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Journal of Clinical Microbiology, November 2000, p. 4242-4245, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Improved Pulsed-Field Gel Electrophoresis for
Typing Vancomycin-Resistant Enterococci
Durmishkhan
Turabelidze,1
Mamuka
Kotetishvili,1,2
Arnold
Kreger,1
J. Glenn
Morris Jr.,1 and
Alexander
Sulakvelidze1,*
Division of Molecular Epidemiology,
Department of Epidemiology and Preventive Medicine, University of
Maryland School of Medicine, Baltimore, Maryland
21201,1 and the Kanchaveli Institute
of Plant Protection, Tbilisi, Georgia 3800772
Received 25 April 2000/Returned for modification 28 June
2000/Accepted 1 September 2000
 |
ABSTRACT |
A rapid protocol for subtyping vancomycin-resistant enterococci by
pulsed-field gel electrophoresis is reported. The procedure is simple
and potentially cost-effective and allows reproducible subtyping of the
strains in approximately 1 day.
| |
TEXT |
Vancomycin-resistant enterococci
(VRE) have been isolated with increased frequency in all major medical
centers in the United States, Canada, and Western Europe
(10). Several typing methods, such as phage typing
(13), serotyping (15), biotyping (4), biochemical fingerprinting (12) and, more recently, DNA
restriction fragment analysis (8), total plasmid profile
analysis (14), random amplified PCR (9),
pulsed-field gel electrophoresis (PFGE) (21), and ribotyping
(6), have been used for epidemiological investigations of
enterococcal outbreaks and for subtyping of enterococcal strains. These
methods vary in their reproducibility and discriminatory ability, with
PFGE reported (2, 3, 6, 16) to be superior to the others.
Therefore, PFGE is currently considered to be the "gold standard"
for subtyping enterococci and has been used extensively for molecular
epidemiological characterization of VRE outbreaks. However, despite
this fact, a standardized, optimal procedure for PFGE typing of VRE is
not available at the present time. In 1990, Murray et al.
(21) developed one of the first PFGE protocols for typing
enterococci, and their procedure has been used, in its original form
(2, 7, 12, 16, 18) or with minor modifications (5, 11,
20, 22), by numerous investigators. However, the studies used
PFGE typing in order to address specific clinical or research issues,
and they were not designed to simplify or improve the PFGE typing
protocol. Therefore, the typing procedures were not necessarily
optimal, since they involved the use of numerous reagents and buffers, were laborious, and required several days before the results could be evaluated.
The goal of the studies described in this paper was to develop a
simple, rapid, and reproducible procedure for PFGE typing of
vancomycin-resistant Enterococcus faecium and
Enterococcus faecalis (the two species that account for
virtually all human VRE infections [10]), which would
also be suitable for subtyping other enterococci, including
vancomycin-susceptible strains and, potentially, other gram-positive bacteria.
Bacterial strains.
Our enterococcal strain collection
contained 70 VRE isolates (4 E. faecalis strains, including
ATCC strain 51299, and 66 E. faecium strains) and 10 vancomycin-sensitive enterococci (all E. faecalis strains,
including ATCC strain 29212). The strains were kept in 30%
glycerol-70% L broth at 
80°C, and they were plated on L-agar
medium before plugs were prepared. The nonenterococcal isolates
analyzed were three strains of Staphylococcus aureus (including ATCC strains 25923 and 29213), two strains of Listeria monocytogenes (including the CDC standard strain H2446), two
strains of Streptococcus pyogenes (ATCC strains 14919 and
12353), and one strain of Lactobacillus casei.
Standard procedure.
We used the procedure of Murray et al.
(21), which has been used by many investigators (2, 7,
12, 16, 18) for PFGE typing of enterococci (including VRE), as
the standard protocol from which we developed (and against which we
evaluated) our procedure. PFGE patterns obtained from eight
enterococcal strains analyzed by the standard procedure are shown in
Fig. 1A.
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FIG. 1.
PFGE patterns of SmaI-digested DNAs of
enterococcal strains. (A) Results obtained using the standard procedure
of Murray et al. (21). (B) Results obtained using our
procedure. Lane 1, low-range PFGE markers; lanes 2 and 3, E. faecalis strains, including the ATCC strains 29212 (lane 2) and
51299 (lane 3); lanes 4 through 9, E. faecium strains; lane
10,  ladder. Molecular sizes are shown in kilobases on the right.
|
|
Optimization of plug preparation.
In order to improve the
typing procedure, we examined the value of, and eliminated, several
reagents (including deoxycholic acid, which is toxic) used by Murray et
al. (21) during plug preparation (Table
1). In addition, we examined the
efficacies of various concentrations of three lytic enzymes (lysozyme,
mutanolysin, and lysostaphin) instead of or in addition to lysozyme
(which is used in the standard procedure) for lysing the VRE strains and the effect of shortening the lysis and proteolysis steps (Table 1).
Furthermore, we determined that eliminating the proteinase K digestion
step (1) did not have a negative effect on the quality of
the DNA in the plugs.
Optimization of restriction digestion and electrophoresis
parameters.
Previous studies (21) established that
digestion with SmaI is best for PFGE analysis of
enterococci; thus, we used that enzyme in all of our PFGE typing
experiments. However, because other investigators (19)
reported that digestion with ApaI may be useful in PFGE
typing of VRE strains, we examined whether the DNA in plugs prepared by
our procedure was suitable for digestion with that enzyme. DNA of 20 randomly selected strains was incubated (25°C; 2 h) with
ApaI, and PFGE typing revealed complete DNA digestion (data
not shown). Thus, the DNA obtained by our procedure is suitable for
analysis by both enzymes routinely used for PFGE typing of enterococci.
