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Journal of Clinical Microbiology, October 2001, p. 3481-3485, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3481-3485.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Development of a Canadian Standardized Protocol for Subtyping
Methicillin-Resistant Staphylococcus aureus Using
Pulsed-Field Gel Electrophoresis
M. R.
Mulvey,1,*
L.
Chui,2
J.
Ismail,3
L.
Louie,4
C.
Murphy,1
N.
Chang,2
M.
Alfa,5 and
the Canadian
Committee for the Standardization of Molecular
Methods
National Microbiology Laboratory, Health
Canada,1 and St. Boniface
Hospital,5 Winnipeg, Manitoba, Alberta
Provincial Laboratory, Edmonton, Alberta,2
LSPQ/INSPQ, SainteAnne de Bellevue,
Quebec,3 and Sunnybrook and Women's
College and Health Science Centre, Toronto,
Ontario,4 Canada
Received 1 May 2001/Returned for modification 30 May 2001/Accepted 22 July 2001
 |
ABSTRACT |
A panel of 24 methicillin-resistant Staphylococcus
aureus strains was distributed to 15 laboratories in Canada to
evaluate their in-house pulsed-field gel electrophoresis (PFGE)
protocols and interpretation criteria. Attempts to compare fingerprint
images using computer-aided analysis were not successful due to
variability in individual laboratory PFGE protocols. In addition,
individual site interpretation of the fingerprint patterns was
inadequate, as 7 of 13 sites (54%) made at least one error in
interpreting the fingerprints from the panel. A 2-day standardized PFGE
protocol (culture to gel image) was developed and distributed to all of the sites. Each site was requested to use the standardized protocol on
five strains from the original panel. Thirteen sites submitted gel
images for comparisons. The protocol demonstrated excellent reproducibility and allowed interlaboratory comparisons with Molecular Analyst DST software (Bio-Rad) and 1.5% band tolerance.
| |
INTRODUCTION |
Methicillin-resistant
Staphylococcus aureus (MRSA) was first reported in England
in 1961, shortly after the introduction of methicillin in 1959 (3, 9). Since that time, MRSA has become a worldwide
problem, causing nosocomial infections in many countries (10, 13,
14, 17, 19, 21, 22). Surveillance programs in the United
Kingdom, Spain, Brazil, Germany, the United States, and Canada have
identified epidemic MRSA strains which appear to possess the ability to
spread rapidly among patients within and between hospitals and nursing
homes (1, 4, 7, 12, 13, 16, 22, 25).
Pulsed-field gel electrophoresis (PFGE) has become the gold standard
for the epidemiological typing of a number of bacterial species
(2). However, multicenter studies comparing PFGE
fingerprints for MRSA clearly have demonstrated the requirement for
standardized conditions (5, 6, 23). Differences in cell
numbers, lysis conditions, PFGE equipment, electrophoresis conditions,
run times, and analytical methods all potentially affect the
intercenter reproducibility of this technique.
The Canadian Committee for the Standardization of Molecular
Methods (CCSMM) is a Health Canada initiative established in 1998 to
deal with issues regarding the standardization of molecular techniques
(11). All known Canadian laboratories currently using molecular techniques for routine epidemiologic surveillance as well as
all provincial public health laboratories were invited to join the
CCSMM. The mission statement, "To standardize molecular methodologies
to ensure quality testing and reporting of results for epidemiologic
purposes," reflected the requirement by Canadian laboratories for
standardized molecular protocols to aid in the molecular surveillance
of many organisms. Realizing the strong potential for monitoring
molecular subtypes of organisms using DNA restriction fragment length
polymorphisms in PFGE, the committee has initially focused its efforts
on this area. One of the subcommittees undertook the task of developing
and implementing protocols for data collection of PFGE fingerprints for MRSA.
In this report, we describe the process involved in establishing a
Canadian standardized PFGE protocol for macrorestriction analysis of
SmaI-digested MRSA. The protocol is rapid, and the quality
assurance program initiated will allow intercenter comparisons of MRSA
DNA fingerprints for national surveillance studies.
| |
MATERIALS AND METHODS |
Bacterial strains.
