Name: preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing
Uploaded: 2014-10-15T14:00:00
Description: Watch this Scientific Journal Video about Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing at JoVE.com

我们感谢洛桑大学医院中心的细菌学实验室的技术人员的帮助，​​以实现在实验室的技术。

该协议已开发和验证实施作为常规工具之前，下面我们机构的研发流程和道德准则。 1，制备血培养菌体通过氯化铵红细胞裂解过程<ol><li>制备血培养阳性随后离心。 <ol><li>消毒血培养上限。添加70％乙醇的瓶盖和焚烧。 注意：不要执行在层流罩这一步。 </li><li>将瓶子在层流罩。收集5毫升血培养有20G的注射器。 </li><li>添加5毫升血培养中装有45毫升无菌水（H 2 O?...

<ol>
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L., Diekema, D. J., Beekmann, S. E., Chapin, K. C., Doern, G. 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G., Drake, S. K., Murray, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+bacteria+in+positive+blood+culture+broths+by+matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry.">Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry.</a> J Clin Microbiol. 48, 444-447 (2010).</li><li>Croxatto, A., Prod&amp;34hom, G., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+MALDI-TOF+mass+spectrometry+in+clinical+diagnostic+microbiology.">Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology.</a> FEMS Microbiol Rev. 36, 380-407 (2012).</li><li>Chen, J. H., 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=Direct+bacterial+identification+in+positive+blood+cultures+by+use+of+two+commercial+matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry+systems.">Direct bacterial identification in positive blood cultures by use of two commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry systems.</a> J Clin Microbiol. 51, 1733-1739 (2013).</li><li>Prod&amp;34hom, G., Bizzini, A., Durussel, C., Bille, J., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry+for+direct+bacterial+identification+from+positive+blood+culture+pellets.">Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets.</a> J Clin Microbiol. 48, 1481-1483 (2010).</li><li>Prod&amp;34hom, G., Durussel, C., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+blood-culture+bacterial+pellet+preparation+for+faster+accurate+direct+bacterial+identification+and+antibiotic+susceptibility+testing+with+the+VITEK+2+system.">A simple blood-culture bacterial pellet preparation for faster accurate direct bacterial identification and antibiotic susceptibility testing with the VITEK 2 system.</a> Journal of medical microbiology. 62, 773-777 (2013).</li><li>Clerc, 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=Matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry+and+PCR-based+rapid+diagnosis+of+Staphylococcus+aureus+bacteraemia.">Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR-based rapid diagnosis of Staphylococcus aureus bacteraemia.</a> Clin Microbiol Infect. , (2013).</li><li>Martiny, 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=Impact+of+rapid+microbial+identification+directly+from+positive+blood+cultures+using+matrix-assisted+laser+desorption/ionization+time-of-flight+mass+spectrometry+on+patient+management.">Impact of rapid microbial identification directly from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on patient management.</a> Clin Microbiol Infect. 19, E568-E581 (2013).</li><li>Stoneking, L. 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相对于传统的血培养阳性的诊断方法，在快速采集细菌沉淀物通过使用氯化铵裂解离心方法减少时间由16至24小时的时间来报告AST由24至48小时（ 图1和报告标识3）。 迅速出台相应的抗生素治疗是关键，以提高患者的血液感染患上的结果。因此，早期识别中1小时，随后在约8至16小时，得到了迅速的AST的传染性病原体的代表在脓毒症患者的临床管理有重?...

