Previous Article | Next Article 
Journal of Clinical Microbiology, November 1999, p. 3491-3496, Vol. 37, No. 11
Institut für Hygiene und Mikrobiologie
der Universität Würzburg1 and
Kinderklinik der Universität
Würzburg,2 97080 Würzburg, Germany
Received 28 April 1999/Returned for modification 17 July
1999/Accepted 23 July 1999
We have isolated one sorbitol-nonfermenting (SNF) Escherichia
coli O157:H7 isolate and five sorbitol-fermenting (SF) E. coli O157:H Shiga toxin (Stx)-producing
Escherichia coli (STEC) of various serotypes has been linked
to a spectrum of disorders, including watery diarrhea, bloody diarrhea
(hemorrhagic colitis), and hemolytic-uremic syndrome (HUS)
(37).
STEC has as its cardinal virulence trait the ability to produce one or
more Stxs (Stx1, Stx2, or Stx2 variants). Stxs are toxic to cultured
human colonic and ileal epithelial cells (25) and
endothelial cells (27). Even in the absence of cytotoxicity, Stxs can stimulate the production of vasoactive factors by endothelial cells (5). Thus, the ability to produce an Stx is quite
plausibly related to the intestinal and extraintestinal manifestations
of human STEC infections.
E. coli strains that express the O157 antigen are the most
commonly isolated STEC strains worldwide. Such organisms are easily detected by toxin-independent detection protocols, such as
sorbitol-MacConkey (SMAC) agar screening (29), or the
immunomagnetic separation (IMS) technique. Unlike approximately 80% of
other E. coli strains, most O157 STEC isolates do not
ferment D-sorbitol after overnight incubation. Therefore,
SMAC agar was developed by substituting the carbohydrate sorbitol for
lactose in MacConkey agar. SMAC agar has proved to be effective for the
isolation of O157 STEC and is the most widely used medium for this purpose.
IMS can isolate sorbitol-fermenting (SF) E. coli
O157:H We have isolated from humans nontoxigenic E. coli strains
that express the O157 antigen and present data suggesting that Stx may
not be obligatorily produced by E. coli O157 strains
associated with human disease, including HUS.
Bacterial strains.
The origins and characteristics of the
E. coli O157 strains used as controls in this study are
described in Table 1. The origins of all
other strains are described in the text.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Escherichia coli O157:H7 and O157:H
Strains That Do Not Produce Shiga Toxin: Phenotypic and Genetic
Characterization of Isolates Associated with Diarrhea and
Hemolytic-Uremic Syndrome
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
isolates that do not contain Shiga
toxin (Stx) genes (stx). Isolates originated from patients
with diarrhea (n = 4) and hemolytic-uremic syndrome
(HUS) (n = 2). All isolates harbored a chromosomal
eae gene encoding gamma-intimin as well as the plasmid
genes E-hly and etp. The E. coli
O157:H7 isolate was katP and espP positive. Respective sera obtained from the patient with HUS contained antibodies to the O157 lipopolysaccharide antigen. The
stx-negative E. coli O157:H7 isolate is
genetically related to stx-positive SNF E. coli
O157:H7. All stx-negative SF E. coli
O157:H isolates belong to the same genetic cluster and
are closely related to stx-positive SF E. coli
O157:H isolates. Our data indicate that
stx-negative E. coli O157:H7/H
variants may occur at a low frequency and cannot be recognized by
diagnostic methods that target Stx.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
as well as sorbitol-nonfermenting (SNF) E. coli O157:H7 (19). However, SF non-O157:H7 STEC can
also cause human disease (17, 38), and because of this, Stx
detection systems have been used to identify such pathogens in human
stools (10, 15, 21, 28).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
E. coli strains used as controls in
this studya
Isolation of E. coli O157 from stool specimens. A total of 2,785 stool specimens from patients with diarrhea or HUS were screened in 1996 and 1997 for the presence of Stx-producing E. coli in the Institute for Hygiene and Microbiology, University of Würzburg, Würzburg, Germany. The majority of the stool samples were obtained from hospitalized children throughout Germany. Detection of E. coli O157 was performed as described below.
