BioVector® Parabacteroides merdae Type Strain / 粪副拟杆菌标准菌株

一 产品基本信息与微生物学背景

• 菌株名称：Parabacteroides merdae（中文学术名：粪副拟杆菌）。

• 经典分类标杆菌株：ATCC 43184 / DSM 19495 / JCM 14815 / CCUG 38531（原分类法中曾被命名为 Bacteroides merdae 粪拟杆菌，后经 16S rRNA 基因组重分类修正独立为副拟杆菌属）。

• 菌株物种来源：最初自健康人类的粪便（Feces）组织中分离获得，是人类肠道核心核心核心核心共生菌群（Gut microbiota）的核心成员。

• 生物学表型与超微特征：

  • 形态与染色：革兰氏阴性（Gram-negative）。在显微镜下表现为细小的专性短杆状（Short rods）或球杆状（Coccobacilli），大小约为 $0.5 - 0.8 \times 1.0 - 2.0\ \mu\text{m}$。无芽孢，通常不具运动性，细胞表面通常包裹有一层微型的多糖荚膜。

  • 气体代谢特性：严格厌氧（Strictly anaerobic）。接触氧气会迅速引发该菌体内发生强烈的氧化应激死亡。

  • 生化代谢谱系：具有极强的糖苷水解酶活性，能高效利用和降解人类摄入的复杂膳食纤维（不溶性多糖），其主要发酵终产物（Metabolic end-products）为乙酸（Acetate）和琥珀酸（Succinate），基本不产生或仅产生微量丁酸。在生化鉴定中，吲哚（Indole）反应通常为阴性，过氧化氢酶（Catalase）多为阳性。

• 生物安全级别：1级（BSL-1）。

二 核心科研价值与肠道微生态医学转化应用

随着近年来全球对“肠道菌群-宿主轴（Gut-Host Axis）”研究的爆发，Parabacteroides merdae 已从传统的普通共生菌跃升为合成生物学与药物开发（LBP, 活体生物药）的明星底盘：

1. 构建人类核心肠道仿真模式菌群（Synthetic Communities）：在建立微生态小鼠模型（如无菌小鼠 Gnotobiotic mice 重新定殖）时，P. merdae 作为拟杆菌目（Bacteroidales）的代表性优势物种，与拟杆菌属、梭菌属等混合组装成简化的多菌组合（如经典选定的 12 菌或 20 菌群），用以解构复杂肠道生态位（Niche）的竞争、排他以及群落稳态维持机制。

2. 调控机体代谢性疾病（肥胖、高脂血症与糖尿病）：临床转录组与代谢组学研究表明，P. merdae 的肠道丰度与机体的脂质代谢、胰岛素敏感性存在强烈的正相关或负相关。它通过分泌特定的小分子代谢物，能够调控宿主远端肠道上皮细胞的法尼醇 X 受体（FXR）以及胆汁酸循环（Bile acid signaling loop），是开发新型减肥、降脂靶向微生态制剂的重点筛选对象。

3. 免疫调节、肠道屏障保护与肿瘤免疫协同（Cancer Immunotherapy）：用于研究宿主免疫耐受的诱导。该菌的特殊外膜成分能够刺激结肠固有层的树突状细胞（DCs），温和诱导调节性 T 细胞（Tregs）的生成，协助维持肠道黏膜屏障的物理完整性。同时，在前沿肿瘤免疫研究中，其在肠道内的特定丰度常被证实能显著重塑全身免疫微环境，成倍提升抗 PD-1/PD-L1 等免疫检查点阻断疗法（ICB）对实体瘤的临床临床杀伤敏感性。

三 实验室厌氧复苏、培养、常规传代与质控标准步骤

极其重要的操作警告：Parabacteroides merdae 属于严格厌氧菌。无论是开封复苏、平板涂布还是液体培养，整个操作流程必须在高效厌氧操作系统（如配置有混合气体的厌氧工作站 Anaerobic Chamber）或严格使用厌氧产气包密封罐（Anaerobic Jar）中进行。暴露在空气中会直接导致复苏失败！

1. 专用培养基、气体配比与环境物理常数

• 基础培养基（推荐以下三种之一）：

  • BHI 脑心浸液培养基（Brain Heart Infusion）：需额外添加 5 $\mu$g/mL 氯化血红素（Hemin） 和 1 $\mu$g/mL 维生素 K1（Vitamin K1） 效果极佳。

  • PYG 培养基（Peptone Yeast Glucose Medium）：专用于厌氧菌发酵代谢分析的标准培养基。

  • 哥伦比亚血琼脂平板（Columbia Blood Agar）：加入 5% 优质脱纤维绵羊血。

• 标准工作气体配比：80% 氮气（$N_2$） + 10% 氢气（$H_2$） + 10% 二氧化碳（$CO_2$）（氢气用作工作站内催化剂除氧的关键还原剂）。

