Shiga toxin Producing Escherichia coli-Nancy Strockbine PhD

Pathogenesis and Detection of Shiga toxin-
producing Escherichia coli ─
Food Safety Issues Related to E. coli O157
and non-O157 Strains

Nancy A. Strockbine, Ph.D.
Chief, Escherichia and Shigella Reference Unit
Enteric Diseases Laboratory Branch

Presented at the Eastern Pennsylvania Branch-ASM 41st Annual Symposium
“Global Movement of Infectious Pathogens and Improved Laboratory Detection Methods”
Philadelphia, Pennsylvania
17 November 2011

Division of Foodborne, Waterborne and Environmental Diseases
National Center for Emerging and Zoonotic Infectious Diseases

Outline
• Pathogenesis
• Epidemiology and surveillance
• Detection
• Food safety

Terminology
• STEC – Escherichia coli that produce one or
more Shiga toxins

• EHEC– A subset of STEC that are capable of
causing diarrheal disease, including bloody
diarrhea and HUS

Clinical Presentation of STEC Disease in Humans
• Asymptomatic infection
• Nonbloody diarrhea
• Bloody diarrhea/hemorrhagic colitis
• Hemolytic uraemic syndrome (6-15%)
– Microangiopathic hemolytic anemia
– Thrombocytopenia
– Acute renal failure
• Chronic kidney failure in 25% of those with HUS
• Neurologic symptoms seen in TTP

Sequence of events in STEC infection
STEC O157 ingested Non-O157 STEC ingested
3 - 4 days 3 - 4 days

non-bloody diarrhea, non-bloody diarrhea,
abdominal cramps abdominal cramps
(short lived fever) (short lived fever)

80% 1 - 2 days 45% 1 - 2 days

bloody bloody
diarrhea diarrhea
94% 6-15% 98% <2%
5 - 6 days 5 - 6 days
(up to 2-3 (up to 2-3
weeks)
resolution HUS resolution weeks) HUS

E. coli Pathotypes
―Flexible Genome‖
• ~ 9,400 genes in pangenome UPEC/
NMEC
EIEC/
Shigell
EAEC a
• ~ 2,200 genes in core EPEC ETEC

• Drivers of genetic diversity
Commensal
– Phages
– Plasmids 4,238 – 5,589 genes per bacterial genome

– Pathogenicity Islands

~ 2,200

Rasko, DA et al. J. Bacteriol. 2008

How big is 1030 ?

1030 phages equals mass of ~106 Blue Whales
106 Blue Whales end-to-end will circle over half the Earth’s
circumference

Shiga toxins

 Phage encoded toxins
 Act locally and systemically O’Brien AD et al. Science 226:694-696, 1984.

 Receptors on intestinal epithelium and kidney endothelium
 Inhibit protein synthesis
 binding of toxin to vascular tissue thought to trigger coagulation
cascade
 Two subgroups (Stx1 and Stx2)
 Strains that produce Stx2 are more virulent
 Necessary but not sufficient to cause disease
 Other virulence factors involved

Potential Virulence Genes
Gene or plasmid Predicted product or phenotype

stx1 Shiga toxin 1
stx2 Shiga toxin 2
eae intimin
EHEC-hlyA (ehxA) EHEC hemolysin (enterohemolysin)
espP serine protease
katP catalase
cdt cytolethal distending toxin
efa-1 EHEC factor of adherence (Efa1)
saa STEC autoagglutinating adhesin (Saa)
iha IrgA homologue adhesin (Iha)
lfpA Major fimbrial subunit of LPF (Long polar Fimbriae)
ent/espL2, nleB, nleE, nleF, genes from genomic islands OI-122 and OI-71
nleH1-2, nleA
irp-2 Iron-repressible protein 2
fyuA Yersiniabactin receptor

Virulence profile and clinical manifestation in
559 Danish STEC patients 1994-2005

Other
100%
D
80%
PD
60%
BD
40%
PBD
20%
HUS
0%
stx2 + stx1 + stx1 + stx2 stx1 + stx1
eae stx2 + eae stx2
eae
Courtesy Flemming Scheutz, WHO Collaborating Center for Escherichia and Klebsiella, SSI

