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Previous Article | Next Article 
Journal of Clinical Microbiology, May 2000, p. 1786-1790, Vol. 38, No. 5
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Sequence Diversity of the Escherichia
coli H7 fliC Genes: Implication for a DNA-Based Typing
Scheme for E. coli O157:H7
Lei
Wang,
Deborah
Rothemund,
Heather
Curd, and
Peter R.
Reeves*
Department of Microbiology, The University of
Sydney, Camperdown, New South Wales 2006, Australia
Received 5 November 1999/Returned for modification 14 January
2000/Accepted 21 February 2000
 |
ABSTRACT |
Flagellar (H) antigens are mostly encoded by genes at the
fliC locus in E. coli. We have sequenced 11 H7
fliC genes from Escherichia coli strains that
belong to seven O serotypes. These sequences, together with those of
nine other H7 fliC genes (from strains of three different O
serotypes) sequenced recently (S. D. Reid, R. K. Selander,
and T. S. Whittam, J. Bacteriol. 181:153-160, 1999), include 10 different sequences. The differences between these 10 sequences range
from 0.06 to 3.12%. By comparison with other E. coli
flagellin genes, we have identified primer length sequences specific
for H7 genes in general and others specific for H7 genes of O157 and
O55 strains: the specificity was confirmed by PCR testing the type
strains for all 53 E. coli H types. We have previously
identified genes specific for the E. coli O157 antigen, and
use of the combination of O157- and H7-specific primers allows the
sensitive and rapid detection of O157:H7 E. coli strains, which cause the majority of hemorrhagic colitis cases.
| |
INTRODUCTION |
Escherichia coli is a
clonal species, with clones normally identified by their combination of
O and H (and sometimes K) antigens. All enterohemorrhagic E. coli strains produce Shiga-like toxins (Stxs), but E. coli O157:H7 strains are the strains most frequently isolated from
humans and are the predominant cause of hemolytic-uremic syndrome
(HUS), with the O157:H7 clone having caused approximately two-thirds of
all recent cases of HUS in North America and Europe (33).
Because of the very low infective dose of this organism (13), bacteria that enter the human food chain can still
pose a health problem after enormous dilution. For example, in January 1993 there was an outbreak due to contamination with E. coli
O157:H7 at a large meat-processing plant that made over 1 million
hamburger patties per day. The affected hamburgers were sold through
one retail chain over 4 U.S. states, and 477 people became ill, of whom
3 died (1, 4). The scale of the plant and its operation are
such that there would have been great dilution of the contaminated meat, and the highest count of O157:H7 found in hamburger patties with
the same production date was 15 organisms per g, although the
hamburgers which caused the infection were not tested.
The flagellar protein constitutes the subunit of the helical filament
which forms the flagellar organelle. The flagellar protein carries the
antigenic determinant(s) for the H antigen. There are 53 H types in
E. coli (note that H50 was deleted from the list [6,
21]), and in most cases the flagellin structural genes are
located at the fliC locus (17, 18, 30). Studies of E. coli and Salmonella enterica flagellar
proteins demonstrated that these proteins are conserved in their
terminal regions, while the central region is variable, giving rise to
serotype-specific epitopes (12, 18, 22, 35).
Great efforts have been made to develop a method for timely and
accurate detection of the O157:H7 strain (see, e.g., references 5, 10, 13, and 23). PCR-based
methods are ideal for rapid detection of organisms at low
concentrations. PCR detection with probes based on the Stx and
eaeA (encoding intimin) genes and an unspecified plasmid
gene have been developed, but each probe gave a positive result with
some other E. coli strains even when only a small number of
strains were tested (10). PCR tests with primers based on
the unique substitution in the O157:H7 uidA gene (7) have also been developed.
Serotyping is routinely used to characterize E. coli
isolates, and strains with the combination of O157 and H7 antigens all appear to belong to the clone that causes HUS in humans. We suggest that a PCR test based on the antigens will provide the specificity required to detect this high-risk organism. A PCR-restriction fragment
length polymorphism test has been developed by using the H7
fliC gene as the target (9), but as discussed
below, it has significant disadvantages compared to a PCR test.
