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Journal of Clinical Microbiology, November 1998, p. 3133-3137, Vol. 36, No. 11
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Fluorescent Amplified-Fragment Length Polymorphism
Analysis of an Outbreak of Group A Streptococcal Invasive
Disease
Meeta
Desai,1
Asha
Tanna,2
Robert
Wall,3
Androulla
Efstratiou,2
Robert
George,2 and
John
Stanley1,*
Molecular Biology
Unit1 and
Respiratory and Systemic Infection
Laboratory,2 Central Public Health Laboratory,
London NW9 5HT, and
Department of Microbiology, Northwick Park
Hospital, Harrow, Middlesex,3 United
Kingdom
Received 13 May 1998/Returned for modification 9 July 1998/Accepted 2 August 1998
 |
ABSTRACT |
Fluorescent amplified-fragment length polymorphism (FAFLP) analysis
was carried out for an outbreak of group A streptococcal (GAS) invasive
disease. Streptococcal genomic DNAs were digested with endonucleases
EcoRI and MseI, site-specific adaptors were ligated, and PCR amplification was carried out with an
EcoRI adaptor-specific primer labelled with fluorescent
dye. Amplified fragments of up to 600 bp in size were separated on a
polyacrylamide sequencing gel which contained internal size markers in
each lane. These data were automatically scanned and analyzed,
fragments were precisely sized (±1 bp), and electropherograms were
generated for each genome with GeneScan 2.1 software. All isolates were
compared in this way. Among 27 GAS isolates examined, we found 18 FAFLP
profiles, compared with 12 macrorestriction profiles by pulsed-field
gel electrophoresis. FAFLP readily distinguished genotypes for two clones of GAS serotype M77 which were responsible for outbreaks of
invasive disease in a care-of-the-elderly system. It provided an
automated analysis of the whole genome of bacterial isolates. It was
reproducible, more discriminatory, and capable of higher throughput
than other molecular typing methods. Given agreed conditions, FAFLP
would be reproducible between laboratories for rapid characterization of outbreak strains.
| |
INTRODUCTION |
Streptococcus pyogenes
(Lancefield group A streptococcus [GAS]) is a major causative agent
of noninvasive and invasive human disease, ranging from sore throat to
the progressively destructive tissue infection necrotizing fasciitis.
Its emm gene encodes the filamentous M protein that is the
antigenic basis for classical (Lancefield) serotyping. This
distinguishes more than 90 M serotypes of GAS (5, 6).
Various molecular methods have been used to characterize GAS isolates.
They include multilocus enzyme electrophoresis (8), restriction endonuclease analysis of genomic DNA (2),
ribotyping (1, 12), random amplified polymorphic DNA
(RAPD) fingerprinting (10), PCR-restriction fragment
length polymorphism (PCR-RFLP) analysis of the emm gene
(13), and pulsed-field gel electrophoresis (PFGE) (11,
12). The most reproducible of these methods, PFGE, is
time-consuming and labor-intensive. While the PCR-based methods are
more rapid, RAPD analysis is poorly reproducible and PCR-RFLP analysis
is restricted to polymorphic analysis of regions of one to several
kilobases. A combination of existing methods has been used for
high-resolution epidemiological analysis of streptococcal outbreaks
(13). Nonetheless, it would be greatly advantageous to have
a single PCR-based technique which could rapidly and reproducibly analyze the whole genome for accurate strain genotyping.
Amplified-fragment length polymorphism (AFLP) analysis is a novel
PCR-based technique (15) which has been used for DNA
fingerprinting of plant genomes. It requires no prior knowledge of
genomic DNA sequences and potentially offers better discriminatory
power and speed than the existing molecular typing techniques described above. As originally proposed, AFLP used radioactively labelled primers
for PCR amplification of small genomic fragments defined by known
restriction sites and adaptors. Several bacterial genera have been
studied by radioactive AFLP; they include Legionella (14), Aeromonas, and Xanthomonas
(3, 4).
