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Journal of Clinical Microbiology, April 2000, p. 1632-1635, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of a Recurrent Clonal Type of
Escherichia coli O157:H7 Causing Major Outbreaks of
Infection in Scotland
Lesley J.
Allison,1
Philip E.
Carter,1 and
Fiona M.
Thomson-Carter2,*
Department of Medical Microbiology,
University of Aberdeen, Foresterhill, Aberdeen AB25
2ZD,1 and Scottish National Reference
Laboratory for Campylobacter and Escherichia coli O157,
Grampian University Hospitals Trust, Foresterhill, Aberdeen AB25
2ZN,2 Scotland
Received 20 October 1999/Returned for modification 22 January
2000/Accepted 29 January 2000
 |
ABSTRACT |
A particular recurrent clonal type of Escherichia coli
O157 has been isolated from multiple clinical, veterinary, food, and environmental sources throughout Scotland since 1989. Significant genotypic variation was detected among isolates from distinct outbreaks, with the presence or absence of single fragments being sufficient to delineate outbreak groups within the clonal type.
| |
TEXT |
Since its first description in 1982, Escherichia coli O157:H7 has caused many major general
outbreaks of infection worldwide including those in Japan (ca. 10,000 cases and 5 deaths) (7) and Scotland (496 cases and 20 deaths) (2).
Pulsed-field gel electrophoresis (PFGE) has been applied in
investigations of several outbreaks of E. coli O157
infection (1, 2, 3). Systematic application of PFGE as
a determinative method for E. coli O157 in the Scottish
Reference Laboratory (SRL) (11) has, for the first time,
enabled identification of recurrent clonal types defined according to
the criteria of Orskov and Orskov (15). The most significant
of these, termed the "West Lothian" (WL) clonal type, has been
isolated from multiple veterinary (cattle and sheep), food (raw and
cooked meat), environmental (dairy machinery and milk tanker), and
clinical (>400 patients) sources throughout Scotland over the last 5 years. It has been responsible for several major outbreaks of
infection, separate both temporally and geographically, including the
largest milk-borne outbreak in the world (West Lothian, Scotland, 1994)
and the most severe outbreak worldwide, the central Scotland outbreak
(November 1996), which caused the deaths of 20 people.
The aim of this study was to further characterize isolates of the
clonal type by enhanced fingerprinting in order to distinguish them.
Sources of bacterial isolates.
During the course of three
major outbreaks caused by the putative recurrent clonal type, in excess
of 600 isolates were analyzed by phenotypic and genotypic methods. The
outbreaks occurred as follows: West Lothian, 1994, 100 cases; Highland,
1994, 8 cases; central Scotland, 1996, 496 cases. Isolates with numbers
318 to 340 inclusive were associated with the West Lothian outbreak, isolates with numbers 522 to 594 inclusive were associated with the
Highland outbreak, and isolates with numbers 1476 to 1717 inclusive
were associated with the central Scotland outbreak. In addition to
isolates from the three outbreak groups, other representative phage
type 2 isolates received by SRL (1992 to November 1996 [onset of
central Scotland outbreak]) and November 1996 to March 1998) were
similarly analyzed.
Standard typing and subtyping.
Isolates were characterized
biochemically and serotyped as E. coli O157:H7 by standard
microbiological methods. Phage typing was performed with the study
group by the method of Khakria et al. (10). The
supplementary phages were also included (R. Khakria, M. Mulvey, R. Ahmed, D. Woodward, and W. Johnson, Abstr. 3rd International Symposium
and Workshop on Shiga Toxin (Verocytotoxin)-Producing Escherichia coli
Infections, abstr. V124/I, 1997). All isolates were tested with primers
specific for the genes encoding Verotoxin type 1 (VT1) and VT2 as
described previously (18). All outbreak isolates were
E. coli O157:H7, phage type 2, VT1 negative, and VT2
positive. The supplementary phages did not further discriminate the
outbreak groups. Phage type 2 is common in Scotland, comprising 58.3%
of the annual SRL sample load as a result of the central Scotland
outbreak (1996-1997) and 18.5% of the annual sample load in
1997-1998 (21).
Enhanced strain fingerprinting.
Enhanced strain fingerprinting
by several genotypic techniques was performed with a selected study
group of 53 isolates from the three outbreaks caused by the phage type
2 WL clonal type. The isolates analyzed included an isolate from milk
and a cattle isolate associated with the West Lothian outbreak and a
food sample associated with the central Scotland outbreak.
