BioVector® 齿垢密螺旋体 ATCC 35405 标准菌株

BioVector® Treponema denticola ATCC 35405 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：齿垢密螺旋体标准菌株（Treponema denticola）。

• 菌株编号与别名：ATCC 35405、DSM 14222、JCM 8225、CIP 105220。

• 物种分类：细菌界（Bacteria），螺旋体门（Spirochaetota），螺旋体纲（Spirochaetia），螺旋体目（Spirochaetales），螺旋体科（Spirochaetaceae），密螺旋体属（Treponema）。

• 生物学与生理特异性特征：

  • 红色复合体（Red Complex）核心成员：齿垢密螺旋体与牙龈卟啉单胞菌（P. gingivalis）和福赛坦纳菌（T. forsythia）共同构成了著名的口腔致病菌“红色复合体”，是导致重度人类牙周炎、牙槽骨破坏及牙龈组织溃烂的关键病原体。

  • 专性厌氧与高运动性：ATCC 35405 是一种革兰氏阴性（Gram-negative）、严格厌氧（Strict anaerobe）的螺旋状柔性细菌。该菌具有独特的周质轴丝（内鞭毛 / Periplasmic flagella），使其在稠密的口腔菌斑、高粘度的结缔组织或粘液中依然具备极强的运动和穿透能力。

  • 全基因组测序模式株：ATCC 35405 是全球首个完成全基因组测序（Genome sequenced）的齿垢密螺旋体品系，其分子背景极其清晰。

• 来源历史：最初由美国弗吉尼亚理工大学（Virginia Polytechnic Institute and State University）从人类牙周炎患者的牙龈下菌斑（Human subgingival plaque）中分离获得，是国际通用的模式参考菌株（Type strain）。

• 生物安全级别：2级（BSL-2）。涉及活菌操作必须在二级生物安全柜及严格的厌氧操作系统内进行。

二 核心科研价值与转化医学应用

齿垢密螺旋体是口腔微生物学和螺旋体生物学研究中极其重要但培养难度极高的标杆菌株：

1. 牙周致病机理与组织降解研究：广泛用于探究其核心毒力因子——Dentilisin（一种具有糜蛋白酶样活性的蛋白酶复合体）、Msp（主要外膜蛋白）以及 1 型多聚糖如何破坏宿主上皮屏障、降解细胞外基质（纤连蛋白、胶原蛋白）、并干扰宿主免疫中性粒细胞的功能。

2. 多菌种牙斑块生物膜协同互作：在体外构建三维口腔微生态或“红色复合体”人工生物膜模型时，ATCC 35405 常作为核心运动与空间穿插组分。研究它如何与牙龈卟啉单胞菌等进行代谢偶联（如利用对方产生的挥发性脂肪酸或短链脂肪酸）。

3. 分子诊断与科研质控（Diagnostics & Quality Control）：作为法定的模式菌株，是开发检测口腔螺旋体定量的常规 PCR/qPCR 试剂盒、临床全基因组宏基因组测序（NGS）分析以及厌氧分类学的官方阳性对照标准物。

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

齿垢密螺旋体对氧气极其敏感，且无法在普通的琼脂固体平板表面直接生长，常规必须在高度富集的液体培养基中进行悬浮培养：

• 核心专用培养基（NOS 肉汤 / New Oral Spirochete Medium）：

  • 基础：心浸液肉汤（Heart Infusion Broth）或脑心浸液（BHI）肉汤，补充 10% 灭菌灭活的优质兔血清（Rabbit Serum）或胎牛血清（兔血清效果更佳）、0.5% 酵母浸膏。

  • 关键还原剂与生化添加剂：必须在无菌条件下加入 L-半胱氨酸（L-Cysteine HCl，作为还原剂，提供极低氧化还原电位）、硫代硫酸钠、挥发性脂肪酸混合物（VFA）、辅酶（Hemin 5 microgram/mL）和维生素 K1（1 microgram/mL）。

