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Updated: Nov 16, 2025
Author Spotlight: Advancing Syphilis Research — Innovations in Treponema pallidum Cultivation and Genetic Engineering
Published on: January 24, 2025
Parameters Affecting Continuous In Vitro Culture of Treponema pallidum Strains
Diane G Edmondson1, Bridget D DeLay2, Lindsay E Kowis2
1Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston, Houston, Texas, USA Diane.G.Edmondson@uth.tmc.edu.
Abstract:
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.IMPORTANCE Syphilis is caused by the bacterium Treponema pallidum subsp. pallidum Until recently, this pathogen could only be maintained through infection of rabbits or other animals, making study of this important human pathogen challenging and costly. T. pallidum subsp. pallidum has now been successfully cultured for over 3 years in a tissue culture system using a medium called TpCM-2. Here, we further define the growth requirements of this important human pathogen, promoting a better understanding of the biology of this fastidious organism.
Insights
Researchers have successfully cultured the syphilis bacterium, Treponema pallidum, in vitro for over three years. This breakthrough defines optimal conditions for sustained growth, advancing the study of this challenging pathogen.
Area of Science:
- Microbiology
- Infectious Diseases
- Cell Biology
Background:
- Syphilis, caused by Treponema pallidum subsp. pallidum, has historically been difficult to study due to challenges in in vitro cultivation.
- Previous methods relied on animal models, limiting research accessibility and increasing costs.
- Establishing a robust in vitro culture system is crucial for understanding syphilis pathogenesis and developing new therapies.
Purpose of the Study:
- To define and optimize the conditions for sustained long-term in vitro culture of Treponema pallidum subsp. pallidum.
- To identify essential components and environmental factors required for the growth and survival of T. pallidum in vitro.
- To establish a reliable and reproducible culture system for this fastidious bacterial pathogen.
Main Methods:
- Coculture of T. pallidum subsp. pallidum with Sf1Ep rabbit epithelial cells in TpCM-2 medium under low-oxygen conditions.
- Evaluation of different basal media (Eagle's minimal essential medium, CMRL 1066, M199) and fetal bovine serum concentrations.
- Assessment of the impact of reactive oxygen species scavengers and alternative incubation methods (Brewer jar).
Main Results:
- Sustained in vitro culture of T. pallidum subsp. pallidum for over 3 years was achieved.
- Optimal growth required Sf1Ep cells, specific basal media (CMRL 1066 or M199), and 20% fetal bovine serum.
- Growth followed a typical bacterial curve, and culture was also successful using a Brewer jar.
Conclusions:
- The study successfully established and characterized a long-term in vitro culture system for T. pallidum subsp. pallidum.
- Key parameters including cell line, medium composition, and serum concentration were identified for optimal growth.
- This advancement provides a vital tool for further research into syphilis biology and treatment strategies.
