Direct Isolation of Treponema pallidum from Syphilis Patients

梅毒的病原体梅毒螺旋体梅毒亚种梅毒螺旋体的研究因无法在体外培养螺旋体而受到多年的阻碍。这一限制也阻碍了隔离新的 梅毒螺旋体的研究结果是，大多数研究菌株都是在上个世纪分离出来的，并已在兔体内繁殖多年。2018年，我们开发了微氧条件下梅毒螺旋体与Sf1Ep兔上皮细胞共培养的体外培养系统，实现了梅毒螺旋体的连续体外培养。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持了完全的毒力。在这里，我们建议使用这个新开发的体外系统来直接从梅毒患者的临床样本中分离出新的梅毒螺旋体菌株。在目标1中，我们将尝试从患者身上分离出新的梅毒螺旋体菌株。最初的实验将集中在收集样本和保持梅毒螺旋体在从临床现场运输过程中的最佳方法上。在确定了这些参数后，我们将继续从患者皮损和疑似先天性梅毒婴儿的胎盘和脐带标本中分离弓形虫/伊杜姆。我们的临床合作调查员将协调患者样本的收集，我们将使用体外培养系统在体外扩大这些样本。在目标2中，将分析新的患者来源的梅毒螺旋体菌株在兔模型中的体外生长速度和感染特性(例如，体内生长速度、传播、组织趋向性和组织病理学)的差异。还将使用成熟的体外培养程序进行抗菌药敏感性检测。最后，将进行基因组测序，以便能够分析分离菌株的序列，以寻找与其生理和感染特性有关的差异，特别是考虑到它们与Nichols和SS14基因簇分布以及阿奇霉素敏感性的持续变化的关系。预计这一项目将大大提高梅毒螺旋体分离的效率，相对于兔接种，从而允许更彻底地表征导致这种重要的性传播感染的菌株。分析现代梅毒菌株中存在的变异性可以深入了解社区感染模式、患者结局和社区中抗菌素耐药性的发展。此外，该项目的成功完成还将建立一个可供研究的梅毒菌株库，大大增加可用于这一重要人类病原体的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.