A total of 10 ml of GN broth Hajna (Difco, Detroit, Mich.) was inoculated with 1 g of the stool sample, and the mixture was incubated for 6 h at 37°C. E. coli O157 was sought from 1 ml of this broth by the IMS technique as described previously (19). Fifty microliters of the bacterium-bead suspension was streaked onto SMAC agar and cefixime-tellurite SMAC (CT-SMAC) agar plates. Up to 1,500 colonies from both plates were scraped off with a sterile swab and were suspended in 1 ml of sterile 0.9% NaCl solution. The bacterial cell concentration was determined and was adjusted with the McFarland no. 3 turbidity standard. Fifteen microliters of a 1:25 dilution (106 bacterial cells) made from this suspension was subjected to PCR with primer pairs KS7 and KS8, LP43 and LP44, and SK1 and SK2 (34), which are specific for stx1, stx2, and eae, respectively. The remaining suspension was stored at 4°C until the PCR results were obtained. PCR-positive samples were processed in the following manner: the bacterial suspension which was stored at 4°C was diluted and restreaked onto three SMAC agar plates to obtain 500 CFU/plate. Colony blot hybridization was then performed with stx1-, stx2-, and eae-specific probes (33) on these plates to detect and isolate STEC colonies.Microbiological methods. Detection of the enterohemolytic phenotype was performed on blood agar plates containing washed sheep erythrocytes (34). Fermentation of sorbitol was detected on SMAC agar plates after overnight incubation (29). Resistance to tellurite was determined on CT-SMAC agar containing 2.5 mg of tellurite per liter (40).
PCR techniques. PCR was conducted with 106 bacteria. Amplification was performed in a total volume of 50 µl containing each deoxynucleoside triphosphate at 200 µM, 30 pmol of each primer, 5 µl of 10-fold-concentrated DNA polymerase synthesis buffer (Perkin-Elmer, Applied Biosystems, Weiterstadt, Germany), 3 µl of a 25 mM MgCl2 stock solution, and 2.0 U of AmpliTaq DNA polymerase (Perkin-Elmer, Applied Biosystems). For detection of stx genes in the isolated strains, primers LP30-LP31 (stxA1), KS7-KS8 (stxB1), and LP43-LP44 (stxA2) were used as described previously (34). JS1 (5'-CAT GAA GAA GAT GTT TAT GGC G-3') and JS2 (5'-CTC AGT CAT TAT TAA ACT GCA C-3') were used for the amplification of the entire stxB2 subunit gene by the protocol described previously for GK5 and GK6 (15). For detection of plasmid genes, PCRs for E-hly (34), etp (34), katP (34), and espP (9) were performed as described previously. The eae genes were amplified with primers SK1-SK2 (32) and with primers LP1-LP2 and LP1-LP3 (31). A 5-µl volume of each PCR sample was analyzed by gel electrophoresis on 1.5% agarose gels. The fliC gene was amplified with primers F-FLIC1 and R-FLIC2, and the PCR product was restricted with RsaI as described by Fields et al. (12). Restriction fragments were separated on a 2.5% (wt/vol) agarose gel.
Analysis of genomic DNA of E. coli O157 strains was performed by random amplification of polymorphic DNA (RAPD) PCR fingerprinting with a single primer, primer 1247 (5'-AAG AGC CCG T-3') (16). Internal standards (PCR products with known sizes) were run in each lane for standardization of gels for analysis. Gels were stained with ethidium bromide and were digitized for computer-aided analysis. The GelCompar software package (Applied Maths, Kortrijk, Belgium) was used for analysis. Calculation of the similarity matrix was performed with the Jacquard algorithm after defining each single band. The hierarchic clustering was achieved by the unweighted pair group method with arithmetic averages clustering algorithm (36).Standard DNA techniques. Restriction endonuclease digestion (New England Biolabs) was performed according to the supplier's instructions. Restriction fragments were separated electrophoretically in 0.6 to 0.9% agarose gels in 0.5-fold TBE (Tris-borate-EDTA) buffer (30), stained in ethidium bromide, and analyzed. For Southern blot hybridization, DNA was prepared by the method of Heuvelink et al. (16): 100 ng of DNA was separated electrophoretically and was transferred from agarose gels to Zeta-probe nylon membranes (Bio-Rad, Munich, Germany) by standard methods (30). For hybridization assays, the nonradioactive DNA Labeling and Detection Kit (Boehringer GmbH, Mannheim, Germany) was used according to the manufacturer's instructions. The specific washing step was performed twice (5 min each time) in SSC buffer (sodium chloride, sodium citrate) and 0.1% (wt/vol) sodium dodecyl sulfate. The concentration of SSC and the temperature of the washings were calculated as described by Meinkoth and Wahl (24) to effect different stringencies. Probes were labeled with digoxigenin-11-dUTP by random hexamer labeling. The probes used included fragments of stx1 (amplified from EDL933 by PCR with primers KS7 and KS8), stx2 (amplified from EDL933 by PCR with primers GK3 and GK4), and eae (amplified from EDL933 with primers SK1 and SK2 [32]).
Vero cell assay. The Vero cell toxicities of bacterial culture supernatants and fecal filtrates were determined as described previously (14, 20, 34). Ten, 100, and 1,000 50% cytotoxic doses of Stx2 per ml were added to filtrates to confirm that no inhibitors of cytotoxicity were present.
