DNA hybridization and contour-clamped
homogeneous electric field electrophoresis for
identification of enterococci to the species
level.
S Donabedian, J W Chow, D M Shlaes, M Green and M J Zervos
J. Clin. Microbiol. 1995, 33(1):141.
These include:
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JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1995, p. 141–145
0095-1137/95/$04.0010
Copyright q 1995, American Society for Microbiology
Vol. 33, No. 1
DNA Hybridization and Contour-Clamped Homogeneous
Electric Field Electrophoresis for Identification
of Enterococci to the Species Level
SUSAN DONABEDIAN,1 JOSEPH W. CHOW,2 DAVID M. SHLAES,3 MICHAEL GREEN,4
1,2
AND MARCUS J. ZERVOS *
Received 19 May 1994/Returned for modification 1 July 1994/Accepted 11 October 1994
In this study, 113 Enterococcus faecium, 37 Enterococcus faecalis, 24 Enterococcus gallinarum, 15 Enterococcus
raffinosus, and 13 Enterococcus casseliflavus clinical isolates and American Type Culture Collection (ATCC)
strains were evaluated by contour-clamped homogeneous electric field electrophoresis. Thirty-one of the E.
faecium, 22 of the E. faecalis, 24 of the E. gallinarum, 15 of the E. raffinosus, and 13 of the E. casseliflavus isolates
were also evaluated by DNA-DNA hybridization. Genomic DNAs from type strains E. faecalis ATCC 19433, E.
faecium ATCC 19434, E. gallinarum ATCC 49573, E. raffinosus ATCC 49427, and E. casseliflavus ATCC 25788
were labeled with biotin for use as probes. E. faecalis differed from all other species in always having a largest
fragment of >400 kb. E. gallinarum was different from all other species in having all SmaI fragments of <200
kb. Biotin-labeled probes showed a high degree of hybridization with genomic DNA from the same species and
a low degree of hybridization when hybridized to genomic DNA from different species for all isolates tested
except for four isolates identified as E. faecium by conventional biochemical methods. The DNA from these four
isolates hybridized strongly to DNA from E. gallinarum ATCC 49573 and weakly to E. faecium ATCC 19434
DNA and had all SmaI fragments of <200 kb in size. These data suggest that these isolates are nonmotile E.
gallinarum. DNA from each ATCC type strain hybridized strongly with itself and had only a low degree of
hybridization with DNA from other ATCC type strains tested. These results suggest that contour-clamped
homogeneous electric field electrophoresis patterns and DNA-DNA hybridization with biotin-labeled probes
may be of use for species differentiation of some enterococci.
reliability for identification of some strains (23, 29). Earlier
studies have shown penicillin-binding protein and bacteriolytic
pattern analysis to be of use for species identification (22, 31).
In this study, we evaluated DNA-DNA hybridization and
contour-clamped homogeneous electric field (CHEF) electrophoresis for species differentiation of enterococci.
Enterococci are now the second leading cause of nosocomial
infections (26). Enterococcus faecalis remains the most common species isolated, but other enterococci such as Enterococcus faecium, Enterococcus raffinosus, and Enterococcus gallinarum have become increasingly prevalent (3, 4, 7, 8, 15).
Identification of an enterococcus isolate to the species level is
clinically relevant since E. faecium is more resistant to penicillins and E. gallinarum is more resistant to glycopeptides than E.
faecalis. Species identification of enterococci is important for
epidemiologic and infection control purposes. Transmission
has been associated with intra- and interhospital spread of
isolates (3, 4, 7, 8, 21, 32), and the results of species identification are often used as first evidence for nosocomial spread.
Conventional biochemical methods involve the testing of
many characteristics, which makes identification complex, time
consuming, and expensive for some isolates. Confirmation of
the identity of organisms in the event that biochemical reactions are equivocal is sometimes needed. Several systems for
identification of gram-positive cocci are commercially available
(2, 6, 11, 16–18, 24), but many are unable to recognize some of
the more recently described enterococcal species because of
phenotypic similarities among these strains (5, 13, 14, 23, 29).
