Seqsphere version 6

Raw FASTQ sequencing reads from the Illumina MiSeq whole-genome sequencing were processed using an assembly pipeline generated with the SeqSphere+ version 6 (Ridom GmbH, Münster, Germany)⁴. FastQC (Andrews et al., 2010 ) was used to perform a quality check of the read files to assess the sequencing quality scores, total number of reads, and GC content. For the removal of the Nextera XT index library adapters, Trimmomatic (Bolger et al., 2014 (link)) was applied to achieve an average Q score of 30 in a sweeping window of 20 bases. The BWA plug-in in the SeqSphere+ software was used for the assembly of genome sequences of each isolate by mapping the paired-end reads to the complete reference genome of E. faecium DO (TX16_NC-017960) (Qin et al., 2012 (link)). The resultant contiguous consensus sequences (contigs) were exported for in silico identification of vancomycin-resistance (van) locus using the ResFinder server on the Centre for Genomic Epidemiology (CGE) online tool⁵. The settings used were a minimum sequence identity threshold of 90% and a genome length identity cut-off of 60%. The assembled genome sequences were queried against the MLST tool⁶ from the CGE database to determine the sequence type of the isolates. The E. faecium MLST database⁷ was also queried to confirm the sequence types.

Isolation of vancomycin-resistant enterococci from samples positive for vanA or vanB gene was completed using selective culture plates, CHROMID^® VRE, Biomerieux, Ballerup Denmark), and/or CHROMID^® CPS^® Elite plates (Biomerieux), to which 5 µg vancomycin Diatabs (Rosco Diagnostica, Taastrup, Denmark) were added.
For Whole Genome Sequencing (WGS) genomic DNA was extracted using DNeasy Blood and Tissue Kit (QIAGEN, Germantown, MD, USA) and fragment libraries were constructed using the Nextera Kit (Illumina, San Diego, CA, USA). Sequencing was completed using a Mieq instrument (Illumina) and 2 × 150 bp paired-end techniques according to the manufacturer’s instructions. MLST Sequence Type (ST) was inferred from the WGS using the MLST webserver (Version 1.7, https://cge.cbs.dtu.dk/services/MLST/ (accessed 15 June 2020)). The isolates were further typed in seqsphere + version 6 (Ridom GmbH, Münster, Germany (seqsphere/">http://www.ridom.de/seqsphere/ (accessed 15 June 2020)) using the cgMLST scheme of Complex Type (CT) by de Been et al. [22 (link)] for E. faecium. The ResFinder web server (https://cge.cbs.dtu.dk/services/ResFinder/ (accessed 15 June 2020)) was used to identify van genes in the assembled genome data, using a threshold of 90% minimum sequence identity and 60% minimum length identity cut-off.