Name: preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing
Uploaded: 2014-10-15T14:00:00.000+00:00
Duration: 11 min 25 s
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）50毫升离心管中，混匀。 </li></ol></li><li>制备血培养菌体裂解通过离心。 <ol><li>离心样品在1000×g离心在室温10分钟。 </li><li>雷莫已经上清液（主要含H 2 O和血细胞）用实验室真空泵。 </li><li>重悬在1ml的自制氯化铵裂解溶液（0.15M的氯化铵 ，1mM的KHCO 3）沉淀。分解的丸粒通过刮削并通过涡旋管和离心的样品在140×g离心在室温10分钟。 </li><li>丢弃它主要含有红细胞碎片的上清液。 <ol><li>如果颗粒仍然出血，重悬沉淀在2ml无菌蒸馏H 2 O中，进一步溶解残存的红血细胞和清洗沉淀。离心样品在140×g离心在室温10分钟，并弃去上清液。 </li></ol></li><li>悬浮颗粒在200微升H 2 O的</li></ol></li><li>血培养物上的各种选择性和非选择性琼脂培养基上继代培养。 <ol><li>收取1毫升血培养有20G的注射器。 </li><li>接种1滴血文化诠释o在血琼脂，巧克力琼脂，McConkey琼脂和Schaedler琼脂四个象限。 </li><li>在37℃下的血琼脂和巧克力琼脂平板上，在5％CO 2气氛中，McConkey琼脂在常压和Schaedler琼脂平板在厌氧条件。 </li><li>按照对不同媒体细菌生长。 </li><li>检查是否有细菌纯度。 </li></ol></li></ol> 2，鉴定通过MALDI-TOF质谱<ol><li>直接标识从血液沉淀。 <ol><li>传输1微升的血液沉淀在MALDI靶盘，让它干在42℃加热平台。 </li><li>覆盖与1微升70％甲酸样品，让它干在42℃加热平台。 </li><li>覆盖与1μl的MALDI基质的样品（饱和α-氰基-4 - 羟基肉桂酸在50％乙腈 - 2.5％三氟乙酸中的溶液），并让它干燥在42℃下加热平台。 </li><li>继续执行MALDI-TOF质谱分析所描述的制造商MALDI-TOF MS系统。 </li><li>如果鉴定成绩是在种的水平（例如，得分&lt;2）无效，执行血液颗粒样品的蛋白提取。 </li></ol></li><li>蛋白质提取的血培养沉淀后鉴定。 <ol><li>混合20微升用1ml 70％乙醇，离心的沉淀物以13,000 xg离心在RT 2分钟。 </li><li>弃去上清液，加入25微升70％甲酸水溶液和25μl的100％乙腈以沉淀。涡大力悬浮颗粒。用吸头打破下来涡旋之前强硬的悬浮颗粒。 </li><li>离心机以13,000 xg离心在RT 2分钟。传输1微升上清液（包含提取的蛋白质）在MALDI靶板上，让它干在42℃加热平台。请按2.1.2所述。 </li></ol></li></ol> 3，细菌IDENTIFication和药敏试验用全自动微生物系统<ol><li>制备细菌悬浮液进行细菌鉴定和AST与全自动微生物系统。 <ol><li>准备装有3毫升无菌0.45％NaCl溶液与全自动微生物系统兼容的塑料管。 </li><li>加血培养粒料的足够体积的0.45％的NaCl溶液中，得到的细菌悬浮液对应于0.6的麦法兰浊度至0.8。测量用密度计机浊度。 请注意:在血培养细菌沉淀的体积为50至200μl的变化以达到0.6的麦法兰浊度至0.8。 </li></ol></li><li>如制造商所述接种的自动化革兰氏阳性（GP）或革兰氏阴性（GN）卡，用于识别和自动AST卡的革兰氏阳性球菌和革兰氏阴性细菌的抗菌敏感性试验。 注:辨识n中具有全科医生或GN卡，如果MALDI-TOF质谱鉴定得分值&gt; 2不适用。通过传代培养的菌悬液于血琼脂（GP和GN）和麦康凯琼脂（GN）板检查菌悬液的纯度。 </li></ol> 4，药敏试验采用纸片扩散分析 纸片扩散法是通过在药敏试验（EUCAST，3.0版，2013年4月，www.eucast.org）欧洲委员会称。 <ol><li>细菌悬液的制备进行纸片扩散法药敏试验。 <ol><li>制备含有3毫升的无菌0.9％氯化钠溶液的玻璃试管中。 </li><li>加血培养粒料的足够体积的0.9％的NaCl溶液中，得到的细菌悬浮液相当于0.5麦克法兰浊度。测量用密度计机浊度。 请注意:在血培养物的体积细菌沉淀从50到200μl的变化以达到0.6的麦法兰浊度至0.8。 </li><li>漩涡的菌悬液。 </li></ol></li><li>细菌悬浮液上的Mueller-Hinton琼脂（MH）或的Mueller-Hinton琼脂，苛养菌琼脂（MHF）的接种（MH补充有5％去纤维蛋白马血和20毫克/升的β-烟酰胺腺嘌呤二核苷酸）。 <ol><li>蘸无菌棉签在细菌悬浮液，并轻轻转动棉签在玻璃管的内壁上除去过量的液体。 </li><li>通过擦拭在三个方向上，或通过使用自动板旋转器均匀分布的菌液到氢/ MHF琼脂板的整个表面上。 </li></ol></li><li>在接种后的琼脂平板的抗微生物磁盘中的应用。 <ol><li>在干燥接种琼脂手动板的表面上或通过使用敏感性盘的分配器应用密切的磁盘。 </li><li>孵育板在适当的坦佩叉涂抹，并作为药敏试验EUCAST纸片扩散法所描述的氛围（3.0版，2013年4月，www.eucast.org）。 </li></ol></li></ol> 5，自动基于PCR的诊断测试为MRSA的检测<ol><li>使用微量，转移50微升血液中的文化沉淀成提供在20秒自动PCR为基础的诊断测试套件和旋涡的MRSA样本的试剂瓶。 </li><li>用1毫升微量，分配所述样本到盒的样品端口。将墨盒插入自动PCR反应体系和所描述​​的制造商开始试验。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