Standardized automated methods provide accurate species
identification of the majority of isolates but have lacked
MATERIALS AND METHODS
Strains. Clinical isolates of enterococci from separate patients in diverse
geographic areas (1986–1993) and American Type Culture Collection (ATCC)
strains were studied. The strains studied included 113 E. faecium, 37 E. faecalis,
24 E. gallinarum, 15 E. raffinosus, and 13 Enterococcus casseliflavus isolates.
Strains were collected from diverse geographic areas in the United States,
Canada, and Italy. Some of these isolates have been reported previously (3, 4, 7,
8–10, 15, 19, 20, 28, 30). These five species of enterococci were selected for study
since they represent the most common infecting isolates of enterococci and have
been most commonly associated with antimicrobial resistance and intrahospital
spread. Type strains E. faecium ATCC 19434, E. faecalis ATCC 19433, E.
gallinarum ATCC 49573, E. raffinosus ATCC 49427, and E. casseliflavus ATCC
25788, and E. faecalis nontype strains ATCC 29200, ATCC 29212, and ATCC
35038 were evaluated by CHEF electrophoresis, and genomic DNAs from type
strains were used as probes in hybridization studies. Genomic DNAs from
Escherichia coli ATCC 25922 and calf thymus were used as negative controls for
hybridizations. Strains were grown for 18 h in brain heart infusion (BHI) broth
(Difco Laboratories, Detroit, Mich.) mixed with an equal volume of sterile
glycerol and stored at 2708C. Conventional biochemical reactions as outlined by
Facklam and Collins were used to identify the organisms (12). Motility was
determined with motility test medium (Difco) modified by adding 4 g of nutrient
broth (Difco) and 1 g of NaCl per liter. Motility test media were incubated at
308C and read at 24 h, 48 h, and 7 days. For four isolates from Pittsburgh, Pa.,
identified as nonmotile E. gallinarum, motility was determined in multiple
experiments in two separate laboratories. The in vitro antibiotic susceptibility of
* Corresponding author. Mailing address: William Beaumont Hospital, 3601 West 13 Mile Rd., Royal Oak, MI 48073. Phone: (810)
551-4040. Fax: (810) 551-8800.
141
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Infectious Diseases Section, Department of Medicine, William Beaumont Hospital, Royal Oak, Michigan 480731;
Wayne State University School of Medicine, Detroit, Michigan 482012; Medical and Research Services,
Infectious Diseases Section, Department of Veterans Affairs Medical Center, Cleveland, Ohio 441063;
and Departments of Pediatrics and Surgery, Children’s Hospital of Pittsburgh, University
of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 152134
142
DONABEDIAN ET AL.
J. CLIN. MICROBIOL.
TABLE 1. Size of largest fragment of SmaI-digested genomic DNA of five species of enterococci and number
of isolates used in hybridization study
Species
E.
E.
E.
E.
E.
a
faecium
faecalis
gallinarum
raffinosus
casseliflavus
No. of isolates evaluated by
CHEF electrophoresis
No. of strain types by
CHEF electrophoresis
Largest SmaI
fragment (kb)
No. of isolates evaluated
by hybridization
No. of strain types
by hybridization
113
37
24
15
13
61
31
22
11
13
,400 (97)a
.400 (100)
,200 (100)
,350 (67)
,300 (77)
31
22
24
15
13
15
21
22
11
13
Values in parentheses are percentages of strains having largest SmaI fragment of this size.
tion solution (50% formamide, 0.75 M NaCl, 75 mM Na citrate, 25 mM
NaH2PO4, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.1% Ficoll,
0.5 mg of sheared denatured salmon sperm DNA per ml). The bag was sealed
and incubated in a shaking water bath at 428C for 4 h. The prehybridization
solution was removed, and 10 ml of hybridization solution (45% formamide, 0.75
M NaCl, 75 mM Na citrate, 20 mM NaH2PO4, 0.02% polyvinylpyrrolidone,
0.02% bovine serum albumin, 0.2% Ficoll, 5.0% dextran sulfate, 0.2 mg of
sheared denatured salmon sperm DNA per ml; and purified biotinylated
genomic DNA [0.1 mg/ml]) was added, and the bag was sealed and incubated in
a shaking water bath at 428C for 18 h. The membrane was then washed in 250 ml
of 23 SSC–0.1% SDS two times for 3 min, 250 ml of 0.23 SSC–0.1% SDS two
times for 3 min, 250 ml of 0.163 SSC–0.1% SDS two times for 15 min at 508C,
and then in 250 ml of 23 SSC at room temperature for 3 min.
