Characterization of a genomic island harbouring a new<i>vanD</i>allele from<i>Enterococcus faecium</i>N15-508 isolated in Canada

Sir, Acquired resistance to vancomycin in enterococci is mediated by the d-alanyl-d-lactate (d-Ala-d-Lac) ligases vanA, vanB, vanD and vanM and by the d-alanyl-d-serine (d-Ala-d-Ser) ligases vanE, vanG, vanL and vanN.1,2 Enterococci harbouring vanD are relatively rare having been found only in a few strains of Enterococcus faecium and Enterococcus faecalis and a few other species.3 Enterococci harbouring vanD share some unique characteristics: a chromosomal location of the vanD operon, its non-transferability, constitutively expressed resistance and host ddl mutations.3 E. faecium N15-508 was isolated from a rectal swab from an 85-year-old female as part of a screening programme for control of VRE and it exhibited vancomycin and teicoplanin MICs of ≥256 and 32 mg/L, respectively (Etest). Primers designed for this study are listed in Table S1 (available as Supplementary data at JAC Online). The high-level resistance to glycopeptides indicated the acquisition of a d-Ala-d-Lac ligase, but multiplex PCR for vanA, vanB, vanD, ddlE. faecium and ddlE. faecalis (Van-MP)4 was positive for only ddlE. faecium and PCR for vanM was negative (primers vanMF/vanMR). To elucidate the mechanism of resistance to glycopeptides, the genome of E. faecium N15-508 was sequenced (Illumina). In silico MLST (https://cge.cbs.dtu.dk/services/MLST) revealed a novel ST, ST1053, a single locus variant of ST18 belonging to the hospital-adapted group BAPS group 3.3a.5 The ddl allele (ddl-75) was novel, containing a 9 bp in-frame deletion (positions 374–382) (accession no. KT877400). The three residue deletion in the Ddl protein would presumably render it non-functional. If this is the case, it is likely the vanD operon is constitutively expressed to allow for synthesis of cell wall precursors, though we did not test this. The contigs were ordered (Mauve)6 against the ST18 genome of E. faecium DO genome (accession no. CP003583) and regions unique to E. faecium N15-508 were analysed. A protein sharing 80%–94% identity to VanD proteins was found encoded on a 98.9 kb contig. The next contig as aligned to the E. faecium DO genome was linked to the 98.9 kb contig by PCR with primers DO10885F and DO10886R (Figure 1). In this way, a novel 66.7 kb region, labelled IMEEfm15508 (where IME stands for integrative mobilizable element), harbouring a vanD operon was characterized (Figure 1; accession no. KT825491). Supplementary Data lists the ORFs and putative products of IMEEfm15508. Analysis revealed IMEEfm15508 to be a genomic island flanked by 10 bp direct repeats (attL and attR) that had inserted into the middle of the tRNA-Arg(CCT) (Figure 1). The GC content of IMEEfm15508 was 48.6% whereas that of the E. faecium genome is 38%, suggesting acquisition by horizontal transfer. Integration that disrupts a tRNA is unusual as genomic islands commonly insert at the 3′-end of target tRNAs and thus do not damage the gene.7 However, in IMEEfm15508 the first gene was a tRNA-Arg(CTT) of different sequence followed by a tyrosine integrase and an excisionase, the latter two normally being the first genes at one extremity of genomic islands.7 The vanD operon was typical in structure, the vanRD-vanSD regulatory genes being followed by the vanYDd,d-carboxypeptidase, vanHD lactate dehydrogenase, vanD ligase and vanXDd,d-dipeptidase. Overall, the E. faecium operon shared highest identity (90%) with the vanD operon found in Enterococcus gallinarum N04-414, with the corresponding proteins sharing between 86% and 95% identity.8 Other products of interest in IMEEfm15508 include a putative bacitracin resistance operon, an ABC transporter system and antirestriction and Rel-type toxin proteins, which may contribute to the maintenance and stability of IMEEfm15508. Also present were a number of conjugal transfer proteins, namely a relaxase and TraE, PrgI, Maff2 and TraG proteins. Failure of the E. faecium N15-508 vanD gene to amplify in the Van-MP PCR was due to 2 and 3 bp differences in the forward and reverse primer-binding sites, respectively. The vanD primer sets in two other published multiplexes for vancomycin resistance genes would also likely fail to amplify vanDN15-508 as there are from 3 to 11 bp differences in the various primers.9,10 A new ‘universal’ vanD primer set (Duni1/Duni2) based on an alignment of all vanD alleles found in enterococci was designed, which successfully amplified vanDN15-508 as well as the vanD alleles from E. faecium strains BM4339 (vanD1), N97-0330 (vanD3), N03-0191 (vanD4) and N03-0072 (vanD5) and vanD from E. gallinarum N04-0414 (data not shown). Nested PCR demonstrated that IMEEfm15508 could be excised and form a circle via an attI site (Supplementary Data). However, transfer of vanD to E. faecium GE-1 could not be successfully demonstrated. Thus, the genomic island appears to be a putative IME. We could not detect a rejoining of the chromosome by nested PCR to reform an attB integration site, which may indicate a rapid reintegration of circularized IMEEfm15508. Schematic diagram of IMEEfm15508 and flanking regions and comparison with the analogous flanking regions in E. faecium DO. ORFs where a putative function has been identified are shown as open boxes and ORFs that encode a hypothetical protein are shown as black boxes. ORFs on top of the solid black line are transcribed from left to right and those on the bottom from right to left. GNAT, Gcn5-related N-acetyltransferase; TR, transcriptional regulator; RR, response regulator; SK, sensor kinase; Mtase, methyltransferase; int, integrase; xis, excisionase; TnpA, transposase; ABC, ABC transport system protein; dUTPase, deoxyuridine triphosphatase; and also see Supplementary Data. Note that the int genes from the two isolates are not related. To our knowledge, this is the first complete genetic characterization of a VanD element from the chromosome of an Enterococcus. The formation of a circular form provides a potential way for vancomycin resistance transfer if IMEEfm15508 could be mobilized by acquisition of a conjugative element that provided full transfer function in trans. This work was carried out as part of the routine work of the Antimicrobial Resistance and Nosocomial Infections Section of the National Microbiology Laboratory. None to declare. We gratefully acknowledge Romeo Hizon and Tim Du for technical assistance and the Genomics Core Facility of the National Microbiology Laboratory for genomic sequencing.

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