Plasmid-free (recipient) strains of Enterococcus faecalis secrete a number of peptide sex pheromones that act as mating (conjugation) signals to donor bacteria harboring certain conjugative plasmids. (See reference 4 for a recent review.) Upon acquisition of such a plasmid, the related pheromone production is shut down or masked; however, other peptides that act as pheromones specific for different families of plasmids continue to be secreted. One enterococcal sex pheromone, cAM373, has been of particular interest because it is also produced by essentially all strains of Staphylococcus aureus (5). Insofar as vancomycin-resistant enterococci have become a serious clinical problem in recent years due to horizontal gene transfer (1, 3,9-13, 15, 17), such a peptide might contribute to staphylococcal acquisition of this resistance.
The association of an enterococcal vancomycin resistance determinant with a plasmid that responds to the S. aureus cAM373 peptide has not yet been reported. In an effort to identify such a plasmid, we examined 18 vancomycin-resistant E. faecalis strains obtained from The University of Michigan Hospital (Ann Arbor). One strain, 368, was found to carry a transferable plasmid, designated pAM368 (107 kb, based on summation of the sizes ofEcoRI-BamHI restriction fragments separated via agarose gel electrophoresis [14]), bearing avanA determinant (based on PCR analysis [2]). When present alone in the host strain OG1SS or FA2-2, pAM368 was found to confer a typical mating/clumping response to synthetic cAM373 (minimum concentration was 1 ng/ml) as well as culture filtrates ofE. faecalis or S. aureus.
Because of containment considerations, we did not try to transfer pAM368 from E. faecalis to S. aureus, although the introduction of vancomycin resistance into S. aureusunder laboratory conditions has been reported elsewhere (16). Alternatively, we made use of the characterized cAM373-responding plasmid pAM373 (8), which had been shown previously not to establish itself in S. aureus(5). We constructed a cointegrate involving the plasmid pAD2 with the notion that replication functions of the latter might be functional in S. aureus. pAD2 is a 26-kb nonconjugative element originally identified in E. faecalis DS16 (6); it carries Tn917 plus determinants for resistance to streptomycin and kanamycin. A pAM373::pAD2 derivative with normal pheromone response properties was generated by a retrotransfer mating (7) between E. faecalis OG1RF/pAM373 and DS16C1 (which carries only pAD2) (6). In overnight filter matings the cointegrate plasmid in OG1SS/pAM373::pAD2 was able to transfer and establish itself in S. aureus 879R4RF (5) at a frequency of 6.5 × 10−6 per donor, with selection for kanamycin resistance and the recipient markers (rifampin and fusidic acid resistance). The staphylococcal transconjugants contained the intact plasmid and no longer produced a detectable pheromone activity, implying that shutdown functions were being expressed. Exposure to synthetic cAM373 did not induce clumping; however, this could be due to any one of several factors specifically related to the staphylococcal host (e.g., inability of the aggregation substance to locate on the bacterial surface or absence of an appropriate surface binding substance).
Our data support the view that the natural acquisition of vancomycin resistance by S. aureus from enterococci via the utilization of a pheromone-responding plasmid may be imminent.
ACKNOWLEDGMENTS
We thank C. Pierson for hospital isolates from University Hospital that included E. faecalis 368; F. An, S. Flannagan and V. Francia for helpful discussions and technical advice; and A. Burtch for technical help.
This work was supported by National Institutes of Health grants GM33956 and AI10318.
- Copyright © 2001 American Society for Microbiology
