脓肿分枝杆菌是一种新兴的人类病原体,导致显著的发病率甚至死亡率。本质上对几乎所有可用的抗生素都有抵抗力,因此可能是一场噩梦。其内在抗性的机制仍未完全理解。这里,我们选择并确认了脓肿分枝杆菌转座子突变体,该突变体对包括利福平在内的多种药物过敏,rifabutin,万古霉素,氯法齐明,利奈唑胺,亚胺培南,左氧氟沙星,头孢西丁,还有克拉霉素.发现编码推定的阿拉伯糖基转移酶C的基因MAB_0189c被破坏,使用新开发的高效策略,结合下一代测序和多重PCR。此外,MAB_0189c的无选择标记缺失概括了超敏表型。MAB_0189c的破坏导致不能合成脂阿拉伯甘露聚糖并显著增强其细胞包膜通透性。补充MAB_0189c或结核分枝杆菌embC恢复了耐药表型。重要的是,用乙胺丁醇治疗脓肿分枝杆菌,针对结核分枝杆菌阿拉伯糖基转移酶的一线抗结核药物,在体外对多种抗生素有很大程度的敏感。我们最终使用持续脓肿分枝杆菌感染的小鼠模型测试了六种选定药物的活性,发现利奈唑胺,rifabutin,亚胺培南对MAB_0189c缺失株有活性。这些结果确定MAB_0189是脓肿分枝杆菌固有抗性的关键决定因素,优化针对MAB_0189的抑制剂可能是解除脓肿分枝杆菌固有的多种抗生素耐药性的策略。重要性脓肿分枝杆菌对大多数抗生素具有内在抗性,和治疗它的感染是非常具有挑战性的。其内在抗性的机制仍未完全理解。在这里,我们发现了对多种药物过敏的转座子突变体,并鉴定了插入MAB_0189c(编码阿拉伯糖基转移酶的直系同源embC,embC)基因采用一种新开发的快速高效的方法。我们进一步证实,MAB_0189c基因通过影响其细胞包膜中脂阿拉伯甘露聚糖的产生而降低细胞包膜通透性,从而在其固有抗性中起重要作用。最后,我们发现了阿拉伯糖基转移酶抑制剂,乙胺丁醇,9种选定药物的体外活性增加。敲除MAB_0189c使脓肿分枝杆菌对小鼠的3种药物敏感。这些发现表明,潜在的强大的脓肿分枝杆菌EmbC抑制剂可能用于逆转脓肿分枝杆菌对多种药物的内在耐药性。
Mycobacterium abscessus is an emerging human pathogen leading to significant morbidity and even mortality, intrinsically resistant to almost all the antibiotics available and so can be a nightmare. Mechanisms of its intrinsic resistance remain not fully understood. Here, we selected and confirmed an M. abscessus transposon mutant that is hypersensitive to multiple drugs including rifampin, rifabutin, vancomycin, clofazimine, linezolid, imipenem, levofloxacin, cefoxitin, and clarithromycin. The gene MAB_0189c encoding a putative arabinosyltransferase C was found to be disrupted, using a newly developed highly-efficient strategy combining next-generation sequencing and multiple PCR. Furthermore, selectable marker-free deletion of MAB_0189c recapitulated the hypersensitive phenotype. Disruption of MAB_0189c resulted in an inability to synthesize lipoarabinomannan and markedly enhanced its cell envelope permeability. Complementing MAB_0189c or M. tuberculosis embC restored the resistance phenotype. Importantly, treatment of M. abscessus with ethambutol, a first-line antituberculosis drug targeting arabinosyltransferases of M. tuberculosis, largely sensitized M. abscessus to multiple antibiotics in vitro. We finally tested activities of six selected drugs using a murine model of sustained M. abscessus infection and found that linezolid, rifabutin, and imipenem were active against the MAB_0189c deletion strain. These results identified MAB_0189 as a crucial determinant of intrinsic resistance of M. abscessus, and optimizing inhibitors targeting MAB_0189 might be a strategy to disarm the intrinsic multiple antibiotic resistance of M. abscessus. IMPORTANCE Mycobacterium abscessus is intrinsically resistant to most antibiotics, and treatment of its infections is highly challenging. The mechanisms of its intrinsic resistance remain not fully understood. Here we found a transposon mutant hypersensitive to a variety of drugs and identified the transposon inserted into the MAB_0189c (orthologous embC coding arabinosyltransferase, EmbC) gene by using a newly developed rapid and efficient approach. We further verified that the MAB_0189c gene played a significant role in its intrinsic resistance by decreasing the cell envelope permeability through affecting the production of lipoarabinomannan in its cell envelope. Lastly, we found the arabinosyltransferases inhibitor, ethambutol, increased activities of nine selected drugs in vitro. Knockout of MAB_0189c made M. abscessus become susceptible to 3 drugs in mice. These findings indicated that potential powerful M. abscessus EmbC inhibitor might be used to reverse the intrinsic resistance of M. abscessus to multiple drugs.