Proof of causation of the disease by Koch’s postulates- with slides from students seminar on some diseases

2. 1843-1910
• Discriminating between organisms and associating
organisms with particular diseases
• formulated a set of conditions that needed to be met in
order to ‘prove’ a causal relationship

3. Koch's postulates
1. The microorganism must be found in abundance in all
organisms suffering from the disease, but not in healthy
organisms.
2. The microorganism must be isolated from a diseased organism
and grown in pure culture
3. The cultured microorganism should cause disease when
introduced into a healthy organism.
4. The microorganism must be re-isolated from the inoculated,
diseased experimental host and identified as being identical to
the original specific causative agent.

4. Which germ?
Which disease?
Signature of organism

5. Why may it be difficult to prove causation using Koch’s
postulates ?
1. chronic or minor conditions
2. multiple causes
3. pathogen can't be grown
4. no suitable animal model
5. Long incubation period

6. "Molecular Koch's Postulates":
Stanley Falklow (1988)
1. Identify gene (or gene product) responsible for virulence determinant
2. Show gene present in strains of bacteria that cause the disease
3. Not present in avirulent strains
4. Disrupting the gene reduces virulence
5. Introduction of cloned gene into avirulent strain confers virulence.
6. The gene is expressed in vivo
7. Specific immune response to gene protects

7. • Guillain Barre Syndrome
• Hepatitis A
• Boil
• Leprosy (Hanson’s disease)
• Tetanus
• Typhoid fever
• Yellow fever
• Rheumatic Fever

8. Guillain Barre Syndrome & Campylobacter jejuni
Scanning electron microscope image of Blood-free, charcoal-based selective
Campylobacter jejuni, illustrating its corkscrew medium agar (CSM) for isolation of
appearance and bipolar flagella. Campylobacter jejuni at 42 C
Evidence of link between campylobacter jejuni and GBS
•For more than 100 years, a variety of preceding infectious diseases have been described
in association with GBS.
•But the relation with Campylobacter jejuni was discovered recently.
•However gastrointestinal illnesses occurring in up to 20% of GBS patients were
recognized many decades ago.
•Campylobacter infection was first reported as a potential cause of GBS in 1982 in a 45-
year-old man who developed severe GBS with irreversible neurologic damage 2 weeks
after a gastrointestinal illness caused by Campylobacter infection .

9. Guillain Barre Syndrome & Koch’s postulates
Koch’s postulates can not be applied to prove the etiological relationship
between GBS and Campylobacter jejuni
Koch's postulates 01
In GBS only 40 % of the cases Campylobacter jejuni can be isolated as an antecedent event
But in the other cases no such association is found.
Campylobacter can be found in patients with no evidence of GBS and in patients with
gastroenteritis.
Koch's postulates 02
Only 40% of cases this can be done. It requires special culture media for culture and special
conditions. (E.g. Blood-free, charcoal-based selective medium agar (CSM) at 420C)
Organism will also be absent in stool by the time the patient gets the disease.
Koch's postulates 03
It does not cause GBS in all cases only few cases progress in to Guillan Barre syndrome.
Only 1:1000 cases will get GBS from C.jejuni infection.
Koch's postulates 04
Development of GBS can be related to the inoculation of C. jejuni in to a healthy
individual or it can be a purely an incidental cause.
So organism isolation is not always definite.

10. Robert Koch
Blood-free, charcoal-based
selective medium agar (CSM) for
isolation of Campylobacter jejuni
at 42 C
Scanning electron microscope image of A patient with Guillain Barre
Campylobacter jejuni, illustrating its syndrome showing acute flaccid
corkscrew appearance and bipolar paralysis of lower limbs with areflexia

11. Koch’s postulates can not be applied to prove the etiological relationship
between GBS and Campylobacter jejuni
1. Koch's postulates 01
The microorganism must be found in abundance in all organisms suffering from the disease, but should
not be found in healthy animals.
In GBS only 40 % of the cases Campylobacter jejuni can be isolated as an antecedent event
but in the other cases no such association is found. Campylobacter can be found in patients
with no evidence of GBS and in patients with gastroenteritis
2. Koch's postulates 02
The microorganism must be isolated from a diseased organism and grown in pure culture.
Only 40% of cases this can be done. It requires special culture media for culture and special
conditions. E.g. Blood-free, charcoal-based selective medium agar (CSM) at 42 C
3. Koch's postulates 03
The cultured microorganism should cause disease when introduced into a healthy organism.
It does not cause GBS in all cases only few cases progress in to Guillan Barre syndrome.
4. Koch's postulates 04
The microorganism must be reisolated from the inoculated, diseased experimental host and identified as
being identical to the original specific causative agent
Development of GBS can be related to the inoculation of C. jejuni in to a healthy
individual or it can be a purely an incidental cause. So organism isolation is not always
definite.