血液感染和败血症住院的病人发病和死亡的一个主要原因。因此，死亡率为血液感染有关的约14％被观察到的住院病人的37％，并可能增加至35％，在重症监护病房的患者1-3。快速鉴定病原体的是枢转，以指导最佳的抗微生物治疗，并增加抗微生物疗法4,5的成功结果。革兰氏染色，从血培养阳性的快速分析已经对抗生素治疗6,7但准确识别病原体的法律适应化显著影响需要提供最适合的抗生素治疗的患者。举例来说，不同的抗生素治疗方案有以下菌血症与肠球菌和链球菌，很难通过革兰氏染色，以区分实施。同样的，在鉴定物种利埃尔需要检测革兰氏阴性肠杆菌编码的染色体AmpC酶基因，其赋予增加的耐β-内酰胺8。 用血培养阳性，常规的诊断方法是传代的传染性病原体的不同琼脂平板上，这就需要采用不同方法，包括生化检查，在不同的选择性培养基上生长和全自动微生物鉴定系统几个小时的额外孵化前鉴定。时间的常规诊断方法的结果是约1至3天。 基质辅助激光解吸/飞行时间的电离质谱（MALDI-TOF）技术快速鉴定微生物的出现提供了一种新的工具，以快速识别从阳性血直接从生长在琼脂平板上的菌落微生物也培养物（ 图1）9-12。日E使用MALDI-TOF的从血培养鉴定传染性病原体的时候结果已经显著减小到几分钟，而不是通过传统的方法所需要的时间和天数。如由Croxatto 等 13所讨论的，MALDI-TOF鉴定的效率依赖于不同的参数，包括微生物的纯度和数量。这两个标准都容易从生长在琼脂平板上的菌落分离得到的，但需要对细菌浓缩和纯化从复杂样品如血液培养，其中含有多种细胞和蛋白质成分可与MALDI-TOF鉴定干扰预分析处理。 从血培养各种微生物&#39;的分离方法中的一些研究，其中包括皂角苷或其它温和的清洁剂的方法进行细菌提取9,14，血清分离方法10已被使用，裂解离心方法12 </s&gt;和商业解决方案，如sepsityper套件。我们的细菌学诊断实验室已经开发出了基于氯化铵红细胞裂解，允许快速识别细菌和酵母通过MALDI-TOF和自动识别系统（ 图2）15一个简单的血液培养菌体的准备。这血培养微丸制剂也为其他直接下游应用，如革兰染色阳性的样本，自动化PCR为基础的诊断测试，如POCT-PCR的快速检测耐甲氧西林金黄色葡萄球菌 （MRSA）和药敏试验用自动化系统的AST和/或通过在琼脂平板上扩散测定（ 图3）。 在这项工作中，我们描述了血培养菌体的制备方法的不同步骤由Prod&#39;hom 等人所说明15（ 图4）。我们也将取消划线为3，可以在血培养粒料进行的主要应用的协议：用自动化系统16，用于肠杆菌和葡萄球菌和自动化PCR-鉴定通过MALDI-TOF 15，标识（ID）和抗生素敏感性试验（AST）基于诊断测试为MRSA 17的检测。

<table border="0" cellpadding="0" cellspacing="0" width="1130">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">20 needle gauge&#160;</td>
			<td style="width:268px;">Terumo, Leuven, Belgium</td>
			<td style="width:239px;">NN-2038R</td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">50 ml Falcon tube</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>352070</td>
			<td>50 ml centrifuge tubes</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ammonium chlorure</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>101145</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Potassium hydrogen carbonate</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>60340</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>F0507</td>
			<td>Flammable, corrosive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-Cyano-4-hydroxycinnamic acid</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>70990</td>
			<td>Acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Acetonitrile</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>271004</td>
			<td>Flammable, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Trifluoroacetic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>T6508</td>
			<td>Corrosive, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 60 instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27202</td>
			<td>automated microbial system instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-positive (GP) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21342</td>
			<td>automated GP&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-P580 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>22233</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-negative (GN) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21341</td>
			<td>automated GN&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-N242 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>413391</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Xpert MRSA</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>&#160;GXMRSA-100N-10</td>
			<td>nucleic acid amplification technology MRSA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">GeneXpert IV instrument</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>GXIV-4-D</td>
			<td>nucleic acid amplification technology instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Microflex LT MALDI-TOF MS instrument</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>BDAL microflex LT/SH</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MSP 96 target&#160; steel BC</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>280799</td>
			<td>MALDI target plate</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Densitometer Densicheck instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27208</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MALDI Sepsityper kit 50</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>8270170</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mac Conkey agar</td>
			<td>Biolife, Milano, Italy</td>
			<td>4016702</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mueller-Hinton agar</td>
			<td>Oxoid, Hampshire, England</td>
			<td>CM0337</td>
			<td>Mueller-Hinton agar (MH)&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MHF agar</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>43901</td>
			<td>Mueller-Hinton agar-fastidious organisms agar (MHF)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD columbia III agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254071</td>
			<td>blood agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD chocolate agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254089</td>
			<td>chocolate agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD schaedler agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254084</td>
			<td>Schaedler agar</td>
		</tr>
	</tbody>
</table>

preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing

血流感染和败血症的发病率和死亡率的主要原因。患者从菌血症患的成功结果依赖于快速鉴定病原体的指导最佳抗生素治疗。从血培养阳性的革兰氏染色的分析，可以迅速进行，并已显著影响抗生素的治疗方案。然而，准确地识别病原体的，仍然需要建立最优针对性的治疗。我们在座的从血培养阳性可作为样本几个基本的下游应用，如鉴定MALDI-TOF MS，药敏试验（AST）采用纸片扩散法或自动化助攻系统简单，快速的细菌沉淀的准备并且通过自动的基于PCR的诊断测试。直接作用于血培养细菌沉淀应用于这些不同的标识和AST系统的性能是非常SImilar的性能通常是从生长在琼脂平板上的菌落分离得到。相较于传统的方法，迅速收购细菌沉淀的显著减少了时间报告均标识和AST。因此，下面的血培养阳性，鉴定通过MALDI-TOF可以在少于1小时，而AST的结果通过自动AST系统或纸片扩散测定法在8至18小时，分别进行报告。类似地，快速的基于PCR的测定法的结果可被传达给临床医生不到2小时以下内容的菌血症的报告。总之，这些结果表明，血培养细菌沉淀的快速制备具有该识别和AST的周转时间，从而对患者的血液感染的患成功结果的显著影响。

在由Prod&#39;hom 等 15进行的研究中，从78例患者由122阳性血培养的氯化铵裂解离心分离得到的细菌粒料通过MALDI-TOF MS进行分析。出了122血培养阳性，95（77.9％）被正确识别的物种水平和一（0.8％），在属的水平。其余26（21.3％）血培养颗粒没有给出可靠的鉴​​定通过MALDI-TOF。其中，21人革兰氏阳性细菌，包括13链球菌和5凝固酶阴性葡萄球菌。重要的是，10出13不明链球菌是肺炎链?...

Watch this Scientific Journal Video about Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing at JoVE.com

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

从血培养阳性的快速细菌沉淀制备可以用作用于诸如鉴定通过MALDI-TOF，革兰氏染色，药敏试验和基于PCR的试验样品。结果能够迅速地传达给临床医生来改善患者的血液感染的痛苦的结果。

为快速细菌鉴定和药敏试验的准备血培养微丸

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Research

JoVE Journal

Immunology and Infection

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

科研

免疫与感染

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

协议RBD - SARS疫苗的重组蛋白的制备，动物疫苗接种和中检测

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

一个1.5小时的程序，鉴定肠球菌直接从血培养的物种

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

结核分枝杆菌综合肉汤稀释药敏试验微孔板格式为一线和二线药物

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Introducing Shear Stress in the Study of Bacterial Adhesion

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

为期一天的血培养的细菌病原体检测和耐药性检测的工作流程计划

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

参考微量稀释法达巴万星<em&gt;体外</em&gt;细菌敏感性测试生长有氧

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

对于血液处理快速的方法，以提高血浆肽水平的人血产量

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...