In the present study, we have used AFLP analysis with a fluorescently
labelled primer (FAFLP) for molecular epidemiological investigation of
S. pyogenes outbreaks. We evaluated the potential of FAFLP
for achieving precise fragment amplification and sizing, compared it
with PFGE, and examined its potential for defining outbreak strain
genotypes and for subtyping. We evaluated the instrumentation (ABI
Prism 377 DNA automated sequencer; Perkin-Elmer Applied Biosystems) and
software (GeneScan 2.1) in terms of data capture and quality and
investigated the systematic use of FAFLP for an outbreak analysis: in
this case, the analysis of an outbreak of invasive disease in a
district general hospital caused by S. pyogenes serotype
M77.
| |
MATERIALS AND METHODS |
Bacterial strains and culture conditions.
Twenty-six
isolates of S. pyogenes from patients with invasive or
noninvasive streptococcal diseases were obtained from the Streptococcus
and Diphtheria Reference Unit, Respiratory and Systemic Infection
Laboratory. They included a number of isolates from two outbreaks of
streptococcal disease in a care-of-the-elderly system; the identities
of these isolates (see Table 1) were not revealed until after FAFLP
analysis (see below) was completed. Isolates were subjected to
conventional M serotyping (5, 6) before and after genotype
analysis. The type strain of serotype M77, NCTC 12057, was obtained
from the National Collection of Type Cultures (Central Public Health
Laboratory, London, United Kingdom). Streptococci were cultured
aerobically at 37°C for 18 to 24 h on horse blood agar plates
and were preserved for reference in blood glycerol (16% [vol/vol])
broth (Oxoid, Basingstoke, United Kingdom) at 
70°C.
Standard nucleic acid methods.
Genomic DNA was extracted
from streptococcal plate cultures as described previously
(12). The concentration of DNA was estimated with a
spectrophotometer (Beckman DU 640) by standard methods (9).
PFGE was carried out following SmaI macrorestriction as described previously (13).
FAFLP.
Genomic DNA (500 ng) was digested in a total volume
of 22 µl, which consisted of 5 U of MseI (New England
BioLabs [NEB], Hertfordshire, England), 2 µl of 10×
MseI buffer (NEB), 0.2 µl of 10-µg/ml bovine serum
albumin (NEB), and 1.0 µl of DNase-free RNase A (10 µg/µl) for 1 h at 37°C. To this digest was added 5 U of
EcoRI (Life Technologies, Paisley, United Kingdom), 1.68 µl of 0.5 M Tris-HCl (pH 7.6), and 2.1 µl of 0.5 M NaCl; the
reaction mixtures were incubated for a further hour at 37°C.
Endonucleases were then inactivated (65°C for 10 min) prior to
ligation.
To the double-digested DNA, a 25-µl solution containing 5 pmol of
EcoRI adaptor (Genosys Biotechnologies, Cambridge, United Kingdom), 50 pmol of MseI adaptor (Genosys Biotechnologies),
40 U of T4 DNA ligase (NEB), and 5 µl of 10× T4 ligase buffer (NEB) was added. The reaction 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 EcoRI adaptor was as follows:
5'- CTCGTAGACTGCGTACC
CATCTGACGCATGGTTAA-5'
The sequence of the
MseI adaptor was as
follows:
5'-
TACTCAGGACTCATC
GAGTCCTGAGTAGCAG-5'
The forward primer (
EcoRI adaptor specific,
termed
EcoRI+O) was labelled with a blue fluorescent dye,
5-FAM (5-carboxyfluorescein).
