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Industry: Email Alert RSS FeedEscherichia coli and community-acquired gastroenteritis, Melbourne, Australia
Emerging Infectious Diseases, Oct, 2004 by Roy M. Robins-Browne, Anne-Marie Bordun, Marija Tauschek, Vicki R. Bennett-Wood, Jacinta Russell, Frances Oppedisano, Nicole A. Lister, Karl A. Bettelheim, Christopher K. Fairley, Martha I. Sinclair, Margaret E. Hellard
Table 1. Characteristics of the polymerase chain reaction (PCR)
primers used to detect pathogenic Escherichia coli in mixed culture
Target gene
or virulence
factor Primer (a) Primer sequence (5' to 3')
[1.sup.1] GACCCGGCACAAGCATAAGC
eae [2.sup.1] CCACCTGCAGCAACAAGAGG
[1.sup.1] GCATCATCAAGCGTACGTTCC
ehxA [2.sup.1] AATGAGCCAAGCTGGTTAAGCT
[1.sup.2] ATAAATCGCCATTCGTTGACTAC
stx1 [2.sup.2] AGAACGCCCACTGAGATCATC
[1.sup.2] GGCACTGTCTGAAACTGCTCC
stx2 [2.sup.2] TCGCCAGTTATCTGACATTCTG
[1.sup.3] GGCGACAGATTATACCGTGC
ltA [2.sup.3] CCGAATTCTGTTATATATGTC
[1.sup.3] TCTGTATTATCTTTCCCCTC
st1A [2.sup.3] ATAACATCCAGCACAGGC
1 CAGCAGATTGCAGCGCATAT
ipaC 2 CAAGAGCAGATGCATAACGC
1 ATTGAATCTGCAATGGTGC
bfpA 2 ATAGCAGTCGATTTAGCAGCC
1 CTGGCGAAAGACTGTATCAT
pCVD432 2 CAATGTATAGAAATCCGCTGTT
1 ATGCATTACTTTGGGTTTAG
aggA 2 TCAACCTTGACACTTGCC
1 TGATTGAAGCAGAAGCCTGC
lacZ 2 CGCCAATCCACATCTGTGAA
Target gene
or virulence
factor Primer (a) PCR program (30 cycles) (b)
[1.sup.1] 95[degrees]C, 30 s;
eae [2.sup.1] 54[degrees]C, 90 s;
72[degrees]C, 90 s
[1.sup.1]
ehxA [2.sup.1]
[1.sup.2] 95[degrees]C, 30 s;
stx1 [2.sup.2] 52[degrees]C, 60 s;
72[degrees]C, 60 s (c)
[1.sup.2]
stx2 [2.sup.2]
[1.sup.3] 94[degrees]C, 60 s;
ltA [2.sup.3] 50[degrees]C, 60 s;
72[degrees]C, 120 s (c)
[1.sup.3]
st1A [2.sup.3]
1 94[degrees]C, 30 s;
ipaC 2 59[degrees]C, 90 s;
72[degrees]C, 90 s
1 95[degrees]C, 40 s;
bfpA 2 55[degrees]C, 40 s;
72[degrees]C, 40 s
1 94[degrees]C, 40 s;
pCVD432 2 53[degrees]C, 60 s;
72[degrees]C, 60 s
1 94[degrees]C, 60 s;
aggA 2 50[degrees]C, 60 s;
72[degrees]C, 120 s (c)
1 94[degrees]C, 30 s;
lacZ 2 59[degrees]C, 90 s;
72[degrees]C, 90 s
Target gene
or virulence Product
factor Primer (a) size (bp) Reference
[1.sup.1] 384 (8)
eae [2.sup.1]
[1.sup.1] 534 (8)
ehxA [2.sup.1]
[1.sup.2] 180 (8)
stx1 [2.sup.2]
[1.sup.2] 255 (8)
stx2 [2.sup.2]
[1.sup.3] 696 (9)
ltA [2.sup.3]
[1.sup.3] 186 (9)
st1A [2.sup.3]
1 811 This study
ipaC 2
1 461 This study
bfpA 2
1 630 (10)
pCVD432 2
1 414 This study
aggA 2
1 1,350 This study
lacZ 2
(a) Primers with the same superscript were used in duplex PCRs.
