Division of Antimicrobial Resistance, Korea Centers for Disease Control and Prevention, Cheongju, South Korea.
Department of Molecular Cell Biology and Samsung Medical Center, Sungkyunkwan University School of Medicine, Suwon, South Korea.
Clin Microbiol Infect. 2019 Sep;25(9):1156.e1-1156.e7. doi: 10.1016/j.cmi.2019.02.004. Epub 2019 Feb 18.
The phenomenon of colistin dependence in Acinetobacter baumannii has been described in a situation in which colistin is now considered as the last resort for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. In this study, we aimed to reveal a gene associated with colistin dependence in A. baumannii.
The colistin-dependent A. baumannii H08-391D strain was isolated from a patient, and target gene-inactivation mutants were constructed. We investigated the effects of target gene on colistin dependence with quantitative real-time PCR and endotoxin assay. Also, we observed the change of cell morphology by electron microscopy.
The expression of ACICU_02898, encoding a soluble lytic transglycosylase associated with cell-wall degradation and recycling, was increased by eight-to 42-fold in colistin-dependent mutants, and deletion of ACICU_02898 in a colistin-dependent strain led to colistin susceptibility (MIC = 8 mg/L). Endotoxin activity was significantly low in a colistin-dependent derivative ACICU_02898-inactivated mutant and a complemented mutant. In addition, the ACICU_02898-inactivated mutant showed a highly reduced growth rate. The colistin-dependent derivative and ACICU_02898-inactivated mutant showed clearly distinguished absorption profiles in the red/green fluorescence dot blot with regard to their membrane potential. Electron microscopy revealed that the deletion mutant cells were elongated compared to the colistin-susceptible wild-type strain and colistin-dependent strain.
A colistin-dependent A. baumannii strain exhibited a deficiency in its outer membrane integrity and high expression of lytic transglycosylase was required for survival. This study reveals why the colistin-dependent mutant can tolerate high antibiotic concentrations.
多药耐药革兰氏阴性菌感染的治疗中，粘菌素已被视为最后一道防线，在这种情况下，鲍曼不动杆菌中粘菌素依赖现象已有相关描述。本研究旨在揭示鲍曼不动杆菌中与粘菌素依赖相关的基因。
从一名患者中分离出依赖粘菌素的鲍曼不动杆菌 H08-391D 菌株，并构建了靶基因失活突变体。我们通过实时定量 PCR 和内毒素检测研究了靶基因对粘菌素依赖的影响。此外，我们还通过电子显微镜观察了细胞形态的变化。
编码与细胞壁降解和循环相关的可溶性溶菌酶的 ACICU_02898 的表达在粘菌素依赖突变体中增加了 8 至 42 倍，并且在依赖粘菌素的菌株中缺失 ACICU_02898 导致粘菌素敏感性（MIC=8 mg/L）。依赖粘菌素的衍生 ACICU_02898 失活突变体和互补突变体的内毒素活性显著降低。此外，ACICU_02898 失活突变体的生长速度明显降低。依赖粘菌素的衍生菌株和 ACICU_02898 失活突变体在膜电位的红色/绿色荧光斑点印迹中显示出明显不同的吸收谱。电子显微镜显示，与粘菌素敏感的野生型菌株和依赖粘菌素的菌株相比，缺失突变体的细胞明显拉长。
依赖粘菌素的鲍曼不动杆菌菌株表现出外膜完整性缺陷，并且需要高表达溶菌酶才能存活。本研究揭示了为什么依赖粘菌素的突变体能耐受高浓度抗生素。
