show Abstracthide AbstractBackground: Enterococcus faecium is a major nosocomial pathogen that causes significant morbidity and mortality worldwide. Assessment of E. faecium using Multi-Locus Sequencing Typing (MLST) is an important component of hospital infection control measures to understand the spread of this organism. Recent studies however suggest that MLST might be inadequate for E. faecium surveillance. Objectives: To use whole genome sequencing (WGS) to characterize recently identified vancomycin resistant E. faecium clones non-typable by MLST that appear to be causing a multi-jurisdictional outbreak in Australia. Methods: Illumina NextSeq and Pacific Biosciences SMRT sequencing platforms were used to determine the genome sequences of 66 non-typable E. faecium isolates. Phylogenetic and bioinformatics analyses were subsequently performed using a number of in silico tools. Results: Sixty-six E. faecium isolates were identified by WGS from multiple health jurisdictions in Australia that could not be typed by MLST due to a missing pstS allele. SMRT sequencing revealed a large chromosomal rearrangement in strain EF_23341, which most likely resulted in the deletion of pstS and several adjacent genes. Phylogenetic analysis suggests that deletion of pstS within E. faecium has arisen independently on multiple occasions. Importantly, 98 % of isolates carried vancomycin resistance genes. Conclusions: We have identified non-typable vancomycin resistant E. faecium isolates that appear to be causing a multi-jurisdictional outbreak in Australia. Identification of these isolates has important implications for MLST-based typing activities designed to monitor the spread of VRE and provides further evidence supporting the use of WGS for routine hospital surveillance of E. faecium.