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Journal of Clinical Microbiology, September 2001, p. 3346-3349, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3346-3349.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Sequence Typing Confirms that Campylobacter
jejuni Strains Associated with Guillain-Barré and
Miller-Fisher Syndromes Are of Diverse Genetic Lineage, Serotype, and
Flagella Type
K. E.
Dingle,1
N.
Van Den Braak,2
F. M.
Colles,1
Lawrence J.
Price,3
David L.
Woodward,3
Frank G.
Rodgers,3
H. P.
Endtz,2
A.
Van
Belkum,2 and
M. C. J.
Maiden1,*
Wellcome Trust Centre for the Epidemiology of
Infectious Disease, Department of Zoology, University of Oxford,
Oxford, OX1 3FY, United Kingdom1;
Department of Medical Microbiology and Infectious Diseases,
Erasmus University Medical Center Rotterdam, Rotterdam, The
Netherlands2; and National Laboratory
for Enteric Pathogens, National Microbiology Laboratory, Winnipeg,
MB R3E 3R2, Canada3
Received 22 May 2001/Returned for modification 26 June
2001/Accepted 28 June 2001
 |
ABSTRACT |
Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome
(MFS) are correlated with prior infection by Campylobacter
jejuni in up to 40% of cases. Nucleotide sequence-based typing
of 25 C. jejuni isolates associated with neuropathy
permitted robust comparisons with equivalent data from approximately
800 C. jejuni isolates not associated with neuropathy. A
total of 13 genetic lineages and 20 flaA short variable
region nucleotide sequences were present among the 25 isolates. A
minority of isolates (4 of 25) had the flaA short variable
region nucleotide sequences that were previously proposed as a marker
for GBS-associated isolates. These 4 isolates probably represented the
Penner serotype 19 lineage, which has been proposed to have an
association with GBS.
| |
TEXT |
Guillain-Barré Syndrome (GBS)
and Miller-Fisher Syndrome (MFS) are autoimmune disorders of the
peripheral nervous system which cause acute flaccid paralysis
(11). Both syndromes occur worldwide, with incidence rates
of 0.5 to 4 per 100,000 for GBS, the more common of the two
diseases (6). Up to 40% of cases are preceded by
enteritis caused by the gram-negative organism Campylobacter
jejuni 1 to 3 weeks before the onset of neurological symptoms
(10, 14). The mechanisms by which C. jejuni
infections cause neuropathy are yet to be fully elucidated; however,
GBS and MFS are probably a consequence of immunological
cross-reactivities of antibodies stimulated against bacterial cell
surface carbohydrates with human gangliosides (5, 13).
Relative to the isolates generally recovered from patients with
C. jejuni enteritis, certain serotypes, especially Penner
serotype 19 (7), are overrepresented among isolates from
patients who develop GBS and MFS. It has been suggested that the Penner
19 serotype is associated with a C. jejuni clone which has a
higher probability of association with neuropathy (4). A
particular variant of the short variable region (SVR) of the
flagellum-encoding gene, flaA, has been proposed as a marker
for C. jejuni strains which are likely to cause GBS or MFS
(12).
Developments in the molecular characterization of C. jejuni
isolates, specifically the application of multilocus sequence typing
(MLST) (1) and nucleotide sequence typing of the
flaA SVR (9), enable precise characterization
and robust comparisons of the genetic relatedness of isolates via the
Internet regardless of where the isolate characterization is done
(8). Nucleotide sequences are especially amenable to
phylogenetic approaches for the investigation of bacterial population
structure and the forces which influence it. A collection of 25 GBS-
and MFS-associated C. jejuni isolates was analyzed by MLST
and flaA gene sequencing, and Penner and heat-labile (HL)
serotyping data were obtained. The data were compared with equivalent
information for more than 800 C. jejuni isolates held in a
World Wide Web-accessible database (1). Eighteen of the
twenty five isolates were previously described and had been shown to be
diverse by a range of serological and genetic fingerprinting methods
(2, 3), but the nature of the techniques used precluded
the precise genetic comparisons of isolates which are possible with
nucleotide sequence data. The remaining seven isolates were previously
undescribed and comprised three from GBS patients from The Netherlands
(isolates 23, 24, and 25), two siblings of a Dutch GBS
patient (isolates 26 and 27), and two derived from patients with GBS
from Curaçao and Bonaire (isolates 22 and 28, respectively).