Strains 1 and 13 were NTCC 8325, and the
remaining strains used in this study were kindly provided by the
Canadian Nosocomial Infection Surveillance Program (CNISP). Stock
cultures were stored at 
70°C in Microbank vials (Pro-Lab
Diagnostics, Richmond Hill, Ontario, Canada). Strains were distributed
on nutrient agar stabs to participating laboratories.
Study design. (i) Phase 1: initial PFGE evaluation.
A panel
of 24 well-characterized MRSA strains collected by the CNISP and
NTCC 8325 (in duplicate) was distributed to each site. Participants
were requested to type the strains on two gels in a specific order
using their in-house PFGE protocol. Laboratories were asked to submit
their protocol, gel images, and interpretation for evaluation.
(a) Development of a standardized MRSA PFGE protocol.
The
CCSMM MRSA subcommittee evaluated the gel images submitted from all 15 sites for band resolution, intensity, reproducibility, and complexity
of the protocol. There was no one method which produced rapid,
well-resolved MRSA fingerprints acceptable for national comparisons;
however, the subcommittee used the information gained from the various
sites to produce a standardized 2-day protocol (culture to gel
image). The subcommittee evaluated growth and lysis conditions,
agarose plug concentrations, wash conditions, restriction enzyme
concentrations and digest time requirements, electrophoresis
temperatures, agarose gel concentrations, electrophoresis buffer
concentrations, and run times to elucidate the optimal conditions
required to establish an effective 2-day procedure (culture to gel
image) necessary for a Canadian standardized protocol. This protocol
was evaluated for reproducibility between subcommittee sites, and the
data suggested that it may serve as a reproducible method (data not
shown). The protocol is detailed below.
Pick one isolated colony of the strain to be subtyped using a sterile
needle or loop and inoculate 3 ml of brain heart infusion broth.
Incubate with gentle agitation at 37°C for 16 to 18 h. On the
next day, take the following steps. Turn on heating blocks or water
baths. Two will be required, with temperatures set at 37 and 55°C.
Label 1.5-ml microcentrifuge tubes with culture numbers. Place 150 µl
of the overnight culture into a microcentrifuge tube. Pellet the cells
by centrifugation at 18,000 × g in a microcentrifuge for 1 min. Store the remaining culture at 4°C if retesting of the
strain is required. Resuspend each pellet in 150 µl of cell suspension buffer (see below).
To prepare casting plugs, take the following steps. Label wells of
disposable PFGE plug molds (Bio-Rad, Hercules, Calif.)
with the
appropriate culture numbers. Cast two plugs for each
sample, as
follows. Prepare 2.0% low-melting-point (LMP) agarose
in
deionized-distilled H
2O (dd H
2O). Melt or
dissolve the LMP
and incubate it in a 50°C water bath until ready to
use. Note
that unused LMP agarose can be stored at room temperature and
reused two or three times. Complete the remaining casting plug
steps
(up to solidification; see below) for each strain before
proceeding to
the next sample. Add 2 µl of lysostaphin (1 mg of
lysostaphin per ml
of H
2O) per tube and mix gently. Add 150 µl
of 2.0% LMP
agarose. Mix gently by pipetting up and down several
times, but avoid
forming bubbles. Immediately fill two disposable
plug molds
(approximately 100 µl per plug). Avoid forming bubbles
in the plug
molds. Discard the remaining 100 µl. Continue with
the next sample
until all are processed. Allow plugs to solidify
for approximately 15 min at room temperature or 5 min at 4°C.
To lyse cells in LMP agarose, take the following steps. Label 1.5-ml
microcentrifuge tubes with culture numbers. Add 500 µl
of lysis
buffer (see below) to each of the 1.5-ml microcentrifuge
tubes.
Transfer the two plugs to the labeled tubes by removing
tape from the
bottom of the molds and pushing the plugs into the
tubes. Incubate the
tubes at 37°C for 1 h. Remove the tubes from
the 37°C water
bath and decant or aspirate the lysis buffer with
a 1-ml pipette tip,
taking care not to damage the plugs. Calculate
the total volumes of
proteinase K (PK) and PK buffer required
to process all samples.
Add 500 µl of PK-PK buffer (50 µ/ml) solution
to each
microcentrifuge tube. Incubate the tubes at 50°C for 0.5
h.
