Clinical and Epidemiological Aspects of Escherichia coli

O157:H7 in Latin America

Alejandro Cravioto, M.D., Ph.DRosario Morales, M.D., Ph.D

Armando Navarro, B.Sc., M.Sc.Faculty of Medicine, UNAM

Mexico City

Dr. Alejandro Cravioto (b. Mexico City, 1947) In 1973, Dr. Cravioto received his Medical Degree with honors from the Faculty of Medicine at the National Autonomous University of Mexico. Between 1973 and 1976, he continued his studies in Pediatrics at the National Institute of Pediatrics in Mexico City. In 1977, he received a Diploma in Tropical Public Health, and in 1981, a Ph.D. from the London School of Hygiene and Tropical medicine at the University of London.

Escherichia coli

• The organism – Gram negative,

non-spore forming rod,

Fam. Enterobacteriacea

(aerobic/facultatively anaerobic)

• Serotypes – O, H & K antigens

Toxins

- ETEC produce LT and/or ST - EHEC produce Stx toxins

• Generally harmless

• Pathogenic groups

- Enteropathogenic (EPEC)

– Enterotoxigenic (ETEC)

– Enterohemorrhagic (EHEC) – HC, HUS, TTP

– Enteroinvasive (EIEC)

– Enteroaggregative (EAEC)

– Diffusely adherent (DAEC)

E. coli O157:H71982 – First recognized as a pathogen1985 – Associated with hemolytic uremic syndrome1990 – Outbreak from drinking water1991 – Outbreak from apple cider1993 – Multi-state outbreak from fast food hamburgers1995 – Outbreak from fresh produce1996 – Outbreak in Japan – Multi-state outbreak from

unpasteurized apple juice

Meat

General

foods

Lacteous

Ground beef

Hamburger

Roast Beef

Cooked beef products

Fermented sausage

Turkey roll

Apple cider

Mayonnaise

Vegetables

Raw milk

Pasteurised milk

Yoghurt

Cheese

USA, Brasil, Argentina,

Uruguay

USA, Canada, England, Japan

USA

Scotland

USA

England

USA

USA

USA, England, Japan

USA, Canada, England and

Scotland

Scotland

England

Scotland

Outbreaks associated with E. coli O157:H7 in food products

Food product Country

E. coli O157:H7

- incubation – 3 to 9 days - bloody diarrhea, renal failure, thrombocytopenia - infective dose – small to large number of organisms - the young & the elderly - dialysis, fluid balance, hypertension

Pathogenesis - EHEC (O157:H7) – HC, HUS & TTP

- Causes adherence & effacing lesions on intestinal cells (HC) - Systemic complications (HUS) – acute renal failure - toxin causes death of endothelial cells of glomeruli & afferent arterioles, narrowing of blood vessels, hemolytic anemia & thrombocytopenia, reduced glomerular filtration & kidney tissue necrosis

1990 1991 1992 1993 Scotland 3.4

(2.9-3.9) 4.0

(3.4-4.6) 2.3

(1.9-2.7) 2.4

(2.0-2.8) Wales 1.1

(0.7-1.5) 1.4

(0.96-1.8) 1.4

(0.96-1.8) 1.1

(0.7-1.5) England 0.5

(0.44-0.56) 0.7

(0.62-.078) 0.9

(0.82-.098) 0.7

(0.62-.078) (95% confidence interval)

Incidence of E. coli O157 infection reported by laboratories

(per 100 000 population)

City Cases HUS

Oku-cho, Okayama (May, 1996)

468 2

Sakai City, Osaka (July, 1996)

6561 101

Kyoto (July, 1996)

47 3

Outbreaks of enterohemorrhagic E. coli reported in Japan

Incidence of hemolytic-uremic syndrome in children 4 years of age or younger

(per 100 000 population)

Country HUS patients Controls P

Canada

Argentina

United Kingdom

Chile

12 / 40 (30)

4 /51 (8)

52 / 196 (26.5)

6 / 20 (30)

0 / 40 (0)

0 /25 (0)

2 /51 (3.9)

2 / 38 (5.3)

0.00019

0.30

0.00012

0.0158

Comparison of EHEC isolation rates from HUS patients in North America,

Europe, and South America

a Number of children with EHEC / number of children with HUS

whose stool cultures yielded E. coli (%).

