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Journal of Clinical Microbiology, September 2001, p. 3409-3413, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3409-3413.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Murine Monoclonal Antibodies against
Escherichia coli O4 Lipopolysaccharide and H5
Flagellin
Mildred
Rivera-Betancourt and
James E.
Keen*
United States Department of Agriculture,
Agricultural Research Service, Roman L. Hruska U.S. Meat Animal
Research Center (MARC), Clay Center, Nebraska 68933
Received 22 December 2000/Returned for modification 22 March
2001/Accepted 17 June 2001
 |
ABSTRACT |
Two murine monoclonal antibodies (MAb), 2C5-F10 and 8D1-H10,
reactive with Escherichia coli O4 and H5 antigens,
respectively, were generated and characterized. Enzyme immunoassays and
immunoblots demonstrated that MAb 2C5-F10 reacted specifically with
lipopolysaccharide O antigen of E. coli O4 isolates,
while MAb 8D1-H10 reacted with E. coli strains
expressing H5 flagella.
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TEXT |
Escherichia coli
expressing O4 lipopolysaccharide (LPS) and/or H5 flagellin frequently
causes extraintestinal infections in humans and domestic animals
(1, 10, 19). E. coli O4 organisms are common
etiologic agents of human, canine, and feline genitourinary tract
infections, manifested as pyelonephritis, cystitis, prostatitis, urosepsis, and asymptomatic bacteriuria (4, 8, 13, 25, 31). In particular, the E. coli O4:H5 serotype is a
potent human uropathogen, especially the disseminated virulent J96
clone (14, 15). E. coli O4 and/or H5 antigens
are also associated with enteric disease. Shiga toxin-producing
E. coli (STEC) O4:H-negative (26), O4:H5
(11), O4:H10 (29), O2:H5 (5,
27), and O75:H5 (5) strains have caused hemorrhagic
colitis and hemolytic uremic syndrome cases, while non-STEC O4 and
enteroaggregative O4 have caused pediatric diarrhea outbreaks (6,
7). STEC O4:H-negative (28), O4:H4
(3), O4:H21 (24), O4:H25 (30),
and O2:H5 (23) have been isolated from healthy cattle,
while STEC O4 and non-STEC O4 have been associated with calves
(18), pigs (9), and lambs (2)
with diarrhea. While the E. coli O4 and H5 antigens are both
markers of strain pathogenic potential, the O4 antigen moiety may
itself function as a urovirulence factor (22).
Bacteria reference laboratories usually perform E. coli O
and H serotyping using agglutination assays and rabbit hyperimmune antisera against reference O (n = 181) and H
(n = 52) antigens (20; R. A. Wilson,
personal communication). However, absorbed anti-E. coli O4
polyclonal antibodies (PAb) may cross-react with E. coli
possessing O antigens 12, 13, 16, 18, 19, and 102 (20). Furthermore, E. coli expressing H antigens 1, 4, 8, 12, 38, 44, and 56 may cross-react with anti-H5 PAb (R. A. Wilson,
personal communication). Anti-E. coli O4 and H5 monoclonal
antibodies (MAb) have not been reported but offer potential diagnostic
specificity, reagent quality, and typing assay format flexibility
advantages over conventional agglutinating PAb. We generated anti-O4
and anti-H5 MAb in order to possess accurate serotyping reagents for use in ongoing and planned epidemiologic surveys for pathogenic and
zoonotic E. coli in livestock.
