Diaz Lorena, Tran Truc T, Munita Jose M, Miller William R, Rincon Sandra, Carvajal Lina P, Wollam Aye, Reyes Jinnethe, Panesso Diana, Rojas Natalia L, Shamoo Yousif, Murray Barbara E, Weinstock George M, Arias Cesar A
Molecular Genetics and Antimicrobial Resistance Unit, Universidad El Bosque, Bogotá, Colombia.
Department of Internal Medicine, Division of Infectious Diseases, University of Texas Medical School at Houston, Houston, Texas, USA University of Houston College of Pharmacy, Houston, Texas, USA.
Antimicrob Agents Chemother. 2014 Aug;58(8):4527-34. doi: 10.1128/AAC.02686-14. Epub 2014 May 27.
Daptomycin (DAP) is a lipopeptide antibiotic frequently used as a "last-resort" antibiotic against vancomycin-resistant Enterococcus faecium (VRE). However, an important limitation for DAP therapy against VRE is the emergence of resistance during therapy. Mutations in regulatory systems involved in cell envelope homeostasis are postulated to be important mediators of DAP resistance in E. faecium. Thus, in order to gain insights into the genetic bases of DAP resistance in E. faecium, we investigated the presence of changes in 43 predicted proteins previously associated with DAP resistance in enterococci and staphylococci using the genomes of 19 E. faecium with different DAP MICs (range, 3 to 48 μg/ml). Bodipy-DAP (BDP-DAP) binding to the cell membrane assays and time-kill curves (DAP alone and with ampicillin) were performed. Genetic changes involving two major pathways were identified: (i) LiaFSR, a regulatory system associated with the cell envelope stress response, and (ii) YycFGHIJ, a system involved in the regulation of cell wall homeostasis. Thr120 → Ala and Trp73 → Cys substitutions in LiaS and LiaR, respectively, were the most common changes identified. DAP bactericidal activity was abolished in the presence of liaFSR or yycFGHIJ mutations regardless of the DAP MIC and was restored in the presence of ampicillin, but only in representatives of the LiaFSR pathway. Reduced binding of BDP-DAP to the cell surface was the predominant finding correlating with resistance in isolates with DAP MICs above the susceptibility breakpoint. Our findings suggest that genotypic information may be crucial to predict response to DAP plus β-lactam combinations and continue to question the DAP breakpoint of 4 μg/ml.
达托霉素（DAP）是一种脂肽类抗生素，常被用作对抗耐万古霉素屎肠球菌（VRE）的“最后手段”抗生素。然而，DAP治疗VRE的一个重要局限性是在治疗过程中会出现耐药性。参与细胞壁稳态的调节系统中的突变被认为是屎肠球菌中DAP耐药性的重要介导因素。因此，为了深入了解屎肠球菌中DAP耐药性的遗传基础，我们使用19株具有不同DAP MIC值（范围为3至48μg/ml）的屎肠球菌基因组，研究了43种先前与肠球菌和葡萄球菌中DAP耐药性相关的预测蛋白的变化情况。进行了Bodipy-DAP（BDP-DAP）与细胞膜结合试验以及时间杀菌曲线试验（单独使用DAP以及与氨苄西林联合使用）。确定了涉及两条主要途径的基因变化：（i）LiaFSR，一种与细胞壁应激反应相关的调节系统，以及（ii）YycFGHIJ，一种参与细胞壁稳态调节的系统。LiaS和LiaR中分别出现的Thr120→Ala和Trp73→Cys替换是最常见的变化。无论DAP MIC值如何，在存在liaFSR或yycFGHIJ突变的情况下，DAP的杀菌活性均被消除，而在存在氨苄西林的情况下可恢复，但仅在LiaFSR途径的代表菌株中恢复。BDP-DAP与细胞表面的结合减少是与DAP MIC高于敏感断点的分离株耐药性相关的主要发现。我们的研究结果表明，基因型信息对于预测DAP加β-内酰胺联合用药的反应可能至关重要，并且继续对4μg/ml 的DAP断点提出质疑。
