Cotransfer of antibiotic resistance genes and a hylEfm-containing virulence plasmid in Enterococcus faecium

Abstract

The hyl(Efm) gene (encoding a putative hyaluronidase) has been found almost exclusively in Enterococcus faecium clinical isolates, and recently, it was shown to be on a plasmid which increased the ability of E. faecium strains to colonize the gastrointestinal tract. In this work, the results of mating experiments between hyl(Efm)-containing strains of E. faecium belonging to clonal cluster 17 and isolated in the United States and Colombia indicated that the hyl(Efm) gene of these strains is also carried on large plasmids (>145 kb) which we showed transfer readily from clinical strains to E. faecium hosts. Cotransfer of resistance to vancomycin and high-level resistance (HLR) to aminoglycosides (gentamicin and streptomycin) and erythromycin was also observed. The vanA gene cluster and gentamicin resistance determinants were genetically linked to hyl(Efm), whereas erm(B) and ant(6)-I, conferring macrolide-lincosamide-streptogramin B resistance and HLR to streptomycin, respectively, were not. A hyl(Efm)-positive transconjugant resulting from a mating between a well-characterized endocarditis strain [TX0016 (DO)] and a derivative of a fecal strain of E. faecium from a healthy human volunteer (TX1330RF) exhibited increased virulence in a mouse peritonitis model. These results indicate that E. faecium strains use a strategy which involves the recruitment into the same genetic unit of antibiotic resistance genes and determinants that increase the ability to produce disease. Our findings indicate that the acquisition of the hyl(Efm) plasmids may explain, at least in part, the recent successful emergence of some E. faecium strains as nosocomial pathogens.

FIG. 1.

Results of S1 nuclease and I-CeuI digests, PFGE, and hybridizations. (A) S1 digestion of total DNA of E. faecium strains was followed by PFGE (left panel) and hybridization with hyl_Efm (right panel). Lane 1, lambda ladder (molecular sizes in kilobases are shown to the left); lanes 2, strain C; lanes 3, transconjugant D344SRF-C6; lanes 4, D344SRF; lanes 5, clinical strain A; lanes 6, transconjugant TX1330RF-A1 (using strain A as donor); lanes 7, TX1330RF (recipient); lanes 8, clinical strain B; lanes 9, transconjugant TX1330RF-B1 (using strain B as donor); lanes 10, transconjugant TX1330RF-B2 (second transconjugant using clinical strain B as donor). Plasmid bands are shown as linearized fragments on the gel; the white arrowheads indicate the plasmid bands hybridizing with hyl_Efm (right panel). (B) I-CeuI digestion of total DNA was followed by PFGE (left panel) and hybridization with 23S rRNA (center panel) and hyl_Efm (right panel) probes. Lane 1, lambda ladder (molecular sizes in kilobases are shown to the left); lanes 2, strain C; lanes 3, transconjugant D344SRF-C6; lanes 4, D344SRF; lanes 5, clinical strain A; lanes 6, transconjugant TX1330RF-A1; lanes 7, TX1330RF; lanes 8, clinical strain B; lanes 9, transconjugant TX1330RF-B1; lanes 10, second transconjugant, TX1330RF-B2. The figure is a composite of gels and hybridizations from different experiments.

FIG. 2.

Results of PFGE (SmaI digestion; leftmost panels) and hybridizations with hyl_Efm, erm(B), ant(6)-I, vanA, and aac(6′)-aph(2") with E. faecium strains. (A) Lane 1, lambda ladder; lanes 2, clinical strain A (TX16), lanes 3, transconjugant TX1330RF-A1 to which the hyl_Efm gene from strain A was transferred; lanes 4, TX1330RF. (B) Lane 1, lambda ladder; lanes 2, clinical strain B; lanes 3, TX1330RF-B1 (transconjugant of TX1330RF to which the hyl_Efm gene, resistance to vancomycin and erythromycin, and HLR to gentamicin and streptomycin from clinical strain B were transferred); lanes 4, TX1330RF-B2 (second transconjugant of TX1330RF, to which the hyl_Efm gene, resistance to erythromycin, and HLR to streptomycin from clinical strain B were transferred); lanes 5, TX1330RF. White arrowheads indicate the bands to which the hyl_Efm probe hybridized. The figure was created using blots from different experiments.

FIG. 3.

Survival of mice after intraperitoneal inoculation with E. faecium strains. Survival curves using strains TX1330RF and TX1330RF-A1 (TX1330RF transconjugant to which the hyl_Efm-containing plasmid was transferred) in the mouse peritonitis model (10 mice per group) at two different inocula in independent experiments are shown.