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Immunologia e infezioni

伯氏疏螺旋体复合体和回归热疏螺旋体的栽培方法

Published: November 25, 2022
doi:
10.3791/64431
, , , , , ,
1Department of Biology,Mt. Allison University, 2Department of Molecular Biology,Umeå University, 3Biology Centre Czech Academy of Sciences,Institute of Parasitology

Summary

体外培养是检测活细菌存在的直接方法。该协议描述了各种螺旋疏螺旋体的培养方法，包括伯氏疏螺旋体复合体，回归热疏螺旋体属和宫本疏螺旋体的培养方法。 这些物种挑剔且生长缓慢，但可以养殖。

Abstract

疏螺旋体由三组物种组成，莱姆疏螺旋体病（LB）组，也称为B. burgdorferi sensu lato（s.l.），最近重新分类为疏螺旋体，回归热（RF）组Borrelia和第三组爬行动物相关的螺旋体。基于培养的方法仍然是实验室检测研究和临床工作中细菌感染的金标准，因为从体液或组织中培养病原体可直接检测复制的病原体并为研究提供源材料。螺旋疏螺旋体和螺旋疏螺旋体挑剔且生长缓慢，因此通常不用于临床目的培养;然而，文化对于研究是必要的。该协议展示了成功培养LB和RF螺旋体所需的方法和配方，包括来自伯氏螺旋体的所有公认物种，包括B. afzelii，B. americana，B. andersonii，B. bavariensis，B. bissettii/bissettiae，B. burgdorferi sensu stricto （s.s.），加利福尼亚双歧杆菌，卡罗利宁双歧杆菌，智利双歧杆菌，芬兰芽孢杆菌，加里尼双歧杆菌，日本双歧杆菌，库尔滕巴奇双歧杆菌，双歧杆菌， 卢西塔尼双歧杆菌、马里蒂玛双歧杆菌、蛋黄双歧杆菌、斯皮尔曼双歧杆菌、狸猫双歧杆菌、图尔迪双歧杆菌、中华双歧杆菌、瓦莱西亚双歧杆菌、长江双歧杆菌和射频螺旋体、安氏双歧杆菌、苜蓿芽孢杆菌、苜蓿科、鳄鱼双歧杆菌、杜托尼双歧杆菌、西班牙双歧杆菌、波斯双歧杆菌、复发双歧杆菌和宫本双歧杆菌。生长LB和RF螺旋体的基本培养基是Barbour-Stoenner-Kelly（BSK-II或BSK-H）培养基，它可靠地支持螺旋体在已建立的培养物中的生长。为了能够从蜱或宿主来源的样品中培养出新分离的疏螺旋体分离株，其中接种物中的初始螺旋体数量较低，首选改良的Kelly-Pettenkofer（MKP）培养基。这种培养基也支持宫本双歧杆菌的生长。射频螺旋体的培养成功也主要取决于成分的质量。

Introduction

疏螺旋体是螺旋体细菌的一个属，包括三个主要分支：莱姆疏螺旋体病（LB）组，回归热（RF）组，以及似乎仅限于爬行动物的特征不太好的组。随着允许基因组和蛋白质组比较的分子方法的出现，疏螺旋体分类法也在不断变化，就像大多数其他分类组1，2，3，4，5，6，7一样。LB组（也称为莱姆病组）传统上被称为伯氏疏螺旋体（Borrelia burgdorferi sensu lato），以其特征最好的成员伯氏疏螺旋体（Borrelia burgdorferi sensu stricto）命名。本文使用目前使用最广泛的术语：LB、RF 和爬行动物相关组，并描述了 LB 和 RF 组的培养协议。

