Sequencing of the rpoB gene and flanking spacers for molecular identification of Acinetobacter species
Sequencing of the rpoB gene and flanking spacers for molecular identification of Acinetobacter species
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DOI：
10.1128/jcm.44.3.827-832.2006
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发表时间：
2006-03-01
影响因子：
9.4
通讯作者：
Raoult, D
中科院分区：
文献类型：
--
作者：
La Scola, B;Gundi, VAKB;Raoult, D
不动杆菌的种类是根据几种表型特征、DNA-DNA同源性的结果以及最近的16S rRNA基因序列的相似性或差异性来定义的。然而，16S rRNA基因的多态性不足以明确区分所有不动杆菌种类。采用基于RNA聚合酶β -亚基基因（rpoB）的鉴定方法对不动杆菌属内的种进行了划分，确定了17种不动杆菌属参考菌株和7种未命名的基因组种的rpoB基因全序列和侧翼间隔序列（rplL-rpoB和rpoB- rpoc）。通过完整的基因序列（4089 bp），我们将所有物种划分为不同的群。使用这些序列构建的系统发育树的bootstrap值高于使用16S rRNA或gyrB或recA基因获得的系统发育树。四对引物使我们能够扩增和测序rpoB基因的两个高度多态性的部分序列（350和450 bp）。设计并测试了这些和侧翼间隔对17种不动杆菌参考菌株和7种未命名基因组种的快速鉴定。这四个可变序列中的每一个都使我们能够描绘出大多数物种。要区分格林不动杆菌、朱尼不动杆菌、贝氏不动杆菌和基因组种9，至少要有两个多态序列。最后，对21株鲍曼不动杆菌临床分离株进行了种内关系检测，并通过比较rpoB基因的部分序列及其侧间隔序列，正确地将其定位为同一种。
Acinetobacter species are defined on the basis of several phenotypic characters, results of DNA-DNA homology, and more recently, similarities or dissimilarities in 16S rRNA gene sequences. However, the 16S rRNA gene is not polymorphic enough to clearly distinguish all Acinetobacter species. We used an RNA polymerase beta-subunit gene (rpoB)-based identification scheme for the delineation of species within the genus Acinetobacter, and towards that end, we determined the complete rpoB gene and flanking spacer (rplL-rpoB and rpoB-rpoC) sequences of the 17 reference strains of Acinetobacter species and 7 unnamed genomospecies. By using complete gene sequences (4,089 bp), we clearly separated all species and grouped them into different clusters. A phylogenetic tree constructed using these sequences was supported by bootstrap values higher than those obtained with 16S rRNA or the gyrB or recA gene. Four pairs of primers enabled us to amplify and sequence two highly polymorphic partial sequences (350 and 450 bp) of the rpoB gene. These and flanking spacers were designed and tested for rapid identification of the 17 reference strains of Acinetobacter species and 7 unnamed genomospecies. Each of these four variable sequences enabled us to delineate most species. Sequences of at least two polymorphic sequences should be used to distinguish Acinetobacter grimontii, Acinetobacter junii, Acinetobacter baylyi, and genomic species 9 from one another. Finally, 21 clinical isolates of Acinetobacter baumannii were tested for intraspecies relationships and assigned correctly to the same species by comparing the partial sequences of the rpoB gene and its flanking spacers.