Stx1 : 4 subtypes a - d
7-8 variants
Pairwise (OG:100%,UG:0%) (FAST:2,10) Gapcost:0%
VT1 translated sequences

100
96

97

98

99
Stx1a-S._dysenteriae-3818T
a
Stx1a-S._sonnei-CB7888

Stx1b-O111-CB168

Stx1b-O157-EDL933
b
Stx1b-O48-94C

Stx1b-O111-PH

Stx1c-O174-DG131-3 c
Stx1d-ONT-MHI813 d


Pairwise (OG:100%,UG:0%) (FAST:2,10) Gapcost:0% Disc. unk.
vtx_TRANSL

100
82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99
vtx2d-O157-7279

d
vtx2d-O174-EC1720a
vtx2d-O91-a-B2F1
vtx2d-O91-b-B2F1
vtx2d-O8-C466-01B

Stx2 : vtx2d-C_freundii-LM76..
vtx2d-O6-NV206
vtx2d-O22-KY-O19
vtx2d-O73-C165-02

7 subtypes vtx2a-O157-EDL933
vtx2a-O26-FD930
vtx2a-O157-SF

a
a-g
vtx2a-O48-94C
vtx2a-O26-126814
vtx2a-E_cloacae-95MV2
vtx2c-O157-E32511
vtx2c-O157-FLY16

c
vtx2c-O157-C394-03
vtx2c-O157-469
vtx2c-O174-b-031

35 variants vtx2g-O2-7v
vtx2g-O2-S86
vtx2g-Out-S-8 g
vtx2b-O111-S-3
vtx2b-O96-S-6
vtx2b-O22-3143-97
vtx2b-ONT-5293-98
vtx2b-O118-EH250
vtx2b-O16-6451-98
b
vtx2b-O174-a-031
vtx2b-O111-PH
vtx2e-O139-412
vtx2e-O22-3615-99
e
f
vtx2e-O101-E-D43
vtx2f-O128-T4-97


Shiga toxin 2 (stx2) subtype and clinical presentation
Subtype Non-HUS * HUS*
stx2a 60 11
stx2c 49 1
stx2d-activatable 4
stx2d 39
stx2e 2
stx2-variant 3
stx2 + stx2c 23 7
stx2 + stx2d 1
2x stx2-activatable 4
stx2c + stx2-activatable 1
Total 186 19

stx2 OR* 32.5 > stx2c OR* 4.7 for HUS
*) OR: odds ratio; multivariant analysis adjusted for age
Ethelberg et al. 2004 EID: vol 10

Lifestyle options of Shiga toxin-converting
bacteriophages
Lysogenic cycle

Phage DNA integrates
into host chromosome

Lytic cycle

Bacteriophages replicate, toxin production
is amplified, cells lyse and release Shiga
toxin and phage progeny

Electron micrographs by R. Hendrix
Slide courtesy Louise Teel, USUHS

Induction of expression of the late gene
cluster of lambdoid phages

Basic lambda genome structure… …toxin genes here

X

att int xis cIII N cI cro cII O P Q stxA/B S R Rz head genes tail genes

C1 repressor
RecA

• Damage to the host cell DNA triggers the SOS response
• Expression of the bacterial RecA protein is up-regulated
• RecA cleaves the phage repressor of the lytic cycle
• Downstream genes, including the toxin genes, get transcribed

Slide courtesy Louise Teel, USUHS

Norfloxacin-induced Stx phage being released
from a bacterium

Allison, HE Future Microbiol. 2:165-174, 2007

Escherichia coli O104:H4 Outbreak in Germany, May 2011
Proposed scheme for the origin of a new E. coli pathotype--
Enteroaggregative hemorrhagic Escherichia coli

Brzuszkiewicz E. et al. Arch Microbiol. 2011

Surveillance systems
 National surveillance: passive
 National Notifiable Disease Surveillance System
 Public Health Laboratory Information System
 CDC National E. coli Reference Laboratory
 PulseNet
 Sentinel surveillance: active
 Foodborne Disease Active Surveillance Network (FoodNet)