Argentina has one of the highest recorded HUS rates (300 cases/year) in Latin America

The risk of HUS in Stx-EC associated bloody diarrhea is about 4-5%, with 14% of children developing incomplete HUS

A total of 80% of Stx-EC isolated from patients with diarrhea belonged to non-0157 serogroups such as O26, O91, 0103, O111, O113, O128, O145

Incidence of Shiga-toxin (Stx)-associated illness in Buenos Aires, Argentina

Period No. of Cases

Mean Range Cases Admitted

1957-1961 32 12.8 4.5-36 6

1962-1966 130 12.2 2-60 26

1967-1972 16 13.2 2-180 86

1982-1986 273 20.4 3-84 55

1987-1996 897 14.8 6-36 50

1997-1998 80 20.8 5-108 40

Children with HUS at the Hospital de Niños, Dr. Ricardo Gutierrez,

Buenos Aires, ArgentinaAge (months)

Origin of Shiga toxin-producing Escherichia coli strains

Country Serotype Number of Origin Time of Strains Isolation

Argentina Non-O157 8 Healthy steers 1997-1998 7 Healthy calves 1999-2000

O157:H7 2 Healthy and diarrheic 1998-1999

Veal Holstein calves Non-O157 9 Frozen Hamburgers 1998-2000 1 soft cheese sample O157:H7 6 Ground beef sample 2000 Non-O157 1 O157:H7 1 0 Animal and meat sample 1996-2000Brazil O157:H7 3 Healthy calves, heifers 1996-1997 and cows Non-O157 4 O157:NM 1 Calves with diarrhea Unknown Non-O157 3 Non-O157 9 Ground beef and 1995-1996 hamburger sample

Country Cases Brazil (2002)

First case of HUS

Brazil (1990, 2001)

3 Sporadic cases

Colombia (1996-1997)

3/39 (8% ) cases HUS 20/307 (7.0% ) from cattle

Costa Rica

5 Isolated from chicken giblets and raw milk

Enterohemorrhagic E. coli O157:H7 in Latin American Countries

In Mexico, there have been no reports of HC or HUS associated with O157:H7 strains

However, studies from our laboratory conducted between 1985 and 2003, isolated O157 non-motile or non-H7 strains in approx. 10% of the children studied

Origin Number of isolation

Serotype Sorbitol Vero-toxin

SLT1-2

Children < 2 years of

age 1985-1987

18 4 1

O157:NM O157:H10 O157:H28

+ + -

- ¼ -

- - -

Children < 5 years of

age 1988

2

O157:NM

+

-

-

Adult emigrant BCN 1995

1

O157:H7

-

+

+

Children < 5 years of

age 1996-2003

8

O157:NM

+

N/D

+

TOTAL 34/263 (13)

ND Not determinated

Escherichia coli O157 strains isolated in Mexico 1985-2003

N= 263

The responses of 605 human serumsamples against O157 LPS and its two cross-reacting LPSs, O7 and O116, were analyzed by ELISA.

Of the 605 samples, 562 came fromadolescents or adults and 43 from children of different age groups.

Human serum responses against different E. coli LPS’s by ELISA test

using a cut-off point of 0.7

Serum samples E. coli LPS

n(%) O7 (%) O116 (%) O157 (%)

Children 43 1 (2) 5 (12) 2 (5)

Adults 562 7 (1) 27 (5) 28 (5)

Total 605 8 (1) 32 (5) 30 (5)

05

101520253035404550

O7 O116 O157

>0.4

>0.7

Comparative responses of sera from children against different E. coli LPS’s

determined by ELISA test using a cut-off point of >0.4-0.69 and >0.7

E. coli LPS

(%)

0

5

10

15

20

25

O7 O116 O157

>0.4

>0.7

Comparative responses of sera from adults against different E. coli LPS’s determined by ELISA test using a cut-off point of >0.4-0.69

and >0.7

E. coli LPS

(%)

Immune response of human serum samples against E. coli O157 LPS by Western blotting using E. coli O157 LPS developed with rabbit and human serum samples.

kDa

Immune response of human breast milk samples against E. coli O157 LPS

kDa

Western blotting analysis of the serum samples with a positive ELISA result (using a cutoff point of >0.7) showed that 86% (24 of 28) reacted with the O157 LPS.

Further studies with breast milk and bovine serum samples showed that 71% and 23% respectively had a similar LPS response to that found in human serum studies.

Origin strain isolation

(%)

Serotype Sorbitol VERO-toxin

SLT1-2

Pork 1995

1/36 (3) O157:NM + - -

Bovine 1996

3/96 (3)

O157:NM

-

2/3

+

Escherichia coli O157 isolated from Animals in Mexico from 1995-1996

N= 132

Heterologous response of bovine serum samples against different LPS’s of E. coli

N= 310 LPS

80 160 320 640 1280 TOTAL (%)

O7 91 91 57 20 1 259 (84)

O116 59 133 82 19 3 296 (96)

O157 108 84 28 220 (71)

Serum dilutions

Positive samples

O7 (%)

90 (29)

O116 (%)

38 (12)

O157 (%)

70 (23)

TOTAL

198

Response of bovine serum samples

against O157 and cross-reacting LPS’s

LPSN=310

ELISA cut-off: 0.7 DO a 405nm.

Serum dilution: 1:800

These results suggest that early colonization by a non-pathogenicE. coli with an LPS that cross-reactswith O157 could be related to a low incidence of HC or HUS in childrenor adults from developing countries.