SBM 2044: Lecture 6

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 • multiple species, some • occasionally pathogenic • Single species • 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. 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 Toxic-shock • Invasive infections

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

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 • Proteases cleave Desmoglein-1 (Dsg-1), found only • on surface of keratinocytes separate • Epidermolytic (exfoliative) toxins ETA and ETB

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 - intacttoxin cross-links MHC class II + TCR MHC class II 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 • Boils • Carbuncles • Abscess • 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 Cell/tissue damage • Cytolytic toxins 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. Coagulase - triggers fibrin deposition - all strains Facilitates spread + contribute to damage • Other excreted factors: • Catalase - evade phagocytic killing - all strains • Staphylokinase - fibrindegradation • Hyaluronidase • 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 67:1086-1092 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 Lipoproteins