B组的小鼠模型<em>链球菌</em>阴道定植
Summary
此协议的目的是模仿人类的B组链球菌 （GBS）阴道定植在小鼠模型。此方法可用于研究宿主的免疫反应，并促进GBS阴道持久细菌因素，以及测试的治疗策略。
Abstract
无乳链球菌 （B组链球菌 ，GBS），是人体胃肠道和10个阴道的革兰氏阳性，无症状殖民者-成人的30％。在免疫受损的个体，包括新生儿，孕妇和老年人，GBS可以切换到病原体侵入引起败血症，关节炎，肺炎和脑膜炎。由于GBS是新生儿的领先细菌病原体，目前预防由妊娠后期筛查GBS阴道定植和GBS阳性母亲的后续围产期抗生素治疗。重GBS阴道负担为两个新生儿疾病和定植的危险因素。不幸的是，鲜为人知的是，主机和促进或允许GBS定植阴道细菌因素。这个协议描述了一种技术，使用单β雌二醇预处理和每日取样，以确定细菌洛阿建立持久的GBS阴道定植ð。它进一步详细信息的方法来管理其它疗法或感兴趣的试剂和收集阴道灌洗液和生殖道组织。这种小鼠模型将进一步阴道环境中的GBS宿主相互作用，这将导致潜在的治疗靶点怀孕期间母体控制阴道定植和避免传染给脆弱的新生儿的理解。它也将是感兴趣的，以增加在女性阴道我们的一般细菌 – 宿主相互作用的理解。
Introduction
无乳链球菌 ，B组链球菌 （GBS），是一个封装的，革兰氏阳性细菌，其经常从肠道和健康成人的泌尿生殖道中分离。在20世纪70年代，GBS成为感染新生儿死亡率的主要代理，拥有超过7000例新生儿疾病每年1。早发性GBS病（EOD）发生在第一个小时内或生命天，出现肺炎或呼吸窘迫，常发展成败血症，而晚发性疾病（LOD）几个月后随之而来，并与菌血症，频繁地呈现进入脑膜炎2。截至2002年，该中心疾病控制和预防建议在妊娠后期和分娩期预防性使用抗生素（IAP），以GBS阳性母亲1 GBS定植阴道普遍筛查。尽管早发性疾病的减少到大约1000例，美国每年因IAP，GBS仍然是早发性新生儿败血症的主要原因，迟发发生不受影响1。无论是在宫内，分娩过程中，甚至迟发性的情况下，新生儿暴露于GBS需要生存，横向通过大量的主机环境和障碍，免疫逃避，并在脑膜炎，高度管制的血 – 的交接的情况下，脑屏障2。新生儿中的这些有毒相互作用的上游是产妇阴道的初始定植。产妇阴道GBS定植率从8-18％，在发达国家和发展中国家，12.7％，3,4估计的平均增长率。怀孕期间阴道内的定植GBS可能是恒定的，间歇性的，或短暂在个别妇女中5性质。有趣的是，产妇年龄>36年与持续定植6有关。为GBS定植阴道大量生物和社会经济风险因素已经确定。生物因素包括胃肠道GBS殖民化和缺乏肠道内乳酸菌 。然而，种族，肥胖，卫生和性活动也与GBS阴道托架7相关联。
虽然臭名昭著，导致新生儿感染，GBS也导致了各种感染的孕产妇围产期都和产后的。 GBS托架在女性阴道炎8呈递增加，并且在某些情况下，甚至可能是疾病实体9。此外，孕期生殖道GBS的提升可能会导致羊膜腔内感染或绒毛膜羊膜炎10。此外，在高达怀孕3.5％，GBS传播到膀胱引起尿路感染或无症状菌11。怀孕期间GBS菌与产热，绒毛膜羊膜炎，早产，和prematur的风险增加有关膜12电子破裂。两者合计，GBS的阴道内的存在被链接到多个宿主组织的感染，并从这个利基消除GBS的能力是必要为孕产妇和新生儿的健康。
直到最近，大多数的工作检查与宫颈道GBS相互作用被限制为体外细胞模型13-15。 这些体外实验已表明，对于粘附重要细菌因素，包括表面蛋白例如一个菌毛和富含丝氨酸重复17,18，以及双组分调控系统15,19和阴道上皮的对全球转录反应GBS 19。然而，为了充分阐明阴道内的宿主 – 微生物相互作用，一个强大的动物模型是必要的。早期的工作表明，GBS可以从接种小鼠20,21和大鼠的阴道内被恢复<SUP>在两个怀孕和未怀孕的条件22。 2005年，短期GBS定植阴道小鼠为蓝本，研究噬菌体裂解酶的功效在24小时的时间内23治疗阴道GBS。事隔多年，长期阴道GBS定植小鼠模型的开发研究规范GBS持久主机和细菌因素。这种模式已经确定促进殖民GBS众多因素，包括地面附属物17,18和GBS双组分系统19,24。这种模式对宿主反应机制19,25的识别作出了贡献，是用来测试几种治疗策略，包括免疫调节肽26和益生菌27。该协议使接种GBS入小鼠阴道并随后跟踪殖民化和收集样品用于进一步分析的必要的指导。
Protocol