Recently, we sequenced the gene cluster that encodes the O157 antigen,
and by PCR testing against representatives of all 166 E. coli O serotypes and a range of gram-negative bacterial strains, including some that cross-react serologically with E. coli
O157 antisera, we have found that certain O-antigen genes are highly specific for E. coli O157 (34). The object of
this study was to identify sequences specific for the H7
fliC gene. To achieve this, we sequenced the fliC
genes from 11 H7 strains, compared them with the sequences of
fliC genes from 38 E. coli H-antigen type
strains, and identified H7-specific sequences. The specificity was
confirmed by PCR testing the type strains for all 53 E. coli H antigens. The identification of O157- and H7-specific sequences provides the basis for a sensitive test for rapid detection of E. coli O157:H7. This is important both for decision making related to patient care, as early treatment may reduce the risk of
life-threatening complications, and for detection of sources of contamination.
| |
MATERIALS AND METHODS |
Bacterial strains.
Standard E. coli H strains
(6, 21) were obtained from the Institute of Medical and
Veterinary Science, Adelaide, Australia, and Karl Bettelheim of the
Victorian Infectious Diseases Reference Laboratory, Victoria,
Australia. The other strains used are listed in Table
1, together with the names of those who
supplied them.
Sequencing and analysis.
The fliC gene was first
PCR amplified, and the PCR product was sequenced. PCR amplification was
performed in a reaction volume of 100 µl consisting of 10 mM Tris-HCl
(pH 8.3); 50 mM KCl; 1.5 mM MgCl2; 0.02% (wt/vol) bovine
serum albumin; dATP, dTTP, dCTP, and dGTP at a concentration of 20 mM
each; 60 pmol of each primer; 1 µl of DNA; and 2.5 U of
Taq DNA polymerase. The PCR was generally carried out under
the following conditions: denaturation at 94°C for 30 s,
annealing at 55°C for 30 s, and extension at 72°C for 1 min
for 30 cycles (exceptions are referred to in the Results section). The
PCR product was purified with the Promega Wizard PCR purification kit
before being sequenced. The sequencing reactions were performed by the
DyeDeoxy Terminator Cycle Sequencing method (Applied Biosystems, Foster
City, Calif.), and the reaction products were analyzed by using
fluorescent dye and an ABI377 automated sequencer (Applied Biosystems).
Sequence data were assembled and analyzed by using the Australian
National Genomic Information Service, which incorporates several sets
of programs (15, 16).
Specificity assay by PCR.
Chromosomal DNA was isolated by
using the Promega Genomic isolation kit and was checked by gel
electrophoresis and PCR amplification of the mdh gene (which
codes for malate dehydrogenase and which is generally present in
E. coli) by using the oligonucleotides designed by Boyd et
al. (3). PCR was carried out in a total volume of 25 µl,
and after PCR 5 µl was run on an agarose gel to check for amplified DNA.
Nucleotide sequence accession numbers.
The DNA sequences
have been deposited in GenBank. See Table 1 for GenBank entry numbers.
| |
RESULTS AND DISCUSSION |
Sequencing.
Primers flanking the fliC gene were
used to amplify PCR by the H7 fliC genes from 10 E. coli strains of O groups O2, O7, O18, O19, O53, O55, and O157. The
two primers used are primer 1575 (5'-GGG TGG AAA CCC AAT ACG) and
primer 1576 (5'-GCG CAT CAG GCA ATT TGG), which are based on sequences
51 to 34 bp upstream and 37 to 54 bp downstream, respectively, of the
E. coli K-12 fliC gene. Each PCR product was
first sequenced by using the primers used for the PCR amplification.
Primers based on the sequence obtained were then used for further
sequencing, and this procedure was repeated until the entire PCR
product was sequenced in both orientations.
All 10 strains have a fliC gene of 1,758 bp, and the
pairwise differences among these 10 genes ranged from 0 to 2.45%. The fliC gene from the H7 type strain (an O1:K1:H7 E. coli strain) has been sequenced previously (GenBank accession no.
L07388), and variation between this sequence and the 10 new sequences
is higher (between 0.8 and 3.07%). We resequenced the fliC
gene from the H7 type strain and found that its sequence is identical
to that of strain M973 but that its sequence differs from the published sequence (GenBank accession no. L07388) at 11 nucleotides.