(b) Before the first cycle, the sample was denatured for 10 min
at 95[degrees]C; after the last cycle, the sample was extended
for 8 min at 72[degrees]C.
(c) 35 cycles.
Table 2. Classification of pathogenic Escherichia coli according
to amplicon(s) generated by polymerase chain reaction for
virulence-associated determinants
Gene or virulence-associated determinant (a)
Interpretation (b) pCVD432 aggA bfpA eae exhA
EAEC (c) - - -
Typical EPEC - - -
Atypical EPEC - - - -
EIEC - - - - -
ETEC (c) - - - - -
EHEC (d) - - -
STEC, not EHEC (e) - - - - -
Gene or virulence-associated determinant (a)
Interpretation (b) ipaC stlA ltA stx1 stx2
EAEC (c) - - - - -
Typical EPEC - - - - -
Atypical EPEC - - - - -
EIEC - - - -
ETEC (c) - - -
EHEC (d) - - -
STEC, not EHEC (e) - - -
Control
Interpretation (b) strain Reference
EAEC (c) O42 (11)
Typical EPEC E2348/69 (12)
Atypical EPEC E128012 (12)
EIEC 223/83 (13)
ETEC (c) H10407 (14)
EHEC (d) EDL933 (15)
STEC, not EHEC (e)
(a) Factors specified by virulence genes: aggA, aggregative
fimbria, AAF/I; bfpA, bundle-forming pilus; eae, intimin;
ipaC, invasion plasmid antigen; stlA, heat-stable enterotoxin;
ltA, heat-labile enterotoxin; stx, Shiga toxin.
(b) EAEC, enteroaggregative E. coli; EPEC, enteropathogenic E.
coli, EIEC, enteroinvasive E. coli; ETEC, enterotoxigenic E. coli;
STEC, Shiga toxin-producing E. coli; EHEC, enterohemorrhagic E. coli.
(c) Either stlA or ltA positive.
(d) Either eae or ehxA and stx1 or stx2 positive.
(e) Either stx1 or stx2 positive.
Table 3. Characteristics of the polymerase chain reaction
(PCR) primers used in this study for strain characterization
PCR program
Primer Primer sequence (5' to 3') Target (30 cycles) (a)
P1 (b) CTGAACGGCGATTACGCGAA
P2 CCAGACGATACGATCCAG eae (c) 94[degrees]C, 30 s,
53[degrees]C, 30 s;
72[degrees]C, 60 s
P3 CTGGAGTTGTCGATGTT eae- 94[degrees]C, 30 s;
[alpha] 53[degrees]C, 30 s;
72[degrees]C, 120 s
P4 GTAATTGTGGCACTCC eae- 94[degrees]C, 30 s;
[beta] 53[degrees]C, 30 s;
72[degrees]C, 120 s
P5 GCCTCTGACATTGTTAC eae- 94[degrees]C, 30 s;
[gamma] 53[degrees]C, 30 s;
72[degrees]C, 120 s
ecsD-lower TATTTTCAAAAAGAATGATGTC eae 94[degrees]C, 30 s;
53[degrees]C, 30 s;
72[degrees]C, 150 s
SKl (e) CCCGAATTCGGCACAAGCATAAGC
LP5 AGCTCACTCGTAGATGACGGCAAGCG eae- 94[degrees]C, 30 s,
