Séamus Fanning, UCD Centre for Food Safety

School of Public Health, Physiotherapy & Population Science, UCD Veterinary Sciences Centre,

University College Dublin, Belfield, Dublin 4, Ireland.

National Zoonoses Conference (NZC), O’Reilly Hall, University College Dublin,

Wednesday-8th June, 2011.

General Characteristics

!! Member of the Enterobacteriaceae family

!! Gram-negative bacilli, non-spore forming

!! Facultative anaerobes

!! Normally motile with peritrichous flagella

!! Often inhabit gastrointestinal tract and also

found in the environment

Mode of infection

!! Ingestion of contaminated food/water

!! Passage through digestive system and invasion of

intestinal epithelia

!! Subsequent passage to vasculature system can

lead to systemic infection

Salmonella –the bacterium

!! rarely lactose fermenting, there are 2,500 serotypes of Salmonella

!! Salmonella serotypes occur worldwide, infecting mammals, birds and reptiles and are excreted in

their faeces

!! Enteritidis and Typhimurium are the serotypes often reported

!! diagnostic protocols aid in elucidating the epidemiology of these pathogens

Salmonella genus

Traditional Method

ISO 6579:2002

25 g food sample in 225 ml

Buffered Peptone Water (37oC, 18 h) DAY 0

XLD & BGA (37oC, 24 h)

0.1 ml culture in

10 ml RVS broth

(41.5oC, 24 h)

1 ml culture in

10 ml MKTTn broth

(37oC, 24 h) DAY 1

DAY 2

TSA (37oC, 24 h) DAY 3

Biochemical & Serological Testing DAY 4

!! standardised and validated protocols are

in place to facilitate the recovery of

Salmonella from a food matrix

!! present in food in low numbers

!! Salmonella are usually stressed and must

be allowed to recover prior to isolation on

selective culture media

!! the strategy can facilitate the culture of

Salmonella

!! for epidemiological purposes additional

tests must be performed to improve the

definition of the isolate

Salm

on

ella

Ag

on

a

Salm

on

ella A

go

na

Sal.

Typ

him

uri

um

Do these isolates have the same phenotype?

Why sub-type bacterial isolates?

!! for quality control purposes, where bacteria are added to foods

!! to define the ecology of a food production site and identify persistent

isolates that have adapted and to locate contamination hot-spots

!! to protect public health

Salmonella in contaminated food

(ingestion)

Attachment to epithelial cells

Injection of effector proteins

Invasion & Production of

chemotactic factors

Macrophage death and/or

Survival inside these cells

Systemic infection

Phagocytosis (macrophages) and/or

Infiltration of PMN’s

Migration of PMN’s

(through epithelial cells)

Detachment of

epithelial cells

Fluid secretion (diarrhoea)

Stomach

(pH 2)

Intestine

(pH 9)

Host cells

Lamina propria

(pH 8.7-9.4)

Host cells

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CCP Step

Bacterial stress challenges-

-! pH food matrix

-! hygiene measures (biocides)

-! moisture (aw)

-! heat

-! antibacterial measures

Stress increases

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It is not the strongest of the species that survives,

nor the most intelligent that survives.

It is the one that is the most adaptable to change

Charles Darwin

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!! :*'5";*

Food

matrix

Year Pathogen No. affected

(country/ies)

Details

Cereal

(puffed)

2008 S. Agona 28 Puffed rice and puffed wheat cereals implicated in the outbreak were manufactured at the same plant

that manufactured toasted oat cereal implicated in a 1998 outbreak of S. Agona infections. S. Agona

was isolated from the plant and from bags of puffed rice cereals

Cereal

(toasted)

1998 S. Agona 209 An opened box of cereal yielded a S. Agona isolate with a PFGE pattern indistinguishable from

the predominant PFGE pattern among outbreak-associated clinical isolates. Cereal from

unopened boxes was also positive for S. Agona

Tea

(aniseed)

2003 S. Agona 42

(Germany)

S. Agona was isolated from six brands of tea containing aniseed. Various serotypes were

isolated from 61 (11%) of 575 tea and other products containing aniseed. S. Agona survived

upon exposure to hot water during tea-making

Snack

(savory)

1994 S. Agona

PT15

>2,200

(Israel; UK &

USA)

Snacks were manufactured on at least seven separate dates over a 4-month period. Levels

were estimated to be 2 - 45 CFU/25 g packet of peanut flavoured snack

common feature in all of these outbreaks –low moisture food

Outbreaks linked to Salmonella Agona

Collection of isolates

[pre-, post-outbreak]

Sub-typing by PFGE

Genetic characterisation

[genotype]

Physiological characterisation

[phenotype]

Optical mapping

Genome sequencing

gap closure

annotation

Biofilm formation

Growth curves

Acid tolerance

Tolerance to biocides

Motlity

Ex vivo cell culture studies

Malt-o-Meal outbreak: 08-0333

Outbreak strain: 08-0024249 Sump strain: R0102/09

Genomic DNA is captured as single DNA

molecules

DNA is digested with a restriction

endonuclease

Fluorescent intensity is measured to

determine fragment sizes

Order is maintained Whole genome restriction map

!! sub-typing supports epidemiological investigation, though these methods can also be

applied in a broader context

!! though isolates may have indistinguishable DNA fingerprints, there phenotypes may

differ, reflecting adaptation to a stress condition

!! failure in a critical CCP may lead to the escape of an adapted pathogen (following pre-

selection at one or more stages)

!! food safety management systems must be integrated with an environmental

management system

!! a structured collaboration between stakeholders can facilitate a better understanding

of these issues

Marta Martens

Matthew McCusker

Sarah Finn Karen Power

Acknowledgements

Evonne McCabe

Denis O’Leary

Kaye Burgess Geraldine Duffy

Alan Reilly

Wayne Anderson Food Institutional Research Measure (FIRM)- Network & Team Building Initiative

grant no.: 06/TNIUCD10