• 培养物理常数：标准 37 摄氏度，绝对无氧，遮光暗培养。

2. 安瓿管/冻存管菌种的厌氧复苏与启动步序

1. 提前除氧预平衡：在转化操作前 24 小时，将配制好的无菌固体平板或液体培养基置于厌氧工作站内进行提前预还原（Pre-reduction），彻底驱离溶于培养基内部的微量游离分子氧。

2. 从液氮罐或 -80 ℃ 冰箱中取出 P. merdae 冻存管，立刻投入 37 ℃ 恒温水浴中摇晃使其在 1 分钟内急速融化（若为玻璃真空安瓿管，则需按规范用酒精棉擦拭、划痕并微力敲开）。

3. 用无菌移液管吸取 0.5 mL 预还原的 BHI 液体培养基，注入冻存管中，极其轻柔地冲洗、吸打并重悬全部菌体泥沉淀。

4. 双向接种接种质控法（强烈推荐）：

   • 固体通道：吸取 100 $\mu$g 悬液均匀涂布于预还原的哥伦比亚血琼脂平板上（用于观察单菌落形态及检测纯度）。

   • 液体通道：将剩余的所有重悬菌液全量接入盛有 5 - 10 mL 预热 BHI（含 Hemin + Vit K1）的密封厌氧培养管或螺口管底部。

5. 将平板和液体管迅速锁入厌氧工作站，或放入装有新鲜厌氧产气包、冷催化除氧剂以及厌氧指示剂（如美蓝指示条，确保指示条呈白色无氧状态）的厌氧罐内，拧紧阀门，整体移入 37 摄氏度普通培养箱。

6. 严格培养 48 - 72 小时。该菌生长相对稳健，一般在 48 小时左右可见液体培养基呈现均匀中度浑浊，琼脂平板上长出饱满的菌落。

3. 日常传代、形态鉴定与纯度质控

• 传代时机：液体发酵管长至对数生长末期（通常在接种后 24 - 36h，$OD_{600}$ 达到 1.2 - 1.5），或固体平板上的单菌落长至饱满（直径约 1 - 2 mm）时进行传代。传代时按照 1% - 3% 的体积比例，将菌液转接至新的预还原液体培养基中。

• 菌落形态特征（质控标杆）：在含有 5% 绵羊血的哥伦比亚琼脂平板上培养 48h 后，P. merdae 的典型单菌落表现为：圆形、凸起、表面光滑湿润、边缘整齐，直径约 1.0 - 1.5 mm，颜色呈微小的灰白色至半透明乳白色。核心质控：该菌在绵羊血平板上表现为 $\gamma$-溶血（无溶血环，Non-hemolytic）。

• 绝对避氧纯度测试（微生态实验红线）：在微生态研究中，为了防止由于操作不当混入外界有氧杂菌（如大肠杆菌或葡萄球菌），必须同时进行有氧对照测试（Aerobic control run）。即在传代时，挑取同一样本的菌液，平行涂布于两块血平板上：一块置于无氧环境培养，另一块直接置于 37 ℃ 普通有氧空气孵箱中培养。若 48 小时后，有氧培养的平板上出现任何菌落生长，说明系统已发生严重污染，该批次菌株必须予以彻底淘汰。

4. 菌株长期保存标准

• 冻存液配方：推荐使用高效的厌氧专用冻存液。典型配方为：预还原的 BHI 液体培养基 混合 30% 灭菌纯甘油（Glycerol），或采用重组的防御级配方：脱脂奶粉（10% Skim milk）+ 15% 甘油。

• 超低温锁死冷冻规范：

  1. 在厌氧工作站内，收集发酵旺盛（通常为 24h 强力培养代）的 P. merdae 高密度菌液。

  2. 将菌液与预先灭菌并除氧的 50% 甘油水溶液按照 1:1 的体积比在厌氧环境下彻底摇匀，使甘油终浓度维持在 25% 左右。

  3. 分装入无菌冻存管，拧紧管盖锁死气密性。

  4. 将冻存管移出工作站，直接投入 -80 ℃ 超低温冰箱中锁死存放，或者直接丢入液氮罐（-196 ℃）中长期封存。该菌在超低温甘油悬液中状态极度稳定，可保存数年。日常使用时切忌反复解冻，必须实行单管单次融化使用。

Part 2 English Section

I General Information and Microbial Taxonomy Background

• Bacterial Nomenclature:Parabacteroides merdae (formerly cataloged in legacy literature under the genus Bacteroides as Bacteroides merdae; subsequently reassigned to the distinct genus Parabacteroides based on high-resolution comparative 16S rRNA phylogenetics).

