Bacteria Causing

Food Poisoning

Microbiology Department
Faculty of Medicine
2017
Introduction
There are two types of microbiological food poisoni
ng (diseases caused by ingesting food):

1.Food intoxication results from ingesting exotoxins

secreted from bacterial cells growing in food (e.g. B
acillus cereus, Clostridium botulinum, Staphylococc
us aureus) including bacterially produced exotoxins.
2.Food infection results from ingesting whole micro
bes that grow in the gut/intestines and produce an i
nfection (e.g. Campylobacter jejuni, Salmonella spp
., Salmonella typhii, Shigella spp., Vibrio cholerae,
Vibrio parahemolyticus, Clostridium perfringens, he
patitis A).
Symptoms

1. Intoxications are often immediate (sometimes wi

thin 20 minutes) with nausea, abdominal cramps
and violent vomiting. Illness is usually rather sho
rt-lived.

2. Infections require an incubation period of genera

lly 2 to 3 days. Typical symptoms: abdominal cra
mps, diarrhea, nausea, but there can be harsher
long-lasting effects.

4
 Classification of Enterobacteriaceae

There are several selective and differential media used to

isolate distinguishes between LF & LNF
The most important media are:
 MacConkey agar
 Eosin Methylene Blue (EMB) agar
 Salmonella Shigella (SS) agar
 In addition to Triple Sugar Iron (TSI) agar
5
Microbiological food infection

Escherichia coli
• Strains causing GI disease have virulence factors.
• Sources: contaminated food, water, examples: caused b
y O157:H7 strain in 2005.
• Enterotoxin production
• Ability to adhere to small intestin
• Diarrhea causing E. coli (classified according to virulenc
e)
– Entertoxigenic E. coli (ETEC)
– Enterpathogenic E. coli (EPEC)
– Enterohemorrhagic E. coli (EHEC)
– Enteroinvasive E. coli (EIEC)
• Domain: Bacteria
• Kingdom: Bacteria
• Phylum: Proteobacteria
• Class: Gamma Proteobacteria
• Order: Enterobacteriales
• Family: Enterobacteriaceae
• Genus: Escherichia
• Species: Escherichia coli (E. coli)
• Morphology Gram - ve Straight rods,
• 1-3 X 0.4 -0.7 microns,
• Appear in singles or in pairs,
• Motile by peritrichate flagella.
• Very few strains non motile
• Not spore forming, Non acid fast.

8
Enterobacteriaceae: Genetic Proper
ties

• Chromosomal DNA has 39-59% guanine-plus-cytosin

e (G+C) content
• Escherichia coli is the type genus and species of the
Enterobacteriaceae
• Species of Enterobacteriaceae more closely related
by evolutionary distance to Escherichia coli than to
organisms of other families (Pseudomonadaceae, Ae
romonadaceae)
 Cultural characters
• Aerobic / Facultative Anaerobic
• Grows between 10 – 40 C optimal at 37 C
• Grown in simple medium
• Produce Large grayish ,Thick white , moist smooth opaque col
onies
• May contain capsule.
• On MacConkey medium Produce Bright pink Lactose fermenter
s.

10
11
 Identification of Enterobacteriaceae
Biochemical reactions
• Oxidase test
– All members of Enterobacteriaceae are oxidase negative

– Pseudomonas is oxidase positive

• O/F test
– All members of Enterobacteriaceae are O+/F+

– Pseudomonas is O+/F-

• Nitrate reductase
– All members of Enterobacteriaceae are nitrate reductase positive

– Pseudomonas is nitrate reductase negative

 E.coli
Biochemical Characters,

Glucose,Lactose,Mannitol,Maltose fermented. with A/G

I,M,Vi,C tests.
Indole +
Methyl Red +
Voges Proskauer – ve
Citrate –ve
Urease not produced.

