BioVector® 福赛坦纳菌 ATCC 43037 标准菌株

BioVector® Tannerella forsythia ATCC 43037 Standard Strain

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：福赛坦纳菌标准菌株（Tannerella forsythia，曾用名 Bacteroides forsythus）。

• 菌株编号与别名：ATCC 43037、JCM 10827、CIP 105218、CCUG 23111、DSM 102835。

• 物种分类：细菌界（Bacteria），拟杆菌门（Bacteroidota），拟杆菌纲（Bacteroidia），拟杆菌目（Bacteroidales），拟杆菌科（Bacteroidaceae），坦纳菌属（Tannerella）。

• 生物学与生理特异性特征：

  • 红色复合体（Red Complex）核心成员：福赛坦纳菌与牙龈卟啉单胞菌（P. gingivalis）和齿垢密螺旋体（T. denticola）共同构成了著名的口腔致病菌“红色复合体”，是导致重度牙周炎、骨吸收和组织破坏的核心病原体。

  • 严格厌氧与专性营养依赖（N-乙酰胞壁酸依赖型）：ATCC 43037 是一种革兰氏阴性（Gram-negative）、无动力、两端呈尖细状的梭形或短杆状细菌。该菌具有极度苛刻的营养要求，其自身基因组缺乏合成 N-乙酰胞壁酸（NAM, N-acetylmuramic acid） 的关键酶，因此在人工培养时必须在培养基中额外添加外源性 NAM 才能生长。

• 来源历史：最初由美国马萨诸塞州波士顿的福赛斯牙科中心（Forsyth Dental Center）从人类高级牙周炎患者的牙龈下菌斑（Human subgingival plaque）中分离获得，是全球广泛接受的福赛坦纳菌模式参考菌株（Type strain）。

• 生物安全级别：2级（BSL-2）。涉及活菌的操作必须在二级生物安全柜内进行。

二 核心科研价值与转化医学应用

福赛坦纳菌是国际口腔微生物学界公认最难培养但极具临床意义的病原菌之一：

1. 牙周病学与骨吸收机制研究（Periodontology & Bone Resorption）：广泛用于研究其特征性的外膜蛋白（表面 S 层蛋白 / S-layer proteins）、BspA 蛋白（富含亮氨酸的重复序列蛋白）如何诱导宿主上皮细胞分泌促炎细胞因子（IL-6, TNF-alpha），以及如何激活破骨细胞导致牙槽骨吸收。

2. 口腔多物种生物膜模型（Multi-species Biofilm Modeling）：在体外构建“红色复合体”或多菌种人工牙斑块（Biofilm）模型时，ATCC 43037 作为核心组分，用于分析其与其他口腔共生菌/致病菌（如宿主相互依赖的协同作用）的共凝集及空间分布特征。

3. 诊断检测试剂验证（Metrology & Molecular Diagnostics）：作为法定的模式菌株，广泛用于各种口腔微生物 PCR 检测试剂盒、高通量测序（NGS）宏基因组学分析以及临床厌氧菌鉴定系统的特异性对照。

三 实验室菌株复苏、扩增传代与冷冻保存标准步骤

1. 专用培养基与严格环境配置

福赛坦纳菌对环境极其敏感，接触氧气会迅速失活：

• 核心专用培养基配方（NAM 补充型血琼脂 / 肉汤）：

  • 基础：大豆酪蛋白消化物（TSB/TSA）或脑心浸液（BHI）培养基，补充 5% 灭菌脱纤维羊血或马血、0.5% 酵母浸膏（Yeast Extract）、氯化血红素（Hemin，5 microgram/mL）和维生素 K1（1 microgram/mL）。

  • 关键添加剂：必须在无菌条件下加入经微孔滤膜过滤除菌的 N-乙酰胞壁酸（NAM），其最终工作浓度必须达到 10 microgram/mL 至 100 microgram/mL（常用 10-20 microgram/mL）。

• 培养环境参数：温度为 37 摄氏度，必须置于严格厌氧环境（无氧加湿环境，如：85% $N_2$ + 10% $CO_2$ + 5% $H_2$）的厌氧培养箱或使用高效厌氧产气包的厌氧罐中孵育。