MAb were produced from splenocytes of BALB/c mice immunized with
E. coli O4:K3:H5 (E. coli Reference Center
[ECRC] U4-41, the O4 and H5 reference strain) (20)
whole-bacterium lysate and boosted with semipurified H5 flagellin
(12). Immunization, hybridoma and ascites production, and
MAb screening, isotyping, and characterization protocols were as
previously described (12, 21). One anti-E. coli
O4 MAb (2C5-F10; immunoglobulin M [IgM] isotype) and one anti-H5 MAb
(8D1-H10; IgG1 isotype) were generated and characterized. MAb
diagnostic sensitivity and specificity were estimated by enzyme-linked
immunosorbent assay (ELISA) reactivity with whole-bacterium lysate
preparations from 350 antigenically diverse gram-negative bacterial
(272 E. coli and 78 non-E. coli) strains. The
E. coli subset consisted of 8 O4:H5 strains, 4 O4:non-H5 strains, 8 non-O4:H5 strains, and 252 non-O4:non-H5 (including 1 non-O4:H autoagglutinable [HA]) strains of various O:H serotypes including 12 non-O4 isolates reported to react with anti-O4 PAb (O12
[n = 2], O16, O18 [n = 5], O19
[n = 3], O102) and 33 non-H5 isolates reported to
react with anti-H5 PAb (H1 [n = 5], H4
[n = 14], H8 [n = 5], H12
[n = 4], H38 [n = 3], H44, and
H56). Overall, E. coli isolates possessed 102 different O
antigens and 51 different H antigens. Non-E. coli strains
consisted of 50 Salmonella spp. and 28 other gram-negative
bacteria from 22 genera. For ELISA, bacterial-antigen-coated plates
were sequentially incubated with MAb (diluted ascites fluid),
horseradish peroxidase (HRP)-conjugated antibody against mouse IgG plus
mouse IgM (anti-mouse IgG+IgM), and
2,2'-azino-di-[3-ethylbenzthiazoline sulfonate(6)] solution (ABTS
peroxidase substrate) (12). ELISA optical density at dual wavelengths of 405 and 490 nm (OD405/490) was
measured, and OD405/490 of >0.200 was considered
positive (12). Dot box plots (16) of MAb
ELISA OD405/490 values for bacterial antigen
subsets were generated (Prism 3.0; Graph Pad Software Inc., San Diego,
Calif.), and MAb diagnostic-sensitivity and -specificity point
estimates with exact binomial 95% confidence intervals (CI) were
calculated (Epi Info 6.0; Centers for Disease Control and Prevention,
Atlanta, Ga.). Sensitivity was defined as the number of MAb
ELISA-positive isolates per the total number of isolates tested
possessing the target (O4 or H5) antigen. Specificity was defined as
the number of MAb ELISA-nonreactive isolates per the total number of
isolates tested that did not possess the target antigen.
MAb 2C5-F10 was ELISA reactive with 12 E. coli O4 isolates
(sensitivity, 100%; 95% CI, 73.5 to 100) and nonreactive with 260 non-O4 E. coli and 78 non-E. coli isolates
(specificity, 100%; 95% CI, 98.9 to 100) (Fig.
1). Significantly, there was no MAb ELISA
reactivity with 12 bacteria that cross-react with anti-O4 PAb.
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FIG. 1.
Dot box plot of ELISA reactivities of 272 whole-bacterium lysates with anti-E. coli O4 MAb
2C5-F10. ELISA plates coated with whole-bacterium antigens were
incubated sequentially with MAb 2C5-F10 (ascites fluid; 1:32,000),
anti-mouse IgG+IgM-HRP conjugate, and ABTS peroxidase substrate. A MAb
ELISA OD405/490 of >0.200 was considered positive. Dots
represent means of duplicate OD values. The bottom and top edges of the
superimposed box plots are the 25th and 75th distribution percentiles,
respectively; the central horizontal line represents the median (50th
percentile), and the central vertical lines extend from the box as far
as the data extend (range). CRX, non-E. coli O4 bacteria
known to cross-react with anti-E. coli O4 PAb. Data for
MAb ELISA reactivity with 78 non-E. coli isolates are
not shown.
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MAb 8D1-H10 reacted with 13 of 16 E. coli H5 isolates
(sensitivity, 81.3%; 95% CI, 54.4 to 96.0) (Fig.
2); the three nonreactive strains were
E. coli O75:H5 isolates. In spite of repeated attempts to
induce and select for motility using flagellum broth and semisolid motility agar (12), these three strains remained nonmotile
in our laboratory both macroscopically in media and microscopically under phase-contrast microscopy. This suggests that these E. coli O75:H5 strains may have been nonreactive with the anti-H5 MAb due to lack of expression of flagellar antigens (12). This
MAb did not react with 78 non-E. coli strains but reacted
with 1 of 255 non-H5 E. coli (specificity, 99.6%; 95% CI,
97.9 to 99.9) strains (Fig. 2). The cross-reactive strain was E. coli O2:HA (ECRC 94-0609), which possesses an unknown H-antigen.
