MIKRO575 Outi Martikainen 071111-Eng

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17.11.2011

Escherichia coli

Food and environmental hygiene and control MIKRO575

7.11.2011

Outi Martikainen / Bacteriology Unit

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The lecture structure

• Escherichia coli bacterial species

• Beneficial and harmful glycols

• E. coli insulation

• E. colityping

• E. coli control



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Escherichia coli bacterial species

• Gramnegative rods

• facultative anaerobic

• The mobile also stationary positions

• Biochemical properties

Lactose Positive (90% of the strains)

Betaglukuronidaasipositiivinen (97% of the strains)

Indolipositiivinen (99% of the strains)

• Position taxonomically

Fyyla: proteobakteerit Class: gammaproteobakteerit sect:Enterobacteriales family: Enterobacteriaceae family: Escherichia

Type: Escherichia coli

. Local species include E. Albert, E. Herman E. Ferguson,

• colonize humans and other mammals and birds in the large intestine, part of thethe normal flora microbiota

• Neonatal intestinal colonized within a few hourschildbirth

• genetically diverse species

. The exchange of genes in Salmonella spp, Shigella spp. and other E. coli with stocks

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Escherichia coli



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Kaper et al 2004 Nature Reviews

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Useful glycols

• Normaalimikrobiston glycols prevent diseasecausing microbes

infecting the host's intestine or reproducing there

• vitamin K2 return in the colon

• Probiotics

E. coli Nissle 1917:

• in the treatment of chronic intestinal diseases

• reduce the pathogenic glycol (invasive and

The adhesive) adhesion to intestinal epithelial cells, and

increases the production of cytokines (Huebner et al, 2011)

• model organism in genetics

Kloonausisäntänä recombinant DNA techniques

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Hazardous glycols

1. Intestinal Infections

2. Urinary tract infections

3. Meningitis / sepsis

• Kommensaalikolit can cause disease

Weakened immune system

The intestinal epithelium damaged

Bacterial "wrong" place

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Suolistopatogeeniset E. coli

= Ripulikolit

• Cause intestinal infections in humans diarrhea, bloody diarrhea, andvomiting, and possible complications such as haemolyticuremic syndrome (HUS)

• Ripulikolit can be divided into at least five classes = dam groups:

Enteropathogenic E. coli (EPEC)

Enterotoxigenic E. coli (ETEC)

Enterohemorraaginen E. coli (EHEC)

Enteroinvasive E. coli (EIEC)

Enteroaggregatiivinen E. coli (EAEC)

• Ripulikolit occur in animals, especially ruminants, intestinessuch as the normal flora E. coli. They have also been found to besoil and water systems.

• Humans can get infected food of animal originthrough drinking or faeces of animals or human infectedwater.



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Ripulikolien virulenssimekanismit

• Different groups of different virulence pathogens

• Some features may occur in more dam group

• the virulence genes are located on mobile elements (integrated

transposons, bacteriophages and Pathogenicity) of the bacterium

chromosome and plasmids

• subject of the virulence genes of intestinal epithelial cells

Intestinal epithelial colonization

Penetration into cells

Return on toxins and excrete epithelial cells

Nonpathogenic glycol may change by having a pathogen

kolikannoilta or other virulence genes from other species.

Various dam groups can benefit from each other virulence genes new

hypervirulentteja positions?

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EHEC bacteria

• AKA

= VTEC Verotoxigenic E. colitoxic for Vero cells (monkeykidney cells)

STEC = shigatoksiiniaproducing E. coli

• virulence factors

Shigatoksiinit stx1 and Stx2 (stx1and stx

2)

= Intimiini adhesion protein (eae)

Enterohemolysiini (EHEC hly / EHX)

STEC autoagglutinoiva adhesin, Make(Gets)

• Diarrhea / bloody diarrhea



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• sequelae haemolytic uraemicsyndrome (HUS)

• EHEC O157 serogroup strains seriousthe clinical picture most wanted serogroup

• Highrisk groups: children under 5 years children, the elderly

Kaper et al 2004 Nature Reviews

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Pato groups virulenssimekanismit

Kaper et al2004 NatureReviews

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EHEC infection routes

• Cattle, sheep (animal

usually asymptomatic)

