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Journal of Clinical Microbiology, October 1999, p. 3198-3203, Vol. 37, No. 10
Molecular Biology
Unit1 and Laboratory of Hospital
Infection,2 Central Public Health
Laboratory, London NW9 5HT, United Kingdom
Received 22 February 1999/Returned for modification 1 June
1999/Accepted 29 June 1999
Fluorescent amplified-fragment length polymorphism (FAFLP) analysis
was investigated for its ability to identify and subtype isolates of an
epidemic methicillin-resistant phage type of Staphylococcus aureus, EMRSA-15. These isolates were also characterized by
PCR-restriction fragment length polymorphism (PCR-RFLP) of the
coagulase gene and pulsed-field gel electrophoresis (PFGE). For FAFLP,
DNA was double digested with restriction enzymes ApaI plus
TaqI or EcoRI plus MseI.
Site-specific adaptors were ligated to one or the other set of
restriction fragments, and PCR amplification was carried out with
adaptor-specific primers. Amplified fragments separated on an ABI 377 automated sequencer and analyzed with Genescan version 2.1 software
generated FAFLP profiles for all the isolates. The presence or absence
of fragments was scored, similarity coefficients were calculated, and
UPGMA (unweighted pair group method using arithmatic averages) cluster
analysis was performed. Either enzyme-primer combination readily
differentiated EMRSA-15 from other methicillin-resistant S. aureus (MRSA) isolates and also revealed heterogeneity within the
phage type. The discriminatory power of FAFLP was high. By combining
both enzyme-primer data sets, 24 isolates were divided into 11 profiles. PCR-RFLP did not discriminate among these EMRSA-15 isolates.
PFGE could discriminate well between isolates but was not as
reproducible as FAFLP. All S. aureus and MRSA isolates in
this study were typeable by FAFLP, which was easy to perform, robust,
and reproducible, with evident potential to subtype MRSA for purposes
of hospital infection control.
Methicillin-resistant
Staphylococcus aureus (MRSA) is a nosocomial pathogen of
worldwide importance (7). Within the United Kingdom, 16 phage types have been epidemic (EMRSA), of which EMRSA-3, -15, and -16 now predominate (4). In 1997 and 1998, an increased incidence of EMRSA-15 was reported. In all, over 1,200 incidents (three
or more patients in a single hospital in 1 month infected with the same
strain) were reported, with a wider geographical distribution than in
previous years (1).
Reliable and rapid typing of EMRSA is needed to implement effective
infection control measures. Typing systems should ideally show good
discriminatory power and be reproducible, capable of typing all
isolates, and easy to use (11). Phage typing has been used
to type isolates of S. aureus for over 45 years
(21). In this phenotypic technique, strains are classified
according to susceptibilities to a set of internationally agreed-upon
phages. This simple technique has a high throughput, but some isolates are phage nontypeable or may produce ambiguous results (2).
Various molecular methods have been described for typing isolates of
MRSA. They include ribotyping (14), random amplification of
polymorphic DNA by PCR (19), insertion sequence profiling (17), PCR-restriction fragment length polymorphism
(PCR-RFLP) (8), and, notably, pulsed-field gel
electrophoresis (PFGE) (18). PFGE, however, does not
reliably produce stable banding patterns for MRSA; variation is seen in
interlaboratory studies of defined strain collections (5,
20). Such variation seems to be specific to S. aureus
and may be accounted for in part by the presence of variable numbers of
lysogenic phage in genomes (10).
The technique of fluorescent amplified-fragment length polymorphism
(FAFLP) analysis requires double digestion of the bacterial genome with
restriction endonucleases, followed by ligation of adaptor sequences to
the ends of restriction fragments. Subsets of fragments can be
amplified by stringent PCR, using fluorescently-labelled primers
complementary to the adaptor sequences. These can be extended into the
restriction fragments by one or two bases to increase their
selectivity. Amplified fragments are separated by electrophoresis in a
polyacrylamide sequencing gel and visualized by the laser detection
system of an ABI automated sequencer. Since different combinations of
restriction enzymes can be used, FAFLP has the potential to type any
bacterial species. For example, it has been used to investigate an
outbreak of invasive disease caused by group A streptococcus
(6), where it had discriminatory power superior to that of
PFGE. The aim of the present study was to establish whether FAFLP would
reproducibly discriminate between isolates of the clinically important
epidemic S. aureus phage type EMRSA-15.