• Gold-Standard Type Strains: ATCC 43184 / DSM 19495 / JCM 14815 / CCUG 38531.

• Organism Isolation Matrix: Originally isolated and cultured from healthy human feces, establishing it as a foundational, highly abundant commensal member of the human distal gut microbiome.

• Morphological Features and Ultrastructural Profiles:

  • Staining and Cellular Configuration:Gram-negative. Under high-magnification phase-contrast profiling, cells present as small obligate short rods or distinct coccobacilli, dimensionally scaling to approximately $0.5 - 0.8 \times 1.0 - 2.0\ \mu\text{m}$. They are non-spore-forming, structurally non-motile, and standardly encapsulated within a delicate, surface-bound polysaccharide capsular layer.

  • Atmospheric Metabolic Index:Strictly anaerobic. Exposure to ambient atmospheric oxygen tension induces rapid, highly destructive oxidative stress cascades, causing zero metabolic activity and immediate vegetative cell death.

  • Biochemical and Secretome Profile: Outfitted with an extensive repertoire of glycoside hydrolases, enabling efficient fermentation of complex, non-digestible dietary polysaccharides (plant fibers). Its principal metabolic end-products include acetate and succinate, with negligible to non-detectable levels of butyrate. Standard clinical indexing yields an indole-negative and characteristically catalase-positive phenotype.

• Biosafety Threshold: Rated at Biosafety Level 1 (BSL-1).

II Strategic Research Value and Gut Microbiome-Host Applications

Driven by the ongoing expansion of the "Gut-Brain-Immune Axis" paradigm,Parabacteroides merdae has emerged as a cornerstone live biotherapeutic product (LBP) candidate and a valuable synthetic biology platform:

1. Assembling SynComs (Synthetic Microbial Communities):When engineering gnotobiotic animal matrices (e.g., re-conventionalizing germ-free mice),P. merdae is standardly selected to represent the dominant order Bacteroidales. It is incorporated into simplified multi-species consortia (such as the standardized 12-strain or 20-strain synthetic frameworks) to investigate niche competition dynamics, competitive exclusion kinetics, and microbiome community stability.

2. Ameliorating Metabolic Disorders (Obesity, Hyperlipidemia, and Type 2 Diabetes):Clinical metatranscriptomic and metabolomic data demonstrate a significant correlation between P. merdae intestinal abundance and host lipid profiling and insulin sensitivity. This organism modulates host distal colonic epithelial signaling cascades by interacting with the Farnesoid X Receptor (FXR) and modifying bile acid pool pools, making it a primary target for developing next-generation anti-obesity and lipid-lowering probiotics.

3. Immunomodulation, Epithelial Barrier Protection, and Oncology Synergy:Utilized to study mucosal immune tolerance mechanisms. Specific outer membrane macromolecules of P. merdae stimulate colonic lamina propria dendritic cells (DCs), inducing the peripheral generation of regulatory T cells (Tregs) to maintain structural intestinal barrier integrity. Furthermore, emerging immuno-oncology screens identify its presence as an ideal biomarker that prime host systemic immunity, augmenting the therapeutic efficacy of anti-PD-1/anti-PD-L1 Immune Checkpoint Blockade (ICB) therapies against recalcitrant solid tumors.

III Laboratory Anaerobic Cultivation, Isolation, Propagation, and Purity Quality Control

MANDATORY OPERATION PROTOCOL: Parabacteroides merdae is an obligate anaerobe. All handling—including cryovial unpacking, streak-plating, and fluid subculturing—MUST be executed inside a qualified anaerobic workstation or within sealed anaerobic jars equipped with active gas-generating chemical packs. Any transient atmospheric exposure will directly cause absolute cell mortality.

1. Basal Media Optimization and Atmospheric Incubator Configuration

• Formulated Production Medium Base (Select from the following standardized matrices):

  • Brain Heart Infusion (BHI) Broth: Must be fortified with 5 $\mu$g/mL Hemin and 1 $\mu$g/mL Vitamin K1 to achieve optimal growth kinetics.

  • Peptone Yeast Glucose (PYG) Medium: Standardized across baseline metabolomic laboratories to evaluate volatile fatty acid profiles.

  • Columbia Blood Agar Base: Fortified with 5% premium defibrinated sheep blood matrix.

• Workstation Gas Matrix Ratios:80% Nitrogen ($N_2$) + 10% Hydrogen ($H_2$) + 10% Carbon Dioxide ($CO_2$) (where $H_2$ drives continuous oxygen scavenging across palladium catalyst arrays).