• Pets



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• Poorly cooked or raw

minced beef

• Unpasteurised

(milk) products

• Fresh fruits, salads, sprouts

• Water; lakes, ponds

• Human transmitted to another

contagion

• Hospitalacquired infections (food;

salad, kotileivokset)

• Laboratory Infections

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The infectious doses

• EHEC 10 100 cells

• EPEC 10 8 cell

• ETEC 10 8 cell

• EIEC 10 10 6 cell

• 10 EAEC 8 10 10 cell

• EHEC, EPEC, ETEC and the APEC (avian pathogenic E.

coli) in animal pathogens

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Infection Treatment

• Diarrhoeas selflimiting

• EHEC antibiotics is not recommended

Lysis Release of shigatoksiinin

Symptoms get worse

• Asymptomatic Carrier

EAECdam groups, as well as suffering from diarrhea that

healthy children in Africa (Bonkoungou et al 2011)

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E. coli isolation

• Food

• Environmental sample

• Human (feces, urine, blood)

• Concentrates

• Selektiivimaljat

SMAC (sorbitolMacConkey)

CTSMAC (cefiximetelluriitti

SMAC)

CLED (cysteine lactose electrolyte

deficient medium)

• Great weakness

The pathogenic glycol is no different in appearance or biochemical

the characteristics of the normal flora

Search for virulence genes (PCR, DNADNA hybridization)

Meat samples peptonisuola

solution.



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Selektiivimaljat

• SMAC sorbitol

Fermentation

EHEC O157 serogroup

positions often

sorbitolinegatiivisia, but

Also sorbitolipositiivisia

strains are often isolated from the

• CLED lactose

Fermentation

• Enterohemolysiinin return

pointing bowl (TBVP)

shows only the enteric

hemolytic strains

SMAC:

Sorbitol

fermentative

stem

SMAC:

Strain which is not

ferments

sorbitol

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Screening of colonies

mixed culture by PCR

• Individual colonies

• Colony "pools" many of colony testing

in one reaction

• It can be a daunting task if the pathogen concentration

sample low



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Colony hybridization

• bowl colonies leave a DNA imprint membrane stamped

probe pairs with pathogenic colony with imprint antibody

+ Color bind pathogen positioning the plate

After hybridization, the membranes.Health replicated hybridisaatiomembraanille.

Positive control Negative control Positive control Positive control

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Immunomagnetic enrichment (IMS)

• serogroups based enrichment method

• serogroups O26, O103, O111, O145, O157



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• The beads on the surface of serogroup antibody sticksbacterial surface structure the bacteria can be searched

recovered with the beads and the other end to

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If you can not isolate ...

• Monialukkeinen PCR method, the mixed culture ripulikolien

to detect

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E. colityping

• Serotyping

serogroup = O structure (O = somatic antigenLPS)

serotype = O structure H (H = a flagellar antigen)

EPEC O26, O55, O86, O111, O125, O126, n.127, O128

EHEC O26, O45, O91, O103, O111, O113, O145, O157(O157: H7, O157: H)

• EHEC O157 phage types, serotyping, more detailed distinctions

• Other fenotyypitysmenetelmät

• genotyping

Virulence genes

Pulsed genetic fingerprint

Sequencing (antigen / total genomic)

Such as the virulence genes stx subtyping of specific primers orThe PCRRFLP method

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Serotyping

• Otyping

• Htyping

• Lasiagglutinaatio

• Realtime PCR

7 most common human pathogens E. coli O group

(O26, O55, O91, O157, O103, O111, and O145) and

flagellar antigen H7



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Phage types,

• EHEC O157 strains

0%10%20%30%40%50%60%

PT2 PT4 PT49 PT8 Other

Occurring in Finland EHEC O157phage types

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Other fenotyypitysmenetelmät

• antimicrobial susceptibility

• Biochemical identifioimistestit

Lactose and sorbitol fermentation

Βglucuronidase return (PGUA)

• Enterohemolysiinin return indication

• Shigatoksiinin return indication

Verosolukoe



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Lateksiagglutinaatio

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Pulsed (PFGE)