To wash LMP agarose plugs after lysis, take the following steps. Remove
the tubes from the 50°C water baths and aspirate the
PK-PK buffer
solution. Rinse the plugs once with 1.4 ml of wash
buffer (see below),
and then wash them three additional times
with 1.4 ml of wash buffer
for 30 min each wash. All washes should
be conducted at room
temperature. Remove the wash buffer from
the final wash of the
preceding step. For long-term storage, resuspend
the plugs in 1.4 ml of
wash buffer. The plugs are stable for at
least 6 months at 4°C.
For restriction enzyme digestion of LMP agarose plugs, take the
following steps. Label 0.5-ml microcentrifuge tubes with culture
numbers. Remove one plug from a microcentrifuge tube. Cut approximately
one-third of the plug and place it into a new 1.5-ml microcentrifuge
tube. Calculate the volume of restriction enzyme buffer (REB)
required
to digest the plugs. The restriction buffer for
SmaI
is
usually sold as a 10× solution and must be diluted to 1× before
use.
(Calculate the total amount of 1× REB required. A total of
450 µl
will be required per sample [300 µl for the equilibration
and 150 µl for the digest]. Prepare 1× REB by diluting the appropriate
amount of 10× REB stock with ddH
2O.) Equilibrate the plugs
by
adding 300 µl of 1× REB to the tube containing one-third of a
plug and incubate the tube at room temperature or 25°C for 10
min.
Remove the 1× REB from the tube with a 1-ml pipette tip,
taking care
not to damage the plug slice. Add 150 µl of 1× REB
containing 25 U
of
SmaI to each tube and incubate the tube at
25°C for
2 h. Prepare a 1% agarose gel in 120 ml of 0.5× TBE (see
below)
while the DNA is being restriction digested. Pour the dissolved
agarose
into a casting tray. Use a 15-well 0.75-mm comb to make
wells. Aspirate
the enzyme-buffer solution and melt the plug at
65 to 70°C for 10 to
15 min. Add a thin slice of lambda molecular
weight markers (NO340; New
England Biolabs, Mississauga, Ontario,
Canada) to lanes 1, 8, and 15. Load 30 µl of the melted plug into
a well with a tip with
approximately 3 mm cut from the end on
an angle. Load the gel dry (on
the benchtop) and place it into
the buffer chamber after all of the
samples and ladder have been
loaded and solidified. Perform
electrophoresis with a CHEF DR-III
apparatus (Bio-Rad) using switch
times of 5.3 to 34.9 for 18 h
(or 20 h for Mapper or
DR-II) (Genepath strain typing system program
STA) at 6.0 V/cm
and 14°C in 0.5× TBE. Stain the gel for 20 min
with 0.5 mg of
ethidium bromide per liter, and destain it with
fresh ddH
2O
for at least 30 min with three changes of water. Use
UV light
transilluminator to visualize
samples.
Solutions should be made as follows: cell suspension buffer, 10 mM
Tris-HCl (pH 7.2)-20 mM NaCl-50 mM EDTA; lysis buffer,
10 mM Tris-HCl
(pH 7.2)-50 mM NaCl-50 mM EDTA-0.2% deoxycholate-0.5%
Sarkosyl;
wash buffer, 10 mM Tris-HCl (pH 7.6)-0.1 mM EDTA; PK
buffer, 250 mM
EDTA (pH 9.0)-1% Sarkosyl; and 10× TBE; 0.89 M
Tris-HCl (pH
8.4)-0.89 M boric acid-0.02 M
EDTA.
(b) Computer-aided analysis.
DNA fingerprints were digitized
using an in-house apparatus and saved as TIFF files. The fingerprints
generated using the standardized protocol were evaluated using
Molecular Analyst DST version 1.6 software (Bio-Rad) or
BioNumerics version 2.0 (Applied-Maths, Sint-Martens-Latem,
Belgium). DNA fragments on each gel were normalized using the
molecular weight standard run on each gel to allow comparisons between
different gels. A 1.5% band tolerance was selected for use during
comparisons of DNA profiles. Cluster analysis was performed by the
unweighted pair-group method using arithmetic averages (UPGMA), and DNA
relatedness was calculated based on the Dice coefficient.
