Huang Chien-Hsun, Li Shiao-Wen, Huang Lina, Watanabe Koichi
Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan.
Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan.
Front Microbiol. 2018 Aug 22;9:1974. doi: 10.3389/fmicb.2018.01974. eCollection 2018.
, and are phenotypically and genotypically closely related, and together comprise the group. Although the strains of this group are commercially valuable as probiotics, the taxonomic status and nomenclature of the group have long been contentious because of the difficulties in identifying these three species by using the most frequently used genotypic methodology of 16S rRNA gene sequencing. Long used as the gold standard for species classification, DNA-DNA hybridization is laborious, requires expert skills, and is difficult to use routinely in laboratories. Currently, genome-based comparisons, including average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH), are commonly applied to bacterial taxonomy as alternatives to the gold standard method for the demarcating phylogenetic relationships. To establish quick and accurate methods for identifying strains in the group at the species and subspecies levels, we developed species- and subspecies-specific identification methods based on housekeeping gene sequences and whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectral pattern analysis. By phylogenetic analysis based on concatenated housekeeping gene sequences (, and ), 53 strains were separated into four clusters corresponding to the four species: and , and sp. nov. A multiplex minisequencing assay using single nucleotide polymorphism (SNP)-specific primers based on the gene sequences and species-specific primers based on the gene sequences provided high resolution that enabled the strains at the species level to be identified as , and . By MALDI-TOF MS analysis coupled with an internal database and ClinProTools software, species- and subspecies-level group strains were identified based on reliable scores and species- and subspecies-specific MS peaks. The strains were distinguished clearly at the subspecies level based on subspecies-specific MS peaks. This article describes the rapid and accurate methods used for identification and classification of strains in the group based on housekeeping gene sequences and MALDI-TOF MS analysis as well as the novel speciation of this group including . sp. nov. and '' by genome-based methods.
[具体物种名称1]、[具体物种名称2]和[具体物种名称3]在表型和基因型上密切相关，共同构成了[该组名称]组。尽管该组菌株作为益生菌具有商业价值，但由于使用最常用的16S rRNA基因测序基因型方法来鉴定这三个物种存在困难，[该组名称]组的分类地位和命名长期存在争议。DNA-DNA杂交长期以来一直用作物种分类的金标准，但其操作繁琐，需要专业技能，且难以在实验室中常规使用。目前，基于基因组的比较，包括平均核苷酸同一性（ANI）和数字DNA-DNA杂交（dDDH），作为划分系统发育关系的金标准方法的替代方法，常用于细菌分类学。为了建立快速准确的方法来鉴定[该组名称]组中菌株的物种和亚种水平，我们基于管家基因序列和全细胞基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）光谱模式分析开发了物种和亚种特异性鉴定方法。通过基于串联管家基因序列（[具体基因1]、[具体基因2]和[具体基因3]）的系统发育分析，53株菌株被分为四个簇，对应四个物种：[具体物种名称1]、[具体物种名称2]、[具体物种名称3]和[新物种名称]。使用基于[具体基因1]基因序列的单核苷酸多态性（SNP）特异性引物和基于[具体基因2]基因序列的物种特异性引物的多重微测序分析提供了高分辨率，能够在物种水平将菌株鉴定为[具体物种名称1]、[具体物种名称2]和[具体物种名称3]。通过结合内部数据库和ClinProTools软件的MALDI-TOF MS分析，基于可靠分数和物种及亚种特异性MS峰鉴定了物种和亚种水平的[该组名称]组菌株。基于亚种特异性MS峰，在亚种水平清楚地区分了[该组名称]组菌株。本文描述了基于管家基因序列和MALDI-TOF MS分析用于鉴定和分类[该组名称]组菌株的快速准确方法，以及通过基于基因组的方法对该组包括[新物种名称]和[另一新物种名称]的新物种形成。
