BioVector® 伴放线聚集杆菌 ATCC 29523 标准菌株

BioVector® Aggregatibacter actinomycetemcomitans ATCC 29523 Standard Strain Manual

第一部分 中文说明

一 产品基本信息与遗传学背景

• 菌株名称：伴放线聚集杆菌标准菌株（Aggregatibacter actinomycetemcomitans）。

• 菌株编号与别名：ATCC 29523、CDC A1916（常写作 CDC A1914）、CIP 101033、JCM 12985、CCUG 37004。

• 物种分类：细菌界（Bacteria），变形菌门（Pseudomonadota），$\gamma$-变形菌纲（Gammaproteobacteria），巴斯德氏菌目（Pasteurellales），巴斯德氏菌科（Pasteurellaceae），聚集杆菌属（Aggregatibacter）。

• 生物学与血清型特征：

  • 血清型 A（Serotype a）：ATCC 29523 是血清型 A 的经典代表菌株。与高毒力的血清型 b（如 JP2 克隆株）相比，血清型 A 表现出较低的白细胞毒素（Leukotoxin）表达水平，通常更广泛地存在于健康的口腔或口腔平稳状态中。

  • 形态与呼吸特性：革兰氏阴性（Gram-negative）、兼性厌氧（Facultative anaerobe）、无动力、短杆状或球杆状细菌。在固体培养基上通常表现出光滑型表型（Smooth phenotype），在液体培养基中呈现非聚集性生长。

• 来源历史：最初由美国科罗拉多州丹佛市的退伍军人医院（VA Hospital, Denver, USA）从人类血液（Human Blood）样本中分离获得，后经美国疾病预防控制中心（CDC）及美国标准生物品收藏中心（ATCC）鉴定并向全球分发。

• 生物安全级别：2级（BSL-2 / 风险群2）。由于其具有一定的人类致病性，必须在二级生物安全柜内进行无菌操作。

二 核心科研价值与转化医学应用

伴放线聚集杆菌是国际公认的严重口腔疾病及全身侵袭性感染的重要致病菌之一：

1. 牙周病学与口腔微生物学（Periodontology）：广泛用于局限性侵袭性牙周炎（Localized Aggressive Periodontitis）和慢性牙周炎的病因学机制研究。ATCC 29523 作为血清型 A 菌株，经常被临床及学术界用作评估细菌定殖（Colonization）、表面粘附（Adhesion）以及生物膜（Biofilm）形成特异性的对照株。

2. 非口腔感染与心内膜炎研究：该菌株分离自血液，常用于构建 HACEK 感染群（引发感染性心内膜炎的一组临床少见革兰氏阴性菌）的体外模型，深入分析其如何跨越口腔屏障进入循环系统。

3. 免疫逃逸与宿主交互（Host-Pathogen Interaction）：用于测试其表面抗原、脂多糖（LPS）、细胞致死性溶胀毒素（CDT）等毒力因子与人牙龈上皮细胞、白细胞的免疫交互反应。

三 实验室菌株复苏、扩增传代与冷冻保存标准步骤

1. 专用培养基与环境配置

伴放线聚集杆菌对营养要求较高，且在含 $CO_2$ 环境中生长更佳：

• 推荐培养基：巧克力琼脂培养基（Chocolate Agar）、哥伦比亚血琼脂培养基（含 5% - 10% 脱纤维马血或羊血），或补充有 0.6% 酵母浸膏的胰蛋白胨大豆肉汤/琼脂（TSYE）。

• 培养环境参数：温度为 37 摄氏度，必须置于 5% 至 10% $CO_2$ 的加湿孵箱或厌氧/微需氧培养罐中进行孵育。

2. 冻干粉菌种复苏步骤（Thawing Freeze-Dried Cultures）

1. 在二级生物安全柜内小心打开含有 ATCC 29523 冻干菌种的小安瓿管。

2. 用无菌移液管吸取约 0.5 mL 预热的 TSYE 肉汤或常规脑心浸液（BHI）肉汤，滴加到冻干菌块上，轻轻吹打使其完全悬浮溶解。

3. 将溶解后的全量菌悬液接种到巧克力琼脂平板或血琼脂平板上，使用接种环进行三区或四区划线以获得单克隆。

4. 极重要：将平板立即放入 37 摄氏度、5% - 10% $CO_2$ 孵箱中，由于该菌株生长较为缓慢，通常需要连续孵育 24 至 48 小时 甚至更久方可肉眼观察到特征性菌落。

3. 菌株传代与纯化（Passaging & Maintenance）

• 特征观察：在血平板上，ATCC 29523 的菌落通常较小、微白至半透明，有时在琼脂内部贴合较紧（光滑型在液体中较难自聚）。

• 传代操作：挑取平板上生长良好的单菌落，重新划线接种至新鲜的血琼脂平板或接种于 TSYE 液体肉汤中。液体培养通常孵育 7 至 15 小时即可进入对数生长期。不建议在平板上连续长期传代，以防其由于自发突变而导致原有毒力因子或表面抗原结构丢失。

4. 菌株长期冷冻保存（Cryopreservation）

• 短期保存：将长有菌落的固体平板用封口膜密封，置于 4 摄氏度冰箱中，可维持 1-2 周（不推荐）。

• 长期甘油冷冻法：采集处于对数生长旺盛期的液体培养物，按照 80% 液体培养物 + 20% 灭菌高纯度甘油 的比例（或使用含 15% 甘油的脱脂奶粉悬液）在冻存管中轻柔混匀。立即置于 -80 摄氏度 超低温冰箱或直接投入 液氮（-196 摄氏度） 中，可实现数年以上的无限期稳定保存。

Part 2 English Section

I General Information and Genetic Architecture

• Organism Name: Aggregatibacter actinomycetemcomitans Standard Reference Strain.