正如螺旋体科成员所预期的那样，疏螺旋体可以采用独特的长而薄的螺旋形状，通常长20-30μm，宽0.2-0.3μm。然而，疏螺旋体细胞具有高度多形性，由于其复杂的细胞和遗传结构，可以在培养物和体内1，8中采用许多其他形状。在其螺旋体形式中，平面正弦波形态是由其轴向内鞭毛在内膜和外膜之间的周质空间中旋转引起的。这种结构使细胞具有高度运动性，外膜含有蛋白质，使细胞能够与宿主组织相互作用9，10。外膜蛋白的表达受到严格调控，不仅影响宿主组织侵袭，还影响与宿主免疫系统的相互作用11。这种复杂的基因表达使疏螺旋体细胞能够在脊椎动物宿主和无脊椎动物载体的不同环境之间穿梭。疏螺旋体的基因组在原核生物中是不寻常的，由线性而不是圆形染色体组成。除线性染色体外，疏螺旋体物种还含有7-21个质粒，有些是线性的，有些是圆形的。质粒含有宿主适应和毒力所需的大部分基因，来自噬菌体的环状质粒被认为是螺旋体细胞之间大部分水平基因流动的原因12，13。与宿主适应中的作用一致，莱姆疏螺旋体病组的一些（可能许多或全部）成员在培养物中丢失质粒14。研究得最好的“实验室适应”伯氏疏螺旋体B. burgdorferi菌株B31在该物种的野生分离株中发现的9个质粒中只有7个15。同样，加里尼双歧杆菌在培养物中丢失质粒16。一些研究表明，RF物种和宫本双歧杆菌在培养时保留质粒14，17，但最近的研究表明，长期体外培养18改变了质粒和感染性。

基于培养的方法仍然是实验室检测细菌感染的金标准，无论是研究和临床工作14，17。从体液或组织中培养病原体可直接检测复制病原体，并为研究提供源材料14，17。该协议展示了成功培养LB组螺旋体以及RF疏螺旋体和宫本芽孢杆菌所需的方法和配方。生长螺旋体疏螺旋体的基本培养基是Barbour-Stoenner-Kelly培养基（BSK-II或市售的BSK-H），有或没有抗生素以减少污染原核生物的生长。该介质改编自最初用于支持RF Borrelia19的介质，由Stoenner20和Barbour21进一步修改。此后开发了许多修饰，每种修饰都对细菌生理学产生影响，可影响生长，感染性和致病性22。该培养基可靠地支持已建立培养物中螺旋体的生长，并已用于从蜱、哺乳动物和临床样品中分离螺旋体23。最近开发的变体，改良的Kelly-Pettenkofer（MKP）培养基，当从环境样品中分离新的疏螺旋体分离物时，当可用于播种培养物的样品中存在的螺旋体数量低时，可以提供更好的分离成功率，形态和运动性23，24。在所有情况下，种植的成功都主要取决于新鲜制备的培养基和适当成分的使用;并非所有商业成分都能生产高质量的培养基。接种的培养物可以方便地在传统的32-34°C培养箱中在少量残留环境氧气存在的情况下孵育，而无需摇动。螺旋疏螺旋体是厌氧菌，但在自然界中暴露于氧气和二氧化碳浓度的波动中，并对基因表达的变化做出反应26，27，28，29。因此，基因表达、生长和其他代谢研究应使用氧控培养箱或厌氧室来控制氧气和二氧化碳水平。在培养中，每周或更频繁地使用暗场显微镜或相差显微镜检查培养物是否存在螺旋体。培养涂片可以用银染、免疫组化或通过使用荧光标记菌株29，30 染色。PCR和DNA测序是一种灵敏和特异性的方法，用于检测和遗传鉴定或确认疏螺旋体物种30，31，32，33。