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><tbody><tr><td>20 G needle</td><td>Terumo, Leuven, Belgium</td><td>NN-2038R</td><td></td></tr><tr><td>50 ml Falcon tube</td><td>BD, Franklin Lakes, NJ, USA</td><td>352070</td><td>50 ml centrifuge tubes</td></tr><tr><td>Ammonium chlorure</td><td>Merck, Darmstadt, Germany</td><td>101145</td><td></td></tr><tr><td>Potassium hydrogen carbonate</td><td>Fluka, St. Louis, MO, USA&amp;nbsp;</td><td>60340</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich, St. Louis, MO, USA&amp;nbsp;</td><td>F0507</td><td>Flammable, corrosive</td></tr><tr><td>&amp;#945;-Cyano-4-hydroxycinnamic acid</td><td>Fluka, St. Louis, MO, USA&amp;nbsp;</td><td>70990</td><td>Acute toxicity</td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich, St. Louis, MO, USA&amp;nbsp;</td><td>271004</td><td>Flammable, acute toxicity</td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich, St. Louis, MO, USA&amp;nbsp;</td><td>T6508</td><td>Corrosive, acute toxicity</td></tr><tr><td>Vitek 2 60 instrument</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>27202</td><td>automated microbial system instrument</td></tr><tr><td>Vitek 2 Gram-positive (GP) card</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>21342</td><td>automated GP&amp;nbsp; identification card</td></tr><tr><td>Vitek 2 AST-P580 card</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>22233</td><td>automated microbial AST system</td></tr><tr><td>Vitek 2 Gram-negative (GN) card</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>21341</td><td>automated GN&amp;nbsp; identification card</td></tr><tr><td>Vitek 2 AST-N242 card</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>413391</td><td>automated microbial AST system</td></tr><tr><td>Xpert MRSA</td><td>Cepheid, Sunnyvale, Ca, USA</td><td>&amp;nbsp;GXMRSA-100N-10</td><td>nucleic acid amplification technology MRSA</td></tr><tr><td>GeneXpert IV instrument</td><td>Cepheid, Sunnyvale, Ca, USA</td><td>GXIV-4-D</td><td>nucleic acid amplification technology instrument</td></tr><tr><td>Microflex LT MALDI-TOF MS instrument</td><td>Bruker Daltonics, Bremen, Germany</td><td>BDAL microflex LT/SH</td><td></td></tr><tr><td>MSP 96 target steel BC</td><td>Bruker Daltonics, Bremen, Germany</td><td>280799</td><td>MALDI target plate</td></tr><tr><td>Densitometer Densicheck instrument</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>27208</td><td></td></tr><tr><td>MALDI Sepsityper kit 50</td><td>Bruker Daltonics, Bremen, Germany</td><td>8270170</td><td></td></tr><tr><td>Mac Conkey agar</td><td>Biolife, Milano, Italy</td><td>4016702</td><td></td></tr><tr><td>Mueller-Hinton agar</td><td>Oxoid, Hampshire, England</td><td>CM0337</td><td>Mueller-Hinton agar (MH)&amp;nbsp;</td></tr><tr><td>MHF agar</td><td>Biom&amp;eacute;rieux, Marcy-l&#039;Etoile, France</td><td>43901</td><td>Mueller-Hinton agar-fastidious organisms agar (MHF)</td></tr><tr><td>BD columbia III agar</td><td>BD, Franklin Lakes, NJ, USA</td><td>254071</td><td>blood agar</td></tr><tr><td>BD chocolate agar</td><td>BD, Franklin Lakes, NJ, USA</td><td>254089</td><td>chocolate agar</td></tr><tr><td>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 ...