• 培养环境参数：温度为 37 摄氏度，必须置于严格无氧加湿环境（如：85% $N_2$ + 10% $CO_2$ + 5% $H_2$）的厌氧工作站或配有高活性催化剂的厌氧罐中孵育。

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

1. 预还原工作（Pre-reduction）：将配置好的 NOS 液体肉汤提前置于严格厌氧箱中进行预还原（至少 24 小时），使液体中的溶解氧彻底被半胱氨酸消耗殆尽。

2. 在生物安全柜内小心打开含有 ATCC 35405 的冻干安瓿管。

3. 用无菌移液管吸取约 1.0 mL 预还原的厌氧 NOS 肉汤，滴加到冻干菌块上，轻柔吹打使其完全悬浮溶解。

4. 将全量菌悬液转入含有 5-8 mL 预还原 NOS 肉汤的无菌螺口试管中，封口紧闭。

5. 极重要：立即将试管送回 37 摄氏度严格厌氧箱 中静置培养。由于该菌复苏较为迟缓，通常需要连续孵育 5 至 7 天。在显微镜下（最好使用暗视野显微镜 / Dark-field microscopy）观察到大量呈现典型波浪状蛇行滑动的活泼螺旋体，且肉眼可见培养基呈现轻微均匀混浊，即代表复苏成功。

3. 菌株传代与日常维持（Passaging & Maintenance）

• 监测方法：由于螺旋体菌落无法在常规有氧平板观察，建议传代前抽取少量培养物置于暗视野显微镜下，确认菌体细长、螺旋规则且运动剧烈。

• 传代操作：按照 10% 的接种量（例如将 1 mL 旺盛生长期的菌液转接至 9 mL 新鲜的、预还原的 NOS 肉汤中）。传代周期通常为 3 至 5 天（当细胞密度达到约 $1 \times 10^8$ cells/mL 且处于对数生长晚期时最佳）。避免让菌液进入衰亡期（菌体会变圆、断裂或失去动力），否则转接极易失败。

4. 菌株长期冷冻保存（Cryopreservation）

• 长期甘油冷冻法：在严格厌氧环境下，采集处于对数生长活跃期（动力极佳）的 NOS 液体培养物。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 NOS 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: Treponema denticola Standard Reference / Type Strain.

• Strain Designations and Aliases: ATCC 35405, DSM 14222, JCM 8225, CIP 105220.

• Taxonomic Classification: Domain Bacteria, Phylum Spirochaetota, Class Spirochaetia, Order Spirochaetales, Family Spirochaetaceae, Genus Treponema.

• Biological and Physiological Core Framework:

  • Red Complex Triad Cornerstone: Alongside Porphyromonas gingivalis and Tannerella forsythia, Treponema denticola represents a defining member of the infamous oral pathogenic "Red Complex." It is universally recognized as a primary clinical driver of advanced human periodontitis, extensive alveolar bone destruction, and tissue necrosis.

  • Strict Anaerobe & High Motility Architecture: ATCC 35405 is a Gram-negative, strictly anaerobic, highly flexible, helical/spirochetal bacterium. It possesses specialized periplasmic flagella (endoflagella) located within the periplasmic space, granting it exceptional swimming and corkscrew-like drilling motility through dense subgingival plaque matrices and highly viscous connective tissues.

  • Fully Sequenced Paradigm: ATCC 35405 serves as the definitive global reference type strain for the species and was the first Treponema denticola line to have its complete genome fully sequenced and mapped.

• Source Isolation History: Originally recovered and isolated from human subgingival plaque samples harvested from patients experiencing active periodontal breakdown at Virginia Polytechnic Institute and State University, USA.

• Biosafety Matrix: Classified under Biosafety Level 2 (BSL-2) containment parameters. All operations involving viable cultures must be carried out within certified biosafety enclosures and strictly anaerobic specialized containment systems.

