Shiga Toxin Producing Escherichia coli: Recent Trends and Facts

E. coli O157:H7 Nomenclature Introduction Outbreaks Non- 0157 STEC Animal Reservoir Transmission Antibiotic Resistance Detection Methods Summary

 Gram negative rod shaped bacterium  Facultative anaerobe  Flagella are encoded by the H7 antigen  Produces Shigatoxin

Lipopolysaccharide (LPS) = O antigen O1-O181

Flagella = H antigen H1-H56

(USDA-ARS, Clay Center, Nebraska)

Verocytotoxin-producing E. coli (VTEC) Shiga toxin-producing E. coli (STEC)

(Konawalchuck et al., 1977) (O Brien et al., 1983)

STEC cause a serious illness with symptoms of severe bloody diarrhea and kidney failure hemolytic uremic syndrome (HUS)

The STEC serotype that causes the most outbreaks and cases of HUS in the United States is E. coli O157:H7

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According to the CDC, an estimated 73,000 E. coli O157:H7 infections and at least 37,000 nonO157:H7 STEC infections occur in U.S. each year

E. coli O157:H7 infections leads hospitalizations and 60 deaths annually

to

2000
(USDA, 2008)

2011 E. coli O157:H7 E. coli O157:H7 2010 E. coli O157:H7 E. coli O145 : E. coli O157:H7 Poultry 2009 E. coli O157:H7 E. coli O157:H7 E. coli O157:H7

Lebanon Bologna Hazelnuts Bravo Farms Cheeses Romaine Lettuce from Beef from National Steak and

Beef from Fairbank Farms Beef from JBS Swift Beef Company Prepackaged Cookie Dough

Non-O157 STEC strains are equally linked to several outbreaks and can be isolated in a similar frequency as E. coli O157:H7 (CDC, 2008)

USDA and beef industry reported at least six other strains of STEC O26, O45, O111, O145, O103 that are equally as virulent as E. coli O157:H7
(Bielaszewska et al., 2005)

Occurrence Non-O157 Serotypes

E. coli O26 E. coli O111 E. coli O103 E. coli O121 E. coli O45 E. coli O145

CDC, 2006

20-70 % of STEC infections throughout the world are due to nonO157 STEC (WHO, 1998) A four fold increase in incidence of non-O157 STEC has been reported from 2000-2006
(FoodNet, CDC)

Non-O157 are underrepresented

This pathogen is widely distributed within bovine populations, where it is a benign commensal of the gastrointestinal tract
(Galland et al., 2001)

STEC isolates have been recovered from a variety of ruminant and non-ruminant: cattle, sheep, goat, pigs, poultry, dogs and cats

There are 3 general modes of STEC transmission :



Foodborne transmission - 52% infection  41% ground beef  21% manufacturing process Person-to-person contact - 14% infection Environmental transmission - 9% infection
(Rangel et al., 2005)

STEC Transmission Model

Meat and Pet Animals

Meat Cow
Contact

Water

Cow

Milk
Water

Human Human

Manure
Deer

Fruits and vegetables

Sulka, 2005

The spread of antibiotic-resistant (ABR) bacteria is serious public health concerns

(WHO, 2003)

Antibiotics resistance for Ampicillin Tetracycline SulphamethoxazoleTrimethoprim, Chloramphenicol has increased among E. coli (Maynard et al., 2004)

http://benefitofmanukahoney.com/tag/vre/

In 1980s, ABR was uncommon in E. coli O157:H7 1% (2/200) of E. coli O157:H7 collected by CDC 1983 1985 were resistant to antibiotics (Bopp et al., 1987)

2.9% (5/174) E. coli O157:H7 strains were resistant to an antibiotic (Ratnam et al., 1988) In more recent years, however, the isolation of antibiotic resistance STEC has shown an increasing trend

Streptomycin-sulfisoxazole-tetracycline (SSxT) resistance was dominant among E. coli O157:H7

(Willshaw et al. 1996)

70% E. coli O157:H7 isolated from ground beef, showed similar resistance (SSxT) (Meng et al. 1998) Kang et al. (2005) reported >80% of 296 commensal E. coli isolates from animals were resistant to Tetracycline sulfamethoxazole and streptomycin

E. coli O26, O103, O111, O128, and O145 isolates. Sulfamethoxazole - 59% Streptomycin -59% Ampicillin -56% Tetracycline -56% Cephalothin -50% Trimethoprim-sulfamethoxazole -38% Chloramphenicol -34%
(Schroeder et al.,2002)

Zero-tolerance policy for the presence of E. coli O157:H7 in ground beef has been in place since 1996


Sorbitol-MacConkey agar supplemented with cefixime and tellurite (ct-SMAC) Chromocult Rainbow agar

 

Specimen

Colorless colony on SMAC agar

Agglutination in O157 antiserum

Pulsed field gel electrophoresis

PFGE Patterns to PulseNet

Stool Specimen

GN broth

Stx EIA

Note: no SMAC plate, no colony, no PFGE, no PulseNet

   

Presence of sorbitol fermenting E. coli O157:H7 No selective medium available Look like normal E. coli Long identification time 2-4 days
Sorbitol MacConkey agar (SMAC)

Enzyme immunoassays (EIA), were first licensed in1995 for being used in labs ELISA, Latex agglutination, IMS Most commercial kits target  O157 antigen  Shiga-toxin antigen

Commercial Stx EIAs

 Premier EHEC  ProSpecT Shiga Toxin  Duopath Verotoxin GLISA  ImmunoCard STAT! EHEC  BioStar OIA SHIGATOX

Stx EIAs cannot differentiate  between E. coli O157:H7 and other STEC serotypes  between Stx1 and Stx2 (more serious symptoms) False positive reactions are not uncommon Many lab tests for only Shiga toxin antigen

PFGE Gold Standard Drawbacks

Its can not differentiate non-O157 serotypes Complicated data analysis may raised reproducibility issues

A number of PCR based methods are available for the detection of E. coli O157:H7 and STEC

Samples are enriched in selective broth PCR for presence of Shiga toxin & virulence genes

USDA-ARS, Clay Center, Nebraska

Recently a Multiplex Taqman Real- Time PCR based assay was developed for the detection: O26, O45, O103, O111, O121, O145, 091. Detection limit 1-10 cfu/ml
(Fratamico et al.,2011; Valadez et al.,2010)

Drawback: They are very expensive

Macroarray system for detection of Non:O157 strains O26, O111, O103,O145 and also O157 A cloth-based hybridization array system Detection limit: 10-60 cfu/ml The advantages of this assay: Polyester cloth is inexpensive and rapid It detects 6 different STEC strain with virulence gene
(Blais et al., 2011)

Diarrhoea/Blood Samples

36 % samples Reference Labs

64% onsite STEC testing

89% Culture based

7% Stx EIA

4% Culture based + EIA

No lab used PCR based methods for detection of STEC

(Hoefer et al., 2011)

 STEC are a natural part of the animal microflora  Non-O157 STEC is probably just as prevalent, maybe more,

than O157 STEC.

 No selective medium for non-O157 STEC  Lack of any test for simultaneous identification STEC and

detection of Virulence and Antibiotic Resistance genes

 Lack of clear guidelines for testing, interpretation of results,

and reporting

Thank you