1. SBM 2044: Lecture 6
AIMS: To provide overview of:
Staphylococcus
S. aureus – diseases and drug-resistance
Multitude of S. aureus virulence factors

2. Staphylococcus Gram-positive cocci Grow in clusters
Gr. “Staphyle” = cluster of grapes)
Natural habitat – resident flora of human or animals
Hardy – can survive well outside host
Divided into 2 groups: coagulase-positive and -negative
Positive
Negative
S. epidermidis - Biofilms on plastic prosthetic implants (e.g. i.v. catheters; heart valves)
S. saprophyticus - UTIs, particularly cystitis
Single species
multiple species, some
occasionally pathogenic
Highly virulent
Much less virulent
e.g.:
S. epidermidis
S. saprophyticus
S. aureus

3. Staphylococcus aureus
Yellow/gold colonies
(aureus = gold)
Haemolytic on blood agar
Coagulase-positive
Ubiquitous – carried on skin and/or anterior nares
> 30% individuals
> 90% of hospital staff
Major problems
nosocomial infections
antibiotic resistance

4. Staphylococcus aureus
The most common pus-forming (pyogenic) bacteria
Can produce focal abscess, from the skin (furuncles, boils) to the lungs, osteomyelitis, kidneys and endocarditis
Include S. aureus, S. epidermidis, S. saprohyticus (UTI)
S. aureus can persist in the body because they have numerous cell surface virulence, exotoxins and enzymes

5. Staphylococci

6. S. aureus Encounter Major reservoirs = humans
Live on skin – grow at high salt and lipid concentrations because they make lipases and glycerol ester hydrolases, that degrade skin lipids
Colonise skin and mucosal surfaces using MSCRAMMs:
Fibronectin–binding proteins (FnbpA, FnbpB)
Collagen-binding
Clumping factors A and B
Spread person-to-person by direct contact or airborne

7. S. aureus pathogenesis Entry
Tissue penetration upon skin or mucosal membrane damaged by cut
Spread and Multiplication
Survival in tissues dependon
no. of entering microorganisms
site involved
speed of body’s inflammatory responses
immunological history of the host

8. S. aureus pathogenesis Damage
Local infections  pus collection, i.e. abscess
Staphylococci can spread into subcutaneous and submucosal tissues and caused cellulitis
Activate acute inflammatory reaction, pouring in chemotactic factors
Damaged area are usually localised by the formation of thick-walled fibrin capsule : center of abscess is necrotic with debris of dead cells
Why many virulence factors?

9. Surface structures:
Capsules – inhibit phagocytosis
Peptidoglycan – interacts with TLR-2, activate alternative pathway
Teichoic acid – C’ activation and adherence to mucosal cells
Protein A – binds to Fc terminus of IgG
Secreted factors:
Catalase – H2O2  H2O
Coagulases – fibrinogen  fibrin
Pore-forming toxins – create channels to disturb cellular homeostasis
Haemolysins –
Leukocidin
Hyaluronidase – hydrolyse matrix of connective tissues
Β-lactamase – hydrolyse penicillin
Penicillin-binding protein (PBP2a)

10. S. epidermidis Normal flora, rarely caused disease
Infections of S. epidermidis with other catalase-negative staphylococci in patients implanted with artificial devices e.g. prosthetic joints or IV catheters
Results in septicaemia and endocarditis
Possibly peptidoglycan or slime layer allows the organisms to stick to the surface of plastics

11. S. saprophyticus
Caused cystitis in young women

12. Staphylococcal toxin diseases
Staphylococcal scalded skin syndrome (SSSS)
Exfoliative toxins A and B – highly tissue specificserine proteases that causes separation of the layers of the epidermis at the desmosomes
Staphylococcal toxic shock syndrome (TSS)
characterised by fever, skin rash, hypotension, peeling of the skin
use tampons – oxygenated vagina and stimulate toxin production
TSST-1, staphylococcal enterotoxins AE
Virulence gene regulation – two-component regulatory systems
Accessory gene regulator (Agr), staphylococcal respiratory response (Srr)

13. Diagnosis
Gram stain and culture
Treatment
Methicillin-sensitive S. aureus – Rx: semi-synthetic penicillins and cephalosporins
Methicillin-resistant S. aureus – Rx: vancomycin
vancomycin-resistant S. aureus –acquired the genes of resistance from vancomycin-resistant Enterococcus species

14. Brief history of drug-resistance in S. aureus
1949-’50: First widespread use of natural penicillins
1951: First penicillin-resistant isolate of S. aureus
produced an enzyme that inactivated penicillin – essentially,
a penicillin-binding protein (PBP) with b-lactamase activity
b-lactamase producing S. aureus isolated increasingly thro’ 50s
Early 1960s: Methicillin introduced
Resistant to b-lactamase
By late 1960:
Methicillin-resistant S. aureus (MRSA)
mec genetic element, encoding a PBP (called PBP2’) with
reduced affinity for methicillin

15. Brief history of drug-resistance in S. aureus
Since 1950s:
As each new antibiotic introduced, resistant S. aureus
strains appeared – leading to multiple drug-resistance
By 1990s:
Depending on location, up to 25% S. aureus isolates
were MRSA strains resistant to all other useful
antibiotics except vancomycin
MRSA now almost synonymous with multi-drug resistance
(Vancomycin: given i.v. – can have side effects)

