Working group participants from eastern Australia and northern New Zealand contributed ST796 E. faecium isolates from their local outbreaks – Fig. 1 shows the geographic distribution and number of isolates per institution. Initial identification of E. faecium was performed by the source hospital microbiology laboratory. Most isolates were originally classified as likely ST796 by Single Nucleotide Polymorphism High Resolution Melt (SNP HRM – in short, a rapid typing system based on the melt curves of common SNPs with variable G + C content) after referral to Austin Health [19]. Subsequently, speciation was re-confirmed by MALDI-TOF mass spectrometry or Vitek® 2 and the vancomycin resistance genotype determined by PCR as previously described [20]. A summary of the isolates with their epidemiological and meta-data is provided in Additional file 1: Table S1 and described in outline below.
Rates of Enterococcus faecium bacteremia at Austin health
Using discharge data collected by Austin Health’s information management system, rates of VRE E. faecium bacteremia by ST over 6-month periods per 1000 discharged patients were calculated from 2011 to 2014. These were compared with previously published rates from 1998 to 2010 [12].
Antibiotic susceptibility testing
Antibiotic susceptibility testing was performed on all Austin Health, Alfred Health and John Hunter Hospital ST796 E. faecium blood culture isolates using the Vitek® 2 and Gram Positive Susceptibility card, AST-P612, according to the manufacturer’s instructions (bioMérieux). Austin Health isolates were further assessed by Etest® according to the manufacturer’s instructions (bioMérieux) for susceptibility to streptomycin, daptomycin, tigecycline, quinupristin-dalfopristin and chloramphenicol. Minimum Inhibitory Concentrations (MICs) were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines where applicable.
Provenance of 131 Enterococcus faecium isolates that underwent WGS
Alfred Health
Alfred Health, a university teaching hospital in Melbourne with solid organ and allogeneic bone marrow transplantation units, experienced a marked increase in VRE BSI in 2014; 17 consecutive VRE BSI isolates (all E. faecium) were investigated as the STs and relatedness of isolates were unknown. Sixteen isolates were found to be ST796 (the other was ST203). One BSI isolate from 2013 identified retrospectively via SNP HRM typing as likely to be ST796 was included in the analysis [21].
Auckland City Hospital
Thirteen isolates were referred from Auckland City Hospital, New Zealand, during a VRE colonization outbreak that affected more than 50 hospitalized patients in 2012. The outbreak appeared to be clonal by pulsed field gel electrophoresis (PFGE, data not shown) performed at the national reference laboratory (ESR, Wellington, New Zealand), and followed the repatriation in 2012 from Monash Health in Melbourne of a patient who had been hospitalized while travelling in Australia and acquired VRE prior to transfer. This patient was found to have two PFGE pulsotypes of colonizing VRE E. faecium, detected serendipitously on antibiotic susceptibility testing, with a double zone of inhibition observed around a streptomycin disc; one matched the outbreak ST796 by MLST, whereas the other pulsotype was ST203. The patient’s earlier rectal colonization and liver abscess VRE E. faecium isolates from Monash Health were identified as ST203.
A single colonizing isolate from a patient in another Auckland hospital, with no known links to Auckland City Hospital, also typed as ST796 by MLST so was included in WGS; this patient had similarly been transferred from a Melbourne hospital in 2012.
Austin Health
To capture the first appearance of ST796 in Austin BSI cases and to monitor its incidence compared to other STs over time, 79 consecutive E. faecium isolates obtained from all episodes of BSI at Austin Health from 1st January 2011 to 31st December 2014 were assessed. Thirty-nine were vanB VRE, and 24 of these ST796. Three additional isolates from early 2015 were subsequently identified as ST796, so were included in phenotypic and phlyogenomic analyses. Additionally, 32 colonizing isolates of E. faecium over the same period obtained from high-risk patients who are routinely screened for VRE rectal colonization, and one environmental isolate detected on surveillance, with matching SNP HRM genotype to the first ST796 BSI isolate, were included – one VSE, 30 vanB, one vanA and one with both vanA and vanB.
John Hunter Hospital
One hundred nine E. faecium blood culture and selected screening isolates from John Hunter Hospital in New South Wales (NSW) obtained between 2007 and 2015 were available for WGS. Of these we identified 12 ST796 isolates, all from 2015. These comprised one VSE and one VRE from adults with positive blood cultures, and ten screening isolates from a NICU colonization outbreak.
Monash Health
A large outbreak of VRE colonization in the NICU and special care nurseries at Monash Health in Melbourne was recognized in October 2013, with VRE never previously detected in these wards [17]. WGS was performed on 26 isolates (two environmental swab isolates, one urine culture, one eye swab, one blood culture and 21 screening samples from more than 40 colonized neonates).
Royal Adelaide Hospital
Two of five patient isolates from a VRE colonization outbreak in September 2013 at the Royal Adelaide Hospital in South Australia matched the SNP HRM type of ST796 so were included in WGS analysis.
WGS and bioinformatic analyses
Short read sequencing libraries were generated from genomic DNA using the Illumina Nextera XT DNA sample preparation kit. Libraries were sequenced on the Illumina platform using either the MiSeq with 250-cycle paired end chemistry or the NextSeq 500 with 150-cycle paired end chemistry according to the manufacturer’s instructions.
Snippy v3.2 (https://github.com/tseemann/snippy) was used to map sequence reads against the fully assembled Ef_aus0233 genome (GenBank accession no. PRJEB14733), a representative of the ST796 lineage [22]. Single nucleotide polymorphisms (SNPs) in core genome positions were used to construct a Maximum Likelihood tree with FastTree v2.1.8 [23]. The tree was used as a guide for ClonalFrame v1.7 to infer regions of recombination [24]. As previously described, a robust and recombination-free tree was generated [22]. Tree branches with less than 70% bootstrap support (500 replicates) were collapsed. Pairwise core non-recombinogenic SNP differences between isolates were tabulated and visualized using a custom R-script (https://github.com/MDU-PHL/pairwise_snp_differences).
Ethics approval
VRE isolates were collected and compared, along with rates of BSI, using non-identifying data as part of standard infection control procedures under appropriately constituted infection control committees at each institution.