Harmsen D, Singer C, Rothgänger J, Tønjum T, de Hoog G S, Shah H, Albert J, Frosch M
Institute of Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Str. 2, Bau 17, 97080 Würzburg, Germany.
J Clin Microbiol. 2001 Mar;39(3):936-42. doi: 10.1128/JCM.39.3.936-942.2001.
Fast and reliable identification of microbial isolates is a fundamental goal of clinical microbiology. However, in the case of some fastidious gram-negative bacterial species, classical phenotype identification based on either metabolic, enzymatic, or serological methods is difficult, time-consuming, and/or inadequate. 16S or 23S ribosomal DNA (rDNA) bacterial sequencing will most often result in accurate speciation of isolates. Therefore, the objective of this study was to find a hypervariable rDNA stretch, flanked by strongly conserved regions, which is suitable for molecular species identification of members of the Neisseriaceae and Moraxellaceae. The inter- and intrageneric relationships were investigated using comparative sequence analysis of PCR-amplified partial 16S and 23S rDNAs from a total of 94 strains. When compared to the type species of the genera Acinetobacter, Moraxella, and Neisseria, an average of 30 polymorphic positions was observed within the partial 16S rDNA investigated (corresponding to Escherichia coli positions 54 to 510) for each species and an average of 11 polymorphic positions was observed within the 202 nucleotides of the 23S rDNA gene (positions 1400 to 1600). Neisseria macacae and Neisseria mucosa subsp. mucosa (ATCC 19696) had identical 16S and 23S rDNA sequences. Species clusters were heterogeneous in both genes in the case of Acinetobacter lwoffii, Moraxella lacunata, and N. mucosa. Neisseria meningitidis isolates failed to cluster only in the 23S rDNA subset. Our data showed that the 16S rDNA region is more suitable than the partial 23S rDNA for the molecular diagnosis of Neisseriaceae and Moraxellaceae and that a reference database should include more than one strain of each species. All sequence chromatograms and taxonomic and disease-related information are available as part of our ribosomal differentiation of medical microorganisms (RIDOM) web-based service (http://www.ridom.hygiene.uni-wuerzburg.de/). Users can submit a sequence and conduct a similarity search against the RIDOM reference database for microbial identification purposes.
快速且可靠地鉴定微生物分离株是临床微生物学的一个基本目标。然而，对于一些苛求的革兰氏阴性菌物种，基于代谢、酶学或血清学方法的经典表型鉴定困难、耗时且/或不充分。16S或23S核糖体DNA（rDNA）细菌测序通常能准确鉴定分离株的物种。因此，本研究的目的是找到一个高变rDNA片段，其两侧为高度保守区域，适用于奈瑟菌科和莫拉菌科成员的分子物种鉴定。使用对来自总共94株菌株的PCR扩增的部分16S和23S rDNA进行比较序列分析，研究了属间和属内关系。与不动杆菌属、莫拉菌属和奈瑟菌属的模式种相比，在所研究的部分16S rDNA（对应于大肠杆菌的第54至510位）中，每个物种平均观察到30个多态性位点，在23S rDNA基因的202个核苷酸（第1400至1600位）内平均观察到11个多态性位点。猕猴奈瑟菌和粘膜炎奈瑟菌粘膜亚种（ATCC 19696）具有相同的16S和23S rDNA序列。在沃氏不动杆菌、腔隙莫拉菌和粘膜炎奈瑟菌的情况下，两个基因中的物种聚类都是异质的。脑膜炎奈瑟菌分离株仅在23S rDNA子集中未能聚类。我们的数据表明，16S rDNA区域比部分23S rDNA更适合用于奈瑟菌科和莫拉菌科的分子诊断，并且参考数据库应包括每个物种的多个菌株。所有序列色谱图以及分类学和疾病相关信息都可作为我们基于网络的医学微生物核糖体分化（RIDOM）服务（http://www.ridom.hygiene.uni-wuerzburg.de/）的一部分获取。用户可以提交一个序列，并针对RIDOM参考数据库进行相似性搜索以进行微生物鉴定。