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FIG. 2.
Dot box plot of ELISA reactivities of 272 whole-cell
bacterium lysates with anti-E. coli H5 MAb 8D1-H10.
ELISA plates coated with whole-bacterium antigens were incubated
sequentially with MAb 8D1-H10 (ascites fluid; 1:50,000), IgG+IgM-HRP,
and ABTS solution. OD values represent means of duplicate wells. For
dot box plot interpretation, see the legend for Fig. 1. CRX,
non-E. coli H5 bacteria known to cross-react with
anti-E. coli H5 PAb; *, datum point for E.
coli O2:HA (autoagglutinable) 92-0609, which was presumptively
H5 based on Western immunoblotting (Fig. 4). Data for MAb ELISA
reactivity with 78 non-E. coli isolates are not shown.
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
followed by Western immunoblotting using whole-bacterium lysates,
semipurified flagellin, and LPS preparations from E. coli O4
and H5 and non-O4 and non-H5 antigens confirmed MAb O4 and H5
specificity (12, 21) (Fig. 3
and 4). Reactive bands from gels
transferred onto polyvinylidene difluoride membranes and incubated with
MAb were revealed with HRP-conjugated rabbit anti-mouse IgG+IgM and
diaminobenzidine substrate. The ladder pattern characteristic of LPS
was observed on silver-stained gels and membranes incubated with
anti-O4 MAb 2C5-F10 (Fig. 3), which reacted only with E. coli O4:H5 (lanes 1 to 3) and O4:H32 (lane 4), but not with four
other whole-bacterium preparations (Fig. 3B). Similar results were
obtained using semipurified LPS as antigen (data not shown).
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FIG. 3.
SDS-PAGE and Western blots of MAb 2C5-F10
(anti-E. coli O4) on whole-bacterium antigens (25 µg
per well). Lane 1, E. coli O4:H5 U4-41; lane 2, E. coli O4:H5 J96; lane 3, E. coli O4:H5
CA002; lane 4, O4:H32 90-1870; lane 5, E. coli
O-negative:NM CDC 3377-85; lane 6, O-negative:H-negative 90-1870; lane
7, E. coli O18:H5 94-0061; lane 8, E.
coli O12:H6 H9; and lane 9, E. coli O13:H11 SU
4321-41. After electrophoresis, one gel was silver stained (A). The
other gel was transferred onto a polyvinylidene difluoride membrane and
incubated with MAb 2C5-F10 (cell culture medium; 1:2) (B). MAb-reactive
bands were revealed with rabbit anti-mouse IgG+IgM-HRP (1:1,000) and
diaminobenzidine substrate. Molecular masses (in kilodaltons) are
indicated on the left.
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FIG. 4.
SDS-PAGE and Western blots of MAb 8D1-H10
(anti-E. coli H5) on partially purified flagellins
(lanes 1 to 3; 2 µg per well) and whole-bacterium antigens (lanes 4 to 9; 24 µg per well). Lane 1, E. coli O157:H7 43895;
lane 2, E. coli O9:H12 Bi316-42; lane 3, E.
coli O4:H5 U4-41; lane 4, E. coli O4:H5 J96;
lane 5, E. coli O75:H5 CL35; lane 6, E.
coli O166:H-negative 94-0287; lane 7, E. coli
O139:H56 SN3N/1; lane 8, E. coli O4:H5 91-1750; and lane
9, E. coli O2:HA 94-0609. After electrophoresis, one gel
was Coomassie brilliant blue stained (A). The other gel was transferred
onto a polyvinylidene difluoride membrane and incubated with MAb
8D1-H10 (cell culture medium; 1:1,000) (B). MAb-reactive bands were
revealed with rabbit anti-mouse IgG+IgM-HRP (1:1,000) and
diaminobenzidine substrate. Molecular masses (in kilodaltons) are
indicated on the left.
