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Journal of Clinical Microbiology, August 1998, p. 2339-2341, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of a Rough Strain of
Escherichia coli O157:H7 That Produces No Detectable
O157 Antigen
Peter
Feng,1,*
Robin C.
Sandlin,2
Choong H.
Park,3
Richard A.
Wilson,4 and
Mitsuaki
Nishibuchi5
Division of Microbiological Studies, Food and
Drug Administration, Washington, D.C.
202041;
Department of Microbiology
and Immunology, Uniformed Services University of the Health
Sciences, Bethesda, Maryland 208142;
INOVA Fairfax Hospital, Falls Church, Virginia
220423;
Escherichia coli Reference
Center, Pennsylvania State University, University Park,
Pennsylvania 168024; and
Center for
Southeast Asian Studies, Kyoto University, Kyoto, Japan
606-015
Received 17 March 1998/Returned for modification 5 May
1998/Accepted 12 May 1998
 |
ABSTRACT |
MA6, an O157:H7-like strain, did not react with most anti-O157 kits
examined; however, it had the rfbE gene that is essential for O157 expression and carried O157:H7 virulence factors.
Lipopolysaccharide analysis showed that MA6 is a rough strain that does
not produce the O157 antigen, but genetically, it belongs in the
O157:H7 clonal group.
| |
TEXT |
Enterohemorrhagic Escherichia
coli (EHEC) of O157:H7 serotype is identified
serologically by its somatic O157 and flagellar H7 antigens. In routine
clinical analysis, fecal specimens are plated onto sorbitol MacConkey
agar and non-sorbitol-fermenting colonies are tested serologically for
the O157 antigen. Only isolates that react with anti-O157 serum are
serotyped further for the H7 antigen and assayed for virulence factors.
Analysis for O157:H7 in foods is done by a similar protocol, except
that samples are first enriched in broth medium before plating and
testing for the sorbitol phenotype and the O157 antigen (5).
Recently, many commercial anti-O157 kits have been introduced, which
assay enrichment broths directly for O157:H7 (6). Therefore,
testing for the O157 antigen is critical in the isolation and
identification of O157:H7 from both clinical and food specimens.
A recent paper reported the isolation of E. coli O157:H7
from beef marketed in Malaysia (13). One of the
isolates, MA6, had the typical O157:H7 phenotype, and an initial
screening using two latex agglutination kits as well as an O:H
serotyping kit showed this strain to be O157:H7. However, when MA6 was
tested with another anti-O157 test (QUIX; Universal HealthWatch, Inc., Columbia, Md.), it was found to be negative. This discrepancy in MA6
reactivity to O157-specific antisera was unexpected and therefore was investigated further in this study. We used serological and genetic assays to characterize MA6 and determined that it was a
rough strain of O157:H7 that does not make the O157 antigen.
Serological analysis.
The discrepancy in serological
reactivity of MA6 could be due to differences in the specificity of
O157 antisera; hence, we examined MA6 with other anti-O157 assays.
These tests, listed in Table 1, were
performed according to the manufacturers' specifications. Using TT12,
an outbreak strain of O157:H7 serotype as the positive control, MA6 was
found not to react with most of the anti-O157 kits examined. It did,
however, react with anti-H7 sera in the Seiken and RIM kits; hence, it
carried the H7 antigen. To resolve this discrepancy regarding its
O-antigen type, MA6 was sent for serotyping to the Centers for Disease
Control and Prevention and to the Escherichia coli Reference
Center, Pennsylvania State University. Both institutions verified the
presence of the H7 antigen; however, O serotyping was inconclusive. The
reference center found that MA6 agglutinated with anti-O157 serum, but
it also reacted with 14 other O sera.
Genetic analysis.
Since the O antigen of strain MA6 could not
be determined, we tested MA6 for O157:H7 traits by PCR, starting with
the rfbE gene that is essential for O157 antigen expression
(1). Primers PF-38 (5'-CTGTTATGTTGTACTGCTTCA-3')
and PF-36 (5'-CAATTCCACCGCCCCACTCG-3') were modified
from the primers of Bilge et al. (1), and they amplify a
1,280-bp DNA sequence that includes the entire rfbE gene.
Both TT12 and MA6 produced identical amplicons of the expected size for
the rfbE fragment (Fig. 1,
lanes 4 and 5); hence, despite the absence of reactivity to most O157
sera, MA6 appears to carry genetic sequences essential for O157 antigen
expression.
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FIG. 1.
Agarose gel electrophoresis of DNA fragments amplified
from strains TT12 (O157:H7) and MA6 by PCR, using primers specific for
various genes (shown in parentheses). Lanes: 1, 123-bp molecular size
ladder; 2, TT12 (stx2,
stx1, and uidA); 3, MA6
(stx2, stx1, and
uidA); 4, TT12 (rfbE); 5, MA6 (rfbE);
6, TT12 (eaeA and EHEC plasmid); 7, MA6 (eaeA and
EHEC plasmid).
|
|
Radu et al. (
13) tested strain MA6 with DNA probe, PCR, and
agarose gel electrophoresis to show the presence of
eaeA,
stx2,
and the EHEC plasmid, respectively. Those
results were verified
in our study by PCR, using primers for these
virulence factors
(
2,
10), as identical amplicons were
obtained from both TT12
and MA6 for
eaeA (397 bp), EHEC
plasmid (166 bp), and
stx2 (584
bp) (Fig.
1).
