BioVector? 伴放線聚集桿菌 ATCC 29523 標(biāo)準(zhǔn)菌株

BioVector? Aggregatibacter actinomycetemcomitans ATCC 29523 Standard Strain Manual

第一部分 中文說(shuō)明

一 產(chǎn)品基本信息與遺傳學(xué)背景

• 菌株名稱：伴放線聚集桿菌標(biāo)準(zhǔn)菌株（Aggregatibacter actinomycetemcomitans）。

• 菌株編號(hào)與別名：ATCC 29523、CDC A1916（常寫作 CDC A1914）、CIP 101033、JCM 12985、CCUG 37004。

• 物種分類：細(xì)菌界（Bacteria），變形菌門（Pseudomonadota），$\gamma$-變形菌綱（Gammaproteobacteria），巴斯德氏菌目（Pasteurellales），巴斯德氏菌科（Pasteurellaceae），聚集桿菌屬（Aggregatibacter）。

• 生物學(xué)與血清型特征：

  • 血清型 A（Serotype a）：ATCC 29523 是血清型 A 的經(jīng)典代表菌株。與高毒力的血清型 b（如 JP2 克隆株）相比，血清型 A 表現(xiàn)出較低的白細(xì)胞毒素（Leukotoxin）表達(dá)水平，通常更廣泛地存在于健康的口腔或口腔平穩(wěn)狀態(tài)中。

  • 形態(tài)與呼吸特性：革蘭氏陰性（Gram-negative）、兼性厭氧（Facultative anaerobe）、無(wú)動(dòng)力、短桿狀或球桿狀細(xì)菌。在固體培養(yǎng)基上通常表現(xiàn)出光滑型表型（Smooth phenotype），在液體培養(yǎng)基中呈現(xiàn)非聚集性生長(zhǎng)。

• 來(lái)源歷史：最初由美國(guó)科羅拉多州丹佛市的退伍軍人醫(yī)院（VA Hospital, Denver, USA）從人類血液（Human Blood）樣本中分離獲得，后經(jīng)美國(guó)疾病預(yù)防控制中心（CDC）及美國(guó)標(biāo)準(zhǔn)生物品收藏中心（ATCC）鑒定并向全球分發(fā)。

• 生物安全級(jí)別：2級(jí)（BSL-2 / 風(fēng)險(xiǎn)群2）。由于其具有一定的人類致病性，必須在二級(jí)生物安全柜內(nèi)進(jìn)行無(wú)菌操作。

二 核心科研價(jià)值與轉(zhuǎn)化醫(yī)學(xué)應(yīng)用

伴放線聚集桿菌是國(guó)際公認(rèn)的嚴(yán)重口腔疾病及全身侵襲性感染的重要致病菌之一：

1. 牙周病學(xué)與口腔微生物學(xué)（Periodontology）：廣泛用于局限性侵襲性牙周炎（Localized Aggressive Periodontitis）和慢性牙周炎的病因?qū)W機(jī)制研究。ATCC 29523 作為血清型 A 菌株，經(jīng)常被臨床及學(xué)術(shù)界用作評(píng)估細(xì)菌定殖（Colonization）、表面粘附（Adhesion）以及生物膜（Biofilm）形成特異性的對(duì)照株。

2. 非口腔感染與心內(nèi)膜炎研究：該菌株分離自血液，常用于構(gòu)建 HACEK 感染群（引發(fā)感染性心內(nèi)膜炎的一組臨床少見革蘭氏陰性菌）的體外模型，深入分析其如何跨越口腔屏障進(jìn)入循環(huán)系統(tǒng)。

3. 免疫逃逸與宿主交互（Host-Pathogen Interaction）：用于測(cè)試其表面抗原、脂多糖（LPS）、細(xì)胞致死性溶脹毒素（CDT）等毒力因子與人牙齦上皮細(xì)胞、白細(xì)胞的免疫交互反應(yīng)。

三 實(shí)驗(yàn)室菌株復(fù)蘇、擴(kuò)增傳代與冷凍保存標(biāo)準(zhǔn)步驟

1. 專用培養(yǎng)基與環(huán)境配置

伴放線聚集桿菌對(duì)營(yíng)養(yǎng)要求較高，且在含 $CO_2$ 環(huán)境中生長(zhǎng)更佳：

• 推薦培養(yǎng)基：巧克力瓊脂培養(yǎng)基（Chocolate Agar）、哥倫比亞血瓊脂培養(yǎng)基（含 5% - 10% 脫纖維馬血或羊血），或補(bǔ)充有 0.6% 酵母浸膏的胰蛋白胨大豆肉湯/瓊脂（TSYE）。

• 培養(yǎng)環(huán)境參數(shù)：溫度為 37 攝氏度，必須置于 5% 至 10% $CO_2$ 的加濕孵箱或厭氧/微需氧培養(yǎng)罐中進(jìn)行孵育。

2. 凍干粉菌種復(fù)蘇步驟（Thawing Freeze-Dried Cultures）

1. 在二級(jí)生物安全柜內(nèi)小心打開含有 ATCC 29523 凍干菌種的小安瓿管。

2. 用無(wú)菌移液管吸取約 0.5 mL 預(yù)熱的 TSYE 肉湯或常規(guī)腦心浸液（BHI）肉湯，滴加到凍干菌塊上，輕輕吹打使其完全懸浮溶解。

3. 將溶解后的全量菌懸液接種到巧克力瓊脂平板或血瓊脂平板上，使用接種環(huán)進(jìn)行三區(qū)或四區(qū)劃線以獲得單克隆。

4. 極重要：將平板立即放入 37 攝氏度、5% - 10% $CO_2$ 孵箱中，由于該菌株生長(zhǎng)較為緩慢，通常需要連續(xù)孵育 24 至 48 小時(shí) 甚至更久方可肉眼觀察到特征性菌落。

3. 菌株傳代與純化（Passaging & Maintenance）

• 特征觀察：在血平板上，ATCC 29523 的菌落通常較小、微白至半透明，有時(shí)在瓊脂內(nèi)部貼合較緊（光滑型在液體中較難自聚）。

• 傳代操作：挑取平板上生長(zhǎng)良好的單菌落，重新劃線接種至新鮮的血瓊脂平板或接種于 TSYE 液體肉湯中。液體培養(yǎng)通常孵育 7 至 15 小時(shí)即可進(jìn)入對(duì)數(shù)生長(zhǎng)期。不建議在平板上連續(xù)長(zhǎng)期傳代，以防其由于自發(fā)突變而導(dǎo)致原有毒力因子或表面抗原結(jié)構(gòu)丟失。

4. 菌株長(zhǎng)期冷凍保存（Cryopreservation）

• 短期保存：將長(zhǎng)有菌落的固體平板用封口膜密封，置于 4 攝氏度冰箱中，可維持 1-2 周（不推薦）。

• 長(zhǎng)期甘油冷凍法：采集處于對(duì)數(shù)生長(zhǎng)旺盛期的液體培養(yǎng)物，按照 80% 液體培養(yǎng)物 + 20% 滅菌高純度甘油 的比例（或使用含 15% 甘油的脫脂奶粉懸液）在凍存管中輕柔混勻。立即置于 -80 攝氏度 超低溫冰箱或直接投入 液氮（-196 攝氏度） 中，可實(shí)現(xiàn)數(shù)年以上的無(wú)限期穩(wěn)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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