Murchan Stephen, Kaufmann Mary Elizabeth, Deplano Ariane, de Ryck Raf, Struelens Marc, Zinn Christina Elsberg, Fussing Vivian, Salmenlinna Saara, Vuopio-Varkila Jaana, El Solh Névine, Cuny Christina, Witte Wolfgang, Tassios Panayotis T, Legakis Nikolas, van Leeuwen Willem, van Belkum Alex, Vindel Anna, Laconcha Idoia, Garaizar Javier, Haeggman Saara, Olsson-Liljequist Barbro, Ransjo Ulrika, Coombes Geoffrey, Cookson Barry
Laboratory of Hospital Infection, Central Public Health Laboratory, London NW9 5HT, United Kingdom.
J Clin Microbiol. 2003 Apr;41(4):1574-85. doi: 10.1128/JCM.41.4.1574-1585.2003.
Pulsed-fieldgel electrophoresis (PFGE) is the most common genotypic method used in reference and clinical laboratories for typing methicillin-resistant Staphylococcus aureus (MRSA). Many different protocols have been developed in laboratories that have extensive experience with the technique and have established national databases. However, the comparabilities of the different European PFGE protocols for MRSA and of the various national MRSA clones themselves had not been addressed until now. This multinational European Union (EU) project has established for the first time a European database of representative epidemic MRSA (EMRSA) strains and has compared them by using a new "harmonized" PFGE protocol developed by a consensus approach that has demonstrated sufficient reproducibility to allow the successful comparison of pulsed-field gels between laboratories and the tracking of strains around the EU. In-house protocols from 10 laboratories in eight European countries were compared by each center with a "gold standard" or initial harmonized protocol in which many of the parameters had been standardized. The group found that it was not important to standardize some elements of the protocol, such as the type of agarose, DNA block preparation, and plug digestion. Other elements were shown to be critical, namely, a standard gel volume and concentration of agarose, the DNA concentration in the plug, the ionic strength and volume of running buffer used, the running temperature, the voltage, and the switching times of electrophoresis. A new harmonized protocol was agreed on, further modified in a pilot study in two laboratories, and finally tested by all others. Seven laboratories' gels were found to be of sufficiently good quality to allow comparison of the strains by using a computer software program, while two gels could not be analyzed because of inadequate destaining and DNA overloading. Good-quality gels and inclusion of an internal quality control strain are essential before attempting intercenter PFGE comparisons. A number of clonally related strains have been shown to be present in multiple countries throughout Europe. The well-known Iberian clone has been demonstrated in Belgium, Finland, France, Germany, and Spain (and from the wider HARMONY collection in Portugal, Slovenia, and Sweden). Strains from the United Kingdom (EMRSA-15 and -16) have been identified in several othercountries, and other clonally related strains have also been identified. This highlights the need for closer international collaboration to monitor the spread of current epidemic strains as well as the emergence of new ones.
脉冲场凝胶电泳（PFGE）是参考实验室和临床实验室用于耐甲氧西林金黄色葡萄球菌（MRSA）分型的最常用基因分型方法。许多经验丰富且已建立国家数据库的实验室开发了多种不同的方案。然而，不同的欧洲MRSA的PFGE方案以及各个国家的MRSA克隆本身的可比性此前尚未得到解决。这个欧盟跨国项目首次建立了一个具有代表性的流行MRSA（EMRSA）菌株的欧洲数据库，并通过使用一种新的“统一”PFGE方案对它们进行比较，该方案是通过共识方法制定的，已证明具有足够的可重复性，能够成功比较不同实验室之间的脉冲场凝胶，并追踪欧盟范围内的菌株。八个欧洲国家的10个实验室的内部方案由每个中心与一种“金标准”或初始统一方案进行比较，在该初始统一方案中许多参数已标准化。该团队发现，对方案的某些要素进行标准化并不重要，如琼脂糖类型、DNA块制备和胶块消化。其他要素则被证明至关重要，即标准凝胶体积和琼脂糖浓度、胶块中的DNA浓度、所用运行缓冲液的离子强度和体积、运行温度、电压以及电泳的切换时间。大家商定了一个新的统一方案，在两个实验室的初步研究中进一步修改，最后由其他所有实验室进行测试。发现七个实验室的凝胶质量足够好，可以使用计算机软件程序对菌株进行比较，而由于脱色不足和DNA过载，有两个凝胶无法分析。在尝试进行中心间PFGE比较之前，高质量的凝胶和纳入内部质量控制菌株至关重要。已证明许多克隆相关菌株在欧洲多个国家都有出现。著名的伊比利亚克隆已在比利时、芬兰、法国、德国和西班牙被发现（以及来自葡萄牙、斯洛文尼亚和瑞典更广泛的HARMONY样本库）。来自英国的菌株（EMRSA - 15和 - 16）在其他几个国家也已被鉴定出来，其他克隆相关菌株也已被识别。这突出表明需要更紧密的国际合作来监测当前流行菌株的传播以及新菌株的出现。
