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INTRODUCTION |
Conjugative plasmids have been
identified in many streptococci and enterococci (4). Two
types of conjugative plasmids in enterococci have been reported. One
type of plasmid, with a molecular size of approximately 30 kb, does not
produce mating aggregates and does not transfer between donor and
recipient cells in liquid broth. The plasmids are able to transfer at
relatively low frequencies on a solid surface, such as during filter
mating (4). These plasmids usually have a broad host range
and transfer between a variety of streptococcal species, and even
between different genera. Macrolide-lincosamide-streptogramin B (MLS)
resistance is frequently associated with this type of plasmid (13,
22, 30). Of the MLS resistance-encoding plasmids, pIP501
(1, 13, 22, 28, 29, 46) and pAM
1 (30) are
representative; the transfer mechanism for these plasmids has been
characterized in detail at the molecular level.
The other type of plasmid is mainly found in Enterococcus
faecalis and is a pheromone-responsive plasmid (4-6,
10-12). The E. faecalis pheromone-related
plasmid conjugation system is unique among the bacteria. The
pheromone-responsive plasmid transfer between E. faecalis strains occurs at a high frequency of 100 to
10
2 per donor cell within a few hours of broth mating. A
plasmid of this type usually has a molecular size greater than 45 kb
and confers a mating response to the small sex pheromones secreted by
potential recipient cells (4-6, 10-12). This mating signal induces the synthesis of a surface aggregation substance that facilitates the formation of mating aggregates. The sex pheromones also
induce a series of genes required for plasmid transfer. Plasmid-free recipients secrete multiple sex pheromones, each specific for a donor
harboring a related pheromone-responsive plasmid. Once a plasmid is
acquired by the recipient, secretion of the related pheromone ceases,
whereas other, unrelated pheromones continue to be produced. The
pheromone (culture filtrate of a plasmid-free strain) induces
self-aggregation of donor cells, as shown by suspension of the donor
cells in a culture filtrate. Determinants encoded on
pheromone-responsive plasmids include those for hemolysin, bacteriocin,
and resistance to antibiotics and UV light (2, 4, 15, 24-26, 35,
41, 43, 44).
Drug-resistant enterococci have been an increasingly significant cause
of nosocomial infection in recent years (34).
Enterococcus faecium clinical isolates usually have
multiple-drug resistance. Of the drug-resistant strains, those
resistant to high levels of penicillin and those resistant to
gentamicin and glycopeptide (vancomycin) cause serious nosocomial
infections (31, 34, 45). Among the resistance traits
acquired by E. faecium, glycopeptide (vancomycin)
resistance has been shown to be encoded on a conjugative plasmid
(9, 31, 32, 38). One of the vancomycin resistance plasmids,
pIP819 (34 kb), which is a relatively small plasmid, transfers by
filter mating to other E. faecium and E. faecalis strains, a variety of streptococcus species, and
Listeria monocytogenes (9, 32). The other
plasmid, pHKK100 (58 kb), which is a relatively large plasmid, is a
pheromone-responsive plasmid and transfers efficiently
(104 per donor cell) from E. faecium to
E. faecalis JH2-2 in broth mating (19).
However, a highly efficient system for plasmid transfer during broth
mating between E. faecium strains has not been
described to date. In this report, we show that a plasmid conferring
resistance to high levels of gentamicin isolated from an E. faecium clinical strain transferred efficiently to E. faecium or E. faecalis during broth mating and
that the plasmid was different from the pheromone-responsive plasmid.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and media.
The bacterial
strains and plasmids used in this study are listed in Table
1. E. faecalis strains
were grown in Todd-Hewitt broth (THB) (Difco Laboratories, Detroit,
Mich.) or N2GT broth (nutrient broth no. 2; Oxoid Ltd., London,
England) supplemented with 0.2% glucose and 100 mM Tris-HCl (pH 7.5).
N2GT broth was also used in the mating experiments. Escherichia
coli strains were grown in Luria-Bertani medium. Agar plates were
prepared by adding 1.5% agar to broth media. All bacterial strains
were grown at 37°C. Antibiotics were used at the following
concentrations: ampicillin, 100 µg/ml; erythromycin, 12.5 µg/ml;
chloramphenicol, 20 µg/ml; fusidic acid, 25 µg/ml; rifampin, 25 µg/ml; tetracycline, 10 µg/ml; streptomycin, 500 µg/ml;
kanamycin, 500 µg/ml; gentamicin, 500 µg/ml.
Mating procedures.