13
 Identification of Enterobacteriaceae
Differentiation between LF & NLF by Growth on MacConkey a
gar
• Method:
– MacConkey agar is inoculated with tested organism
using streak plate technique
– Incubate the plate in incubator at 37 C/24 hrs
• Results:
– LF organism appears as pink colonies (e.g. E. coli)
– NLF organism appears as colorless colonies (e.g. Shi
gella)
Flame & Cool

1 2
3
4 Flame & Cool
5
14
Flame & Cool
 E.coli
Antigenic Structure

• Somatic 0 170
• Capsular K 100
• Flagella H 75
• Virulence factors
Surface Antigens Toxins
O Endotoxic activity
K protects against the phagocytosis
Fimbriae promote virulence ( important in UTI )

15
 Escherichia coli Toxins

• E.coli produce Exotoxins

• Hemolysins, Enterotoxins causes Diarrheas,

• Enterotoxins – produced by enterotoxigenic strains of E. coli (E

TEC). Causes a movement of water and ions from the tissues t
o the bowel resulting in watery diarrhea.

• There are two types of enterotoxin: LT and ST

• Heat labile (LT) Heat stable (ST)

Vero toxins VT Like Shigella toxins

16
 Escherichia coli Toxins (cont’)

• Produce Enterotoxin L T and S T

• Labile toxin 1956 De experiments in Rabbit illeal loop causes o
utpouring of fluids
• E.coli Labile toxin like Cholera toxin
• L T contains component A and B
• A = Active B= Binding
• B causes Binding with Gm I Ganglioside receptor on Intestinal
epithelial cells

17
 Escherichia coli Toxins (cont’)

• Both enterotoxins are comp

osed of five beta subunits (f
or binding) and 1 alpha sub
unit (has the toxic enzymati
c activity).

18
 Escherichia coli Toxins (cont’)

Labile toxin

•Component A Activated to A1 and A2

•A1 Activates adenyl cyclase in the enterocytes to for
m cyclic adenosine 5 monophosphate
•Causes to increase outflow of water and electrolytes i
n the gut lumen causes Diarrhea

19
 Escherichia coli Toxins (cont’)

Stable Toxin

•ST A and ST B
•ST A Acts by activation of Cyclic guano sine monophosphate.( C
GMP )
•Causes fluid accumulation in Intestine.
•E.coli ( Some ) produce Verocytotoxin causes cytotoxicity to Vero
cells.
•Acts like Shigella dysentery toxin

20
Escherichia coli Toxins (cont’) Mechanis
m of action of Toxins

• Increased cAMP alters the

activity of sodium and
chloride transporters
producing an ion imbalance
that results in fluid
transport into the bowel

21
 E.coli a Complex Microbe

• More than 700 serotypes of

E. coli have been
identified. The different E.
coli serotypes are
distinguished by their
“O” and “H” antigens
on their bodies and flagella,
respectively.

22
 Classification of E.coli

1.Enteropathogenic EPEC
2.Enterotoxigenic ETEC
3.Enteroinvasive EIEC
4.Enterohemorrhagic EHEC
5.Enteroaggresive EAEC

23
Pathogenic group
Enteropatogenic E coli (EPEC)
Enterotoxigenic E coli
(ETEC)
Enterohemorrhagic (verotoxin
producing) E coli (EHEC)
Enteroinvasive E coli (EIEC)
Enteroaggregative E coli (EAEC)
Diffuse aggregative E coli
(DAEC)
epidemiology
EPEC strains belong to particular O
serotypes cause sporadic cases and
outbreaks of infection in babies and
young children importance in adults less
clear
Most important bacteria cause of
diarrhea in children in developing
countries most common cause of
traveler’s diarrhea water contaminated
by human or animal sewage may be
important in spread
Serotype O157 most important EHEC in
human infections
Outbreaks and sporadic cases occur
worldwide
Food and unpasteurized milk impotant in
spread
May cause haemolytic uremic syndrome
(HUS)
Important cause of diarrhea in areas of
poor hygiene
Ionfection usually foodborne, no
evidence of animal or environmental
reservoir
Characterisitic attachment to tissue
culture cells
Cause diarrhea in children in developing
counties
Role of toxins uncertain
Laboratory diagnosis
Isolate organism from feces
Determine serotype of several colonies
withpolyvalent antisera for known EPEC
types
Adhesion to tissue culture cells can be
demonstrated
By a fluorescence actin staining test
DNA-based assays fro detection of
attacment
(virulence) factors
Isolate organisms from feces
Tests commercially available for
immunologic detection of toxins from
culture supernatants
Genes probes specific for LT and ST genes
available for detection of ETEC in feces
and in food and water samples
Isolate organisms from feces
Proportion of EHEC in fecal sample may be
very low (often <1% of E coli colonies)
Usually sorbitol non-fermenters
Shiga toxin production and associated genes
detected by biological, immunological and
nucleid acid based assays
Isolate organisms from feces
Test for enteroinvasive potential in tissue
culture cells or nucleid acid based assays
for invasion associated genes
Issue culture assays for aggregative of
diffuse adherence
Microbiological food infection

