Staphylococcus and Streptococcus

1. Staphylococcus and Streptococcus

2. Nightmare bacteria Super bugs Flesh eating bacteria Streptococcal necrotizing fasciitis MRSA

3. Common characteristics Staphylococcus Streptococcus • Gram positive cocci • Facultative anaerobes • Non-sporing, non-motile • Important pathogens • Pyogenic –suppurative infections • Commensals of mucous membranes and skin

4. Staphylococci • Opaque, creamy colonies • Produce catalase • Resistant to bile salts (grow on MacConkey agar) • Grow on simple media (Nutrient agar) • Generally survive well in the environment Streptococci • Translucent, greyish colonies • Don’t produce catalase • Sensitive to bile salts (don’t grow on MacConkey agar) • Don’t grow on simple media (Nutrient agar) • Generally survive poorly in the environment Differentiating characteristics

5. Staphylococci - EM

6. Staphylococci of veterinary importance

7. Laboratory diagnosis • Specimens: • May include exudates, pus, mastatic milk, skin scraping, etc • Direct microscopy: • To examine gram stained smears (pus/exudates) • Isolation: • Routine medium is blood agar (24 hours). • MacConkey agar in parallel, to detect any gram negative bacteria.

8. toxin toxin S.aureus cultural characteristics

9. Staphylococcal haemolysins

10. Differentiating pathogenic from non pathogenic Staphylococci • Coagulase correlates with pathogenecity. • Micrococci are O-F positive, Streptococci & enterococci are … negative?

11. Coagulase and DNA tests DNAse agar. Not definitive

12. Necrotising “flesh eating” Pneumonia Furuncle Impetigo Endocarditis & Septicaemia Osteomyelitis Scalded Skin S. aureus Infections Toxic Shock Syndrome

13. Terminology Impetigo: A contagious skin infection that is characterized by the eruption of superficial pustules and the formation of thick yellow crusts, commonly on the face. Septicaemia: blood poisoning caused by pathogenic microorganisms and their toxic products in the bloodstream Osteomyelitis: infection of bone and bone marrow in which the resulting inflammation can lead to a reduction of blood supply to the bone

14. Strategies of extracellular pathogens Counteract complement Counteract phagocytes Acquire nutrients

15. Adherence • Fibronectin-binding protein • Extracellular adhesion • protein (Eap) • Anti-chemotaxis • Chemotaxis inhibitory protein • of staphylococci (CHIPS) • Toxins • Exfoliative toxins • Superantigens • -Toxic shock toxins • - Enterotoxins • Degradative enzymes • DNAse • Lipases • Proteases • Hyaluronidase Staphylococcus virulence factors • Anti-opsonic & anti-phagocytic • Staphylococcal complement • inhibitor (SCIN) • Protein A • Capsule • Staphylokinase • Fibrinogen-binding proteins • Haemolysins + Leukocidins Iron-uptake systems Staphyloferrin B

16. 1. Binding of the Fc portion of antibody to protein A Interferes with phagocytosis 2. Staphylococcal complement inhibitor (SCIN) inhibits complement activation - 3. Capsule production Inhibits all complement (C’) activation pathways C’ SCIN Activates and binds complement, but not opsonised or phagocytosed 4. Fibrinogen binding protein -surface bound fibrinogen Inhibits opsonisation 5. Haemolysins and leukocidin Inhibit or lyse phagocytes Staphylococcal resistance to phagocytosis

17. S.aureus disease in animals Mastitis Bumble foot

18. Staphylococcal scalded skin syndrome (SSSS)

19. Staphylococcal skin diseases Pyogenic infections following penetration of skin, boils etc –many species. Exfoliative skin diseases. Humans Pigs Dogs S.aureus S.hyicus S.intermedius Scalded skin syndrome Greasy pig disease Pyoderma

20. Exfoliative conditions skin diseases • Prevalent Staph on skin of particular host • Humans = S.aureus • Pigs = S.hyicus • Dogs = S.intermedius • Virulence factors: Exfoliative toxin (ET). • S.aureus – at least 4 ET • Protease – cleaves desmoglein 1 in desmosomes • Separation of the upper epidermis • S.hyicus • At least 5 ET • S.intermedius • An ET-like toxin has been described

21. “Tampon disease” • Staphylococcal toxic shock syndrome (TSS): high fever, hypotension, renal failure • Increase in staphylococcal TSS in menstruating women in early 1980’s in USA • Particular S.aureus strains producing toxic shock syndrome toxin-1 (TSST-1) is a SUPERANTIGEN • Associated with super absorbent tampons • ? Absorb Mg++ low [Mg++] act as cue for S.aureus to produce TTST-1.

