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Antimicrobial Agents and Chemotherapy, December 2003, p. 3954-3959, Vol. 47, No. 12
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.12.3954-3959.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Plasmid Content of a Vancomycin-Resistant Enterococcus faecalis Isolate from a Patient Also Colonized by Staphylococcus aureus with a VanA Phenotype

Susan E. Flannagan,1 Joseph W. Chow,2,3 Susan M. Donabedian,4 William J. Brown,5,6 Mary B. Perri,4 Marcus J. Zervos,2,4,7 Yoshiyuki Ozawa,1 and Don B. Clewell1,8*

Department of Biologic and Materials Sciences, School of Dentistry,1 Department of Microbiology and Immunology, School of Medicine, The University of Michigan, Ann Arbor, Michigan 48109,8 Division of Infectious Diseases,2 Department of Pathology,Wayne State University School of Medicine,5 Detroit Medical Center University Laboratories,6 John D. Dingell VA Medical Center, Detroit, Michigan 48201,3 Division of Infectious Disease, Department of Medicine,7 Research Institute, William Beaumont Hospital, Royal Oak, Michigan 480734

Received 19 June 2003/ Returned for modification 24 August 2003/ Accepted 10 September 2003


   ABSTRACT
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Vancomycin-resistant Enterococcus faecalis coisolated with vancomycin-resistant (VanA) Staphylococcus aureus was found to contain two plasmids, designated pAM830 (45 kb) and pAM831 (95 kb). pAM830, found to be conjugative and closely related to the Inc18 family of broad-host-range conjugative plasmids, encodes resistances to vancomycin (via a Tn1546-like element) and erythromycin; pAM831 encodes resistances to gentamicin, streptomycin, and erythromycin.


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The first example of a vancomycin-resistant Staphylococcus aureus (VRSA) strain with the VanA phenotype was isolated during the summer of 2002 from a diabetic patient in Michigan (3, 28). Interestingly, a vancomycin-resistant Enterococcus faecalis (VRE) strain with a similar VanA phenotype was coisolated with the VRSA strain (coisolates were obtained from a foot ulcer and from the tip of a dialysis catheter), thus raising the question of whether the S. aureus isolate acquired resistance from the VRE strain. Enterococci and staphylococci are known to exchange genetic information, as was demonstrated previously (4, 26) with the transfer of the broad-host-range erythromycin-resistance plasmids pAMß1 and related (Inc18) elements (16, 20, 32). There is even a report of plasmid-encoded transfer, in the laboratory, of vanA from E. faecalis to S. aureus (23). In addition, S. aureus is known to secrete a peptide with an activity resembling a known E. faecalis sex pheromone, cAM373 (4, 13). Indeed, an E. faecalis vanA-carrying plasmid, pAM368, was recently found to encode a response to cAM373 (27), thus raising concern about the potential uptake of vanA from enterococci by a pheromone-related process. The data reported here address the nature of the VanA-related trait in the VRE strain and show that it involves a Tn1546-like element (1) associated with a conjugative plasmid of the Inc18 family. We also present data relating to a vancomycin-sensitive, methicillin-resistant S. aureus (MRSA) nasal isolate believed to have been the host that acquired the vanA gene associated with the VRSA strain.

The strains and plasmids used or identified in the study are listed in Table 1. Plasmid characterization made use of CsCl-ethidium bromide buoyant density centrifugation and other previously described standard methodologies (13, 14, 25). With regard to the enterococcal strains, the MICs for various antibiotics are indicated in Table 2. The resistances of primary significance were to vancomycin, erythromycin, gentamicin, and streptomycin. The VRE strain was also hemolytic on horse blood agar and exhibited a bacteriocin activity using E. faecalis OG1X as the indicator. Figure 1A shows the results of pulsed-field gel electrophoresis analysis of chromosomal DNA preparations from both the E. faecalis foot isolate (DMC83006B) and the catheter isolate (WBH27862), as well as a vancomycin-sensitive derivative (discussed below) of the foot isolate; all are seen to be isogenic. (Fig. 1A also shows that key transconjugants generated in the study described below are isogenic with the recipient strain JH2-2.)


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  		TABLE 1. Bacterial strains and plasmids used in this study

 

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  		TABLE 2. Antibiotic resistance levels for E. faecalis strains

 


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  		FIG. 1. Comparison of DNA from enterococcal strains. (A) Pulsed-field gel electrophoresis of genomic DNA digested with SmaI. (B) Plasmid DNA digested with HindIII. Lanes 1, molecular size marker; lanes 2, WBH27862; lanes 3, DMC83006B; lanes 4, SF830Vs; lanes 5, SFG1; lanes 6, SFV1; lane 7, JH2-2.

