Lecture 1 Flashcards
Give a simple definition of a pathogen
An organism capable of causing disease
Define disease
The clinical signs and symptoms of damage that occur in a host as a result of its interaction with an infectious agent
What are the six stages of infection of pathogens and what do these involve?
- Transmission (vector, environment, host restricted)
- Infection (how to enter and breach host barriers)
- Establishment (colonise, survive and spread)
- Persist (Evade the immune system)
- Disease (damage host cells, tissues)
- Antibiotic resistance
What is the importance of bacteria as a area of research?
The rising threat of bacterial antibiotic resistance, creating a global drive in bacterial research. As infections could occur after routine surgery and become fatal.
How does the status of a host affect the progression of disease?
- The immune status of the host
- Whether they have had prior exposure
- Whether they have a genetic predisposition
- immune status (age, stress, diet, surpressed)
What is the progression of disease largely dependent on?
Pathogen/host interations
What features of the pathogen interact with that of the host to determine disease progression?
- Site of infection (related to the pathogen’s properties)
- Specific traits of the bacterial isolate (virulence factors, metabolism, growth characteristics)
- Route of inoculum (tissue specificity of pathogen, host defences)
- Size of inoculum (LD50 = lethal dose 50, the dosage that causes 50% mortality in the animal model.)
What was the inportance of Koch in bacterial studies?
Koch linked pathogens to disease as the causative agents. He proved that specific microorganisms caused a specific disease
What are the purpose of Koch’s postulates?
1890 To prove that a specific microorganism causes a specific disease
What is the one-microbe one-disease concept?
the idea that one microbe should be linked to one disease directly. “it is essential that following isolation of a pure culture of the suspected pathogen, a laboratory culture of the organism should both initiate the disease and be recovered from the animal.”
What are Koch’s postulates?
- The microorganism must be found in abundance in all organisms suffering from the disease but not in healthy organisms
- The microorganism must be isolated from a diseased organism and grown in pure culture
- The cultures microorganism should cause disease when introduced into a healthy organism
- The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as identical to the original specific causative agent
By what process would Koch demonstrate experimentally that a specific microbe causes a specific disease?
- Take a diseased animal and take a smaple of its blood
- Culture the suspected pathogen in pure culture
- Input into a healthy organism and observe disease
- Take blood samples from the experimental host and culture the pathogen
- Confirm that is the same as the original specific causative agent
- Must take samples from healthy individual and not see suspected pathogen
What are the limitations of Koch’s postulates?
They make implicit assumptions that may not translate to reality:
- The pathogen is lab culturable
- The right conditions are known and being used The bacteria may require certain nurtients (fastidious), may have certain O2 requirements or may be unculturable
Give an example of an unculturable pathogen
Catscratch disease In 1990 the pathogen was visualised at large densities around the infected area but culturing failed so the identigy of the pathogen was unknown Bartonella species take 45 days to grow if they do at all as they are fastidious. Involved in emerging diseases, very comnplex and there are many species: 16s rRNA sequencing used to confirm involvment
How have technological advances overcome the limitations of Koch’s postulates for unculturable pathogens?
- The application of 16s rRNA to determine the presence and sequence of the pathogen
- Took four patients with symptoms and visible clusters of bacteria (upon staining) in the affected tissues
- The DNA was extracted from frozen or paraffin embedded tissue
- Using 16s rRNA “universal” eubacterial primers DNA amplified by PCR DNA was cloned and sequenced
- More specific primers were designed and more patients tested, alongside healthy individuals
What are three strategies of baterial pathogenesis?
- Extracellular pathogen
- invasive
- can spread
- e.g. Streptococcus pneumoniae
- Toxin producing pathogens (extracellular)
- e.g. Clostridium botulinum, Vibrio chloera, Escherichia coli O157::H7
- Intracellular pathogen (non-obligate)
- e.g. Salmonella (enterica) serovar Typhimurium, Listeria monocytogenes, Chlamydia spp.
Give examples of virulence factors
- Adhesins (fimbriae, pili, outer membrane proteins)
- Flagella (motility, penetrate mucin)
- Proteins to help obtain rain/essential nutrients (siderophores-iron)
- Toxins
- Capsule
- Immune modulatory proteins
- Type III secretion system
What are the functions of virulence factors?