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Specificity of MAb 8D1-H10 for H5 antigen was confirmed by SDS-PAGE of
semipurified flagellins and whole-bacterium preparations followed by
immunoblotting (Fig. 4). MAb 8D1-H10 reacted predominantly with
purified flagellins (lane 3) and whole-bacterium preparations (lanes 4, 5, and 8) of E. coli H5 at the expected molecular mass of 46 kDa (17). MAb 8D1-H10 immunoblotting of ELISA-reactive E. coli O2:HA 94-0609 produced a doublet of reactive bands
at Mr of 
46 and 37 kDa. The
46-kDa reactive band of this autoagglutinable strain was of a size
consistent with H5 flagellin; the 37-kDa band could be an H5
degradation product or a non-H5 flagellin.
To validate MAb performance, a second panel of 37 E. coli
O4, H5, non-O4, and non-H5 human clinical isolates (kindly provided by
J. R. Johnson, University of Minnesota, St. Paul) was tested blindly for ELISA reactivity separately with each MAb (Table
1). The anti-O4 MAb reacted with 11 of 12 E. coli O4 strains and with 0 of 25 non-O4 strains. The
nonreactive E. coli O4:H-multiple strain Z was serotyped as
O82:H-multiple when submitted to the ECRC after MAb ELISA testing. The
anti-H5 MAb reacted only with the four E. coli H5 strains.
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TABLE 1.
ELISA reactivities of MAb 2C5-F12 (anti-O4) and 8D1-H10
(anti-H5) to 37 E. coli whole-bacterium antigens
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In conclusion, we produced MAb against the E. coli O4 and H5
antigens that appeared to be sensitive and specific as assessed by
ELISA and immunoblotting. These MAb have potential clinical use as
immunodiagnostic or serotyping reagents, especially if used in
combination with other phenotypic or genotypic pathogenicity markers.
They may be of particular utility for experimental or epidemiologic
studies of extraintestinal E. coli O4:H5 infections.
| |
ACKNOWLEDGMENTS |
We thank R. A. Wilson, ECRC, Pennsylvania State University,
University Park, and J. R. Johnson, University of Minnesota, St. Paul, for providing bacterial strains. We also acknowledge S. Fryda-Bradley, R. Mlejnek, and S. Yang for technical assistance and J. Rosch for manuscript preparation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: USDA, ARS, U.S.
Meat Animal Research Center, P.O. Box 166, State Spur 18D, Clay Center, NE 68933. Phone: (402) 762-4343. Fax: (402) 762-4375. E-mail: keen{at}emailmarc.usda.gov.
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REFERENCES |
| 1.
|
Blanco, J. E.,
J. Blanco,
M. Blanco,
M. P. Alonso, and W. H. Jansen.
1994.
Serotypes of CNF1-producing Escherichia coli strains that cause extraintestinal infections in humans.
Eur. J. Epidemiol.
10:707-711[CrossRef][Medline].
|
| 2.
|
Blanco, J.,
D. Cid,
J. E. Blanco,
M. Blanco,
J. A. Ruiz Santa Quiteira, and R. de la Fuente.
1996.
Serogroups, toxins and antibiotic resistance of Escherichia coli strains isolated from diarrhoeic lambs in Spain.
Vet. Microbiol.
49:209-217[CrossRef][Medline].
|
| 3.
|
Blanco, M.,
J. Blanco,
J. E. Blanco,
E. A. Gonzalez,
T. A. Gomes,
L. F. Zerbini,
T. Yano, and A. F. de Castro.
1994.
Genes coding for Shiga-like toxins in bovine verotoxin-producing Escherichia coli (VTEC) strains belonging to different O:K:H serotypes.
Vet. Microbiol.
42:105-110[CrossRef][Medline].
|
| 4.
|
Blanco, M.,
J. E. Blanco,
M. P. Alonso, and J. Blanco.
1996.
Virulence factors and O groups of Escherichia coli isolates from patients with acute pyelonephritis, cystitis and asymptomatic bacteriuria.
Eur. J. Epidemiol.
12:191-198[CrossRef][Medline].
|
| 5.
|
Bockemuhl, 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.
Zentbl. Bakteriol.
