BIOS2060 Microbial mechanisms of foodborne disease

The genus Salmonella

Dr Lim Yin Sze

Lecture materials are provided by Prof. Christine Dodd
Learning outcomes

• Understand the characteristics of Salmonella spp.

• Understand the symptoms and mode of action for Salmonella
poisoning and infection
• Understand the source of Salmonella spp. and their route of
transmission
Family Enterobacteriaceae
• Found primarily in the gastrointestinal system of animals
and therefore in faeces.
• Cause contamination of animal products but also any source
where faecal cross-contamination occurs
Characteristics:
• Gram negative rods
• Facultative anaerobes
• Non-spore former
• Growth optimum 37oC
• Bile salt tolerant

Genera:
• Salmonella, Escherichia, Shigella, Yersinia 3
 Salmonella
and
Escherichia
Escherichia coli
Salmonella spp.

Both E. coli and Salmonella

are Gram negative rods

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 Differentiating Enterobacteriaceae
Biochemical tests:
Carbohydrate fermentation (lactose) splits Enterobacteriaceae
into two groups:
• Coliform: lactose-fermenting bacterium
• Escherichia, Klebsiella, Enterobacter
• Non-coliform: lactose non-fermenting bacterium
• Salmonella, Shigella, Yersinia

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 lactose non-fermenting
MacConkey
non-coliforms:
Salmonella agar
• Selective
agent:
bile salts

• Diagnostic
agents: lactose
plus phenol
red pH
indicator

lactose-fermenting Coliforms: E. coli 6

 Xylose lysine deoxycholate (XLD) agar
Salmonella

E. coli

• Salmonella ferment xylose, decarboxylate lysine and metabolise thiosulphate

to produce hydrogen sulphide, which leads to the formation of colonies with
black centered.
• Coliforms such as E. coli will ferment lactose present in the medium and acidify
the medium turning it yellow. 7
Serotype or Serovar
A group of intimately related microorganisms distinguished by a common set of
antigens
The various Antigens possible on a bacterial cell
 Serotyping
Distinction of species/sub-types (serovar/serotype) based on
antibody testing:
• O antigen: lipopolysaccharide (LPS) of cell surface
• H antigen: flagella
• Vi antigen: capsule (Salmonella)

Simple slide agglutination reaction:

reacting known antiserum (antibody)
with bacteria to determine if they have
the reacting antigen

+  9
 Use of serotyping
• In most species, different serotypes/ serovars are reported by
number
• E. coli O157:H7
• In Salmonella each serotype was given a species name;
• > 2200 serovars (Kauffman-White scheme)
• Salmonella typhimurium O1, 4, [5],12:Hi;1,2
• Salmonella lagos O1, 4, [5],12:Hi;1,5

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 Evolutionary relatedness of Salmonella

Salmonella enterica I
(S. enterica)

S. enterica II
(S. salamae)
S. enterica IV
(S. houtenae)
S. enterica IIIb
(S. diarizonae)

S. enterica VI
(S. indica)

S. enterica IIIa
(S. arizonae)
Salmonella bongori

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New taxonomy:
– Most Salmonella causing food borne diseases are serovars of the species Sal. enterica and
subspecies enterica.
– Sal. enterica subspecies enterica serovar Typhimurium or shortened to Sal. Typhimurium
 Salmonella physiology
• Typical members of the Enterobacteriaceae
• Catalase positive, oxidase negative and generally motile with
peritrichous flagella.
• Growth range: 5 - 47 oC (opt. 35 - 37 oC)
• Sensitive to heat, usually killed at temperature ≥ 70oC (eg
pasteurisation temperature 72oC < 0.1min)
 but heat resistance might increase by sub-lethal heat shocking
(e.g. 48 oC 30 min) or low aw media.
• pH range for growth: 4.0 - 9.0 (opt. 6.5 – 7.5)
• Required high Aw for growth: 0.94 – 0.99
• Causing following clinical syndromes:
– Enteritis – infection localized to intestine
– Enteric (Typhoid) fever – systemic disease
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Enteritis
• Most common type of Salmonellosis
• Gastrointestinal infections due to ingestion of live cells (high dose of 10 6
cells but also depends on serotype, food vehicles or susceptibility of
individual) (Infection!)
• Such as S. Enteritidis, S. Typhimurium
Symptoms:
• nausea, diarrhoea, vomiting; mild fever and abdominal pain are also
common.
• Blood in stool is not uncommon
• Degree of symptoms varies between patients
• Incubation: 6 – 48 h
• Self limiting: 1-7 days
• Mortality: ~ 0.1%
• After symptoms subside, some patients continue to secrete organism in
their stools for up to 3 months; 1-3 % may excrete for more than a year
(asymptomatic carrier) 13
 Enteric fever
• Host adapted serotypes
• Humans: Sal. Typhi
Sal. Paratyphi
• Cause severe systemic disease
• Enteric fever (typhoid/paratyphoid)
• Most significant route - water borne but person to person
transmission also a factor
• 10-15% mortality rate untreated

