孟加拉虎粪便放线菌的分离及其多样性

【目的】建立有效分离动物粪便放线菌的方法,认识孟加拉虎粪便放线菌的多样性。【方法】从预处理、抑制剂、培养基三个方面考虑,采用平板稀释的分离方法。用细菌通用引物扩增获得放线菌菌株的16SrRNA基因,根据系统发育分析进行鉴定。【结果】孟加拉虎粪便样品中,可培养放线菌的菌落平均数量为1.10×108cfu/g(粪便湿重);对分离到的110株纯培养放线菌的16S rRNA基因部分序列分析表明:它们分布于10个科、12个属,主要是一些菌丝分化程度低的放线菌,如:Arthrobacter、Dietzia、Kocuria、Corynebacterium、Microbacterium等;产丝的放线菌主要以Streptomyces占优势,约占分离到放线菌总数的64%。【结论】干热处理粪便样品可以大大提高放线菌的出菌率;添加多种抑制剂及不含天然成分的组合培养基较适合粪便放线菌的分离;孟加拉虎粪便中可培养放线菌的多样性较丰富。本研究提供的分离动物粪便放线菌的有效方法,为动物粪便放线菌资源的研究和开发利用提供了借鉴作用。

Isolation methods and diversity of culturable fecal actinobacteria associated with Panthera tigris tigris in Yunnan Safari Park

Objective] We studied the isolation methods and diversity of culturable fecal actinobacteria associated with Panthera tigris tigris by using culture-dependent approaches.Methods] Fresh fecal samples of healthy Panthera tigris tigris were collected from Yunnan Safari Park.Pretreatment of the samples,isolation media and inhibitors were tested for actinobacteria isolation.16S rRNA genes of actinobacteria were sequenced and subjected to phylogenetic analysis.Results] The abundance of culturable actinobacteria was 1.10×108 cfu/g colony forming units(CFU) per gram of feces(wet weight).We obtained 110 purified cultural actinobacterium strains.The analysis based on 16S rRNA gene sequences showed that these strains were distributed in 10 different families and 12 genera of actinobacteria at least,and most of them were non-filamentous,such as Arthrobacter,Dietzia,Kocuria,Corynebacterium and Microbacterium.Streptomyces was the mainly classical filamentous actinobacteria,and up to 64% of total.Conclusion] Drying and heating up the fecal samples can greatly increase the rate of the actinobacteria.Many kinds of inhibitors and chemical defined media are suitable for isolation of fecal actinobacteria.The culturable actinobacteria are abundant in Panthera tigris tigris feces.Our study found an effective method to isolate animals’ fecal actinobacteria and it’s useful for studying and exploiting animals’ fecal actinobacteria.