(ii) Phase 2: PFGE using the standardized protocol.
Laboratories were requested to perform PFGE using the standardized
protocol developed by the CCSMM MRSA subcommittee. A subset of the
originally distributed panel of MRSA strains was used by participating
sites for quality assurance and included strains 1, 2, 3, 9, and 17. These five strains were chosen in part to limit the workload of
participating laboratories in phase 2 of the study; to test the ability
of a site to differentiate slight band deviations (strains 2 and 3), to
differentiate smaller bands (under 150 kb) (strain 9), and to resolve
large bands (strain 17); and for comparability with other methods by
using NCTC 8325 (strain 1). Laboratories were required to fingerprint
these strains in order of the strain number and flank them with lambda markers.
| |
RESULTS AND DISCUSSION |
Assessment of protocols.
A total of 15 laboratories
representing 7 provinces participated in phase 1 of the study. A panel
including 24 well-characterized MRSA strains collected by the
CNISP and NTCC 8325 was distributed to each site (Table
1). Participants were requested to type
the strains in a specific order on two gels using their in-house PFGE protocol and to submit the protocol, gel images, and interpretation for
evaluation. A sample gel of the strains distributed and the order in
which they were requested to be loaded is shown in Fig. 1. Protocols varied considerably between
sites (data not shown) with respect to length of protocol, pulse times,
and duration of electrophoresis. Not surprisingly, the fingerprints
from the gel images submitted from the 15 sites could not be compared
using Molecular Analyst DST version 1.6 software due to
different variables described previously (5, 6, 23; data not shown).
Thirteen of the 15 laboratories interpreted the fingerprints using
current guidelines or a modification thereof (20). The
intralaboratory fingerprint analysis was unreliable, as 7 of 13 sites
(54%) made at least one error in interpreting the 24 fingerprints
(Table 1). Fingerprint analyses for identical strains were correct for
76.6 and 76.9% of the profiles on different and same gels,
respectively. For unrelated strains, analyses at the 13 sites were
correct for 90.4% of the profiles on same or different gels (Table 1).
Analysis of the interpretations revealed that the errors were primarily linked to failure to recognize matching fingerprints (human error) or
failure to resolve differences in the fingerprints due to run conditions. These data highlight the need for a standardized PFGE protocol and a quality assurance program to ensure data integrity.
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FIG. 1.
DNA fingerprints obtained with PFGE for 24 MRSA strains
distributed in the panel. Numbers above the lanes indicate strain
numbers; M, lambda concatamers.
|
|
Interlaboratory standardization.
The standardized protocol was
distributed to all participating laboratories. Five of the original 24 MRSA strains (1, 2, 3, 9, and 17) were selected as quality
control strains for the purposes of evaluating the standardized
protocol (Fig. 2A). Although it may have
been more reliable to use all 24 MRSA strains used in phase 1, we
decided to use a subset for quality control and certification
purposes. Thirteen of the original sites submitted gel images for
analysis using the standardized protocol and control strains.
Comparisons of the normalized gel images with Molecular Analyst DST
version 1.6 software using the Dice coefficient and a UPGMA-derived
dendrogram are shown in Fig. 2B for 12 sites. The fingerprints from the
13th site (site M) were not comparable due to poor resolution of the
lambda markers in addition to problems with band resolution which may
have been associated with plug washing conditions and overloading.
Attempts are under way to modify that site's procedures to produce
comparable fingerprints.
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|
FIG. 2.
PFGE analysis using the standardized protocol described
in the text. (A) Sample gel showing five strains used in the quality
control panel. Numbers above the lanes indicate strain numbers. (B)
Dendrogram comparing fingerprints from 12 sites using the
standardized protocol.
|
|
Using a band tolerance of 1.5% and an optimization of 4%, all strains
from the 12 sites clustered in their respective strain
groups; this
result is not unexpected for strains 1, 9, and 17,
since the patterns
are not related (
7 bands) to each other. However,
strain 2 and strain
3 vary by only a single band shift, of less
than 20 kb (Fig.
1 and
2),
and they also correctly clustered in
their respective strain
groups.
