抗耐甲氧西林金黄色葡萄球菌菌株的筛选、鉴定及其活性物质初步研究

从海南红树林土壤分离筛选抗耐甲氧西林金黄色葡萄球菌（MRSA）菌株，通过形态学观察、生理生化试验及分子生物学进行菌种鉴定。采用不同培养基优选抑菌活性物质生成条件；以有机溶剂萃取抑菌活性物质，采用气相色谱-质谱（GC-MS）分析抑菌物质化学成分，采用微量稀释法测定最低抑菌浓度与最低杀菌浓度。结果表明，筛选得到一株抗MRSA菌株，编号为NEL-002，被鉴定为萎缩芽孢杆菌（Bacillus atrophaeus）。菌株NEL-002采用LB液体培养基得到发酵液经石油醚萃取粗提物抗MRSA菌活性最强，且在40～80 ℃、pH在5～10较稳定，对多种蛋白酶不敏感；最低抑菌质量浓度为62.5 μg/mL，最低杀菌质量浓度为500 μg/mL。GC-MS鉴定出抗菌活性物质为十甲基环戊硅氧烷、十二甲基环六硅氧烷及邻苯二甲酸二丁酯。

Screening and identification of methicillin-resistant Staphylococcus aureus resistant strain and preliminary study of their active substances

Methicillin-resistant Staphylococcus aureus (MRSA) resistant strains were isolated and screened from Hainan mangrove soil, and identified by morphological observation, physiological and biochemical tests and molecular biology. Production conditions of antibacterial active substances were optimized and screened by different media. The antibacterial active substances were extracted with organic solvent, the chemical constituents of the antibacterial substances were analyzed by GC-MS, and the minimum inhibitory concentration and minimum bactericidal concentration were determined by microdilution method. The results showed that a MRSA resistant strain was screened, numbered NEL-002, and identified as Bacillus atrophaeus. The crude extract of strain NEL-002 fermentation broth in LB liquid medium extracted by petroleum ether had the strongest anti-MRSA activity, which was stable at 40-80 ℃, pH 5-10, and insensitive to multiple proteases. The minimum inhibitory mass concentration was 62.5 μg/ml, and the minimum bactericidal mass concentration was 500 μg/ml. The antibacterial active substances were identified as decamylcyclopentasiloxane, dodecamylcyclohexasiloxane and dibutyl phthalate by GC-MS.