Serological and immunological techniques. Isolation of lipopolysaccharide (LPS) and electroblotting were performed as described previously (4). For detection of immunoglobulin M (IgM) antibodies against O157 LPS by immunoblotting, the techniques of Bitzan et al. (4) were used. The E. coli O and H antigens were determined with the Wellcolex E. coli O157:H7 Rapid Latex Test Kit (Murex Diagnostica/Abbott Laboratories, Wiesbaden, Germany) according to the manufacturer's instructions. E. coli serotypes were confirmed by the method described by Bockemühl et al. (6).
An enzyme immunoassay (Premier EHEC EIA; Meridian Diagnostics Inc., Cincinnati, Ohio) was used to detect the expression of Stxs from isolated organisms according to the manufacturer's instructions, with minor modifications. Briefly, 4 ml of tryptic soy broth was supplemented with 0.05 µg of mitomycin C per ml and was inoculated with the test strain, and the mixture was incubated overnight at 37°C with shaking. A total of 50 µl of the overnight culture was mixed with 200 µl of sample dilution buffer, and 100 µl of this mixture was transferred to the well of a microtiter plate coated with polyclonal anti-Stx IgG antibodies. After 1 h of incubation at room temperature, the well was washed five times with washing buffer. Detection of bound antigen was performed as described in the manual of the Premier EHEC EIA. Stx-positive strain E. coli O157:H7 EDL933 and stx-negative strain E. coli O157:H19 693/91 were used as controls.| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Identification of stx-negative E. coli O157
strains.
During routine diagnostic work in 1996 and 1997, tests
for the detection of STEC were performed with 2,875 stool specimens. From 145 of these stool specimens, 145 STEC O157 (118 sorbitol-negative and 27 sorbitol-positive strains) could be isolated in our laboratory. Eighty-five strains were from patients with HUS and 60 were from patients with diarrhea without HUS. In addition, five stool specimens from five different patients (Table 2)
did not show toxic activity for Vero cells, and PCR screening with
stx1 and stx2-specific primers (stx PCRs) after enrichment by the IMS technique was
negative. However, the PCR of the five stool specimens with
eae-specific primers (eae PCR) was positive.
Stool samples from diarrheal patients were taken 1 to 2 days after the
onset of diarrhea and from both HUS patients during the acute phase of
disease 7 days after the onset of diarrhea.
|
.
Sera from the two children with HUS were obtained during the acute
phase of HUS 8 days after the onset of diarrhea and were tested by
immunoblot analysis with O157 LPS. Both sera were positive by the IgM
immunoblot assay.
Strain characteristics.
One of the Stx-negative isolates was
of serotype O157:H7, and the other five were of serotype
O157:H. All isolates were from patients with primary
cases of infection, and no temporal or geographic clustering could be
observed. The origins of these isolates are described in Table 2. PCR
was performed to characterize the fliC gene of the E. coli O157:H7 isolates and selected controls (see Fig.
1). Restriction fragment length polymorphism (RFLP) analysis of the PCR products after digestion with
RsaI demonstrated a pattern identical to that obtained by the fliC PCR with strains of the H7 or H type
as described by Fields et al. (12). The results of
fliC PCR and RFLP analysis of all tested strains are
depicted in Fig. 1. E. coli O157:H7 strains EDL933 and 19685 and E. coli O157:H control strains 702/88,
1533/97, 3817/96, and E32511 as well as stx-negative SF
O157:H isolates 2937, 6790, 431, 659, and 2576 demonstrated identical RFLP patterns after digestion with
RsaI. This pattern is identical to that described by Fields
et al. (12) and confirmed the presence of an
H7/H antigen in the respective isolates. E. coli control strains 693/91 (O157:H19), 241/88 (O157:H43), 1083/87
(O157:H45), and 904/90 (O157:H45) showed different patterns after
digestion with RsaI, and these patterns were distinguishable
from that of E. coli O157:H7/H.
|
Virulence determinants of the stx-negative E. coli O157 strains. None of the E. coli O157 strains gave a PCR product with primers LP30-LP31 and LP43-LP44, which are complementary to the A-subunit genes of stx1 and stx2, respectively. To test the hypothesis that A-subunit genes different from stxA1 and stxA2 were present, stx PCR was repeated with primers KS7-KS8 and JS1-JS2, which are complementary to the B-subunit genes of stx1 and stx2, respectively, but these PCRs were also negative. Moreover, the chromosomal DNAs of all isolates were hybridized with probes complementary to stx1 and stx2 sequences under low-stringency conditions, but no signal could be demonstrated (Fig. 2B and C). Control hybridization with an eae-specific probe showed a signal in all cases (Fig. 2A). None of the strains reacted in the Premier EHEC EIA.
|
isolates, however,
contained only E-hly and etp and lacked
espP and katP.
