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The bacterium that causes syphilis,  Treponema pallidum  subsp.  pallidum , has now been cultured  in vitro  continuously for periods exceeding 3 years using a system consisting of coculture with Sf1Ep rabbit epithelial cells in TpCM-2 medium and a low-oxygen environment. In addition, long-term culture of several other syphilis isolates (SS14, Mexico A, UW231B, and UW249B) and the  T. pallidum  subsp.  endemicum  Bosnia A strain has been achieved. During  in vitro  passage,  T. pallidum  subsp.  pallidum  exhibited a typical bacterial growth curve with logarithmic and stationary phases. Sf1Ep cells are required for sustained growth and motility; however, high initial Sf1Ep cell numbers resulted in reduced multiplication and survival. Use of Eagle’s minimal essential medium as the basal medium was not effective in sustaining growth of  T. pallidum  subsp.  pallidum  beyond the first passage, whereas CMRL 1066 or M199 supported long-term culture, confirming that additional nutrients present in these more complex basal media are required for long-term culture.  T. pallidum  subsp.  pallidum  growth was dependent upon the presence of fetal bovine serum, with 20% (vol/vol) being the optimal concentration. Omission of reactive oxygen species scavengers dithiothreitol,  d -mannitol, or  l -histidine did not dramatically affect survival or growth. Additionally,  T. pallidum  subsp.  pallidum  can be successfully cultured in a Brewer jar instead of a specialized low-oxygen incubator. Phosphomycin or amphotericin B can be added to the medium to aid in the prevention of bacterial or fungal contamination, respectively. These results help define the parameters of the  T. pallidum  subsp.  pallidum  culture system that are required for sustained, long-term survival and multiplication.

Parameters Affecting Continuous In Vitro Culture of Treponema pallidum Strains