Shiga toxin Producing Escherichia coli-Nancy Strockbine PhD

Nancy A. Strockbine, Ph.D.

Chief, Escherichia and Shigella Reference Unit

Enteric Diseases Laboratory Branch

Pathogenesis and Detection of Shiga toxin-

producing Escherichia coli ─

Food Safety Issues Related to E. coli O157

and non-O157 Strains

Division of Foodborne, Waterborne and Environmental Diseases

National Center for Emerging and Zoonotic Infectious Diseases

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

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

WHAT DO THEY CAUSE?

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

3 - 4 days

non-bloody diarrhea,

abdominal cramps (short lived fever)

5 - 6 days (up to 2-3

weeks) resolution

94%

bloody

diarrhea

1 - 2 days 80%

Non-O157 STEC ingested

3 - 4 days

5 - 6 days (up to 2-3

weeks) resolution

98%

bloody

diarrhea

1 - 2 days 45%

<2%

HUS HUS

non-bloody diarrhea,

abdominal cramps (short lived fever)

6-15%

E. coli Pathotypes

―Flexible Genome‖

• ~ 9,400 genes in pangenome

• ~ 2,200 genes in core

• Drivers of genetic diversity

– Phages

– Plasmids

– Pathogenicity Islands

~ 2,200

EPEC ETEC

UPEC/

NMEC

EAEC

EIEC/

Shigella

Commensal

4,238 – 5,589 genes per bacterial genome

Rasko, DA et al. J. Bacteriol. 2008

1030 phages equals mass of ~106 Blue Whales

106 Blue Whales end-to-end will circle over half the Earth’s

circumference

How big is 1030 ?

Shiga toxins

Phage encoded toxins

Act locally and systemically

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

O’Brien AD et al. Science 226:694-696, 1984.

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,

nleH1-2, nleA

genes from genomic islands OI-122 and OI-71

irp-2 Iron-repressible protein 2

fyuA Yersiniabactin receptor

Virulence profile and clinical manifestation in

559 Danish STEC patients 1994-2005

0%

20%

40%

60%

80%

100%

stx2 +

eae

stx1 +

stx2 +

eae

stx1 +

eae

stx2 stx1 +

stx2

stx1

Other

D

PD

BD

PBD

HUS

Courtesy Flemming Scheutz, WHO Collaborating Center for Escherichia and Klebsiella, SSI

Stx1 : 4 subtypes a - d

7-8 variants

d

b

c

a Stx1a-S._dysenteriae-3818T

Stx1a-S._sonnei-CB7888

Stx1b-O111-CB168

Stx1b-O157-EDL933

Stx1b-O48-94C

Stx1b-O111-PH

Stx1c-O174-DG131-3

Stx1d-ONT-MHI813

Pairwise (OG:100%,UG:0%) (FAST:2,10) Gapcost:0%

VT1 translated sequences

100

99

98

97

96


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

vtx_TRANSL

100

999897969594939291908988878685848382vtx2d-O157-7279

vtx2d-O174-EC1720a

vtx2d-O91-a-B2F1

vtx2d-O91-b-B2F1

vtx2d-O8-C466-01B

vtx2d-C_freundii-LM76..

vtx2d-O6-NV206

vtx2d-O22-KY-O19

vtx2d-O73-C165-02

vtx2a-O157-EDL933

vtx2a-O26-FD930

vtx2a-O157-SF

vtx2a-O48-94C

vtx2a-O26-126814

vtx2a-E_cloacae-95MV2

vtx2c-O157-E32511

vtx2c-O157-FLY16

vtx2c-O157-C394-03

vtx2c-O157-469

vtx2c-O174-b-031

vtx2g-O2-7v

vtx2g-O2-S86

vtx2g-Out-S-8

vtx2b-O111-S-3

vtx2b-O96-S-6

vtx2b-O22-3143-97

vtx2b-ONT-5293-98

vtx2b-O118-EH250

vtx2b-O16-6451-98

vtx2b-O174-a-031

vtx2b-O111-PH

vtx2e-O139-412

vtx2e-O22-3615-99

vtx2e-O101-E-D43

vtx2f-O128-T4-97

d

f

b

g

c

a

e

Stx2 :

7 subtypes

a - g

35 variants


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

Ethelberg et al. 2004 EID: vol 10

Shiga toxin 2 (stx2) subtype and clinical presentation

stx2 OR* 32.5 > stx2c OR* 4.7 for HUS *) OR: odds ratio; multivariant analysis adjusted for age


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

Lytic cycle

Phage DNA integrates into host chromosome

Lysogenic cycle

Electron micrographs by R. Hendrix

Slide courtesy Louise Teel, USUHS

Lifestyle options of Shiga toxin-converting

bacteriophages

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

X

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

C1 repressor

Basic lambda genome structure… …toxin genes here

Induction of expression of the late gene

cluster of lambdoid phages

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

HOW COMMON ARE NON-O157 STEC?

