp veya FU olarak ifade edilen tüm gözle ...

Staphylococcus (S. aureus) sığır 1 meme içi enfeksiyon (IMI) dünya çapında sorumlu en önemli patojen olarak bilinir. 12 Avrupa ülkesinde bunun ineklerde Cosandey ve ark., 3 ve Boss ve diğ. 4 tarafından Fournier ve ark. 2 İsviçre ineklerde gösterdi ve çalışmalarla çalışma S. aureus IMI genetik olarak heterojen bir grup büyükbaş izole. 16S-23S rRNA intergenik aralayıcı bölge (RS-PCR), PCR amplifikasyonu ile, 17 genotip toplam başlangıçta 101 epidemiyolojik bağımsız izolatların 2 tespit edilmiştir. Diğer 15 genotipleri (GTs) her 1 tüm izolatların% 4, yapım nadiren meydana oysa Genotip B ve genotip C (GTC), (% 80) en sık görülen idi. Avrupa 3. seviyede, GTB, GTC, GTF, GTI ve GTR tüm analiz suşları (n = 456)% 76 içeren en önemli genotipleri vardı. GTB w Orta Avrupa&#39;da yer alıyorduhereas diğer genotip yaygın dağıtılmıştır. Suşların kalan% 24 birçoğu, ancak, sadece bir kez gözlendi 41 genotipleri dahil. Genotipleri çok Avrupa düzeyinde olduğu Swiss&#39;te yanı sıra, onların virülans gen modeli ile ilişkili olduğunu Fournier ve ark. 2, Cosandey vd. 3 ve Graber vd. 5 ile çalışmalar daha gösterdi. Çalışmalar daha S. arasında IMI prevalansının büyük farklılıklar ortaya aureus GTB, GTC ve diğer genotipleri 2,5: GTB dikkate alınarak, bir sürüdeki ineklerin% 87&#39;ye kadar bu genotip ile enfekte edildi. GTC ve diğer genotiplerinin durumda, ancak, IMI sadece sürünün 1 ila 2 ineklerde tespit edildi. IMI S. neden aureus, normal gelen meme bezlerinin iltihaplanması süt iltihaplı hücrelerin bir artış ile sonuçlanır. Bunun bir sonucu olarak, somatik hücre koSütteki unts (SCC), çoğu ülkede süt kalitesinin önemli bir göstergedir, azalan süt fiyatı ya da teslim durağına giden artar. Fournier ve ark., 2 RS-PCR yanı sıra, diğer yazma yöntemleri S. sığır suşları alt tiplerinin için tarif edilmiştir aureus 8-11. İki ortak olanları spa yazarak 12 ve Multi Odağı Sırası Yazma (MLST) 13 içerir. Ikinci bir 7 hazırlık geninin sıralama gerektirir, oysa önceki bir protein A stafilokok Spa kodlayan genin değişken aralayıcı bölgesini DNA dizilimi dayanır. Bu, bir 12 ve yedi PCR 13, sırasıyla özel ekipman kullanılarak amplikon saflaştırma, sıralama reaksiyonunun ve analiz, ardından yapılması gerektiği anlamına gelir. RS-PCR ile karşılaştırıldığında, bu yöntem, düşük seviyede örnek ve yüksek maliyetleri ile sonuçlanan laboratuarda önemli bir ek bir çaba gerektirir.verim PFGE için özellikle düşüktür. S. sığır suşları için bu yöntemlerin çözünürlüğü göz önüne alındığında aureus, RS-PCR, en azından MLST 4 veya PFGE 15 den 4 ve daha iyi yazarak spa kadar iyidir. İkili yazma 13 dahil olmak üzere tüm bu yöntemler S. neden sığır IMI patogenezinde içine daha fazla bilgi elde etkinliğini gösterdi aureus, ancak çünkü söz konusu kısıtlamalar onlar büyük klinik araştırmalar için daha az uygundur. Buna ek olarak, RS-PCR yöntemi ile genotipleme Fournier ve arkadaşları tarafından tarif edildiği gibi. 2 epidemiyolojik ve S patojenik potansiyeli güvenilir bir tahmin sağlar aureus, sığır IMI klinik veteriner hekimlikte iki anahtar değerleri çıkıyor.

<table border="0" cellpadding="0" cellspacing="0" width="616">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris(hydroxymethyl)-aminomethan</td>
			<td style="width:100px;">Merck</td>
			<td style="width:119px;">1.08382.0500</td>
			<td style="width:181px;">Mw =121.14g/mol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Titriplex III</td>
			<td style="width:100px;">Merck</td>
			<td style="width:119px;">1.08418.0250</td>
			<td>Mw = 372.24g/mol; Substance is equivalent to Na2EDTA&#183;2H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrochloric acid 5mol/l</td>
			<td style="width:100px;">Merck</td>
			<td style="width:119px;">1.09911.0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Columbia agar+5% sheep blood</td>
			<td style="width:100px;">bioM&#233;rieux</td>
			<td style="width:119px;">43049</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Agilent DNA7500 Kit</td>
			<td style="width:100px;">Agilent Technologies</td>
			<td style="width:119px;">5067-1504</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Agilent 2100 Bioanalyzer</td>
			<td style="width:100px;">Agilent Technologies</td>
			<td style="width:119px;">G2940CA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Agilent 2100 expert sofware</td>
			<td style="width:100px;">Agilent Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HotStarTaq master mix&#160;</td>
			<td style="width:100px;">Qiagen</td>
			<td align="right" style="width:119px;">203445</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primer L1</td>
			<td style="width:100px;">Mycrosinth</td>
			<td style="width:119px;"></td>
			<td>5&#39;CAA GGC ATC CAC CGT3&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primer G1</td>
			<td style="width:100px;">Mycrosinth</td>
			<td style="width:119px;"></td>
			<td>5&#39;GAA GTC GTA ACA AGG3&#39;</td>
		</tr>
	</tbody>
</table>

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.

Bir genotip ve türevleri tanımı belirlenen tüm genotipler birden fazla bant farklı bir elektroforetik profilini yeni genotip olarak kabul edilir. Yeni genotipleri adlandırılır ve genişletilmiş Fournier ve diğerlerine göre. 5 GTAZ genotiplerin GTAA ardından GTZ genotiplerin GTA giden ve GTBA GTBZ için ve GTCC için GTCA düzeydedir. sadece bir bant değişimin, bir genotip bir varyasyon olarak ...

Watch this Scientific Journal Video about Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR at JoVE.com

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, ...

genotyping-of-staphylococcus-aureus-by-ribosomal-spacer-pcr-rs-pcr

Research

JoVE Journal

Immunology and Infection

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

9.5K Görüntüleme.  Agroscope. 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.

Video: Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

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 ...

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 ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nano PCR ile Hayvan Örnekler Solunum Patojenler yüksek verimlilik Algılama

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

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 ...