 
Transferable resistance traits of the VRE strain. Vancomycin resistance (Vmr) was observed to transfer to E. faecalis JH2-2 from DMC83006B in overnight filter matings (4) at a frequency of 1.2 x 10-3 per donor (Table 3). A similar frequency was observed when selection was for erythromycin resistance (Emr). The frequency was an order of magnitude higher when the recipient was Enterococcus faecium. When JH2-2 transconjugants selected on vancomycin were examined for unselected uptake of Emr, all were found to have acquired this trait as well. In contrast, the transconjugants were sensitive to gentamicin. When the selected transconjugant SFV1 was used as a donor for a second round of transfer, movement of Vmr occurred at a frequency of 2.1 x 10-4 per donor and again resulted in cotransfer of Emr. When the SFV1 strain, as well as five additional transconjugants, were examined for plasmid content, they were found to harbor a 45-kb plasmid that was subsequently designated pAM830 (Fig. 1B, lane 6). Plasmid sizes were determined by summation of restriction fragments using agarose gel electrophoresis following separate digestions with BamHI, EcoR1, or PstI.


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  		TABLE 3. Transfer of resistance

 
The pAM830 plasmid could also be visualized in the original DMC83006B host, where it appears together with additional plasmid DNA (Fig. 1B, lane 3). It is noted that while the plasmid content of the two clinical VRE isolates is very similar, a few additional restriction fragments are present in the case of WBH27862. Sensitivity to vancomycin appeared spontaneously during growth of DMC83006B in the absence of drug; one of 25 colonies from nonselective medium represented a derivative that had lost the Vmr trait and concomitantly lost the pAM830 plasmid. This strain, designated SF830Vs, remained resistant to gentamicin, erythromycin, and streptomycin and maintained a 95-kb plasmid (Fig. 1B, lane 4) that was subsequently designated pAM831. The latter DNA was found to transfer from SF830Vs to JH2-2 at a relatively low frequency (about 10-7) when selection was for Gmr or Emr (Table 3). Considering the low transfer frequency, it remains unclear whether an unknown mobilizing factor in the donor aids in transfer of pAM831. Plasmid DNA from a transconjugant, SFG1, of such a mating is shown in Fig. 1B (lane 5) and is identical to plasmid DNA of eight additional Gmr transconjugants examined, four from the DMC83006B donor and four from donor SF830Vs (data not shown). The data are consistent with the view that whereas pAM830 encodes Vmr and Emr, pAM831 encodes Gmr, Emr, and Smr. (There are Emr determinants on both plasmids.) Bacteriocin activity was associated with pAM831, whereas an additional bacteriocin to which pAM831 does not provide immunity, hemolysin, and levofloxacin resistance traits of DMC83006B were not associated with either plasmid and thus are believed to be encoded on the host chromosome.

The vanA determinant of pAM830 is associated with a Tn1546-like transposon. Eight pairs of primers designed to generate PCR products that, taken together, overlap to span the entire sequence of the known VanA transposon Tn1546 (1) produced data reflecting the expected size (10.8 kb) of this element on pAM830 (data not shown). Primers corresponding to regions close to the ends were used for sequencing outward to determine the presence of inverted repeats, the junctions, and adjacent DNA. The ends of the element are identical to Tn1546, and the transposon is flanked by 5-bp direct repeats (TTCTT) presumed to reflect target site duplication. Blast analysis of the adjacent DNA revealed near-identity with sequences known to be present in the plasmids pAMß1 and pIP501.

pAM830 is closely related to the Inc18 family of plasmids. pAM830 and pAM831 were used separately as probes in Southern analyses to determine their relationship to the following: (i) the pheromone-responding plasmids pAD1 and pAM373, (ii) the Inc18-type plasmids pAMß1 and pIP501, and (iii) pMG1, representative of a group of conjugative plasmids commonly found to be associated with gentamicin and/or vancomycin resistance in E. faecium (18, 31). As shown in Fig. 2B, pAM830 exhibited strong homology with pIP501 (lane 4) and pAMß1 (lane 5). In the case of pIP501, which was cleaved with HindIII (the other plasmids were cut with EcoRI), hybridization over the entire element is clearly evident. pAM831 did not exhibit such homology with the two Inc18 plasmids; only one to two bands were detected and with relatively low intensity. pAM830 also exhibited limited homology with pAM373 (one band; lane 3), and pAM831 exhibited limited homology with both pAD1 (one band; lane 2) and pAM373 (two bands; lane 3). pAM830 and pAM831 exhibited very limited homology to each other, and that which was observed may in part reflect that both carry Emr determinants. Little, if any, homology with pMG1 was observed (lane 6).



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  		FIG. 2. Hybridization analysis of conjugative plasmids. An agarose gel with identically loaded halves, one of which is shown in panel A, was blotted to nitrocellulose, and separate halves (B) were probed with 32P-labeled plasmids pAM830 and pAM831, as indicated. Lanes 1, molecular size marker; lanes 2, pAD1; lanes 3, pAM373; lanes 4, pIP501; lanes 5, pAMß1; lanes 6, pMG1; lanes 7, pAM831; lanes 8, pAM830; lanes 9, pAM829. pIP501 was digested with HindIII; all others were digested with EcoRI.