By PCR with primers LP1 and LP3 it could be shown that eae
encoding gamma-intimin, which is typically found in Stx-producing E. coli O157, is present in all strains. The E. coli O157:H7 strain showed the typical enterohemolytic phenotype
on blood agar plates after 18 to 24 h of incubation. However, of
the E. coli O157:H isolates, only 30% of the
colonies were enterohemolytic when they were streaked for isolation
onto blood agar plates.
Clonal analysis. We compared the genetic relatedness of these strains by RAPD analysis. Figure 3 depicts a dendrogram constructed with GelCompar software. The dendrogram shows the genetic relatedness of the stx-negative E. coli O157 isolates and stx-positive E. coli O157:H7 and O157 isolates with other H antigens.
|
strains
than to the SF O157:H isolates. Moreover, as
expected, the SF stx-negative E. coli O157:H strains are clonal and are most closely related to
the stx-positive E. coli
O157:H strains. E. coli O157 strains of
the H and H7 types are more closely related to each other
than to E. coli strains of other H types.
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Our finding of clinical E. coli O157:H7/H
isolates that lack stx genes was surprising. Such
stx-negative E. coli O157 strains have rarely
been identified, presumably because in strains of this serotype, unlike
other toxigenic E. coli strains that produce Stx, the toxin
genes are retained after multiple replications. It is probable that a
progenitor of the organisms that we detected contained stx
genes that were subsequently lost. The emergence of pathogenic E. coli O157:H7 proposed by Feng et al. (11) does not
accommodate the existence of a nontoxigenic E. coli strain that expresses the O157 LPS antigen.
We are unable to state with certainty that Stx had no role in the diseases observed in the patients who we report on here. Specifically, we cannot prove that we did not inadvertently overlook the true toxigenic pathogens in the isolation process, and we cannot affirm that the organisms that were initially shed did not contain toxin genes that were lost during isolation or subculture, a phenomenon that has been seen among STEC strains belonging to serotypes O2:H5, O26:H11, O73:H34, and O100:H32 (18).
Several lines of evidence suggest that these explanations do not apply.
First, had we neglected to recover toxin-producing E. coli
O157:H7/H strains that were actually present or if
stx genes were lost on subculture, the probability of
identification of toxin in the stool by Vero cell assay should have
been high, especially as the stools from early in the course of the
illness were analyzed. Second, our isolation protocol contains no
obvious bias against the isolation of toxin-producing organisms. In
addition, stx-negative E. coli
O157:H7/H presumably contains many of the virulence
traits contained by nontoxigenic, enteropathogenic E. coli
O55:H7 (11), an organism closely related to E. coli O157:H7/H.
E. coli strains of this serotype have been recovered from children with nonbloody diarrhea in North America (8), and it is plausible that nontoxigenic E. coli O157:H7 is similarly pathogenic.
Our data have a variety of implications for the diagnosis of E. coli O157:H7/H infections and for our understanding
of the pathogenesis of diseases caused by this organism. First,
screening on SMAC agar is inadequate for the detection of SF
non-O157:H7 STEC strains, including
stx2-positive SF E. coli
O157:H. To identify these organisms, it is necessary to
detect the Stx phenotype by cytotoxicity assay or antigen detection or
to detect stx genes. However, the presumptively pathogenic
strains that we reported on above would be overlooked by protocols that
rely on toxin detection. Thus, we strongly urge the performance of parallel and complementary tests that address a variety of nontoxin phenotypes and loci possessed by such organisms when performing thorough enteric microbiologic evaluations.
Second, our data support the role of Stx as a cause of bloody diarrhea in humans, in contrast to its questionable role as a cause of nonbloody diarrhea. None of the patients described in this report developed bloody diarrhea, suggesting that toxin production is necessary for hemorrhagic colitis in most patients.
Third, Stx does not appear to be necessary for all manifestations of the diseases associated with E. coli O157:H7. It is interesting that Shigella dysenteriae serotype 1, which does not have the ability to produce Stx, causes nonbloody diarrhea in monkeys (13) and that Stx production is not needed for diarrhea in gnotobiotic piglets challenged with STEC (39). Also, Li et al. (23) demonstrated that E. coli O157:H7 strains deficient in the ability to produce Stxs were able to induce abnormalities of colonic structure and ion transport in New Zealand White rabbits.
Fourth, HUS might result from non-Stx factors produced by E. coli O157:H7/H. Other bacteria that do not produce
Stx, such as Streptococcus pneumoniae and Neisseria
meningitidis, have occasionally been associated with HUS (2,
22). Our data raise the possibility that other properties of
E. coli O157:H7 might also be sufficient to produce HUS in
susceptible hosts. However, we observed HUS caused by an Stx-negative
E. coli O157 strain without evidence of the presence of
other pathogenic E. coli strains in only a single patient,
and we urge clinicians to use caution before attributing HUS to
nontoxigenic E. coli. Certainly, additional reports of the
isolation of such potential pathogens from children with HUS, without
evidence of the presence of fecal Stx, would lend support to our speculation.