Salmonella
• Gram negative rod, facultative anaerobe, peritrichous flagella
• More than 1000 strains base on the O and H antigen, but for human
the most important is Salmonella enterica.
• There are thousands of Salmonella serotypes, but clinically divided i
nto 3 groups: Salmonella typhi, Salmonella cholerae-suis, and Salm
onella enteritidis
• There is a large contaminated food, especially poultry, and daily pro
ducts: eggs, raw milk, raw beef, unwashed fruit.
• Salmonellas are almost always acquired orally in food or drink that i
s contaminated.
• Most Salmonella killed by acid so need to ingest large numbers to s
urvive stomach acid in order to cause infection.
• Incubation time: 12-36 hours
• Laboratory: culture (isolate) and identify lactose
negative & H2S positive
Pathogenesis :
Infection by ingestion --- small intestinal via lymphatics ---
mesenteric glands -- multiplication --- blood via thoracic duc
t --- bacteriaemic phase ( 1 - 10 days) : infection liver, gall b
ladder, spleen, kidney & bone marrow.
Gall bladder --- invasion lymphoid tissue -- Peyer’s patch
es & lymphoid follicles -- acute inflammatory reactions --- u
lcer haemorrhage -- perforation & necrosis

Mochammad Hatta@2013
POSITIVE CULTURE AND IgM ANTIBODY RESPONS IN TYPHOID
FEVER

100
90
80
70 Blood
% Positive

60
Faeces
50
40 Urine
30 IgM
20
10
0
1 2 3 4 5 6 7 8
Weeks

Mochammad Hatta@2013
 Typhoid fever
Laboratory diagnosis

• Polymerase Chain Reaction (PCR)

• Culture of blood or bone marrow
– 80% during first week
• Culture of urine or stool
– in presence of clinical picture characteristics
• Serologic test
– antibody test against somatic (O) or flagellar (H) a
ntigen

Mochammad Hatta@2013
Prinsip tehnik Polymerase Chain Reaction (PCR)

DNA yang akan 5' -------------------------------------- 3'

diamplifikasi 3' _________________________ 5’
ß Denaturasi + primer

____ ------------------------- 3’
3' __________________ ____
ßtambah Taq polymerase <>
ß dNTPs
ß
_____________ --------------------------3’
3'___________<>____ <>_________
ß
ß
siklus diulangi sehingga menghasilkan copy DNA
yang spesifik secara eksponensial

Mochammad Hatta@2013
Deteksi Salmonella typhi dengan Nested PCR

Mochammad Hatta@2013
 Typhoid fever
(nested)

ST1 : 5’-ACT GCT AAA ACC ACT ACT-3’

ST2 : 5’-TTA ACG CAG TAA AGA GAG-3’
ST3 : 5’-AGA TGG TAE TGG CGT TGC TC-3’
ST4 : 5’-TGG AGA CTT CGG TCG CGT AG-3’

(M. Hatta & Henk L Smits. Annals Tropical Medicine & Parasitology (Liverpool), 2006)

Mochammad Hatta@2013
Hasil nested PCR S.typhi dari penderita
demam tifoid

Mochammad Hatta@2013
MDR PCR product S.typhi Vietnam and Indonesian isolated

Vietnam Indonesia

Mochammad Hatta@2013
MDR PCR product S.typhi Vietnam isolated

941 bp
819
639

310

Mochammad Hatta@2013
PCR for the detection of S. typhi specific DNA in blood, stool and urine
samples from patients with suspected typhoid fever .

