34. Bacterial Pathogens and Diseases I

Question 1

Define pathogen

Answer 1

A microorganism capable of causing disease.

Question 2

Define pathogenicity

Answer 2

The ability of an infectious agent to cause disease.

Question 3

Define virulence

Answer 3

The quantitative ability of an agent to cause disease.

Question 4

Define toxigenicity

Answer 4

The ability of a microorganism to produce a toxin that contributes to the development of disease.

Question 5

List some virulence mechanisms

Answer 5

• Adherence Factors
• Biofilms
• Invasion of Host Cells and Tissues
• Toxins ~ endotoxins and exotoxins

Question 6

What are exotoxins?

Answer 6

• 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.

Question 7

What selective advantages do exotoxins give to the bacteria?

Answer 7

• 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.

Question 8

Looking at a specific disease, in the case of Staphylococcus aureus, what toxins does it produce and what are their functions?

Answer 8

• 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

Question 9

In the case of Staphylococcus aureus, why does it have these toxins?

Answer 9

• 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.

Question 10

Expand on the genetics of exotoxins

Answer 10

• 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.

Question 11

Describe the classification of exotoxins

Answer 11

• 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

Question 12

Describe membrane acting toxins – type I

Answer 12

• 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

Question 13

Describe membrane damaging toxins – type II

Answer 13

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

2. Enzymatical damage e.g. S. aureus β- haemolysin, PSM

OR

1. Receptor mediated
2. Receptor Independent

Question 14

Describe intracellular toxins – type III

Answer 14

• 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 15

In intracellular toxins, the enzymatic component A has a. variety of activities. List some of them.

Answer 15

• 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

Question 16

Expand on superantigens and inflammatory cytokines

Answer 16

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.

Question 17

Expand on toxoids ,vaccine and antibodies

Answer 17

• 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

1. Diphtheria antitoxin – horse antibodies.
2. Tetanus – pooled human immunoglobulin. Specific or normal.
3. Botulism – horse antibodies

• Experimental and research – monoclonal antibodies ~ not used routinely at the moment.

Question 18

Describe the microbiology of Clostridium difficile

Answer 18

• gram-positive bacillus.
• anaerobic.
• spore-forming.
• toxin-producing.
• Can be carried asymptomatically in the gut.
• 3 toxins.

Question 19

Describe the epidemiology of Clostridium difficile

Answer 19

• 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 20

How do antibiotics affect Clostridium difficile?

Answer 20

• 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 21

Describe the toxins in Clostridium difficile

Answer 21

• 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.

Question 22

Describe the mechanism of action of TcdA/TcdB toxin

Answer 22

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

Question 23

What are the effects of the TcdA/TcdB toxins in the cell?

Answer 23

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

Question 24

What are some symptoms of the C.difficile disease?

Answer 24

It can range from:

• Asymptomatic
• Watery Diarrhoea
• Dysentery
• Pseudomembranous Colitis
• Toxic megacolon and peritonitis

Question 25

How would you diagnose C.difficile?

Answer 25

•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

Question 26

How would you treat C.difficile?

Answer 26

• 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.

Question 27

What is the VTEC/STEC disease?

Answer 27

• 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.

Question 28

What is the epidemiology of VTEC/STEC?

Answer 28

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 29

What is the pathogenesis of the toxins of VTEC?

Answer 29

• 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

Question 30

Describe the mechanism of the shigatoxin

Answer 30

•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 31

Describe the pathogenesis of VTEC

Answer 31

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 32

Describe the symptoms and disease of VTEC

Answer 32

• 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 33

How would we diagnose and treat VTEC?

Answer 33

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