Long-Term In Vitro Culture of the Syphilis Spirochete Treponema pallidum subsp. pallidum

Abstract

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.

Keywords: Treponema pallidum; cell culture; cell structure; electron microscopy; infectivity; physiology; spirochetes.

FIG 1

Long-term cultivation of T. pallidum subsp. pallidum Nichols in a tissue culture system (experiment 1). Primary cultures represent the results obtained without subculture. The 500-µl passages used transfer of that volume for each subculture, whereas the low-inoculum passages used lower volumes (100 µl to 250 µl). (A) In this “sawtooth” plot, the number of T. pallidum organisms per culture upon harvest and the number inoculated in the next subculture are shown for each time point. (B) Percent motility provides a measure of viability. (C) Average (Ave.) generation time, representing the time in hours divided by log₂(fold increase) for each time point. T. pallidum per-culture and percent motility values represent means ± SE of results from three biological replicates.

FIG 2

Cumulative generations during in vitro culture of T. pallidum subsp. pallidum Nichols in experiment 1. Increases in the primary cultures occurred only within the first 12 days. The generations accumulated more rapidly in the low-inoculum cultures than in the 500-µl inoculum cultures until the latter were discontinued on day 116.

FIG 3

Multiplication and genome replication of T. pallidum Nichols in Sf1Ep cocultures and axenic cultures. Fold increases are shown in parentheses. The axenic cultures were initiated in parallel with the Sf1Ep cocultures in experiment 1 (Table 2) (Fig. 1), using the same frozen stock of T. pallidum. (A) The number of T. pallidum organisms per culture increased only ~2-fold in axenic cultures but increased over 55-fold for cocultures with Sf1Ep cells. Genome equivalent (Equiv.) increases were similar to bacterial cell number increases for the Sf1Ep cocultures but were somewhat higher than the cell number increases for the axenic cultures. (B) Numbers of genome Equiv. per cell remained stable for the exponentially growing cocultures but increased for the axenic cultures. T. pallidum per-culture and genome equivalent values represent means ± SE of results from triplicate cultures.

FIG 4

Consistency of T. pallidum multiplication in vitro, as illustrated by cumulative generation values from experiments 1 to 5.

FIG 5

T. pallidum subsp. pallidum Nichols organisms cultured in vitro for 116 days were fully infectious in the rabbit intradermal infection model. This photograph of one of the three rabbits in infectivity experiment A (Table 3) was taken on day 45 postinoculation. Paired sites were inoculated intradermally with the dosages shown. Sites inoculated with higher doses had undergone ulceration, as is typical of intradermal infections in rabbits.

FIG 6

Appearance of in vitro-cultured T. pallidum subsp. pallidum Nichols by dark-field light microscopy. This specimen was from a 200-day culture from experiment 1. (A) Four organisms with typical morphology, showing regions with planar, “flat wave” morphology (arrowheads). (B) Example of the inherent flexibility of T. pallidum. (C) Ring-shaped organism in which the two ends are joined. All of these forms are also commonly seen in T. pallidum organisms freshly extracted from rabbit tissue.

FIG 7

Structure of T. pallidum subsp. pallidum Nichols following 54 days of in vitro culture, as determined by cryo-electron microscopy. (A and D) Two T. pallidum cells were imaged at low magnification (bar, 1 µm). (B, C, E, and F) Highlighted areas in panels A and D were imaged at high magnification (bar, 200 nm). B, intact region in the middle of the cell, showing the binding of small (~10-nm) particles or vesicles to the outer membrane surface; C, region of the cell in panel A in which the outer membrane has been stripped off. Note the lack of bound particles. E, end of the cell in panel D, showing the conical tip structure and a prominent membrane bleb, both typical of T. pallidum structure; F, intact region in middle of cell. The outlines of periplasmic flagella are clearly visible in this and other panels.

FIG 8

Effect of medium volume on T. pallidum multiplication in the Sf1Ep coculture system. Equal quantities of T. pallidum Nichols were inoculated into triplicate 9-cm² Sf1Ep cell cocultures containing 2, 3, or 4 ml of TpCM-2. The cultures were harvested and evaluated for T. pallidum fold increase and motility on day 7. (A) Increased culture yield with higher medium volumes. (B) High retention of motility in all cultures.