Parameters Affecting Continuous In Vitro Culture of Treponema pallidum Strains

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.

Keywords: Treponema pallidum; bejel; culture; epithelial cells; growth requirements; oxygen; physiology; syphilis.

FIG 1

Long-term in vitro culture of T. pallidum, as exemplified by a lineage of T. pallidum subsp. pallidum Nichols maintained continuously for over 3 years. (A) Sawtooth plot showing the numbers of T. pallidum per culture and the number transferred to new cultures at each time point. Results represent the mean ± SEM for three biological replicates. (B) Percent of motile organisms, expressed as mean of three biological replicates. (C) Average generation time in hours for each time point. Decreases in T. pallidum per culture and percent motility and corresponding increases in generation time correspond to times of decreased growth and viability related to Sf1Ep cell culture status or medium composition issues (see the text). Representative of two long-term experiments (Table 1).

FIG 2

Growth curves of T. pallidum subsp. pallidum Nichols cultured in the Sf1Ep system with TpCM-2 medium. The changes in T. pallidum per culture and percent motility are shown for experiment 1 (A, B), experiment 2 (C, D), and experiment 3 (E, F). Experiment 1 consisted of a single condition in which 1 × 10⁵ Sf1Ep cells were added per culture. In experiments 2 and 3, parallel triplicate cultures were seeded with 3 × 10⁴ Sf1Ep cells (low inoculum) or 1 × 10⁵ Sf1Ep cells (high inoculum). The T. pallidum inoculum was adjusted to 1 × 10⁶ per culture in these experiments, so in experiments 2 and 3, the multiplicity of infection (MOI) was 33 and 10 in the low and high Sf1Ep concentration cultures, respectively. In each experiment and condition, three biological replicates were harvested at each time point; the results represent the mean ± SEM for these cultures.

FIG 3

Relationship between the initial Sf1Ep cell numbers and multiplication of T. pallidum subsp. pallidum Nichols over a 7-day period of incubation. T. pallidum used for inoculating cultures were harvested from in vitro cultures, frozen at −80°C in 15% glycerol, and then thawed; this process effectively kills >99% of the Sf1Ep cells present in the inoculum. Triplicate cultures seeded with the number of Sf1Ep cells shown were inoculated with the same T. pallidum preparation (7.1 × 10⁶/culture) and harvested after 7 days of incubation. MOI ranged from 18 T. pallidum/cell at 400,000 Sf1Ep cells seeded to 1,131 T. pallidum/cell at 6,250 Sf1Ep cells seeded. (A) Cumulative number of T. pallidum generations (log₂-fold increases). (B) Percent motile T. pallidum. (C) Number of Sf1Ep cells per culture at the time of harvest. All values represent the mean ± SEM for triplicate cultures.

FIG 4

Long-term survival and multiplication of T. pallidum subsp. pallidum Nichols is dependent on the nutrient composition of the basal medium. In this study, the same preparation of spirochetes (from TpCM-2 in vitro cultures) was passaged in parallel in T. pallidum culture media containing Eagle’s MEM (TpCM-1), CMRL 1066 (TpCM-2), and M199 (TpCM-3) as the basal medium and examined for up to 7 in vitro passages. Error bars represent SEM between triplicate cultures. (A) Sawtooth plot shows continued multiplication in TpCM-2 and TpCM-3 but the rapid decline of T. pallidum cultured in TpCM-1 after the first passage. (B) Percent motile T. pallidum in the cultures. (C) Cumulative number of T. pallidum generations in the three media.

FIG 5

Requirement for fetal bovine serum (FBS) in TpCM-2 for support of T. pallidum subsp. pallidum Nichols long-term in vitro survival and multiplication. Triplicate parallel cultures at each serum concentration were monitored over three 7-day passages. (A) T. pallidum per culture, showing the culture yield and inoculum for the next passage at each time point. (B) Percent motility of T. pallidum. Values represent the mean ± SEM from three biological replicates.

FIG 6

T. pallidum subsp. pallidum Nichols can be cultured effectively long-term in vitro in Brewer jars. A T. pallidum preparation was used to inoculate duplicate 6-well plates containing Sf1Ep cells and TpCM-2 medium. The cultures were incubated in parallel in a Brewer jar equilibrated with 1.5% O₂: 5% CO₂: balance N₂ or a tri-gas incubator containing the same gas mixture at 34°C and were passaged in the same manner at 7-day intervals. Results represent the average T. pallidum per culture and percent motility ± SEM for triplicate cultures in each environment.

FIG 7

The absence of dithiothreitol (DTT) does not dramatically affect the multiplication or motility of T. pallidum under standard passage conditions. T. pallidum subsp. pallidum Nichols freshly harvested from in vitro cultures was transferred to new cultures with Sf1Ep cells and TpCM-2 containing either no DTT, the usual concentration of DTT (0.52 mM, 1× DTT), or double the concentration of DTT (1.04 mM, 2× DTT). T. pallidum concentration (A) and motility (B) were monitored over a total of four passages in which the no DTT, 1× DTT, and 2× DTT conditions were maintained. (C) Cumulative number of T. pallidum generations. Results represent the mean of three replicate cultures under each condition (± SEM for T. pallidum per culture and percent motile values).

FIG 8

The presence of the reactive oxygen species (ROS)-scavenging medium components dithiothreitol (DTT), d-mannitol (Man), and l-histidine (His) is not required for in vitro survival and multiplication of T. pallidum subsp. pallidum Nichols. Cultures were inoculated with T. pallidum either freshly harvested from in vitro cultures (A, experiment 1) or frozen and then thawed immediately prior to inoculation (B and C, experiments 2 and 3). Parallel cultures contained Sf1Ep cells and either TpCM-2 (control) or the same medium preparation lacking Man and His (−Man −His) or Man, His, and DTT (−Man −His −DTT). The average numbers of cumulative generations for triplicate cultures under each condition are shown. Note that the first passage of cultures in experiment 3 was performed at 6 days rather than the standard 7 days.

FIG 9

T. pallidum growth in vitro is reduced upon addition of rifampicin but not phosphomycin. Triplicate cultures at each antibiotic concentration were inoculated with T. pallidum subsp. pallidum Nichols and incubated for 7 days in the Sf1Ep culture system with TpCM-2 under standard conditions. The effects of phosphomycin (A, B) and rifampicin (C, D) were examined in two separate experiments. (A, C) Average number of T. pallidum per culture ± SEM from three samples. Dashed line indicates the inoculum level. (B, D). Average percent motility of T. pallidum ± SEM from three samples.

FIG 10

Effects of the presence of amphotericin B and phosphomycin on the long-term culture of T. pallidum subsp. pallidum Nichols. Subculture was performed at 7-day intervals in the constant presence or absence of 2.5 μg/ml amphotericin B (A), 20 μg/ml phosphomycin (B), or both 2.5 μg/ml amphotericin B and 20 μg/ml phosphomycin (C). Data are shown as the cumulative number of generations over the 28-d period of the experiments.