BioVector? Parabacteroides merdae Type Strain / 糞副擬桿菌標準菌株

一 產(chǎn)品基本信息與微生物學(xué)背景

• 菌株名稱：Parabacteroides merdae（中文學(xué)術(shù)名：糞副擬桿菌）。

• 經(jīng)典分類標桿菌株：ATCC 43184 / DSM 19495 / JCM 14815 / CCUG 38531（原分類法中曾被命名為 Bacteroides merdae 糞擬桿菌，后經(jīng) 16S rRNA 基因組重分類修正獨立為副擬桿菌屬）。

• 菌株物種來源：最初自健康人類的糞便（Feces）組織中分離獲得，是人類腸道核心核心核心核心共生菌群（Gut microbiota）的核心成員。

• 生物學(xué)表型與超微特征：

  • 形態(tài)與染色：革蘭氏陰性（Gram-negative）。在顯微鏡下表現(xiàn)為細小的專性短桿狀（Short rods）或球桿狀（Coccobacilli），大小約為 $0.5 - 0.8 \times 1.0 - 2.0\ \mu\text{m}$。無芽孢，通常不具運動性，細胞表面通常包裹有一層微型的多糖莢膜。

  • 氣體代謝特性：嚴格厭氧（Strictly anaerobic）。接觸氧氣會迅速引發(fā)該菌體內(nèi)發(fā)生強烈的氧化應(yīng)激死亡。

  • 生化代謝譜系：具有極強的糖苷水解酶活性，能高效利用和降解人類攝入的復(fù)雜膳食纖維（不溶性多糖），其主要發(fā)酵終產(chǎn)物（Metabolic end-products）為乙酸（Acetate）和琥珀酸（Succinate），基本不產(chǎn)生或僅產(chǎn)生微量丁酸。在生化鑒定中，吲哚（Indole）反應(yīng)通常為陰性，過氧化氫酶（Catalase）多為陽性。

• 生物安全級別：1級（BSL-1）。

二 核心科研價值與腸道微生態(tài)醫(yī)學(xué)轉(zhuǎn)化應(yīng)用

隨著近年來全球?qū)Α澳c道菌群-宿主軸（Gut-Host Axis）”研究的爆發(fā)，Parabacteroides merdae 已從傳統(tǒng)的普通共生菌躍升為合成生物學(xué)與藥物開發(fā)（LBP, 活體生物藥）的明星底盤：

1. 構(gòu)建人類核心腸道仿真模式菌群（Synthetic Communities）：在建立微生態(tài)小鼠模型（如無菌小鼠 Gnotobiotic mice 重新定殖）時，P. merdae 作為擬桿菌目（Bacteroidales）的代表性優(yōu)勢物種，與擬桿菌屬、梭菌屬等混合組裝成簡化的多菌組合（如經(jīng)典選定的 12 菌或 20 菌群），用以解構(gòu)復(fù)雜腸道生態(tài)位（Niche）的競爭、排他以及群落穩(wěn)態(tài)維持機制。

2. 調(diào)控機體代謝性疾?。ǚ逝?、高脂血癥與糖尿?。?/strong>：臨床轉(zhuǎn)錄組與代謝組學(xué)研究表明，P. merdae 的腸道豐度與機體的脂質(zhì)代謝、胰島素敏感性存在強烈的正相關(guān)或負相關(guān)。它通過分泌特定的小分子代謝物，能夠調(diào)控宿主遠端腸道上皮細胞的法尼醇 X 受體（FXR）以及膽汁酸循環(huán)（Bile acid signaling loop），是開發(fā)新型減肥、降脂靶向微生態(tài)制劑的重點篩選對象。

3. 免疫調(diào)節(jié)、腸道屏障保護與腫瘤免疫協(xié)同（Cancer Immunotherapy）：用于研究宿主免疫耐受的誘導(dǎo)。該菌的特殊外膜成分能夠刺激結(jié)腸固有層的樹突狀細胞（DCs），溫和誘導(dǎo)調(diào)節(jié)性 T 細胞（Tregs）的生成，協(xié)助維持腸道黏膜屏障的物理完整性。同時，在前沿腫瘤免疫研究中，其在腸道內(nèi)的特定豐度常被證實能顯著重塑全身免疫微環(huán)境，成倍提升抗 PD-1/PD-L1 等免疫檢查點阻斷療法（ICB）對實體瘤的臨床臨床殺傷敏感性。

三 實驗室厭氧復(fù)蘇、培養(yǎng)、常規(guī)傳代與質(zhì)控標準步驟

極其重要的操作警告：Parabacteroides merdae 屬于嚴格厭氧菌。無論是開封復(fù)蘇、平板涂布還是液體培養(yǎng)，整個操作流程必須在高效厭氧操作系統(tǒng)（如配置有混合氣體的厭氧工作站 Anaerobic Chamber）或嚴格使用厭氧產(chǎn)氣包密封罐（Anaerobic Jar）中進行。暴露在空氣中會直接導(dǎo)致復(fù)蘇失?。?/strong>

1. 專用培養(yǎng)基、氣體配比與環(huán)境物理常數(shù)

• 基礎(chǔ)培養(yǎng)基（推薦以下三種之一）：

  • BHI 腦心浸液培養(yǎng)基（Brain Heart Infusion）：需額外添加 5 $\mu$g/mL 氯化血紅素（Hemin） 和 1 $\mu$g/mL 維生素 K1（Vitamin K1） 效果極佳。