The BluGene nonradioactive nucleic acid detection system (BRL) was used to
detect biotinylated probe as described in the manufacturer’s directions. The
degree of hybridization of biotin-labeled genomic DNA from each ATCC type
strain with genomic DNA from clinical isolates was determined by comparing
these reactions with those of the labeled ATCC strain DNA with itself (positive
control) and with E. coli and calf thymus DNA (negative controls). Biotinylated
DNA from each ATCC strain was also hybridized with unlabeled DNA from all
other ATCC strains in this study. The degree of hybridization was determined by
visual inspection. All hybridizations were done in duplicate to determine
reproducibility.
RESULTS
CHEF electrophoresis. By CHEF electrophoresis, E. faecalis
differed from all other enterococcal species studied in that its
largest SmaI fragment was always greater than 400 kb (Table
1); 78% of E. faecalis isolates had fragments larger than 500 kb.
Ninety-seven percent of E. faecium isolates had a largest SmaI
fragment of ,400 kb (Table 1), and 85% had a largest SmaI
fragment of ,350 kb. Of all isolates identified by biochemical
reactions as E. faecium, four isolates from Pittsburgh had SmaI
fragments all smaller than 200 kb (Fig. 1). Three of these
isolates had identical SmaI restriction enzyme patterns by
CHEF electrophoresis, and the fourth differed by only one
band. All isolates had ampicillin MICs of 1.0 mg/ml and
vancomycin MICs of 8.0 mg/ml. E. gallinarum always had a
largest fragment of ,200 kb. Sixty-seven percent of E. raffinosus isolates had a largest SmaI fragment of ,350 kb, and 77%
of E. casseliflavus isolates had a largest SmaI restriction
fragment of ,300 kb. Under the CHEF conditions used in this
study, the SmaI restriction fragments of E. faecalis isolates
were spread out across the entire length of the gel (,48.5 to
500 kb), those of E. faecium and E. raffinosus isolates were
concentrated at the bottom two-thirds of the gel (85% of E.
faecium and 67% of E. raffinosus isolates ranged between
,48.5 and ,350 kb), and E. gallinarum SmaI fragments were
grouped tightly at the bottom one-third of the gel (,200 kb)
(Fig. 1). By changing to a ramped switching time of 0.1 to 20 s,
these tightly grouped fragments could be further separated for
DNA typing purposes. E. casseliflavus fragment patterns were
similar to those of E. gallinarum, but 62% of the strains also
had one to three bands larger than 200 kb.
Hybridization studies. Biotin-labeled genomic DNA from E.
faecium type strain ATCC 19434 hybridized strongly to unla-
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these four strains to ampicillin and vancomycin was determined by use of
techniques (10, 20) described previously.
DNA methods. (i) Preparation of genomic DNA for CHEF electrophoresis.
Genomic DNA for CHEF electrophoresis was prepared by modifying procedures described previously (10, 27). All reagents were from Sigma Chemical Co.
unless otherwise noted. Cells were grown in 5 ml of BHI broth (Difco) for 18 h
and collected by centrifugation. The cell pellet was suspended in 2 ml of PIV
buffer (1 M NaCl, 10 mM Tris [pH 7.6]) and mixed with 2 ml of 1.6%
low-melting-point agarose (Bethesda Research Laboratories [BRL], Gaithersburg, Md.) at 558C. The mixture was transferred into sample plug molds
(Bio-Rad, Hercules, Calif.) and refrigerated for 1 h. Sample plugs were
incubated at 378C for 18 h in 5 ml of lysis buffer (6 mM Tris, 1 M NaCl, 100 mM
EDTA, 0.5% Brij 58, 0.2% deoxycholate, 0.5% sarcosine, 20 mg of RNase per ml,
1 mg of lysozyme per ml [pH 7.6]), incubated at 558C for 18 h in 5 ml of ESP
solution (0.5 M EDTA [pH 9 to 9.5], 1.0% sarcosine, 50 mg of proteinase K per
ml), incubated with 5 ml of TE (10 mM Tris, 0.1 mM EDTA [pH 7.5]) at 378C
three times (30 min each time), and stored in TE at 48C. To digest the DNA,
one-quarter of a sample plug was placed in a microcentrifuge tube containing 0.3
ml of sterilized deionized water, 30 ml of React 4 buffer (BRL), and 30 U of SmaI
(BRL) and incubated overnight at room temperature. The plug was then
incubated in 1 ml of TE at 378C for 1 h. Sample plugs were loaded in 0.8%
agarose gel in 0.53 TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA),
electrophoresed on a CHEF DR II apparatus (Bio-Rad; initial switch time, 5 s;
final switch time, 35 s; start ratio, 1; voltage, 200; run time, 21 h; temperature,
148C), and stained with ethidium bromide. The sizes of fragments were determined by comparing them with lambda ladder standards (New England Biolabs,
Beverly, Mass.).