We also determined the conditions required for the most rapid, optimal
digestion of the enterococcal DNA and the value of using various
agarose concentrations and electrophoresis conditions for optimal
resolution of the SmaI-generated DNA fragments (Table 1).
Optimization of staining and destaining.
Primarily because of
safety and environmental concerns, we chose not to consider the
approach (17) of including ethidium bromide in the
agarose-electrophoresis buffer in order to eliminate the time required
to stain gels after electrophoresis. However, we determined the
shortest staining and destaining times yielding optimal results (Table
1).
Modified PFGE protocol.
The results of the above-described
studies optimized the PFGE protocol for typing VRE. Briefly, bacteria
from overnight L-agar cultures were harvested and washed twice with
cell suspension buffer (100 mM Tris-HCl [pH 8] and 100 mM EDTA), and
the suspensions were diluted with cell suspension buffer to a final
optical density at 610 nm (1-cm light path) of 3.7 to 4.0 (ca. 2.5 × 109 CFU/ml). Aliquots (0.2 ml each) of the suspensions
were lysed (Table 2), an equal volume of
1.2% molten SeaKem Gold agarose (FMC BioProducts, Rockland, Maine)
containing 1% sodium dodecyl sulfate was added, and the mixtures were
poured into 2-cm by 1-cm by 1.5-mm reusable plug molds (Bio-Rad
Laboratories, Hercules, Calif.) and allowed to solidify at 4°C for 10 min.
The proteolysis, washing, electrophoresis (performed using the CHEF
Mapper or CHEF DR III apparatus [Bio-Rad]), and staining and
destaining steps were performed as described in Table 2. This modified
PFGE protocol, which includes a significantly faster method for making
plugs (ca. 4 h instead of the usual 3 to 4 days) and a shorter
electrophoresis time (20 h instead of 30 to 40 h) than does the
standard protocol, allowed VRE typing to be performed in ca. 28 h
instead of the 3 to 7 days required for the standard PFGE protocol (the
major differences between the standard protocol and our modified
protocol are summarized in Table 2).
Discriminatory ability and reproducibility of our procedure.
PFGE patterns obtained from eight enterococcal strains analyzed by our
modified procedure are shown in Fig. 1B. The bands in Fig. 1A are
better separated than the bands in Fig. 1B, and the improved resolution
probably is due to the significantly longer electrophoresis time used
in the standard procedure (30 h, versus 20 h in our modified
protocol). However, the resolution achieved by our modified procedure
provides the same level of discrimination among the VRE strains as does
the standard procedure (i.e., the strains identified as separate clones
by the standard procedure were also differentiated by our protocol
[Fig. 1]). Thus, we found that our modified electrophoresis
parameters are sufficient for generating easily interpretable PFGE
banding patterns of VRE strains. The electrophoresis time can be
shortened further if the results are urgently required (e.g., 16 h
of electrophoresis provides good separation; data not shown) or can be
prolonged if improved resolution of the bands is desired.
Analysis of the entire enterococcal strain collection by our modified
procedure yielded clear banding patterns in all cases (data not shown).
In addition, repeated typing (at least three times per strain) of 10 randomly chosen VRE isolates yielded identical patterns.
Applicability for typing of other gram-positive bacteria.
We
analyzed a small collection of gram-positive, nonenterococcal bacteria
by our modified procedure, without custom selecting the restriction
enzyme or optimizing the PFGE parameters for each species. In all
instances, a complete digest of plug-embedded DNA was obtained (Fig.
2), which suggests that the modified
protocol also can be used (with some species-specific modifications in bacterial cell density, restriction enzymes, and fine-tuning of electrophoresis conditions) for molecular epidemiological analysis of
gram-positive bacteria other than enterococci.
View larger version (104K):
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FIG. 2.
PFGE patterns of SmaI-digested DNAs of
various gram-positive, nonenterococcal bacteria analyzed by our
protocol. Lane 1, ladder; lanes 2 and 3, L. monocytogenes strains; lanes 4 and 5, S. pyogenes
strains; lane 6, L. casei strain; lanes 7, 8, and 9, S. aureus strains; lane 10, low-range PFGE marker. Molecular
sizes are shown in kilobases on the right.
|
|
In conclusion, the protocol described in this article allows rapid (ca.
28 h) and reproducible PFGE typing of enterococci and,
potentially, other gram-positive bacteria. Moreover, although we have
not calculated the actual cost of performing PFGE typing by our
simplified procedure, it is likely, because of the reduction in
reagents and personnel time, that the procedure is a cost-saving alternative to previously published PFGE typing protocols. We expect
that the procedure will be of value during epidemiological investigations of VRE outbreaks and for comparative characterization of
VRE strains isolated in different geographic loci.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge Judith Johnson, Connie Mackinson,
Zemphira Alavidze, and Ekaterine Chighladze for their help in subculturing and cataloguing the bacterial strains used in this study.
M.K. was supported by an International Training and Research in
Emerging Infectious Diseases grant from the Fogarty International Center, National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Molecular Epidemiology, Department of Epidemiology and Preventive
Medicine, University of Maryland School of Medicine, MSTF Bldg., Room
9-34, 10 S. Pine St., Baltimore, MD 21201. Phone: (410) 706-4587. Fax: (410) 706-4581. E-mail: asulakve{at}medicine.umaryland.edu.
| |
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Journal of Clinical Microbiology, November 2000, p. 4242-4245, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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