FoodNet
10 sites , 46 million persons (15% of US population)

Incidence of reported STEC O157 and non-O157 STEC
infections, by year, FoodNet, 1996-2009

2.5
STEC O157 Non-O157 STEC
Cases per 100,000 population

2

1.5

1

0.5

0
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

Year

Healthy People 2010 objective is 1 case/100,000 persons

Incidence of O157 STEC and non-O157 STEC
Cases at FoodNet Sites, 2009
3

2.5
Number of cases/100,000 population

2

STEC**
1.5 O157

STEC non-
1 O157

0.5

0
CA CO CT GA MD MN NM NY OR TN Overall
2009

Burden of Illness
 Surveillance detects the tip of the
iceberg
 Detecting an illness depends on
probability of…
 ill person seeking medical care
 stool sample requested
 stool sample received
 necessary tests performed
 test result positive
 infection reported

Proportion of Annual Foodborne Illness in the
United States by Pathogen
Salmonella spp., nontyphoidal
Clostridium perfringens, foodborne
Campylobacter spp.
Staphylococcus aureus, foodborne
Shigella spp.
STEC non-O157
Yersinia enterocolitica
Bacillus cereus, foodborne
STEC O157
V. parahaemolyticus
ETEC, foodborne
Vibrio spp., other
Other diarrheagenic E. coli
Streptococcus spp. group A, foodborne
S. enterica serotype Typhi
Listeria monocytogenes
Brucella spp.
V. vulnificus
Vibrio cholerae, toxigenic
Mycobacterium bovis
Clostridium botulinum, foodborne
0 200,000 400,000 600,000 800,000 1,000,000 1,200,000

Number of illnesses

Scallan et al. 2011 EID 17(1)7-15

Hospitalization rate, %
V. vulnificus
Mycobacterium bovis
Brucella spp.
STEC O157
Vibrio spp., other
V. parahaemolyticus
Shigella spp.
Campylobacter spp.
STEC non-O157
ETEC, foodborne
0 10 20 30 40 50 60 70 80 90 100


Death Rate, %
V. vulnificus
Mycobacterium bovis
Vibrio spp., other
V. parahaemolyticus
Brucella spp.
STEC O157
STEC non-O157
Shigella spp.
Campylobacter spp.
ETEC, foodborne
0 5 10 15 20 25 30 35 40


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Client

BNServer
with database

Client

• Upload & download of information
• Internet based

Shiga toxin gene distribution among 19,402
STEC from the US, 2006-2010 by Serogroup
No. isolates from PulseNet and CDC Ref Lab

Serogroup
* Includes 120 O groups

Prevalence of STEC Serogroups in the US
from 2006-2010 n = 19,402
4500
No. isolates from PulseNet and CDC Ref Lab

4000
O157
3500
O26
3000
O103
2500
O111
2000
1500 O45

1000 O121
500 O145
0
Other
2006 2007 2008 2009 2010 *
year Total
* Includes 120 O groups

Geographic Distribution of 1342 STEC O157
isolates from 2006-2010

= 1-250 isolates
= 251-500 isolates
= 501-750 isolates
= 751-1000 isolates
= > 1001 isolates


= none reported
= 1-30 isolates
= 31-60 isolates
= 61-90 isolates
= 91-120 isolates
= > 121 isolates


= none reported
= 1-25 isolates
= 26--50 isolates
= 50--75 isolates
= 75-100 isolates
= > 101 isolates


= none reported
= 1-10 isolates
= 11-20 isolates
= 21-30 isolates
= 31-40 isolates
= 41-50 isolates

Key factors in STEC transmission
 Reservoir is the intestinal tract of animals
 Especially cattle
 Very low infectious dose
 <100 organisms
 Multiple modes of transmission
 Foodborne
 Animal contact
 Waterborne
 Person-to-person contact
 Most infections are not outbreak-related
 ~19% of E. coli O157 infections, ~9% of non-O157 STEC infections