Broth matings were performed as
previously described (11) with a donor/recipient ratio of
1:10. Overnight cultures of 0.05 ml of the donor and 0.45 ml of the
recipient were each added to 4.5 ml of fresh N2GT broth, and the
mixtures were incubated at 37°C with gentle agitation for the
appropriate times and then vortexed. Portions of the mixed cultures
were then plated on solid media with appropriate selective antibiotics.
Colonies were counted after 48 h of incubation at 37°C.
Pheromone induction and the mating experiments were performed as
previously described (5, 6, 10, 23).
Clumping assay.
Detection of aggregation (clumping) was as
previously described (4, 10, 11). The pheromone corresponded
to a culture filtrate of plasmid-free strain FA2-2. Generally, 0.5 ml
of culture filtrate from late-log-phase cells was mixed with 0.5 ml of
fresh N2GT broth and 20 µl of the overnight-cultured cells to be
tested for their ability to respond. The mixtures were cultured for 2 to 4 h at 37°C with shaking and were examined for clumping.
Isolation and manipulation of plasmid DNA.
Plasmid DNA was
isolated by the alkaline lysis method (36). Plasmid DNA was
treated with restriction enzymes and subjected to agarose gel
electrophoresis for analysis of DNA fragments, etc. Restriction enzymes
were obtained from Nippon Gene (Toyama, Japan), New England Biolabs,
Inc., and Takara (Tokyo, Japan) and were used in accordance with the
supplier's specifications. Agarose was obtained from Wako Chemicals,
Osaka, Japan. Gels with a 0.8% agarose concentration were used for
size determination of large DNA fragments (greater than 0.5 kb), and
2.0% agarose gels were used to determine the sizes of smaller
fragments (less than 0.5 kb) (15, 43). A "glass milk"
kit (Gene Clean II kit; Bio 101, Inc., La Jolla, Calif.) or
low-melting-point agarose and -agarase I (Nippon Gene) were used for
the elution of the DNA fragments from agarose gels. The eluted
fragments were ligated to dephosphorylated, restriction enzyme-digested
vector DNA with T4 DNA ligase and were then introduced into
E. coli by electrotransformation (14). Transformants were selected on Luria-Bertani agar medium containing suitable antibiotics.
Determination of the pMG1 restriction map.
Initially, pMG1
DNA was digested with EcoRI, and the sizes of the
EcoRI fragments were determined. The molecular sizes of EcoRI fragments A to E were 50.2, 11.5, 2.0, 0.7, and 0.7 kb, respectively. To determine the order of the EcoRI
fragments, pMG1 DNA was partially digested with a low concentration of
EcoRI or was completely digested with EcoRI,
KpnI, XbaI, EcoRV, or NdeI and one of the other enzymes examined (double digestion). The digested
DNAs were examined by agarose gel electrophoresis (data not shown), and
the order of the fragments determined by these methods was ACDEB.
To determine the order of EcoRI fragments, the relational
clone method was also used and a relational clone set was obtained by
methods previously described (15). After agarose gel
electrophoresis of an EcoRI partial digest of pMG1 DNA, the
fragments were eluted and used for cloning. The cloning vector and host
strain were pAM401 and E. coli DH1, respectively.
Southern hybridization.
Southern hybridization was performed
with the digoxigenin-based nonradioisotope system of Boehringer GmbH
(Mannheim, Germany), and all procedures were mainly based on the
manufacturer's manual and common protocols (36).
Hybridization was performed at 42°C overnight in the presence of 50%
formamide. Signals were detected with the DIG chemiluminescence
detection kit (Boehringer GmbH). CSPD (Boehringer, GmbH) was used as a
substrate for alkali phosphatase conjugated to antidigoxigenin
antibody.
| |
RESULTS AND DISCUSSION |
Isolation of plasmid pMG1 from an E. faecium
clinical isolate.
Thirty-three E. faecium clinical
isolates were examined for resistance to gentamicin. Two of the 33 E. faecium clinical isolates showed resistance
to concentrations of gentamicin greater than 1,000 µg/ml. The two
strains were designated GF112 and GF113. GF112 was resistant to
erythromycin, tetracycline, and gentamicin, and GF113 was resistant to
ampicillin, tetracycline, and gentamicin. To examine the
transferability of the gentamicin resistance trait, broth mating
experiments were performed between each of the Gmr
E. faecium strains and an E. faecium
KTRF recipient strain. The mixture of donor and recipient was incubated
at 37°C with gentle agitation for 4 h and then vortexed. A
0.1-ml aliquot of the mixed culture was plated out on THB agar
containing 500 µg of gentamicin per ml for selection of the plasmid
plus 25 µg of rifampin and fusidic acid per ml for counterselection.