PFGE was performed in a CHEF DR-II apparatus (Bio-Rad, Hemel Hempstead,
United Kingdom) by the method of Krause et al. (11). Cleavage of the agarose-embedded DNA was achieved with XbaI
(Promega, Southampton, United Kingdom), NotI (Promega),
SfiI (Promega), XhoI (Boehringer Mannheim, Lewes,
United Kingdom), and AvrII (Boehringer Mannheim) according
to the manufacturer's instructions. Run times and pulse times were
optimized for each enzyme, as follows: for XbaI, pulse times
of 37 to 47 s for 22 h or, alternatively, 15 to 50 s for
22 h; for NotI, pulse times of 10 to 30 s for
23 h; for SfiI, pulse times of 15 to 50 s for
22 h; for XhoI, pulse times of 1 to 15 s for
18 h; and for AvrII, pulse times of 5 to 50 s for
22 h, all with linear ramping. To assess variations in the small
(<150-kb)-fragment profiles, inserts containing DNA cleaved with
XbaI were run at pulse times of 6 to 30 s for 29 h
or with AvrII at a pulse times of 6.75 to 26.29 s for 30.46 h.
Systematic reference laboratory analyses have demonstrated that
disparate isolates that share the same phage type (phage type
2) can be
subdivided into at least 10 distinct pulsed-field groups
by the
standard PFGE method (
XbaI digestion, pulse times of 37
to
47 s for 22 h) (
1). During the course of the
outbreaks SRL
applied these run parameters in real-time analyses and
found the
macrorestriction patterns to be indistinguishable for
isolates
from each of the three outbreaks (Fig.
1). The patterns were stable
during
primary analysis and reproducible on subsequent repeated
subculture. In
this study the maximum period that elapsed between
receipt of the
isolate and the most recent subculture was 38 months.
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FIG. 1.
PFGE profiles of genomic DNAs West Lothian, Highland,
and central Scotland outbreak isolates cleaved with XbaI
(lanes 2 to 4), XhoI (lanes 5 to 7), NotI (lanes
8 to 10), and SfiI (lanes 11 to 13). Lanes 1 and 14, bacteriophage lambda concatemer size markers. The unnumbered lanes
correspond to lanes 1 to 14 from left to right, respectively.
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Indistinguishable profiles were also observed when genomic DNA was
cleaved with
XhoI,
SfiI, and
NotI
(Fig.
1). When DNA was
cleaved with
AvrII and run with a
pulse time of 5 to 50 s, differences
were observed in the
particular region of the macrorestriction
profile where small fragments
(<150 kb) are resolved. Electrophoretic
conditions were altered (pulse
times of 6.75 to 26.29 s for 30.46
h) to optimize separation of the
small fragments in this region.
It was then possible to distinguish the
central Scotland outbreak
isolates from other outbreak isolates by the
presence of a triplet
of fragments in the size range of 97 to 145 kb.
Isolates from
the Highland and West Lothian outbreaks also possessed a
triplet,
but the profiles varied by the absence of fragment A and the
presence
of fragment B (Fig.
2). The
profile for isolate 1551 differed
from the profiles for the rest of the
central Scotland group by
a single band (Fig.
2, lane 7).
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FIG. 2.
PFGE profiles of AvrII-cleaved genomic DNAs
from West Lothian (lanes 1 to 3), Highland (lanes 4 and 5), and central
Scotland (lanes 6 to 9) outbreak isolates. Lane 10, bacteriophage
lambda concatemer size markers. The fragment identifying central
Scotland isolates is indicated by A. The fragment absent from central
Scotland isolates but present in both West Lothian and Highland
isolates is indicated by B. The unnumbered lanes correspond to lanes 1 to 10 from left to right, respectively.
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Following resolution of
XbaI-cleaved fragments with
parameters that optimize separation of fragments in the size range of
50 to 300 kb, a single band difference was observed in the isolates
from the Highland outbreak (Fig.
3). The
profiles for the isolates
from the West Lothian and central Scotland
outbreaks remained
indistinguishable with the exception of that for
isolate 340,
whose profile was indistinguishable from those of the
Highland
outbreak isolates.
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FIG. 3.
PFGE profiles of XbaI-cleaved genomic DNA
from West Lothian (lanes 2 to 4), Highland (lanes 5 to 7), and central
Scotland (lanes 8 to 10) outbreak isolates. Lanes 1 and 11, bacteriophage lambda concatemer size markers. The unnumbered lanes
correspond to lanes 1 to 11 from left to right, respectively.
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Similar PFGE analyses of a group of representative phage type 2 strains
isolated either before or after the onset of the central
Scotland
outbreak demonstrated that the central Scotland outbreak
clonal type,
as identified by its macrorestriction profile, had
been isolated on
only one previous occasion in Scotland, from
a single cluster of
infection in a family in April 1995. From
November 1996 to March 1998, however, the clonal type was isolated
from 12 separate incidents
(excluding the central Scotland outbreak)
of infection from patients
throughout
Scotland.