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)

0

0.5

1

1.5

2

2.5

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

STEC O157 Non-O157 STEC

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

Year

Ca

ses

pe

r 1

00

,00

0 p

op

ula

tio

n

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

0

0.5

1

1.5

2

2.5

3

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

STEC** O157

STEC non-O157

Incidence of O157 STEC and non-O157 STEC

Cases at FoodNet Sites, 2009

Nu

mb

er o

f ca

ses/

10

0,0

00

po

pu

lati

on

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

http://www.brooks.edu/careers.htm

0 200,000 400,000 600,000 800,000 1,000,000 1,200,000

Clostridium botulinum, foodborne

Mycobacterium bovis

Vibrio cholerae, toxigenic

V. vulnificus

Brucella spp.

Listeria monocytogenes

S. enterica serotype Typhi

Streptococcus spp. group A, foodborne

Other diarrheagenic E. coli

Vibrio spp., other

ETEC, foodborne

V. parahaemolyticus

STEC O157

Bacillus cereus, foodborne

Yersinia enterocolitica

STEC non-O157

Shigella spp.

Staphylococcus aureus, foodborne

Campylobacter spp.

Clostridium perfringens, foodborne

Salmonella spp., nontyphoidal

Proportion of Annual Foodborne Illness in the

United States by Pathogen

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

Number of illnesses

0 10 20 30 40 50 60 70 80 90 100




ETEC, foodborne



STEC non-O157

Campylobacter spp.

Shigella spp.

V. parahaemolyticus



Vibrio spp., other


STEC O157

Brucella spp.

Mycobacterium bovis



V. vulnificus


Hospitalization rate, %


0 5 10 15 20 25 30 35 40


ETEC, foodborne





Campylobacter spp.


Shigella spp.


STEC non-O157

STEC O157


Brucella spp.

V. parahaemolyticus


Vibrio spp., other

Mycobacterium bovis



V. vulnificus

Death Rate, %


E. coli O157:H7 Gel

BioNumerics Server

BNServer

with database

Client

Client

• Upload & download of information

• Internet based

Shiga toxin gene distribution among 19,402

STEC from the US, 2006-2010 by Serogroup

No

. is

ola

tes

fro

m P

uls

eNet

an

d C

DC

Ref

La

b

Serogroup * Includes 120 O groups

Prevalence of STEC Serogroups in the US

from 2006-2010 n = 19,402

0

500

1000

1500

2000

2500

3000

3500

4000

4500

2006 2007 2008 2009 2010

O157

O26

O103

O111

O45

O121

O145

Other*Total

No

. is

ola

tes

fro

m P

uls

eNet

an

d C

DC

Ref

La

b

year * Includes 120 O groups

= 501-750 isolates

= 1-250 isolates

= 251-500 isolates

= 751-1000 isolates

= > 1001 isolates

Geographic Distribution of 1342 STEC O157

isolates from 2006-2010

= 61-90 isolates

= none reported

= 1-30 isolates

= 31-60 isolates

= 91-120 isolates

= > 121 isolates



= 50--75 isolates

= none reported

= 1-25 isolates

= 26--50 isolates

= 75-100 isolates

= > 101 isolates



= 21-30 isolates

= none reported

= 1-10 isolates

= 11-20 isolates

= 31-40 isolates

= 41-50 isolates



HOW ARE STEC TRANSMITTED?

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

Mode of transmission

Illnesses in outbreaks

(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

Mode of transmission

Non-

O157

No.

Non-

O157

%

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

HOW SHOULD LABS DETECT STEC?

