我们要感谢脑膜炎信托基金和MRC为这项工作提供资金。

1. 通过SOE-PCR23 生成突变扩增子和扩增壮观霉素盒
<ol><li>首先进行PCR以扩增菌株519/43中层基因侧翼区域的同源臂（分别为ply 5'（488 bp）和ply3'（715 bp））。Use primers plyFw1_NOTI （TTT GCGGCCGCCAGTAAATGACTTTATACTAGCTATG）， ply5'R1_BamHI （CGAAATATAGACCAAAGGACGC GGATCC AGAACCAAACTTGACCTTGA）， ply3'F1_BamHI （TCAAGGTCAAGTTTGGTTCTGGATCC GCGTCCTTTGGTTTCG） 和plyRv2_NotI （TTTGCGGCCGCCATTTTCTACCTTATCCTCTACC）.</li>
	<li>对ply5'使用以下PCR条件:在94°C下变性60秒，步骤2:在94°C下变性30秒，步骤3:在58°C下退火30秒，步骤4:在72°C下延伸30秒，步骤5:返回步骤2并重复35个循环，步骤6:在72°C下最终延伸30秒。<ol><li>对 ply3' 使用相同的 PCR 条件，但步骤 4 和步骤 6 的延长时间应为 60 秒。</li>
	</ol></li>
	<li>通过凝胶电泳分析PCR产物并从凝胶中切除扩增子。</li>
	<li>按照制造商说明（材料表）中描述的方案纯化PCR扩增子。</li>
	<li>在SOE-PCR中使用等摩尔量的两个同源臂作为模板。使用引物plyFw1_NOTI （TTT GCGGCCGCCAGTAAATGACTTTATACTAGCTATG）和plyRv2_NotI（TTTGCGGCCGCCATTTTCTACCTTATCCTCTACC）的引物融合两个扩增子。</li>
	<li>使用以下SOE-PCR条件（步骤1:在94°C变性2分钟，步骤2:在94°C变性30秒，步骤3:退火58°C退火30秒，步骤4:在68°C下延伸60秒，步骤5:返回步骤2并重复25个循环，步骤6:最终在68°C下延伸90秒）。</li>
	<li>通过凝胶电泳分析SOE-PCR产物。使用凝胶提取试剂盒将其从凝胶中切除，并按照提供的说明进行操作。</li>
	<li>使用以下PCR条件扩增质粒pR412中的大观霉素盒:步骤1:在94°C变性60秒，步骤2:在94°C变性30秒，步骤3:在55°C下退火30秒，步骤4:在68°C下延伸60秒，步骤5:返回步骤2并重复25个循环， 步骤6:在68°C下最终延伸60秒。<ol><li>使用引物BamHI_SP2F2（GGATCC CTA GAA CTA GTG GAT CCC CC）和BamHI_SP2R2（GGATCC AAT TCT GCA GAT TTT AC ATG ATC）。质粒pR412购自Marc PrudHomme博士（法国国家科学研究中心保罗·萨巴蒂埃图卢兹大学）。</li>
	</ol></li>
	<li>通过凝胶电泳分析PCR扩增子。如上所述切除和纯化所得的PCR扩增子。</li>
</ol>2. 质粒pSD1的生成和 大肠杆菌 Dh5α的化学转化
<ol><li>按照pGEMT简易系统I制造商说明（材料表）进行连接。在微量离心管中，加入 5 μL 2x 连接缓冲液、1 μL pGEMTeasy、2 μL ply_SOE产物、1 μL T4 DNA 连接酶和水，总体积为 20 μL。在4°C孵育过夜。 生成质粒pSD1。</li>
	<li>用pSD1转化化学含量 的大肠杆菌 Dh5α。首先将 50 μL 化学含量大 肠杆菌 Dh5α 与 3 μL pSD1 连接反应在冰上孵育 15 分钟。然后继续将细胞暴露于热冲击（42°C，30秒）。将细胞放在冰上2分钟。</li>
	<li>从冰上取出细胞并加入 350 μL S.O.C 培养基。将培养物在37°C，120rpm下孵育2小时。</li>
	<li>将转化接种在补充有 0.4 mM IPTG、0.24 mg/mL X-Gal 用于蓝/白选择和 100 μg/mL 氨苄青霉素的 Luria Bertani 琼脂 （LBA） 上，以确保平板中生长的所有菌落都具有质粒骨架。白色菌落含有pSD1。</li>
	<li>挑选三个白色菌落，在 10 mL LB 中建立过夜生长，并补充有 100 μg/mL 氨苄青霉素。将培养物在37°C振荡孵育过夜。</li>
	<li>第二天以3，082× g 离心培养物，并使用沉淀进行质粒提取。</li>
