Multi-Loci Gene Sequencing and Identification of Bifidobacteria Strains Isolated from Dairy and Pharmaceutical Sources in South Africa

The 16S rRNA gene sequence analysis of Bifidobacterium species reveals high interspecies sequence similarity in the range of 87.7–99.5%. This study illustrated the extent of superiority of a multigenic approach, involving protein-coding genes, in comparison to the 16S rRNA gene, to precisely delineate presumptive Bifidobacterium isolates obtained from probiotic milk beverages, culture collections and pharmaceutical probiotic preparations. Oligonucleotide pairs PurF-rev/PurF-uni; RpoC-uni/RpoC-rev; DnaB-uni/DnaB-rev; DnaG-uni/DnaG-rev; and ClpC-uni/ClpC-rev amplified housekeeping genes while 27F/1492R amplified the 16S rRNA gene of the presumptive bifidobacteria in a polymerase chain reaction. Sequences of 16S rRNA gene and some protein-coding genes effectively identified the isolates. Phylogenetic analysis together with concatenation showed that clpC, purF and dnaG genes had over 8-fold better discriminatory power than the 16S rRNA gene in discriminating between Bifidobacterium isolates. However, phylogenetic analysis involving dnaB and rpoC gene sequences or their concatenated trees showed discrepancies in clustering isolates with designated type strains.对双歧杆菌（Bifidobacterium）物种的16S rRNA基因序列分析结果显示，其种间序列相似性介于87.7%~99.5%之间。本研究阐明了多基因分析策略相较于16S rRNA基因的优势程度：该策略涉及蛋白编码基因，可精准区分从益生菌乳饮料、菌种保藏中心以及药用益生菌制剂中分离得到的疑似双歧杆菌菌株。实验中，寡核苷酸引物对PurF-rev/PurF-uni、RpoC-uni/RpoC-rev、DnaB-uni/DnaB-rev、DnaG-uni/DnaG-rev以及ClpC-uni/ClpC-rev可扩增持家基因，而引物对27F/1492R则通过聚合酶链式反应（polymerase chain reaction, PCR）扩增疑似双歧杆菌的16S rRNA基因。16S rRNA基因与部分蛋白编码基因的序列信息可有效完成分离株的鉴定工作。系统发育分析结合序列拼接结果表明，clpC、purF与dnaG基因的区分能力较16S rRNA基因高出8倍以上。不过，基于dnaB和rpoC基因序列或其拼接构建的系统发育树，在对分离株与指定模式菌株进行聚类分析时出现了结果偏差。