16. Salyers & Whitt First edition (1994) page 106
Vancomycin
Salyers & Whitt First edition (1994) page 106
“Vancomycin is virtually the only antibiotic left that is effective
in treating infections caused by such strains, and no one knows
how long it will be before the first vancomycin resistant MRSA
strains appear”
Japan, May 1996:
4 month old boy - MRSA infection after surgery
Vancomycin given for 29 days – no improvement
Strain found to have ‘intermediate’ level resistance
to vancomycin (VISA)
Regulatory mutants producing more copies of the target
Since then, VISA has been isolated across globe

17. VISA infections - treatable with high vancomycin dose
Salyers & Whitt 2nd edition (2002) page 169
“Keep in mind that MRSA strains are still treatable with
vancomycin. Imagine the carnage if vancomycin-resistant
MRSA strains appear”
Michigan USA, July 2002
First fully vancomycin-resistant S.aureus (VRSA)
Acquired van genes – probably from Enterococcus
Pennsylvania USA, Nov. 6, 2002
Second reported case of VRSA - unrelated to above

18. Staphylococcus aureus
Versatile pathogen
Toxinogenic diseases
Staphylococcal food-posioning
Scalded-skin syndrome
Toxic-shock syndrome
Suppurative infections – wide variety
Localised skin infections
Septicaemia
abscess, boils, furuncles, impetigo
Serious wound infections
Invasive infections
Toxic-shock

19. Staphylococcal food-poisoning
Staphylococcal enterotoxins (SE):
SEA, B, C1, C2, C3, D & E
Ingestion of food containing SE
symptoms 1 – 6 h after ingestion
Vomiting
Abdominal
cramps
Diarrhoea

20. Staphylococcal food-poisoning
Very common, very unpleasant, but usually self-limiting
(only rarely fatal)
“At first you think you are going to die, then you know
that you won’t, but wish that you could”
Enterotoxicity limited to primates
Exps. in monkeys, suggest emetic response involves
stimulation of nerves in stomach or gut, signalling
emetic centre of brain
Mechanism unknown – no in vitro models
Suggestions for link with superantigenic activities speculative

21. Scalded-skin syndrome
Epidermolytic (exfoliative) toxins ETA and ETB
Proteases cleave Desmoglein-1 (Dsg-1), found only
on surface of keratinocytes separate

22. Toxic-shock syndrome (TSS)
Originally recognised (1970s) as tampon-associated
toxic-shock
Led to identification of TSST
Later recognised SEs could also induce shock if
sufficient quantities reached bloodstream
TSST and SEs act as “superantigens” to activate large
‘families’ of T cells leading to overproduction of
cytokines and shock

23. Conventional antigen presentation to CD4 T cells
MHC class II
Antigen-presenting
cell (APC)
T cell receptors (TCR)
Ag uptake +
processing
Peptide associates
with ‘groove’ at top
of MHC class II
Recognition only by
epitope-specific TCR a b
Small subset
of T cells
activated
Epitope recognized ‘in context’ of MHC class II,
by an epitope-specific hypervariable sequence

24. Superantigens - intact toxin cross-links MHC class II + TCR
Antigen-presenting
cell (APC)
T cell receptors (TCR)
NO Ag uptake +
processing required
Can bind to TCRs
with various epitope-
specificities Va Vb
Many T cells
activated
May be specific for certain Vb families of TCR,
but each Vb family includes many diff. TCRs

25. S. aureus Most common cause of localised skin infections Abscess Boils
Carbuncles
Impetigo

26. Example of S. aureus invasive infections
Mass of
fibrin/debris
Fluid ?
S. aureus grown
from aspirated
exudate
> 500 ml
Still not drained completely

27. S. aureus virulence factors
Cytolytic toxins
Cell/tissue damage
Lysis of infiltrating PMNs
Suppuration
Amplifies mediator production
PMN infiltration increases
a-haemolysin - 85% clinical isolates
b-haemolysin - bovine isolates only (hlb - phage att site)
g-haemolysin - 99% clinical isolates
d-haemolysin - 100% strains
Leucocidin – 2% clinical isolates

28. Other excreted factors:
Catalase - evade phagocytic killing - all strains
Coagulase - triggers fibrin deposition
- all strains
Staphylokinase - fibrin degradation
Hyaluronidase
Facilitates spread +
contribute to damage
Collagenase
Various proteases
Lipase
Siderophores – at least 2 different siderophores

29. S. aureus virulence factors Cell-surface
Capsules:
much less than other encapsulated species
however expressed by 80% fresh clinical isolates
Transferrin receptor protein (Tpn)
role in iron acquisition
lack N-terminal signal peptides & wall-associating
domains - secreted or leakage ??
GADPH !!
Modun & Williams (1999) Infect Immun 67:
N-terminal sequencing of Tpn revealed that it was a
GAPDH (gyyceraldehyde-3-phosphate dehydrogenase)

30. MSCRAMMs: microbial surface components recognising adhesive
matrix molecules

31. Gram-positive bacteria
Anionic polymers
Teichoic
acid
LTA
Cell wall carbohydrate
(some species)
Wall-associated
proteins
Peptidoglycan
ALSO: Mention flagella, pili and fimbriae
Lipoproteins