Proportion of illnesses by mode of transmission in
344 STEC O157 outbreaks, 1998-2007

Illnesses in outbreaks
Mode of transmission (n=7,864 illnesses)
%
Foodborne 69
Waterborne 18
Animals or their environment 8
Person-to-person 6

Non-O157 STEC outbreaks: modes of
Transmission—United States, 1990-2008
Non- Non-
Mode of transmission O157 O157
No. %

Foodborne 9 33
Person-to-person 7 26
Water 4 15
Animal contact 4 15
Mixed modes 1 4
Unknown 2 7
Total 27 100

Outbreak of STEC O145 Infections
May 2010
 33 cases in 5 states
 Michigan, New York, Ohio, Pennsylvania, and Tennessee
 First recognized multistate outbreak of non-O157 STEC
 40% hospitalized, 10% developed HUS
 As severe as illness caused by E. coli O157:H7
 Caused by contaminated Romaine lettuce

Exposures associated with sporadic non-O157
STEC infections
 Australia
 Corned beef, camping, occupational contact with animals
 Germany
 Children: touching a ruminant, playing in a sandbox
 Adults: eating lamb and spreadable sausage
 United States
 Minnesota: recent international travel?
 FoodNet: study under development

Clinical laboratory recommendations, 2009
 Simultaneously culture all stools
submitted from patients with
acute community-acquired
diarrhea or suspected HUS for
O157 and assay for non-O157
STEC with a test that detects Shiga
toxin
 Report and send E. coli O157
isolates and Stx+ broths to a public
health laboratory as soon as
possible

Why test all stool samples for STEC?
 Selective testing practices miss many STEC infections
 Children
• Over half of infections occur in older adolescents and adults
• Highest mortality rate in persons ≥60 years old
 Summer months
• ~50% of infections occur in non-summer months
• Outbreaks can occur year round
 Bloody diarrhea
• Some patients do not have bloody diarrhea
 STEC might be detected as often as other bacterial
enteric pathogens

Why simultaneously culture for E. coli O157 and assay
for Shiga toxin?

 Most sensitive approach to detect all STEC infections
 Rapidly distinguishes O157 from non-O157 STEC infections
 Isolates are obtained in a timely manner

Proposed best practice benefits patient care and
public health
 Patient care
 Facilitates early clinical management decisions to reduce risk of HUS
• Avoidance of antibiotics and anti-diarrheals
 Early identification of E. coli O157 can further influence management
decisions
 Avoidance of unnecessary procedures
 Public health
 Allows for prompt confirmation and subtyping by public health labs
to detect and control of outbreaks
 Allows for monitoring of epidemiological trends

Clinical Diagnosis of STEC infection
Stool
Specimen
Test
Culture • Shiga toxin
for O157 or H7
STEC • ID as E. coli

Streak to Test in
Culture for Selective/differential O157 latex Send STEC O157 and
non-O157 agar reagent positive broths to public
STEC health lab

16-24
hours

Shiga toxin or stx gene
Enrichment broth
detection Stx/stx+ broth

Isolation of STEC from Stx-positive broths by PHLs
Shiga toxin-positive broth

Selective plate: CT-SMAC or CHROM O157
Nonselective plate: SMAC or WSBM

Screen suspect colonies in O157 latex reagent
IF NEGATIVE
SMAC or WSBM

Sweep of Growth or Isolated colonies (or pool 5 colonies)

Shiga toxin assay or PCR for stx1, stx2

Serogrouping and PFGE

•Detects E. coli O104
• Available in Europe;
not yet in the US
•Tests for 15 bacteria,
viruses and parasites in
under 5 hours

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Seegene Diarrhea ACE Detection for Stool
1 Viral and 2 Bacterial Panels (14 Agents in 6 hr)
C ||Diarrhea-B2 ACE Detection
C Diarrhea-B2 ACE Detection
11 Aeromonas spp.
Aeromonas spp.