Nucleotide sequence variation within the H7 fliC
genes.
DNA sequences were aligned by using the CLUSTALW program,
and phylogenetic analysis was performed with MULTICOMP software (31).
Reid et al. (32) recently sequenced the H7 fliC
genes from nine E. coli strains of three serotypes (O157:H7,
O55:H7, and O128:H7). They found four distinct sequences, which were
named H7-1 to H7-4 (32). The H7-1 sequence represents the
fliC genes from two O55:H7 strains and a nonmotile O157
strain, the H7-2 sequence represents the H7 fliC genes from
three O157:H7 strains, and the H7-3 and H7-4 sequences represent the H7
fliC genes from a nonmotile O157 and an O128:H7 strain,
respectively (32). Alignment of the 11 newly obtained H7
fliC sequences with the four representative sequences of
Reid et al. (32) revealed six more distinct sequences, H7-5
to H7-10 (Table 1).
Sixty-two polymorphic sites are available within the 10 H7
fliC sequences (Fig. 1), with
46 occurring at the third base of a codon, 5 occurring at the second
base, and 11 occurring at the first base. Sixteen nonsynonymous
substitutions, which represented 16 of the 585 codons, were found. Of
the 62 polymorphic sites, 47 were phylogenetically informative (at
least 2 bases were present in 2 or more of the 10 sequences).
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FIG. 1.
Polymorphic sites within the 10 H7 fliC
alleles. Numbering of the polymorphic sites (vertical format) is from
the first nucleotide of the gene. The position within the codon for
each polymorphic site is shown below the sequences.
|
|
H7-1, H7-2, and H7-5 represent H7 sequences of O55 and O157 strains and
are almost identical, with 1- or 2-nucleotide pairwise differences
between them (Fig. 1). These sequences are distinct from those of other
H7 alleles at three "signature" nucleotides at positions 192, 195, and 1683 (Fig. 1). Fields et al. (9) conducted a
PCR-restriction fragment length polymorphism analysis of
fliC genes from 70 O157:H7 and 18 O55:H7 strains and found that the H7 genes from all 70 O157:H7 strains and 16 of the 18 O55:H7
strains gave identical patterns. Thus, it is highly likely that the H7
genes from all O157:H7 strains are closely related to the H7 gene of
the O55:H7 clone. It has been found by multilocus enzyme
electrophoresis that the O157:H7 and O55:H7 clones form a small group
which stands apart from most other E. coli strains (8,
24, 36-40).
H7-3 (32) was found only in a nonmotile O157 strain. It
differs from H7-1 by two frameshift insertions at positions 12 and 17 (not shown in Fig. 1) and seven additional mutations (all of which are
nonsynonymous, five of which are clustered at the 5' end from positions
68 to 133, and two of which are clustered at the 3' end at positions
1727 and 1728) (Fig. 1). It has been suggested that these seven
mutations were fixed after the clone lost motility, which would remove
the selective constraints on amino acid-altering mutations
(32). However, this does not explain why the mutations are
clustered at the ends of fliC, which is indicative of recombination.
Alleles H7-8, H7-9, and H7-10 are closely related, with pairwise
differences of 0.06 and 0.46% (Fig. 1), and they differ from other H7
alleles by 2.28 to 3.12%. These three alleles represent H7 sequences
from O1, O2, O7, and O10 strains. Alleles H7-4, H7-6, and H7-7 (which
represent the H7 genes from O19, O53, and O128 strains) are more
similar to O157 and O55:H7 alleles (Fig. 1), with pairwise differences
ranging from 0.28 to 0.9%. We suggest that there are two groups of H7
genes and that these comprise alleles H7-1 to H7-6 and alleles H7-8 to
H7-10, respectively.
Oligonucleotide primers specific to H7.
We have sequenced the
fliC genes from 39 H type strains (unpublished data).
Comparing the sequences of H7 strains with those of the other 38 H type
strains, we have identified two primer sequences (primer 1806 [5'-GCT
GCA ACG GTA AGT GAT] at positions 553 to 570 of H7-10 and primer 1809 [5'-GGC AGC AAG CGG GTT GGT] at positions 1500 to 1483 of H7-10)
(Fig. 2) which are conserved in all the
10 H7 alleles but which have less than 85% similarity with any other
fliC gene of the 39 H type strains.