Bacteriophage lambda restriction fragment length polymorphism (RFLP)
analysis was performed with purified genomic DNA (2 µg)
cleaved with
either
PvuII or
HindIII. The DNA fragments
were separated,
blotted, and probed with digoxigenin-labeled
bacteriophage lambda
(Bio-Rad) (
16). Variations in RFLP
profiles, which differentiated
each of the three outbreak groups, were
observed (Fig.
4). All
central Scotland
outbreak isolates possessed an extra band (denoted
by an arrowhead in
Fig.
4) not present in the other outbreak isolates.
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FIG. 4.
Bacteriophage lambda RFLP analysis of
PvuII-cleaved genomic DNA from West Lothian (lanes 2 to 10, and 18), Highland (lane 11), and central Scotland (lanes 12 to 17)
outbreak isolates. Lanes 1 and 19, DNA molecular weight standards. The
unnumbered lanes correspond to lanes 1 to 19 from left to right,
respectively.
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One particular fragment in the smaller size range was absent from all
isolates from the Highland outbreak but was present
in most isolates of
the other outbreak groups (Fig.
4, lane 11;
the missing band is denoted
by a period). Two West Lothian isolates
(isolates 325 and 340) were
indistinguishable from the Highland
isolates by this method. Isolate
325 could be differentiated from
the Highland isolates by PFGE of
XbaI-cleaved DNA, but isolate
340 could not. Similar
experiments performed on
HindIII-cleaved
DNA
demonstrated indistinguishable RFLP profiles for all isolates
in all
outbreak
groups.
Supplementary genotypic methods were applied to further
distinguish isolates. Insertion sequence analysis (IS
1) was
performed
with purified DNA (2 µg) cleaved with
RsaI
(Boehringer Mannheim).
The DNA fragments were separated
electrophoretically in 1% (wt/vol)
agarose in 0.5× TBE
(Tris-borate-EDTA) and were transferred to
a positively charged nylon
membrane (
20). The IS
1 insertion
sequence was
amplified (
12) and the product was labeled with
digoxigenin
(DIG-High Prime; Boehringer Mannheim). High-stringency
hybridization
and detection of fragments were carried out according
to the
manufacturer's instructions. Hybridization demonstrated
that all
isolates possessed a single hybridizing fragment that
was identical
among the isolates. Identical results were obtained
with different
restriction enzymes (data not
shown).
A range of DNA amplification-based fingerprinting methods was also
applied. All amplifications were performed on a DNA Thermocycler
480 (Perkin-Elmer Biosystems, Warrington, United Kingdom), and
reaction
mixtures contained 1.5 mM MgCl
2, 0.2 mM deoxynucleoside
triphosphates, 1.25 U of
Taq DNA polymerase, buffer
(Bioline),
2 ng of purified bacterial genomic DNA per µl
(
17), and 0.25
µM
primers.
Arbitrarily primed PCR (AP-PCR) was performed with two primers selected
for their superior discriminatory abilities: 5'-GTGGATGCGA
(
14) and AB7-10 (5'-AAGAGGCCAG; Applied
Biotechnologies, Epsom,
United Kingdom) (L. J. Allison and F. M. Thomson-Carter, unpublished
data). Thermocycler reaction conditions
were an initial cycle
of 94°C for 5 min, then 30 cycles of 94°C for
1 min, 30°C for
1 min, and 72°C for 2 min, and then an extension
cycle at 72°C
for 5
min.
Enterobacterial repetitive intergenic consensus (ERIC) PCR with the
standard ERIC primers (
13) was performed. Amplification
conditions were an initial cycle of 94°C for 5 min and then 34
cycles
of 94°C for 1 min, 37°C for 1 min, and 72°C for 2 min,
followed
by 72°C for 5
min.
Amplification of the 16S-23S spacer regions was performed with primers
described previously (
5). Amplification conditions
were an
initial cycle of 95°C for 1 min and then 30 cycles at
94°C for 1 min and 70°C for 2 min. A final extension cycle of
70°C for 5 min
was performed. From the known genome sequence of
E. coli
K-12 (
4), the expected size range for PCR products
would be
963 to 1,066 bp; the sizes of the products observed in
these
experiments fell within this range, and the products were
subsequently
cleaved with either
HaeIII or
TaqI.
Amplified fragment length polymorphism (AFLP) analysis was performed
with
EcoRI and
MseI or
TaqI. Ligation
of adapters, preselective
and selective amplification, and labeling of
the fragments were
performed essentially as described previously
(
8). Fluorescently
labeled fragments were separated on an
ABI 377 XL automated DNA
sequencer running Genescan application
software.
None of the PCR-based assays (AP-PCR, ERIC PCR, 16S-23S spacer region
RFLP analysis, and AFLP analysis) could discriminate
the outbreak
groups. Additionally, all WL clonal type isolates
possessed copies of
the normal and the variant VT2 gene (
22),
as determined by
cleavage of the PCR-amplified B subunit with
HaeIII.