22 C. perfringens
C. perfringens

33 Aeromonas spp.
Aeromonas spp.

44 E.coli : H7
E.coli : H7

E.coli : O157
E.coli : O157
55 E.coli : H7
E.coli : H7

E.coli : O157
E.coli : O157
E.coli : H7
E.coli : H7
66 VTEC
VTEC

Y.enterocolitica
Y.enterocolitica
1~7: Clinical
1~7: Clinical
7 7 samples
samples

Detection of STEC in Foods
http://www.fsis.usda.gov/PDF/MLG_5B_00.pdf

Sample enrichment

Genomic DNA extraction

TaqMan-based multiplex real-time PCR assay:
stx1, stx1, eae (intimin) and 16S rRNA
If positive

O-antigen identification (real-time PCR)

Immunomagnetic separation

Selective plating confirmation

Food Safety
• September 20, 2011 FSIS announced six STEC serogroups
(O26, O45, O103, O111, O121 and O145) will be adulterants
on raw, non-intact beef products in the same manner as
E. coli O157:H7
• FSIS will apply its adulteration decision when testing is
initiated March 5, 2012

Summary
 STEC can cause non-bloody or bloody diarrhea and HUS
 Horizontal gene transfer is common -- phage play an
important role
 Prevalence varies geographically
 Primary reservoir ruminants, especially cattle
 Simultaneous culture for E. coli O157:H7 and an assay that
detects Stx or stx genes is the most sensitive approach for all
STEC
 STEC O26, O45, O103, O111, O121 and O145 FSIS will be
regulated by FSIS like E. coli O157:H7 starting March 2012

Pathogenesis and Detection of Shiga toxin-producing Escherichia coli ─
Food Safety Issues Related to E. coli O157
and non-O157 Strains

Nancy A. Strockbine, Ph.D.
Chief, Escherichia and Shigella Reference Unit
Division of Foodborne, Waterborne and Environmental Diseases
National Center for Emerging and Zoonotic Infectious Diseases
Centers for Disease Control and Prevention
Phone: (404) 639-4186
FAX: (404) 639-3333
E-mail: Nancy.Strockbine@cdc.hhs.gov

The findings and conclusions in this report are those of the author and do not necessarily
represent the official position of the Centers for Disease Control and Prevention.


Peter Gerner-Smidt, M.D., D.M.S., Branch Chief
John Besser, Ph.D., Deputy Branch Chief
Sherricka Simington, Branch manager
Nicole Rankine, QMS manager
4 FTE, 2 non-FTE

National Enteric National Antimicrobial National Botulism PulseNet USA Team National Enteric
Reference Laboratory Resistance Surveillance Laboratory Laboratory Diagnostics
Team Team Preparedness Team and Outbreak Team
Patricia Fields, Ph.D. Jean Whichard, D.V.M., Ph.D. Susan Maslanka, Ph.D. Efrain Ribot, Ph.D. Deborah Talkington, Ph.D.
12 FTE, 8 non-FTE 4 FTE, 5 non-FTE 5 FTE, 3 non-FTE 13 FTE, 7 non-FTE 7 FTE, 1 non-FTE

Campylobacter and NARMS Surveillance Botulism Public Health PulseNet Database Unit Epidemic
Helicobacter Unit Unit Research Unit Kelley Hise, M.P.H. Investigations
Collette Fitzgerald, Ph.D. Kevin Joyce Brian Raphael, Ph.D. Laboratory Unit
Cheryl Bopp, M.S.
Escherichia and Shigella,
Unit NARMS Applied Botulism Outbreak PulseNet Methods Immunodiagnostics
Nancy Strockbine, Ph.D. Research Unit Investigation Unit Development and Unit
Jean Whichard, D.V.M., Carolina Luquéz, Ph.D. Reference Unit Deborah Talkington, Ph.D.
Ph.D. Efrain Ribot, Ph.D.
Salmonella Unit
Patricia Fields, Ph.D.

Listeria ,Yersinia , Vibrio
and other
Enterobacteriaceae Unit 2-1-2010
Cheryl Tarr, Ph.D.

Shiga toxin Producing Escherichia coli-Nancy Strockbine PhD

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