Koreen Larry, Ramaswamy Srinivas V, Naidich Steven, Koreen Irina V, Graff Gavin R, Graviss Edward A, Kreiswirth Barry N
New Jersey Medical School and Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.
J Clin Microbiol. 2005 Aug;43(8):3985-94. doi: 10.1128/JCM.43.8.3985-3994.2005.
Molecular techniques such as spa typing and multilocus sequence typing use DNA sequence data for differentiating Staphylococcus aureus isolates. Although spa typing is capable of detecting both genetic micro- and macrovariation, it has less discriminatory power than the more labor-intensive pulsed-field gel electrophoresis (PFGE) and costly genomic DNA microarray analyses. This limitation hinders strain interrogation for newly emerging clones and outbreak investigations in hospital or community settings where robust clones are endemic. To overcome this constraint, we developed a typing system using DNA sequence analysis of the serine-aspartate (SD) repeat-encoding region within the gene encoding the keratin- and fibrinogen-binding clumping factor B (clfB typing) and tested whether it is capable of discriminating within clonal groups. We analyzed 116 S. aureus strains, and the repeat region was present in all isolates, varying in sequence and in length from 420 to 804 bp. In a sample of 36 well-characterized genetically diverse isolates, clfB typing subdivided identical spa and PFGE clusters which had been discriminated by whole-genome DNA microarray mapping. The combination of spa typing and clfB typing resulted in a discriminatory power (99.5%) substantially higher than that of spa typing alone and closely approached that of the whole-genome microarray (100.0%). clfB typing also successfully resolved genetic differences among isolates differentiated by PFGE that had been collected over short periods of time from single hospitals and that belonged to the most prevalent S. aureus clone in the United States. clfB typing demonstrated in vivo, in vitro, and interpatient transmission stability yet revealed that this locus may be recombinogenic in a primarily clonal population structure. Taken together, these data show that the SD repeat-encoding region of clfB is a highly stable marker of microvariation, that in conjunction with spa typing it may serve as a DNA sequence-based alternative to PFGE for investigating genetically similar strains, and that it is useful for analyzing collections of isolates in both long-term population-based and local epidemiologic studies.
诸如葡萄球菌蛋白A（spa）分型和多位点序列分型等分子技术利用DNA序列数据来区分金黄色葡萄球菌分离株。尽管spa分型能够检测到遗传上的微观和宏观变异，但与劳动强度更大的脉冲场凝胶电泳（PFGE）以及成本更高的基因组DNA微阵列分析相比，它的鉴别能力较弱。这一局限性阻碍了对医院或社区环境中新兴克隆株的菌株鉴定以及暴发调查，在这些环境中存在着流行的优势克隆株。为克服这一限制，我们开发了一种分型系统，该系统通过对编码角蛋白和纤维蛋白原结合凝聚因子B的基因内丝氨酸 - 天冬氨酸（SD）重复编码区域进行DNA序列分析（clfB分型），并测试其是否能够在克隆组内进行鉴别。我们分析了116株金黄色葡萄球菌菌株，所有分离株中均存在重复区域，其序列和长度在420至804 bp之间变化。在36株特征明确、遗传背景多样的分离株样本中，clfB分型细分了通过全基因组DNA微阵列图谱已鉴别出的相同spa和PFGE聚类。spa分型与clfB分型相结合产生的鉴别能力（99.5%）显著高于单独的spa分型，且与全基因组微阵列的鉴别能力（100.0%）相近。clfB分型还成功解析了通过PFGE区分的分离株之间的遗传差异，这些分离株是在短时间内从单一医院收集的，并且属于美国最常见的金黄色葡萄球菌克隆株。clfB分型在体内、体外以及患者间传播中均显示出稳定性，但也揭示了该位点在主要为克隆性的群体结构中可能具有重组性。综上所述，这些数据表明clfB的SD重复编码区域是微观变异的高度稳定标记，与spa分型结合，它可作为基于DNA序列的替代PFGE的方法用于研究遗传相似菌株，并且在长期基于人群的研究和局部流行病学研究中分析分离株集合时均很有用。
