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

1. 金黄色葡萄球菌菌株<ol><li>价差10微升的S.金黄色葡萄球菌的库存培养或一个菌落生长在含5％绵羊血（BA）哥伦比亚琼脂板上的选择性培养基上。 </li><li>在37℃需氧温育18小时至24小时。 </li></ol> 2. DNA提取<ol><li>制备含10mM的Tris-HCl和10mM 的 Na 2 EDTA，pH 8.5的TEL缓冲液中。在121℃制备等分试样并高压灭菌15分钟。 </li><li>用100微升TEL无菌塑料环或无菌的木制牙签，重悬收集BA 1殖民地。应用1.5 mL管与螺旋盖。 </li><li>孵育在95℃下进行10分钟。随后，立即将样品在湿冰。 </li><li>稀释样品1:100在适合的PCR（=模板DNA用于PCR） 的 H 2 O操作。非稀释和稀释的样品可以保持在-20℃下1个月。 </li></ol> 3. RS-PCR <ol><lI&gt;对于每个样品吸管成PCR管1.5微升的 H 2 0，12.5微升的Taq主混合物，2微升G1引物10μM的，2微升的L1引物10微米，7微升，对应于约30毫微克纯的模板DNA DNA 2。 </li><li>在标准PCR循环仪运行下面循环的程序:15分钟95℃，随后27个循环，包括94℃1分钟，随后在2分钟坡道和退火在55℃7分钟。再过2分钟斜坡后，在72℃延伸2分钟。通过在72℃最后延伸10分钟，然后冷却至4℃终止的PCR。 注:PCR产物可被储存在-20℃下5天。 </li><li>对于每次运行的RS-PCR包括一个阳性对照， 即，与已知的基因型（通常GTB）的样品，和一个阴性对照（H 2 O）。 </li></ol> 4.电泳<ol><li>使用脱氧核糖核酸（救护包）商用微型电泳试剂盒一起连接到PC和安装软件生物分析仪。仔细研究制造商的相应手册。 </li><li>设置在芯片涂刷站并根据制造商的手册调节注射器剪辑。 </li><li>根据制造商的手册制备凝胶染料混合。 </li><li>载入凝胶染料混合物（根据制造商的手册）。 <ol><li>平衡凝胶染料混合至RT使用前30分钟。 </li><li>提出了新的DNA芯片的芯片吸站。吸管9微升凝胶染料在井标记G. </li><li>确保柱塞被定位以1ml，然后关闭该芯片涂刷站。按柱塞，直到它被注射器夹子保持。正是等待30秒再松开注射器夹。 </li><li>等待5秒。活塞慢慢回拉至1ml的位置。 </li><li>打开芯片吸站和吸管9微升凝胶染料在井都标有&#39;G&#39;。 </li></ol></li><li>加载并购arker <ol><li>吸取5微升标记在所有12个样品孔，并在梯井。不要让这些13口井空的。 </li></ol></li><li>加载梯和样品。 <ol><li>吸管1μl的DNA梯状到良好标有阶梯符号。 </li><li>吸取1微升样品（用井）或1微升去离子水（未使用的水井）的。 </li><li>横过来时，芯片的适配器和涡旋1分钟2400转。 </li><li> 5分钟内运行在生物分析仪芯片。 </li></ol></li><li>运行分析<ol><li>启动生物分析仪和电脑相连。打开软件。 </li><li>打开生物分析仪的盖和芯片放入芯片保持器（芯片只适合单程）。仔细盖上盖子。 </li><li>在软件的仪器上下文菜单中选择"电泳"&gt;"双链"&gt;"DNA 7500"。接受软件的当前文件前缀或修改。</li><li>点击存在于软件的启动按钮。在软件中输入样本名。当芯片运行结束（后约30分钟） 运行邮件的末尾出现在生物分析仪软件。 </li><li>根据制造商的指示删除该芯片和清洁生物分析仪的电极。 </li></ol></li></ol> 5.从电泳图谱推断基因型<ol><li>加载泰姬陵软件（软件是免费提供从作者）。 </li><li>进口参考数据到泰姬陵软件:"文件"&gt;"导入参考数据" </li><li>打开在生物分析仪软件的数据上下文中运行电泳。选择一个样品的电泳轮廓。 </li><li>检查相关的峰值。 <ol><li>使用泰姬陵软件来检查相关峰:"工具"&gt;"平均荧光"。插入"荧光"框荧光值表示为荧光单位（FU），因为它们可以从生物分析仪软件被读出。将4（3）峰升序排列最高的荧光，以逗号分隔。例如:126130132137。 注意:如果不是所有的这些峰FU的由生物分析仪软件的指示，就需要通过从积读出来进行估计。 </li></ol></li><li>按"计算"按钮。如果观察到的荧光超过盒子"最小峰高"给出的值峰值是相关的。 </li><li>推断基因型。 <ol><li>插入文本框泰姬陵"输入峰（BP）"，在升序排列的所有相关峰基点大小和以逗号分隔。例如:440,462,519,579,637。 注意:如果不是所有这些峰的大小是由生物分析仪软件的指示，就需要通过从积读出来进行估计。 </li></ol></li><li>按"计算"按钮。 </li><li>在列表中，查找最小平方马氏距离"D2M"。如果相应的P值给它是≥0.05则概率为≥5％，该零假设H 0被拒绝（H 0:所观察到的与参考轮廓是相同的），这意味着所观察到的信息被接受，以完全相同指定基因型的基准轮廓。 </li><li>如果D2M是最小的，但相应的P值的字段是空的，拒绝H 0的概率为&lt;5％。 注意:这意味着该轮廓是不相等的，尽管D2M是最小的。这样的结果，可能会导致从非平均的电泳条件。 <ol><li>为了纠正这些偏差，插入"观察峰（BP）"值395的文本框，然后按"计算"按钮。再次检查最小D2M和相应的P值≥0.05。如果不成功，尝试值405，415，和425。 注意:有时更小的步骤，甚至是表示。如果仍然没有成功，所观察到的信息中不存在的参考数据，它可能是一个新的基因型或变体。 </li></ol></li><li>各种型材<ol><li>重复步骤对每个电泳图谱5.6至5.9分开。 </li></ol></li></ol>