</ol>3. 质粒DNA提取、pSD1和壮观霉素基因的限制性酶切及pSD2组装
<ol><li>按照商业试剂盒随附的说明提取质粒DNA（材料表）。</li>
	<li>为先前扩增和纯化的pSD1质粒和大观霉素盒设置BamHI限制性酶切。使用以下条件和数量如 表1所示。</li>
	<li>将限制性酶切反应和对照在37°C孵育3小时。</li>
	<li>通过电泳分析限制性内切物，切除条带并按照商业试剂盒提供的说明进行纯化（材料表）。</li>
	<li>接下来，按照制造商说明（材料表）使用BamHI-消化的pSD1和大观霉素（来自步骤3.2）制备连接反应。在微量离心管中，加入以下反应组分:5 μL 2x 连接缓冲液、2 μL pSD1、2 μL 大观霉素盒、1 μL T4 DNA 连接酶，并在 4 °C 下孵育过夜。 生成质粒pSD2。</li>
	<li>如步骤2.2中所述，将质粒pSD2转化为化学感受态大 肠杆菌 Dh5α。</li>
	<li>根据携带质粒pSD2的转化体在补充有100μg/mL大观霉素和氨苄青霉素的LBA中生长的能力来选择转化体。</li>
	<li>按照上述说明并按照制造商的说明（μL）进行质粒DNA提取（pSD2）。</li>
</ol>4. 肺炎链球菌 菌株的转化 519/43
<ol><li>在BHI中制备肺炎链球菌519/43的过夜培养物，并使其在37°C，5%CO2下静态生长。</li>
	<li>第二天，将培养物在 10 mL 新鲜 BHI 肉汤中以 1:50 和 1:100 稀释。在37°C下静态孵育培养物，直到OD595nm 在0.05和0.1之间（最佳DNA采集接近0.1 OD）。</li>
	<li>一旦OD达到0.1，取860μL并转移到微量离心管中。在同一微量离心管中，添加:100 μL 100 mM NaOH、10 μL 20% （w/v） BSA、10 μL 100 mM CaCl 2、2 μL 50 ng/mL CSP124 和 500 ng pSD2。</li>
	<li>将反应在37°C下静态孵育3小时。</li>
	<li>将 330 μL 板铺在补充有 100 μg/mL 壮观霉素的 5% 血琼脂平板 （BA） 上，在 3 个孵育小时内每小时一次。</li>
	<li>将板在37°C，5%CO2下孵育过夜。将大观霉素耐药菌落贴在补充有 100 μg/mL 大观霉素的另一块 BA 平板上以及补充有 100 μg/mL 氨苄西林的 BA 平板上。在上述条件下孵育两组板过夜。氨苄西林板用于测试质粒骨架的存在。</li>
	<li>使用NOTI plyFw1_（TTT GCGGCCGCCAGTAAATGACTTTATACTAGCTATGG） 和SPEC_REV（TAATTCCTCTAAGTCATAATTTCCG）的引物通过PCR确认大观霉素盒的存在。使用附着在突变区域外的引物plySCN1（CCAATGGAAATCGCTAGGCAAGAGATAA）和plySCN2（ATTACTTAGTCCAACCACGGCTGAT）通过PCR确认突变。</li>
	<li>通过使用引物plySCN1（CCAATGGAAATCGCTAGGCAAGAGATAA），plySCN2（ATTACTTAGTCCAACCACGGCTGAT）以及引物及其在大观霉素盒sqr1（CCTGATCCAACATGTAGTAGTACC）sqf2（CGTAGTTATCTTGGAGAGAATA）spec_sqf1（GGTACTTACATGTGTGGATCAGG）和spec_sqr2 TATTCTCTCCAAGATAACTACG中的引物测序，确认基因组正确位置的整合。</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+pneumolysin+deficient+mutant+in+Streptococcus+pneumoniae+serotype+1+strain+519/43+and+phenotypic+characterisation.">Construction of a pneumolysin deficient mutant in Streptococcus pneumoniae serotype 1 strain 519/43 and phenotypic characterisation.</a> Microbial Pathogenesis. 141, 103999 (2020).</li></ol>

