Direct Isolation of Treponema pallidum from Syphilis Patients

梅毒螺旋体（Treponema pallidum subspecies pallidum，T.梅毒的病原体-梅毒螺旋体（pallidum），由于不能在体外培养螺旋体，多年来一直受到阻碍。这一限制也阻碍了新的隔离 T.结果，大多数研究菌株是在上个世纪分离的，并且已经在兔子中繁殖多年。2018年，我们开发了一种涉及T. pallidum与Sf 1 Ep兔上皮细胞在微需氧条件下的体外培养，导致T.苍白球目前，我们已经在体外培养了三年多，这些培养物在兔模型中保留了完整的毒力。在这里，我们建议使用这种新开发的体外系统，以分离新菌株的T。直接从梅毒患者的临床样品中分离梅毒螺旋体。目的1：尝试分离新的T.患者的苍白球。最初的实验将集中在收集样本和维持T。苍白球活力从临床站点运输过程中。在定义了这些参数之后，我们将继续分离T。pal/p我们的临床合作研究者将协调患者样本的采集，我们将使用体外培养系统在体外扩增这些样本。在目标2中，新的患者来源的T.将分析苍白球菌株在兔模型中的体外生长速率和感染特性（例如，体内生长速率、传播、组织嗜性和组织病理学）的差异。还将使用成熟的体外培养程序检查抗菌药物敏感性。最后，将进行基因组测序，以允许分析分离菌株的序列，以确定与其生理和感染特性相关的差异，特别是考虑到其与Nichols和SS 14遗传簇分布和阿奇霉素敏感性的持续变化的关系。预计该项目将大大提高T。苍白球分离相对于兔接种，从而允许更彻底的表征菌株引起这一重要的性传播感染。对现代梅毒菌株中存在的变异性进行分析，可以深入了解社区感染模式、患者结局以及社区中抗生素耐药性的发展。此外，该项目的成功完成还将有助于建立一个随时可用的梅毒菌株研究库，大大增加可用于对这一重要人类病原体进行生理和遗传分析的菌株数量。

Research on Treponema pallidum subspecies pallidum (T. pallidum), the causative agent of syphilis, was impeded for years by inability to cultivate the spirochete in vitro. This limitation also hampered isolation of new T. pallidum strains with the result that most research strains were isolated in the last century and have been propagated in rabbits for many years. In 2018, we developed an in vitro culture system involving co-culture of T. pallidum with Sf1Ep rabbit epithelial cells under microaerobic conditions that has resulted in continuous in vitro cultivation of T. pallidum. Currently, we have maintained cultures in vitro for over three years, and these cultures have retained full virulence in the rabbit model. Here, we propose to use this newly developed in vitro system to isolate novel strains of T. pallidum directly from clinical samples derived from syphilis patients. In the Aim 1, we will attempt to isolate new strains of T. pallidum from patients. Initial experiments will focus on the best approaches to collect samples and maintain T. pallidum viability during transport from clinical sites. After definition of these parameters, we will proceed with isolation of T. pal/idum from patient lesions and from placenta and umbilical cord specimens from infants with suspected congenital syphilis. Our clinical co-investigators will coordinate collection of patient samples and we will use the in vitro culture system to expand those samples in vitro. In Aim 2, the new patient-derived T. pallidum strains will be analyzed for differences in in vitro growth rates and infection properties in the rabbit model (e.g. in vivo growth rates, dissemination, tissue tropism, and histopathology). Antimicrobial susceptibility will also be examined using a well-developed in vitro culture procedure. Lastly, genomic sequencing will be performed to permit analysis of the sequences of the isolated strains for differences related to their physiologic and infection properties, particularly in view of their relationship to the ongoing changes in the distribution of the Nichols and SS14 genetic clusters and of azithromycin susceptibility. It is anticipated that this project will lead to greatly increased efficiency of T. pallidum isolation relative to rabbit inoculation, thereby permitting a much more thorough characterization of strains causing this important sexually transmitted infection. Analyses of variability present in modern syphilis strains can lead to insight into community infection patterns, patient outcomes, and the development of antimicrobial resistance in the community. In addition, successful completion of this project will also allow for the creation of a readily available bank of syphilis strains for research, greatly increasing the number of strains that are available for the physiologic and genetic analysis of this important human pathogen.