• Comparison of the same serotype

(OH structure) strains

between

• bacterial chromosomal

DNA cleavage

restriction enzyme

EHEC XbaI

• Electrophoresis, wherein

electric current changes direction

at certain intervals

• Epidemiological studies

eg. food poisoning

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PFGE genetic strains

fingerprint



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EHEC diagnostics Taba in

TABA of posted

Faecal Primary culture /

Pure culture

Where

+

API 20E

O: H serotypes

Phage types (O157)

Stx1 / Stx2 return

Ehly

Antibiogrammi

Sorbitol fermentation

Betaglucuronidase

Final answers

+ EHEC (O157)

VTEC + (nonO157)

EHEC

EPEC +

PCR positive colony

isolated and

identified by

PCR

stx1, Stx

2, Eae,

ehly may

PFGE

(Xba I)

Stx2subtypes

Eae subtypes

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Ripulikolidiagnostiikka



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• Find Finland and many other countries

usually only the EHEC pathogen O157 serogroup Group

dangerous serogroup (HUS)

• Many studies breakdown of EHEC O157 and

VTEC nonO157, although there are also other serotypes

has been found to be highly virulent (e.g. O26,

O103, O111, O145)

• Other ripulikoliryhmiä usually are not searched when

explaining the cause of intestinal infections

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0

500

1000

1500

2000

2500

3000

3500

4000

4500

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

Campylobacter Salmonella Yersinia Shigella EHEC

Food

bacterial infections in 20002010

Cases Details: THL / TTRegister



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EHEC cases annually

Certified EHEC cases in 19962010

2

51

29

11 6 July 7 6 4 5 6 93

13 930

1

1

6

0 January 1 0 010

2 00

30

57

9

13

21

12 July 7 96

6

March 65

13

11 159

61

4338

1815 15 15

10

21

14 128

29

2023

0

10

20

30

40

50

60

70

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

NonO157 (n = 135)

sor + O157: H7 / H(n = 25)

sorO157: H7 / H(n = 168)

Total (n = 351)

Photo: Lord of the Lienemann / THL

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EHEC epidemic in Finland

• Donegal, in 1997

Serogroup O157

N. 15 infection

Bathing water

• Southern Finland, 19982002

Serogroup O157

Minor epidemic clusters

Minced meat, cattle



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German EHEC O104: H4 outbreak

"The killer cucumbers"

• The outbreak strain of EHEC + EAEC features

Stx2, AGGR

Intimiininegative, negative enterohemolysiini

Multiresistant

• uncommon serotype O104: H4

• Affected 2684, 39 dead (by 20.7.2011)

• HUSsyndrome sufferers 1/3

• EFSA (European Food Safety Authority) and the

ECDC (European Centre for Disease) risk assessment Germany and

French epidemics probable common factor

Egyptian origin fenugreek seed sprouts

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Ripulikolit in Burkina Faso

• The Academy of Finland project

20082011: Food &

waterborne

enteropatogeenisten

bacteria Epidemiology

Burkina Faso



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• microbiological research

sustainable

public health

In support of the strategy

• No previous knowledge

enteric

epidemiology

• Detection of Ripulikolien

food and people

stools, EHEC strains

Isolation and typing

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Food "hygiene" in Ouagadougou reason

ripulikolien high incidence raa'oissa meats

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Comparison of the prevalence of virulence genes

ripulikolipositiivisissa meat samples, and



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children's stool samples

16PCR primers in a mixed culture

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Burkina Faso isolated EHEC strains

• Eristysmenetelminä colony hybridization and PCR screening

• 21 positions: 20 meat (beef and veal products, mutton) and 1

a child suffering from diarrhea stool

• All the heels of stx1, Side of the stx

2stx

2more virulent.