Bacterial strains and culture conditions.
Twenty-four
isolates of EMRSA-15 obtained from the Laboratory of Hospital Infection
had been assigned to this phage type on the basis of phage lytic
patterns, resistance to methicillin and penicillin, and variable
resistance to erythromycin and ciprofloxacin. Isolates had been
submitted for typing between 1990 and 1997 by different hospital
infection control laboratories with a diverse geographical
distribution. Isolates listed as from the same region (Table
1) were from the same hospital but from
different patients. In one hospital, transmission was known to have
occurred between two patients (isolates 499 and 501). In all other
instances, the isolates were not epidemiologically related. Three
non-EMRSA-15 isolates and the Oxford S. aureus strain NCTC
6731 (isolates 16, 30, 299, and T) served as controls.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Genotyping of Epidemic Methicillin-Resistant
Staphylococcus aureus Phage Type 15 Isolates by Fluorescent
Amplified-Fragment Length Polymorphism Analysis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
MRSA
isolate data
70°C. Genomic DNA was isolated from plate
cultures with lysostaphin-sodium chloride-cetyltrimethylammonium
bromide, as described previously (8). The concentration of
DNA was estimated by UV spectrophotometry at
A260 (with a Beckman DU 640) by standard methods
(15).
Phage typing. Phage susceptibilities determined by the method of Blair and Williams (3) at 100 times routine test dilution with the current set of international typing phages are shown in Table 1. The isolates were ascribed to EMRSA-15 on the basis of three lytic patterns: the classic pattern of weak susceptibility to phage 75 (75 wk) (13), a 42E variant pattern, and an 83C variant pattern.
PCR-RFLP of the coagulase (coa) gene. The coa gene was amplified from all isolates with a RoboCycler gradient 96 platform (Stratagene Ltd., Cambridge, United Kingdom). In a final volume of 50 µl, each reaction mixture contained 75 pmol of both primers 1513 and 2168 (8), 200 µM deoxynucleoside triphosphates, 1× PCR buffer, 3 mM MgCl2, 1.25 U of Taq DNA polymerase (all from Life Technologies, Paisley, United Kingdom), and 1× bovine serum albumin (New England Biolabs, Hertfordshire, United Kingdom [NEB]). After an initial denaturation step of 94°C for 2 min, the cycling profile was 94°C (30 s), 57°C (30 s), and 72°C (1 min) for 35 cycles. The last cycle had a 5-min extension step at 72°C. Restriction endonuclease analysis of PCR products was performed with AluI and CfoI (Boehringer Mannheim, Lewes, United Kingdom) as described previously (8).
PFGE. SmaI macrorestriction of genomic DNA was carried out as described for isolates of Streptococcus pyogenes (16). PFGE gel photographs were scanned into the Taxotron software package (Institut Pasteur, Paris, France), and fragments were sized with reference to internal lane markers, using its programs RestrictoScan and RestrictoTyper.
FAFLP. The enzymes EcoRI and MseI were used to digest approximately 500 ng of genomic DNA from each isolate, and fragments were ligated to double-stranded adaptors as previously described (6). PCR was performed in a 20-µl volume containing 1.5 µl of ligated DNA, 15 µl of Amplification Core mix (Perkin-Elmer Applied Biosystems, Warrington, Cheshire, United Kingdom), 5 pmol of MseI adaptor-specific primer (Perkin-Elmer Applied Biosystems), and 1 pmol of 5-carboxyfluoroscein-labelled EcoRI adaptor-specific primer (EcoRI+0) (Perkin-Elmer Applied Biosystems). The MseI primer contained the extra selective base C (MseI+C).
