我们要感谢他们的帮助梅奥诊所的分枝杆菌实验室的化验师在研究过程中。

每个实验室都需要执行，其机构的生物安全主任的合作的风险评估，以确定相应的生物安全水平的接种准备和移液的接种入盘。在我们的实验室，我们利用生物安全3级实验室（BSL3）的做法，直到微孔板接种，并用塑料的粘接密封密封。这些板块，然后放在一个塑料袋，这是热也密封。在一个生物安全2级实验室（BSL2）进行孵化和解释微孔板读数。 1。材料的标签<ol><li>标签的血琼脂平板上，三个Middlebrook 7H10板，Middlebrook 7H9肉汤，一个盐水之间的玻璃珠管和AST与相应的标识符（例如，病人姓名，病历号）的BSL2一个酶标板。 </li><li>标签在血琼脂和7H10镀金的“纯度检查”。定期接种菌落计数的建议，以确保生物体的适当集中是在测试中使用。执行时的菌落计数将需要有两个“1 / 1菌落计数”和“1 / 50菌落计数”标记7H10板。 </li></ol> 2。技师的准备工作进入了BSL3 <ol><li>适当的个人防护设备是必要的工作在BSL3的，包括前坚实的礼服，头盖，手套，鞋套和口罩。每个机构都应该建立在自己的呼吸保护计划与工业卫生机构的主任。在我们的实验室，我们有几个呼吸器，包括动力的空气净化呼吸器（PAPRs）和装有HEPA过滤，N95半面呼吸器。 </li></ol> 3。制备的生物安全柜<ol><li>在BSL3，准备通过生物安全柜（BSC）的表面进行消毒，并把一纸毛巾浸泡消毒剂约6英寸风口面板。 </li><li>放置在左侧的垃圾容器，并在右侧的小nephlometer vortexer。 </li><li>接种循环，pipetors和技巧都放在旁边的纸巾。 </li><li>将与有机体（肉汤或固体培养基上）和麦克法兰0.5标准纸巾的架子。 </li></ol> 4。校准的浊度<ol><li>生理盐水之间的玻璃珠管放置在浊度和仪器设置为零，使用生理盐水之间的空白。 </li><li>广场麦克法兰0.5标准，在浊度和校准根据制造商的指示。 </li></ol> 5。接种物的制备<ol><li>工作中的BSC，从固体培养基上刮去使用适当标记的生理盐水之间的玻璃珠管无菌循环，直到获得一个≥0.5麦克法兰标准菌落。 </li><li>目视检查样品，并麦克法兰标准进行比较。 </li><li>涡样品为30-60秒。 </li><li>允许在接种15分钟定居（设置一个计时器，）。 </li></ol> 6。微孔板接种<ol><li>调整用生理盐水之间使用校准步骤4.1和4.2麦克法兰0.5浊度肉汤接种。这必须完成在30分钟内（每生产厂家的规格）。 </li><li>使用200μLpipetter和一个LONG气溶胶耐吸管尖删除生理盐水珠之间的管前1 / 3 路 100μL接种。接种Middlebrook 7H9肉汤。慢慢涡接种20秒7H9肉汤。 </li><li>从制造商的包中取出的AST微孔板。不要使用如果干燥剂是蓝色或铝箔包损坏。 </li><li>准备使用1:50稀释管编制的1:50稀释后吹打成无菌管50μL无菌水。 </li><li>小心倒入一个低谷7H9肉汤接种。避免产生气溶胶。 </li><li>与标签朝上放置AST微孔板。 </li><li>使用12通道pipetter，每孔加100μL接种。 </li><li>准备菌落计数稀释： <ol type="a"><li> 1 / 1稀释准备裸奔1μL环上标有“1 / 2”7H10板以及阳性对照（H12）。 </li><li> 1 / 50稀释的准备以及阳性对照（H12）接种1μL循环和漩入管50μL准备以上的水管。此稀释液连续板标有“1 / 50”。 </li></ol></li><li>接种纯度板从低谷到一个适当标记的血琼脂和7H10板和隔离连胜吹打50微升。 </li><li>地点AST微孔板上的粘接密封。按牢牢掌握每口井，以保证足够的密封使用的密封的白回来。避免折痕。 </li><li>微孔板与tuberculocidal消毒剂和一个塑料袋，并关闭到位微孔板与热封或磁带浸湿的纸巾擦拭消毒。 <ol type="a"><li>作为一个辅助容器的塑料袋供应，以确保所有的肉汤中应该有泄漏或溢出的主要板块的。 </li></ol></li></ol> 7。材料和生物安全柜消毒<ol><li>为了避免产生气溶胶之前，将它轻轻放入丢弃的容器接种槽上方的另一个低谷。 </li><li>与tuberculocidal消毒剂擦拭消毒所有包括nephlometer，vortexer，板，管等材料，并等待适当的时机，根据制造商的指示，以确保消毒，然后删除从BSC。例如，Prospray，使用的tuberculocidal消毒剂，我们需要15分钟，杀灭分枝杆菌。 </li><li>将丢弃在生物危害袋和密封容器前高压灭菌。 </li></ol> 8。孵化<ol><li>板与媒体应培养下，应小心，以避免任何液体溢出或溅水。 </li><li>在一个35-37 ° C培养箱中孵育5-10％5-10％CO 2在BSL2实验室CO 2的AST微孔板板，7H10和血琼脂平板。 <ol class="lalpha"><li>在我们的实验室，这是可以接受的，因为该板块良好的密封在一个密封的塑料袋。 </li></ol></li><li>不超过2的酶标助攻板应堆放在孵化器中，根据制造商的指示。 </li></ol> 9。代表性的成果结果的解释： 密封助攻微孔板可以读取使用手动镜作为一种辅助手段，或用自动酶标仪Vizion（郊游诊断）等。该板可尽快接种后7天的检查。检查位置H11和H12的生长控制井，以确定如果他们是积极的（例如，有一个细胞在井底的存款）。如果不积极的增长控制井，reinc​​ubate板，并定期重新审视（例如，第10天，14天），直到增长的控制井是积极的。 <ol type="a"><li>镜像读取： <ol type="i"><li>密封放置到平台AST微孔板; </li><li>阅读增长的控制井7天的潜伏期后，如果这是消极的，reinc​​ubate助攻微孔板; </li><li>如果增长的控制是可以接受的的，然后读取含药井。对于每种药物，记录第一稀释与生长控制药物井没有增长。 </li><li>增长出现浑浊或细胞在井底的存款。 </li></ol></li><li>仪器阅读：按照制造商的说明，下面的说明是如何将Vizion平台阅读的例子： <ol type="i"><li>登录。 </li><li>使用“MYCOTB”AST板软件。 </li><li>将面向用户的条形码密封板。增长出现浑浊或细胞在井底的存款。 </li><li>生长控制;如果这是消极的，reinc​​ubate助攻微孔板，如果是正读板，通过触摸屏幕上的每一种药物，这并不表明增长的第一口井。 </li></ol></li></ol>阅读纯度板。血琼脂平板上应显示在48小时后没有增长，7H10纯度板应该有一个生物类型的融合发展。如果被污染，AST的酶标测试需要从一个纯粹的文化重复。 计算菌落计数，从积极的控制以及使用下表： 菌落计数键<table border="1"><tbody><tr><td> 算的上标有“1 / 1＃殖民地”板块</td><td> ＃殖民地算的上标有“1 / 50”板</td><td> 接种菌落数（CFU / ml为） </td></tr><tr><td> &lt;50 </td><td> 0 </td><td> &lt;5 × 10 4 </td></tr><tr><td> 50-100 </td><td> 0-2 </td><td> 5 × 10 4 1 × 10 5 </td></tr><tr><td> &gt; 100 </td><td> ≤10 </td><td> 1 × 10 5至5 × 10 5 </td></tr><tr><td> &gt; 100 </td><td> &gt; 10 </td><td> &gt; 5 × 10 5 </td></tr></tbody></table>有可能是尾随段-氨基水杨酸在结核分枝杆菌tuberculo的某些菌株SIS。这可以解释，在所有的水井，有颗粒和使用的第一口井，旁边的终点稀释同样大小的颗粒。抵抗这种药物是罕见的。 <img src="/files/ftp_upload/3094/3094fig1.jpg" alt="图1"/> 图1。代表性的结果，使用MYCOTB板 。 <table border="1"><tbody><tr><td> 药品缩写 </td><td> 药物 </td><td> MICμ/毫升 </td></tr><tr><td> OFL </td><td>氧氟沙星</td><td> 16 </td></tr><tr><td> MXF </td><td>莫西沙星</td><td> 8 </td></tr><tr><td> RIF </td><td>利福平</td><td> &gt; 16 </td></tr><tr><td>急性心肌梗死</td><td>丁胺卡那霉素</td><td> 0.5 </td></tr><tr><td> STR </td><td>链霉素</td><td> 16 </td></tr><tr><td> RFB </td><td>利福布丁</td><td> 8 </td></tr><tr><td>保安局首席助理局长</td><td> P -氨基水杨酸</td><td> ≤0.5 </td></tr><tr><td>理工学院</td><td> ethionamide </td><td> 5 </td></tr><tr><td>公益少年团</td><td>环丝氨酸</td><td> 4 </td></tr><tr><td>异烟肼</td><td>异烟肼</td><td> 2 </td></tr><tr><td>简</td><td>卡那霉素</td><td> 1.2 </td></tr><tr><td>教统局</td><td>乙胺丁醇</td><td> 4.0 </td></tr></tbody></table>