No (%) of patients with the following result

Patient group Blood Faeces Urine
Neg Pos Neg Pos Neg Pos

Culture positive 1 (1) 71 (99) 16 (67) 8 (33) 22 (38) 36 (62)

Culture negative 21 (45) 26 (55) 7 (41) 10 (59) 11 (28) 28 (72)

Non-typhoid patients 12 (100) 0 (0) 2 (100) 0 (0) 10 (100) 0 (0)

(INCO-DC EC Research project Report, 2002)

Mochammad Hatta@2013
 Salmonella bacteria
on MacConkey agar

Lactose-positive ba
cteria show pink co
lonies (upper left)

Lactose-negative b
acteria have colorle
ss colonies (lower r
ight)

Mochammad Hatta@2013
Biochemical reactions for identification of S. t
yphi by the API 20E procedure

Mochammad Hatta@2013
Black coloni
es of Salmo
nella typhi af
ter growth o
n bismuth s
ulfite agar

Mochammad Hatta@2013
Dipstick for Typhoid Fever
Procedure
– Add 5µl serum to 250µl detection reagent
– Incubate dipstick for 3 hours
– Rinse with tap water
– Read by visual inspection

Result

(Mochammad Hatta, et al. American J. Tropical Medicine & Hygiene, 2002)

Mochammad Hatta@2013
Mochammad Hatta@2013
 Dipstick for Typhoid Fever
Procedure
– Add 5µl serum to 250µl detection reagent
– Incubate dipstick for 3 hours
– Rinse with tap water
– Read by visual inspection

Result Patients with cl

inical suspicion
of typhoid feve
r from Makass
ar, Indonesia

Control
Mochammad Hatta@2013 Test
 Typhoid Fever Dipstick
CTD, Ho Chi Minh City, Vietnam
Comparison of tests

Test Sensitivity (%) Specificity (%)

IgM ELISA 1:400 75 94

IgG ELISA 1:1.600 68 92
IgA ELISA 1:200 52 95

Widal O 1:400 47 93
Widal H 1:200 60 98

Dipstick 77 95

Mochammad Hatta@2013
Typhoid Fever Dipstick
Semarang, Indonesia

Patient group, Number positive (%) / total

culture result

Dr. Kariadi Hospital

(bone marrow culture)
S. typhi positive 38 (70.4) / 54
S. typhi negative 0 (0) / 2

3 district hospitals
(blood culture)
S. typhi positive 32 (86.5) / 37
S. typhi negative 2 (7.7) / 26

Mochammad Hatta@2013
 Typhoid Fever Dipstick
Makassar, Indonesia

Patient group No. positive (%) /

total
Suspects
Clinical diagnosis: typhoid 85 (47.5) / 179
S. typhi culture positive 73 (65.2) / 112
S. paratyphi culture positive 4 (66.6) / 6
Culture negative 8 (13.1) / 61

Clinical diagnosis: other

Culture negative 0 (0) / 64

Hospital controls 0 (0) / 259

School children 2 (1) / 194

Mochammad Hatta@2013
 Typhoid Fever Dipstick
Makassar, Indonesia
Follow-up

Sample DPO No. positive(%)/

Total

S. typhi culturepositive
First 8 30(76.9) /39
Second 15 32(82.1) /39
Third 29 38(97.4) /39

S.typhi culturenegative
First 6 2(4.3) /47
Second 13 36(76.6) /47
Third 27 39(83.0) /47

Mochammad Hatta@2013
 Typhoid fever
Culture and Dipstick
Assay Sensitivity Specificity PPV NPV

Culture 65.9% 100% 100% 74%

Dipstick 47.5% 95% 92% 65%

Dipstick: finger prick blood, same day result

Mochammad Hatta@2013
 Rapid test for typhoid fever
• 80% sensitivity compared with blood culture
• PPV (92%) and NPV (64%) somewhat lower tha
n that of culture
• Same day result
• Easy to perform
• High stability of components

Mochammad Hatta@2013
DIPSTICK FOR DETECT IGM ANTIBODIES
1. Simple and rapid
2. Required no equipment
3. Highly stable reagents
4. Low cost
5. Easy to applied in field

Mochammad Hatta@2013
 TYPHOID Lateral Flow
Principle

I
m m
un
oc
hr
oma
to
gr
aph
i
c s
tr
ipa
ss
ay
T
es
t C
on
t
rol

Samplepad / Co
nju
gat
e D
et
ect
ion
st
rip S
in
k
blo
o dce
ll p a
d
s
epar
atio
nfilt
er