<ol>
	<li>Sears, P. M., McCarthy, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+and+treatment+of+staphylococcal+mastitis.">Management and treatment of staphylococcal mastitis.</a> Vet.Clin.North Am.Food Anim.Pract. 19 (1), 171-185 (2003).</li><li>Fournier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+Staphylococcus+aureus:+association+of+virulence+genes,+genotypes+and+clinical+outcome.">Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome.</a> Res.Vet.Sci. 85 (3), 439-448 (2008).</li><li>Cosandey, A., et al. <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+genotype+B+and+other+genotypes+isolated+from+cow+milk+in+European+countries.">Staphylococcus aureus genotype B and other genotypes isolated from cow milk in European countries.</a> J.Dairy Sci. 99 (1), 529-540 (2016).</li><li>Boss, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+Staphylococcus+aureus:+Subtyping,+evolution,+and+zoonotic+transfer.">Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer.</a> J.Dairy Sci. 99 (1), 515-528 (2016).</li><li>Graber, H. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mastitis-related+subtypes+of+bovine+Staphylococcus+aureus+are+characterized+by+different+clinical+properties.">Mastitis-related subtypes of bovine Staphylococcus aureus are characterized by different clinical properties.</a> J.Dairy Sci. 92 (4), 1442-1451 (2009).</li><li>Heiniger, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kosten-Nutzen-Analyse+einer+Intervention+zur+Verbesserung+der+Eutergesundheit+in+Schweizer+Milchviehbetrieben.">Kosten-Nutzen-Analyse einer Intervention zur Verbesserung der Eutergesundheit in Schweizer Milchviehbetrieben.</a> Schweiz.Arch.Tierheilkd. 156 (10), 473-481 (2014).</li><li>Hamann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition+of+the+physiological+cell+count+threshold+based+on+changes+in+milk+composition.">Definition of the physiological cell count threshold based on changes in milk composition.</a> IDF Mastitis Newsletter. 25, 9-12 (2003).</li><li>Hwang, S. Y., Park, Y. K., Koo, H. C., Park, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=spa+typing+and+enterotoxin+gene+profile+of+Staphylococcus+aureus+isolated+from+bovine+raw+milk+in+Korea.">spa typing and enterotoxin gene profile of Staphylococcus aureus isolated from bovine raw milk in Korea.</a> J.Vet.Sci. 11 (2), 125-131 (2010).</li><li>Sakwinska, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Link+between+genotype+and+antimicrobial+resistance+in+bovine+mastitis-related+Staphylococcus+aureus+strains,+determined+by+comparing+Swiss+and+French+isolates+from+the+Rhone+Valley.">Link between genotype and antimicrobial resistance in bovine mastitis-related Staphylococcus aureus strains, determined by comparing Swiss and French isolates from the Rhone Valley.</a> Appl.Environ.Microbiol. 77 (10), 3428-3432 (2011).</li><li>Rabello, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilocus+sequence+typing+of+Staphylococcus+aureus+isolates+recovered+from+cows+with+mastitis+in+Brazilian+dairy+herds.">Multilocus sequence typing of Staphylococcus aureus isolates recovered from cows with mastitis in Brazilian dairy herds.</a> J.Med.Microbiol. 56, 1505-1511 (2007).</li><li>Tavakol, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine-associated+MRSA+ST398+in+the+Netherlands.">Bovine-associated MRSA ST398 in the Netherlands.</a> Acta Vet.Scand. 54, 28 (2012).</li><li>Harmsen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Typing+of+methicillin-resistant+Staphylococcus+aureus+in+a+university+hospital+setting+by+using+novel+software+for+spa+repeat+determination+and+database+management.">Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management.</a> J.Clin.Microbiol. 41 (12), 5442-5448 (2003).</li><li>Zadoks, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+pulsed-field+gel+electrophoresis+and+binary+typing+as+tools+in+veterinary+clinical+microbiology+and+molecular+epidemiologic+analysis+of+bovine+and+human+Staphylococcus+aureus+isolates.">Application of pulsed-field gel electrophoresis and binary typing as tools in veterinary clinical microbiology and molecular epidemiologic analysis of bovine and human Staphylococcus aureus isolates.</a> J.Clin.Microbiol. 38 (5), 1931-1939 (2000).</li><li>Cremonesi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+characteristics+of+Staphylococcus+aureus+strains+associated+with+high+within-herd+prevalence+of+intramammary+infections+in+dairy+cows.">Genomic characteristics of Staphylococcus aureus strains associated with high within-herd prevalence of intramammary infections in dairy cows.</a> J.Dairy Sci. 98 (10), 6828-6838 (2015).</li><li>Enright, M. C., Day, N. P., Davies, C. E., Peacock, S. J., Spratt, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilocus+sequence+typing+for+characterization+of+methicillin-resistant+and+methicillin-susceptible+clones+of+Staphylococcus+aureus.">Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus.</a> J.Clin.Microbiol. 38 (3), 1008-1015 (2000).</li></ol>