preparation-blood-culture-pellet-for-rapid-bacterial-identification

Research

JoVE Journal

Immunology and Infection

35.5K 次观看.  University Hospital Center and University of Lausanne. 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。 

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

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

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

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

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

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 ...

科研

免疫与感染

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

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

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. ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

检测使用荧光脱氧核酶的细菌

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

水性抗生素耐药细菌的分离和鉴定及其抗生素耐药基因的分子表征

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

环境科学

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

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

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 ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

喷墨打印机辅助自动跳棋阵列和手动消时法的抗菌协同试验

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Liquid-grown Streptomyces cultures are characterized by mycelial pellets that are heterogeneous in size. We here describe a method to analyze and sort such pellets in a high-throughput manner. These pellets can be used for further analyses, which will provide leads to understand and control growth heterogeneity.

排序<em&gt;链霉菌</em&gt;细胞沉淀用一个复杂的对象参数分析仪和分选机

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these ...

生物学

血培养瓶中的加速药敏试验

Direct Microbial Identification using An Automated Microbial Identification System to Facilitate the EUCAST RAST Method Without Mass Spectrometry

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques providing results in 3-4 days. Recognizing this delay in turnaround time (TAT), both EUCAST and CLSI have developed protocols for determining AST results directly from positively flagged automated blood culture bottles (+aBCs). EUCAST rapid AST (RAST) protocol was first introduced in 2018, where zone diameter breakpoints for four common etiological agents of GN sepsis, i.e., Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex can be reported. However, those clinical laboratories that have implemented this method in their routine workflow rely on mass spectrometry-based microbial identification, which is not easily available, thus precluding its implementation in resource-limited settings. To circumvent it, we evaluated a direct inoculum protocol (DIP) using a commercial automated microbial identification and antimicrobial susceptibility testing system (aMIAST) to enable early microbial identification within 8 h of positive flagging of aBC. We evaluated this protocol from January to October 2023 to identify the four RAST reportable GN (RR-GN) in the positively flagged aBC. The microbial identification results in DIP were compared with the standard inoculum preparation protocol (SIP) in aMIAST. Of 204 +aBCs with monomorphic GN (+naBC), one of the 4 RR-GN was identified in 105 +naBCs by SIP (E. coli: 50, K. pneumoniae: 20, P. aeruginosa: 9 and A. baumannii complex: 26). Of these, 94% (98/105) were correctly identified by DIP whereas major error and very major error rates were 6% (7/105) and 1.7% (4/240), respectively. When DIP for microbial identification is done using the EUCAST RAST method, provisional clinical reports can be provided within 24 h of receiving the sample. This approach has the potential to significantly reduce the TAT, enabling early institution of appropriate antimicrobial therapy.

作者聚焦：通过直接鉴定血培养瓶中的革兰氏阴性菌来提高诊断准确性

EUCAST has developed a direct antimicrobial susceptibility testing (AST) protocol for automated blood cultures. However, its dependence on mass spectrometry-based microbial identification can be obviated by using a direct inoculum preparation protocol in an automated microbial identification system. This approach can provide AST reports within 24 h of sample collection.

使用自动微生物鉴定系统进行直接微生物鉴定，以促进 EUCAST RAST 方法，无需质谱法

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques ...

为快速细菌鉴定和药敏试验的准备血培养微丸

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一个1.5小时的程序，鉴定肠球菌直接从血培养的物种

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

检测使用荧光脱氧核酶的细菌

水性抗生素耐药细菌的分离和鉴定及其抗生素耐药基因的分子表征

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

喷墨打印机辅助自动跳棋阵列和手动消时法的抗菌协同试验

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使用自动微生物鉴定系统进行直接微生物鉴定，以促进 EUCAST RAST 方法，无需质谱法

使用自动微生物鉴定系统进行直接微生物鉴定，以促进 EUCAST RAST 方法，无需质谱法

使用自动微生物鉴定系统进行直接微生物鉴定，以促进 EUCAST RAST 方法，无需质谱法