Identification of <b> <i>vat</i> (E-3), a Novel Gene Encoding Resistance to Quinupristin-Dalfopristin in a Strain of <i>Enterococcus faecium</i> from a Hospital Patient in the United Kingdom </b>

Quinupristin-dalfopristin is a mixture of semisynthetic streptogramins A and B that was recently licensed for clinical use in the United States and Europe (3). A related antibiotic, virginiamycin, has been used as a growth promoter for production animals in Europe and the United States, although its use was banned in the European Union in July 1999. Virginiamycin-resistant Enterococcus faecium strains have been isolated from exposed farm animals, raw meat, and hospital patients and are cross-resistant to quinupristin-dalfopristin (1, 2, 5; L. B. Jensen, A. M. Hammerum, F. M. Aarestrup, A. E. van den Bogaard, and E. E. Stobberingh, Letter, Antimicrob. Agents Chemother. 42:3330–3331, 1998; G. Werner, I. Klare, and W. Witte, Letter, Eur. J. Clin. Microbiol. Infect. Dis. 17:401–402, 1998). Resistance to streptogramin A is a prerequisite for resistance to quinupristin-dalfopristin and virginiamycin and is mediated in E. faecium by vat(D) (previously satA) or vat(E) (previously satG), two plasmid-mediated genes that encode acetyltransferases that inactivate streptogramin A (4; G. Werner and W. Witte, Letter, Antimicrob. Agents Chemother. 43:1813–1814, 1999). Available vat(E) sequences are not identical, and we propose designating the alleles in the order of their deposition in the GenBank database: vat(E-1) (accession numbers {type:entrez-nucleotide,attrs:{text:AF139735,term_id:5353576}}AF139735, {type:entrez-nucleotide,attrs:{text:AF229200,term_id:7595743}}AF229200, and {type:entrez-nucleotide,attrs:{text:AF242872,term_id:9246952}}AF242872) and vat(E-2) ({type:entrez-nucleotide,attrs:{text:AF153312,term_id:6690519}}AF153312). vat(E-2) differs from vat(E-1) by three nucleotides (99.5% identity), which are predicted to result in two amino acid substitutions (Fig. ​(Fig.1).1). Two other alleles, each differing from vat(E-1) by two nucleotides, have been reported but have not yet been deposited in GenBank (6). FIG. 1 Comparison of the amino acid sequences of alleles of the Vat(E) streptogramin A acetyltransferase (see the text for GenBank accession numbers). We have previously detected vat(E) by PCR in isolates of quinupristin-dalfopristin-resistant E. faecium (MIC ≥ 32 μg/ml) from animals and raw meat (n = 10) and also from hospital patients in the United Kingdom (n = 4) (5). A 512-bp internal fragment of vat(E) was amplified from these isolates (5) and subjected to direct cycle sequencing using an ALFexpress DNA sequencer (Amersham Pharmacia Biotech, St. Albans, United Kingdom) and a Thermo Sequenase fluorescence-labeled primer cycle sequencing kit (Amersham Pharmacia Biotech). The sequences, which represented 80% of the vat(E) gene, were compared with those of vat(E-1) and vat(E-2). The sequences of the PCR products from 13 isolates were identical to vat(E-1). However, one isolate, designated E. faecium A41, from a hospital patient, yielded a distinct sequence. Two overlapping fragments of the vat(E) allele from this strain were amplified and cloned into pCR2.1-TOPO (Invitrogen, Groningen, The Netherlands) to yield recombinant plasmid pARL00.31, containing the 512-bp fragment, and pARL00.38, containing a 300-bp fragment spanning the 3′ end of vat(E) and extending 137 bp downstream of the stop codon. The latter fragment was amplified with primers 5′-CCA ATT CAA CTC ATC GGA CC-3′ and 5′-TAC GAG TAG AGT ACC GCC AG-3′ and corresponded to nucleotides 4063 to 4362 of GenBank sequence {type:entrez-nucleotide,attrs:{text:AF242872,term_id:9246952}}AF242872. For each fragment, the inserts of three separate clones were sequenced in both directions using a Dye-Labeled ddNTP Terminator Cycle Sequencing Kit (Beckman Coulter UK Ltd., High Wycombe, United Kingdom) and samples were analyzed on a CEQ 2000 automated sequencer (Beckman). Fragments were assembled with ContigExpress (InforMax Inc., Oxford, United Kingdom). The vat(E) allele from E. faecium A41 has been designated vat(E-3) and deposited in GenBank under the accession number {type:entrez-nucleotide,attrs:{text:AY008284,term_id:12383031}}AY008284. It had 20 nucleotide changes (4% divergence) compared with vat(E-1). Fifteen of these changes were silent, but the others resulted in five previously undescribed amino acid substitutions (Fig. ​(Fig.1).1). The predicted Vat(E-3) peptide had 97% amino acid identity with Vat(E-1) and 96% identity with Vat(E-2). In comparison with sequences downstream of vat(E-1) and vat(E-2), the sequence immediately downstream of the vat(E-3) stop codon had a single base insertion (an additional C after nucleotide 4235 of GenBank sequence {type:entrez-nucleotide,attrs:{text:AF242872,term_id:9246952}}AF242872) and two substitutions (both T→C changes at nucleotides 4227 and 4253 of GenBank sequence {type:entrez-nucleotide,attrs:{text:AF242872,term_id:9246952}}AF242872). We have confirmed the allelic nature of vat(E) apparent in GenBank submissions and previous reports (2, 6). The vat(E-1) allele was present in all vat(E) PCR-positive E. faecium strains from nonhuman sources studied here and in three of the four clinical isolates. The fourth clinical isolate harbored vat(E-3), which showed greater sequence divergence from vat(E-1) than other previously reported alleles (20 versus 2 or 3 nucleotide changes). In conclusion, isolates of quinupristin-dalfopristin- and virginiamycin-resistant E. faecium that give a vat(E)-specific PCR product should not be assumed to carry identical alleles. Furthermore, we suggest that the epidemiological significance of a vat(E)-positive PCR result cannot be judged accurately in the absence of sequence data.

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