- Attachment and entry into the body
- Local or general spread in the body
- Multiplication
- Evasion of host defences
- Shedding from the body
- Causing damage in the host
When was the first virulence factor cloned?
1984 A single gene conferring virulence traits on a non-pathogenic E.coli
Define and describe an endotoxin
- lipopolysaccharide of gram negative bacteria
- cell bound
- heat stable
- weakly toxic
- detected by TLR4
What are the general symptoms of an endotoxin?
Fever, diarrhea, vomiting
What is TRL4?
(toll-like receptor) A protein in humans that detects lipopolysaccharides from gram-negative bacteria in order to activate the innate immune system
Define and describe an exotoxin
Proteins released extracellulary produced by certain gram negative and gram positive species
- generally heat liable
- has specific targets
- usually highly toxic
Define and describe an enterotoxin
group of exotoxins that act on the small intestine causing change in the interesting permeability leading to diarrhea
Give three examples of enterotoxins
C.difficile toxin Cholera toxin E.coli toxin
Define a toxoid
an inactivated toxin useful as a vaccine
How have single virulence factors been involved in establishing the elements of acute infection?
The first virulence factor to be confirmed as a single gene (1985 - Isberg and Falkow) -Yersinia pseudotuberculosis -single gene identified as responsible for the invasions of cultured epithelial cells
How did Isberg and Falkow confirm the first virulence factor as a single gene?
- Cloned large Yersinia pseudotuberculosis genome fragments in E.coli.
- Resulted in enriched clones that allowed e.coli to invade host cells.
- Pinpointed gene using tranposon mutagenesis and identified a loss of invasion mediated by the clone
How did Falkow modernise Koch’s postulates?
- Determine the phenotype associated with pathogenic strains of species
- The inactivation of the gene should lead to a measurable reduction in virulence OR The isolated gene can confer trait
Describe the Cholera toxin as an example of a gram negative exotoxin
- Vibrio cholerae (gram-negative bacteria)
- Transmitted: water-oral
- Colonises the small intestine
- An exotoxin
What is the molecular effect of the cholera toxin in the small intestine?
Causes increased adenylate cyclase activity cAMP levels are increased changes the sodium/chloride flux in and out of cells
Aside from the cholera toxin what are the additional virulence factors of Vibrio cholerae?
- adhesin
- mucinase
- flagella In human volunteers
- even after knocking out the cholera exotoxin gene (V.cholerae toxin mutant) the volunteers still experienced mild diarrhea
What are the physical symptoms of infection with the Cholera toxin?
loss of fluid and electrolytes (diarrhea)
Define a commensal
A symbiotic relationship between two organisms of different species in which one derives some benefit while the other is unaffected (e.g. species present in the microbiome)
What are the benefits of the microbiome?
- stimulate the immune system
- prevent colonisation with pathogens
- produce beneficial nutrients/components
Give 5 examples of areas on the body with a microbiome community
- Anterior nares
- Buccal mucosa
- Supragingival plaque
- Faeces
- Posterior fornix
Give an outline of the structure, function and diversity of the healthy human microbiome
- Bacterial and viral cells on our body outnumber human cells
- Species diversity varies between areas on the body, and the number of microbial genes varies even higher
- Carriage of the microbial taxa varies but the metabolic pathways remain stable within a healthy population (c. huttenhower et al. 2012)
What is the species # and microbial gene # of the Anterior nares?
species #: 900 microbial gene #: 30 000
What is the species # and microbial gene # of the Supragingival plaque?
species #: 1300 microbial gene #: 20 000
What is the species # and microbial gene # of the faeces (distal gut)?
species #: 4 000 microbial gene #: 800 000
What is the species # and microbial gene # of the Buccal mucosa?
species #: 800 microbial gene #: 70 000
What is the species # and microbial gene # of the Posterior fornix?
species #: 300 microbial gene #: 70 000
What experiment determined the involvement of the gut microbiome on the propensity of an individual to be obese?
