摘要
目的 筛选对多重耐药菌具有广谱抗性的放线菌,发掘新型抗生素,为人类和动物细菌性疾病防控提供物质储备。方法 采集江苏省部分地区土壤样品20份,稀释涂布于改良高氏一号固体培养基进行分离纯化,Chelex-100法提取放线菌基因组DNA,PCR测定16S rRNA基因序列,确定菌属。应用垂直条纹法检测放线菌对标准菌株和临床多重耐药分离菌株的抗菌效果;PCR检测放线菌抗菌活性和抗生素合成基因之间是否存在相关性;筛选抗菌活性最强的放线菌,检测不同时间抗菌物质释放量,测定临床分离病原菌的抗菌谱;基于16S rRNA基因确定其系统进化关系;对抑菌活性最好的放线菌进行基因组测序,应用antiSMASH在线工具分析次级代谢产物生物合成基因簇,预测其产生新颖次级代谢产物的潜力。结果 从20份土壤样品中分离纯化237株放线菌,分布于放线菌纲的10个目13个科19个属,优势菌属为链霉菌属,占分离总数的57.8%;抗生素合成基因检测实验表明放线菌的抗菌作用与抗生素合成基因的存在可能呈正相关;抗菌活性最强的链霉菌命名为Y94,在第七天产生具有抗菌活性的次级代谢产物最多;Y94产生的次级代谢产物对临床分离的238株不同种属的病原菌均有良好的抗菌效果;对Y94进行系统发育分析,结果表明其与甲酸链霉菌(Streptomyces formicae)1H-GS9的相似度为95.83%,可能为潜在新种;Y94全基因组中有多种抗生素相关的次级代谢产物基因簇,有巨大的生物合成潜能。结论 链霉菌Y94株具有抗多重耐药菌活性,可能属于1个新的种属,其抗菌活性物质可能是1种新的物质,具有潜在的开发价值。
Objective Screening of positive actinomycetal strains with broad-spectrum resistance to multidrug-resistant bacteria,discovering new antibiotics for the prevention and control of bacterial infections of human-being and animals.Methods Twenty soil samples were collected from some areas in Jiangsu Province.For isolation and purification of actinomycetes,the samples were diluted and plated on the modified GS 1 agar.Chelex-100 was used to extract the genomic DNA from actinomycetal strains,and PCR was used to identify the genus based on the 16S rRNA gene sequence of the purified colonies.The antibacterial activity of actinomycetes was detected using the vertical stripe method with standard strains and isolates in animal clinic.PCR was used to detect the correlation of actinomycetal strains between the antibacterial activity and antibiotic synthesis genes.The actinomycetal strains with the highest antibacterial activity were evaluated,antibacterial substances release were detected in different time after inoculated in liquid medium,and the antibacterial spectrum of clinically isolates were determined;The 16S rRNA genes were used to establish the phylogenetic connection;The genome of the actinomycetal strain with highest antibiotic activity was further sequenced.The secondary metabolite biosynthetic gene clusters were analyzed by antiSMASH.Results 237 actinomycetal strains were obtained from 20 samples of soil,which could be categorized into 10 orders,13 families,and 19 genera.Most of them were identified as Streptomyces spp.with 57.8%of the total isolates.PCR results showed that the antibacterial activity of actinomycetal strains connected with the presence of antibiotic synthesis genes.The strain of Streptomyces with the highest antibacterial activity to the bacteria tested was designated as Y94.Streptomyces Y94 products had the highest antibacterial activity on the seventh day;The biotics of Y94 against to 238 isolates of pathogenic bacteria from different species of animal collected in our lab.Phylogenetic analysis
作者
罗健雅
黄紫贝
赵以恒
郭鑫
李蒙
苏思元
李自盼
徐昀眺
王海燕
刘文博
Luo Jianya;Huang Zibei;Zhao Yiheng;Guo Xin;Li Meng;Su Siyuan;Li Zipan;Xu Yuntiao;Wang Haiyan;Liu Wenbo(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225009;Jiangsu Vocational College of Agricultural and Animal Husbandry,Taizhou 225300)
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2023年第11期1229-1241,共13页
Chinese Journal of Antibiotics
基金
江苏高校优势学科建设工程资助项目(No.PAPD)
江苏现代农业(水禽)产业技术体系(No.JATS[2021])
江苏省农业科技自主创新资金项目(No.CX(20)2012)。
关键词
土壤
放线菌
多重耐药菌
抗菌活性
抗生素合成基因
全基因组
Soil
Actinomycetal strains
Multi-drug resistance bacteria
Whole genome
Antibacterial activity
Antibiotic synthesis genes
