BioVector? 齒垢密螺旋體 ATCC 35405 標(biāo)準(zhǔn)菌株

BioVector? Treponema denticola ATCC 35405 Standard Strain Manual

第一部分 中文說明

一 產(chǎn)品基本信息與遺傳學(xué)背景

• 菌株名稱：齒垢密螺旋體標(biāo)準(zhǔn)菌株（Treponema denticola）。

• 菌株編號(hào)與別名：ATCC 35405、DSM 14222、JCM 8225、CIP 105220。

• 物種分類：細(xì)菌界（Bacteria），螺旋體門（Spirochaetota），螺旋體綱（Spirochaetia），螺旋體目（Spirochaetales），螺旋體科（Spirochaetaceae），密螺旋體屬（Treponema）。

• 生物學(xué)與生理特異性特征：

  • 紅色復(fù)合體（Red Complex）核心成員：齒垢密螺旋體與牙齦卟啉單胞菌（P. gingivalis）和福賽坦納菌（T. forsythia）共同構(gòu)成了著名的口腔致病菌“紅色復(fù)合體”，是導(dǎo)致重度人類牙周炎、牙槽骨破壞及牙齦組織潰爛的關(guān)鍵病原體。

  • 專性厭氧與高運(yùn)動(dòng)性：ATCC 35405 是一種革蘭氏陰性（Gram-negative）、嚴(yán)格厭氧（Strict anaerobe）的螺旋狀柔性細(xì)菌。該菌具有獨(dú)特的周質(zhì)軸絲（內(nèi)鞭毛 / Periplasmic flagella），使其在稠密的口腔菌斑、高粘度的結(jié)締組織或粘液中依然具備極強(qiáng)的運(yùn)動(dòng)和穿透能力。

  • 全基因組測(cè)序模式株：ATCC 35405 是全球首個(gè)完成全基因組測(cè)序（Genome sequenced）的齒垢密螺旋體品系，其分子背景極其清晰。

• 來源歷史：最初由美國弗吉尼亞理工大學(xué)（Virginia Polytechnic Institute and State University）從人類牙周炎患者的牙齦下菌斑（Human subgingival plaque）中分離獲得，是國際通用的模式參考菌株（Type strain）。

• 生物安全級(jí)別：2級(jí)（BSL-2）。涉及活菌操作必須在二級(jí)生物安全柜及嚴(yán)格的厭氧操作系統(tǒng)內(nèi)進(jìn)行。

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

齒垢密螺旋體是口腔微生物學(xué)和螺旋體生物學(xué)研究中極其重要但培養(yǎng)難度極高的標(biāo)桿菌株：

1. 牙周致病機(jī)理與組織降解研究：廣泛用于探究其核心毒力因子——Dentilisin（一種具有糜蛋白酶樣活性的蛋白酶復(fù)合體）、Msp（主要外膜蛋白）以及 1 型多聚糖如何破壞宿主上皮屏障、降解細(xì)胞外基質(zhì)（纖連蛋白、膠原蛋白）、并干擾宿主免疫中性粒細(xì)胞的功能。

2. 多菌種牙斑塊生物膜協(xié)同互作：在體外構(gòu)建三維口腔微生態(tài)或“紅色復(fù)合體”人工生物膜模型時(shí)，ATCC 35405 常作為核心運(yùn)動(dòng)與空間穿插組分。研究它如何與牙齦卟啉單胞菌等進(jìn)行代謝偶聯(lián)（如利用對(duì)方產(chǎn)生的揮發(fā)性脂肪酸或短鏈脂肪酸）。

3. 分子診斷與科研質(zhì)控（Diagnostics & Quality Control）：作為法定的模式菌株，是開發(fā)檢測(cè)口腔螺旋體定量的常規(guī) PCR/qPCR 試劑盒、臨床全基因組宏基因組測(cè)序（NGS）分析以及厭氧分類學(xué)的官方陽性對(duì)照標(biāo)準(zhǔn)物。

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)基與嚴(yán)格環(huán)境配置

齒垢密螺旋體對(duì)氧氣極其敏感，且無法在普通的瓊脂固體平板表面直接生長(zhǎng)，常規(guī)必須在高度富集的液體培養(yǎng)基中進(jìn)行懸浮培養(yǎng)：