The reverse primer (
MseI
adaptor specific) contained an extra
selective base
(
MseI+T). PCRs were performed in 25-µl volumes
containing
2.5 µl of ligated DNA, 16.6 pmol of FAM-labelled
EcoRI
primer (5'-GACTGCGTACCAATTC-3'; Genosys Biotechnologies),
100
pmol of
MseI+T primer
(5'-GATGAGTCCTGAGTAA
T-3'; Genosys
Biotechnologies), 2.5 µl of 10×
Taq polymerase
buffer (Life Technologies),
1.5 mM MgCl
2 (Life
Technologies), each of the four deoxynucleoside
triphosphates at a
concentration of 10 mM (Life Technologies),
1 µl of 10-µg/ml bovine
serum albumin (NEB), and 0.625 U of
Taq DNA polymerase
(Life Technologies). Touchdown PCR cycling conditions
were used for
amplification as follows: denaturation for 2 min
at 94°C (1 cycle),
followed by 30 cycles of denaturation at 94°C
for 20 s, a 30-s
annealing step (see below), and a 2-min extension
step at 72°C. The
annealing temperature for the first cycle was
66°C; for the next nine
cycles, the temperature was decreased
by 1°C at each cycle. The
annealing temperature for the remaining
20 cycles was 56°C. This was
followed by a final extension at
60°C for 30 min. PCR was performed
in a PE-9600 thermocycler (Perkin-Elmer
Corp., Norwalk, Conn.). FAFLP
reaction mixtures were stored at
20°C.
Gel analysis.
Amplification products were separated on a 5%
denaturing (sequencing) polyacrylamide gel on an ABI Prism 377 DNA
automated sequencer (Perkin-Elmer Corp.). The gel was prepared from 5%
acrylamide-bisacrylamide (19:1; Amresco, Solon, Ohio) and 6.0 M urea in
1× TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA). To 50 ml of gel
solution was added 250 µl of 10% ammonium persulfate and 35 µl of
N,N,N',N'-tetramethylethylenediamine (Amresco). Spacers and sharks-tooth combs were 0.2 mm in thickness. Gels were poured with the ABI 377 gel cassette and gel injection device
and were allowed to polymerize at room temperature for at least 2 h. FAFLP reaction mixtures were electrophoresed directly or were
diluted; 1.5 µl of the reaction mixture was added to 1.5 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 0.5 µl of the
internal size marker [GeneScan-2500 labelled with the red fluorescent
dye ROX {6-carboxy-x-rhodamine}; Perkin-Elmer]). The
sample mixture was heated at 95°C for 2 min, cooled on ice, and
immediately loaded onto the gel. Running buffer was 1× TBE buffer, and
the electrophoresis conditions were 2.5 kV at 51°C for 5 h. The
well-to-read distance was 36 cm.
| |
RESULTS |
Epidemiology of the outbreak.
Two clusters of S. pyogenes infections affected elderly patients in North London in
1997. Cluster 1 occurred in January and involved two residents of a
nursing home. The first patient presented with signs of cellulitis and
septicemia, and blood cultures yielded GAS (isolate SA10, Table
1). Subsequent screening of staff and another resident yielded GAS (isolate SA78, Table 1) from that resident's leg ulcer. Both isolates were serotyped as T13 M77.
Cluster 2 occurred over a 6-week period, from April to June, and
involved 10 inpatients on three of four care-of-the-elderly
wards at
the local hospital (Northwick Park Hospital, London,
United Kingdom).
After the first four patients were identified,
all staff, other
patients, and the environment were screened,
but no reservoirs or
sources of infection could be identified.
Three new isolates were obtained on 1 day, one via the general
practitioner of a patient who had been recently hospitalized
and two
from inpatients. All four wards were then closed for admissions
or
transfers, and sampling and screening of all patients, staff,
and the
environment were undertaken. Patients recently discharged
from any of
the relevant wards were identified, and samples for
screening cultures
were requested via their general practitioners.
Four further
S. pyogenes isolates were obtained, including one
from a member of
the staff. All the patient isolates were serotyped
as T13 M77, while
that from the staff member was T12 M12 (not
included in this study).
There was regular transfer of patients between the nursing home
involved in cluster 1 and the care-of-the-elderly wards at
Northwick
Park Hospital, raising the possibility that the two
clusters could be
linked.
Macrorestriction and PFGE.
Twenty-six S. pyogenes
isolates and the type strain for serotype M77 were analyzed in a
"blinded" manner, with their identities concealed until completion
of genotypic analysis. The discriminatory power of PFGE was compared
with that of FAFLP. Macrorestriction with SmaI generated 10 to 13 genomic fragments ranging in size from 40 to 500 kbp. However,
because of their large size and the limitations of sizing from agarose
gels, these fragments could only be indirectly and approximately sized.