<ol>
	<li>Wengenack, N., Hall, L., Labombardi, V., Parrish, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+New+Sensititre+(MYCOTB)+MIC+Plate+for+the+Susceptibility+Testing+of+Mycobacterium+tuberculosis+(Mtb)+to+First+and+Second+Line+Drugs.">Evaluation of the New Sensititre (MYCOTB) MIC Plate for the Susceptibility Testing of Mycobacterium tuberculosis (Mtb) to First and Second Line Drugs.</a> , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HHS+Publication+No.+(CDC)+21-112:+Biosafety+in+Microbiological+and+Biomedical+Laboratories+(BMBL).">HHS Publication No. (CDC) 21-112: Biosafety in Microbiological and Biomedical Laboratories (BMBL).</a> U.S. Department of Health and Human Services Centers for Disease and Preventions. , (2009).</li><li>Morcillo, N., Imperiale, B., Digiulio, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+MGIT+960+and+the+colorimetric-based+method+for+tuberculosis+drug+susceptibility+testing.">Evaluation of MGIT 960 and the colorimetric-based method for tuberculosis drug susceptibility testing.</a> Int J Tuberc Lung Dis. 14, 1169-1175 (2010).</li><li>Bemer, P., Bodmer, T., Munzinger, J., Perrin, M., Vincent, V., Drugeon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+Evaluation+of+the+MB/BACT+System+for+Susceptibility+Testing+of+Mycobacterium+tuberculosis.">Multicenter Evaluation of the MB/BACT System for Susceptibility Testing of Mycobacterium tuberculosis.</a> J Clin Microbiol. 42, 1030-1034 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SENSITITRE+MYCOTB+Susceptibility+Plate:+For+Mycobacterium+Susceptibility+Testing.">SENSITITRE MYCOTB Susceptibility Plate: For Mycobacterium Susceptibility Testing.</a> Product Insert. , (2010).</li><li>CLSI. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Susceptiblity Testing of Mycobacteria, Nocardia, and Other Aerobic Actinomycetes; Approved Standard. Document M24-A. , (2003).</li><li>Sullivan, N., Sotos, J., Anhalt, K., Allen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+results+from+a+new+TREK+Sensititre+Mycobacterium+tuberculosis+MIC+plate+(MYCOTB).">Preliminary results from a new TREK Sensititre Mycobacterium tuberculosis MIC plate (MYCOTB).</a> , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VersaTREK+MYCO+SUSCEPTIBLITY+KIT.">VersaTREK MYCO SUSCEPTIBLITY KIT.</a> Product insert. , 7115-7160 (2008).</li><li>Parrish, N. M., Carroll, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+Clinical+Mycobacteriology+Laboratory+in+the+Diagnosis+and+Management+of+Tuberculosis+in+Low+Prevalance+Settings.">The Role of the Clinical Mycobacteriology Laboratory in the Diagnosis and Management of Tuberculosis in Low Prevalance Settings.</a> J Clin Microbiol. , (2010).</li><li>Kam, K. M., Sloutsky, A., Yip, C. W., Bulled, N., Zingol, M., Espinal, M., Kim, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+critical+concentrations+of+second-line+anti-tuberculosis+drugs+with+clinical+and+microbiological+relevance.">Determination of critical concentrations of second-line anti-tuberculosis drugs with clinical and microbiological relevance.</a> Int J Tubercl Lung Dis. 14, 282-288 (2010).</li><li>Woods, G. L., Warren, N. G., Inderlied, C. B., Murray, P. R., Barron, E. J., Jorgensen, J. H., Landry, M. L., Pfaller, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+Test+Methods:+Mycobacteria,+Nocardia,+and+Other+Actinomycetes.">Susceptibility Test Methods: Mycobacteria, Nocardia, and Other Actinomycetes.</a> Manual of Clinical Microbiology. , 1223-1247 (2007).</li><li>Bergmann, J. S., Woods, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+ESP+Culture+System+II+for+Testing+Susceptibilities+of+Mycobacterium+tuberculosis+Isolates+to+Four+Primary+Antituberculosis+Drugs.">Evaluation of the ESP Culture System II for Testing Susceptibilities of Mycobacterium tuberculosis Isolates to Four Primary Antituberculosis Drugs.</a> J Clin Microbiol. 36, 2940-2943 (1998).</li></ol>

