34. Bacterial Pathogens and Diseases I Flashcards
Define pathogen
A microorganism capable of causing disease.
Define pathogenicity
The ability of an infectious agent to cause disease.
Define virulence
The quantitative ability of an agent to cause disease.
Define toxigenicity
The ability of a microorganism to produce a toxin that contributes to the development of disease.
List some virulence mechanisms
- Adherence Factors
- Biofilms
- Invasion of Host Cells and Tissues
- Toxins ~ endotoxins and exotoxins
What are exotoxins?
- Heterogeneous group of proteins produced and secreted by living bacterial cells.
- Produced by both gram negative and gram positive bacteria.
- Cause disease symptoms in host during disease.
- Act via a variety of diverse mechanisms.
What selective advantages do exotoxins give to the bacteria?
- Cause disease? – may help transmission of disease, however in severe disease host may be a literal and evolutionary dead end.
- However with many toxins the disease causing activity may be not be the primary function.
- Other activities
- Evade immune response
- Enable biofilm formation
- Enable attachment to host cells.
- Escape from phagosomes
• All allowing for colonisation, niche establishment and carriage ~ Evolutionary
advantage.
Looking at a specific disease, in the case of Staphylococcus aureus, what toxins does it produce and what are their functions?
• Haemolytic toxins
• cause cells to lyse by forming pores
• Important cause of features of S. aureus disease.
• E.g. α,β,δ, toxins ,Panton Vlentine Leukocidin (PVL), LukAB, LukED, LukMF
• Phenol soluble modulins PSM
- Aggregate the lipid bilayer of host cells - lysis
In the case of Staphylococcus aureus, why does it have these toxins?
- The α-toxins inhibit the attachment of the phagosome to the lysosome so Staphylococcus aureus can survive longer
- PSMs they kill other bacteria, so less competition for the Staphylococcus aureus for resources ~ PSM allows the Staphylococcus aureus to slide along making it mobile
- In terms of the infection itself, the α-toxin helps in its initial attachment, the β-toxin aids in the accumulation of the bacteria, and the PSMs help in the detachment when needed.
Expand on the genetics of exotoxins
• Can be encoded by chromosomal genes
- Shiga toxin in Shigella dysenteriae
- TcdA and TcdB in C. difficile
• Many toxins coded by extrachromosomal genes
- Plasmids – Bacillus anthracis toxin, tetanus toxin
- Lysogenic bacteriophage – e.g. streptococcal pyrogenic exotoxins in Scarlet Fever, Diphtheria toxin.
Describe the classification of exotoxins
• As very diverse group of proteins and many ways to classify.
• Classification can be by the toxins activity .
1. Membrane Acting Toxins – Type I
2. Membrane Damaging Toxins – Type II
3. Intracellular Toxins – Type III
• This classification has its problems:
- Many toxins may have more than one type activity.
- As mechanisms better understood this classification tends to break down
Describe membrane acting toxins – type I
• ACT:
- Act from without the cell.
• INTERFERE:
- Interfere with host cell signaling by inappropriate activation of host cell receptors.
• TARGET: - Target receptors include:
- Guanylyl cyclase → ↑
- intracellular cGMP
- Adenyl cyclase → ↑
- intracellular cAMP
- Rho proteins
- Ras proteins
Describe membrane damaging toxins – type II
Cause damage to the host cell membrane.
1. Insert channels into host cell membrane.
• β sheet toxins e.g. S.aureus α – toxin, δ toxin, PVL
• α helix toxins – e.g. diphtheria toxin
- Enzymatical damage e.g. S. aureus β- haemolysin, PSM
OR
- Receptor mediated
- Receptor Independent
Describe intracellular toxins – type III
• Active within the cell – must gain access to the cell
• Usually 2 components – AB Toxins
- Receptor binding and translocation function – B
- Toxigenic (enzymatic) – A
- May be single or multiple B units e.g. Cholera toxin AB5
In intracellular toxins, the enzymatic component A has a. variety of activities. List some of them.