(ii) Preparation of DNA for hybridizations. Genomic DNA from all strains
was prepared by a method described previously (1). Genomic DNA from calf
thymus was from Sigma. Isolates were grown in 2 ml of BHI broth for 18 h at
378C. One and one-half milliliters of this culture was transferred to a microcentrifuge tube, and the pellet was collected by centrifugation for 2 min. The cells
were suspended in 565 ml of TE containing 10 mg of lysozyme per ml, 5 U of
mutanolysin was added, and the solution was incubated at 378C for 30 min. Three
microliters of a 20-mg/ml concentration of proteinase K and 30 ml of 10% sodium
dodecyl sulfate (SDS) were added, and the solution was incubated at 378C for 1
h. One-hundred microliters of 5 M NaCl was added, the solution was mixed
thoroughly, and then 80 ml of 10% CTAB (cetyl-trimethylammonium bromide)
in 0.7 M NaCl was added and the mixture was incubated at 658C for 10 min. The
solution was then extracted with a 24:1 mixture of chloroform-isoamyl alcohol,
mixed thoroughly, and spun in a microcentrifuge for 5 min. The aqueous layer
was removed to a fresh microcentrifuge tube, extracted with a 25:24:1 mixture of
phenol-chloroform-isoamyl alcohol, mixed thoroughly, and spun in a microcentrifuge for 5 min. The aqueous phase was transferred to a fresh microcentrifuge
tube, and the DNA was precipitated by adding 0.6 volume of isopropanol. The
DNA was pelleted in a microcentrifuge, washed with 70% ethanol, dried, and
suspended in 100 ml of TE containing 2 mg of RNase.
Agarose gel electrophoresis and Southern blot. One microgram of DNA from
all strains was digested with 10 U of EcoRI (BRL), run on a 0.7% vertical
agarose gel in TAE (0.4 M Tris, 0.2 M Na citrate, 9 mM EDTA [pH 8.2]) at 60
V for 2 h, and stained with ethidium bromide. The DNA was nicked on a 312-nm
UV transilluminator for 5 min, denatured by soaking the gel in 200 ml of 0.5 N
NaOH–1.5 M NaCl two times for 10 min, and then neutralized by soaking the gel
in 200 ml of 1 M Tris–1.5 M NaCl (pH 8) two times for 10 min. The gel was then
rinsed in 103 SSC (13 SSC is 0.015 M Na citrate plus 0.15 M NaCl [pH 7]),
blotted by the capillary transfer method (25) onto a MagnaCharge nylon
membrane (Micron Separations, Inc., Westboro, Mass.), and then baked in a
vacuum oven at 808C for 1.5 h.
Labeling of probes. Two micrograms of DNA from all ATCC type strains was
labeled with biotin-7-dATP (BRL) by the Nick Translation System (BRL).
Biotin-labeled probe was purified on a Sephadex G-75 column, precipitated by
adding 1/10 its volume of 3 M Na acetate and 2 volumes of ethanol, and kept at
2208C for 18 h. The purified biotinylated genomic DNA was suspended in 0.3 ml
of deionized water.
Hybridization and detection of biotinylated probe. The membrane was soaked
in 23 SSC and placed in a hybridization bag (BRL) with 10 ml of prehybridiza-
VOL. 33, 1995
PROBES FOR SPECIES IDENTIFICATION OF ENTEROCOCCI
143
beled, EcoRI-digested genomic DNA from itself (Fig. 2A and
B, lane 1) and to DNA from 108 clinical isolates identified as
E. faecium by conventional biochemical testing. DNA from all
other enterococcal ATCC strains and clinical isolates tested
hybridized only weakly with the E. faecium ATCC 19434
probe, including four isolates from Pittsburgh which were
identified as E. faecium by biochemical testing (Fig. 3A, lanes
7 through 10). Biotin-labeled genomic DNA from E. gallinarum type strain ATCC 49573 hybridized strongly with unlabeled EcoRI-digested genomic DNA from itself (Fig. 2C, lane
2), from all other clinical isolates identified as E. gallinarum by
biochemical tests, and from the four isolates from Pittsburgh
which were identified as E. faecium by conventional biochemical testing (Fig. 3B, lanes 7 through 10) and had SmaI bands
all smaller than 200 kb. DNA from one strain, which was
identified as an E. gallinarum by biochemical testing, hybridized only weakly to the ATCC 49573 probe but was previously
described as being a nonpigmented E. casseliflavus isolate (30).
