Investigation of Treponema pallidum subsp. pallidum, the spirochete that causes syphilis, has been hindered by an inability to culture the organism continuously in vitro despite more than a century of effort. In this study, long-term logarithmic multiplication of T. pallidum was attained through subculture every 6 to 7 days and periodic feeding using a modified medium (T. pallidum culture medium 2 [TpCM-2]) with a previously described microaerobic, rabbit epithelial cell coincubation system. Currently, cultures have maintained continuous growth for over 6 months with full retention of viability as measured by motility and rabbit infectivity. This system has been applied successfully to the well-studied Nichols strain of T. pallidum, as well as to two recent syphilis isolates, UW231B and UW249B. Light microscopy and cryo-electron microscopy showed that in vitro-cultured T. pallidum retains wild-type morphology. Further refinement of this long-term subculture system is expected to facilitate study of the physiological, genetic, pathological, immunologic, and antimicrobial susceptibility properties of T. pallidum subsp. pallidum and closely related pathogenic Treponema species and subspecies.IMPORTANCE Syphilis, a sexually transmitted disease with a global distribution, is caused by a spiral-shaped bacterium called Treponema pallidum subspecies pallidum Previously, T. pallidum was one of the few major bacterial pathogens that had not been cultured long-term in vitro (in a test tube), greatly hindering efforts to better understand this organism and the disease that it causes. In this article, we report the successful long-term cultivation of T. pallidum in a tissue culture system, a finding that is likely to enhance our ability to obtain new information applicable to the diagnosis, treatment, and prevention of syphilis.

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