作者没有什么可透露的。

肺炎链球菌，特别是血清1型，仍然是引起侵袭性肺炎球菌疾病和脑膜炎的全球威胁。尽管在非洲引入了各种疫苗，可以预防血清型1，但这种血清型仍然能够引起导致高发病率和死亡率的暴发13。由于其临床相关性，遗传操纵这种血清型的能力至关重要。本研究中描述的方法允许对该血清型中的代表性菌株进行遗传操作。侵袭性菌株 519/43 （ST5316），一种来自丹麦脑膜炎患?...

肺炎链球菌（肺炎链球菌，肺炎球菌）是全球发病和死亡的主要原因之一。直到最近，已经发现了近100种肺炎链球菌血清型1，2，3，4，5，6，7。侵袭性肺炎球菌病（IPD）每年造成约70万人死亡，其中5岁以下儿童8。肺炎链球菌是全球细菌性肺炎、中耳炎、脑膜炎和败血症的主要原因9。
在非洲脑膜炎地带，血清型1是脑膜炎暴发的原因，其中序列型（ST）ST217是一种毒性极强的序列类型，占主导地位10，11，12，13，14，15。它在脑膜炎病理学中的重要性被比作非洲脑膜炎带脑膜炎奈瑟菌的重要性16。血清 1 型通常是 IPD 的主要原因;然而，它在运输中很少发现。事实上，在冈比亚，这种血清型占所有侵袭性疾病的20%，但仅在0.5%的健康携带者中发现14，17，18，19。感受态肺炎球菌的遗传交换和重组通常发生在携带中，而不是在侵袭性疾病中发生20。此外，血清型 1 已被证明是肺炎球菌中描述的最短携带率之一（仅 9 天）。因此，有人提出这种血清型的重组率可能比其他血清型低得多21。
有必要进行深入研究，以了解血清型1菌株携带率低的原因及其在撒哈拉以南非洲侵袭性疾病中的重要性。
在这里，我们报告了一种协议，该协议允许特定血清型1菌株的全基因组诱变，519/43。这种菌株可以很容易地获得新的DNA并将其重组到其基因组中。这种方法还不是菌株间，但在519/43背景下完成时非常有效（其他靶标已经突变，手稿正在准备中）。通过简单地使用519/43菌株，并利用其自然能力，以及替代提供外源DNA的方式，我们能够突变该血清型1菌株中的肺溶血素基因（ply）。该方法代表了Harvey等人提出的方法的改进22 ，因为它是一步完成的，无需通过不同的血清型传代DNA。然而，由于菌株间变异性，没有一种方法适用于所有菌株。突变特定基因并观察其影响的能力将使人们深入了解血清型 1型肺炎链球菌 菌株，并将为这些菌株在撒哈拉以南非洲脑膜炎中的作用提供答案。

<table>
	<tbody>
		<tr>
			<td>AccuPrime Pfx DNA polymerase</td>
			<td>Invitrogen</td>
			<td>12344024</td>
			<td>Used for amplification of the fragments</td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>A9518</td>
			<td>Used for bacterial selection on stage 1(pSD1)</td>
		</tr>
		<tr>
			<td>Blood Agar Base</td>
			<td>Oxoid</td>
			<td>CM0055</td>
			<td>Used to plate S. pneumoniae transformants</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumine</td>
			<td>sigma</td>
			<td>55470</td>
			<td>used for S. pneumoniae Transformation</td>
		</tr>
		<tr>
			<td>Brain Heart Infusion</td>
			<td>Oxoid</td>
			<td>CM1135</td>
			<td>used to grow S. pneumoniae cells</td>
		</tr>
		<tr>
			<td>Calcium Chloride Cacl2</td>
			<td>Sigma</td>
			<td>449709</td>
			<td>used for S. pneumoniae Transformation</td>
		</tr>
		<tr>
			<td>Competence stimulating peptide 1</td>
			<td>AnaSpec</td>
			<td>AS-63779</td>
			<td>used for S. pneumoniae Transformation</td>
		</tr>
		<tr>
			<td>Luria Broth Agar</td>
			<td>Gibco</td>
			<td>22700025</td>
			<td>used for plating and selection of pSD1 and pSD2</td>
		</tr>
		<tr>
			<td>Luria Broth Base (Miller&#39;s formulation)</td>
			<td>Gibco</td>
			<td>12795027</td>
			<td>used for plating and selection of pSD1 and pSD2</td>
		</tr>
		<tr>
			<td>Monarch Gel Extraction Kit</td>
			<td>NEB</td>
			<td>T1020S</td>
			<td>Used to extract the bands from the DNA gel</td>
		</tr>
		<tr>
			<td>Monarch Plasmid Miniprep Kit</td>
			<td>NEB</td>
			<td>T1010S</td>
			<td>Used to extract plasmid from the cells</td>
		</tr>
		<tr>
			<td>pGEM T-easy</td>
			<td>Promega</td>
			<td>A1360</td>
			<td>used as suicide plasmid</td>
		</tr>
		<tr>
			<td>S.O.C.</td>
			<td>Invitrogen</td>
			<td>15544034</td>
			<td>used for recovery of cells after transformation</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH)</td>
			<td>Sigma</td>
			<td>S0899</td>
			<td>used for S.pneumoniae Transformation</td>
		</tr>
		<tr>
			<td>Spectinomycin Hydrochloride</td>
			<td>SigmaAldrich</td>
			<td>PHR1426</td>
			<td>Used for bacterial selection</td>
		</tr>
		<tr>
			<td>Subcloning Efficiency DH5&#945; Competent Cells</td>
			<td>Invitrogen</td>
			<td>18265017</td>
			<td>used for the creation of pSD1 and pSD2</td>
		</tr>
	</tbody>
</table>