[epsilon] 53[degrees]C, 60 s;
72[degrees]C, 120 s
LP6B TAGTTGTACTCCCCTTATCC eae- 94[degrees]C, 30 s;
[zeta] 53[degrees]C, 60 s;
72[degrees]C, 150 s
LP7 TTTATCCTGCTCCGTTTGCT eae- 94[degrees]C, 30 s;
[iota] 53[degrees]C, 60 s;
72[degrees]C, 150 s
LP8 TAGATGACGGTAAGCGAC eae-[eta] 94[degrees]C, 30 s;
53[degrees]C, 60 s;
72[degrees]C, 150 s
LP10 GGCATTGTTATCTGTTGTCT eae- 94[degrees]C, 30 s;
[kappa] 53[degrees]C, 60 s;
72[degrees]C, 150 s
LP11B GTTGATAACTCCTGATATTTTA eae- 94[degrees]C, 30 s;
[theta] 53[degrees]C, 60 s;
72[degrees]C, 150 s
LPFDF GAACTGTAGATGGGTAC lpfD 94[degrees]C, 30 s;
LPFDR AGCAGGCATAACGCAAG 53[degrees]C, 50 s;
72[degrees]C, 60 s
Donne-280 CGGAACAGTAGGTTCACCTTC efa1 94[degrees]C, 30 s,
Donne-281 AGTGCCCGTGTTCTTGAACTG 50[degrees]C, 30 s;
72[degrees]C, 120 s
EASTOS1 GCCATCAACACAGTATATCCG astA 94[degrees]C, 30 s,
EASTOS2 CGCGAGTGACGGCTTTGTAG 50[degrees]C, 60 s;
72[degrees]C, 90 s
AggRksl GTATACACAAAAGAAGGAAGC aggR 94[degrees]C, 30 s,
AggRkas2 ACAGAATCGTCAGCATCAGC 50[degrees]C, 60 s;
72[degrees]C, 45 s
(f)
Product
Primer Primer sequence (5' to 3') size (bp) Reference
P1 (b) CTGAACGGCGATTACGCGAA
P2 CCAGACGATACGATCCAG 917 (17)
P3 CTGGAGTTGTCGATGTT 1,648 (17)
P4 GTAATTGTGGCACTCC 1,926 (17)
P5 GCCTCTGACATTGTTAC 1,770 (17)
ecsD-lower TATTTTCAAAAAGAATGATGTC [approximately (18)
equal to] 2,990
(d)
SKl (e) CCCGAATTCGGCACAAGCATAAGC
LP5 AGCTCACTCGTAGATGACGGCAAGCG 2,608 (18)
LP6B TAGTTGTACTCCCCTTATCC 2,430 (18)
LP7 TTTATCCTGCTCCGTTTGCT 2,685 (18)
LP8 TAGATGACGGTAAGCGAC 2,590 (18)
LP10 GGCATTGTTATCTGTTGTCT 2,769 (18)
LP11B GTTGATAACTCCTGATATTTTA 2,686 (18)
LPFDF GAACTGTAGATGGGTAC 798 (19)
LPFDR AGCAGGCATAACGCAAG
Donne-280 CGGAACAGTAGGTTCACCTTC 2,226 (20)
Donne-281 AGTGCCCGTGTTCTTGAACTG
EASTOS1 GCCATCAACACAGTATATCCG 109 (10)
EASTOS2 CGCGAGTGACGGCTTTGTAG
AggRksl GTATACACAAAAGAAGGAAGC 254 (21)
AggRkas2 ACAGAATCGTCAGCATCAGC
(a) Before the first cycle, the sample was denatured for 10 min at
95[degrees]C; after the last cycle, the sample was extended for 7
min at 72[degrees]C.
(b) Primer P1 was used as forward primer in all PCR reactions in
combination with P2, P3, P4, and escD-lower.
(c) Conserved region of eae.
(d) Amplicon sequenced.
(e) Primer SK1 was used as forward primer in all PCR reactions
in combination with LP5, LP68, LP7, LP8, LP10, and LP11 B.
(f) 35 cycles.
