Escherichia coli
• Commensal found in
large bowel in most
mammals.
• Certain strains may cause
disease:
– Urinary tract infections
– Sepsis/meningitis
– Diarrhea
Diarrheagenic groups
•
•
•
•
•
•
Enterotoxigenic E. coli (ETEC)
Enteroinvasive E. coli (EIEC)
Enteropathogenic E. coli (EPEC)
Enteroaggregative E. coli (EAEC)
Diffusely adherent E. coli (DAEC)
Enterohemorrhagic E. coli (EHEC)
– Escherichia coli O157:H7
EHEC
• Group defined by
those strains that
produce shiga-toxin
(Stx1, Stx2) and cause
hemorrhagic colitis
(HC) and/or hemolytic
uremic syndrome
(HUS).
EHEC serotypes
• O157:H7-50%
• Non-O157 serotypes
–
–
–
–
–
–
O26:H11-21%
O111:NM-19%
O103:H2-10%
O121-8%
O145-6%
O45-6%
Virulence factors
• Shiga-like toxin
– Stx1 and Stx2
– Main virulence factor
– associated with HUS
• Intimin
– Mediates attachment
• EHEC plasmid
– Enterohemolysin
– Catalase
From: Whittam, T.S. 1998. Escherichia coli O157:H7 and other ShigaToxin producing E. coli strains. J.B. Kaper and A. D. O’Brien ed.
Attaching and effacing
(A/E) pathology
Shiga Toxin
• Encoded on Stx bacteriophage
• Originally discovered in
Shigella dysenteriae (Stx1like)
• Multiple variants-Stx1, Stx2
(Stx2c, d, e, f, g)
• AB-5 toxin (5 B components
and one A component)
Shiga Toxin
• Toxin enters blood stream
• 5 B subunits bind to
GB3/CD77 glycolipid
receptor (Kidney).
• Translocates A subunit which
is cleaved into an A1 peptide
• A1 peptide has N-glycosidase
activity that inhibits protein
synthesis through cleavage of
28S ribosomal RNA.
Disease associated with EHEC
• Phase 1: Presymptomatic stage
– Acquisition of infection
• Ingestion of undercooked beef is major risk factor
• Many other vehicles for infection and reservoirs: water,
vegetables, other mammals, etc.
• Very low infectious dose: 10-100 bacteria.
– Incubation period
• 1-10 days
• Average ~4 days after ingestion
Disease associated with EHEC
• Phase 2: Symptomatic phase
– Before bloody diarrhea
• Cramp-like abdominal pains
• Clingliness to a parent-lethargy
• Irritability and vomiting
– Bloody Diarrhea (82%; O157: 38%; non-O157)
• Supportive therapy to monitor development of HUS
• HUS (7%; O157: 1.5% non-O157) occurs on average day
6.5 after bloody diarrhea begins.
Disease associated with EHEC
• Phase 3:
– Microangiopathic sequelae
• Development of complete or incomplete HUS
• Approximately 15% of children with culture confirmed
EHEC.
• Low platelet count is usually first sign of HUS
• 3-5% mortality rate of patients with HUS
Disease associated with EHEC
• Phase 4: Postsymptomatic stage.
– E. coli O157:H7 can be excreted for up to a month.
– For a child to return to day care or school, it is
recommended that that patient have two negative stool
cultures beforehand.
Laboratory Detection Methods
• Culture methods-Sorbital MacConkey agar
– Only detects O157:H7
– Does NOT detect other EHEC serotypes
• Tests to detect shiga-toxin (detects all EHEC
serotypes)
– EIA (rapid kits available)
– PCR (Test available at UNMC-Commercial kits)
Public Health Questions-1997
• What is the prevalence of E. coli O157:H7 in
persons with diarrhea?
• What is the prevalence of non-O157:H7 STEC in
persons with diarrhea?
• Develop a shiga-toxin PCR test to detect shigatoxin from stool specimens. Test developed to
use at NPHL.
• Funding from LB-1206
Why ask these questions-1997
• Some clinical laboratories do not screen for
O157:H7 in routine stool cultures.
• No clinical laboratories screen for non-O157:H7
STEC.
• Develop a cost-effective method to detect nonO157:H7 STEC from stools.
Study Design
• Collaborated with 9 regional clinical laboratories
in Nebraska.
• NPHL was sent (through NPHL courier system)
stool samples from patients with diarrhea.
• Analysis:
– CT-SMAC culture
– Meridian EHEC EIA
– stx PCR
Results
-335 specimens were received from May 98-October 98
-5/9 laboratories had positive samples
-14 samples were positive by at least one of the methods (4.2%)
-Isolates from 13/14 positive samples were obtained
-6/13: O157:H7 or O157:NM (1.8%)
-7/13: non-O157 serotypes (2.1%)
Conclusions of Nebraska Study
-4.2% EHEC prevalence rate.
-1.8% O157:H7
-2.2% non-O157:H7
-O111:NM, O26:H11, O145:NM, O103:H2 have previously been
associated with HUS.