In summary, we have identified stx-deficient E. coli O157 strains from infected humans with diarrhea and HUS using
stx-independent recovery techniques. Moreover, these
patients had no evidence of fecal Stx. The infecting isolates had other
characteristics of E. coli O157:H7/H and were
closely related to pathogenic toxigenic E. coli O157. Our
findings suggest that Stx- and stx gene-based detection
systems should be complemented by additional methods for the
identification of stx-negative E. coli O157 in microbiologic
evaluations and that the diseases in humans caused by E. coli O157:H7/H do not uniformly require the
production of Stx by these pathogens.
| |
ACKNOWLEDGMENTS |
|---|
We thank Phillip I. Tarr for helpful discussions and critical reading of the manuscript and Barbara Plaschke for excellent technical assistance.
This work was supported by grant Ka 717/3-1 (Ökologie bakterieller Krankheitserreger: Molekulare und evolutionäre Aspekte) from the Deutsche Forschungsgemeinschaft.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Institut für Hygiene und Mikrobiologie der Universität Würzburg, Bau 17, Josef-Schneider-Str. 2, D-97080 Würzburg. Phone: 49/931/201/5162. Fax: 49/931/201-5166. E-mail: hkarch{at}hygiene.uni-wuerzburg.de.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Aleksic, S., H. Karch, and J. Bockemühl. 1992. A biotyping scheme for Shiga-like (Vero) toxin-producing Escherichia coli O157 and a list of serological cross-reactions between O157 and other gram-negative bacteria. Int. J. Med. Microbiol. Virol. Parasitol. Infect. Dis. 276:221-230. |
| 2. |
Alon, U.,
S. P. Adler, and J. C. Chan.
1984.
Hemolytic-uremic syndrome associated with Streptococcus pneumoniae. Report of a case and review of the literature.
Am. J. Dis. Child.
138:496-499 |
| 3. |
Bielaszewska, M.,
H. Schmidt,
M. A. Karmali,
R. Khakhria,
J. Janda,
K. Bláhová, and H. Karch.
1998.
Isolation and characterization of sorbitol-fermenting Shiga toxin (Verocytotoxin)-producing Escherichia coli O157:H strains in the Czech Republic.
J. Clin. Microbiol.
36:2135-2137 |
| 4. | Bitzan, M., E. Moebius, K. Ludwig, D. E. Müller Wiefel, J. Heesemann, and H. Karch. 1991. High incidence of serum antibodies to Escherichia coli O157 lipopolysaccharide in children with hemolytic-uremic syndrome. J. Pediatr. 119:380-385[Medline]. |
| 5. | Bitzan, M. M., Y. Wang, J. Lin, and P. A. Marsden. 1998. Verotoxin and ricin have novel effects on preproendothelin-1 expression but fail to modify nitric oxide synthase (ecNOS) expression and NO production in vascular endothelium. J. Clin. Invest. 101:372-382[Medline]. |
| 6. | Bockemühl, J., S. Aleksic, and H. Karch. 1992. Serological and biochemical properties of Shiga-like toxin (verocytotoxin)-producing strains of Escherichia coli, other than O-group 157, from patients in Germany. Int. J. Med. Microbiol. Virol. Parasitol. Infect. Dis. 276:189-195. |
| 7. | Bockemühl, J., H. Karch, and H. Tschäpe. 1997. Infektionen des Menschen durch enterohämorrhagische Escherichia coli (EHEC) in Deutschland, 1996. Bundesgesundhbl. 6:194-197. |
| 8. | Bokete, T. N., T. S. Whittam, R. A. Wilson, C. R. Clausen, C. M. O'Callahan, S. L. Moseley, T. R. Frotsche, and P. I. Tarr. 1997. Genetic and phenotypic analysis of Escherichia coli with enteropathogenic characteristics isolated from Seattle children. J. Infect. Dis. 175:1382-1389[Medline]. |
| 9. |
Brunder, W.,
H. Schmidt,
M. Frosch, and H. Karch.
1999.
The large plasmids of Shiga-toxin-producing Escherichia coli (STEC) are highly variable genetic elements.
Microbiology
145:1005-1014 |
| 10. | Cebula, T. A., W. L. Payne, and P. Feng. 1995. Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR. J. Clin. Microbiol. 33:248-250[Abstract]. |
| 11. | Feng, P., K. A. Lampel, H. Karch, and T. S. Whittam. 1998. Genotypic and phenotypic changes in the emergence of Escherichia coli O157:H7. J. Infect. Dis. 177:1750-1753[Medline]. |
| 12. | Fields, P. I., K. Blom, H. J. Hughes, L. O. Helsel, P. Feng, and B. Swaminathan. 1997. Molecular characterization of the gene encoding H antigen in Escherichia coli and development of a PCR-restriction fragment length polymorphism test for identification of E. coli O157:H7 and O157:NM. J. Clin. Microbiol. 35:1066-1070[Abstract]. |
| 13. |
Fontaine, A.,
J. Arondel, and P. J. Sansonetti.
1988.
