Commensal found in large bowel in most mammals.

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Escherichia coli

• Commensal found in large bowel in most mammals.
• Certain strains may cause disease
• Urinary tract infections
• Sepsis/meningitis
• Diarrhea

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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 O157H7

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EHEC

• Group defined by those strains that produce
  shiga-toxin (Stx1, Stx2) and cause hemorrhagic
  colitis (HC) and/or hemolytic uremic syndrome
  (HUS).

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EHEC serotypes

• O157H7-50
• Non-O157 serotypes
• O26H11-21
• O111NM-19
• O103H2-10
• O121-8
• O145-6
• O45-6

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Virulence factors

• Shiga-like toxin
• Stx1 and Stx2
• Main virulence factor
• associated with HUS
• Intimin
• Mediates attachment
• EHEC plasmid
• Enterohemolysin
• Catalase

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From Whittam, T.S. 1998. Escherichia coli
O157H7 and other Shiga- Toxin producing E. coli
strains. J.B. Kaper and A. D. OBrien ed.
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Attaching and effacing (A/E) pathology
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Shiga Toxin

• Encoded on Stx bacteriophage
• Originally discovered in Shigella dysenteriae
  (Stx1-like)
• Multiple variants-Stx1, Stx2 (Stx2c, d, e, f, g)
• AB-5 toxin (5 B components and one A component)

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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.

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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

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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.

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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

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Disease associated with EHEC

• Phase 4 Postsymptomatic stage.
• E. coli O157H7 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.

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Laboratory Detection Methods

• Culture methods-Sorbital MacConkey agar
• Only detects O157H7
• 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)

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Public Health Questions-1997

• What is the prevalence of E. coli O157H7 in
  persons with diarrhea?
• What is the prevalence of non-O157H7 STEC in
  persons with diarrhea?
• Develop a shiga-toxin PCR test to detect
  shiga-toxin from stool specimens. Test developed
  to use at NPHL.
• Funding from LB-1206

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Why ask these questions-1997

• Some clinical laboratories do not screen for
  O157H7 in routine stool cultures.
• No clinical laboratories screen for non-O157H7
  STEC.
• Develop a cost-effective method to detect
  non-O157H7 STEC from stools.

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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

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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 O157H7 or O157NM
(1.8) -7/13 non-O157 serotypes (2.1)
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Conclusions of Nebraska Study
-4.2 EHEC prevalence rate. -1.8 O157H7 -2.2
non-O157H7 -O111NM, O26H11, O145NM, O103H2
have previously been associated with HUS. -Two
O111NM 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
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Prevalence of other bacterial diarrheal diseases

• Camplobacteriosis
• Salmonellosis
• Shigellosis
• E. coli O157
• Yersiniosis
• Listeriosis
• Vibrio

EHEC
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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. 2000NEJM 342
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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

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Molecular Epidemiology
Genomic Bar Code Fingerprinting Assesses
Relatedness of Different Isolates Is Strain A
related to Strain B
992523
4357
0 2
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Typical questions addressed through molecular
epidemiology

• Are the Escherichia coli O157H7 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 isolatethe same strain??

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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

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PFGE-Pulsed-Field Gel Electrophoresis
THE CHROMOSOME IS THE MOST FUNDAMENTAL MOLECULE
OF IDENTITY IN THE CELL
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Molecular Epidemiology-NPHL

• Escherichia coli O157H7
• Salmonella, Campylobacter, Listeria.
• Nosocomial-MRSA, VRE, Pseudomonas aeruginosa,
  Klebsiella pneumoniae and other enterics.

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E. coli O157H7
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Reporting procedure

• Step 1--Compare PFGE patterns to Nebraska
  database
• EC157x.001-EC157x.0240

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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
  mannerstandardized protocol. Detects inter-and
  intrastate outbreaks. Program called
  Pulsenet-managed by CDC

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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.

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Top 5 E. coli O157H7 PFGE patterns
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2006 fresh spinach outbreak
-199 person infected from 26 states -102 were
hospitalized (51) 3 deaths (one from
Nebraska) -31 (16) developed HUS -Both O157H7
and O26H11 isolated from ill patients and spinach
1-4 5-9 10-14 gt 15
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Richard Goering, Ph.D. Creighton University