constructing mutants in serotype 1 streptococcus pneumoniae strain 519/43

肺炎链球菌血清1型仍然是低收入和中等收入国家的一个大问题，特别是在撒哈拉以南非洲。尽管它很重要，但由于缺乏遗传工具来修饰它，这种血清型的研究受到了阻碍。在这项研究中，我们描述了一种对血清型1临床分离株（菌株519/43）进行基因修饰的方法。有趣的是，这是通过利用肺炎球菌自然获取DNA的能力来实现的。然而，与大多数肺炎球菌不同，线性DNA的使用并不成功;为了突变这种重要的菌株，必须使用自杀质粒。这种方法为更深入地了解这种难以捉摸的血清型提供了手段，无论是在生物学上还是在致病性方面。为了验证该方法，已知的主要肺炎球菌毒素肺溶血素发生了突变，因为它具有众所周知且易于遵循的表型。我们表明，正如预期的那样，突变体失去了裂解红细胞的能力。通过能够突变感兴趣的血清型中的重要基因，我们能够观察到腹膜内和鼻内感染时功能丧失突变体的不同表型与其他血清型观察到的表型不同。综上所述，本研究证明菌株519/43（血清型1）可以进行基因改造。

这里描述的方案首先使用PCR扩增左右同源臂，同时从 层 基因的中间区域删除191 bp。在进行PCR时，在左同源臂的3'和右同源臂的5'端引入BamHI位点（图1A）。接下来是PCR-SOE，其中左和右同源臂融合成一个扩增子（图1B）。然后将该SOE-PCR扩增子克隆到pGEMTeasy中使用TA克隆以生成质粒pSD1（图1C）。成功转化将产生对氨苄青霉素耐药...

Watch this Scientific Journal Video about Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43 at JoVE.com

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

在这里，我们描述了一种 肺炎链球菌 血清型1菌株519/43，该菌株可以通过利用其自然获取DNA和自杀质粒的能力进行基因改造。作为原理证明，在肺溶血素（ply）基因中制造了同基因突变体。

在血清型 1 型肺炎链球菌 菌株 519/43 中构建突变体

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...

constructing-mutants-serotype-1-streptococcus-pneumoniae-strain

Research

JoVE Journal

Immunology and Infection

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

1.6K 次观看.  London School of Hygiene and Tropical Medicine. 在这里，我们描述了一种 肺炎链球菌 血清型1菌株519/43，该菌株可以通过利用其自然获取DNA和自杀质粒的能力进行基因改造。作为原理证明，在肺溶血素（ply）基因中制造了同基因突变体。

视频: Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

荚膜血清型<em&gt;肺炎链球菌</em&gt;使用荚膜肿胀反应

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

荚膜血清型<em&gt;肺炎链球菌</em&gt;乳胶凝集

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对<i&gt;秒。肺炎</i&gt;<i&gt;小时。流感</i&gt;<i&gt; K。肺炎</i&gt;<em&gt;点。铜绿假单胞菌</em&gt;或<em&gt;一个。鲍曼不动杆菌</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

复制的有序, 非冗余图书馆的铜绿假单胞菌应变 PA14 座子插入突变体

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

在血清型 1 型肺炎链球菌 菌株 519/43 中构建突变体

在血清型 1 型肺炎链球菌菌株 519/43 中构建突变体