Nucleotide sequence accession number.
The nucleotide sequences
of the flaA SVRs of the isolates analyzed in this study are
available under the GenBank accession numbers listed in Table 1.
The MLST profiles demonstrated that the 25 isolates were genetically
diverse, with 20 different sequence types (STs); these were assigned
names and grouped into 13 distinct clonal complexes, which are thought
to correspond to genetic lineages, using the previously published
definitions (1) (Table 1).
The high level of genetic diversity among these isolates was also
reflected phenotypically in their large range of Penner and HL
serotypes (Table 1). The level of diversity observed among the 25 isolates was similar to that present in the C. jejuni MLST
database (http://mlst.zoo.ox.ac.uk/), which contained 814 isolates of
human, animal, and environmental origin at the time of comparison. The
most common lineage or clonal complex present in the MLST database was
the ST-21 complex, which made up 260 of the 814 isolates (32%) at the
time of comparison. While no representatives of ST-21 itself were
present among the collection of 25 isolates, 6 of the 25 isolates were
considered to be variants of this sequence type (Table 1). Of these 6 variants, 2 STs, ST-19 and ST-53, were frequently found in the
database, with 30 and 19 isolates, respectively. A total of 4 isolates
belonged to the ST-22 complex, which is associated with Penner serotype 19 and HL 36 and 77 (Table 1); this lineage is comparatively poorly
represented in the database (12 of 814 isolates [1.5%]). The
ST-61 and ST-48 complexes were represented twice, and the ST-42, ST-51,
ST-52, ST-206, and ST-403 complexes once each
in all of these examples
the abundance of these lineages among the neuropathy-associated
isolates was consistent with their abundance in the database as a
whole. The absence of members of the ST-45 complex, which was the
second-most-common lineage represented in the database (74 of 814 isolates), may indicate that members of this complex have a low
probability of association with GBS or MFS. The MLST data were
compatible with previous analyses of these isolates (2).
Isolates 13 and 14, which were previously reported to exhibit whole
genome homology (4), belonged to distinct STs of the ST-21
complex, differing by only one nucleotide in the gltA locus,
probably corresponding to a point mutation. Isolates MF20 and GB25 had
identical STs and may both belong to an as yet undefined clonal
complex. The same may be true for isolates GB22 and GB21, which shared
six of the seven MLST loci.
The nucleotide sequences of the
flaA SVR present among the
25 isolates were diverse, with 20 distinct SVR nucleotide sequences
(Table
1). The two most diverse sequences differed at 63 of 321
nucleotide sites (19.6%), corresponding to 16 differences (15.0%)
among the 107 amino acids. The
flaA sequences from the four
ST-22
complex isolates were similar to each other and to the
flaA sequences
reported to be associated with GBS previously
(0.3 to 1.6% nucleotide
sequence differences with only one amino acid
change [Fig.
1])
(
12).
These SVR sequences differed at 17 to 62 nucleotide sites
(5.3 to
19.3%) and 6 to 16 amino acids (5.6 to 15.0%) from the
remaining SVR
sequences obtained for the isolates described here.
The
flaA
SVR nucleotide sequence data provided slightly more discrimination
than
PCR-restriction fragment length polymorphism (RFLP) fingerprint
patterns, with 14 unique sequences compared to 12 patterns by
PCR-RFLP
among the 18 previously described isolates (
3). Isolates
with identical PCR-RFLP patterns either had identical SVR sequences
or
differed at one nucleotide. Identical
fla SVR sequences have
been described for isolates which were not associated with GBS
or MFS
(data not shown).
