Differential Growth Characteristics and Streptomycin Susceptibility of Virulent and Avirulent Mycobacterium tuberculosis Strains in a Novel Fibroblast-Mycobacterium Microcolony Assay

The ability to spread from cell to cell may be an important virulence determinant of Mycobacterium tuberculosis. An in vitro assay was developed to characterize this ability among four strains of M. tuberculosis: the attenuated strain H37Ra, the virulent strains H37Rv and Erdman, and a virulent clinical isolate (Stew). Confluent monolayers of human skin fibroblasts were infected with these strains and overlaid with agar-medium. M. tuberculosis infection developed over 21 days as microcolonies originating within the plane of the fibroblasts. Microcolonies of the virulent strains had an elongated appearance and exhibited extensive cording. The cords appeared to invade adjacent cells within the plane of the monolayer. Microcolony diameter of the Erdman strain was significantly larger than that of the other virulent strains, indicating that virulent strains can have distinguishing phenotypes in this assay. In contrast, avirulent H37Ra microcolonies were rounded and noncorded. H37Ra microcolonies were significantly smaller than those of the virulent strains. Microcolony diameter of the virulent strains was not reduced by the extracellularly acting antibiotic streptomycin at concentrations of up to 5.0 μg/ml. In contrast, H37Ra microcolony size was reduced at concentrations as low as 0.5 μg/ml. Growth of all strains was similarly inhibited by 1.0 μg of streptomycin per ml in fibroblast-conditioned tissue culture medium alone. When fibroblasts were infected with the M. tuberculosis strains without an agar overlay, with and without streptomycin, numbers of CFU mirrored the changes observed in the microcolony assay. There was a statistically significant decrease in H37Ra CFU compared to virulent strains after treatment with streptomycin. These differences between H37Ra and virulent strains in human fibroblasts suggest that H37Ra may be lacking a virulence determinant involved in cell-to-cell spread of M. tuberculosis.

细胞间传播能力或许是结核分枝杆菌（Mycobacterium tuberculosis）重要的毒力决定因子。本研究开发了一种体外实验方法，以表征四株结核分枝杆菌的该传播能力：减毒株H37Ra、强毒株H37Rv与Erdman，以及一株强毒临床分离株（Stew）。将人皮肤成纤维细胞的汇合单层培养物用上述菌株感染，并以琼脂培养基进行覆盖培养。结核分枝杆菌的感染会在21天内于成纤维细胞单层平面内形成微菌落。强毒株的微菌落呈细长形态，且呈现广泛的索状生长特征；这些索状结构似乎可侵入单层平面内的相邻细胞。Erdman株的微菌落直径显著大于其他强毒株，表明在该实验体系中，不同强毒株可展现出具有区分度的表型。与之相反，减毒株H37Ra的微菌落呈圆形，且无索状生长特征，其微菌落直径显著小于各强毒株。在浓度高达5.0 μg/ml的胞外作用抗生素链霉素处理下，强毒株的微菌落直径未出现缩减；而H37Ra的微菌落尺寸在链霉素浓度低至0.5 μg/ml时即出现减小。仅在成纤维细胞条件化组织培养基中，1.0 μg/ml的链霉素即可对所有菌株的生长产生相似的抑制作用。当不使用琼脂覆盖、分别添加或不添加链霉素感染成纤维细胞时，菌落形成单位（CFU，Colony-Forming Unit）的数量变化与微菌落实验中观测到的结果一致。经链霉素处理后，H37Ra的CFU数量相较于强毒株出现了具有统计学意义的下降。上述H37Ra与强毒株在人成纤维细胞中的差异表明，H37Ra可能缺失了参与结核分枝杆菌细胞间传播的毒力决定因子。