Koch's Postulates Flashcards
What were the theories behind factors that cause disease in the 1800s? (2)
-miasma theory: inhaled vapors cause disease
- germ theory: microorganisms cause disease
Koch’s Postulate: First Postulate
- the microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms
Koch’s Postulates: Second Postulate
- the microorganism must be isolated from a diseased organism and grown in pure culture
Koch’s Postulate: Third Postulate
- the cultured microorganism should cause disease when introduced into a healthy host
Koch’s Postulates: Fourth Postulate
- microorganism must be re-isolated from the inoculated diseased experimental host and identified as being identical to the specific causative agent
Challenges to Koch’s Postulates: First (3)
- host susceptibility varies in human and animal populations due to a variety of factors
- opportunistic bacteria can be found in healthy hosts too
- may be difficult to isolate bacteria from certain areas, such as brain and bones
Challenges to Koch’s Postulates: Second Postulate (2)
- not all bacteria can be cultured in pure form
- many bacteria undergo genetic and phenotypic changes in culture environments
Challenges to Koch’s Postulates: Third Postulate (2)
- not all bacterial species are equally virulent
- closely related bacterial strains can be very different
Challenges to Koch’s Postulates: Fourth Postulate
- re-inoculation of a host may not be possible or ethical
Example challenge to Koch’s Postulates: First Postulate (2)
- Mycobacterium tuberculosis
- H. pylori
Example challenge to Koch’s Postulates: First Postulate and Mycobacterium tuberculosis (2)
- could not be visualized as it did not gram stain due to differences in cell wall structure
- new cultivation and staining methods had to be developed
Example challenge to Koch’s Postulates: First Postulate and H. pylori (2)
- causes gastric ulcers, but not all of the ulcers contain H. pylori
- not all hosts develop disease even if they are carriers
Example challenge to Koch’s Postulates: Second Postulate
- syphilis bacterium
Example challenge to Koch’s Postulates: Second Postulate and Syphilis bacterium (2)
- bacterium has never been cultivated as a pure culture in lab medium
- rabbit testicles are used as a growth chamber
Example challenge to Koch’s Postulates: Third Postulate (2)
- Helicobacter
- Salmonella
Example challenge to Koch’s Postulates: Third Postulate and Helicobacter (2)
- Helicobacter mustelae causes disease in animal models, while Helicobacter pylori causes diseases in humans
- must determine if it is okay to use different bacterial species to satisfy this postulate
Example challenge to Koch’s Postulates: Third Postulate and Salmonella (2)
- Salmonella enterica serovar Typhimurium causes disease in mice while Salmonella enterica serovar Typhi causes disease in humans
- must determine if it is okay to use different bacterial species to satisfy this postulate
Example challenge to Koch’s Postulates: Fourth Postulate
- is the bacterium directly responsible for the host damage
- can the bacterium be isolated directly from the site of host damage?
Modern Approaches to Satisfy Koch’s Postulates (4)
- PCR
- immunohistochemistry and immunofluorescence
- ELISA
- eliminate the pathogen and prevent/cure the disease
Modern Approaches to Satisfy Koch’s Postulates: PCR
- PCR and 16S sequencing can be used to amplify and detect bacteria during culturing
Modern Approaches to Satisfy Koch’s Postulates: PCR/16S Sequencing steps (2)
- 16S RNA primers are designed to detect conserved regions, with unique regions in-between that act as barcodes to identify the bacteria
- PCR is done to amplify the bacterial nucleic acid for detection
Modern Approaches to Satisfy Koch’s Postulates: Immunohistochemistry (2)
- microscopic images of immunohistochemical staining can be used to visualize the pathogen in the tissue directly
- uses of antibodies against the bacteria with attached reporter enzymes
Modern Approaches to Satisfy Koch’s Postulates: Immunofluorescence (2)
- micrographs of cells with fluorescently labelled antibodies against cell markers and bacterial
- cells infected with bacteria will exhibit colocalization of fluorescent markers, while healthy cells will not
Modern Approaches to Satisfy Koch’s Postulates: ELISA (2)
- use of fluorescently labelled antibodies against bacteria to detect pathogens in infection site without culturing
- Western Blot/SDS-PAGE can be used subsequently
ELISA methods (3)
- direct ELISA
- indirect ELISA
- capture/sandwich ELISA
Modern Approaches to Satisfy Koch’s Postulates: Eliminate the Pathogen and Prevent/Cure the Disease; Why didn’t Koch try this?