Role of Shiga toxin in the pathogenesis of bacillary dysentery, studied by using a Tox mutant of Shigella dysenteriae 1.
Infect. Immun.
56:3099-3109 |
| 14. |
Gentry, M. K., and J. M. Dalrymple.
1980.
Quantitative microtiter cytotoxicity assay for Shigella toxin.
J. Clin. Microbiol.
12:361-366 |
| 15. |
Gunzer, F.,
H. Böhm,
H. Rüssmann,
M. Bitzan,
S. Aleksic, and H. Karch.
1992.
Molecular detection of sorbitol-fermenting Escherichia coli O157 in patients with hemolytic-uremic syndrome.
J. Clin. Microbiol.
30:1807-1810 |
| 16. | Heuvelink, A. E., N. C. van de Kar, J. F. Meis, L. A. Monnens, and W. J. Melchers. 1995. Characterization of verocytotoxin-producing Escherichia coli O157 isolates from patients with haemolytic uraemic syndrome in Western Europe. Epidemiol. Infect. 15:1-14. |
| 17. | Johnson, R. P., R. C. Clarke, J. B. Wilson, S. C. Read, K. Rahn, S. A. Renwick, K. A. Sandhu, D. Alves, M. A. Karmali, H. Lior, S. A. McEwen, J. S. Spika, and C. L. Gyles. 1996. Growing concerns and recent outbreaks involving non-O157:H7 serotypes of verotoxigenic Escherichia coli. J. Food. Prot. 59:1112-1122. |
| 18. |
Karch, H.,
T. Meyer,
H. Rüssmann, and J. Heesemann.
1992.
Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coli upon subcultivation.
Infect. Immun.
60:3464-3467 |
| 19. | Karch, H., C. Janetzki-Mittmann, S. Aleksic, and M. Datz. 1996. Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods, and direct culture. J. Clin. Microbiol. 34:516-519[Abstract]. |
| 20. | Karmali, M. A., M. Petric, C. Lim, P. C. Fleming, G. S. Arbus, and H. Lior. 1985. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J. Infect. Dis. 151:775-782[Medline]. |
| 21. | Kehl, K. S., P. Havens, C. E. Behnke, and D. W. Acheson. 1997. Evaluation of the premier EHEC assay for detection of Shiga toxin-producing Escherichia coli. J. Clin. Microbiol. 35:2051-2054[Abstract]. |
| 22. | Khodasevich, L. S., and K. G. Dobrodeev. 1991. Hemolytic-uremic syndrome in meningococcal infection. Pediatriia 1:86-88. |
| 23. | Li, Z., C. Bell, A. Buret, R. Robins Browne, D. Stiel, and E. O'Loughlin. 1993. The effect of enterohemorrhagic Escherichia coli O157:H7 on intestinal structure and solute transport in rabbits. Gastroenterology 104:467-474[Medline]. |
| 24. | Meinkoth, J., and G. Wahl. 1984. Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem. 138:267-284[Medline]. |
| 25. |
Moyer, M. P.,
P. S. Dixon,
S. W. Rothman, and J. E. Brown.
1987.
Cytotoxicity of Shiga toxin for primary cultures of human colonic and ileal epithelial cells.
Infect. Immun.
55:1533-1535 |
| 26. | O'Brien, A. D., T. A. Lively, M. E. Chen, S. W. Rothman, and S. B. Formal. 1983. Escherichia coli O157:H7 strains associated with haemorrhagic colitis in the United States produce a Shigella dysenteriae 1 (SHIGA) like cytotoxin. Lancet i:702. |
| 27. | Obrig, T. G. 1998. Interaction of Shiga toxins with endothelial cells, p. 303-311. In J. B. Kaper, and A. D. O'Brien (ed.), Escherichia coli O157:H7 and other Shiga toxin-producing E. coli strains. American Society for Microbiology, Washington, D.C |
| 28. | Ramotar, K., B. Waldhart, D. Church, R. Szumski, and T. J. Louie. 1995. Direct detection of verotoxin-producing Escherichia coli in stool samples by PCR. J. Clin. Microbiol. 33:519-524[Abstract]. |
| 29. |
Ratnam, S.,
S. B. March,
R. Ahmed,
G. S. Bezanson, and S. Kasatiya.
1988.
Characterization of Escherichia coli serotype O157:H7.
J. Clin. Microbiol.
26:2006-2012 |
| 30. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y |
| 31. |
Schmidt, H.,
H. Rüssmann, and H. Karch.
1993.
