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.