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.

梅毒的致病药物（T. pallidum）的毛颗粒亚种（T. pallidum）的研究受到了多年的影响多年。这种限制也阻碍了新的隔离 T. pallidum菌株的结果是，大多数研究菌株在上个世纪被隔离，并且在兔子中已经繁殖了很多年。在2018年，我们开发了一种体外培养系统，涉及pallidum t. pallidum与SF1EP兔上皮细胞的共培养，在微恐惧条件下，导致了pallidum的体外培养。目前，我们已经在体外维持了三年多的文化，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，将新型的T. pallidum菌株直接从源自梅毒患者的临床样本中分离出来。在AIM 1中，我们将尝试将新的T. pallidum菌株与患者分离。最初的实验将集中在收集样品并维持从临床部位运输过程中的最佳方法。在定义了这些参数之后，我们将从患者病变中分离出T. PAL/IDUM，以及胎盘和脐带标本的胎盘和脐带标本。我们的临床共同评估者将协调患者样品的收集，我们将使用体外培养系统在体外扩展这些样品。在AIM 2中，将分析新的源自患者的粒子菌株，以分析兔模型中体外生长速率和感染特性的差异（例如，体内生长速率，传播速率，组织疗法和组织病理学）。还将使用良好发达的体外培养程序检查抗菌敏感性。最后，将进行基因组测序，以允许分析与其生理和感染特性相关的差异序列，尤其是鉴于它们与Nichols和nichols和SS14遗传簇的持续变化以及Azithromycin敏感性的持续变化。可以预料，该项目将大大提高与兔接种相对于兔子接种的T. pallidum隔离的效率，从而允许对菌株的更彻底的特征，从而导致这种重要的性传播感染。现代梅毒菌株中存在的可变性的分析可能会导致人们对社区感染模式，患者结局以及社区中抗菌素耐药性的发展。此外，该项目的成功完成还将允许创建一个易于使用的梅毒菌株来进行研究，从而大大增加了可用于对这种重要人类病原体的生理和遗传分析的菌株数量。