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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.
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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
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!! 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
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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
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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
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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
,-.+"*/0$/'1-2/$
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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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!! !"#$%"&'()*"+,"%-./.%"0123"4156"'"!"&'()*"+,"7/8%9"
!! :*'5";*
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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
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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
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Malt-o-Meal outbreak: 08-0333
Outbreak strain: 08-0024249 Sump strain: R0102/09
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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
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!! 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
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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