Zhang Jie, Xu Lu-Lu, Gan Dan, Zhang Xingping
Clin Lab. 2018 Jun 1;64(6):1021-1030. doi: 10.7754/Clin.Lab.2018.180342.
The increase in the prevalence of drug-resistant Acinetobacter baumannii is a serious public health concern, which is closely linked to the formation of biofilm. It is reported that the bacteriophage and its endolysin have a good ability to degrade biofilms. The goals of this study were to compare the ability of A. baumannii bacteriophage AB3, its endolysin AB3, and three antibiotics to degrade A. baumannii biofilm and biofilm-bound A. baumannii and to understand the antibacterial mechanism of LysAB3.
The 558-bp sequence of the LysAB3 gene was amplified by polymerase chain reaction (PCR); the fragment was cloned into pET28a (+) to construct the recombinant plasmid pET28a-LysAB3, which was then expressed in E. coli BL21 (DE3) to obtain the LysAB3. Differences in A. baumannii biofilm and biofilm-bound A. baumannii after treatment with bacteriophage AB3, LysAB3 or three antibiotics were examined using the crystal violet staining method and an MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay. Changes in biofilm morphology and thickness in each treatment group were observed by laser scanning confocal microscopy. In addition, a LysAB3 construct with the amphiphilic peptide structural region removed (LysAB3-D) was assessed for its antibacterial activity.
After 24-hour treatment with either bacteriophage AB3 and its LysAB3, A. baumannii biofilms were significantly degraded, and the number of viable biofilm-bound A. baumannii were also significantly decreased. After removing the amphiphilic peptide structure motif from LysAB3, the antibacterial activity decreased from 95.8% to 33.3%.
Thus, LysAB3 can effectively degrade A. baumannii biofilm and biofilm-bound A. baumannii in vitro. The antibacterial mechanism of LysAB3 may be associated with the ability of the amphiphilic peptide structural region to enhance the permeability of cytoplasmic membrane of A. baumannii by degradation of bacterial wall peptidoglycan.
耐多药鲍曼不动杆菌患病率的增加是一个严重的公共卫生问题，这与生物膜的形成密切相关。据报道，噬菌体及其溶菌酶具有良好的生物膜降解能力。本研究的目的是比较鲍曼不动杆菌噬菌体AB3、其溶菌酶AB3和三种抗生素对鲍曼不动杆菌生物膜及生物膜结合的鲍曼不动杆菌的降解能力，并了解LysAB3的抗菌机制。
通过聚合酶链反应（PCR）扩增LysAB3基因的558 bp序列；将该片段克隆到pET28a(+)中构建重组质粒pET28a-LysAB3，然后在大肠杆菌BL21(DE3)中表达以获得LysAB3。使用结晶紫染色法和MTT（3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐）测定法检测用噬菌体AB3、LysAB3或三种抗生素处理后鲍曼不动杆菌生物膜及生物膜结合的鲍曼不动杆菌的差异。通过激光扫描共聚焦显微镜观察各处理组生物膜形态和厚度的变化。此外，评估去除两亲性肽结构区域的LysAB3构建体（LysAB3-D）的抗菌活性。
用噬菌体AB3及其LysAB3处理24小时后，鲍曼不动杆菌生物膜显著降解，生物膜结合的鲍曼不动杆菌活菌数也显著减少。从LysAB3中去除两亲性肽结构基序后，抗菌活性从95.8%降至33.3%。
因此，LysAB3可在体外有效降解鲍曼不动杆菌生物膜及生物膜结合的鲍曼不动杆菌。LysAB3的抗菌机制可能与两亲性肽结构区域通过降解细菌细胞壁肽聚糖增强鲍曼不动杆菌细胞质膜通透性的能力有关。