276:189-195.
|
| 6.
|
Cobeljic, M.,
B. Miljkovic-Selimovic,
D. Paunovic-Todosijevic,
Z. Velickovic,
Z. Lepsanovic,
N. Zec,
D. Savic,
R. Ilic,
S. Konstantinovic,
B. Jovanovic, and V. Kostic.
1996.
Enteroaggregative Escherichia coli associated with an outbreak of diarrhoea in a neonatal nursery ward.
Epidemiol. Infect.
117:11-16[Medline].
|
| 7.
|
Colonna, B.,
L. Ranucci,
P. A. Fradiani,
M. Casalino,
A. Calconi, and M. Nicoletti.
1992.
Organization of aerobactin, hemolysin, and antibacterial resistance genes in lactose-negative Escherichia coli strains of serotype O4 isolated from children with diarrhea.
Infect. Immun.
60:5224-5233[Abstract/Free Full Text].
|
| 8.
|
Donneberg, M. S., and R. A. Welch.
1996.
Virulence determinants of uropathogenic Escherichia coli, p. 135-174.
In
H. L. T. Mobley, and J. W. Warren (ed.), Urinary tract infections: molecular pathogenesis and clinical management. American Society for Microbiology, Washington D.C.
|
| 9.
|
Garabal, J. I.,
E. A. Gonzalez,
F. Vazquez,
J. Blanco,
M. Blanco, and J. E. Blanco.
1996.
Serogroups of Escherichia coli isolated from piglets in Spain.
Vet. Microbiol.
48:113-123[CrossRef][Medline].
|
| 10.
|
Gransden, W. R.,
S. J. Eykyn,
L. Phillips, and B. Rowe.
1991.
Bacteremia due to Escherichia coli: a study of 861 episodes.
Rev. Infect. Dis.
12:1008-1018.
|
| 11.
|
Gunzburg, S.,
M. Gracey,
D. Forbes,
L. Hewitt, and K. Bettelheim.
1988.
Haemolytic-uraemic syndrome and verocytotoxigenic Esch. coli.
Med. J. Aust.
149:54-55[Medline].
|
| 12.
|
He, Y.,
J. E. Keen,
R. B. Westerman,
E. T. Littledike, and J. Kwang.
1996.
Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.
Appl. Environ. Microbiol.
62:3325-3332[Abstract].
|
| 13.
|
Johnson, J. R.
1991.
Virulence factors in Escherichia coli urinary tract infection.
Clin. Microbiol. Rev.
4:80-128[Abstract/Free Full Text].
|
| 14.
|
Johnson, J. R.,
T. A. Russo,
F. Scheutz,
J. J. Brown,
L. Zhang,
K. Palin,
C. Rode,
C. Bloch,
C. F. Marrs, and B. Foxman.
1997.
Discovery of disseminated J96-like strains of uropathogenic Escherichia coli O4:H5 containing genes for both PapGJ96 (class I) and PrsGJ96 (class III) Gal( 1-4) Gal-binding adhesin.
J. Infect. Dis.
175:983-988[Medline].
|
| 15.
|
Johnson, J. R.,
A. E. Stapleton,
T. A. Russo,
F. Scheutz,
J. J. Brown, and J. N. Maslow.
1997.
Characteristics and prevalence within serogroup O4 of a J96-like clonal group of uropathogenic Escherichia coli O4:H5 containing the class I and class III alleles of papG.
Infect. Immun.
65:2153-2159[Abstract].
|
| 16.
|
Krieg, A. F.,
J. R. Beck, and M. B. Bongiovanni.
1988.
The dot plot: a starting point for evaluating test performance.
JAMA
260:3309-3312[Abstract/Free Full Text].
|
| 17.
|
Lawn, A. M.
1977.
Comparison of the flagellins from different morphotypes of Escherichia coli.
J. Gen. Microbiol.
101:121-131.
|
| 18.
|
Orden, J. A.,
J. A. Ruiz-Santa-Quiteria,
D. Cid,
S. Garcia,
R. Sanz, and R. de la Fuente.
1998.
Verotoxin-producing Escherichia coli (VTEC) and eae-positive non-VTEC in 1-30-days-old diarrhoeic dairy calves.
Vet. Microbiol.
63:239-248[CrossRef][Medline].
|
| 19.
|
Ørskov, F., and I. Ørskov.
