广西蚕沙细菌组成多样性解析和VBNC菌群的发掘

Bacterial diversity and VBNC bacteria in silkworm excrement from Guangxi

摘要:

[目的]解析广西蚕沙中细菌群落组成与多样性，为蚕沙中菌种资源发掘和蚕沙的综合利用提供科学依据。[方法]通过高通量测序技术研究细菌群落组成特征，同时利用常规稀释涂布平板法和基于复苏促进因子（Rpf）的MPN培养系统解析并筛选蚕沙中可培养和活的非可培养（VBNC）状态的优势菌群，并经16S rRNA基因测序对筛选得到的菌株作初步分类鉴定。[结果]高通量测序表明，广西蚕沙样品中细菌归属于10个门、18个纲、27个目、57个科、96个属，其中4个属的丰度达1%以上，优势菌群为变形菌门（Proteobacteria）肠杆菌属（Enterobacter）；通过稀释涂布平板法共获得14个属的33株可培养细菌，其中4个属（Citrobacter、Weissella、Chitinophaga、Pseudoclavibacter）在高通量测序中未被检测到；而在MPN培养系统中，基于复苏促进因子的处理组细菌总数最大检出丰度提高了100倍，并从中共检出21株对Rpf敏感的VBNC菌株，其中6个属（Paenibacillus、Caulobacter、Roseomonas、Pantoea、Erwinia、Acinetobacter）为传统分离法中未能获得的菌属，2个属（Paenibacillus和Caulobacter）在高通量测序中未被检测到。[结论]蚕沙细菌多样性丰富，不同方法解析蚕沙细菌组成多样性存在一定差异，而且通过添加Rpf首次发现了蚕沙中存在大量过去未被认知的VBNC状态细菌，其中从16S rRNA基因序列同源性推测有3个VBNC菌株为潜在新种。此研究结果为深入挖掘蚕沙中微生物资源提供了新的视角与途径，为蚕沙的综合处理提供了研究基础。

Abstract:

[Objective] To analyze the composition and diversity of bacterial in silkworm excrement from Guangxi, and to explore microbial resource of silkworm excrement. [Methods] Through high-throughput sequencing technology, we analyzed the composition of bacterial communities in silkworm excrement. Meanwhile, we isolated culturable bacteria and those in viable but non-culturable (VBNC) state in silkworm excrement using spread-plate method and resuscitation promoting factor (Rpf)-based MPN method, and then classified and identified the isolated strains through 16S rRNA gene sequence analysis. [Results] The bacteria detected from silkworm excrement of Guangxi belonged to 10 phyla, 18 classes, 27 orders, 57 families and 96 genera, in which the bacteria abundance of 4 genera was greater than 1% and the dominant microflora was Enterobacter (Proteobacteria). We obtained 33 culturable bacteria from 14 genera through the spread-plate method, in which 4 genera (Citrobacter, Weissella, Chitinophaga, Pseudoclavibacter) were not detected by high-throughput sequencing. The detection abundance of bacteria increased by up to 100 times by Rpf-based MPN method. Also, we found 21 Rpf-sensitive and resuscitation-cultivable VBNC strains from the Rpf-treatment group, in which 6 genera (Paenibacillus, Caulobacter, Roseomonas, Pantoea, Erwinia, Acinetobacter) and 2 genera (Paenibacillus and Caulobacter) were not found through spread-plate method and high-throughput sequencing, respectively. [Conclusion] Our results provide a new perspective and a new approach for exploring and utilizing the microbial resources in silkworm excrement.