BSK-II存在许多小的变体，有些是商业上可获得的。第1节中描述的协议改编自Barbour（1984）21。液体MKP培养基是最近开发的培养基，在第2节中描述。它是根据先前报道的方案33，34制备的，该方案与BSK培养基类似，由两个步骤组成：碱性培养基的制备和完整培养基的制备。疏螺旋体培养基可以制备有或没有抗生素，如第3节所述;抗生素的作用是减少接种临床或环境样本时引入的污染细菌，如第4节所述;如果接种纯疏螺旋体培养物，则可能不需要抗生素。制作长期疏螺旋体库存通常很重要，第 5 节描述了这样做的方案。第6节描述了使用这些培养基从临床或环境样品中分离纯感性疏螺旋体克隆。有许多可能的方法36;下面是一个发现有效的。本协议中使用的电镀介质是BSK-II电镀介质37和MKP培养基34的修改（兔血清增加到10%38）。

Protocol

所有涉及从人类受试者获得的样本的研究均已获得相关大学和/或医疗机构的机构审查委员会的批准，并在收集样本之前获得参与者的书面知情同意。所有涉及从动物身上获得的样本的研究均在机构动物伦理委员会的指导下获得批准和进行。在相关情况下，已获得环境取样的批准。 注意：培养的成功在很大程度上取决于成分的质量。商业BSK培养基可用，可有力地支持实验室适?…

Representative Results

疏螺旋体 培养基BSK和MKP及其变体是丰富的培养基，其成分需要按顺序制备和灭菌。正确制备后，BSK培养基应为红橙色和透明（图1）。变暖后持续存在的浊度和沉淀表明成分有问题、培养基生产或污染;这种介质最好丢弃。如果将明胶添加到BSK和MKP中，则冷藏时培养基会呈凝胶状;加热后，它将是液体，尽管略带粘性。 纯 疏螺旋体 培养物的?…

Discussion

细菌的实验室培养是研究的跳板。培养能力所赋予的深远优势体现在一个多世纪以来，直到最近才取得成功的梅毒螺旋体（梅毒螺旋体）的斗争中，44.螺旋疏螺旋体也对培养具有挑战性，但培养是可能的23，24，34，44，45，46，47，48<…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了加拿大自然科学和工程研究理事会和加拿大莱姆病基金会（AB和VL），瑞典研究理事会（SB，M-LF 和IN），TAČR GAMA 2项目-“支持生物中心CAS的应用潜力验证2.0”（TP01010022）（MG和NR）的部分支持，以及捷克共和国卫生部NV19-05-00191（MG和NR）的赠款。我们感谢 S. Vranjes（2021 年，鲁登科实验室）提供图 4 中的图像，感谢 J. Thomas-Ebbett（2021 年，劳埃德实验室）提供图 5 中的图像。我们感谢所有为该领域做出贡献的研究人员，并向那些由于篇幅限制而无法引用其工作的人道歉。