-Two O111:NM isolates were indistinguishable by PFGE, which
suggests a possible outbreak which was not detected.
-Developed a shiga-toxin PCR test which is in use at the NPHL
for physician use.
Fey et al. 2000 EID
Prevalence of other bacterial diarrheal
diseases
•
•
•
•
•
•
•
Camplobacteriosis
Salmonellosis
Shigellosis
E. coli O157
Yersiniosis
Listeriosis
Vibrio
EHEC
Treatment of EHEC
• 71 children with culture confirmed O157 infection.
– 9 patients had HUS
– 10 patients were treated with antibiotics
• 5/10 patients receiving antibiotics came down with HUS
– 4/61 patients not receiving antibiotics came down with
HUS.
• Treatment is supportive, no antibiotics are given
Wong et al. 2000
NEJM; 342
What is the current Nebraska state
protocol?
• All Microbiology laboratories should be
performing shiga-toxin test on routine stool
samples for bacterial pathogens. (CDC MMWR
2006)
• If laboratory does not isolate STEC, then stool
sample is sent to NPHL for STEC isolation.
– Imperative for molecular epidemiology program
Molecular Epidemiology
Genomic “Bar Code” Fingerprinting
0
992523
4357
2
Assesses Relatedness of Different Isolates
Is Strain “A” related to Strain “B”
Typical questions addressed through
molecular epidemiology
• Are the Escherichia coli
O157:H7 isolates obtained
from beef the same “strain” as
that obtained from the
patient(s)?
• Are the 7 MRSA isolates
obtained from the ICU the
same “strain?”
• Pre- and post-treatment
isolate…the same strain??
Methods used in Molecular
Epidemiology
• First Generation-Plasmid typing
• Second Generation-Restriction enzymes and
probes
• Third Generation-PCR methods and PFGE
• Fourth Generation-Sequencing methods
PFGE Gold Standard in almost all cases when
molecular epidemiology is in question
PFGE-Pulsed-Field Gel Electrophoresis
THE CHROMOSOME IS
THE MOST FUNDAMENTAL
MOLECULE OF IDENTITY
IN THE CELL
Molecular Epidemiology-NPHL
• Escherichia coli O157:H7
• Salmonella,
Campylobacter, Listeria.
• Nosocomial-MRSA,
VRE, Pseudomonas
aeruginosa, Klebsiella
pneumoniae and other
enterics.
E. coli O157:H7
Reporting procedure
• Step 1--Compare PFGE patterns to
Nebraska database
–EC157x.001-EC157x.0240
Dice (Opt:1.50%) (Tol 1.5%-1.5%) (H>0.0% S>0.0%) [0.0%-98.3%]
100
95
90
PFGE-XbaI
85
80
75
PFGE-XbaI
REF 6511
x.0185
REF 6528
x.0104
REF 6550
x.0055
REF 6463
x.0068
REF 6557
x.0188
REF 6443
x.0091
REF 6337
x.0175
REF 6576
x.0187
REF 6514
x.0155
REF 6666
x.0190
REF 6336
x.0174
REF 6510
x.0184
REF 6436
x.0180
REF 6313
x.0002
REF 6314
x.0002
REF 6312
x.0002
REF 6398
x.0181
REF 6405
x.0002
REF 6603
x.0189
REF 6537
x.0186
REF 6433
x.0179
REF 6411
x.0099
REF 6421
x.0177
REF 6373
x.0182
REF 6403
x.0176
REF 6431
x.0178
REF 6489
x.0183
REF 6498
x.0183
REF 6542
Reporting procedure
• Step 2: Compare
Nebraska PFGE pattern
with National database at
Centers for Disease
Control and Prevention
(CDC)
– Every state laboratory
performs PFGE in identical
manner—standardized
protocol. Detects inter-and
intrastate outbreaks.
Program called Pulsenetmanaged by CDC
Reporting procedure
• Step 3--Send Report to epidemiologists at State
level as well as Douglas and Lancaster County
–
–
–
–
Name, PFGE pattern, site/date of isolation
Has the PFGE pattern been seen in Nebraska recently?
Has the PFGE pattern been seen ever in Nebraska?
Has the PFGE pattern been seen in the US within the
last 60 days?
– The NPHL receives information from epidemiologists
office regarding epidemiological information.
Top 5 E. coli O157:H7 PFGE patterns
Dice (Opt:1.50%) (Tol 1.5%-1.5%) (H>0.0% S>0.0%) [0.0%-98.3%]
100
95
PFGE-XbaI
90
85
80
PFGE-XbaI
REF 2781
x.0004
REF 5144
x.0117
REF 5500
x.0048
REF 5804
x.0082
REF 5833
x.0055
2006 fresh spinach outbreak
1-4
5-9
10-14
> 15
-199 person infected from 26 states
-102 were hospitalized (51%) 3 deaths (one from Nebraska)
-31 (16%) developed HUS
-Both O157:H7 and O26:H11 isolated from ill patients and spinach
Richard Goering, Ph.D.
Creighton University