1992.
Escherichia coli serotyping and disease in man and animals.
Can. J. Microbiol.
38:699-704[Medline].
|
| 20.
|
Ørskov, F., and I. Ørskov.
1984.
Serotyping of Escherichia coli.
Methods Microbiol.
14:43-112.
|
| 21.
|
Rivera-Betancourt, M., and J. E. Keen.
2000.
Murine monoclonal antibodies specific for Escherichia coli O26 and O111.
Appl. Environ. Microbiol.
66:4124-4127[Abstract/Free Full Text].
|
| 22.
|
Russo, T. A.,
J. J. Brown,
S. T. Jodush, and J. R. Johnson.
1996.
The O4 specific antigen moiety of lipopolysaccharide but not the K54 group 2 capsule is important for urovirulence of an extraintestinal isolate of Escherichia coli.
Infect. Immun.
64:2343-2348[Abstract].
|
| 23.
|
Sandhu, K. S.,
R. C. Clarke,
K. McFadden,
A. Brouwer,
M. Louie,
J. Wilson,
H. Lior, and C. L. Gyles.
1996.
Prevalence of the eaeA gene in verotoxigenic Escherichia coli strains from dairy cattle in Southwest Ontario.
Epidemiol. Infect.
116:1-7[Medline].
|
| 24.
|
Suthienkul, O.,
J. Edward Brown,
J. Seriwatana,
S. Tienthongdee,
S. Sastravaha, and P. Echeverria.
1990.
Shiga-like-toxin-producing Escherichia coli in retail meats and cattle in Thailand.
Appl. Environ. Microbiol.
56:1135-1139[Abstract/Free Full Text].
|
| 25.
|
Terai, A.,
S. Yamamoto,
K. Mitsumori,
Y. Okada,
H. Kurazono,
Y. Takeda, and O. Yoshida.
1997.
Escherichia coli virulence factors and serotypes in acute bacterial prostatitis.
Int. J. Urol.
4:289-294[CrossRef][Medline].
|
| 26.
|
Tzipori, S.,
K. I. Wachsmuth,
J. Smithers, and C. Jackson.
1988.
Studies in gnotobiotic piglets on non-O157:H7 Escherichia coli serotypes isolated from patients with hemorrhagic colitis.
Gastroenterology
94:590-597[Medline].
|
| 27.
|
von Wulffen, H.,
H. Russmann,
H. Karch,
T. Meyer,
M. Bitzan,
T. C. Kohrt, and S. Aleksic.
1989.
Verocytotoxin-producing Escherichia coli O2:H5 isolated from patients with ulcerative colitis.
Lancet
i:1449-1450.
|
| 28.
|
Wieler, L. H.,
E. Vieler,
C. Erpenstein,
T. Schlapp,
H. Steinrück,
R. Bauerfeind,
A. Byomi, and G. Baljer.
1996.
Shiga toxin-producing Escherichia coli strains from bovines: association of adhesion with carriage of eae and other genes.
J. Clin. Microbiol.
34:2980-2984[Abstract].
|
| 29.
|
Willshaw, G. A.,
S. M. Scotland,
H. R. Smith, and B. Rowe.
1992.
Properties of vero cytotoxin-producing Escherichia coli of human origin of O serogroups other than O157.
J. Infect. Dis.
166:797-802[Medline].
|
| 30.
|
Wilson, J. B.,
R. C. Clarke,
S. A. Renwick,
K. Bahn,
R. P. Johnson,
M. A. Karmali,
H. Lior,
D. Alves,
C. L. Gyles,
K. S. Sandhu,
S. A. McEwen, and J. S. Spika.
1996.
Vero cytotoxigenic Escherichia coli infection in dairy farm families.
J. Infect. Dis.
174:1021-1027[Medline].
|
| 31.
|
Yuri, K.,
K. Nakata,
H. Katae,
T. Tsukamoto, and A. Hasegawa.
1999.
Serotypes and virulence factors of Escherichia coli strains isolated from dogs and cats.
J. Vet. Med.
61:37-40[CrossRef].
|
Journal of Clinical Microbiology, September 2001, p. 3409-3413, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3409-3413.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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