The aim of the study described here was to differentiate isolates of a
recurrent clonal type by enhanced DNA fingerprinting.
The conventional
method of subtyping of
E. coli O157, bacteriophage
typing,
provided little useful information in investigation of
the central
Scotland outbreak since in excess of 58% of all bacterial
isolates
received by SRL during the period from 1 April 1996 to
31 March 1997 were phage type
2.
PFGE analyses with an extended panel of endonucleases demonstrated that
the study group of isolates representing the three
major outbreaks
could be discriminated under strictly defined
conditions by using
XbaI and
AvrII. Other restriction enzymes
could
not differentiate the clonal type. Different run parameters
for
XbaI-cleaved DNA distinguished Highland outbreak isolates
from the other two groups, while
AvrII distinguished the
central
Scotland outbreak group from the others. Two isolates (isolates
340 and 1551) did not share the indistinguishable profile for
the
outbreak isolates, with the profile for isolates 340 and 1551
differing
from the profile for the outbreak isolates by one fragment.
Isolate 340 from the West Lothian outbreak had a profile indistinguishable
from
that for the Highland isolates following cleavage with both
XbaI and
AvrII. The majority of isolates formed
distinct identifiable
outbreak groups that distinguished them from the
remainder of
the broader WL clonal type. Overreliance on one particular
endonuclease
should be avoided in PFGE analysis of
E. coli O157.
Bacteriophage lambda RFLP analysis also differentiated the three
outbreak groups. However, as with PFGE, certain isolates
from different
outbreaks were indistinguishable: West Lothian
isolates 325 and 340 and
Highland outbreak isolates. Isolate 340
could not be distinguished from
Highland outbreak isolates by
any method. If certain genetic events
gave rise to the differences
observed between the West Lothian and
Highland outbreak groups,
then conversely, their nonoccurrence or
reversal in this particular
isolate could lead to this
observation.
A minor variation in PFGE profiles has previously been attributed to
clonal turnover in individual patients shedding
E. coli O157
for up to 62 days (
9). In the present study, however,
analyses were performed with multiple isolates from disparate
sources
while the outbreaks were
ongoing.
The significance of the genomic differences elicited by PFGE and
bacteriophage lambda RFLP analysis is not clear. Certainly,
these are
minor variations, at most a few small fragments, of
a basically similar
macrorestriction profile compared with, for
example, the major
differences observed between the PFGE profiles
of phage types 2 and 49 (
11). During the recent outbreaks in
Japan analogous
small-fragment differences were observed and found
to be insignificant:
on hybridization a minor fragment (75 kb)
was found to be derived
directly from a larger chromosomal fragment
of the macrorestriction
profile for the outbreak strain. Therefore,
the discrepant fragment did
not materially affect the grouping
of the outbreak strains
(
7). However, the reasons why these
observed differences
arise remain as yet undetermined. The proposed
interpretative criteria
for PFGE profiles (
20) do not include
experimental evidence
describing the means by which such variation
occurs or the degree of
variation seen, providing only a general
outline of common genetic
events and their potential effect on
the PFGE profiles of various
bacterial genera. It may be postulated
from the evidence from this
study that since the majority (80%)
of genotypic analyses performed
could not discriminate the outbreak
groups, the isolates were
genetically homogeneous. Overreliance
on minor, arguably insignificant
differences in genomic structure
as evinced by the PFGE
macrorestriction profiles should perhaps
be resisted, particularly in
light of the variation in genomic
size apparent among certain isolates
(
6). Conversely, both
PFGE and bacteriophage lambda RFLP
analyses were able to separate
members of the WL clonal type into
discrete outbreak groups, indicating
that whatever genetic alterations
have occurred, although apparently
minor, they can give rise to
distinctive subsets of isolates.
These variations may be exploited as
epidemiological markers in
the real-time investigation of outbreaks of
E. coli O157
infection.
| |
ACKNOWLEDGMENTS |
The expert technical assistance of the SRL staff is gratefully
acknowledged. The continuing cooperation of colleagues in submission of
isolates to SRL is gratefully acknowledged.
This study was funded by the Scottish Office Agriculture, Environment
and Fisheries Department (project UAB/004/97) and the Acute Healthcare
Research Commitee, Chief Scientist Office, Scottish Office Department
of Health (project K/MRS/C50/2376). The Reference Laboratory for
Campylobacter and Escherichia coli O157 is funded by the National Services Division, Scottish Office Department of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Microbiology, Grampian University Hospitals NHS Trust,
Foresterhill, Aberdeen AB25 2ZN, Scotland. Phone: 44 1224 553819. Fax:
44 1224 840632. E-mail: mmb049{at}abdn.ac.uk.
| |
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Journal of Clinical Microbiology, April 2000, p. 1632-1635, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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