• 核心專用培養(yǎng)基（NOS 肉湯 / New Oral Spirochete Medium）：

  • 基礎(chǔ)：心浸液肉湯（Heart Infusion Broth）或腦心浸液（BHI）肉湯，補(bǔ)充 10% 滅菌滅活的優(yōu)質(zhì)兔血清（Rabbit Serum）或胎牛血清（兔血清效果更佳）、0.5% 酵母浸膏。

  • 關(guān)鍵還原劑與生化添加劑：必須在無菌條件下加入 L-半胱氨酸（L-Cysteine HCl，作為還原劑，提供極低氧化還原電位）、硫代硫酸鈉、揮發(fā)性脂肪酸混合物（VFA）、輔酶（Hemin 5 microgram/mL）和維生素 K1（1 microgram/mL）。

• 培養(yǎng)環(huán)境參數(shù)：溫度為 37 攝氏度，必須置于嚴(yán)格無氧加濕環(huán)境（如：85% $N_2$ + 10% $CO_2$ + 5% $H_2$）的厭氧工作站或配有高活性催化劑的厭氧罐中孵育。

2. 凍干粉/凍存菌種復(fù)蘇步驟（Thawing & Revitalization）

1. 預(yù)還原工作（Pre-reduction）：將配置好的 NOS 液體肉湯提前置于嚴(yán)格厭氧箱中進(jìn)行預(yù)還原（至少 24 小時(shí)），使液體中的溶解氧徹底被半胱氨酸消耗殆盡。

2. 在生物安全柜內(nèi)小心打開含有 ATCC 35405 的凍干安瓿管。

3. 用無菌移液管吸取約 1.0 mL 預(yù)還原的厭氧 NOS 肉湯，滴加到凍干菌塊上，輕柔吹打使其完全懸浮溶解。

4. 將全量菌懸液轉(zhuǎn)入含有 5-8 mL 預(yù)還原 NOS 肉湯的無菌螺口試管中，封口緊閉。

5. 極重要：立即將試管送回 37 攝氏度嚴(yán)格厭氧箱 中靜置培養(yǎng)。由于該菌復(fù)蘇較為遲緩，通常需要連續(xù)孵育 5 至 7 天。在顯微鏡下（最好使用暗視野顯微鏡 / Dark-field microscopy）觀察到大量呈現(xiàn)典型波浪狀蛇行滑動(dòng)的活潑螺旋體，且肉眼可見培養(yǎng)基呈現(xiàn)輕微均勻混濁，即代表復(fù)蘇成功。

3. 菌株傳代與日常維持（Passaging & Maintenance）

• 監(jiān)測(cè)方法：由于螺旋體菌落無法在常規(guī)有氧平板觀察，建議傳代前抽取少量培養(yǎng)物置于暗視野顯微鏡下，確認(rèn)菌體細(xì)長(zhǎng)、螺旋規(guī)則且運(yùn)動(dòng)劇烈。

• 傳代操作：按照 10% 的接種量（例如將 1 mL 旺盛生長(zhǎng)期的菌液轉(zhuǎn)接至 9 mL 新鮮的、預(yù)還原的 NOS 肉湯中）。傳代周期通常為 3 至 5 天（當(dāng)細(xì)胞密度達(dá)到約 $1 \times 10^8$ cells/mL 且處于對(duì)數(shù)生長(zhǎng)晚期時(shí)最佳）。避免讓菌液進(jìn)入衰亡期（菌體會(huì)變圓、斷裂或失去動(dòng)力），否則轉(zhuǎn)接極易失敗。

4. 菌株長(zhǎng)期冷凍保存（Cryopreservation）

• 長(zhǎng)期甘油冷凍法：在嚴(yán)格厭氧環(huán)境下，采集處于對(duì)數(shù)生長(zhǎng)活躍期（動(dòng)力極佳）的 NOS 液體培養(yǎng)物。按照 80% 液體菌懸液 + 20% 滅菌高純度甘油 的比例在凍存管中輕柔混勻（或使用含 15% 甘油的預(yù)還原 NOS 肉湯）。

• 保存條件：混勻后，立即將凍存管移出并投入 -80 攝氏度 超低溫冰箱，或者直接放入 液氮（-196 攝氏度） 中。在連續(xù)穩(wěn)定的超低溫下，可實(shí)現(xiàn)數(shù)年以上的無限期穩(wěn)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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