Werner Guido, Willems Rob J L, Hildebrandt Bianca, Klare Ingo, Witte Wolfgang
Robert Koch Institute, Wernigerode Branch, 38855 Wernigerode, Germany.
J Clin Microbiol. 2003 Apr;41(4):1499-506. doi: 10.1128/JCM.41.4.1499-1506.2003.
A variety of methods is used for a molecular typing of Enterococcus spp. and related gram-positive bacteria including macrorestriction analysis using pulsed-field gel electrophoresis (PFGE), ribotyping, rapid amplification of polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP). To test the influence of transferable determinants on the outcome of different typing methods commonly used for enterococci, we established a homogenous strain collection of 24 transconjugants resulting from filter matings with antibiotic-resistant Enterococcus faecium. As expected, AFLP, RAPD, and PFGE all identified our model bacteria as strongly related. However, distinct differences in the resolving and discriminatory power of the tested methods could be clearly addressed. In PFGE, 22 of 24 transconjugants possessed less than a three-band difference to the recipient pattern and would be regarded as strongly related. Three different RAPD PCRs were tested; in two reactions, identical patterns for all transconjugants and the recipient were produced. One RAPD PCR produced an identical pattern for 18 transconjugants and the recipient and a clearly different pattern for the remaining 6 transconjugants due to a newly appearing fragment resulting from acquisition of the tetL gene. AFLP clusters all transconjugants into a group of major relatedness. Percent similarities were highly dependent on the method used for calculating the similarity coefficient (curve-based versus band-based similarity coefficient). Fragment patterns of digested plasmids showed the possession of nonidentical plasmids in most transconjugants. PFGE still could be recommended as the method of choice. Nevertheless, the more-modern AFLP approach produces patterns of comparable discriminatory power while possessing some advantages over PFGE (less-time-consuming internal standards). Plasmid fingerprints can be included to subdifferentiate enterococcal isolates possessing identical macrorestriction and PCR typing patterns.
多种方法可用于肠球菌属及相关革兰氏阳性菌的分子分型，包括使用脉冲场凝胶电泳（PFGE）的宏观限制性分析、核糖体分型、多态性DNA快速扩增（RAPD）以及扩增片段长度多态性（AFLP）。为了测试可转移决定簇对肠球菌常用不同分型方法结果的影响，我们建立了一个由24个转接合子组成的同质菌株库，这些转接合子是通过与耐抗生素屎肠球菌进行滤膜交配产生的。正如预期的那样，AFLP、RAPD和PFGE均将我们的模型细菌鉴定为高度相关。然而，测试方法在分辨力和鉴别力方面的明显差异可以得到明确体现。在PFGE中，24个转接合子中有22个与受体模式的条带差异小于三条，会被视为高度相关。测试了三种不同的RAPD PCR；在两个反应中，所有转接合子和受体产生了相同的模式。一种RAPD PCR为18个转接合子和受体产生了相同的模式，而其余6个转接合子由于获得tetL基因产生了一个新出现的片段，从而产生了明显不同的模式。AFLP将所有转接合子聚类为一个主要相关性组。相似百分比高度依赖于用于计算相似系数的方法（基于曲线与基于条带的相似系数）。消化质粒的片段模式显示大多数转接合子拥有不同的质粒。PFGE仍然可以被推荐为首选方法。尽管如此，更现代的AFLP方法产生的模式具有相当的鉴别力，同时相对于PFGE具有一些优势（内标耗时更少）。可以纳入质粒指纹图谱来对具有相同宏观限制性和PCR分型模式的肠球菌分离株进行亚区分。