Virulence determinants in nontoxinogenic Escherichia coli O157 strains that cause infantile diarrhea.
Infect. Immun.
61:4894-4898 |
| 32. | Schmidt, H., B. Plaschke, S. Franke, H. Rüssmann, A. Schwarzkopf, and H. Karch. 1994. Differentiation in virulence patterns of Escherichia coli possessing eae genes. Med. Microbiol. Immunol. Berl. 183:23-31[Medline]. |
| 33. | Schmidt, H., H. Rüssmann, A. Schwarzkopf, S. Aleksic, J. Heesemann, and H. Karch. 1994. Prevalence of attaching and effacing Escherichia coli in stool samples from patients and controls. Int. J. Med. Microbiol. Virol. Parasitol. Infect. Dis. 281:201-213. |
| 34. | Schmidt, H., C. Geitz, P. I. Tarr, M. Frosch, and H. Karch. 1999. Non-O157:H7 pathogenic Shiga toxin-producing Escherichia coli: phenotypic and genetic profiling of virulence traits and evidence for clonality. J. Infect. Dis. 179:115-123[Medline]. |
| 35. |
Schmitt, C. K.,
M. L. McKee, and A. D. O'Brien.
1991.
Two copies of Shiga-like toxin II-related genes common in enterohemorrhagic Escherichia coli strains are responsible for the antigenic heterogeneity of the O157:H strain E32511.
Infect. Immun.
59:1065-1073 |
| 36. | Sneath, P. H., and R. R. Sokal. 1973. Numerical taxonomy. The principles and practice of numerical classification. W. H. Freeman & Co., San Francisco, Calif |
| 37. | Tarr, P. I. 1995. Escherichia coli O157:H7: clinical, diagnostic, and epidemiological aspects of human infection. Clin. Infect. Dis. 20:1-8[Medline]. |
| 38. | Tarr, P. I., and M. A. Neill. 1996. Perspective: the problem of non-O157:H7 Shiga toxin (Verocytotoxin)-producing Escherichia coli. J. Infect. Dis. 174:1136-1139[Medline]. |
| 39. |
Tzipori, S.,
H. Karch,
K. I. Wachsmuth,
R. M. Robins-Browne,
A. D. O'Brien,
H. Lior,
M. L. Cohen,
J. Smithers, and M. M. Levine.
1987.
Role of a 60-megadalton plasmid and Shiga-like toxins in the pathogenesis of infection caused by enterohemorrhagic Escherichia coli O157:H7 in gnotobiotic piglets.
Infect. Immun.
55:3117-3125 |
| 40. |
Zadik, P. M.,
P. A. Chapman, and C. A. Siddons.
1993.
use of tellurite for the selection of verocytotoxigenic Escherichia coli O157.
J. Med. Microbiol.
39:155-158 |
Journal of Clinical Microbiology, November 1999, p. 3491-3496, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Garcia-Aljaro, C., Muniesa, M., Jofre, J., Blanch, A. R.
(2009). Genotypic and Phenotypic Diversity among Induced, stx2-Carrying Bacteriophages from Environmental Escherichia coli Strains. Appl. Environ. Microbiol.
75: 329-336
[Abstract] [Full Text] -
Brockmeyer, J., Bielaszewska, M., Fruth, A., Bonn, M. L., Mellmann, A., Humpf, H.-U., Karch, H.
(2007). Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity. Appl. Environ. Microbiol.
73: 6351-6359
[Abstract] [Full Text] -
Cookson, A. L., Bennett, J., Thomson-Carter, F., Attwood, G. T.
(2007). Molecular Subtyping and Genetic Analysis of the Enterohemolysin Gene (ehxA) from Shiga Toxin-Producing Escherichia coli and Atypical Enteropathogenic E. coli. Appl. Environ. Microbiol.
73: 6360-6369
[Abstract] [Full Text] -
Aktan, I., La Ragione, R. M., Pritchard, G. C., Woodward, M. J.
(2007). Prevalence of attaching and effacing Escherichia coli serogroup O103 in orphan lambs on an open farm in eastern England. Vet Rec.
161: 386-387
[Full Text] -
Aktan, I., La Ragione, R. M., Woodward, M. J.
(2007). Colonization, Persistence, and Tissue Tropism of Escherichia coli O26 in Conventionally Reared Weaned Lambs. Appl. Environ. Microbiol.
73: 691-698
[Abstract] [Full Text] -
Morinaga, N., Yahiro, K., Matsuura, G., Watanabe, M., Nomura, F., Moss, J., Noda, M.
(2007). Two Distinct Cytotoxic Activities of Subtilase Cytotoxin Produced by Shiga-Toxigenic Escherichia coli. Infect. Immun.
75: 488-496
[Abstract] [Full Text] -
Maldonado, Y., Fiser, J. C., Nakatsu, C. H., Bhunia, A. K.