DNA from all other enterococcal ATCC strains and clinical
isolates hybridized only weakly with the E. gallinarum 49573
probe. Biotin-labeled genomic DNA from E. casseliflavus type
strain ATCC 25788 hybridized strongly to unlabeled, EcoRIdigested genomic DNA from itself (Fig. 2D, lane 3) and to
DNA contained in all other clinical isolates identified as E.
casseliflavus, including the nonpigmented E. casseliflavus isolate mentioned above. DNA from all other enterococcal
ATCC strains and clinical isolates hybridized only weakly with
the E. casseliflavus ATCC 25788 probe. Biotin-labeled genomic
DNA from E. faecalis type strain ATCC 19433 hybridized
strongly to unlabeled, EcoRI-digested genomic DNA from
itself (Fig. 2E, lane 4), to DNA from E. faecalis ATCC 29200,
ATCC 29212, and ATCC 35038, and to DNA from all clinical
isolates identified as E. faecalis and only weakly to DNA
contained in all other enterococcal ATCC strains and clinical
isolates in this study. Biotin-labeled genomic DNA from E.
raffinosus type strain ATCC 49427 hybridized strongly to
unlabeled, EcoRI-digested genomic DNA from itself (Fig. 2F,
FIG. 2. (A) Agarose gel electrophoresis of genomic DNA digested with
EcoRI. (B to F) Southern blots probed with biotin-labeled genomic DNA from
E. faecium ATCC 19434 (B), E. gallinarum ATCC 49573 (C), E. casseliflavus
ATCC 25788 (D), E. faecalis ATCC 19433 (E), and E. raffinosus ATCC 49427
(F). Lanes: 1, E. faecium ATCC 19434; 2, E. gallinarum ATCC 49573; 3, E.
casseliflavus ATCC 25788; 4, E. faecalis ATCC 19433; 5, E. raffinosus ATCC
49427; 6, E. coli ATCC 25922; 7, calf thymus.
lane 5) and to DNA from all other clinical isolates identified as
E. raffinosus and only weakly to DNA contained in other
enterococcal ATCC strains and clinical isolates in this study.
DNA from the ATCC enterococcal strains tested did not
hybridize with genomic DNA from E. coli ATCC 25922 or calf
thymus DNA to any detectable degree (Figs. 2A to F, lanes 6
and 7). Results of all hybridizations were the same in parallel
experiments.
DISCUSSION
The taxonomy of Enterococcus spp. has not been defined
completely. With increasing recognition of enterococci other
than E. faecalis as the causes of serious nosocomial infection,
identification of these organisms by the clinical microbiology
laboratory for clinical, epidemiologic, or infection control
purposes has become necessary.
The results of this study suggest that CHEF electrophoresis
patterns of SmaI-digested genomic DNA are characteristic of
some enterococcal species and can be helpful when used with
Downloaded from http://jcm.asm.org/ on April 30, 2014 by PENN STATE UNIV
FIG. 1. CHEF electrophoresis of SmaI-digested genomic DNA from enterococcal strains. Lanes: 1 and 16, lambda phage DNA ladder standard; 2, E. faecalis
ATCC 19433; 3, E. faecalis clinical isolate; 4, E. faecium ATCC 19434; 5, E.
faecium clinical isolate; 6, E. gallinarum ATCC 49573; 7, E. gallinarum clinical
isolate; 8, E. raffinosus ATCC 49427; 9, E. raffinosus clinical isolate; 10, E.
casseliflavus ATCC 25788; 11, E. casseliflavus clinical isolate; 12 to 15, four
isolates from Pittsburgh identified as E. faecium by conventional biochemical
reactions and as E. gallinarum by hybridization analysis.