Twelve PFGE profiles were observed among the 27 isolates; 2 of these
shared the same profile, profile P1. Profile P2 was shared
by nine
isolates, while profile P6 was shared by six isolates
(22%).
Comparative data for PFGE and FAFLP analysis for the 27
isolates are
presented in Table
1.
FAFLP.
FAFLP gel data consisted of precisely sized amplified
fragments in the size range of 90 to 600 bp. Sequencing gel data were transformed with GeneScan 2.1 software (ABI) into electropherograms; these were color coded, overlaid, and visually inspected for
polymorphisms. Two kinds of experiments were carried out to test the
reproducibilities of the FAFLP profiles and the precision of sizing of
amplified fragments. First, the same DNA preparation was used in
different FAFLP reaction mixtures and was then analyzed on the same gel and also on different gels. Second, different DNA preparations from the
same strain or isolate were subjected to the same FAFLP reaction
(conditions) and were analyzed as described above. Under all these
experimental conditions, the characteristic amplified fragment profile
(size range, 90 to 600 bp) was reproducibly detected, and none of the
fragment sizes varied in any instance by more than 1.0 bp. The FAFLP
procedure was rapid and easy to use and could discriminate diverse
strain genotypes among isolates.
FAFLP generated 100 to 120 fragments upon amplification with the
selective primers
EcoRI+0 and
MseI+T. The sizes
of the amplified
fragments ranged from 60 to 600 bp; fragments of less
than 90
bp were discounted from GeneScan 2.1 analysis due to inadequate
resolution in this size range. Peak height (Fig.
1) indicates
the relative fluorescence of
the detected fragments. Peak height
thresholds were set at 125; any
peak heights of less than this
value were not included in the analysis.
Peak height was not found
to vary between replicate runs with identical
DNAs. Examples of
the electropherograms derived by GeneScan 2.1 analysis for three
EcoRI+0 plus
MseI+T
amplifications (i.e., FAFLP profiles for three
different strains) are
shown in Fig.
1.
View larger version (41K):
[in this window]
[in a new window]
|
FIG. 1.
GeneScan 2.1 software-derived electropherograms (FAFLP
profiles) for EcoRI+0 plus MseI+T amplifications
of three GAS genomes. Panel 1, profile A1; the first outbreak strain
(Table 1); the two sections represent FAFLP fragments from 90 to 275 bp
and from 276 to 600 bp, respectively. Panel 2, profile A2; the second
outbreak strain. Panel 3, profile A10; a non-outbreak-related isolate
which had the same PFGE profile as that of the second outbreak strain
shown in panel 2. The solid arrowheads and peaks indicate a fragment
characteristic of that profile (sizes are indicated in base pairs).
Open arrowheads indicate the absence of a polymorphic fragment from
that profile.
|
|
The GeneScan 2.1 software could overlay up to 16 electropherograms for
comparison between isolates. We thereby identified
two genotypes, to
which we could assign two (Fig.
1, panel 1)
and eight (Fig.
1, panel 2)
isolates, respectively. The first
FAFLP genotype (profile A1; Fig.
1,
panel 1) was characterized
by a unique combination of 18 polymorphic
fragments. It contained
5 characteristic fragments of 164, 217, 253, 365, and 583 bp (solid
arrowheads and peaks, Fig.
1) and lacked 13 polymorphic fragments
(open arrowheads, Fig.
1) found in other
profiles. We assigned
eight isolates to a second FAFLP genotype
(profile A2; Fig.
1,
panel 2). This contained four fragments of 97, 253, 294, and 365
bp (solid arrowheads and peaks, Fig.
1) and did not
contain any
of 14 polymorphic fragments (open arrowheads, Fig.
1) found
in
other profiles.
When the experiment was unblinded, profiles A1 and A2 were found to
include 10 serotype M77 isolates from the two North London
outbreaks.
Profile A1 was shared by two isolates from the first
outbreak, while
profile A2 was shared by eight isolates from the
second outbreak.
