High-Resolution Melting Genotyping of Enterococcus faecium Based on Multilocus Sequence Typing Derived Single Nucleotide Polymorphisms

Steven Y C Tong,Elizabeth A Grabsch,Shirley Xie,Farshid Dakh,M Lindsay Grayson,Susan A Ballard,Paul D R Johnson,Benjamin P Howden,Leisha J Richardson,Philip M Giffard,Tarek Msadek

doi:10.1371/journal.pone.0029189

Abstract

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 “melting types” (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.

Highlights

Enterococcus faecium emerged as a hospital-associated pathogen in the 1990s in the USA, Europe and Australia
In silico analysis based on predicted Tm differences suggested that these eight fragments would partition the 567 sequence types (STs) currently in the multilocus sequence typing (MLST) database into 230 Melting Types (MelTs) with a D of 0.991, where the MLST database is regarded as providing D = 1
We have developed and validated an high-resolution melting (HRM) based method for genotyping E. faecium that can be performed at low cost and high throughput

Summary

Introduction

Enterococcus faecium emerged as a hospital-associated pathogen in the 1990s in the USA, Europe and Australia. The acquisition of resistance to ampicillin and vancomycin and spread of successful vancomycin-resistant clones has resulted in an increasing burden of vancomycin-resistant E. faecium (VREfm) as a difficult to treat nosocomial infection [1]. Pulsed-field gel electrophoresis (PFGE) has been considered the reference method for tracking outbreaks due to its high discriminatory power [2]. PFGE remains difficult to standardize between laboratories, and the high genomic plasticity of E. faecium means that using PFGE data to track strains over long time periods and to infer the large scale population structure can be problematic [1]. Multilocus sequence typing (MLST) provides portable genotyping data that unambiguously reveal the population structure and long-term patterns of dissemination [3].

Methods

Results

Conclusion