bacterial pathogens and disease I

Question 1

what are exotoxins

Answer 1

heterogeneous group of proteins produced and secreted by living bacterial cells

produced by both gram negative and positive bacteria

cause disease symptoms in host during disease

act via a variety of diverse mechanism

Question 2

why have exotoxins

Answer 2

may help transmission of disease

evade immune response
enable biofilm formation
enable attachment to host cells 
escape from phagosomes 
all allow for colonisation, niche establishment and carriage

Question 3

why have exotoxins?

staphylococcus aureus case

Answer 3

Haemolytic toxins
cause cells to lyse by forming pores
Important cause of features of S. aureus disease.
α,β,, toxins ,Panton Valentine Leukocidin (PVL), LukAB, LukED, LukMF

Phenol soluble modulins PSM
Aggregate the lipid bilayer of host cells - lysis

Majority of S. aureus in humans is asymptomatic carriage in the nose.
So what are those toxins doing in the nose?

Question 4

genetics of exotoxins

Answer 4

Can be encoded by chromosomal genes Shiga toxin in Shigella dysenteriae, TcdA & 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.

Question 5

classification of exotoxins

Answer 5

s very diverse group of proteins and many ways to classify.
Classification can be by the toxins activity .

Membrane Acting Toxins – Type I
Membrane Damaging Toxins – Type II
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.

Question 6

membrane acting toxins i

Answer 6

act from without the cell

interefere with host cell signalling by inappropriate activation of host cell receptors

target receptors include: Guanylyl cyclase → ↑ intracellular cGMP
Adenyl cyclase → ↑ intracellular cAMP
Rho proteins
Ras proteins

Question 7

membrane damaging toxins - type II

Answer 7

Cause damage to the host cell membrane.

Insert channels into host cell membrane.
β sheet toxins e.g. S.aureus α – toxin,  toxin, PVL
α helix toxins – e.g. diphtheria toxin
Enzymatical damage e.g. S. aureus β- haemolysin, PSM

OR

Receptor mediated

Receptor Independent

Question 8

intracellular toxins - type III

Answer 8

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

Question 9

intracellular toxins - type III - AB toxins

Answer 9

Enzymatic component A – wide variety of activities.

ADP – ribosyl transferases - e.g. Exotoxin A of Pseudomonas aeruginosa, pertussis toxin.
Glucosyltransferases – e.g. TcdA and TcdB of Clostridium difficile
Deamidase – e.g. dermonecrotic toxin of Bordetella pertussis.
Protease – e.g. Clostridial neurotoxins: botulism & tetanus
Adenylcyclase - e.g. EF (Edema factor) toxin of Bacillus anthracis

Question 10

superantigens and inflammatory cytokines

Answer 10

Exotoxins are able to induce inflammatory cytokine release
IL1, IL1β, TNF, IL 6, 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.

Question 11

clostridium dificile

Answer 11

Epidemiology
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.

Question 12

clostridium dificile - antibiotics

Answer 12

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

Question 13

clostridium difficile disease

Answer 13

Cytopathic & Cytotoxic
Patchy necrosis with neutrophil infiltration
Epithelial ulcers
Pseudomembranes – leucocytes, fibrin, mucous, cell debris.

Question 14

c. difficile - diagnosis

Answer 14

Clinical signs and symptoms
Raised white cell count in blood.
Detection of organisms and toxins in stool
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

Question 15

c. difficile treatment

Answer 15

Treatment dependent on severity and presence of surgical complications
Ideally removal of offending antibiotic – not always possible
Antibiotics fidaxomicin or metronidazole or vancomycin
Surgery – partial, total colectomy
Recurrent – faecal transplant.

Question 16

verocyotoxin escherichia coli disease

Answer 16

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 O 157: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.

Question 17

epidemiology

Answer 17

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

Question 18

pathogenesis toxin

Answer 18

toxin – Shiga like toxin (SLT) = shigatoxin (Stx) = verocytotoxin (VTEC)
Stx, Stx1, Stx1a, 1c, 1d Stx2a, 2c, 2d – variations in a.a. sequence
Gene carried on lysogenic virus
Type III exotoxin – AB5
Enzymatic component A = N-Glycosidase
Bound to 5 B subunits

Question 19

mechansim

Answer 19

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.

Question 20

STEC pathogenesis

Answer 20

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.

Question 21

STEC disease

Answer 21

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.

Question 22

STEC diagnosis and treatment

Answer 22

diagnosis = clinical signs and symptoms
haematological and biochemical evidence
stool culture - growth on SMac
PCR for Stx genes

treatment:
- supportive including renal dialysis and blood product transfusion

antibitoics have little to no role