不同年龄大熊猫肠道可培养芽孢杆菌的多样性及部分功能特性分析

Diversity and functional characteristics of Bacillus isolated from gut of giant panda (Ailuropoda melanoleuca) at different age

摘要:

【背景】大熊猫的肠道内微生物类群丰富，种群结构与宿主的年龄、生存环境、季节变化等因素有关，其中年龄是影响肠道菌群组成的重要因素之一。【目的】以不同年龄阶段的大熊猫粪便为研究对象，旨在了解不同年龄大熊猫肠道内芽孢杆菌的多样性，探究大熊猫肠道芽孢杆菌种类与年龄段之间的关系，并为优良益生菌剂的开发提供菌种资源。【方法】用稀释涂布法分离大熊猫粪便中的芽孢杆菌，对分离的菌株进行BOXA1R-PCR、16S rRNA基因系统发育及主成分分析，揭示大熊猫肠道中可培养芽孢杆菌的多样性；采用对峙生长法和药敏纸片琼脂扩散法分别检测菌株的抗菌能力和药敏性。【结果】从大熊猫粪便中共分离出90株芽孢杆菌，基于BOXA1R-PCR分析菌株的遗传多样性并从中选取41株代表菌株，经16S rRNA基因测序分析后，结果显示归属于枯草芽孢杆菌(Bacillus subtilis)、萎缩芽孢杆菌(Bacillus atrophaeus)、贝莱斯芽孢杆菌(Bacillus velezensis)、甲基营养型芽孢杆菌(Bacillus methylotrophicus)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和短小芽孢杆菌(Bacillus pumilus) 6个种；主成分分析结果表明大熊猫肠道芽孢杆菌种群组成与年龄存在一定的相关性。所有的供试菌株都具有纤维素降解潜力，大部分菌株对病原菌具有不同程度的抑制作用；除对青霉素具有耐药性外，供试菌株对常见的抗生素耐受性低。【结论】年龄是影响大熊猫肠道芽孢杆菌分布的重要因素，成年大熊猫肠道芽孢杆菌种类多样性最丰富，这为大熊猫益生菌制剂的开发提供了菌种资源。

Abstract:

[Background] The intestinal microflora of giant panda are abundant, and the population structure is related to the age of the host, living environment, seasonal changes and other factors, among which age is one of the important factors affecting the composition of intestinal microflora. [Objective] Study the diversity of Bacillus isolated from giant panda gut in different age, in order to explore the relationship between the diversity of Bacillus isolated from giant panda gut and age, which provide strain resources for the development of new probiotic. [Methods] Spread plate method was employed to isolate Bacillus. BOXA1R-PCR, 16S rRNA gene phylogenetic analysis and principal component analysis (PCA) were used to analyze the diversity of culturable Bacillus. Confrontation growth method and disk diffusion test were employed to test the antibacterial activity and antibiotic susceptibility of isolates. [Results] Total 90 strains belong to Bacillus genera were isolated. Based on the result of BOXA1R-PCR analysis, 41 representative strains were selected for further 16S rRNA gene sequencing. The result showed the isolates were affiliated to 6 species, including Bacillus subtilis, B. atrophaeus, B. velezensis, B. methylotrophicus, B. amyloliquefaciens, B. pumilus. The result of PCA showed the population composition of Bacillus in giant panda gut was influenced by age. All of representative isolates possessed CMCase ability, most of isolates exhibited antimicrobial activity to pathogens. All the 41 representative strains showed resistance to Penicillin G and susceptible to the other 9 antibiotics. [Conclusion] This study indicated that Bacillus isolated from giant panda gut was abundant and the distribution was influenced by age, which possessed great potential in developing probiotics and can be further explored.