  • PYG 培養(yǎng)基（Peptone Yeast Glucose Medium）：專用于厭氧菌發(fā)酵代謝分析的標準培養(yǎng)基。

  • 哥倫比亞血瓊脂平板（Columbia Blood Agar）：加入 5% 優(yōu)質(zhì)脫纖維綿羊血。

• 標準工作氣體配比：80% 氮氣（$N_2$） + 10% 氫氣（$H_2$） + 10% 二氧化碳（$CO_2$）（氫氣用作工作站內(nèi)催化劑除氧的關(guān)鍵還原劑）。

• 培養(yǎng)物理常數(shù)：標準 37 攝氏度，絕對無氧，遮光暗培養(yǎng)。

2. 安瓿管/凍存管菌種的厭氧復(fù)蘇與啟動步序

1. 提前除氧預(yù)平衡：在轉(zhuǎn)化操作前 24 小時，將配制好的無菌固體平板或液體培養(yǎng)基置于厭氧工作站內(nèi)進行提前預(yù)還原（Pre-reduction），徹底驅(qū)離溶于培養(yǎng)基內(nèi)部的微量游離分子氧。

2. 從液氮罐或 -80 ℃ 冰箱中取出 P. merdae 凍存管，立刻投入 37 ℃ 恒溫水浴中搖晃使其在 1 分鐘內(nèi)急速融化（若為玻璃真空安瓿管，則需按規(guī)范用酒精棉擦拭、劃痕并微力敲開）。

3. 用無菌移液管吸取 0.5 mL 預(yù)還原的 BHI 液體培養(yǎng)基，注入凍存管中，極其輕柔地沖洗、吸打并重懸全部菌體泥沉淀。

4. 雙向接種接種質(zhì)控法（強烈推薦）：

   • 固體通道：吸取 100 $\mu$g 懸液均勻涂布于預(yù)還原的哥倫比亞血瓊脂平板上（用于觀察單菌落形態(tài)及檢測純度）。

   • 液體通道：將剩余的所有重懸菌液全量接入盛有 5 - 10 mL 預(yù)熱 BHI（含 Hemin + Vit K1）的密封厭氧培養(yǎng)管或螺口管底部。

5. 將平板和液體管迅速鎖入?yún)捬豕ぷ髡荆?/span>或放入裝有新鮮厭氧產(chǎn)氣包、冷催化除氧劑以及厭氧指示劑（如美藍指示條，確保指示條呈白色無氧狀態(tài)）的厭氧罐內(nèi)，擰緊閥門，整體移入 37 攝氏度普通培養(yǎng)箱。

6. 嚴格培養(yǎng) 48 - 72 小時。該菌生長相對穩(wěn)健，一般在 48 小時左右可見液體培養(yǎng)基呈現(xiàn)均勻中度渾濁，瓊脂平板上長出飽滿的菌落。

3. 日常傳代、形態(tài)鑒定與純度質(zhì)控

• 傳代時機：液體發(fā)酵管長至對數(shù)生長末期（通常在接種后 24 - 36h，$OD_{600}$ 達到 1.2 - 1.5），或固體平板上的單菌落長至飽滿（直徑約 1 - 2 mm）時進行傳代。傳代時按照 1% - 3% 的體積比例，將菌液轉(zhuǎn)接至新的預(yù)還原液體培養(yǎng)基中。

• 菌落形態(tài)特征（質(zhì)控標桿）：在含有 5% 綿羊血的哥倫比亞瓊脂平板上培養(yǎng) 48h 后，P. merdae 的典型單菌落表現(xiàn)為：圓形、凸起、表面光滑濕潤、邊緣整齊，直徑約 1.0 - 1.5 mm，顏色呈微小的灰白色至半透明乳白色。核心質(zhì)控：該菌在綿羊血平板上表現(xiàn)為 $\gamma$-溶血（無溶血環(huán)，Non-hemolytic）。

• 絕對避氧純度測試（微生態(tài)實驗紅線）：在微生態(tài)研究中，為了防止由于操作不當(dāng)混入外界有氧雜菌（如大腸桿菌或葡萄球菌），必須同時進行有氧對照測試（Aerobic control run）。即在傳代時，挑取同一樣本的菌液，平行涂布于兩塊血平板上：一塊置于無氧環(huán)境培養(yǎng)，另一塊直接置于 37 ℃ 普通有氧空氣孵箱中培養(yǎng)。若 48 小時后，有氧培養(yǎng)的平板上出現(xiàn)任何菌落生長，說明系統(tǒng)已發(fā)生嚴重污染，該批次菌株必須予以徹底淘汰。

4. 菌株長期保存標準

• 凍存液配方：推薦使用高效的厭氧專用凍存液。典型配方為：預(yù)還原的 BHI 液體培養(yǎng)基 混合 30% 滅菌純甘油（Glycerol），或采用重組的防御級配方：脫脂奶粉（10% Skim milk）+ 15% 甘油。

• 超低溫鎖死冷凍規(guī)范：

  1. 在厭氧工作站內(nèi)，收集發(fā)酵旺盛（通常為 24h 強力培養(yǎng)代）的 P. merdae 高密度菌液。

  2. 將菌液與預(yù)先滅菌并除氧的 50% 甘油水溶液按照 1:1 的體積比在厭氧環(huán)境下徹底搖勻，使甘油終濃度維持在 25% 左右。

  3. 分裝入無菌凍存管，擰緊管蓋鎖死氣密性。

  4. 將凍存管移出工作站，直接投入 -80 ℃ 超低溫冰箱中鎖死存放，或者直接丟入液氮罐（-196 ℃）中長期封存。該菌在超低溫甘油懸液中狀態(tài)極度穩(wěn)定，可保存數(shù)年。日常使用時切忌反復(fù)解凍，必須實行單管單次融化使用。

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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