(2005). Cytotoxicity Potential and Genotypic Characterization of Escherichia coli Isolates from Environmental and Food Sources. Appl. Environ. Microbiol.
71: 1890-1898
[Abstract] [Full Text] -
Beutin, L., Kaulfuss, S., Herold, S., Oswald, E., Schmidt, H.
(2005). Genetic Analysis of Enteropathogenic and Enterohemorrhagic Escherichia coli Serogroup O103 Strains by Molecular Typing of Virulence and Housekeeping Genes and Pulsed-Field Gel Electrophoresis. J. Clin. Microbiol.
43: 1552-1563
[Abstract] [Full Text] -
Creuzburg, K., Kohler, B., Hempel, H., Schreier, P., Jacobs, E., Schmidt, H.
(2005). Genetic structure and chromosomal integration site of the cryptic prophage CP-1639 encoding Shiga toxin 1. Microbiology
151: 941-950
[Abstract] [Full Text] -
Paton, A. W., Srimanote, P., Talbot, U. M., Wang, H., Paton, J. C.
(2004). A New Family of Potent AB5 Cytotoxins Produced by Shiga Toxigenic Escherichia coli. JEM
0: jem.20040392--C12
[Abstract] [Full Text] -
Friedrich, A. W., Nierhoff, K. V., Bielaszewska, M., Mellmann, A., Karch, H.
(2004). Phylogeny, Clinical Associations, and Diagnostic Utility of the Pilin Subunit Gene (sfpA) of Sorbitol-Fermenting, Enterohemorrhagic Escherichia coli O157:H-. J. Clin. Microbiol.
42: 4697-4701
[Abstract] [Full Text] -
Dunn, J. R., Keen, J. E., Moreland, D., Thompson, R. A.
(2004). Prevalence of Escherichia coli O157:H7 in White-tailed Deer from Louisiana. J Wildl Dis
40: 361-365
[Abstract] [Full Text] -
Ritchie, J. M., Campbell, G. R., Shepherd, J., Beaton, Y., Jones, D., Killham, K., Artz, R. R. E.
(2003). A Stable Bioluminescent Construct of Escherichia coli O157:H7 for Hazard Assessments of Long-Term Survival in the Environment. Appl. Environ. Microbiol.
69: 3359-3367
[Abstract] [Full Text] -
Kehl, S. C.
(2002). Role of the Laboratory in the Diagnosis of Enterohemorrhagic Escherichia coli Infections. J. Clin. Microbiol.
40: 2711-2715
[Full Text] -
Feng, P., Dey, M., Abe, A., Takeda, T.
(2001). Isogenic Strain of Escherichia coli O157:H7 That Has Lost both Shiga Toxin 1 and 2 Genes. CVI
8: 711-717
[Abstract] [Full Text] -
Karch, H., Bielaszewska, M.
(2001). Sorbitol-Fermenting Shiga Toxin-Producing Escherichia coli O157:H{-} Strains: Epidemiology, Phenotypic and Molecular Characteristics, and Microbiological Diagnosis. J. Clin. Microbiol.
39: 2043-2049
[Full Text] -
Muniesa, M., Recktenwald, J., Bielaszewska, M., Karch, H., Schmidt, H.
(2000). Characterization of a Shiga Toxin 2e-Converting Bacteriophage from an Escherichia coli Strain of Human Origin. Infect. Immun.
68: 4850-4855
[Abstract] [Full Text] -
Unkmeir, A., Schmidt, H.
(2000). Structural Analysis of Phage-Borne stx Genes and Their Flanking Sequences in Shiga Toxin-Producing Escherichia coli and Shigella dysenteriae Type 1 Strains. Infect. Immun.
68: 4856-4864
[Abstract] [Full Text] -
Zhang, W.-L., Bielaszewska, M., Bockemühl, J., Schmidt, H., Scheutz, F., Karch, H.
(2000). Molecular Analysis of H Antigens Reveals that Human Diarrheagenic Escherichia coli O26 Strains That Carry the eae Gene Belong to the H11 Clonal Complex. J. Clin. Microbiol.
38: 2989-2993
[Abstract] [Full Text] -
Zhang, W.-L., Bielaszewska, M., Liesegang, A., Tschäpe, H., Schmidt, H., Bitzan, M., Karch, H.
(2000). Molecular Characteristics and Epidemiological Significance of Shiga Toxin-Producing Escherichia coli O26 Strains. J. Clin. Microbiol.
38: 2134-2140
[Abstract] [Full Text] -
Köhler, B., Karch, H., Schmidt, H.
(2000). Antibacterials that are used as growth promoters in animal husbandry can affect the release of Shiga-toxin-2-converting bacteriophages and Shiga toxin 2 from Escherichia coli strains. Microbiology
146: 1085-1090
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»