2. 冻干粉/冻存菌种复苏步骤（Thawing & Revitalization）

1. 准备工作：将补充有 NAM、血红素和维生素 K1 的固体血琼脂平板或液体肉汤提前置于厌氧箱中进行预还原（Pre-reduction，至少 12-24 小时），以彻底驱除培养基内的游离氧。

2. 在生物安全柜内小心打开含有 ATCC 43037 的冻干安瓿管。

3. 用无菌移液管吸取约 0.5 mL 预还原的厌氧肉汤，滴加到冻干菌块上使其完全溶解。

4. 将全量菌悬液迅速接种到预还原的 NAM 补充型血琼脂平板上（进行密集区域密集划线，由于其生长微弱，早期建议集中接种）。

5. 极重要：接种后立即将平板送回 37 摄氏度严格厌氧箱 中。由于福赛坦纳菌生长速度极慢，通常需要连续孵育 5 至 7 天（甚至更久）方可看到针尖大小的微小菌落。

3. 菌株传代与日常维持（Passaging & Maintenance）

• 形态观察：在固体平板上，ATCC 43037 形成极微小、凸起、光滑、微白至半透明的菌落。

• 传代操作：挑取平板上生长良好的微小菌落，接种至新鲜的预还原 NAM 补充型液体肉汤或固体平板上。液体培养通常需要 3 至 5 天方可达到对数生长晚期。建议每次传代时间控制在菌落刚长出的活跃期，严禁使用干枯或长期暴露在室温环境下的老旧平板进行转接。

4. 菌株长期冷冻保存（Cryopreservation）

• 长期甘油冷冻法：采集处于对数生长活跃期的液体培养物。在严格厌氧环境下，按照 80% 液体菌悬液 + 20% 灭菌高纯度甘油 的比例在冻存管中轻柔混匀，或使用含 15% 甘油的预还原厌氧肉汤。

• 保存条件：混匀后，立即将冻存管移出并投入 -80 摄氏度 超低温冰箱，或者直接放入 液氮（-196 摄氏度） 中。在连续稳定的超低温下，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: Tannerella forsythia Standard Reference / Type Strain (formerly known as Bacteroides forsythus).

• Strain Designations and Aliases: ATCC 43037, JCM 10827, CIP 105218, CCUG 23111, DSM 102835.

• Taxonomic Classification: Domain Bacteria, Phylum Bacteroidota, Class Bacteroidia, Order Bacteroidales, Family Bacteroidaceae, Genus Tannerella.

• Biological and Physiological Core Framework:

  • Red Complex Cornerstone: Along with Porphyromonas gingivalis and Treponema denticola, Tannerella forsythia belongs to the infamous "Red Complex" group of oral anaerobic pathogens, globally recognized as the main clinical drivers of advanced periodontitis, tissue loss, and alveolar bone destruction.

  • Strict Anaerobe & Auxotrophic Requirement (N-Acetylmuramic Acid Dependent): ATCC 43037 is a Gram-negative, non-motile, spindle-shaped or fusiform rod-like bacillus with tapered ends. This species exhibits an extremely fastidious nutritional profile; its genome lacks the essential biosynthetic machinery to produce N-acetylmuramic acid (NAM), a vital element for cell wall peptidoglycan assembly. Consequently, it absolutely requires exogenous NAM supplementation to proliferate in artificial media.

• Source Isolation History: Originally recovered and isolated from human subgingival plaque samples harvested from patients diagnosed with severe periodontitis at the Forsyth Dental Center in Boston, Massachusetts, USA. It serves as the official international taxonomic reference type strain for the species.

• Biosafety Matrix: Classified under Biosafety Level 2 (BSL-2) containment parameters. All workflows with viable cultures require certified biosafety enclosures and anaerobically controlled systems.

II Strategic Research Value and Translational Fields

Tannerella forsythia is universally regarded as one of the most fastidious yet pathologically significant oral microbes:

1. Periodontitis Etiology and Alveolar Bone Resorption: Widely used to characterize its unique outer cell surface layer (S-layer proteins) and the BspA virulence factor (a leucine-rich repeat protein). Researchers assess how these surface components trigger host epithelial arrays to secrete pro-inflammatory cytokines (such as IL-6 and TNF-alpha) and drive osteoclast differentiation.

