Johansson A, Ibrahim A, Göransson I, Eriksson U, Gurycova D, Clarridge J E, Sjöstedt A
Infectious Diseases, Umeå University, and Department of Microbiology, Defence Research Establishment, Sweden.
J Clin Microbiol. 2000 Nov;38(11):4180-5. doi: 10.1128/JCM.38.11.4180-4185.2000.
Previous studies have demonstrated that the four subspecies of the human pathogen Francisella tularensis, despite showing marked variations in their virulence for mammals and originating from different regions in the Northern Hemisphere, display a very close phylogenetic relationship. This property has hampered the development of generally applicable typing methods. To overcome this problem, we evaluated the use of PCR for discrimination of the subspecies using various forms of long arbitrary primers or primers specific for repetitive extragenic palindromic sequences (REP) or enterobacterial repetitive intragenic consensus (ERIC) sequences. Patterns generated by use of REP, ERIC, or long arbitrary primers allowed differentiation at the species level and of the four subspecies of F. tularensis. With each of these three methods, similar or identical clustering of strains was found, and groups of strains of different geographical origins or differing in virulence showed distinct patterns. The discriminatory indices of the methods varied from 0.57 to 0.65; thus, the patterns were not sufficiently discriminatory to distinguish individual strains. The sequence of a fragment generated by amplification with an arbitrary primer was determined, and a region showing interstrain heterogeneity was identified. Specific primers were designed, and a PCR was developed that distinguished strains of F. tularensis subsp. holarctica from strains of other F. tularensis subspecies, including strains of the highly virulent F. tularensis subsp. tularensis. Notably, one European isolate showed the genetic pattern typical of the highly virulent F. tularensis subsp. tularensis, generally believed to exist only in North America. It is proposed that a combination of the specific PCR together with one method generating subspecies-specific patterns is suitable as a rapid and relatively simple strategy for discrimination of Francisella species and subspecies.
先前的研究表明，人类病原体土拉弗朗西斯菌的四个亚种，尽管在对哺乳动物的毒力上表现出显著差异且起源于北半球的不同地区，但显示出非常密切的系统发育关系。这一特性阻碍了通用分型方法的发展。为克服这一问题，我们评估了使用聚合酶链反应（PCR），通过各种形式的长任意引物或针对重复基因外回文序列（REP）或肠杆菌重复基因内共有序列（ERIC）的引物来区分亚种。使用REP、ERIC或长任意引物产生的图谱能够在种水平以及土拉弗朗西斯菌的四个亚种水平上进行区分。使用这三种方法中的每一种，都发现菌株有相似或相同的聚类情况，不同地理起源或毒力不同的菌株组显示出不同的图谱。这些方法的鉴别指数在0.57至0.65之间；因此，这些图谱的鉴别力不足以区分单个菌株。测定了用任意引物扩增产生的片段的序列，并鉴定出一个显示菌株间异质性的区域。设计了特异性引物，并开发了一种PCR方法，该方法可区分土拉弗朗西斯菌全北区亚种的菌株与其他土拉弗朗西斯菌亚种的菌株，包括高毒力的土拉弗朗西斯菌土拉热亚种的菌株。值得注意的是，一株欧洲分离株显示出高毒力的土拉弗朗西斯菌土拉热亚种的典型遗传图谱，而通常认为该亚种仅存在于北美。有人提出，将特异性PCR与一种产生亚种特异性图谱的方法相结合，适合作为一种快速且相对简单的策略来区分弗朗西斯菌的种和亚种。