 
The VRE strains do not exhibit a "pheromone" response to staphylococcal or enterococcal culture supernatants. Conjugative plasmids that confer a pheromone response in enterococci exhibit a characteristic clumping response that, using a microtiter dilution assay (10), can be used to quantitate the pheromone present in a given culture supernatant. Using this method, culture supernatants of the plasmid-free strains E. faecalis JH2-2 and E. faecalis OG1X did not generate a clumping response by the VRE isolates DMC83006B or WBH27862 or the transconjugant strain SFV1 carrying pAM830. These strains also did not respond to supernatants of the MRSA nasal isolate, although cAM373 activity (titer of 8) was detected, as well as activity (titer of 2) similar to that of cAD1 (10) produced by pSK41-type plasmids (11). The data indicate that a pheromone-responding plasmid is not present in the VRE strains.

Identification of a pSK41/pGO1-type plasmid in the S. aureus MRSA strain. Like the VRSA strain (J. M. Mohammed, L. Weigel, N. Clark, L. McDougal, P. Raney, A. Whitney, S. McAllister, M. Kellum, L. Jevitt, and F. C. Tenover, Abstr. Intersci. Conf. Antimicrob. Agents Chemother., abstr. LB-7, 2002), a vancomycin-sensitive nasal isolate of S. aureus (DMC82991A) exhibited the atypical characteristic of being weakly esculin positive. And in addition to being resistant to oxacillin, it was resistant to gentamicin (MIC, >100 µg/ml), kanamycin (MIC, >50 µg/ml), erythromycin (MIC, >10 µg/ml), and rifampin (MIC, >25 µg/ml). The strain harbors a 47-kb plasmid that has been designated pAM829 (Fig. 2A, lane 9). The VRSA strain contains a single plasmid that is essentially identical but with an additional segment carrying a vanA gene (F. Tenover and L. Weigel, personal communication). The pAM829 element showed limited homology when probed with pAM831, perhaps due to the presence of Gmr genes on both, but no homology was detected with the VanA plasmid pAM830 (Fig. 2B, lane 9). When the strain was mated (overnight filter mating) with the S. aureus recipient 879R4RF with selection for transfer of the Gmr trait, transconjugants were detected at a very low frequency (6.6 x 10-9). Transconjugants were also kanamycin resistant but were sensitive to erythromycin.

pGO1 and pSK41 are members of a family of conjugative plasmids known to commonly carry Gmr determinants (5, 12, 21). Therefore, the relationship of these plasmids to pAM829 was examined by performing a hybridization analysis using pSK41 and pAM829 as probes. As shown in Fig. 3B, strong homology between the plasmids is evident. Indeed, the agarose gel (Fig. 3A)showed a number of bands of common size for pAM829, pSK41, and pGO1. In addition, use of a pair of primers designed to amplify a 952-bp portion of the traA gene of pSK41 (2) resulted in generation of a 0.9-kb amplicon from pAM829 as well as from pSK41 and pGO1 (data not shown). The data are consistent with pAM829 being a member of the pSK41/pGO1 family of conjugative staphylococcal plasmids.



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  		FIG. 3. Analysis of S. aureus plasmids. An agarose gel with identically loaded halves, one of which is shown in panel A, was blotted to nitrocellulose, and separate halves (B) were probed with the 32P-labeled plasmids pAM829 and pSK41 as indicated. Lanes 1, molecular size marker; lanes 2, pAM829; lanes 3, pSK41; lanes 4, pGO1. All plasmids were digested with HindIII.

 
Conclusions. The Inc18-type conjugative element carrying the Tn1546-like transposon in the VRE strain is unrelated to the plasmid in the vancomycin-sensitive staphylococcal nasal isolate or, by inference, to the plasmid in the VRSA strain representing the same element but with additional DNA carrying vanA. The data do not conclusively show that the vanA determinant traveled on a plasmid from the VRE strain to the MRSA strain (e.g., on pAM830); however such an event can easily be envisioned to have occurred, followed by transposition of the resistance determinant to pAM829 and subsequent segregation of pAM830.


   ACKNOWLEDGMENTS
 
Support for this research came from the National Institutes of Health Grant GM33956.

We thank Y. Ike for strain BM4105-RF/pMG1, R. Skurray for strain SK5428, and P. Trieu-Cuot for strain BM4105-RF.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biologic and Materials Sciences, The University of Michigan School of Dentistry, 1011 N. University Ave., Ann Arbor, MI 48109-1078. Phone: (734) 763-0117. Fax: (734) 763-9905. E-mail: dclewell{at}umich.edu. Back


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Antimicrobial Agents and Chemotherapy, December 2003, p. 3954-3959, Vol. 47, No. 12
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.12.3954-3959.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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