12.  HAV is a Heat stable, Picornavirus (ssRNA)
 Mode of transmission by Fecal-oral route
 Water & food borne disease
 HAV causes inflammation of the liver
 Signs & symptoms
Jaundice, Fever, Abdominal pain, Nausea ,Diarrhoea,
Sharp pain in right upper quadrant of the abdomen.
 During Hippocrates's era - Infectious icterus
 1956, Dr. Saul Krugmann - Labelled the of two forms of hepatitis
By their aetiological agents
(In 1956 Dr. Krugman conduct a study among mentally retarded, disabled children in
Willow brook State School (New York), and he fed & injected infected serum to the
children. And ethics of his experiment aroused controversy & it was censured by NY
senate.)
 1973, Feinstone & Purcell - identified Hepatitis A virus –
1st visualized by Immune electron microscopy

13. HAV & Koch’s postulates
• First postulate –
90% of infected children <= 5 yrs are Asymptomatic
Not all the subjects who have infected get the symptoms, infants
• Second postulate –
Pure cultures for the viruses were not available during that time.
Nowadays HAV grows in primate cell cultures.
(chimpanzees/marmosets)
• Third postulate –
No evidence of disease by Pure cultured virus.
BUT ;
1944, McCollam, Transmission of the infected serum to volunteers
1956, Dr.Saul Krugman, feed & inject infected serum to the mentally
retarded, disabled children in Willow brook State School (New York)
and they developed the hepatitis
• Fourth postulate –
HAV grows in primate cell cultures. After serial passage, strains may
lose virulence for the liver.

14. BOIL
• Sub-epidermal collection of
pus
• Usually arises around a hair
follicle
• This is caused by a bacteria
called staphylococcus
aureus
Staphylococcus aureus

15. Koch’s postulates - Proved
1. The Koch saw -must be presentpusall cases of
1878 – organism staphylococci in in from
disease. boils .
2. The bacteria must be isolated from culture
1884 – Rosenbach studied through pure the
and identified Staphylococcusin pure culture.
diseased host and grown aureus
3. The Garre rubbed Staphylococcus aureus on the
1885 –
specific disease must be reproduced
skin of hisa pure culture of bacteria is inoculated
when arm and was able to produce boils.
into a healthy susceptible host.
4. The bacteria must be recoverable from the
experimentally infected host.

16. Leprosy
(Hanson’s disease)

17. Koch’s Postulates Evidence/Exceptions
1. The micro-organism must 1. 1873- Dr.Gerhard Hansen
regularly be isolated from cause discovered Mycobacterium
of the illness. leprae.
2. It must be grown in pure culture 2. 1968- Research started on in vivo
in vitro. culturing of the bacterium, in
3. When such a pure culture is Armadillos.
inoculated in to susceptible 3. When such an in vivo-cultured
animal species, the typical organism is inoculated into
disease must result. susceptible species the particular
4. From such experimentally disease results.
induced disease, the micro- 4. The particular microbe can be
organism must again be isolated. isolated from such a diseased
animal.

18. Disease :- Tetanus
Name of Microbe :- Clostridium tetani
Genus of the microbe :- Genus Clotridium
Spores (cultivated fields)
Common Features Contamination
(Genus - Clostridum )
Necrotic & Poorly
Colonization
perfused wounds
• Widely distributed
• Rod shaped Infection
• Spore forming Vegetative microbe
• Gram-positive
• Anaerobic
Toxin – tetanospasmin
(Tetanospasmin toxemia)

20. Postulate 01 :- agree
- Not included in ‘Normal Microbiota’ (Primary pathogen)
- Production of Highly toxic Tetanospasmin
- When colonized always cause Tetanus
Postulate 02 :- agree
- Can be grown in ‘Pure Culture’ – Needs special conditions
- Very little number of organisms
- Difficulty to locate the place of colonization
Postulate 03 :- agree
Should be inoculated under special conditions
Well perfused and
living tissue
tissue O2 No Colonization

21. Postulate 04 :- agree
- Can be grown again by specimens taken from experimentally
. inoculated patients
- (same principles apply as postulate 02)
Postulate 05 :- Does not agree
Very small amount of toxin Disease or death
Little or Inadequate No immune response
production of the when inoculated again
antigen

22. Typhoid Fever (Salmonella typhi)
• Characterised by • Number of organisms
– Sustained fever needed - 103
– Headache • Transmitted by
– Malaise – Faecally contaminated water
– Anorexia and food
– Constipation – Via the fecal-oral route
– Diarrhea • Affects the
– Non productive cough – Reticuloendothelial system
– Rose spots
Typhoid Mary !

23. Koch’s Postulates & Typhoid
Koch’s Postulate Agree/ Reason
Disagree
1. Presence of (Agree) Typhoid Mary
microbe at the lesion Tony Labella
site and absent in all
healthy animals
2. Ability to isolate Agree HEK agar
from the site and MacConkey’s agar
culture Red slant and yellow butt in TSI
3. Ability to reproduce Agree Chimpanzee’s throat
the disease in a By Elie Metchnikoff (1911)
susceptible
experimental animal
4. Ability to recover - -
from the experimental
animal

24. • Introduction
• History
• Virus & vector KOCH
• How it affects humans
• Clinical features

25. Virus:
• Found in both affected & unaffected people
• Is grown in a variety of cultures.
• May or may not produce disease.
• Isolated from diseased persons.

26. Can it be proved by Koch's postulates?
Can't.........
Why cannot it be proved by Koch's postulates?
Is the organism (Streptococcus pyogenes) present in
all the affected sites (joints, heart, CNS)?
Can the organism be isolated from the affected sites
(joints, heart, CNS)?
If the isolated and cultured organism inoculated
in to a susceptible person or animal, does it always
cause rheumatic fever?

27. How was causation proved?
Epidemiological studies
1960 – Stollerman noted
Outbreak of type A streptococcal upper respiratory
tract infection followed by outbreak of Rheumatic fever ¹
Another observation:
Streptococcus is not resistant to penicillin and
it has not developed into resistant forms!
But...
Penicillin has clearly failed to eradicate Rheumatic fever ²
Pathogenesis of Rheumatic fever?
Is Streptococcus pyogenes the only causative agent?
Reference: 1 & 2 http://www.heart.bmjjournals.com/cgi/content/full/91/1/3