姥鲨共附生微生物抗菌活性筛选及系统发育分析

Screening and phylogenetic analysis of symbiotic and epiphyte microorganisms with Cetorhinus maximus

目的以深海姥鲨鱼鳃和胃肠道共附生微生物为研究对象，对可培养微生物进行分离、纯化和初步鉴定，筛选抗菌活性，为寻找天然活性产物奠定基础。方法采用倍比稀释涂布和划线法结合分离纯化可培养微生物；以枯草芽孢杆菌、金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌、产气肠杆菌、白色念珠菌为抗菌活性筛选模型，利用琼脂块法和滤纸片法筛选纯培养菌株抗菌活性；利用形态观察、生理生化反应和16S rRNA技术对抑菌活性进行初步鉴定。结果从姥鲨鱼鳃和胃肠道分离得到的可培养微生物36株，其中来自鱼鳃5株，来自胃肠道31株；菌株统计表明：真菌7株，占19％；放线菌1株，占3％；细菌28株，占78％。对23株来自姥鲨胃肠道的细菌进行抗菌活性筛选，15种表现抗（抑）菌活性，占分离菌株的65％。他们分别隶属于芽孢杆菌属（Bacillus）5株，葡萄球菌属（Staphylococcus）6株，短波单胞菌属（Brevundimonas）2株，嗜冷杆菌属（Psychrobacter）2株。结论利用常规微生物学技术可以对姥鲨胃肠道和鱼鳃等部位存在共附生微生物分离和纯化，其中超过50％的菌株表现抗（抑）菌效果。

Objective To investigate, purify, screen and identify antimicrobial activities of the strains of cuhurable microorganisms from gills, stomachs or intestines. Methods A total of 452 culturable microorganisms and 36 strains were isolated from gills, stomachs or intestines of Cetorhinus maximus （ Basking shark）, of which 5 strains were from gills and 31 strains were from stomachs or intestines. In all, there were 7 strains of fungi （19%） , 1 strain of actinomycetes （ 3% ） and 28 strains of bacteria （ 78% ）. Molecular identification and classification of 15 isolates based on 16S rRNA sequences indicated that they belonged to the Bacillus, Brevundimonas, Staphylococcus, Staphylococcus xylosus, Bacillus pumilus. Antibacterial activity was screened for the culturable bacteria. Results Laboratory experiments revealed that in total 23 strains were isolated from gills, stomachs or intestines, and 15 strains showed ant/microbial activities against Bacillus subtilis , Staphylococcus aureus , Escherichia coli , Pseudomonas aeruginose , Enterobacter aerogenes , Candida albicans （ with the method of agar piece and disk paper tests）, accounting for 65% of the isolated strains. They belonged to Bacillus （5 strains）, Staphylococcus （6 strains）, Brevundimonas （2 strains）, and Psychrobacter （2 strains） respectively. Conclusions The isolation and purification of symbiotic microorganisms from gills, stomachs or intestines of Cetorhinus maximus could be realized via routine microbiology. Results indicated that 50% of the isolated strains showed bacteriostatic effects to various degrees. There is great probability that active antimicrobial substances from these strains might be found through further research.