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Stage 1:
Enteric fever
• Ingestion of live cells  invade from intestine to lymph nodes
• Released from macrophage into blood stream
• Disseminated to organs (liver esp. gall bladder, spleen, kidneys, bone
marrow) after 24-72h - Septicaemia
• Slow onset of rising fever, red spots which fade on pressure, abdominal pain.

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Stage 2:
Enteric fever
• Further multiplication and shedding
• Reach gall bladder and multiplies in bile, re-infect small intestine
• causing inflammation and ulceration.
• Fever persists but with onset of diarrhoea (“pea soup” stool) or
constipation.
• In severe cases, haemorrhage of the ulcer and perforation of the small
intestine and leading to periodontitis.

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 Typhoid/paratyphoid fever symptoms
• longer incubation period: 8-15 d (can be from 3 - 56 days)
• Symptoms: Rising fever, rose red spots which fade on pressure (below chest,
abdominal pain, splenomegaly, abdominal complications of intestinal
haemorrhage or perforation.
• Most patients also reported headache, constipation (adult) or diarrhoea
(children)
• Small proportion report nausea, vomiting, abdominal cramps and cough.

• After remission of symptoms, a carrier state can persists for several months
or occasionally years
• Gall bladder still colonised by the bacteria and discharged into faeces.

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Infectious dose
• How many cells do you need?
106 cells/g usually quoted but it depends on following:
• Age: very young or old more susceptible
• Strain of Salmonella: some are more virulent than others

Food sources detected in a number of outbreaks:

• Cheddar cheese  1.5 – 9.1 cells/100g
• Chocolate  1 cell/g

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Treatments
• Normally self-limiting
• Only treatment is fluid and electrolyte replacement
• Antibiotics normally only given when systemic disease occurs
such as Chloramphenicol, ampicillin and amoxicillin.
• Chronic carriers can be treated with antibiotics, but in cases of
relapse  Cholecystectomy (surgical removal of the gall
bladder) is needed.

Prevention
• Safe drinking water
• Effective sewage disposal
• Hygienic food preparation
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 S. Typhimurium DT104
• Salmonella Typhimurium DT104 emerged initially in cattle in the UK
• Now detected in poultry, sheeps, pigs and horses in several countries
worldwide including: the United States, the United Kingdom (UK),
Canada, Germany, France, Austria, and Denmark.
• Of particular concern with DT104 is the presence of a multiple
antimicrobial resistance pattern (R-Type ACSSuSpT) to Ampicillin,
Chloramphenicol, Streptomycin, Sulphonamide, Spectinomycin and
Tetracycline

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 Mechanisms of
Salmonella pathogenicity

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 Mechanisms of Salmonella pathogenicity
Gastroenteritis:
• microorganism attaches to and penetrates epithelium of small
intestine causing disease by killing epithelial cells and inducing
fluid accumulation (diarrhoea)
• Various virulence factors involved.