Materials

1.7 mL tubes VWR 87003-294 Standard item – any supplier will do
0.2 µm Sterile syringe filter  VWR 28145-501 Standard item – any supplier will do
10 µL barrier pipette tip Neptune BT10XLS3 Standard item – any supplier will do
10 mL Serological pipettes  Celltreat 229011B Standard item – any supplier will do
1000 µL barrier pipette tip Neptune BT1000.96 Standard item – any supplier will do
15 mL tube Celltreat 188261 Standard item – any supplier will do
20 µL barrier pipette tip Neptune BT20 Standard item – any supplier will do
20 mL Sterile syringe  BD 309661 Standard item – any supplier will do
200 µL barrier pipette tip Neptune BT200 Standard item – any supplier will do
25 mL Screw Cap Culture Tubes Fisher Scientific 14-933C Standard item – any supplier will do
25 mL Serological pipettes Celltreat 229025B Standard item – any supplier will do
3 mL Sterile syringe BD 309657 Standard item – any supplier will do
35% BSA  Sigma A-7409 Source is important – see note
5 mL Serological pipettes  Celltreat 229006B Standard item – any supplier will do
50 mL tube Celltreat 229421 Standard item – any supplier will do
6.5 ml MKP glass tubes  Schott Schott Nr. 26 135 115 Standard item – any supplier will do
Amikacine Sigma PHR1654 Standard item – any supplier will do
Amphotericin B Sigma A9528-100MG Standard item – any supplier will do
Bactrim/rimethoprim/sulfamethoxazole Sigma PHR1126-1G Standard item – any supplier will do
BBL Brucella broth  BD 211088 Standard item – any supplier will do
Biosafety Cabinet Labconco 302419100 Standard item – any supplier will do
Blood collection tubes (yellow top – ACD) Fisher Scientific BD Vacutainer Glass Blood Collection Tubes with Acid Citrate Dextrose (ACD) Standard item – any supplier will do
BSK-H Medium [w 6% Rabbit serum]  Darlynn biologicals BB83-500 Standard item – any supplier will do
centrifuge  Eppendorf model 5430 Standard item – any supplier will do
Citric acid TrisodiumSaltDihydrate Sigma C-8532 100 g Standard item – any supplier will do
CMRL Gibco BRL 21540 500 mL Standard item – any supplier will do
CMRL-1066 Gibco 21-510-018 Standard item – any supplier will do
Cryogenic Tubes (Nalgene) Fisher Scientific 5000-0020 Standard item – any supplier will do
Deep Petri with stacking ring 100 mm × 25 mm Sigma P7741 Standard item – any supplier will do
Digital Incubator VWR model 1545 Standard item – any supplier will do
DMSO ThermoFisher D12345 Standard item – any supplier will do
Filters for filter sterilization Millipore 0.22μm GPExpressPLUS Membrane SCGPU05RE Standard item – any supplier will do
Gelatin Difco BD 214340 500 g Standard item – any supplier will do
Glass Culture Tubes Fisher Scientific 99449-20 Standard item – any supplier will do
Glucose Sigma G-7021 1 kg Standard item – any supplier will do
Glycerol Sigma G5516 Standard item – any supplier will do
Hemafuge (Hematocrit & Immuno hematology centrifuge ) Labwissen Model 3220 Standard item – any supplier will do
HEPES Sigma  H-3784 100 g Standard item – any supplier will do
N-acetylglucoseamine Sigma  A-3286 25 g Standard item – any supplier will do
Neopeptone Difco  BD 211681 500 g Standard item – any supplier will do
Neubauer Hematocytometer Sigma  Z359629 Standard item – any supplier will do
Phase contrast microscope  Leitz Standard item – any supplier will do
Phosphomycin Sigma P5396-1G Standard item – any supplier will do
Phosphomycine Sigma P5396 Standard item – any supplier will do
Pipetboy Integra Standard item – any supplier will do
Precision Standard Balance OHAUS model TS200S Standard item – any supplier will do
Pyruvic acid (Na salt) Sigma P-8574 25 g Standard item – any supplier will do
Rabbit Serum  Gibco 16-120-032 Source is important 
Rabbit Serum  Sigma R-4505  100 mL Source is important 
Rifampicin Sigma R3501-1G Standard item – any supplier will do
Sodium bicarbonate Sigma S-5761     500 g Standard item – any supplier will do
Sufametaxazole  Sigma PHR1126 Standard item – any supplier will do
TC Yeastolate Difco  BD 255752 100 g Standard item – any supplier will do
Transfer Pipettes VWR 470225-044 Standard item – any supplier will do
Trimethoprim Sigma PHR1056 Standard item – any supplier will do
DOWNLOAD MATERIALS LIST

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SpirochetesBorrelia BurgdorferiRelapsing Fever BorreliaBorrelia MiyamotoiCultureBSK-II MediumBacterial GrowthRabbit SerumGelatinAnaerobic AtmosphereIncubation Temperature

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Berthold, A., Faucillion, M., Nilsson, I., Golovchenko, M., Lloyd, V., Bergström, S., Rudenko, N. Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia. J. Vis. Exp. (189), e64431, doi:10.3791/64431 (2022).
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