• Strain Designations and Aliases: ATCC 29523, CDC A1916 (also referred to as CDC A1914), CIP 101033, JCM 12985, CCUG 37004.

• Taxonomic Classification: Domain Bacteria, Phylum Pseudomonadota, Class Gammaproteobacteria, Order Pasteurellales, Family Pasteurellaceae, Genus Aggregatibacter, Species Aggregatibacter actinomycetemcomitans.

• Biological and Serological Profile:

  • Serotype A Core Identity: ATCC 29523 serves as a globally recognized standard indexing archetype for Serotype a. Compared to highly aggressive Serotype b strains (such as the JP2 clonal lineages), Serotype a variants demonstrate lower baseline leukotoxin expression metrics and are regularly documented within stable, healthy oral microenvironments.

  • Morphology and Metabolic Trait: A Gram-negative, non-motile, facultative anaerobic coccobacillus/short rod bacterium.It exhibits a characteristic smooth phenotype when cultivated on premium solid agar plates and preserves a non-aggregative phenotype in continuous liquid broth suspensions.

• Source Isolation History: Originally recovered and isolated from human blood profiles at the VA Hospital in Denver, Colorado, USA.The isolate was sequentially verified and accessioned by the Centers for Disease Control and Prevention (CDC) and the American Type Culture Collection (ATCC) for international deployment.

• Biosafety Matrix: Classified under Biosafety Level 2 (BSL-2) containment parameters.Due to its clinically demonstrated status as an opportunistic human pathogen, handling requires dedicated Class II biosafety containment frameworks.

II Strategic Research Value and Translational Fields

Aggregatibacter actinomycetemcomitans is universally benchmarked as a critical etiologic pathogen mediating aggressive oral infections and specific extra-oral systemic diseases:

1. Periodontology and Oral Microbiome Modeling: Extensively utilized in mapping the downstream molecular pathways governing localized aggressive periodontitis and chronic adult periodontitis.Researchers deploy ATCC 29523 as a valuable serotype-specific control baseline to evaluate bacterial tissue colonization, surface adhesion profiles, and dense biofilm architecture assembly.

2. Extra-Oral Systemic Pathogenesis: Given its direct human blood isolation provenance, this strain remains central to modeling the HACEK group syndrome (a cluster of fastidious Gram-negative bacilli driving culture-negative infective endocarditis), illustrating how oral pathobionts bypass mucosal barriers to enter central circulatory loops.

3. Host-Immune Escape and Virulence Metrology: Serves as a screening platform to evaluate how surface lipopolysaccharides (LPS), heat shock proteins, and cytolethal distending toxins (CDT) modulate interaction profiles with immortalized human gingival epithelial cells and suppress local white blood cell defenses.

III Thawing, Proliferation, Passaging, and Cryopreservation Routines

1. Medium Formulations and Ambient Specifications

This fastidious species dictates complex nutritional matrices and a carbon-dioxide-fortified environment to trigger optimal logarithmic division:

• Recommended Medium Matrix: Chocolate Agar Medium, Columbia Blood Agar (supplemented with 5% to 10% defibrinated horse or sheep blood), or Tryptic Soy Broth/Agar supplemented with 0.6% yeast extract (TSYE Matrix).

• Incubation Parameters: Temperature must be tightly locked at 37 degrees Celsius under a humidified atmosphere containing 5% to 10% $CO_2$. Alternatively, use dedicated microaerophilic or anaerobic jars configured for gas exchange.

2. Thawing Freeze-Dried Cultures (Lyophilized Pellet Revitalization)

1. Within an approved BSL-2 biosafety cabinet, carefully open the sealed outer glass ampoule containing the lyophilized ATCC 29523 pellet.

2. Utilizing a sterile pasteur pipette, introduce approximately 0.5 mL of pre-warmed sterile TSYE broth or Brain Heart Infusion (BHI) broth directly onto the pellet. Gently aspirate to achieve complete dissolution.

3. Transfer the concentrated suspension onto chocolate agar plates or blood agar plates, executing a standard three-zone or four-zone streak configuration to ensure isolated single colonies.

4. Crucial Parameter: Immediately move the plates into a 37 degrees Celsius incubator supplemented with 5% to 10% $CO_2$. Because this strain grows relatively slowly, distinct colonies typically emerge after 24 to 48 hours of continuous incubation.

3. Subculturing and Strain Passaging

• Colony Morphology: On premium blood matrix, ATCC 29523 colonies appear small, translucent to slightly opaque-white, and can strongly adhere to the agar subsurface.

• Passaging Routine: Pick distinct, well-isolated single colonies using a sterile loop and re-streak onto fresh solid agar surfaces or inoculate directly into liquid TSYE broth systems.Liquid cultures generally require 7 to 15 hours of incubation to achieve mid-log phase density. Continuous, prolonged passaging on solid plates should be restricted to prevent spontaneous mutations that diminish target virulence markers or surface antigen configurations.

4. Cryopreservation Protocol

• Short-Term Maintenance: Wrap solid plates containing active colonies with laboratory parafilm and store at 4 degrees Celsius. This method is only viable for 1 to 2 weeks and is discouraged for reference stocks.

• Long-Term Cryo-Freezing Routine: Harvest liquid cultures during their optimal log-growth phase. Combine the bacterial suspension in a cryovial containing 80% active culture slurry and 20% sterile high-purity glycerol (or 15% glycerol-fortified skim milk solution). Mix gently, and transfer immediately to an ultra-low temperature freezer at -80 degrees Celsius or directly into liquid nitrogen (-196 degrees Celsius) for indefinite functional preservation across decades.

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