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Antimicrobial Agents and Chemotherapy, April 1998, p. 963-964, Vol. 42, No. 4
Infectious Diseases Section and Research
Service,
Received 8 July 1997/Returned for modification 19 November
1997/Accepted 24 January 1998
An approximately 60-kb transferable, vanB-carrying
plasmid has been identified in a clinical Enterococcus
faecium strain. A similar plasmid has been observed in an
unrelated E. faecium strain, suggesting that plasmid
transfer of vanB operons occurs in nature and plays a role
in the dissemination of VanB-type resistance among strains of E. faecium.
The spread of glycopeptide-resistant
enterococci through hospitals in the United States has been rapid and
dramatic (3). To date, two types of transferable
glycopeptide resistance phenotypes, VanA and VanB, have been described.
The vanB operon is chromosomally located in most cases and
in one case has been found within a 65-kb transposon (13).
Three previous reports indicate that the vanB operon may be
plasmid carried, but none have provided evidence suggesting that
vanB-carrying plasmids have spread between clinical strains
(2, 18, 19). We report herein transferable vanB
plasmids from strains of Enterococcus faecium isolated in northeast Ohio.
The strains described in this report (E. faecium U37 and
H11) were clinical isolates from two geographically distinct hospitals in northeast Ohio. Species identification was performed with the Vitek
automated system. MICs of vancomycin, ampicillin, tetracycline, and
minocycline were determined in duplicate by broth macrodilution with
brain heart infusion (BHI) broth (12). Screening for
associated resistances was performed by streaking clinical isolates (or
transconjugants) onto BHI agar containing chloramphenicol (10 µg/ml),
erythromycin (10 µg/ml), gentamicin (2,000 µg/ml) or streptomycin
(2,000 µg/ml). Conjugation experiments were performed on
nitrocellulose filters as described by Christie et al. (4).
The fusidic acid- and rifampin-resistant recipients for conjugation
experiments with clinical strains as donors were E. faecium
GE-1 (7), E. faecium SF68 (9),
Enterococcus faecalis JH2-2, and E. faecalis
JH2-7 (10). Transconjugants were selected on BHI agar plates
containing vancomycin (6 µg/ml) or tetracycline (10 µg/ml), fusidic
acid (25 µg/ml), and rifampin (100 µg/ml). Plasmid stability was
evaluated by growth of strains for 30 generations (starting from a
single bacterium) and comparing colony counts on BHI agar plates or BHI agar plates with vancomycin (10 µg/ml).
Plasmid DNA was extracted as described by Ehrenfeld and Clewell
(6). Genomic DNA was extracted as described by Storrs et al.
(15). Digested DNA was transferred from agarose gels to nylon membranes by using a vacuum blotting apparatus (Pharmacia LKB,
Uppsala, Sweden). Hybridization with digoxigenin-labeled probes was
performed according to the specifications of the manufacturer (Boehringer-Mannheim Biochemicals, Indianapolis, Ind.). Probes included
a 2.1-kb EcoRV fragment internal to the
vanHBBXB operon (8), a
1.8-kb AccI fragment from plasmid pLRM19 (this study), and a
restriction fragment from within transposon Tn5384 identical to the replication genes of broad-host-range plasmid pIP501 (1, 16). Amplification products derived from the joint of the
circularized form of Tn916 (14) and from an
internal region of the Tn916 tet(M) gene (11)
were used as probes for Tn916-related sequences.
E. faecium U37 and H11 were confirmed as clonally distinct
by IS6770 hybridization of genomic digests (data not shown)
(17). MICs of vancomycin, teicoplanin, and ampicillin for
U37 and H11 were 32 µg/liter, <0.5 µg/ml, and 256 µg/ml,
respectively (Table 1). The two strains
also expressed resistance to erythromycin, gentamicin, streptomycin,
and tetracycline but were susceptible to chloramphenicol. Vancomycin
resistance was transferable from both donors to GE-1 at rates of
10 Plasmid preparations revealed a ca. 60-kb plasmid in transconjugants
from matings involving U37 (plasmid designated pLRM19) and H11 (plasmid
designated pLRM20). In addition to pLRM20, transconjugants from matings
involving H11 possessed four additional plasmids (data not shown). A
comparison of HincII digests of plasmid preparations from
transconjugants from both U37 and H11 is shown in Fig.
