Parameters Affecting Continuous In Vitro Culture of Treponema pallidum Strains.
Parameters Affecting Continuous In Vitro Culture of Treponema pallidum Strains.
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DOI：
10.1128/mbio.03536-20
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发表时间：
2021-02-23
期刊：
影响因子：
6.4
通讯作者：
Norris SJ
中科院分区：
文献类型：
--
作者：
Edmondson DG;DeLay BD;Kowis LE;Norris SJ
引起梅毒的细菌，梅毒螺旋体亚种。pallidum，现在已经在体外连续培养超过3年的时间，使用由与Sf 1 Ep兔上皮细胞在TpCM-2培养基和低氧环境中共培养组成的系统。此外，对其他几种梅毒分离株（SS 14、Mexico A、UW 231 B和UW 249 B）和T.苍白球亚种获得了波斯尼亚特有菌A菌株。在体外传代过程中，T.苍白球亚种苍白球呈现典型的细菌生长曲线，具有对数期和稳定期。Sf 1 Ep细胞是持续生长和运动所必需的;然而，高初始Sf 1 Ep细胞数量导致增殖和存活减少。使用Eagle基本培养基作为基础培养基不能有效地维持T.苍白球亚种在第一代之后，CMRL 1066或M199支持长期培养，证实了这些更复杂的基础培养基中存在的额外营养素是长期培养所需的。T.苍白球亚种苍白球的生长依赖于胎牛血清的存在，20%（体积/体积）是最佳浓度。省略活性氧清除剂二硫苏糖醇，d-甘露醇，或l-组氨酸没有显着影响生存或生长。此外，T.苍白球亚种苍白球可以成功地培养在布鲁尔瓶，而不是一个专门的低氧培养箱。可向培养基中加入磷霉素或阿替霉素B以分别帮助防止细菌或真菌污染。这些结果有助于确定T.苍白球亚种pallidum培养系统，需要持续，长期生存和繁殖。
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.