The authors have nothing to disclose.

整个过程中最关键的步骤是当在RS-PCR（&gt; 30纳克纯的DNA /测定）过量的DNA的2。在一般情况下，一个轮廓的分辨率为最佳，如果所有其峰在基线开始和结束。如果两个峰值靠得太近，分辨率是不完整的。在这些条件下，从而需要人工识别的生物分析仪软件可能导致不适当的峰鉴定。 所有表现为BP或FU的明显分离，相关峰作进一步的分析。太多模板DNA RS-PCR结果在宽峰，因...

葡萄球菌 （ 金黄色葡萄球菌 ）被称为全球用于在牛1乳房内感染（IMI）负责的最重要的病原体。在12个欧洲国家的奶牛研究由Fournier 等人 2瑞士奶牛示范和研究通过Cosandey 等 3和老板等 4 S.金黄色葡萄球菌从牛隔离IMI是一种遗传异质组。由16S-23S rRNA基因间区（RS-PCR）的PCR扩增，共有17个基因型在101流行病学独立的分离2最初检测。基因型B和C型（GTC）是最常见的（80％），而其它15个基因型（GTS）发生很少，各使所有的菌株1至4％。在欧洲3级，GTB，GTC，GTF，GTI和GTR是由所有分析的株（N = 456）的76％，最重要的基因型。 GTB是位于欧洲中部W¯¯hereas其他基因型广泛传播。菌株的其余24％包含41种基因型，其中许多，但是，观察到只有一次。 通过Fournier 等人的 2，Cosandey 等 3和格雷伯等人 5的研究进一步表明，基因型与它们的毒力基因图案在瑞士高度相关的，以及如在欧洲一级。该研究进一步揭示之间S.在IMI患病率差异巨大的金黄色葡萄球菌 GTB，GTC和其他基因型2,5:考虑GTB，最多在牛群奶牛的87％是由这种基因型感染。在GTC和其他基因型的情况下，然而，IMI仅在1至2的奶牛畜群的发现。 IMI引起S.黄色葡萄球菌 ，通常会导致在相应的乳腺炎症和牛奶中的增加的炎性细胞。因此，体细胞共牛奶UNTS（SCC），牛奶的品质在大多数国家的关键指标，增加从而导致降低牛奶价格甚至停止发货。 除了Fournier 等人 2的RS-PCR其他分型方法已为亚型S的牛菌株描述金黄色葡萄球菌 8-11。两种最常见的包括水疗打字12多位点序列分型（MLST）13。前者是基于DNA测序葡萄球菌温泉基因编码蛋白质A的可变间隔区，而后者要求7管家基因的测序。这意味着一个12和七个PCR的13，分别需要执行，随后扩增子纯化，测序反应和分析使用特定的设备。相比的RS-PCR，这些方法需要在导致低的样品通量和成本高的实验室一个相当大的额外的努力。该吞吐量是特别低PFGE。考虑到这些方法对S的牛株分辨率金黄色葡萄球菌 ，RS-PCR是至少作为温泉打字4和优于MLST 4或电泳15一样好。 所有这些方法包括二进制打字13表现在获得进一步的深入了解造成S.牛IMI的发病机制及其有效性所提到的限制的金黄色葡萄球菌 ，但因为它们是用于大型临床研究较少为宜。另外，作为由Fournier 等人描述由RS-PCR基因分型。2允许流行病学和S的致病潜力的可靠的预测金黄色葡萄球菌参与牛IMI，在临床兽医学两个关键值。

<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.

基因型及其变体的定义电泳轮廓从所有确定的基因型在一个以上的频带不同的被认为是新的基因型。新基因型根据Fournier 等人的命名和扩展。5通往基因型GTA到GTZ，其次是基因型GTAA到GTAZ，并GTBA到GTBZ和GTCA到GTCC目前。只在一个频带变化被视为一个基因型变体。它的标志是基因型（如 GTRI，GTRII）的名?...

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.

基因分型<em&gt;金黄色葡萄球菌</em&gt;由核糖体垫片的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 次观看.  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.

视频: 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.

滴流和旋转盘反应器使用金黄色葡萄球菌生物膜分析

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

科研

免疫与感染

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.

皮下感染耐甲氧西林金黄色葡萄球菌（MRSA）的

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.

耐甲氧西林实验性心内膜炎模型<em&gt;金黄色葡萄球菌</em&gt;（MRSA）的大鼠

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.

金黄色葡萄球菌生长使用人血红蛋白作为铁源

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.

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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.

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

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.

针对相关生物膜<em&gt;金黄色葡萄球菌</em&gt;使用基于刃天青药物敏感性测定

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.

在动物标本呼吸道病原体的高通量检测通过纳米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 ...

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.

方法基因水平转移的研究<em&gt;金黄色葡萄球菌</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.

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

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