144
DONABEDIAN ET AL.
biochemical tests for identifying these organisms to the species
level. CHEF analysis has proved invaluable in strain typing of
enterococci for epidemiologic and infection control purposes.
For epidemiologic studies, enterococci are considered to be
the same strain if they have all SmaI restriction fragments in
common by CHEF electrophoresis and clonally related if they
differ by no more than three fragments. When different species
of enterococci are compared, as in this study, we found
multiple differences in restriction fragment size. This can also
occur within an individual species; however, the fragments are
usually within a certain size range for each species. Under the
CHEF conditions in this study, the differences between E.
faecalis, E. faecium, and E. gallinarum were most striking. For
instance, all E. gallinarum isolates tested had all SmaI fragments of ,200 kb and all E. faecium isolates tested had many
SmaI fragments of .200 kb, and yet their biochemical profiles
are very similar. E. faecalis isolates tested always had at least
one SmaI fragment of $400, while only 3% of E. faecium, 8%
of E. casseliflavus, 7% of E. raffinosus, and 0% of E. gallinarum
isolates had SmaI fragments of .400 kb. CHEF electrophoresis is now routinely used in epidemiologic studies and is not a
labor-intensive procedure. It is not an unequivocal method for
determining the species of enterococci but can provide valu-
able information in cases where biochemical tests are ambiguous.
This study also shows DNA probes constructed from
genomic DNA contained in ATCC enterococcal type strains to
be useful for species differentiation of enterococci. Probes
derived from E. faecalis, E. faecium, E. gallinarum, E. raffinosus, and E. casseliflavus ATCC strains hybridized strongly to
DNA from clinical isolates of the corresponding species.
Results were reproducible and easy to interpret. Although the
evaluation is not quantitative, it is a way to visually assess the
approximate degree of DNA homology between isolates and
may provide more accurate species identification. The practical
advantage and feasibility of DNA probes make the use of DNA
probes a preferred method compared with CHEF electrophoresis for species identification of enterococci.
According to the typing scheme of Facklam and Collins (12),
motility is a characteristic of both E. gallinarum and E.
casseliflavus. The primary differentiating characteristic between these two species is the production of yellow pigment by
E. casseliflavus (5, 12). Pigment production and motility may
occasionally be misleading criteria for definitive identification
of E. gallinarum and E. casseliflavus, since these properties may
not be stable in some isolates. In an earlier study, isolates
identified as E. gallinarum by conventional tests were shown to
be non-pigment-producing E. casseliflavus isolates on the basis
of penicillin-binding protein profiles and DNA homology (30).
One of these isolates was tested in this study, and our data
support this conclusion. Similarly, E. casseliflavus ATCC
strains which were nonmotile showed DNA homology with the
motile E. casseliflavus clinical isolates with which they were
compared (30). On the basis of the typing scheme of Facklam
and Collins (12), motility is the primary biochemical characteristic differentiating E. gallinarum from E. faecium. Four
isolates in this study (from Pittsburgh) were identified as E.
faecium by conventional biochemical tests since they were
nonmotile; however, DNA from these isolates hybridized only
weakly to the E. faecium ATCC 19434 probe. The E. gallinarum ATCC 49573 probe hybridized strongly to DNA from
these isolates. Three of these isolates had identical SmaI
restriction enzyme patterns, and the fourth differed by only one
band, indicating that they are clonally related. All SmaI
fragments were less than 200 kb in size as were those of all E.
gallinarum strains tested. No other E. faecium strain tested had
all SmaI fragments of ,200 kb. In addition, these isolates were
ampicillin susceptible, unlike most other vancomycin-resistant
E. faecium isolates and typical of E. gallinarum. These results
suggest that these isolates could be nonmotile E. gallinarum
isolates and that motility may therefore not always be a reliable
trait in itself to distinguish E. gallinarum from E. faecium. DNA
probes and CHEF electrophoresis will not replace standard
biochemical identification of enterococci but may provide
information which can be of use in identifying some isolates to
the species level.
ACKNOWLEDGMENTS
This work was funded in part by the William Beaumont Hospital
Research Institute.
We thank John Boyce, Lynn Steele-Moore, Robert McCabe, Valerie
Chirurgi, Sheldon Markowitz, Philip Coudron, Alex Kuritza, Carl
Pierson, Daniel Sahm, Mary Hayden, Andrew Simor, and Lizzie
Harrell for contributing bacterial isolates used in this study.
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