Mochammad Hatta@2013
 TYPHOID Lateral Flow
Method

• Add 5l serum

• Add 130l sample fluid
• Wait 10 minutes Control line
Test line
• Read result

Sample well

Mochammad Hatta@2013
Typhoid Fever Latex Agglutination

5 seconds 15 seconds

45 seconds > 60 seconds

Mochammad Hatta@2013
 Shigella
 Coliform bacilli (enteric rods)
 Nonmotile gram-negative facultative anaerobes
 Four species
 Shigella sonnei (most common in industrial world)
 Shigella flexneri (most common in developing countries)
 Shigella boydii
 Shigella dysenteriae
 Non-lactose fermenting
 Resistant to bile salts
 Epidemiology and Clinical Syndromes of Shigella
Shigellosis = Generic term for disease

 Low infectious dose (102-104 CFU)

 Humans are only reservoir
 Transmission by fecal-oral route
 Incubation period = 1-3 days
 Watery diarrhea with fever; changing to dysentery
 Major cause of bacillary dysentery (severe 2nd stage) in pediatric age grou
p (1-10 yrs) via fecal-oral route
 Outbreaks in daycare centers, nurseries, institutions
 Estimated 15% of pediatric diarrhea in U.S.
 Leading cause of infant diarrhea and mortality (death) in developing count
ries
 Pathogenesis of Shigella
Shigellosis

Two-stage disease:
 Early stage:
 Watery diarrhea attributed to the enterotoxic activity of
Shiga toxin following ingestion and noninvasive coloniz
ation, multiplication, and production of enterotoxin in the
small intestine
 Fever attributed to neurotoxic activity of toxin

 Second stage:
 Adherence to and tissue invasion of large intestine with
typical symptoms of dysentery
 Cytotoxic activity of Shiga toxin increases severity
 Pathogenesis and Virulence Factors (cont.)

Virulence attributable to:

 Invasiveness
 Attachment (adherence) and internalization with complex
genetic control
 Large multi-gene virulence plasmid regulated by multiple
chromosomal genes
 Exotoxin (Shiga toxin)
 Intracellular survival & multiplication
Pathogenesis and Virulence Factors (cont.)

Invasiveness in Shigella-Associated Dysentery

 Penetrate through mucosal surface of colon (colonic mucosa)
and invade and multiply in the colonic epithelium but do not t
ypically invade beyond the epithelium into the lamina propria
(thin layer of fibrous connective tissue immediately beneath t
he surface epithelium of mucous membranes)

 Preferentially attach to and invade into M cells in Peyer’s pat

ches (lymphoid tissue, i.e., lymphatic system) of small intesti
ne
Pathogenesis and Virulence Factors (cont.)

Invasiveness in Shigella-Associated Dysentery(cont.)

 M cells typically transport foreign antigens from the intestine to underl
ying macrophages, but Shigella can lyse the phagocytic vacuole (pha
gosome) and replicate in the cytoplasm
 Note: This contrasts with Salmonella which multiplies in the pha
gocytic vacuole
 Actin filaments propel the bacteria through the cytoplasm and into adj
acent epithelial cells with cell-to-cell passage, thereby effectively avoi
ding antibody-mediated humoral immunity (similar to Listeria monocy
togenes)
Pathogenesis and Virulence Factors (cont.)

Characteristics of Shiga Toxin

 Enterotoxic, neurotoxic and cytotoxic
 Encoded by chromosomal genes
 Two domain (A-5B) structure
 Similar to the Shiga-like toxin of enterohemorrhagi
c E. coli (EHEC)
 NOTE: except that Shiga-like toxin is encoded by lysog
enic bacteriophage
Pathogenesis and Virulence Factors (cont.)

Shiga Toxin Effects in Shigellosis

Enterotoxic Effect:
 Adheres to small intestine receptors
 Blocks absorption (uptake) of electrolytes, glucose, and amino acids
from the intestinal lumen
 Note: This contrasts with the effects of cholera toxin (Vibrio chol
erae) and labile toxin (LT) of enterotoxigenic E. coli (ETEC) whi
ch act by blocking absorption of Na+, but also cause hypersecre
tion of water and ions of Cl-, K+ (low potassium = hypokalemia),
and HCO3- (loss of bicarbonate buffering capacity leads to meta
bolic acidosis) out of the intestine and into the lumen
Pathogenesis and Virulence Factors (cont.)

