Direct Isolation of Treponema pallidum from Syphilis Patients

从梅毒患者中直接分离梅毒螺旋体

• 批准号: 10652662
• 负责人: Diane G Edmondson
• 金额: $ 19.5万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-23 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10652662
• 关键词: Adult; Aerobic; Antimicrobial Resistance; Antimicrobial susceptibility; Azithromycin; Bacteria; Biological Availability; Biology; Blood; Body Fluids; Characteristics; Clinical; Coculture Techniques; Collection; Communities; Congenital Syphilis; Development; Disease; Epithelial Cells; Gene Expression Profile; Genetic; Genomics; Goals; Growth; Histopathology; In Vitro; Infant; Infection; Laboratory culture; Latent Syphilis; Length; Lesion; Methods; Modeling; Modernization; Morphology; Order Spirochaetales; Organism; Oryctolagus cuniculus; Pathogenesis; Patient-Focused Outcomes; Patients; Pattern; Phenotype; Physiological; Placenta; Predisposition; Primary Lesion; Property; Research; Research Personnel; Sampling; Secondary Lesion; Sequence Analysis; Sexually Transmitted Diseases; Specimen; Syphilis; Syphilitic chancre; System; Temperature; Testing; Testis; Tissues; Treponema pallidum; Treponema pallidum subspecies pallidum; Tube; Umbilical cord structure; Variant; Virulence; antimicrobial drug; clinical research site; experimental study; genetic analysis; human pathogen; in vivo; insight; novel; skin lesion; tissue tropism

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.

由于无法在体外培养螺旋体，对梅毒病原体梅毒螺旋体亚种（T. pallidum）的研究多年来一直受到阻碍。这种限制也阻碍了新产品的隔离 梅毒螺旋体菌株的结果是大多数研究菌株是在上个世纪分离出来的，并且已经在兔子中繁殖了很多年。 2018年，我们开发了一种体外培养系统，将梅毒螺旋体与Sf1Ep兔上皮细胞在微氧条件下共培养，实现了梅毒螺旋体的连续体外培养。目前，我们已在体外维持培养物三年多，这些培养物在兔子模型中保留了完全的毒力。在这里，我们建议使用这种新开发的体外系统直接从梅毒患者的临床样本中分离出梅毒螺旋体的新菌株。在目标 1 中，我们将尝试从患者体内分离出新的梅毒螺旋体菌株。初步实验将重点关注收集样本并在从临床地点运输过程中保持梅毒螺旋体活力的最佳方法。定义这些参数后，我们将继续从患者病灶以及疑似先天性梅毒婴儿的胎盘和脐带标本中分离出 T. pal/idum。我们的临床联合研究人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展这些样本。在目标 2 中，将分析源自患者的新梅毒螺旋体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、组织向性和组织病理学）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，以分析分离菌株的序列，以了解与其生理和感染特性相关的差异，特别是考虑到它们与 Nichols 和 SS14 基因簇以及阿奇霉素分布的持续变化的关系易感性。预计该项目将大大提高相对于兔子接种而言的梅毒螺旋体分离效率，从而可以对引起这种重要性传播感染的菌株进行更彻底的表征。对现代梅毒菌株中存在的变异性的分析可以深入了解社区感染模式、患者结果以及社区抗菌素耐药性的发展。此外，该项目的成功完成还将有助于创建一个可供研究的现成梅毒菌株库，大大增加可用于这种重要人类病原体的生理和遗传分析的菌株数量。