Primers 1806 and 1809 were tested by PCR against all 53 standard H type
strains and the strains listed in Table 1. The PCR was carried out as
described above with an annealing temperature of 58°C. A PCR product
of about 1 kb was generated from all the H7 strains, and none was
generated with these primers by using DNA from any other H type strain
(data not shown).
Until recently, all 53 forms of the H antigen in E. coli
were thought to be encoded at the fliC locus, as has been
shown for E. coli K-12. However, Ratiner (26-30)
showed that some H-antigen genes are at loci other than
fliC, and fllA, fllK, and
fllM have been identified. We have confirmed that at least
39 of the 53 H-antigen genes from type strains map to fliC
(unpublished data), and the primers were chosen after comparison of the
primer sequences with the sequences of the 39 H-antigen genes. The PCR
result included all H-antigen specificities and indicates that even
when tested with type strains whose H-antigen genes we have not
sequenced and/or whose H-antigen gene is at a locus other than
fliC, these primers are still specific for H7.
Oligonucleotide primers specific for H7 genes of O157 and O55
strains.
The fliC genes of O55:H7 and O157:H7 strains
are closely related (see above), and we have identified two
oligonucleotides (oligonucleotides 1696 [5'-GGC CTG ACT CAG GCG GCC]
at positions 178 to 195 in H7-5 and oligonucleotide 1697 [5'-GAG TTA
CCG GCC TGC TGA] at positions 1700 to 1683 in H7-5) (Fig. 2) which are unique to H7 of O157 and O55. Although not identical to any part of the
fliC sequences of any other H7 strain, the sequences of these two primers are identical or have high-level similarity to the
sequences of fliC genes of some other H types. However, a
combination of one of these primers with one of the H7-specific primers
can give specificity for the H7 genes of E. coli O157 and O55.
Primer pairs 1696-1809 and 1697-1806 were used to carry out PCR with
chromosomal DNA samples of all the H type strains and the H7 strains
listed in Table 1. PCRs were carried out as described above with
annealing temperatures of 61°C (for 1696-1809) and 59°C (for
1697-1806). Both primer pairs produced a band of the predicted size
with the O157:H7 strains (strains M1004 and M527; see Table 1) and the
O55:H7 strain (strain M1686; see Table 1) but gave no band with the
other strains tested (data not shown).
Oligonucleotide primers based on genes flanking fliC.
We
have also chosen two primers (primer 2648 [5'-CAA TGC TTC GTG ACG
CAC] and primer 2650 [5'-CAG CGA TGA AAT ACT TGC CAT]) (Fig. 2)
which were based on the E. coli K-12 fliA and
fliD genes (19, 25), respectively.
fliA and fliD flank the fliC gene in
E. coli K-12 (2, 19, 25) and encode the 
factor for transcription initiation of class 3 operons and a filament
cap for filament assembly, respectively (18). Each of these
two primers was paired with the one of the two H7-specific primers (2648-1806 and 2650-1809) to carry out PCR with chromosomal DNA samples
of all the H type strains and the H7 strains listed in Table 1. PCRs
were carried out as described above with annealing temperatures of
56°C (for 2648-1806) and 58°C (for 2650-1809). Both primer pairs
produced bands of the predicted size with all of the H7 strains but
gave no band with the other strains tested (see Fig.
3 for the result obtained with primer
pair 2648-1806).
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FIG. 3.
Agarose gel electrophoresis of PCR product obtained by
PCR assay with oligonucleotide primer 2648-1806 with DNAs from E. coli strains. DNAs from E. coli H type strains are
labeled with the H type numbers, and DNAs from other strains are
labeled with the strain numbers. GeneRuler (1-kb DNA ladder) from Gibco
Life Technologies was used as the molecular size marker.
|
|
The variation among the 10 H7 alleles is very low, but it is
nevertheless possible that our H7-specific primers may fail to give
positive results with some H7 isolates because of the low level of
variation that affects a primer site. The low level of variation makes
it extremely unlikely that the two H7-specific regions (primers 1806 and 1809) in any H7 isolate would both have undergone change, and thus
other primers (whether or not they are H7 specific, such as primers
2648 and primers 2650) can be used to pair with each of 1806 and 1809 to confirm a negative result. H7-specific sequences other than those
for the two primers that we have chosen are also located in the H7
fliC central region, and thus, other H7-specific primers can
also be used.