- antibiotics did not exist at the time, so treatment was a lot more complicated and time-consuming
why is T. pallidum (syphilis bacterium) hard to study using Koch’s Postulates (3)
- bacteria has never been cultivated in the lab
- does not cause human-like disease in other animals
- bacterium has several disease stages, each with different symptoms
Syphilis: Stage 1 (2)
- painless lesions
- can be cured with antibiotics
Syphilis: Stage 2 (4)
- skin rash on hands and feet
- spreads across body
- lasts 2-6 weeks
- clears on its own without antibiotics
Syphilis: Stage 3 (3)
- gumma (large granuloma) forms
- CNS, eye and heart problems
- can be cured with antibiotics
What does Koch’s Postulates address?
- who/what is causing the disease
How do we find out HOW the pathogen is causing the disease? (2)
- must identify mechanism/strategies employed by the bacterium causing the disease
- must study mechanistic understanding, identification, and infection models
virulence factors
- bacterial product or strategy that contributes to the ability of the bacterium to survive in the host/cause infection
what are examples of virulence factors from H. pylori (8)
- flagella
- urease
- lipopolysaccharides
- outer proteins for adhesion
- effector molecules
- type IV secretion system
- secretory enzymes
- exotoxins
How can we identify virulence factors?
- genome maps as virulence factors are encoded by genes
Molecular Version of Koch’s Postulates: First Postulate
- gene for virulence should be present in the strain of bacteria that cause disease and absent in avirulent strains
Molecular Version of Koch’s Postulates: Second Postulate
- (i) knocking out or disruption the gene should reduce virulence, and (ii) introduction of the cloned gene into an avirulent strain should render the avirulent strain virulent
Molecular Version of Koch’s Postulates: Third Postulate
- expression of the gene should be demonstrated in human or a relevant model
Molecular Version of Koch’s Postulates: Fourth Postulate
- antibodies or a cell-mediated immune response to a virulence factor should be protective
Considerations of Models (3)
- relevant experimental system should be chosen as the model
- the assay that is used only defines the virulence of that assay
- if the system is flawed, it cannot be extrapolated to humans or other hosts
what should we consider before choosing bacterial strains (5)
- representative strains
- clinical isolates that can be sequences and be tested with genetic tools
- prototypical wild type strains
- maintenance of virulence factors that may be metabolically expensive
- appropriate mutants
how can we encourage the maintenance of strain virulence?
- freezing and taking small amounts to study instead of breeding the bacteria for long periods of time
what mutants can be used for a study (3)
- spontaneous mutants
- randomly generated mutants
- directed mutants
spontaneous mutants
- unstable genetic mutants, capable of converting back
direct mutants
- more stable mutants
what should we consider when choosing hosts for models (3)
- the infection model should be similar; aerosol infection should use an aerosol model
- tissue distribution of pathology should be similar in both hosts
- identify closely related pathogens that infect different species
advantages of rodent host models (2)
- variability of experiment is smaller due to inbreeding
- knockouts and transgenic animals exist or can be made easily
disadvantages of rodent host models (4)
- lack of variability
- mice can have different microbiota depending on source
- mice have different physiologies, habits, and genetics compared to humans
- human pathogens may not be able to infect rodent model by same route of infections
cell lines (2)
- simple model compared to whole organisms
- generally use epithelial cells, fibroblasts, or monocytes
cell lines: advantages (2)
- simple, more controlled, and cheaper than using whole organisms
- often immortalized by using tumour cells
cell lines: disadvantages (3)
- repeated culturing of cell lines can change their properties
- genes expressed in an organ may not be expressed in tissue culture
- cell lines are usually not polarized, while cells in organs are
polarization of intestinal cells (2)
- cell receptors are asymmetrical
- apical side (lumen) and basolateral size (tissue/blood) express differential receptors
organoids (2)
- new host model that has great potential as it aims to replicate actual organization of human organs
- derived from human stem cells
frequently used host models (7)
- rodents
- worms (C. elegans)
- frogs
- yeast
- slime mold
- bees
- flies (drosophila)
