Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Leuven, Belgium.
J Appl Microbiol. 2011 Mar;110(3):778-85. doi: 10.1111/j.1365-2672.2010.04931.x. Epub 2011 Jan 17.
To select and evaluate an appropriate outer membrane (OM) permeabilizer to use in combination with the highly muralytic bacteriophage endolysin EL188 to inactivate (multi-resistant) Pseudomonas aeruginosa.
We tested the combination of endolysin EL188 and several OM permeabilizing compounds on three selected Ps. aeruginosa strains with varying antibiotic resistance. We analysed OM permeabilization using the hydrophobic probe N-phenylnaphtylamine and a recombinant fusion protein of a peptidoglycan binding domain and green fluorescent protein on the one hand and cell lysis assays on the other hand. Antibacterial assays showed that incubation of 10(6) Ps. aeruginosa cells ml(-1) in presence of 10 mmol l(-1) ethylene diamine tetraacetic acid disodium salt dihydrate (EDTA) and 50 μg ml(-1) endolysin EL188 led to a strain-dependent inactivation between 3·01 ± 0·17 and 4·27 ± 0·11 log units in 30 min. Increasing the EL188 concentration to 250 μg ml(-1) further increased the inactivation of the most antibiotic resistant strain Br667 (4·07 ± 0·09 log units).
Ethylene diamine tetraacetic acid disodium salt dihydrate was selected as the most suitable component to combine with EL188 in order to reduce Ps. aeruginosa with up to 4 log units in a time interval of 30 min.
This in vitro study demonstrates that the application range of bacteriophage encoded endolysins as 'enzybiotics' must not be limited to gram-positive pathogens.
选择和评估一种合适的外膜（OM）通透剂，与高度溶壁噬菌体内溶素 EL188 联合使用，以灭活（多耐药）铜绿假单胞菌。
我们测试了内溶素 EL188 与几种 OM 通透化合物组合对三种具有不同抗生素耐药性的选定铜绿假单胞菌菌株的作用。我们一方面使用疏水性探针 N-苯基萘胺和肽聚糖结合域与绿色荧光蛋白的重组融合蛋白，另一方面使用细胞裂解测定法来分析 OM 通透化。抗菌测定表明，在 10 mmol l(-1) 乙二胺四乙酸二钠二水合物（EDTA）和 50 μg ml(-1) 内溶素 EL188 的存在下孵育 10(6) 个铜绿假单胞菌细胞 ml(-1)，导致菌株依赖性失活，在 30 分钟内达到 3.01 ± 0.17 至 4.27 ± 0.11 对数单位之间。将 EL188 浓度增加到 250 μg ml(-1) 进一步增加了最具抗生素耐药性的菌株 Br667 的失活（4.07 ± 0.09 对数单位）。
乙二胺四乙酸二钠二水合物被选为与 EL188 结合的最合适成分，以便在 30 分钟的时间间隔内将铜绿假单胞菌减少多达 4 个对数单位。
这项体外研究表明，噬菌体编码的内溶素作为“酶生素”的应用范围不应仅限于革兰氏阳性病原体。