The two strains were found to transfer the gentamicin resistance trait.
GF112 was used for further analysis. E. faecium GF112
harbored several plasmids, and the E. faecium KTRF
transconjugant also harbored a number of plasmids (data not shown). To
identify the Gmr plasmid, repeated transfer experiments
between E. faecium KTRF and KTSS were performed by
short mating (30-min mating). To examine the plasmid content of the
transconjugant, plasmid DNA was isolated from 30 transconjugants
obtained from each mating experiment; the DNA was digested with
EcoRI and examined by agarose gel electrophoresis. After
repeated transfer experiments, one gentamicin-resistant transconjugant of E. faecium KTRF was found to
harbor a single plasmid. The plasmid was designated pMG1 and conferred
gentamicin resistance. The restriction map of pMG1 was constructed by
the relational clone method described in Materials and Methods.
EcoRI restriction of pMG1 gave five fragments, A
through E, with molecular sizes of 50.2, 11.5, 2.0, 0.7, and
0.7 kb, respectively. The clockwise order of the fragments
was ACDEB (Fig. 1). The cleavage sites for KpnI, XbaI, EcoRV, and
NdeI are also shown in Fig. 1.
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FIG. 1.
Physical map of pMG1. Fragments produced by restriction
endonuclease restriction of pMG1 DNA are denoted by letters.
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Conjugative transfer in Enterococcus strains.
Plasmid pMG1 was examined for conjugative transfer in broth. As shown
in Table 2, pMG1 transferred between
E. faecium strains, between E. faecalis
strains, and between E. faecium and E. faecalis strains. The transfer frequencies were around
104 per donor cell after 3 h of mating (Table 2).
pMG1 also transferred from E. faecium to E. hirae 9790 at a frequency of about 104 per donor
cell (data not shown). Plasmid pAM714 is an erythromycin resistance-conferring derivative of pAD1::Tn917
(23). Plasmid pAD1 (58 kb) (5, 6, 8) is a
pheromone-responsive conjugative hemolysin/bacteriocin plasmid found in
E. faecalis. Pheromone-responsive conjugative plasmid
pAM714 transfers between E. faecalis strains at a
frequency of 102 per donor cell. However, the plasmid did
not transfer to E. faecium in broth.
Figure 2 shows the kinetics of pMG1 and
pAM714 plasmid transfer. pMG1 showed normal transfer between
E. faecium strains and between E. faecalis strains. Approximately 60 min of mating was required
before a significant level of transfer occurred, and an increase in the
number of transconjugants was observed between 2 and 3 h after the
start of the mating experiment. In the early stage of mating, e.g.,
between 15 and 30 min, a few transconjugants grew on the selective agar
plates. Pheromone-responsive plasmid pAM714 also showed normal transfer
(23). The maximum transfer frequency of pMG1 was about
5 × 104 after 3 h of mating, which was about a
factor of 1 × 102 less than that of pAM714. For the
pheromone-responsive plasmid, a sex pheromone secreted into the broth
culture by the recipient induces the synthesis of an aggregation
substance on the donor cell surface, and donor and recipient then form
a mating aggregate (4, 10, 11). Usually, the transconjugant
of the pheromone-responsive plasmid is not obtained in a short mating
period, such as 15 min.
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FIG. 2.
Kinetics of transfer for pheromone-independent plasmid
pMG1 and pheromone-dependent plasmid pAM714. Matings were carried out
with mixtures that each consisted initially of 0.05 ml of the donor,
0.45 ml of the recipient, and 4.5 ml of fresh N2GT broth. The mixtures
were incubated at 37°C with gentle shaking. At the indicated time
points, 0.1-ml samples were removed, diluted appropriately, and plated
on THB agar plates containing gentamicin or erythromycin for the
selection of pMG1 or pAM714 transconjugants, respectively, plus drugs
for selection of the recipient strains. Symbols:  , transfer from
E. faecalis FA2-2(pMG1) to E. faecalis
JH2SS;  , transfer from E. faecium KTSS(pMG1) to
E. faecium KTRF;  , transfer from E. faecalis JH2SS(pAM714) to E. faecalis FA2-2.
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The mating kinetics of pMG1 suggest that mating aggregates occur by the
random collision of donor and recipient cells, with little or no
transfer of the plasmid occurring in the first 60 min of mating.
Increased transfer occurs after this period. In the early stage of the
mating experiment, a few mating cells survived and completed mating on
the selective agar plate, and the resulting transconjugants then grew.
