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

David M Engelthaler,Jesse Trujillo,James M Schupp,Carl F Eberhard,Frederic Decruyenaere,Joel Mossong,Elizabeth M Driebe,Jolene Bowers,Jos Even,Erin Kelley,Paul Keim

doi:10.1186/1471-2334-13-339

Abstract

Backgroundspa 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).MethodsPhylogenetically 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 techniques.ResultsFourteen real-time PCR assays were developed and validated, including two assays to determine presence of spa t003 or t008. The other twelve assays successfully provided a high degree of resolution within the t003 subtype. WGST analysis of the LTC facility isolates provided greater resolution than other subtyping tools, identifying clusters indicative of ongoing transmission within LTC facilities.ConclusionscanSNP-based PCR assays are useful for local level MRSA phylotyping, especially in the presence of one or more dominant clones. The assays designed here can be easily adapted for investigating t003 MRSA strains in other regions in Western Europe. WGST provides substantially better resolution than other typing methods.

Highlights

The evolution of methicillin resistant Staphylococcus aureus (MRSA) is well documented to be heavily influenced by the success of apparently highly fit clones [1]
Like multi-locus sequence typing (MLST), spa typing is limited in its resolving power; for instance in a recent survey of spa types throughout Europe, five spa types accounted for nearly half of all MRSA strains identified [8]
We describe efforts to develop improved genotyping for MRSA isolates, within the spa t003 clone in Luxembourg, based initially on whole genome sequencing to precisely define the population structure within this narrow clonal complex

Summary

Introduction

The evolution of methicillin resistant Staphylococcus aureus (MRSA) is well documented to be heavily influenced by the success of apparently highly fit clones [1]. Local MRSA populations are frequently dominated by such successful clones, which will vary depending on geographic location [2] These dominant clones typically shape much of the endemic and epidemic spread of this pathogen. Like MLST, spa typing is limited in its resolving power; for instance in a recent survey of spa types throughout Europe, five spa types accounted for nearly half of all MRSA strains identified [8]. These methods are unable to provide sufficient utility for resolving isolates in local epidemic situations [6]. The negative impact of homoplasies, (e.g., character state conflict resulting from convergent evolution) has been well established in spa typing [2,9]

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Discussion

Conclusion