• Physical Processing Constants: Maintain an absolute temperature threshold of 37 °C, zero dissolved oxygen, and complete darkness.

2. Unpacking and Recovering Cryopreserved Type Material

1. Pre-Reduction Synchronization: Place all sterile solid agar plates and liquid tubes inside the active anaerobic chamber at least 24 hours prior to inoculation. This pre-reduction phase ensures the complete evacuation of dissolved residual oxygen molecules from the media.

2. Retrieve the P. merdae cryovial from liquid nitrogen storage and submerge it instantly within a 37 °C water bath, agitating continuously to melt the matrix within 60 seconds.

3. Transfer the vial into the anaerobic workspace, disinfecting the exterior shell with 75% ethanol.

4. Using a pre-reduced pipette, dispense 0.5 mL of pre-reduced BHI broth directly into the cryovial, gently aspirating to resuspend the compacted bacterial pellet.

5. Dual-Channel Cultivation Protocol (Strict Quality Control Standard):

   • Solid Tracking Line: Inoculate a 100 $\mu$L aliquot of the suspension onto a pre-reduced Columbia blood agar plate, streaking for single isolated colonies to assess purity.

   • Liquid Scale Line: Transfer the remaining volume of resuspended bacterial matrix directly into the bottom of a screw-capped tube containing 5 - 10 mL of pre-reduced BHI broth (supplemented with Hemin and Vit K1).

6. Secure the inoculated plates and tubes inside the anaerobic workstation or seal them within an anaerobic jar containing a fresh gas generator envelope and a chemical indicator strip (e.g., methylene blue, which must remain colorless to confirm an anaerobic status). Incubate at 37 °C.

7. Maintain incubation statically for 48 - 72 hours.Parabacteroides merdae exhibits predictable growth kinetics; uniform medium turbidity in the liquid phase and distinct colonies on the solid phase typically emerge by 48 hours.

3. Routine Passaging Metrics and Purity Verification

• Passaging Thresholds: Execute subculturing when liquid cultures approach late-log phase growth (typically 24 - 36 hours post-inoculation, tracking an $OD_{600}$ threshold of 1.2 - 1.5), or when solid-plate single colonies achieve fully mature diameters (1 - 2 mm). Transfer the liquid suspension into fresh pre-reduced media utilizing a 1% to 3% v/v inoculation ratio.

• Colony Morphology Assessment (Identity Markers): Following 48 hours of static anaerobic incubation on Columbia sheep blood agar, mature P. merdae colonies present as circular, convex, smooth, moist, and entire-edged structures measuring 1.0 - 1.5 mm in diameter, exhibiting a greyish-white to semi-translucent cream-like color profile. Crucial Identification Metric: The strain displays distinct $\gamma$-hemolysis (absolute non-hemolytic profile) on sheep blood matrices.

• Aerobic Exposure Purity Test (Critical Experimental Control):To confirm that the anaerobic workspace has not been compromised by contamination with ubiquitous facultative or aerobic organisms (e.g.,Escherichia coli or Staphylococcus species),investigators must execute a parallel aerobic control run during every passaging cycle. Pick an identical aliquot of the active culture and streak it onto two independent blood agar plates: place one inside the anaerobic chamber and incubate the other inside a standard aerobic atmospheric incubator at 37 °C. If any colony formation emerges on the aerobic plate after 48 hours, the entire culture line must be classified as contaminated and discarded immediately.

4. Long-Term Cryopreservation Matrix Parameters

• Cryoprotectant Protective Matrix Formula: Pre-reduced BHI broth supplemented with 30% v/v analytical-grade sterile Glycerol, or a highly protective storage matrix comprising 10% w/v skim milk blended with 15% v/v glycerol.

• Ultra-Low Temperature Freezing Execution:

  1. Inside the anaerobic workspace, harvest high-density vegetative cultures from active, 24-hour log-phase liquid media.

  2. Blend the bacterial fluid in a 1:1 ratio with the pre-sterilized, pre-reduced 50% glycerol stock solution, ensuring a final cryoprotectant concentration of approximately 25% glycerol.

  3. Transfer the mixture into sterile cryovials, tightening the caps completely to maintain an airtight seal.

  4. Move the cryovials out of the chamber and place them immediately into an ultra-low freezer calibrated to -80 °C, or submerge them directly within a liquid nitrogen storage tank (-196 °C) for long-term preservation.Parabacteroides merdae retains high viability under these cryopreservation conditions for several years. To avoid loss of viability, avoid repeated freeze-thaw cycles; thaw each vial only once for direct experimental activation.

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