1. Several identical bands are present in
the two plasmids' digests. vanB-hybridizing
HincII fragments were identical in size (ca. 15 kb) in the
two plasmids (Fig. 1), as were EcoRI fragments (data not
shown). A 1.8-kb AccI fragment (external to the
vanB operon) from pLRM19 hybridized to a similarly sized
HincII band in both preparations, suggesting that pLRM19 and
pLRM20 are related and possibly identical. Neither plasmid hybridized
to the pIP501 replication region or to tet(M), and only very
weak hybridization of the Tn916 joint to a single band
(instead of the expected two) was observed. We therefore conclude that
the tetracycline-minocycline resistance conferred by this plasmid is
not due to the presence of a Tn916-like element.
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Transferable, Plasmid-Mediated VanB-Type
Glycopeptide Resistance in Enterococcus faecium
ABSTRACT
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7 to 2 × 107/recipient CFU.
Transfer was also observed to E. faecium SF68. Attempts to
transfer resistance to E. faecalis JH2-2 or JH2-7 were
unsuccessful. E. faecium transconjugants from matings
involving both donors expressed resistance to vancomycin and
tetracycline-minocycline (Table 1) but were susceptible to ampicillin,
erythromycin, gentamicin, and streptomycin. Vancomycin MICs for U37 and
its transconjugant GE-1(pLRM19) correlated well. In contrast, the
level of vancomycin resistance expressed by transconjugant
GE-1(pLRM20) was less than observed in the donor strain H11 (Table 1).
TABLE 1.
Broth dilution MICs for recipient and
vancomycin-resistant transconjugants
View larger version (69K):
[in a new window]
FIG. 1.
Comparison of HincII digestions of plasmid
preparations from GE-1(pLRM19) and GE-1(pLRM20). Lane 1, bacteriophage
lambda digested with HindIII (size standard 
sizes
denoted on left side of figure); lane 2, GE-1(pLRM20); lane 3, GE-1(pLRM19); lane 4, High-molecular-weight size standards (Bethesda
Research Laboratories, Gaithersburg, Md.) (relevant sizes denoted on
right side of figure); lanes 5 and 6, Southern transfer of digests at
left hybridized with 2.1-kb EcoRV fragment internal to the
vanHBBXB operon; lanes 7 and 8, Southern transfer of digests at left hybridized with 1.8-kb
AccI fragment of pLRM19 (see text).
The presence of highly similar vanB-carrying plasmids in clonally distinct E. faecium strains isolated at different hospitals suggests that plasmid-mediated transfer of vanB operons occurs in nature. Previous reports have confirmed or suggested the presence of the vanB operon on a plasmid in single E. faecalis or E. faecium isolates or in clonally related E. faecium strains (2, 19). No evidence that the plasmids had disseminated among different strains was presented in these reports. The data presented in this paper are consistent with the inter-enterococcal transfer of a vanB plasmid in nature.
The difference in expression of vancomycin resistance between clinical isolate H11 and its transconjugant GE-1(pLRM20) was not explainable by differences in plasmid stability between the two strains (data not shown). Hybridization studies indicated that H11 had an additional vanB operon located on the chromosome, which was not transferred with the plasmids. It is likely that this additional operon is the explanation for the increased expression of resistance in the donor compared to the transconjugant.
The rate of transfer of these plasmids is not consistent with rates
reported for E. faecalis pheromone-responsive plasmids, which transfer to enterococcal recipients at rates of 101
to 102/recipient CFU (5). The
failure to hybridize with an internal fragment from the repE
gene of pIP501 and the failure of transfer to E. faecalis
recipients argue against either plasmid belonging to the
broad-host-range family. It is unclear why we were unable to transfer
these plasmids to E. faecalis. It is possible that the host
range of the plasmids is limited to E. faecium. A more extensive functional analysis of these plasmids is ongoing.
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ACKNOWLEDGMENTS |
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This study was supported in part by the medical research service of the Department of Veterans Affairs (L.B.R.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Infectious Diseases Section 1110(W), VA Medical Center, 10701 East Blvd., Cleveland, OH 44106. Phone: (216) 791-3800 x4399. Fax: (216) 231-3482. E-mail: lbr{at}po.cwru.edu.
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Antimicrobial Agents and Chemotherapy, April 1998, p. 963-964, Vol. 42, No. 4
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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