Vancomycin-resistant Enterococcus faecium harbouring vanN in Canada: a case and complete sequence of pEfm12493 harbouring the vanN operon.

Sir, Resistance to vancomycin in Enterococcus faecium and Enterococcus faecalis is due to acquired operon structures of two types, namely the D-alanyl-D-lactate and D-alanyl-D-serine operons named for the specific ligase they harbour. The former group consists of the vanA, vanB, vanD and vanM operons and the latter group consists of the vanE, vanG, vanL and vanN operons. – 5 The D-Ala-D-Lac operons can be carried on plasmids or on the chromosome while the D-Ala-D-Ser operons are chromosomally located except for vanN, which has been found on a plasmid. To date vanN has only been described twice, from E. faecium isolated in 2008 from a patient in France and from E. faecium isolated from a chicken meat sample collected in Japan in 2011. The vanN operons were identical except for a single non-synonymous substitution in the vanSN gene. A 51-year-old male with tibial and femoral artery occlusion on the right ankle was scheduled for angiography with revascularization. Three weeks later the patient presented at hospital with cellulitis in the right heel and ischaemia in the right ankle wound, which on microbial sampling yielded Escherichia coli, Stenotrophomonas maltophilia and Pantoea agglomerans. Ertapenem treatment was begun and 4 days later trimethoprim/sulfamethoxazole was added. Treatment continued for a further 15 days; the patient was then admitted for revascularization, and the infection was cured successfully. At admission screening (rectal) E. faecium N12-0493 was isolated and was determined by Etest to have a vancomycin MIC of 16 mg/L and a teicoplanin MIC of 0.5 mg/L. Additional testing by Vitek 2 (AST-GP67) showed E. faecium N12-493 was resistant to vancomycin and clindamycin, had intermediate susceptibility to ciprofloxacin, quinupristin/dalfopristin and nitrofurantoin, and was susceptible to ampicillin, high-level gentamicin, levofloxacin, linezolid, tetracycline and tigecycline. No further antimicrobial therapy was started after the procedure. MLST analysis revealed a unique profile, which was assigned ST955 (www.efaecium.mlst.net). ST955 did not cluster with the classical hospital-associated strain types (formerly known as CC17). PCR analysis revealed E. faecium N12-0493 was positive for the E. faecium ddl gene and vanN genes. The putative translation product (149 residues) of the vanN amplicon sequence shared 94% identity with the VanN protein. To identify the genetic context of the complete vanN operon, the genome of E. faecium N12-0493 was completely sequenced using Illumina technology. The assembled reads and the partial vanN sequence of E. faecium N12-0493 were further assembled using the Seqman Pro module of Lasergene 10 (DNASTAR, Madison, WI, USA). In this way a single 137 817 bp circular sequence, designated pEfm12493, was assembled from five contigs, ranging from 2222 to 90 738 bp (mean1⁄427.6 kb), with confirmation by standard PCR and sequencing (Figure 1). A total of 127 ORFs were annotated using Prokka and manual BLAST analysis (http://blast. ncbi.nlm.nih.gov/Blast.cgi) and are listed in Table S1 (available as Supplementary data at JAC Online). Plasmid pEfm12493 was assigned GenBank accession number KP342511. Approximately half of pEfm12493 has synteny with and shares 86%–92% nucleotide identity with large parts of the related plasmids p1 from E. faecium AUS0004, p2 from E. faecium AUS0085 and pQY082 from Enterococcus mundtii QU 25 (Figure 1). This area includes genes involved in replication (repR, prgN), genes involved in conjugative transfer (traE1, virD4, virB4, pilus assembly protein genes), genes encoding three sortases and genes encoding two topoisomerases. Some regions in this area not related to the above-mentioned plasmids include a 13 kb region harbouring genes encoding a carboxypeptidase, a two-component system, a XerS-like recombinase and ParA1, and two different regions with group II intron reverse transcriptase genes, the first of which (rt1) interrupts the virB4 gene and the second of which (rt2) interrupts a peptidase gene. The other half of pEfm12493 includes the vanN operon, a sulphur metabolism operon, several cell surface protein genes, genes encoding an ABC transporter and its cognate regulator, a second different parA gene (parA2) and seven defective ISs. The ISs are presumed to be defective due to being only partial (remnant) or due to mutation in the transposase(s) that causes frameshifts and/or internal stop codons. The E. faecium N12-0493 vanN operon itself shares 93% nucleotide identity with previously described vanN operons, with the proteins sharing between 92% and 98% identity, and thus pEfm12493 encodes a vanN variant, vanN12493. There are also two complete type II toxin–antitoxin (TA) loci (MazEF-like and RelBE-like) and a separate RelB-like antitoxin gene in this area. The TA loci can act as plasmid addiction systems to ensure the stable maintenance and inheritance of pEfm12493.