• All strains intimiininegatiivisia (eae gene), but

seven in favor of intimiinia potential substitute STEC

autoagglutinoiva adhesin (shall gene) (adhesion to the intestinal

epithelial cells)

• Vero cell experiment tested strain produced shigatoksiinia

• Serotypes O8: H, O38: H26, O82: H8, O179: H and

OX183: H18 has been previously isolated from patients of diarrhea

• serotype O2: H2 is previously isolated from a patient HUS



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The study conclusions

• Ripulikoleja occurred in 43% of the openair markets

forsale, as well as in raw meats

59% of diarrhea patients

young children in Burkina Faso

• Baby food is often prepared by

under the same conditions with the crude

processed meat Contaminated meat

may have young children ripulikoli

infections in the back

• EPEC and ETECdam groups

often performed as well as faecal that

meat samples potential

infectious access

• meat and sickly child

isolated EHEC strains are

a possible human

pathogenic

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E. coli control to improve the

• Food control

Studies related to food quality are

primarily the responsibility of the operator

Monitoring of selfsupervision

E. Using coli as an indicator (products, production facilities)

indicates faecal contamination;

The reduction of contamination of farm animals

After the slaughter and processing of carcasses washing with diluted

acid + cold chain

• Water quality monitoring Thermotolerant coliforms, E. coli



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Household water

Bathing water

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Mikrobikriteeriasetuksen (2073/2005)

studies in accordance with

• E. coli Look for:

Meats: minced meat, meat preparations, mechanically separatedmeat (cattle and sheep: EHEC additional research)

PISCES: shelled and cooked crustaceans and molluscs,live bivalve molluscs

MILK: destined for consumption in packedraw milk, raw milk pasteurization or weakerheattreated milk made from cream and butter,pasteurisation or a stronger heattreated milkunripened soft cheeses obtained from whey, oraged cheeses

VEGETABLES AND FRUIT: Precut fruit andvegetables, when intended for consumption (eg.salads, grated, frozen vegetables), fruit andvegetable juices (unpasteurised, such drinkable)

• additional study to look for EHEC bacteria

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EHEC surveillance

• Infectious Diseases Act (989/2006) and Decree (1376/2006)

People EHEC infections are classified as dangerous for the generalCommunicable diseases

• Food control

Meat Inspection

. For example, based on the ELISA method as a rapid test (antigenantibody complex formation) of EHEC O157: a



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• THL

Human strains typing

Epidemics statistics

• EVIRA in

Foodstuffs and typing of strains isolated from animals

Escherichia coli O157, O145, O111, O103 and O26 serogroups demonstration of cultivation method andby realtime PCR method

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Ministry of Agriculture and Forestry Ministry's regulation of bovine EHEC

studies in a slaughterhouse and venue

(24 / EEO / 2006)

" N accordance with 20 § of slaughterhouses and small slaughterhouses Food Acthouse control plan shall be included in the sampling EHECbacteriafighting measures to the slaughterhouse or a smallreceived from the faeces of cattle in accordance with Annex 1. Samplesmust be sent to the admissibility of the Food Act § 39 subsection 2.the laboratory under. The samples must be examined E. coli O157stocks in case. Research laboratory, samples shall be submittedhave isolated E. coli O157 strains of bacteria Food safetyAgency. Finnish Food Safety Authority shall ensure that theThe bacterial strains are also provided. "

" Finnish Food Safety Authority must be drawn up each year slaughterhouseson a sampling plan for the EHEC surveillance.The plan indicated the samples based on the slaughter volumesNumber of teurastamokohtaisesti. At the slaughterhouse must be sampledin accordance with the sampling plan. The samples must besporadic and be distributed as evenly as possible throughout the yearperiod. Small place of at least two samples must be takenper year. If your small abattoirs to slaughter less than 100 head of cattle a year,samples do not need to take. "

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Authorities: food poisoning The study,



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THL TART, Taba

Infectious diseases

register

Hospital districts

The National Food Agency

Food poisoning

register

Veterinary and food

Research equipment

The provincial government

Local Authority

Food Laboratory

Health protection

Authority

Health Centre

Food poisoning investigation group

Niemi et al. 2004

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Summary

• EHEC (O157 serogroup) the most important causes of food poisoning

glycol

Cases annually few other food borne pathogen

in comparison, but cause severe symptoms

• Ripulikolikantojen transformation as a threat Serious infections

• Prevention of infections through appropriate monitoring

• Tracing outbreaks requires cooperation between authorities



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THANK YOU!

[email protected]

MIKRO575 Outi Martikainen 071111-Eng

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