There have been previous reports of primers specific for H7. However,
the primers were based on many fewer sequence data and were not tested
against all E. coli H-antigen type strains. For example,
Gannon et al. (11) reported the use of H7 as a target in a
PCR assay system and used two H7-based primers (primers flicH7-F and
flicH7-R) based on the alignment of two H7 sequences. We compared the
sequences of these two primers with the 39 fliC sequences: the sequences of primer flicH7-F is identical to the sequence of a
region present in 24 fliC genes that encode other H antigens but is different from the sequence of one of the H7 alleles (H7-4) at
one base; the sequence of primer flicH7-R is different from those of
four H7 alleles (H7-5 to H7-8) at 1 base. The first primer is not H7
specific, and the second one is specific to 4 of the 10 H7 sequences,
including 2 of the 3 sequences found in O157:H7 strains, making neither
suitable for a routine assay.
Our sequence data confirm that the PCR-restriction fragment length
polymorphism test for H7 fliC genes developed by Fields et
al. (9) is specific for the H7 genes of O157 and O55
strains. However, because this test is based on the restriction pattern of the PCR-amplified fliC gene, it can be applied only to
pure bacterial cultures and is not suitable for use with environmental samples, in which there could be several E. coli strains
with the O157:H7 strain in the minority. Also, the restriction
enzyme-digested PCR fragments need to be run on an agarose gel to see
the pattern, which makes this test not amenable to the systems
currently used in clinical laboratories, such as the TaqMan assay
(14, 20).
The comprehensive analysis that we have undertaken, which involved more
fliC genes and all H type strains, has shown that sequences
previously thought to be specific for H7 do not have the desired
specificity and has identified primers with better specificity for H7
genes and the H7 genes of O157 and O55 strains. It is nevertheless
possible that with the routine use of the primers identified in this
study, other discrepancies would be found. However, we believe that the
use of two H7-specific primers, each of which can be used with one of
two nonspecific primers, will ensure that the proposed test will have a
very high degree of reliability in detecting the H7 gene of the O157:H7 clone.
There are many O157 or H7 clones, and it is for the O157:H7 clone for
which there is a major need for detection methods. We have previously
identified O-antigen genes specific for O157 and here have identified
H7-specific primers. Primers specific for these two genes provide a
good basis for the development of a PCR-based method that can be used
to identify O157:H7 strains and that can replace the time-consuming
plating and serotyping methods. The use of O157- and H7-specific tests
for screening for this organism is highly desirable, as in the
traditional serologic tests for detection of O157 and H7 strains, tests
specific for the combination of O157 and H7 are used for identification
of this clone.
For example, in routine use, separate PCRs with H7- and O157-specific
primers can be carried out. For H7, one can use primer pairs 2648-1806, 2650-1809, and 1806-1809 separately. Positive PCR results with all the
three primer pairs would indicate the presence of strains that carry
the H7 antigen. Positive PCR results with one or two primer pairs would
indicate the presence of strains that carry the H7 antigen and that
have a variation(s) at a priming site(s). Negative PCR results with all
three primer pairs would indicate the absence of strains that carry the
H7 antigen. The second result is predicted to be very rare and could be
confirmed by sequencing. For O157, one can use previously identified
O157-specific primers (34), and the test can be carried out
by the method of Wang and Reeves (34).
| |
ACKNOWLEDGMENTS |
We thank all the people and institutes listed in Table 1 and Karl
Bettelheim for kindly supplying strains.
This study was supported by the Australian Research Council.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Room 501, Biochemistry & Microbiology Bldg., Maze
Crescent City Rd. Entrance, Darlington Campus, The University of
Sydney, Camperdown, NSW 2006, Australia. Phone: (612) 9351 2536. Fax: (612) 9351 4571. E-mail: reeves{at}angis.org.au.
| |
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Journal of Clinical Microbiology, May 2000, p. 1786-1790, Vol. 38, No. 5
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
