Bosák Juraj, Hrala Matěj, Skokan Filip, Nečas Miroslav, Strnadel Radim, Vrbová Eliška, Pospíšilová Petra, Jedličková Hana, Šmajs David
Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
First Department of Dermatovenerology, St. Anne's University Hospital and Faculty of Medicine, Masaryk University, Brno, Czech Republic.
Clin Microbiol Infect. 2025 Jun;31(6):1061-1065. doi: 10.1016/j.cmi.2025.03.017. Epub 2025 Mar 25.
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.
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. Samples were then 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 × 10 treponemes) were achieved by subculturing every 7 days into fresh well.
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 to 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.
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.
梅毒螺旋体苍白亚种（T. pallidum）是梅毒的病原体，梅毒是一种具有全球公共卫生重要性的性传播疾病。本研究的目的是引入一种新的体外方案，用于直接从患者临床样本中分离梅毒螺旋体，无需进行兔传代培养。
在捷克布尔诺的两家医院，从9名流行病学上无关联的患者中采集了4份口腔拭子和5份生殖器拭子。拭子浸没在TpCM - 2培养基中进行转运。然后将样本置于0.4μm滤膜上，孵育2.5小时。在此期间，螺旋形的梅毒螺旋体细胞通过滤膜孔进入含有TpCM - 2培养基和兔饲养细胞（Sf1Ep）的孔中。通过每7天传代至新鲜孔中，获得了稳定的梅毒螺旋体培养物（含>1×10个梅毒螺旋体）。
建立了一种成功的体外分离梅毒螺旋体的方案。在处理的9份临床标本中，经过14至112天的培养，获得了6株梅毒螺旋体培养物（MU1 - MU6）。其中5株菌株（MU1 - MU5）属于SS14样簇，具有相同的等位基因谱1.3.1。其余菌株（MU6）被鉴定为具有等位基因谱9.16.3的Nichols样菌株。
所引入的体外方案能够从临床材料（包括冷冻样本）中分离梅毒螺旋体，无需实验兔。该方法有助于分离当代具有临床相关性的梅毒螺旋体菌株。