The enzymes ApaI and TaqI were used to digest DNA as follows: approximately 500 ng of DNA was incubated with 4 U of ApaI (NEB), 1× buffer 4 (NEB), 1× bovine serum albumin (NEB), and 0.5 mg of DNase-free RNase A ml
1 in a final
volume of 20 µl at 25°C for 1 h. Five units of TaqI (NEB) was subsequently added to each reaction mixture, and the mixtures
were incubated for a further 1 h at 65°C.
A 20-µl solution containing 4 pmol of ApaI adaptors
(MWG-Biotech UK Ltd., Milton Keynes, United Kingdom), 40 pmol of
TaqI adaptors (MWG-Biotech), 1× T4 ligase buffer (NEB), and
40 U of T4 DNA ligase (NEB) was added to 20 µl of double-digested
DNA. The mixture was incubated at 12°C for 17 h, heated at
65°C for 10 min to inactivate the ligase, and stored at 20°C. The
sequence of the ApaI adaptor was 3' CATCTGACGCATGT, 5'
TCGTAGACTGCGTACAGGCC. The sequence of the TaqI adaptor
was 3' TACTCAGGACTGGC, 5'GACGATGAGTCCTGAC.
PCR was performed in a 25-µl volume containing 2 µl of ligated
sample, 1× PCR buffer, 2 mM deoxynucleoside triphosphates, 1.5 mM
MgCl2, 0.625 U of Taq DNA polymerase (all from
Life Technologies), 5 pmol of TaqI adapter-specific primer
(Genosys Biotechnologies, Cambridge, United Kingdom), and 1 pmol of
5-carboxyfluorescein labelled ApaI adaptor-specific primer
(ApaI+0) (Genosys). The TaqI primer contained the
extra selective base G (TaqI+G). The sequences of the
primers are 5' CGATGAGTCCTGACCGA 3' (TaqI) and 5' GACTGCGTACAGGCCC 3' (ApaI).
For both enzyme-primer combinations, touchdown PCR cycling conditions
were employed as described previously (6). FAFLP products
were stored at 20°C prior to gel electrophoresis.
Gel analysis.
FAFLP fragments were separated in 5%
denaturing (sequencing) polyacrylamide gels on an ABI Prism 377 automated DNA sequencer. Gels 0.2 mm thick were prepared from
LongRanger Singel (Flowgen, Lichfield, Staffordshire, United Kingdom)
according to the manufacturer's instructions. FAFLP products (1 µl)
were added to 1.75 µl of loading dye (a mixture containing 1.25 µl
of formamide, 0.25 µl of loading solution [dextran blue in 50 mM
EDTA], and 0.25 µl of the internal size marker [Perkin-Elmer]). To
size ApaI/TaqI fragments accurately, GeneScan-2500 markers labelled with the red fluorescent dye
6-carboxy-
-rhodamine (ROX) were used as internal size standards,
while for EcoRI/MseI fragments, GeneScan-500
markers (ROX labelled) were used. The sample mixture was heated to
95°C for 2 min, cooled on ice, and immediately loaded into the gel.
Running conditions were 2.5 kV at 51°C for 10 h for
ApaI/TaqI fragments or 1.68 kV at 51°C for 10 h for EcoRI/MseI fragments. The
well-to-read distance was 48 cm in both cases.
Fragment analysis. Fragments were sized with GeneScan version 2.1 software. Peak height thresholds were set at 50; any peak heights less than this value were not included in the analysis. Electropherograms of all fragment profiles were visually inspected for polymorphisms, with the presence and absence of fragments scored in a binary matrix, and were recorded as a text (tab-delimited) file in Excel version 5.0 (Microsoft). Dice coefficients of similarity were calculated with in-house software. Cluster analysis was performed by UPGMA (NEIGHBOR program of PHYLIP) and then displayed with TREEVIEW (12).
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Characterization by existing molecular methods. Twenty-four EMRSA-15 isolates had identical PCR-RFLP patterns following CfoI or AluI digestion of a PCR amplicon corresponding to a fragment of approximately 660 bp from the coagulase gene. This was RFLP pattern 2 (8). The four non-EMRSA-15 isolates had distinct coa gene RFLP patterns corresponding to patterns 1 (isolate 299), 4 (isolate T), 5 (isolate 30), and 6 (isolate 16).