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

Enrichment broth Shiga toxin or stx gene

detection

16-24

hours

Send STEC O157 and

positive broths to public

health lab

Culture

for O157

STEC

Streak to

Selective/differential

agar Culture for

non-O157

STEC

Test in

O157 latex

reagent

Test

• Shiga toxin

or H7

• ID as E. coli

Stx/stx+ broth Enrichment broth

16-24

hours

Enrichment broth

16-24

hours

Enrichment broth

16-24

hours

Enrichment broth

16-24

hours

Enrichment broth

16-24

hours

Enrichment broth

16-24

hours

Enrichment broth

16-24

hours

Enrichment broth

16-24

hours

Enrichment broth

Send STEC O157 and


health lab

16-24

hours

Enrichment broth

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Enrichment broth

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Enrichment broth

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Enrichment broth

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Enrichment broth

Culture

for O157

STEC

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Enrichment broth

Culture

for O157

STEC

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Enrichment broth

Stool

Specimen

Culture

for O157

STEC

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Enrichment broth

Culture for

non-O157

STEC

Stool

Specimen

Culture

for O157

STEC

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Enrichment broth

Culture for

non-O157

STEC

Stool

Specimen

Culture

for O157

STEC

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Enrichment broth

Streak to


agar Culture for

non-O157

STEC

Stool

Specimen

Culture

for O157

STEC

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Test in

O157 latex

reagent

Streak to


agar Culture for

non-O157

STEC

Stool

Specimen

Culture

for O157

STEC

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Shiga toxin or stx gene

detection

Test in

O157 latex

reagent

Streak to


agar Culture for

non-O157

STEC

Stool

Specimen

Culture

for O157

STEC

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Stx/stx+ broth

Shiga toxin or stx gene

detection

Test in

O157 latex

reagent

Streak to


agar

Stool

Specimen

Culture

for O157

STEC

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Test

• Shiga toxin

or H7

• ID as E. coli

Send STEC O157 and


health lab

16-24

hours

Isolation of STEC from Stx-positive broths by PHLs

Selective plate: CT-SMAC or CHROM O157

Nonselective plate: SMAC or WSBM

Screen suspect colonies in O157 latex reagent

Sweep of Growth

Shiga toxin assay or PCR for stx1, stx2

Isolated colonies (or pool 5 colonies)

IF NEGATIVE

or

Shiga toxin-positive broth

SMAC or WSBM

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

Seeplex® System

Seeplex® is a breakthrough multiplexing PCR technology that enables a new standard in simultaneous multi-

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consistently high specificity. The strength and utility of this DPO™ technology can be successfully incorporated into

molecular diagnostics systems such as multiplex diagnostics and SNP genotyping systems.

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molecular diagnostics systems such as multiplex diagnostics and SNP genotyping systems.

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C | Diarrhea-B2 ACE Detection

Y.enterocolitica

1~7: Clinical

samples

7

E.coli : O157

E.coli : H7

VTEC 6

E.coli : O157

E.coli : H7 5

E.coli : H7 4

Aeromonas spp. 3

C. perfringens 2

Aeromonas spp. 1


Y.enterocolitica

1~7: Clinical

samples

7

E.coli : O157

E.coli : H7

VTEC 6

E.coli : O157

E.coli : H7 5

E.coli : H7 4

Aeromonas spp. 3

C. perfringens 2

Aeromonas spp. 1

C | Diarrhea-B2 ACE Detection C | Diarrhea-B2 ACE Detection

E.coli : O157

E.coli : H7


Seegene Diarrhea ACE Detection for Stool 1 Viral and 2 Bacterial Panels (14 Agents in 6 hr)


Y.enterocolitica

1~7: Clinical

samples

7

E.coli : O157

E.coli : H7

VTEC 6

E.coli : O157

E.coli : H7 5

E.coli : H7 4

Aeromonas spp. 3

C. perfringens 2

Aeromonas spp. 1


Y.enterocolitica

1~7: Clinical

samples

7

E.coli : O157

E.coli : H7

VTEC 6

E.coli : O157

E.coli : H7 5

E.coli : H7 4

Aeromonas spp. 3

C. perfringens 2

Aeromonas spp. 1

C | Diarrhea-B2 ACE Detection C | Diarrhea-B2 ACE Detection

E.coli : O157

E.coli : H7


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

O-antigen identification (real-time PCR)

Immunomagnetic separation

Selective plating

If positive

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

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: [email protected]

Pathogenesis and Detection of Shiga toxin-producing Escherichia coli ─

Food Safety Issues Related to E. coli O157

and non-O157 Strains


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.

National Enteric

Reference Laboratory

Team Patricia Fields, Ph.D.

12 FTE, 8 non-FTE

Escherichia and Shigella,

Unit

Nancy Strockbine, Ph.D.

Salmonella Unit

Patricia Fields, Ph.D.

Campylobacter and

Helicobacter Unit

Collette Fitzgerald, Ph.D.

National Botulism

Laboratory

Preparedness Team Susan Maslanka, Ph.D.

5 FTE, 3 non-FTE

NARMS Surveillance

Unit

Kevin Joyce

NARMS Applied

Research Unit

Jean Whichard, D.V.M., Ph.D.

Listeria ,Yersinia , Vibrio

and other

Enterobacteriaceae Unit

Cheryl Tarr, Ph.D.

PulseNet Database Unit Kelley Hise, M.P.H.

PulseNet Methods Development and

Reference Unit Efrain Ribot, Ph.D.

Epidemic Investigations

Laboratory Unit Cheryl Bopp, M.S.

Immunodiagnostics Unit

Deborah Talkington, Ph.D.

Enteric Diseases Laboratory Branch 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 Antimicrobial

Resistance Surveillance

Team Jean Whichard, D.V.M., Ph.D.

4 FTE, 5 non-FTE

National Enteric

Laboratory Diagnostics

and Outbreak Team Deborah Talkington, Ph.D.

7 FTE, 1 non-FTE

PulseNet USA Team

Efrain Ribot, Ph.D.

13 FTE, 7 non-FTE

Botulism Public Health

Research Unit

Brian Raphael, Ph.D.

Botulism Outbreak

Investigation Unit

Carolina Luquéz, Ph.D.

2-1-2010

http://blogs.inside.cdc.gov/ncezid/wp-content/uploads/design_element_640px.jpg

Shiga toxin Producing Escherichia coli-Nancy Strockbine PhD

Health & Medicine

Transcript of Shiga toxin Producing Escherichia coli-Nancy Strockbine PhD