所有的动物工作是经实验室动物护理在州立圣迭戈大学办公室和下接受兽医标准进行。雌性小鼠，年龄8 – 16周，用于该方法的发展。 1.β雌二醇的制备和腹腔注射测量出的称量纸而穿着适当的个人防护装备（PPE）β雌二醇（0.5毫克/鼠标）。 注意：β雌二醇可通过皮肤和粘膜表面吸收。 转移β雌二醇到15毫升锥形管中，涡旋，直到所有的团块被去除和β雌二醇是细粉…
Representative Results
在这种模式的发展，多个观测结果就影响GBS定植阴道的时间因素使得。为了确定在接种的影响GBS细菌的持久性如何动情期，小鼠上演通过阴道灌洗液接种的一天。 图1示出的鼠标发情周期的四个阶段，由以下方法测定湿贴装阴道灌洗液，行之有效的方法29。小鼠分为基于该初始阶段组，并且经由阴道拭子随时间监测GBS持久性。在动情前期阶段接种的小?…
Discussion
进一步的与主机的环境内的主机和其他微生物都GBS相互作用的认识的提高，需要的动物模型。这部作品描述了在小鼠建立阴道GBS定植的技术方面。该协议实现小鼠> 90％定植不使用麻醉剂的接种细菌或收集拭子样品，免疫抑制剂，使定植，阴道预清洗，或添加剂增稠接种物。此外，该模型表明健壮再现性，具有适度的间实验变异GBS持久的长度和细菌负担。在这项研究中证明了代表性结果是独立的…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
We would like to thank the vivarium manager and staff at San Diego State University for support with animal husbandry. During this work, K.A.P. was supported by an ARCS scholarship and a fellowship from the Inamori Foundation. K.S.D. is supported by an R01 grant, NS051247, from the National Institutes of Health.
Materials
| Sesame oil | Sigma Aldrich | S3547-250ML | |
| β-Estradiol | Sigma Aldrich | E8875-1G | CAUTION: Wear appropriate PPE. β-estradiol can be absorbed through the skin and mucosal surfaces. |
| 200 μL gel loading pipette tips | USA Scientific | 1252-0610 | |
| Urethro-genital, sterile, calcium alginate swabs | Puritan | 25-801 A 50 | |
| CHROMagar StrepB | DRG International | SB282 | |
| Todd Hewitt Broth | Hardy Diagnostics | 7161C | |
| 18 G, 1.5 inch needles | BD | 305199 | |
| 26 G, 0.5 inch needles | BD | 305111 | |
| 10 mL syringes | BD | 309604 | |
| 1 mL syringes | BD | 309659 | |
| 0.45 μm PVDF syringe filters | Whatman | 6900-2504 | |
| Dulbecco's Phosphate-Buffered Salt Solution 1X | Corning | 21-031-CV |
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Citar este artículo
Patras, K. A., Doran, K. S. A Murine Model of Group B Streptococcus Vaginal Colonization. J. Vis. Exp. (117), e54708, doi:10.3791/54708 (2016).