2. Polymicrobial Oral Biofilm Architecture: Deployed as a key component in multi-species oral biofilm platforms (mimicking the Red Complex environment), illuminating spatial arrangement patterns, co-aggregation arrays, and synergistic nutritional interdependencies with other oral pathobionts.

3. Diagnostic Validation and Baseline Metrology: Serves as the definitive global reference control strain to benchmark the analytical sensitivity, target specificity, and alignment metrics of novel periodontal PCR diagnostic arrays, microfluidic chips, and next-generation sequencing (NGS) metagenomic profiling platforms.

III Thawing, Proliferation, Passaging, and Cryopreservation Routines

1. Medium Formulations and Controlled Ambient Specifications

This strict anaerobe is highly sensitive to trace oxygen; brief atmospheric exposure during active growth can cause irreversible loss of viability:

• Specialized Enriched Growth Media (NAM-Fortified Blood Agar/Broth):

  • Basal Matrix: Premium Tryptic Soy Broth/Agar (TSB/TSA) or Brain Heart Infusion (BHI) matrix, fortified with 5% defibrinated sheep or horse blood, 0.5% yeast extract, hemin (5 microgram/mL), and coenzyme vitamin K1 (1 microgram/mL).

  • Critical Nutrient Additive: Supplement the media sterilely with filter-sterilized N-acetylmuramic acid (NAM) to achieve a definitive final work concentration of 10 microgram/mL to 100 microgram/mL (typically optimized at 10–20 microgram/mL).

• Atmospheric Parameters: Regulated strictly at 37 degrees Celsius within a rigidly anaerobic workspace (e.g., 85% $N_2$ + 10% $CO_2$ + 5% $H_2$) or inside specialized anaerobic jars configured with palladium catalysts and active gas-scavenging pouches.

2. Thawing and Revitalization Routine (Lyophilized Pellet Revitalization)

1. Pre-reduction Workflow: Solid blood agar plates and liquid media supplemented with NAM must be placed inside the anaerobic workstation for pre-reduction (minimum 12–24 hours) prior to inoculation to deplete dissolved residual oxygen within the media matrix.

2. Inside an approved biosafety enclosure, open the sealed outer glass ampoule containing the lyophilized ATCC 43037 pellet.

3. Utilizing a sterile pipette, deliver approximately 0.5 mL of the pre-reduced anaerobic broth onto the pellet. Mix gently until completely dissolved.

4. Promptly transfer the suspension onto the pre-reduced NAM-fortified blood agar plates, using a dense quadrant streak configuration to concentrate cell density.

5. Critical Instruction: Immediately return the inoculated plates to the 37 degrees Celsius strict anaerobic workspace. Because this strain grows very slowly, initial pinpoint colonies typically emerge only after 5 to 7 days of continuous incubation.

3. Subculturing and Routine Maintenance

• Colony Morphology: On solid matrices, ATCC 43037 colonies present as small, smooth, convex, translucent-to-white pinpoint dots.

• Passaging Routine: Pick distinct colonies using a sterile loop and re-streak onto fresh pre-reduced solid NAM-fortified media or inoculate into liquid broth arrays. Liquid cultures typically require 3 to 5 days to reach stable late-log phase density. Subculturing must be performed using active, freshly emerged colonies; do not attempt to transfer cultures from aged, desiccated plates or plates left at room temperature.

4. Cryopreservation Protocol

• Long-Term Cryo-Freezing Routine: Harvest active liquid cultures during their peak logarithmic phase. Under anaerobic conditions, blend the cell suspension in a sterile cryovial filled with 80% active culture slurry and 20% sterile high-purity glycerol (or utilize a 15% glycerol-fortified pre-reduced anaerobic broth matrix).

• Storage Configuration: Mix thoroughly, extract from the anaerobic hood, and flash-freeze instantly at -80 degrees Celsius or drop directly into liquid nitrogen (-196 degrees Celsius) for indefinite functional preservation across decades.

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