Shiga Toxin Effects in Shigellosis (cont.)

Cytotoxic Effect:
 B subunit of Shiga toxin binds host cell glycolipid
 A domain is internalized via receptor-mediated endocytosis (coated pits)
 Causes irreversible inactivation of the 60S ribosomal subunit, thereby c
ausing:
 Inhibition of protein synthesis
 Cell death
 Microvasculature damage to the intestine
 Hemorrhage (blood & fecal leukocytes in stool)

Neurotoxic Effect: Fever, abdominal cramping are

considered signs of neurotoxicity
Heparin-binding epidermal growth fact
or on heart & nerve surfaces
 Klebsiella
• Also called as Friedlander’s bacillus mucosus ca
psulatus.
• The most popular species: Kl. aeroginosa, Kl. pne
umoniae, Kl. edwarsiella, Kl. atalantae, Kl. ozeanea
e, Kl.rhinosecleromantis.
• Morphology: gram negative, capsulated, non-mo
tile bacillus, measures about 1-2 x 0,5-0,8 micro
meter in size.
• Culture: grows well on ordinary media, optimum
temperature 37 C, mucoid nature of colonies are
found in the medium due to the accumulation of
slime produced by Klebsiella.
Biochemical reactions:
•Indole –ve
•MR –ve
•VP +ve
•Forms urease

Antigenic structure:
•Capsular K
•Somatic O
Pathogenesis: Lab diagnosis:
Second most popular aerobic
bacterial flora of colon  Cultures on blood agar and
 Causes: MacConkey agar
•severe bronchopnumonia  Gram staining
•UTI’s  Biochemical reactions
•Nosocomial infections
•Wound infections
•Septicemia
•Meningitis
•Rarely diarrhea
Treatment:
•Sensitive to cephalosporin, trimethoprim, nitrofura
ntoin.
•Many strains carry plasmids determining MDR.
Selected Clinical and Epidemiologic Characteristics of Typical Illness Cau
sed By Common Food Poisoning Pathogens
Typical Inc
Typical Clinical Assorted Foo
Pathogen ubation Per Duration
Presentation ds
iod
Bacteria
Salmonella spe 1-3 Days 4-7 Days Gastroenteritis Undercooked e
cies ggs or poultry
, produce

Campylobacter 2-5 Days 2-10 Days Gastroenteritis Undercooked

jejuni poultry, unpas
teurized dairy
products
E. coli, Enterot 1-3 Days 3-7 Days Gastroenteritis Many foods
oxigenic

Shigella specie 1-2 Days 4-7 Days Gastroenteritis Produce, egg 68

s
s alad
Listeria monocyt 2-6 weeks Variable Gastroenteritis, Deli meat, hotd
ogenes meningitis abor ogs, unpasteuri
tion zed dairy prod
ucts
Bacillus cereus 1-6 hour <24 hour Vomiting, Gastr Fried rice, meat
oenteritis s

Clostridium bot 12-72 hour Days-months Blurred vision, Home-canned

ulinum paralysis foods, fermente
d fish

Staphylococcus 1-6 hour 1-2 Days Gastroenteritis, Meats, potato

aureus particularly nau & pork, unpaste
sea urized dairy pr
oducts.

Yersinia enteroc 1-2 Days 1-3 weeks Gastroenteritis, Undercooked p

olitica appendicitis-li ork, unpasteuri
ke syndrome zed dairy prod
ucts.
69
Virus Typical inc Duration Typical clinic Assorted foo
ubation per al presentatio ds
iod n

Norovirus 1-2 Days 12-60 Hr Gastroenteritis Under

cooked shell
fish

Hepatitis 15-50 Day Weeks-m Hepatitis Produce, un

A virus s onths dercooked s
hellfish

70
General principles of management:
1. Maintenance of fluid and electrolyte balance alw
ays important.
2. Antimicrobial therapy is primarily for invasive dis
ease.
THANK YOU