Core Genome Multilocus Sequence Typing Scheme for High-Resolution Typing of Enterococcus faecium

Abstract

Enterococcus faecium, a common inhabitant of the human gut, has emerged in the last 2 decades as an important multidrugresistant nosocomial pathogen. Since the start of the 21st century, multilocus sequence typing (MLST) has been used to study the molecular epidemiology of E. faecium. However, due to the use of a small number of genes, the resolution of MLST is limited. Whole-genome sequencing (WGS) now allows for high-resolution tracing of outbreaks, but current WGS-based approaches lack standardization, rendering them less suitable for interlaboratory prospective surveillance. To overcome this limitation, we developed a core genome MLST (cgMLST) scheme for E. faecium. cgMLST transfers genome-wide single nucleotide polymorphism (SNP) diversity into a standardized and portable allele numbering system that is far less computationally intensive than SNPbased analysis of WGS data. The E. faecium cgMLST scheme was built using 40 genome sequences that represented the diversity of the species. The scheme consists of 1,423 cgMLST target genes. To test the performance of the scheme, we performed WGS analysis of 103 outbreak isolates from five different hospitals in the Netherlands, Denmark, and Germany. The cgMLST scheme performed well in distinguishing between epidemiologically related and unrelated isolates, even between those that had the same sequence type (ST), which denotes the higher discriminatory power of this cgMLST scheme over that of conventional MLST. We also show that in terms of resolution, the performance of the E. faecium cgMLST scheme is equivalent to that of an SNP-based approach. In conclusion, the cgMLST scheme developed in this study facilitates rapid, standardized, and high-resolution tracing of E. faecium outbreaks. In the last 2 decades, Enterococcus faecium has emerged as an important multidrug-resistant nosocomial pathogen causing an increasing number of bloodstream infections, mainly in debilitated patients (1-3). Currently, Ͼ90% of the E. faecium strains causing infections have acquired ampicillin resistance, and an increasing number of health care-associated infections and outbreaks are caused by E. faecium strains that are resistant to both ampicillin and vancomycin (4). The global emergence of ampicillin-and vancomycin-resistant E. faecium (VRE) as nosocomial pathogens started in the United States in the late 1980s/early 1990s and occurred in other parts in the world 1 or 2 decades later. In Europe, more particularly in France, Germany, Denmark, and the Netherlands, VRE first spread among livestock due to the use of the vancomycin analog avoparcin as a growth promoter, and it only relatively recently became an important nosocomial pathogen (5, 6). Different molecular typing methods have been used to study the epidemiology of E. faecium, ranging from fingerprint-based methods, like pulsed-field gel electrophoresis (PFGE) (7), ribotyping (8), and amplified fragment length polymorphism (9), to PCR-based methods, like multilocus variable-number tandemrepeat analysis (10) and the sequenced-bas...

Whole-Genome Multilocus Sequence Typing of Extended-Spectrum-Beta-Lactamase-Producing Enterobacteriaceae

“…wgMLST was shown to have higher discriminatory power and typeability than in silico MLST. After the introduction of WGS as a typing tool with high potential, gene-by-gene comparison of WGS data has been applied successfully for several species in outbreak settings (22)(23)(24)(25)(26)(27). To our knowledge, this large multicenter study is the first to apply wgMLST for typing of Enterobacteriaceae in nonoutbreak settings and to include a comprehensive evaluation of its performance in a large collection of isolates without a clear epidemiological link.…”

“…Whole-genome multilocus sequence typing (wgMLST) is a WGS-based typing method that extends MLST to the genome level (20). Today, such gene-bygene comparison of WGS data has been applied successfully for several species in outbreak settings, but data for Enterobacteriaceae and nonoutbreak settings are still limited (22)(23)(24)(25)(26)(27). The aim of this study was to develop representative wgMLST schemes for the Citation Kluytmans-van den Bergh MFQ, Rossen JWA, Bruijning-Verhagen PCJ, Bonten MJM, Friedrich AW, Vandenbroucke-Grauls CMJE, Willems RJL, Kluytmans JAJW, on behalf of the SoM Study Group.…”

“…However, sequence conservation in housekeeping genes limits its discriminatory power and thus its use in outbreak investigations (16)(17)(18). Whole-genome sequencing (WGS) has emerged as an ultimate typing tool that fits any bacterial species, study type, and laboratory (19)(20)(21)(22)(23)(24)(25)(26)(27). Knowledge of the sequence of the entire genome, rather than of only a few loci, has greatly increased the precision of molecular epidemiologic data and allows its use in infection control, where questions of similarity of isolates focus on recently diverged isolates.…”

Genomic Analysis of Bacterial Outbreaks

“…In light of the very large genome plasticity of many bacterial species, fixed compositions of the core and auxiliary genomes are almost impossible, which creates an additional problem for the stability of the scheme. Nevertheless, this approach has gained some popularity and cgMLST ("core genome MLST", a reduced version of wgMLST as described above) schemes are now available for several pathogens including S. aureus, Listeria monocytogenes, Enterococcus faecium (de Been et al, 2015), and S. enterica (Taylor et al, 2015), among others.…”

Vancomycin-resistant Enterococcus faecium high-resolution typing by core genome multilocus sequence typing

“…Recently, a core genome MLST (cgMLST) scheme for E. faecium strains, consisting of 1,423 target genes, was developed by de Been et al, and compared by the authors to a single-nucleotide polymorphism (SNP)-based approach since it allows a high-resolution tracing of E. faecium clones [12].…”