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

【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16SrRNA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。

[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.