Following SmaI macrorestriction of genomic DNA, fragments ranging in size from 75 to 700 kbp were produced from all isolates by PFGE. Eleven EMRSA-15 isolates had identical macrorestriction profiles containing eight fragments (Fig. 1). This profile was designated P1 (Table 1). Eleven EMRSA-15 isolates differed from this predominant profile by one to three fragments (profiles P2 to P8). Two EMRSA-15 isolates and the four non-EMRSA-15 isolates had four or more differences (profiles P9 to P14). When PFGE was repeated, two isolates (isolate 3 and isolate A) exhibited one- to two-band differences compared to their initial PFGE profiles (Fig. 1).
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FAFLP: general considerations. The selective primers used for each FAFLP were chosen empirically. In preliminary experiments, the primers ApaI+0 and TaqI+G and the primers EcoRI+0 and MseI+C produced profiles that could be easily scored and that revealed differences between isolates. Other nucleotide substitutions in the TaqI or MseI selective primers produced profiles with large numbers of small fragments. These were not sufficiently resolved to score easily and revealed fewer differences between isolates.
DNA was extracted from all isolates and subjected to FAFLP with both enzyme-primer combinations. Isolates were later recovered from stocks frozen at70°C, the DNA was reextracted, and the samples were again
subjected to FAFLP. The fragment profiles from different DNA extracts
of the same isolate were shown to be reproducible. It was found that
profiles resulting from degraded DNA extracts (visualized by agarose
gel electrophoresis) contained certain extra-low-intensity fragments.
This phenomenon was not seen in profiles generated from DNA
preparations with undegraded DNA of the same isolate. Profiles
exhibiting such fragments were discarded.
FAFLP with ApaI+0 and TaqI+G. The range and size of fragments produced by FAFLP with the enzyme-primer combination ApaI+0 and TaqI+G was approximately 80 fragments per isolate, ranging from 55 to 820 bp. Nineteen EMRSA-15 isolates had identical fragment profiles (Fig. 2a). Five others had profiles very similar to this predominant profile, containing one extra fragment or lacking one or two different fragments. The extra fragment present in isolates 496 and 497 was 557 bp; in isolate V it was 192 bp, and in isolate K it was 578 bp. The following fragments, characteristic of the predominant profile, were not found in certain isolates: 556 bp (isolates 496 and 497), 607 bp (isolates 497 and J), 721 bp (isolate 496), and 733 bp (isolates 497 and J). The four non-EMRSA-15 isolates each exhibited over 60 fragment differences with respect to the predominant EMRSA-15 profile.
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FAFLP with EcoRI+0 and MseI+C. Approximately 70 fragments per isolate, ranging from 55 to 320 bp, were produced by EcoRI+0 and MseI+C. To resolve the large number of fragments smaller than 200 bp (Fig. 2b), gels (48-cm well-to-read distance) were run at a lower voltage (1.68 kV). Fifteen EMRSA-15 isolates had identical profiles. Nine others had profiles which differed from this predominant profile by one to five fragments. The extra fragment present in isolate Q was 152 bp; in isolate K there were fragments of 228 and 246 bp, and in isolate 496 there were fragments of 240 and 302 bp. The following fragments, characteristic of the predominant profile, were not found in certain isolates: 100 bp (isolates 496, 497, A, and P), 108 bp (isolates A, F, and L), 111 bp (isolate P), 121 bp (isolates A, F, and L), 204 bp (isolates 496, 497, A, F, and L), and 296 bp (isolates 496, 497, A, P, and U). The four non-EMRSA-15 isolates each exhibited over 20 fragment differences with respect to the predominant EMRSA-15 profile.