View larger version (41K):
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|
FIG. 1.
Comparison of the unique flaA SVR
nucleotide sequences present in the ST-22 complex isolates with SVR
sequences proposed to be associated with GBS (12).
Sequences are identified by their GenBank number, and the name of the
isolate(s) in which the sequences have been described is given in
brackets. The sequences are compared to the sequence under accession
no. AF290501: a period (.) indicates identity with this sequence, a
nucleotide substitution being represented by the appropriate letter.
The amino acid translations of these sequences are shown by
single-letter amino acid code, and the single nonsynonymous base change
(asparagine to lysine) is indicated thus: K*. The identical sequences
for isolates 86-1, 86-2, and 86-3 are separately stored in GenBank
under numbers AF290501, AF290502, and AF290503, respectively.
|
|
There was no detectable allele bias among the GBS- and MFS-associated
isolates for MLST loci when they were compared with
the database of
more than 800 isolates (data not shown). Isolates
GB 19 and GB 16 contained an
uncA allele (
uncA-17) which
exhibited
~90% nucleotide sequence identity with the other 11
uncA alleles
present in the isolate collection, which shared
~97% nucleotide
sequence identity. This was likely to be due to the
recruitment
of the allele sequence from another related species, as has
been
observed previously with
glyA alleles with high
homology to
Campylobacter coli sequences being found in some
C. jejuni isolates (
1).
The allele
uncA-17 has been identified in two
C. coli
isolates
(data not shown). The allele sequences found at each locus of
the GBS- and MFS-associated isolates were distributed among the
C. jejuni alleles present in the MLST database in
phylogenetic
reconstructions made by several techniques (data not
shown).
These data support the view that diverse
C. jejuni
genotypes, as measured by MLST and
flaA SVR nucleotide
sequences, and serotypes
reflecting capsular (Penner) and flagellar
(HL) antigens can be
associated with neuropathy. The most common
lineage was the ST-21
complex, which was also the most common lineage
in the collection
of mainly human enteritis-associated isolates
available in the
MLST database (
http://mlst.zoo.ox.ac.uk). The
next-most-common
lineage among the isolates examined was the ST-22
complex, and,
although the numbers are small, this complex was
apparently overrepresented
in the neuropathy-associated isolate
collection relative to the
isolates which are described in the database
as a whole. It is
possible from these data that the ST-45 complex is
relatively
underrepresented among neuropathy-associated isolates. These
observations
are consistent with the ST-22 complex being the Penner
serotype
19 virulent clone proposed to be associated with GBS
(
4). It
appears likely that the
flaA SVR
sequence that was proposed as
a marker for GBS-associated
C. jejuni strains may be a marker
for this clonal complex; however,
since this sequence was present
in only 4 of the 25 neuropathy-associated isolates examined here,
it is not a useful marker
for all
C. jejuni strains likely to
cause
GBS.
| |
ACKNOWLEDGMENTS |
This work was funded by the United Kingdom Ministry of Agriculture
Fisheries and Food, contract number OZ0604. This publication made use
of the Multi Locus Sequence Typing website
(http://mlst.zoo.ox.ac.uk) developed by Man-Suen Chan and sited at
the University of Oxford. The development of this site is funded by the
Wellcome Trust.
| |
FOOTNOTES |
*
Corresponding author. Present address: The Peter
Medawar Building for Pathogen Research, University of Oxford, South
Parks Rd., Oxford, OX1 3FY, United Kingdom Phone: (44) (1865) 271284. Fax: (44) (1865) 271284. E-mail:
martin.maiden{at}zoo.ox.ac.uk.
| |
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Journal of Clinical Microbiology, September 2001, p. 3346-3349, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3346-3349.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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