- ADP – ribosyl transferases - e.g. Exotoxin A of Pseudomonas aeruginosa, pertussis toxin.
- Glucosyltransferases – e.g. TcdA and TcdB of Clostridium difficile
- Deamidate – e.g. dermonecrotic toxin of Bordetella pertussis.
- Protease – e.g. Clostridial neurotoxins: botulism & tetanus
- Adenylcyclase - e.g. EF toxin of Bacillus anthracis
Expand on superantigens and inflammatory cytokines
Exotoxins are able to induce inflammatory cytokine release such as IL1, IL1β, TNF, IL 6,δ interferon, IL18
MECHANISMS:
- Superantigen – non specific bridging of the MHC Class II and T- cell receptor leading to cytokine production. E.g. Staphylococcal Exfoliative Toxin A, Toxic Shock Syndrome Toxin 1 (TSST1)
- Via activation of the different inflammasome leading to release IL1 β and IL18 e.g. S. aureus toxin A, PVL.
Expand on toxoids ,vaccine and antibodies
• Toxins can be inactivated using formaldehyde or glutaraldehyde = toxoids
• Toxoids are inactive proteins but still highly immunogenic – form the basis for
vaccines. The following are toxoid based:
1. Tetanus Vaccine
2. Diphtheria
3. Pertussis (acellular).*
• Treatment of toxin mediated disease can be affected by administering preformed antibodies to the toxin
- Diphtheria antitoxin – horse antibodies.
- Tetanus – pooled human immunoglobulin. Specific or normal.
- Botulism – horse antibodies
• Experimental and research – monoclonal antibodies ~ not used routinely at the moment.
Describe the microbiology of Clostridium difficile
- gram-positive bacillus.
- anaerobic.
- spore-forming.
- toxin-producing.
- Can be carried asymptomatically in the gut.
- 3 toxins.
Describe the epidemiology of Clostridium difficile
- Common hospital acquired infection worldwide.
- Spread by ingestion of spores – remain dormant in environment.
- Coloniser of the human gut up to 5% in adults.
- Risk factors – antibiotic use, age, antacids & prolonged hospital stay.
How do antibiotics affect Clostridium difficile?
- Thought to act by disrupting the microbial ecosystem within the gut.
- Antibiotics provide a competitive advantage to spore forming anaerobes over non spore forming anaerobes.
- Allows C. difficile colonisation and growth.
- All antibiotics have potential for causing disease.
•Some antibiotics worse than others
- 2nd and 3rd generation cephalosporins
- Quinolones
- Clindamycin?
•Others less likely
- Aminoglycosides
- Trimethoprim
- Vancomycin
Describe the toxins in Clostridium difficile
- Cytotoxin A - TcdA coded by tcdA gene
- Cytotoxin B – TcdB coded by tcdB gene
- Binary toxin – C. diff transferase (CDT) – minor role in disease
- Tcd A and Tcd B – Type III AB toxins.
- The A component of toxins are glycosylating enzymes.
Describe the mechanism of action of TcdA/TcdB toxin
1) Toxins bind to specific host cell receptors
2) The toxins are internalised by the cell
3) The endosome in which the toxin is in is acidified
4) There is pore formation in the endosome
5) GTD is released from the endosome to the host cell cytoplasm
6) Rho GTPases are inactivated by glycosylation
7) There are downstream effects within the host cell
What are the effects of the TcdA/TcdB toxins in the cell?
CYTOPATHIC EFFECTS:
- cytoskeleton breakdown
- loss of cell-cell contacts
- increased epithelial permeability
CYTOTOXIC EFFECTS:
- activation of the inflammasome
- increase in ROS levels
- induction of programmed cell death
What are some symptoms of the C.difficile disease?
It can range from:
- Asymptomatic
- Watery Diarrhoea
- Dysentery
- Pseudomembranous Colitis
- Toxic megacolon and peritonitis
How would you diagnose C.difficile?