These observations suggest that some factor necessary for plasmid
transfer is induced during coincubation of the donor and recipients and
that induction of this factor might require contact between the donor
and recipient.
Pheromone response.
The pheromone-responsive plasmids of
E. faecalis induce the synthesis of a proteinaceous
surface adhesion substance (i.e., aggregation substance) on the donor
cell surface, which facilitates the formation of mating aggregates upon
random collision between donor and recipient cells (4, 10,
11). The donor cells can be induced to self-aggregate by
induction with the pheromone of recipient cells (4, 10, 11).
Mating aggregates are observed by the naked eye during broth matings
between E. faecalis donor cells containing pAM714 and
an E. faecalis recipient cell. The self-aggregates of
the donor cells were also observed by induction with a culture
filtrate (pheromone) of FA2-2 (23).
In mating experiments between donor cells containing pMG1
and recipient cells, mating aggregates were not observed by the naked eye during broth mating. During broth mating, small mating aggregates of several cells were observed by microscopy (Fig. 3A), while aggregates of donor cells were
not even observable by microscopy after exposure to a culture
filtrate of the recipient strain, E. faecalis FA2-2 or
E. faecium KTRF (Fig. 3B).
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FIG. 3.
Microscopic observation. The mating mixture (A) and
pheromone-exposed donor cells (B) were observed by microscopy (Nikon
Optiphoto 2 at ×1,000 magnification) after 2 h of mating between
E. faecalis FA2-2(pMG1) donor cells and E. faecalis JH2SS recipient cells, as shown in Fig. 2 (A), and after
2 h of exposure of E. faecalis FA2-2(pMG1) to
FA2-2 culture filtrate (pheromone), as shown in Table 3 (B).
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We examined whether the mating aggregate is dispersed by the addition
of EDTA (49). Mating aggregates of E. faecalis JH2SS containing pheromone-responsive plasmid pAM714 and
the recipient FA2-2 were dispersed by the addition of 25 mM EDTA,
whereas mating aggregates of E. faecalis JH2SS
containing pMG1 and the recipient FA2-2 were not dispersed by the
addition of 25 mM EDTA (data not shown). These results suggest that a
mating substance encoded by pMG1 may be produced and may be located on
the donor cell surface; this mating substance is different from the
aggregation substance produced by the E. faecalis
strain containing the pheromone- responsive plasmid.
Pheromone induction and a mating experiment were performed to determine
whether plasmid pMG1 responded to the pheromone of E. faecalis (5, 6, 10, 23). Donor cells of E. faecalis FA2-2(pMG1) or E. faecium KTRF(pMG1) and
E. faecalis FA2-2(pAM714) were exposed (for 120 min) to
an FA2-2 culture filtrate (pheromone) to induce aggregation and mating
functions before a short (10-min) mating period. The short
mating was carried out between the induced or uninduced donor cells
and the recipient cells. As shown in Table
3, the transfer frequency for the induced
and uninduced plasmid pMG1 was less than 107 per donor
cell. On the other hand, the transfer frequency of induced
pheromone-responsive plasmid pAM714 was 103 per donor
cell and that of the uninduced plasmid was less than 107
per donor cell. These results indicated that plasmid pMG1 did not
respond to the pheromone.
DNA-DNA hybridization.
Several pheromone-responsive plasmids
have been reported. Among these plasmids, the pheromone-related
conjugation systems are well studied for pAD1 (5, 6, 16, 17, 39,
40, 47), pCF10 (3, 12, 20, 33), and pPD1 (15,
17), which confer responses to the sex pheromones cAD1, cCF10,
and cPD1, respectively. The genes involved in the regulation of the pheromone response have been identified and are known to be clustered in a 7-kb region on each plasmid. There is homology between the genes
of these plasmids (15, 17, 21). There is also homology between the genes of the putative surface exclusion protein and those
of the aggregation substance, which are located downstream of the
regulatory region (15, 17). For pAD1, the genes of the
pheromone response regulatory region are located in EcoRI fragment B and the genes for the aggregation substance are located in
EcoRI fragments A and E (5, 17, 40). For pPD1,
the genes of the pheromone response regulatory region are located in
EcoRI fragment A and the genes of the aggregation substance
are located in EcoRI fragments A, C, and G (15,
17). In a previous study, 18 pheromone-responsive plasmids,
including pAD1, pPD1, and pCF10, were analyzed for homology by Southern
blotting (21). In that study, plasmid pAM373 did not show
homology to the aggregation substance genes of the other
pheromone-responsive plasmids (21).