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 Pathogenicity islands: SPI-1
• Contain 25 genes
– 40kb region of DNA absent from E.
coli genome
• promote invasion of epithelial cells
• some genes show relationship to similar
genes on invasion plasmid of Shigella
• present in ALL Salmonella

Pathogenicity islands: SPI-2

• Contain 17 genes
• Systemic infection - spread from intestinal
tissue via lymphatics into bloodstream
• present in Sal. enterica subgroups
– eg Sal. Typhi, Sal. Typhimurium

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H antigen: flagellum

• Bacteria can have >1 flagellin protein

– Typically considered alternatives BUT even in a
single culture subpopulations can express different
phases
• Each of these can be tested for by antibodies

• In Salmonella:
• Phase 1 antigen (fliC gene) is either specific for a serovar or shared by a few
serovars only.
– Hence phase 1 is called the 'specific' phase.
• Phase 2 antigens (fljB gene) are widely shared.
– Hence it is called the 'non-specific' or 'group' phase.
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 Capsular antigens

• These tend to be specific to

particular organisms,
– eg Vi antigen in Salmonella
• Acidic polysaccharide; covering
layer outside the cell wall

Genes/operons: adhesins
• Type 1 fimbriae (fim)
– found in all Salmonella
• Long polar fimbriae (lpf)
– present only in some serotypes;
– thought to have been lost by some
Sal. enterica subgroups by later
deletion event
• Responsible for initial attachment 26
events
 Genes/operons: Enterotoxin
• Mediated by stn gene
• biological activity similar to the
cholera toxin
• increases vascular permeability
• cause fluid loss

Salmonella virulence plasmid

• Coded by spv operon
• needed for systemic infection
• host range factor needed for
intracellular multiplication in liver
and spleen
• found in Sal. Typhimurium, Sal.
Enteritidis, Sal. Choleraesuis, Sal.
Dublin
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 Epidemiology of
Salmonella serotypes

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 Foodborne outbreaks in 2009
implicated food vehicle by pathogen
VTEC
Food vehicle Salmonella spp. Norovirus Campylobacter
O157
L. monocytogenes Other^ Unknown Total

Poultry meat 9 - 9 - 1 2 1 22
Red meat 2 - - 4 1 2 - 9
Finfish - - - - - 5 - 5

Crustacea & shellfish - 12 - - - - - 12

Vegetables & fruit 3 - - - - 1 - 4

Dessert, cakes,
confectionery
3 - 1 - - - 1 5

Milk & dairy products - - - 1 - - - 1

Condiments & sauces 2 - - 1 - 1 - 4

Composite / mixed foods 8 - - 1 - 4 1 14

Eggs & egg dishes 6 - - - - - - 6

Rice 2 - - - - 1 - 3
Total 35 12 10 7 2 16 3 85

Other pathogens/toxins include Bacillus spp., C. perfringens, Scombrotoxin, Shigella spp., S. aureus,
mixed outbreaks, and outbreaks where the organism was unknown (all with representation of ≤5
outbreaks).
Health Protection Report News Archives Volume 4 No 19; 14 May 2010 30
http://www.hpa.org.uk/hpr/archives/2010/news1910.htm#efoss
Trends in Salmonella case incidence 1990- 2008

Salmonella

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 Food sources
• Cells heat sensitive
• Presence in food often because of undercooking or
contamination of product after heating process
• Commonest food vehicles:
• poultry and poultry products
• eggs esp. undercooked
• meat and meat products
• milk and milk products
• vegetables especially raw
• chocolate

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 New food sources for Salmonella
• Salad vegetables increasingly recognised as a source of Salmonellosis
• Previously a number of incidents with bagged ready-to-eat salads
– Salmonella Typhimurium (multi-antibiotic resistant strain) and S. Newport
• Currently Salmonella Bareilly cases are being linked to the consumption of
raw/under-cooked bean sprouts.
• Halva:
– Turkish candy made with ground
sesame seeds and honey, often with
added fruit and nuts
– S. Typhimurium DT104
• Peanuts: S. Stanley and S. Newport
• Fresh herbs: S. Senftenberg

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Salmonellosis could be due to cross contamination via
Faecal – Oral route

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Learning outcomes
• Understand the characteristics of Salmonella spp.
• Understand the symptoms and mode of action for Salmonella
poisoning and infection
• Understand the source of Salmonella spp. and their route of
transmission

Additional readings on Salmonella:

https://www.aphis.usda.gov/.../emergingissues
/downloads/dt104.pdf
https://wwwnc.cdc.gov/travel/diseases/typhoid
https://www.nhs.uk/conditions/typhoid-fever/