Fighting obesity with science (2013) -
- germ-free mice innoculated with microbiota from obese or lean human twins take on the microbiota characteristics of the donor
- those recieving the obese microbiota had an increase in adiposity
- those recieving the lean microbiota remained lean
- if fed an appropriate diet, mice habouring the obese microbiota when housed with the mice harboring the lean microbiota are invaded by the lean microbiota and do not develop increased adiposity
- in contrast, the obese microbiota does not effectively colonise the lean microbiota and these remain lean
Outline David Relman’s modern supplement to Koch’s postulate
“In some cases, human disease is more apt to reflect a disturbed microbial community structure instead of the singluar mischief of a single pathogenic species”
Define an overt or strict pathogen
Only associated with human disease and are not found as members of the normal, healthy microbial flora
Define an opportunistic pathogen
members of the normal flora that only cause disease when introduced into unprotected sites -particularly concerning when normal defence mechanisms are compromised (immunocompromised individuals)
Define a facultative pathogen
can grow and survive in the environment as well as the host
Give an example of an overt/strict pathogen
Neisseria gonorrhoeae
Give an example of an oppourtunistic pathogen and the site of infection
Pseudomonas aeuruginosa at burn wounds
Give and example of a facultatitve pathogen and site of infection
Bacillus anthracis, rare on the skin and the lungs
Outline the process of signature tagged mutagenesis as an exmaple of a process that has undergone the 3R’s (rplace reduce refine)
- 1creation of unique DNA tag with restriction sites for the insertion into a plasmid and reoval of invariant arms
- Incorporation of tags into transposon plasmids
- Individually tagged mutants are organised into 96 well plates for experiments
- mutants are pooled are grown overnight and used for the input pool
- pool injected into an animal model of interest and the infected organs removed
- mutants that survive the screen and recover will be used for output pools
- DNA tags are labelled and released from invariant arms, probes from dot blobs and the input pool are compared to the output pool for non-colonising mutants
- Amplification of IP and OP using plasmid specific primers and tag specific primers. Comparison of input pool and output pool to identify non-colonising mutants
What are the 3R’s of animal research?
Replace reduce refine`
Give some examples and activities of S. pneumoniae virulence factors
- Capsule
- prevents phagocytosis
- Surface adhesions
- attach to the respiratory lining
- Secretrory IgA protease
- cleaves IgA, promotes spread of infection
- Neuroamidamase
- cleaves terminal acetylated neuramic acids from sugar residues
- pneumolysins
- pore forming, tissue damage
Give an example of the number of bacteria that reside in/on our bodies
- Over 400 species in the oral cavity
- between 10 000 and 10 million individual bacterial cells on each hand
Give examples of the bacteria found on the upper respiratory tract
- Haemophilus influenza
- Staphyloccus epidermis, auerus, pneumoniae
Give examples of the bacteria found on the skin
- Staphyloccus epidermis, pneumoniae
- Lactobascillus species
What areas of the body are supposed to be bacteria free?
- Fluids
- blood
- cerebrospinal fluid
- urine
- Tissues/organs
- muscles
- brain
How can commensals cause diseae?
- enter parts of the body normally sterile
- overgrow if the normal balance is disturbed as a result of:
- pH imbalance mucus, immune deficiency, antibiotics
- e.g. Clostridium difficile
use clostridium difficile as a commensal bacterial that can cause disease
- Gr+
- anarobic
- spore former
- part of the normal gut flora
- spread person-person through faecal-oral route
- fluorishes under antibiotic selective pressure
- causes antibiotic associated diarrhea
What are the challenges of identifying genes needed to survive during infection?>
- Many genes
- Many steps of infection
Give an example of differential fluoresence induction as an example of finding genes linked to stages of infeection
Differential Fluorescence induction
- clone: random promoters drive GFP
- screen for strains fluorescent during infection model
- check which are turned off in lab
How can thousands of mutatns be screened following a random mutagenesis?
signiture tagged mutagenesis
How has 2nd generation sequencing been applied to STM pools ?
TraDis:Transposon Directed Insertion site Sequencing
- Deep sequencing of the unique transposon flanking sequences
- Advantage:
- high coverage and quantitative information
- Degree of fitness of mutants.