有正增长，控制一个AST微孔板的一个例子是如图1所示，每种药物端点MIC（μg/ ​​ml的）盘旋。在我们的实验室进行的一项研究表明，AST的微孔板方法相当于金标准琼脂比例法药敏检测结核分枝杆菌 。酶标AST微孔板获得更快的结果的报告，分别助攻微孔板相比，传统的琼脂比例法与AST微孔板和琼脂比例法，在14和21天的最终解释权时，测试第一的优势二线药物，同时它可以帮助防止耐药菌株的过度拖延。 AST微孔板也容易得多比APM成立，并宣读和手动镜和半自动化系统，如VIZION，可以用来读取的板块。为结核病耐药性测试的MIC值的可用性可能提供有价值的新信息，医生相对于传统的临界浓度值，尤其是对那些与“边缘”的易感性株。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> AST的酶标板和粘接密封</td><td>郊游诊断，俄亥俄州克利夫兰</td><td> MYCOTB </td></tr><tr><td>麦克法兰0.5标准</td><td>郊游诊断，克利夫兰，俄亥俄</td><td> E1041 </td></tr><tr><td> Middlebrook 7H10板</td><td>费舍尔</td><td></td></tr><tr><td>血琼脂平板</td><td>费舍尔</td><td></td></tr><tr><td>无菌循环1微升</td><td>费舍尔</td><td></td></tr><tr><td>无菌循环10μL， </td><td>费舍尔</td><td></td></tr><tr><td> Sensititre盐水吐温玻璃珠肉汤</td><td>郊游诊断，克利夫兰，俄亥俄</td><td> 27143 </td></tr><tr><td> 7H9 OADC肉汤</td><td>郊游诊断，克利夫兰，俄亥俄</td><td> T3440 </td></tr><tr><td>龙吸取技巧</td><td>费舍尔</td><td></td></tr><tr><td>无菌水槽</td><td>郊游诊断，克利夫兰，俄亥俄</td><td></td></tr><tr><td> 结核分枝杆菌 </td><td> ATCC </td><td> 27294 </td></tr><tr><td> Pipeters：1000，200，20μL </td><td>费舍尔</td><td></td></tr><tr><td>提示1000，20，20μL </td><td>费舍尔</td><td></td></tr><tr><td>多通道Pipetter </td><td>费舍尔</td><td></td></tr><tr><td>孵化5-10％的CO 2 </td><td>费舍尔</td><td></td></tr><tr><td>塑料包装袋，小，CO 2透水</td><td>保利塑料，德拉诺，PA </td><td></td></tr><tr><td>结核病消毒剂（如ProSpray的C - 60） </td><td> Certol国际商贸城，CO </td><td> PSC60/32 </td></tr><tr><td>镜盒阅读器</td><td>费舍尔</td><td></td></tr></tbody></table>