Thirteen non-outbreak-related serotype M77 isolates
also included in
the study were circulating isolates found in
diverse geographical areas
of the United Kingdom. In Table
2 we
summarize the polymorphisms observed in the FAFLP profiles
for all
serotype M77 isolates studied. The discriminatory power
of FAFLP
exceeded that of PFGE (80 to 90 FAFLP fragments versus
10 to 13 PFGE
fragments). For example, the circulating serotype
M77 isolate SA1558
showed the same PFGE profile as the second
outbreak strain but
exhibited a distinct FAFLP profile, profile
A10 (Fig.
1, panel 3). It
contained a unique combination of eight
fragments of 97, 128, 204, 253, 258, 294, 365, and 509 bp and
did not contain 10 polymorphic fragments
found in other profiles.
Similarly, six circulating isolates of
serotype M77 sharing the
same PFGE profile were each assigned a unique
FAFLP profile (Table
1).
Single isolates of three other GAS serotypes which had been included
generated distinct FAFLP profiles. The polymorphisms
observed between
the isolates of other serotypes (serotypes M4,
M5, and M6) were
different from those observed between isolates
of serotype M77. Two
contemporaneous isolates of serotype M4,
also from Northwick Park
Hospital and with identical PFGE profiles,
had the same FAFLP profile,
profile A17.
| |
DISCUSSION |
We have demonstrated that FAFLP can define the genotype of an
outbreak strain by reproducibly detecting and precisely sizing amplified fragments unique to that genome. In this respect, FAFLP qualifies as a true "typing" technique, since different
laboratories using the same reagents and instrumentation could
determine that FAFLP genotypes are identical, even when the isolates
are from different sources and times. The FAFLP data presented here are intrinsically more reproducible than those obtained by other PCR-based methodologies, such as RAPD analysis, that are based on amplification with degenerate primers and that do not generate fingerprints reproducible enough for interlaboratory comparison (7).
We found that the discriminatory power of FAFLP was considerably higher
than that of PFGE. For example, FAFLP could differentiate the genotypes
of the two outbreak strains from those of the circulating isolates of
the same serotype. Among 13 circulating isolates of serotype M77, FAFLP
could assign unique profiles to each, whereas PFGE could not
differentiate between 6 of them. The number of datum points (fragments)
available for comparison and definition of strain genotype is almost
eight times more for FAFLP than for PFGE. The sizing of the fragments
by FAFLP is also much more precise (±1 bp) than that by PFGE. FAFLP
was less labor intensive, could be performed more rapidly (2 to 2.5 days for FAFLP versus 3.5 to 4 days for PFGE from pure culture), and
was easier to perform than PFGE.
As well as subtyping isolates within an M serotype (M77), FAFLP
distinguished between different M serotypes of GAS. This application is
documented by other preliminary studies in our laboratory (data not
shown) and may allow organisms with homogeneous serotypes to be rapidly
grouped together.
The discriminatory power of FAFLP can be systematically varied by
performing the amplification with primers of specified selectivity to
produce different numbers and sizes of amplified fragments. The
technique can thereby be tailored to the level of typing discrimination required. FAFLP profiles are suitable for rapid electronic transmission for interlaboratory comparison and are well suited for storage in
epidemiological databases for future comparison. The capacity of the
automated equipment to scan amplified fragments in gels is high: 36 lanes can be loaded with different FAFLP reaction mixtures, and
fragments of from 50 to 600 bp can be read in 5 h. We consider
that these features make FAFLP an excellent tool for the rapid and
definitive analysis of outbreaks, even some due to apparently clonal
subtypes that are not susceptible to subdivision by existing phenotypic
techniques or by other currently used molecular techniques.
| |
ACKNOWLEDGMENT |
We thank Philip Mortimer for critical reading of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Molecular
Biology Unit, Central Public Health Laboratory, 61 Colindale Ave.,
London NW9 5HT, United Kingdom. Phone: 0181 200 4400, ext. 3071. Fax: 0181 200 1569. E-mail: mdesai{at}hgmp.mrc.ac.uk.
| |
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Journal of Clinical Microbiology, November 1998, p. 3133-3137, Vol. 36, No. 11
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