•Clinical signs and symptoms
•Raised white cell count in blood.
•Detection of organisms and toxins in stool. It’s a 2 phase test:
- Glutamate dehydrogenase ~ detects if C. difficile organism present.
- Toxin enzyme linked immunosorbent assay (ELISA) for TcdA and TcdB toxins.
- Detection of tcdA and tcdb genes – PCR
- Colonoscopy – pseudomembranous colitis
How would you treat C.difficile?
- Treatment dependent on severity and presence of surgical complications
- Ideally removal of offending antibiotic – not always possible
- Antibiotics that will act against C. difficile are; fidaxomicin or metronidazole or vancomycin (also mentioned as one of the causes)
- Surgery – partial, total colectomy
- Recurrent – faecal transplant.
What is the VTEC/STEC disease?
• Verocytotoxin Escherichia coli (VTEC) disease
• VTEC, or Shiga-toxin (Stx) producing E.coli (STEC) can cause disease mild to life threatening disease.
• Stx carried by some E. coli – most commonly O157:H7 ~ Identified usually by growth on sorbitol MacConkey agar (SMac)
– does not ferment sorbitol and hence is clear. ~ Other less common types not identified using SMac.
What is the epidemiology of VTEC/STEC?
E. coli O157:H7 naturally colonizes the gastrointestinal tracts of cattle who are generally asymptomatic.
TRANSMISSION:
- Predominantly via consumption of contaminated food and water
- Person to person, particularly in child day-care facilities, and from
- Animal to person. E.g. in petting zoos, dairy farms, or camp grounds.
Very low infectious dose
What is the pathogenesis of the toxins of VTEC?
- Toxin – Shiga like toxin (SLT) = shigatoxin (Stx) = verocytotoxin (VTEC)
- Many types: Stx, Stx1, Stx1a, 1c, 1d Stx2a, 2c, 2d – variations in a.a. Sequence ~ all act the same way
- Gene carried on lysogenic virus.
Type III exotoxin – AB5
- Enzymatic component A = N-Glycosidase.~ It is bound to 5 B subunits
Describe the mechanism of the shigatoxin
•Bind to receptor globotriaosylceramide Gb3 or globotetraosylceramide (Gb4) on host cell membrane
•Bound toxin internalised by receptor mediated endocytosis.
•Carried by retrograde trafficking via the Golgi
apparatus to the endoplasmic reticulum.
•The A subunit is cleaved off by membrane bound proteases
•Once in the cytoplasm A1 and A2 disassociate
•A1 binds to 28S RNA subunit – blocks protein
synthesis.
Describe the pathogenesis of VTEC
STEC closely adheres to the epithelial cells of the gut mucosa.
The route by which Stx is transported from the intestine to the kidney and other tissues is debated, possibly polymorphonuclear neutrophils (PMNs)
Bind to glomerular endothelial cells of kidney, cardiovascular and central nervous system.
Very high levels of Gb3 in kidney so kidneys most affected.
Thought that Stx favours inflammation resulting in microvascular thrombosis and inhibition of fibrinolysis.
Describe the symptoms and disease of VTEC
- Can be severe and life threatening. Children < 5 years greatest risk.
- Abdominal cramps, watery or bloody diarrhoea – may not be present
Haemolytic uraemic syndrome
- Anaemia
- Renal Failure
- Thrombocytopaenia
Less common are neurological symptoms
- lethargy,
- severe headache,
- convulsions,
- encephalopathy.
How would we diagnose and treat VTEC?
DIAGNOSIS:
•Clinical signs and symptoms
•Haematological and biochemical evidence.
•Stool culture – Growth on Smac ~ agar
•PCR for Stx genes ~ do screening test, if positive then we grow it
TREATMENT:
•Supportive including renal dialysis and blood product transfusion
•Antibiotics have little to no role
