procedures-for-vitro-cultivation-treponema-pallidum-syphilis

Procedures for In Vitro Cultivation of <i>Treponema pallidum</i>, the Syphilis Spirochete

For over a century, Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, could only be propagated by inoculation and ...

University of Texas Health Science Center at Houston - McGovern Medical School

Doxycycline is regarded as an effective therapy for early syphilis, and there is increasing interest in using doxycycline for prophylaxis of this infection. However, the MIC of doxycycline for  subsp.  has not been reported previously. In this study, an  culture system was utilized to determine that the MIC of doxycycline is 0.06 to 0.10 μg/ml for four strains of  subsp.  (Nichols, SS14, UW231B, and UW249B). The Nichols strain cultured  with doxycycline was also tested for infectivity in rabbits, and the minimum bactericidal concentration (MBC) was found to be ≤0.1 μg/ml using this method. The low MIC and MBC values are consistent with the previously demonstrated clinical efficacy of doxycycline for the treatment of early syphilis. This study represents the first report of the  susceptibility of  to doxycycline, and the resulting information may be useful in the consideration of doxycycline for use in prevention of syphilis.

 Susceptibility of Treponema pallidum subsp.  to Doxycycline.

The bacterium that causes syphilis,  subsp. , has now been cultured  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  subsp.  Bosnia A strain has been achieved. During  passage,  subsp.  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  subsp.  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.  subsp.  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,  subsp.  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  subsp.  culture system that are required for sustained, long-term survival and multiplication. Syphilis is caused by the bacterium  subsp.  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.  subsp.  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.

Diane G. Edmondson

University of Texas Health Science Center at Houston - McGovern Medical School

Procedures for In Vitro Cultivation of Treponema pallidum, the Syphilis Spirochete