antimicrobial susceptibility testing of mycobacterium tuberculosis complex for first and second line drugs by broth dilution in a microtiter plate format

在努力控制耐药结核分枝杆菌（MTB）的增加是重要的抗生素耐药性的快速检测。药敏试验的MTB（AST），传统上一直由琼脂比例法或macrobroth测试仪器，如BACTEC（Becton Dickinson公司，火花，MD），VersaTREK（郊游诊断，美国俄亥俄州克里夫兰市）或BACT /警报（生物梅里埃，黑兹尔伍德，MO）。琼脂比例法，同时考虑“黄金”助攻的标准，是劳动密集型和要求执行含药琼脂菌落计数相比，无毒琼脂阻力计算。如果有≥1％的含药培养基上的增长相比，无毒介质，机体被认为是这种药物的抗药性。 macrobroth方法需要的仪器和测试突破点（“批判”）的第一线药物的药物浓度（异烟肼，乙胺丁醇，利福平和吡嗪酰胺）。这里介绍的方法是在96孔微孔板格式市售[MYCOTB（郊游诊断）]，并包含12抗菌剂，用于治疗结核病的浓度的增加，包括第一（异烟肼，利福平，乙胺丁醇）和二线药物（丁胺卡那霉素， ethionamide，环丝氨酸，卡那霉素，莫西沙星，氧氟沙星，段 - 氨基水杨酸，利福布丁和链霉素）。吡嗪酰胺，第一线药物，不包括在微孔板由于其酸性测试条件的需要。酶标系统的优点包括缓解设置和更快的转身时间（14天）相比，与传统的琼脂比例（21天）。此外，该板块可以从准备使用或者肉汤或固体培养基接种成立。由于微孔板格式是新的，因为结核杆菌呈现独特的安全挑战，在实验室中，这个协议将介绍如何安全地设置，培育和阅读微孔板。

Watch this Scientific Journal Video about Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format at J

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

结核分枝杆菌药敏试验是具有挑战性的，但对病人的护理至关重要。这微孔板格式提供12种antimycobacterial药物进行测试，并提供最低抑菌浓度（MIC）值，这将有助于在适当的治疗。

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). ...

antimicrobial-susceptibility-testing-mycobacterium-tuberculosis

Research

JoVE Journal

Immunology and Infection

38.0K 次观看.  Mayo Clinic. 结核分枝杆菌药敏试验是具有挑战性的，但对病人的护理至关重要。这微孔板格式提供12种antimycobacterial药物进行测试，并提供最低抑菌浓度（MIC）值，这将有助于在适当的治疗。

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

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

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

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

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within 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

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

样品制备<em&gt;结核分枝杆菌</em提取物对核磁共振的代谢组学研究

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

增长结核分枝杆菌生物膜

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

展现着多药耐药<em&gt;结核分枝杆菌</em&gt;扩增基因芯片

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

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

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

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 ...

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

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

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

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

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 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

关于功能研究的三维人体肺组织模型<em&gt;结核分枝杆菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

分析18葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...