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The streptogramin complex quinupristin-dalfopristin is used for treatment of infections with multiresistant Enterococcus faecium in humans (4). In contrast to humans, where streptogramins have been used only rarely, another streptogramin complex, virginiamycin, has been used widely as a growth promoter for broilers, pigs, and cattle in Europe and is still used in the United States (8). In Denmark streptogramins have not yet been used for human therapy, but virginiamycin has been used for growth promotion in food animals for decades (58,696 kg of active compound from 1989 to 1997) (2), and the occurrence of resistance to virginiamycin has frequently been observed among E. faecium isolates from Danish livestock (1, 2).

(Part of this study was presented at the First International ASM Conference on Enterococci: Pathogenesis, Biology, and Antibiotic Resistance, Banff, Alberta, Canada, 27 February to 2 March 2000.)

The vat(D) gene (formerly called satA) is known to encode resistance to streptogramin A in E. faecium, and the erm(B) gene encodes resistance to streptogramin B together with lincosamines and macrolides (6, 9).

An E. faecium strain, F9631160-1 (here called 160-1), was isolated from chicken feces in the Danish surveillance program (DANMAP) (3). E. faecium 160-1 is resistant to both erythromycin (MIC > 32) and quinupristin-dalfopristin (MIC  32). The vat(D) and erm(B) genes were detected on a large plasmid (>150 kb).

The streptogramin resistance was transferred by filter mating to a plasmid-free recipient, E. faecium BM4105-RF (3). One transconjugant, E. faecium AHA15, was use as a donor in a new filter mating with the recipient, E. faecium BM4105-Str.

A second transconjugant, E. faecium 7.1, was selected and studied further together with AHA15. Both AHA15 and 7.1 were resistant to erythromycin and quinupristin-dalfopristin. Neither transcojugants contained the large plasmid previously detected in E. faecium 160-1, but the presence of the erm(B) and vat(D) genes was confirmed in each strain by PCR analysis according to Hammerum et al. (3).

Total DNA from 160-1, AHA15, 7.1, BM4105-Str, and BM4105-RF was analyzed. Hybridization with the erm(B) and vat(D) probes to EcoRI-digested DNA (Fig. 1) showed that both the vat(D) and the erm(B) genes were located on an approximately 6.6-kb fragment in 160-1 and on an approximately 12-kb fragment in AHA15 and 7.1, suggesting a transposition event. None of the probes hybridized to EcoRI-digested DNA from BM4105-RF and BM4105-Str. DNAs from five different transconjugants were cleaved with SmaI. Pulsed-field gel electrophoresis and Southern hybridization with the vat(D) probe indicated a location on bands of three different sizes, again suggesting a transposition event (data not shown).

View larger version (69K): [in this window] [in a new window]   FIG. 1.   Southern blot of EcoRI-digested total DNA of different E. faecium transconjugants from F9631160-1 hybridized with (A) the vat(D) probe or (B) the erm(B) probe. Lane 1 and lane 7, lambda-HindIII marker (0.5, 2.0, 2.3, 4.4, 6.6, 9.4, and 23.1 kb); lane 2, BM4105-RF; lane 3, F9631160-1; lane 4, AHA15; lane 5, 7.1; lane 6, BM4105-Str.

The link between vat(D) and erm(B) was confirmed in E. faecium 160-1 by PCR amplification using one primer that binds within the vat(D) gene and one that binds within the erm(B) gene. This PCR was preformed by using the Expand long PCR system (Roche Diagnostics Gmbh, Mannheim, Germany). Use of primers ErmB-1R and satA-418 (Table 1) resulted in an amplicon of 2.3 kb.

A 4,010-bp fragment from E. faecium 160-1, obtained from the extended PCR and PCR, was sequenced (Fig. 2) (GenBank accession no. AF368302). A segment 1,417 bp in length was 100% identical to a previously cloned vat(D) gene (GenBank accession no. L12033). Upstream of vat(D), a 1,249-bp segment was found with 95% similarity to a gene encoding a putative transposase detected in E. faecium (GenBank accession no. Y16413). The same segment had 95% homology to a transposase gene from the multiresistance Enterococcus faecalis plasmid pRE25 (GenBank accession no. X92945). The transposase should not be functional, because a termination is present after 334 bp (Fig. 2). Upstream of the terminated transposase, a 1,175-bp segment with 100% homology to erm(B) from the Streptococcus agalactiae plasmid pIP501 and multiresistance E. faecalis plasmid pRE25 was detected (GenBank accession nos. X72021 and X92945) (Fig. 2).

View larger version (10K): [in this window] [in a new window]   FIG. 2.   Structure of the sequenced plasmid fragment of E. faecium F9631160-1 (GenBank accession no. AF368302). (A) The small arrows indicate the position of the primers used for PCR (see Table 1). (B) The sizes and positions of genes are indicated. Open reading frame 3 (ORF3) is similar to two putative transposases but is terminated in position 1506. (C) Lines indicate identities (given in percentages) with DNA sequences identified below by their GenBank associations numbers.

The primers in Table 1 were also used for PCR amplification of another E. faecium chicken isolate from the DANMAP study (F9630230-1, here called 230-1). Amplicons of the same size as those in E. faecium 160-1 were found in E. faecium 230-1, indicating the same general structure. The vat(D) and erm(B) genes were located on a large plasmid in strain 230-1; this plasmid could be transferred into both E. faecium and E. faecalis recipients and maintain the plasmid (data not shown).

In a previous work on an E. faecium isolate from humane urine, both the vat(D) gene and the erm(B) gene were located on a 10-kb fragment from a plasmid. This plasmid could not be transferred by conjugation (B. Bozdogan, R. Leclercq, A. Lozniewski, and M. Weber, Letter, Antimicrob. Agents Chemother. 43:2097-2098, 1999). In the present study, the vat(D) and erm(B) genes could be transferred together either by conjugation of a plasmid or by transposition of a putative transposon, and futhermore, a direct physical link between erm(B) and vat(D) was detected. A terminated open reading frame, similar to two published transposases, was detected between vat(D) and erm(B) in both 160-1 and 230-1. Its presence in both strains could indicate the presence of a larger conserved fragment associated with the vat(D) and erm(B) genes. The erm(B)-vat(D) gene cluster might be integrated into a composite transposon like Tn1547 (5) or Tn5385 (7), but further studies should define the limits of this mobile element and its prevalence among streptogramin-resistant E. faecium isolates.

Nucleotide sequence accession numbers. The 4,010-bp fragment from E. faecium 160-1 is registered as Genbank accession no. AF368302.