Cluster analysis. Three dendrograms were constructed on the basis of the ApaI+0-TaqI+G and EcoRI+0-MseI+C FAFLP data (Fig. 3a and b). All show clear separation between the EMRSA-15 isolate and the four unrelated isolates. Nine isolates with the classical EMRSA-15 phage pattern (75 wk) and three with variant patterns showed the predominant FAFLP profile A1 (see below). However, seven isolates with the classical phage pattern had unique FAFLP profiles.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Phage typing and PFGE are currently used to type MRSA. However, phage typing has poor discrimination and fails to type all S. aureus isolates, with 15 to 20% found to be nontypeable (2). In this study, for example, phage typing failed to distinguish EMRSA-15 isolate R from the non-EMRSA-15 isolate 30. The reproducibility of PFGE for S. aureus has also been questioned (5, 20).
The high-resolution genotyping technique of FAFLP has been successfully used to investigate an outbreak of invasive disease by the gram-positive pathogen S. pyogenes (6). In the present study, we asked whether FAFLP was applicable to S. aureus and whether it could reproducibly identify and subtype a major epidemic phage type of MRSA. We provided evidence that S. aureus is fully typeable by FAFLP. In this and other studies, we have not found any S. aureus isolates (>100 tested, including different EMRSA phage types and sporadic strains) that fail to generate FAFLP profiles. This compares well with phage typing (2).
FAFLP is easy to perform for S. aureus, but the electrophoresis run (up to 10 h) is longer than that required to separate FAFLP profiles of S. pyogenes (6). This is due to the presence of many fragments of less than 100 bp that need to be resolved in S. aureus profiles. Digestion of genomic DNA and overnight ligation of adaptor sequences is performed on day 1, PCR followed by electrophoresis of amplified fragments in the polyacrylamide sequencing gel is completed on day 2, and the profiles are analyzed on day 3. In our hands, PFGE of S. aureus, which takes 3.5 to 4 days, is not as robust and is more labor-intensive than FAFLP.
The discriminatory power of FAFLP is determined by the choice of restriction enzymes and by the chosen selectivity of the primers. In the present study, we were able to further increase its discriminatory power by combining data from different enzyme-primer combinations. The range of fragments for the primer pair ApaI+0 and TaqI+G (55 to 820 bp) was greater than that for the primer pair EcoRI+0 and MseI+C (55 to 320 bp). Nonetheless, the discriminatory power for ApaI+0 and TaqI+G was lower. The dendrograms derived from either primer pair (Fig. 3a and b) demonstrate that FAFLP distinguishes isolates of EMRSA-15 from non-EMRSA-15 isolates. Moreover, the dendrogram derived from combined data (Fig. 3c) divided the 24-phage-type EMRSA-15 isolates into 11 profiles. The enzyme-primer combinations used in this study are suitable for FAFLP investigations of other S. aureus isolates.
FAFLP for all isolates in this study was reproducible over time:
profiles were stable for different DNAs extracted successively from an
isolate. For S. aureus this reproducibility was superior to
that of PFGE
our results and other studies (5, 20) show that PFGE of S. aureus can be insufficiently reproducible.
For example, on repetition of PFGE, isolate A yielded one extra band, while isolate 3 yielded one extra band and one absent band. PFGE has
been adopted in preference to phage typing for subtyping S. aureus (2). However, we suggest that it may be
inherently less suitable than FAFLP for genotyping S. aureus, since its display of a single rare-cutter digest of the
whole genome is easily distorted by loss or gain of lysogenized
prophages from the genome (10). By contrast, FAFLP
simultaneously samples approximately 80 loci distributed randomly
across the S. aureus genome, and FAFLP profiles are
apparently not affected in the same way by large mobile DNAs. Thus, in
our study, while the discriminatory power of FAFLP approximated that of
PFGE for EMRSA-15, PFGE was less reproducible.
In summary, we have demonstrated that FAFLP can identify and subtype MRSA. In this study it readily defined a genotype for the epidemic phage-type EMRSA-15, distinguished it from those of other MRSA, and revealed EMRSA-15 to be a clone complex.
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FOOTNOTES |
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* Corresponding author. Mailing address: Molecular Biology Unit, Central Public Health Laboratory, 61 Colindale Ave., London NW9 5HT, United Kingdom. Phone: (44) 181 200 4400, ext. 3090. Fax: (44) 181 200 1569. E-mail: rgrady{at}phls.nhs.uk.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, October 1999, p. 3198-3203, Vol. 37, No. 10
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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