Serological test for toxins (Verotox-F; Denka Seiken,
Tokyo, Japan)
confirmed that MA6 produced only Stx2. Our PCR studies
also showed that
MA6 had the 252-bp amplicon from
uidA (
2),
which
specifically detects a T-to-G base change at +92 in the
uidA
allele (
8). This base mutation is unique to the O157:H7
serotype (
4) and is highly conserved in the O157:H7 clonal
complex (
9); therefore, the presence of this
uidA
base mutation
in MA6 strongly suggests that it is of the O157:H7
serotype. This
assumption was confirmed by testing for genetic
relatedness by
multilocus enzyme electrophoresis (
17), which
showed that MA6
had the ET1 electrophoretic profile that is
characteristic of
O157:H7 (data not shown); hence, it is in the O157:H7
clonal group
(
9).
LPS analysis.
Strain MA6 is genetically like O157:H7 and
carries the rfbE gene required for O157 antigen expression,
yet it did not react with most anti-O157 sera. Hence, we examined the
lipopolysaccharide (LPS) of MA6 for the presence of the O side chain.
The LPSs of MA6, TT12, and DH5
pir (negative control)
were isolated, fractionated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, and silver stained as described previously
(14). All three isolates had low-molecular-weight LPS of the
core region, but the high-molecular-weight ladder, characteristic of
the O-polysaccharide side chain of smooth LPS (3), was
present only in TT12 (Fig. 2A). DH5
pir is a rough mutant of Escherichia coli,
which has a LPS that is truncated at the core and therefore does not
make the O side chain (11). The LPS profile of MA6 also
lacks the O side chain and closely resembled that of DH5
pir; hence, it appears that MA6 is also a rough strain.
Rough mutants of E. coli (O-rough:K1:H7) that produce Stx1
have been isolated from patients with traveler's diarrhea, but those
strains were distinct phenotypically from O157:H7 and did not carry
other EHEC virulence factors (16).
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FIG. 2.
Sodium dodecyl sulfate-polyacrylamide gel (A) and
Western blot (B) of bacterial LPS. Lanes: 1, prestained Kaleidoscope
molecular weight standards (Bio-Rad, Hercules, Calif.); 2, DH5
pir; 3, MA6; 4, TT12. Blot was probed with anti-O157
serum from the Escherichia coli Reference Center,
Pennsylvania State University.
|
|
To confirm the absence of the O157 antigen, a companion gel was Western
blotted onto a polyvinylidine fluoride membrane (Schleicher
& Schuell
Inc., Keene, N.H.) (
14) and probed with a 1:500 dilution
of
anti-O157 rabbit serum (
Escherichia coli Reference Center,
Pennsylvania State University). Antigen-antibody binding was detected
by using a 1:1,000 dilution of alkaline phosphatase-conjugated
goat
anti-rabbit immunoglobulin G (Kirkegaard & Perry Laboratories,
Inc.,
Gaithersburg, Md.) and a precipitable substrate as reported
previously
(
7). The results show that the high-molecular-weight
O side
chain of TT12 (O157:H7) reacted with the anti-O157 serum
but so did
some components in the core (Fig.
2B), which suggests
that the
anti-O157 serum had traces of antibodies to the LPS core.
Neither
DH5
pir nor MA6 showed an antibody-reactive O side
chain,
but minor components in the core also reacted weakly with the
serum. Since this anti-O157 serum was used by the reference center
for
serotyping, it is probable that the agglutination they observed
with
MA6 was due to these traces of antibodies to core LPS in
the serum. A
duplicate blot was probed with a cocktail of 4 of
the 14 antisera
(O124, O158, O57, and O87) from the reference
center that had
agglutinated with MA6. These antibodies also reacted
weakly with the
core LPS of MA6 and DH5
pir (data not shown),
which
confirms that their reactivity with MA6 is also due to antibodies
to
the core LPS.
The absence of an O side chain in strain MA6 would account for the lack
of its serological reactivity with most anti-O157
kits, but the
positive reaction seen with some of the kits cannot
be explained.
Perhaps those kits that are positive with MA6 contain
antibodies
directed to other O157:H7-specific epitopes in addition
to anti-O157
or, like the serum we used, contained traces of antibodies
to the LPS
core. Another possibility, at least with latex agglutination
assays,
may be the use of a heavy inoculum. False-positive latex
agglutination
reactions can occur if a sweep of growth is used
for analysis instead
of a single colony as recommended by the
manufacturer (
15).
Regardless, this study shows the importance
of evaluating commercial
kits before using them routinely for
screening for O157:H7 strains, as
the specificity of some sera
may not have been fully characterized.
In conclusion, we have identified a rough strain of
E. coli
O157:H7 that does not produce O side chain. MA6 has O157:H7-like
phenotypes and virulence factors as well as the
rfbE gene
for
O157 expression and is genetically O157:H7, but the absence of
the
O157 antigen would make this strain undetectable or unidentifiable
with
most serological assays used in the analysis of clinical
or food
samples for
E. coli O157:H7.
| |
ACKNOWLEDGMENTS |
We thank P. Fratamico for providing eaeA and EHEC
plasmid PCR primers, M. Davis and E. Sowers for serotyping, S. Cianci
for graphics, and T. Whittam for clonal analysis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: HFS-516, FDA,
200 C St. SW, Washington, DC 20204. Phone: (202) 205-4518. Fax: (202) 401-7740. E-mail: pxf{at}fdacf.ssw.dhhs.gov.
| |
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Journal of Clinical Microbiology, August 1998, p. 2339-2341, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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