Table 4. Frequency of Escherichia coli pathotypes in
study participants with and without gastroenteritis
Source
Symptomatic Symptom-free
E. coli pathotype (a) (n = 696) (%) (n = 489) (%) p (b)
EAEC 45 (6.5) 15 (3.1) 0.02
Typical EPEC 2 (0.3) 1 (0.2) NS
Atypical EPEC 89 (12.8) 11 (2.3) <0.0001
EIEC 0 0 NS
ETEC 2 (0.3) 0 NS
EHEC 4 (0.6) 0 NS
STEC not EHEC 0 1 (0.2) NS
(a) EAEC, enteroaggregative E. coli; EPEC, enteropathogenic E. coli;
EIEC, enteroinvasive E. coli; ETEC, enterotoxigenic E. coli; STEC,
Shiga toxin-producing E. coli, EHEC, enterohemorrhagic E. coli;
NS, not significant (p > 0.1).
(b) Fisher exact test, 2-tailed.
Table 5. Characteristics of 22 isolates of atypical
enteropathogenic Escherichia coli identified during
this study
Intimin
Strain Sources Serotype type
W7 N 0161:H40 [beta]
W85-2 N OR:H- [gamma]
W114 N 0107:H8 [iota]
W143 N Ont:H5 [epsilon]
W145 N 055:H6 [gamma]
W154 N 0139:H14 [beta]
W185 N Ont:H21 [theta]
W208 N Ont:H4 [beta]
W761 N 0124:H40 [lambda]
W902 N 0125:H6 [alpha]2
W914 N 0125:H6 [alpha]
W1040 G 015:Hnt [beta]
W1056 G 055:H7 [gamma]
W1068 G 051:H49 [alpha]
W1082 G Ont:H6 [beta]
W1092 G OR:H- [eta]/[epsilon]
W1108 G 0172:H4 [theta]
W1118 G 0126:H6 [alpha]
W1120 G Ont:H34 [alpha]
W1134 G Ont:H6 [theta]
W1585 G Ont:H40 [theta]
W1706 G Ont:H6 [alpha]
E128012 C 0114:H2 [beta]
Result of PCR (b)
Strain bfpA aggR efa1 astA lpfD
W7 - - - - -
W85-2 - - - - -
W114 - - - - -
W143 - - - - -
W145 - - -
W154 - - - - -
W185 - - - - -
W208 - - - - -
W761 - - - - -
W902 - - - - -
W914 - - - - -
W1040 - - -
W1056 - - -
W1068 - - - - -
W1082 - - - - -
W1092 - - - -
W1108 - - - - -
W1118 - - - - -
W1120 - - - - -
W1134 - - - - -
W1585 - - - - -
W1706 - - - - -
E128012 - - -
Adhesion FAS HEp-2 cell
Strain pattern (a) assay invasion (c)
W7 NA - 0.01
W85-2 AA 0.02
W114 IA 0.02
W143 IA <0.01
W145 AA 0.03
W154 AA <0.01
W185 IA 0.02
W208 IA 0.08
W761 NA - 0.07
W902 IA 0.06
W914 AA 0.07
W1040 LAL 0.10
W1056 IA 0.77
W1068 AA <0.01
W1082 AA 0.01
W1092 IA <0.01
W1108 IA 0.03
W1118 IA 0.01
W1120 IA 0.01
W1134 NA - 0.01
W1585 NA - 0.02
W1706 AA 0.01
E128012 IA 0.32
(a) N, no symptoms; G, gastroenteritis; C, control strain;
Ont, O nontypable (O1-O181); Hnt, H nontypable (H1-H56);
NA, nonadherent; AA, aggregative adherence pattern; IA,
indeterminate adherence pattern; LAL, localized-like
adherence pattern; FAS, fluorescent actin staining.
(b) PCR, polymerase chain reaction; , positive; -, negative.
(c) Data are the number of bacteria recovered from HEp-2 cells
after treatment with gentamicin as a percentage of the total
number of cell-associated bacteria and are the mean of two
separate assays.
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