II Strategic Research Value and Translational Fields

Treponema denticola is universally utilized as the benchmark model for studying oral spirochete biology and polymicrobial synergy:

1. Periodontal Pathogenesis and Tissue Degradation: Extensively harnessed to dissect its core virulence arsenal, specifically Dentilisin (a high-molecular-weight chymotrypsin-like protease complex), the Major Surface Protein (Msp), and Type 1 phosphoglycan loops. Researchers evaluate how these factors degrade host extracellular matrix barriers (fibronectin, collagen) and suppress local neutrophil immune responses.

2. Polymicrobial Oral Biofilm Architecture: Deployed as a key structural component in multi-species oral biofilm matrices mimicking the subgingival pocket. It serves to illuminate metabolic coupling pathways (such as its reliance on volatile or short-chain fatty acids manufactured by P. gingivalis) and physical co-aggregation patterns with other oral pathobionts.

3. Diagnostic Validation and Quality Control: Serves as the global reference type strain to benchmark the analytical sensitivity, multiplex specificity, and alignment metrics of novel periodontal quantitative PCR (qPCR) assays, microfluidic diagnostic arrays, and next-generation sequencing (NGS) metagenomic reference pipelines.

III Thawing, Proliferation, Passaging, and Cryopreservation Routines

1. Medium Formulations and Controlled Ambient Specifications

This fastidious spirochete is highly sensitive to trace oxygen and cannot form colonies on standard solid agar plate surfaces under routine setups; it must be grown in specialized liquid suspension cultures:

• Specialized Enriched Media (NOS Broth / New Oral Spirochete Matrix):

  • Basal Matrix: Heart Infusion Broth or Brain Heart Infusion (BHI) matrix, fortified with 10% sterile, heat-inactivated premium Rabbit Serum (or fetal bovine serum, though rabbit serum is highly preferred) and 0.5% yeast extract.

  • Critical Reducing and Biochemical Additives: Must be supplemented sterilely with L-Cysteine HCl (acting as a heavy reducing agent to drop redox potential), sodium thiosulfate, a volatile fatty acid (VFA) mixture, hemin (5 microgram/mL), and vitamin K1 (1 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 active palladium oxygen-scavenging systems.

2. Thawing and Revitalization Routine (Lyophilized Pellet Revitalization)

1. Pre-reduction Workflow: Formulated liquid NOS broth tubes must be transferred into the strict anaerobic workstation for pre-reduction (minimum 24 hours) prior to inoculation to ensure any dissolved residual oxygen is fully neutralized by the L-cysteine.

2. Inside an approved biosafety enclosure, open the sealed outer glass ampoule containing the lyophilized ATCC 35405 pellet.

3. Utilizing a sterile pipette, deliver approximately 1.0 mL of the pre-reduced liquid NOS broth onto the pellet. Mix gently until completely dissolved.

4. Promptly transfer the entire suspension into a sterile screw-cap culture tube containing 5–8 mL of pre-reduced anaerobic NOS broth, tightening the cap firmly.

5. Critical Instruction: Immediately place the inoculated tube into the 37 degrees Celsius strict anaerobic workspace. Because initial revitalization is slow, incubate undisturbed for 5 to 7 days. Revitalization is confirmed when the medium exhibits uniform, faint turbidity and observation under a dark-field microscope reveals abundant, actively swimming spirochetes showing typical wave-like motility.

3. Subculturing and Routine Maintenance

• Monitoring Method: Because solid colony tracking is unavailable, evaluate the culture prior to passaging using dark-field microscopy to verify that the spirochetes are elongated, properly helical, and actively motile.

• Passaging Routine: Inoculate fresh pre-reduced NOS broth arrays using a 10% inoculation volume (e.g., transferring 1 mL of active culture into 9 mL of fresh medium). The standard passaging interval tracks between 3 to 5 days, ideally targeting the late-exponential growth phase when cell density strikes approximately $1 \times 10^8$ cells/mL. Do not allow cultures to enter the stationary or decline phase, as cells will round up, fragment, lose motility, and fail to pass successfully.

4. Cryopreservation Protocol

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

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

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