In vitro isolation of contemporary Treponema pallidum strains directly from clinical samples of syphilis patients.

Objectives: Treponema pallidum subsp. pallidum (T. pallidum) is the etiological agent of syphilis, a sexually transmitted disease of global public health importance. The objective of this study was to introduce a novel in vitro protocol for isolation of T. pallidum directly from patients' clinical samples, eliminating the need for rabbit propagation.

Methods: Four oral and five genital swabs were collected from nine epidemiologically unrelated patients at two hospitals in Brno, Czech Republic. Swabs were submerged in TpCM-2 medium for transport. Then, samples were placed on a 0.4 μm filters and incubated for 2.5 hours. During this period, spiral T. pallidum cells passed through the filter pores to the well containing TpCM-2 medium and rabbit feeder cells (Sf1Ep). Stable T. pallidum cultures (containing >1 × 10⁷ treponemes) were achieved through subculturing every 7 days into fresh well.

Results: A successful protocol for in vitro isolation of T. pallidum was established. From the nine clinical specimens processed, six T. pallidum cultures (MU1-MU6) were derived after 14-112 days of cultivation. Five of these strains (MU1-MU5) belonged to SS14-like cluster and shared the same allelic profile 1.3.1. The remaining strain (MU6) was identified as a Nichols-like strain with an allelic profile 9.16.3.

Conclusions: The introduced in vitro protocol enables isolation of T. pallidum from clinical material, including frozen samples, without the need for experimental rabbits. This method facilitates the isolation of contemporary, clinically relevant treponemal strains.