Centre for Infectious Diseases and Microbiology, Level 3, ICPMR Building, Westmead Hospital, Darcy Road, Westmead, New South Wales 2145, Australia.
J Clin Microbiol. 2010 Aug;48(8):2741-8. doi: 10.1128/JCM.02201-09. Epub 2010 Jun 2.
The relatively high-level clonality of methicillin-resistant Staphylococcus aureus (MRSA) and its frequent high-level endemicity in nosocomial settings complicate the development of methods for rapid subtyping of MRSA strains that are capable of identifying person-to-person transmission in hospitals. Phage-derived open reading frame (PDORF) typing is an MRSA typing method targeting mobile genetic elements that was recently described and evaluated using a geographically restricted set of isolates. The objective of this study was to develop a multiplex PCR-reverse line blot (mPCR/RLB) assay for PDORF typing and to test its applicability on a broad range of isolates and in an environment where MRSA is highly endemic. The 16 targets were identified using a 23-primer-pair mPCR/RLB assay with two probes for each target. The method was evaluated using 42 MRSA reference strains, including those representing major international clones, and 35 isolates from episodes of suspected nosocomial transmission. In vivo stability was explored using 81 isolate pairs. Pulsed-field gel electrophoresis (PFGE) and spa typing were performed for comparison. Among the 42 reference strains, there were 33 PFGE subtypes, 30 PDORF types, and 22 spa types. Simpson's index of diversity was 0.987, 0.971, and 0.926 for PFGE subtyping, PDORF typing, and spa typing, respectively. Typing of clinical isolates by PDORF typing and PFGE demonstrated concordant results. mPCR/RLB-based PDORF typing has similar discriminatory power to that of PFGE, can assist in tracking MRSA transmission events in a setting of high-level endemicity, and has the advantage of being a high-throughput technique.
耐甲氧西林金黄色葡萄球菌（MRSA）的相对高水平克隆性及其在医院环境中的频繁高水平地方性，使得能够识别医院内人与人之间传播的 MRSA 菌株的快速亚型分型方法的发展变得复杂。噬菌体衍生的开放阅读框（PDORF）分型是一种针对移动遗传元件的 MRSA 分型方法，最近已经描述并使用地理上受限的分离物进行了评估。本研究的目的是开发一种用于 PDORF 分型的多重 PCR-反向线印迹（mPCR/RLB）检测方法，并在广泛的分离物中以及在 MRSA 高度地方性的环境中测试其适用性。使用包含两对探针的 23 对引物的 mPCR/RLB 检测方法，鉴定了 16 个靶标。该方法使用 42 株 MRSA 参考菌株进行了评估，包括代表主要国际克隆的菌株，以及 35 株疑似医院内传播事件的分离株。使用 81 对分离株进行了体内稳定性评估。进行了脉冲场凝胶电泳（PFGE）和 spa 分型以进行比较。在 42 株参考菌株中，有 33 种 PFGE 亚型、30 种 PDORF 型和 22 种 spa 型。PFGE 亚型分型、PDORF 型分型和 spa 型分型的 Simpson 多样性指数分别为 0.987、0.971 和 0.926。PDORF 分型和 PFGE 对临床分离株的分型结果一致。基于 mPCR/RLB 的 PDORF 分型具有与 PFGE 相似的区分能力，可协助追踪高水平地方性环境中的 MRSA 传播事件，并且具有高通量技术的优势。