Plasmid pMG326, which contains a part (11.5 kb) of EcoRI
fragment A of pPD1, contains the regulatory region for the pheromone response of pPD1, a possible surface exclusion protein gene, and the
gene encoding the N-terminal region of the aggregation substance (37). Plasmid pMG326 DNA was studied for homology with that of plasmid pMG1 (Fig. 4B). The pMG326 DNA
probe hybridized to specific EcoRI fragments of
pheromone-responsive plasmids pAD1, pPD1, and pAM373 and
broad-host-range plasmid pAM1. The pMG326 DNA probe hybridized to
EcoRI fragments B and E of pAD1 and to EcoRI
fragment A of pPD1. But the pMG326 DNA probe did not hybridize with any
EcoRI fragments of pMG1 and broad-host-range plasmid pIP501.
These results indicate that pMG1 did not contain any sequences homologous with the consensus sequence found in the
pheromone-responsive plasmids.
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FIG. 4.
Agarose gel electrophoresis of restriction
endonuclease-digested plasmid DNAs and hybridization with plasmid
pMG326, which contains a part (11.5 kb) of EcoRI fragment A,
or the pMG1 probe. (A) Agarose gel electrophoresis of restriction
endonuclease-digested plasmid DNAs. Plasmid pIP501 was digested with
KpnI, and the other plasmids were digested with
EcoRI. Lanes: 1, pAM1; 2, pAM373; 3, pIP501; 4, pMG1; 5, pAD1; 6, pPD1; 7, HindIII-digested lambda DNA.
Duplicate gels were Southern blotted and hybridized to pMG326 DNA (B)
or pMG1 DNA (C).
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Plasmid pMG1 DNA was also studied for homology with that of
pheromone-responsive plasmids pAD1, pPD1, and pAM373 and
broad-host-range plasmids pIP501 and pAM1 (Fig. 4C). The pMG1 DNA
probe hybridized to all EcoRI fragments of pMG1 DNA, whereas
the pMG1 DNA probe did not hybridize with any EcoRI
fragments of other plasmid DNAs.
The hybridization studies indicate that pMG1 did not contain any
sequences homologous with those of the pheromone-responsive plasmids or
the broad-host-range plasmids; thus plasmid pMG1 was different from
these conjugative plasmids with respect to its DNA sequence.
Conjugative transposon Tn916 (16.5 kb; encodes tetracycline
resistance) can be transferred from a donor to a recipient strain during conjugative transposition. The transfer of Tn916
occurs via mating on a solid surface (filter mating). Jaworski and
Clewell (27) reported that mutants of Tn916 can
transfer efficiently via liquid mating. Thus, conjugative
transposon Tn916 was studied for homology with plasmid pMG1.
Plasmid pAM120 (18), consisting of Tn916
cloned into pBR322-derived vector pGL101, was used for the
hybridization study with EcoRI fragments of pMG1 DNA. pAM120 did not hybridize with any EcoRI fragment of pMG1 (data not
shown), indicating that pMG1 did not contain any sequence homologous
with that of conjugative transposon Tn916.
Among the gram-positive bacteria, a highly efficient transfer system
which functions during broth mating has only been found in
pheromone-responsive plasmids (4-6, 10, 12). The majority of pheromone-responsive plasmids have been found in E. faecalis and transfer efficiently in broth between E. faecalis strains (4, 6). One pheromone-responsive
plasmid is found in E. faecium and also transfers
efficiently via broth mating from E. faecium to
E. faecalis (38). pMG1 is a relatively large
plasmid (65.1 kb), which transfers efficiently in broth between
E. faecium strains, between E. faecalis
strains, between E. faecium and E. faecalis strains, and also between E. faecium and
Enterococcus hirae 9790, indicating that pMG1 has great
ability to transfer among the enterococci.
In contrast to what was found for the pheromone-responsive plasmids,
E. faecalis or E. faecium strains
carrying pMG1 were not induced to form mating aggregates or to transfer
the plasmid by the E. faecalis pheromone (culture
filtrate of E. faecalis FA2-2), indicating that plasmid
pMG1 does not require a pheromone.
This work was supported in part by grants for the study of
drug-resistant bacteria from the Japanese Ministry of Health and Welfare in 1996, 1997, and 1998 and by a grant from the Japanese Ministry of Education, Science, and Culture.
We thank F. L. Macrina for providing plasmid pIP501 and D. B. Clewell for providing plasmids pAM1 and pAM373. We thank E. Kamei for helpful advice on the manuscript.
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Abstract
Introduction