Under the band tolerance and optimization settings applied, the site C
patterns for strains 1 and 2, the site H pattern for
strain 9, and the
site A pattern for strain 17 did not show 100%
similarity within each
cluster. Percent similarities were as follows:
90.9% for strain 1 at
site C with strains 1 at sites E and F;
90.9% for strain 2 at site A
with strain 2 at site C; 90.9% for
strain 9 at site B with strain 9 at
site H; and 88.9% for strain
17 at site A with strain 17 at sites C,
J, and K (Fig.
2B). All
other comparisons yielded similarities of 100%
under the conditions
described. Identical results were obtained when
the images were
analyzed with BioNumerics version 2.0.
An algorithm in Molecular Analyst DST version 1.6 software, called band
tolerance statistics, allows the comparison of identical
fingerprint
from a number of different experiments to an averaged
fingerprint. A
comparison of the fingerprints using this algorithm
is shown in Table
2. The numbers indicate the mean
deviations
of band positions from an averaged fingerprint for all the
sites
for a specific strain. The majority of the sites displayed
average
deviations of less than 1.7%, suggesting that the standardized
PFGE protocol was reproducible. However, site C had a significantly
higher deviation, 2.51%. Examination of the gel image from site
C
suggested that variability in the PFGE apparatus might have
caused the
deviation, assuming that the standardized protocol
was followed. In
addition, a similar explanation could explain
the difficulties
associated with site M, the results from which
could not be
normalized using Molecular Analyst DST software.
Efforts are under way
to alter the electrophoresis conditions
at these two sites to allow
comparisons.
This procedure has been used to subtype over 2,000 MRSA isolates
collected by the CNISP (data not shown). No isolates to date
have been
identified as nontypeable using this procedure. The
advent of clinical
MRSA isolates displaying reduced susceptibility
to vancomycin in
numerous countries raises the distinct possibility
that
vancomycin-resistant MRSA could emerge in patient populations
(
8,
15,
18,
24). This fact, along with the recent identification
of
epidemic strains in Canada (
16), highlights the need for
a
rapid molecular-based surveillance mechanism in Canada to aid
in
infection control efforts. The recent development of server-based
data
collection systems for fingerprint information (BioNumerics)
and the
standardization of PFGE methods such as the methods described
in this
report, will be required to collect and disseminate accurate,
timely
information to infection control practitioners and microbiologists
to
limit the spread of MRSA. A quality assurance program is currently
being developed to ensure that accurate data are generated from
participating sites as the standardized method develops into a
national
surveillance
program.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: National
Microbiology Laboratory, Nosocomial Infections, 1015 Arlington St.,
Winnipeg, Manitoba, Canada R3E 3R2. Phone: (204) 789-2133. Fax: (204)
789-2018. E-mail: michael_mulvey{at}he-sc.gc.ca.
Participants of The Canadian Committee for the Standardization of
Molecular Methods: L. Abbott, Charlottetown, Prince Edward Island; M. Alfa, Winnipeg, Manitoba; S. Byrne, Vancouver, British Columbia; B. Ciebin, Toronto, Ontario; L. Chui, Edmonton, Alberta; C. Clark,
Winnipeg, Manitoba; J. Conly, Toronto, Ontario; R. Davidson, Halifax,
Nova Scotia; J. Farber, Ottowa, Ontario; K. Fonseca, Calgary, Alberta;
G. Horsman, Regina, Saskatchwan; J. Ismail, Ste-Anne de Bellevue,
Quebec; P. Jayaratne, Hamilton, Ontario; A. Kabani, Winnipeg, Manitoba;
M. Kanchana, Saskatoon, Saskatchewan; L. Louie, Toronto, Ontario; A. McGeer, Toronto, Ontario; M. Mulvey, Winnipeg, Manitoba; G. Norris, St.
John's, Newfoundland; M. Peppler, Edmonton, Alberta; G. Peters,
London, Ontario; K. Ramotar, Ottowa, Ontario; J. Wylie, Winnipeg,
Manitoba; and K. Ziebell, Guelph, Ontario.
| |
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Journal of Clinical Microbiology, October 2001, p. 3481-3485, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3481-3485.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
