Rapid and robust phylotyping of spa t003, a dominant MRSA clone in Luxembourg and other European countries

Background: spa typing is a common genotyping tool for methicillin-resistant Staphylococcus aureus (MRSA) in Europe. Given the high prevalence of dominant clones, spa-typing is proving to be limited in its ability to distinguish outbreak isolates from background isolates. New molecular tools need to be employed to improve subtyping of dominant local MRSA strains (e.g., spa type t003). Methods: Phylogenetically critical, or canonical, SNPs (can-SNPs) were identified as subtyping targets through sequence analysis of 40 MRSA whole genomes from Luxembourg. Real-time PCR assays were designed around target SNPs and validated using a repository of 240 previously sub-typed and epidemiologically characterized Luxembourg MRSA isolates, including 153 community and hospital isolates, 69 isolates from long term care (LTC) facilities, and 21 prospectively analyzed MRSA isolates. Selected isolates were also analyzed by whole genome SNP typing (WGST) for comparison to the SNP assays and other subtyping...研究背景：spa分型（spa typing）是欧洲地区用于耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus, MRSA）的常用基因分型工具。鉴于优势克隆的高流行率，spa分型在区分暴发分离株与背景分离株时能力有限，亟需开发新型分子工具以优化当地优势MRSA菌株（如spa型别t003）的亚型分型工作。 研究方法：通过对卢森堡地区40株MRSA全基因组开展序列分析，筛选得到系统发育关键的标准单核苷酸多态性（canonical single nucleotide polymorphisms, can-SNPs）作为亚型分型靶点。围绕目标单核苷酸多态性（single nucleotide polymorphism, SNP）设计实时聚合酶链式反应（real-time PCR）检测体系，并利用240株已完成亚型分型与流行病学特征鉴定的卢森堡MRSA分离株库对该体系进行验证，该菌株库包含153株社区与医院分离株、69株来自长期护理（long term care, LTC）机构的分离株，以及21株经前瞻性分析的MRSA分离株。同时选取部分分离株开展全基因组SNP分型（whole genome SNP typing, WGST），以与该SNP检测方法及其他亚型分型技术进行对比……