Ackart David F, Hascall-Dove Laurel, Caceres Silvia M, Kirk Natalie M, Podell Brendan K, Melander Christian, Orme Ian M, Leid Jeff G, Nick Jerry A, Basaraba Randall J
Department of Microbiology, Immunology and Pathology, Mycobacterial Research Laboratories, Colorado State University, Fort Collins, CO, USA.
Pathog Dis. 2014 Apr;70(3):359-69. doi: 10.1111/2049-632X.12144. Epub 2014 Feb 24.
There is an urgent need to improve methods used to screen antituberculosis drugs. An in vitro assay was developed to test drug treatment strategies that specifically target drug-tolerant Mycobacterium tuberculosis. The H37Rv strain of M. tuberculosis survived antimicrobial treatment as attached microbial communities when maintained in tissue culture media (RPMI-1640) with or without lysed human peripheral blood leukocytes. When cultured planktonically in the presence of Tween-80, bacilli failed to form microbial communities or reach logarithmic phase growth yet remained highly susceptible to antimicrobial drugs. In the absence of Tween, bacilli tolerated drug therapy by forming complex microbial communities attached to untreated well surfaces or to the extracellular matrix derived from lysed human leukocytes. Treatment of microbial communities with DNase I or Tween effectively dispersed bacilli and restored drug susceptibility. These data demonstrate that in vitro expression of drug tolerance by M. tuberculosis is linked to the establishment of attached microbial communities and that dispersion of bacilli targeting the extracellular matrix including DNA restores drug susceptibility. Modifications of this in vitro assay may prove beneficial in a high-throughput platform to screen new antituberculosis drugs especially those that target drug-tolerant bacilli.
迫切需要改进用于筛选抗结核药物的方法。开发了一种体外试验来测试专门针对耐药物结核分枝杆菌的药物治疗策略。当在含有或不含有裂解的人外周血白细胞的组织培养基（RPMI-1640）中培养时，结核分枝杆菌的H37Rv菌株作为附着的微生物群落存活于抗菌治疗中。当在吐温-80存在下进行浮游培养时，杆菌无法形成微生物群落或达到对数期生长，但仍对抗菌药物高度敏感。在没有吐温的情况下，杆菌通过形成附着在未处理的孔表面或来自裂解的人白细胞的细胞外基质上的复杂微生物群落来耐受药物治疗。用DNA酶I或吐温处理微生物群落可有效分散杆菌并恢复药物敏感性。这些数据表明，结核分枝杆菌在体外对药物的耐受性表达与附着的微生物群落的建立有关，并且针对包括DNA在内的细胞外基质的杆菌分散可恢复药物敏感性。这种体外试验的改进可能在高通量平台中证明对筛选新的抗结核药物特别是那些针对耐药物杆菌的药物有益。