22. Superantigen Antigen Stimulate 2-20 % of T cells Stimulate < 0.01% of T cells Massive release of pro-inflammatory cytokines Shock Superantigens (SA) Superantigens (SAgs) are a class of antigens which cause non-specific activation of T-cells resulting in polyclonal T cell activation and massive cytokine release. SAgs can be produced by pathogenic microbes as a defense mechanism against the immune system. S.aureus and Streptococcus pyogenes produce many SA

23. TLR-2 TLR-4 TLR-5 TLR-9 Pattern-recognition receptors Toll-like receptors (TLRs) Peptidoglycan, Lipoproteins Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They recognize structurally conserved molecules derived from microbes. Once these microbes have breached physical barriers such as the skin or intestinal tract mucosa, they are recognized by TLRs which activates immune cell responses. LPS, lipid A Flagellin Bacterial DNA

24. Host defence against Staphylococcus aureus infection require recognition of bacterial lipoproteins Juliane Bubeck Wardenburg, Wade A. Williams, and Dominique MissiakasHost defenses against Staphylococcus aureus infection require recognition of bacterial lipoproteinsPNAS 2006 103: 13831-13836 S.aureus mutant can not make lipoproteins Infection of mice Survivors S.aureus 90% S.aureus mut 20%

25. MRSA in pets

26. Streptococci

27. Haemolysis

28. Streptococi cultural characteristics S.zooepidemicus S.equi S.uberis MacConkey No growth

29. Typing of Streptococci Superficially grouped based on their growth on blood agar plates into: • Alpha haemolytic: produce green discoloration around the colonies, but RBCs are intact. • Beta haemolytic: lyse RBCs and produce clear zone around the colonies. • Gamma haemolytic: non-haemolytic. Most are not pathogenic.

30. Laboratory diagnosis • Specimens: • May include exudates, pus, mastatic milk, skin scraping, etc • Direct microscopy: • Smears (pus/exudates) are fixed and gram stained. • Isolation: • Routine medium is blood agar (18-24 hours). • MacConkey agar as a selective media (to distinguish from Staph). • Identification: • Colonial appearance, G-stained smears, Catalase, Lansfield grouping, etc.

31. CAMP test • Based on the haemolytic synergism between S. aureus toxins and group B Streptococci. • A culture of Staph aureus is streaked across the centre of a blood agar plate. • A streak of the suspect of group B Streptococci is streaked at right angle (90 degree). • After 24 hour incubation, a positive CAMP test is indicated by an arrow-head of complete haemolysis.

32. Typing of Streptococci Lansfield serologic typing – place streptococci in 22 (Lansfield) groups (A-V), based on differences in carbohydrate antigen in cell wall. Use antisera against different antigens to type.

33. Streptococci of veterinary importance

34. Pathogenicity factors of pyogenic (-haemolytic) streptococci) • Adherence to epithelium etc: • Fibronectin binding proteins • Facilitate internalisation and subsequent localisation in the sub-epithelial space • Anti-chemotaxis: • C5a peptidase • IL-8 protease (ScpC) Systemic toxicity Streptococcal superantigens • Anti-opsonic & • anti-phagocytic factors: • Capsule • M-protein + M-like proteins • Haemolysins • (Streptolysins O & S) • Degradative enzymes: • DNAases • Proteases • Hyaluronidase • Streptokinase • Cysteine protease

35. M-protein - structure & function

36. M-protein - structure & function Cell wall Hypervariable region Fibrinogen binding Cell membrane Host cross-reactive epitopes Factor H binding (destroy C3 convertase and preventing opsonization)

37. Bovine mastitis incidence (2004) S. agalactiae 2% S. dysgalactiae 7% S. uberis 28% Staphylococcus aureus 25% E. coli 25%

38. Streptococcal mastitis

39. Equine Group C streptococci S. equisimilis Nasopharyngeal commensal Opportunistic sepsis e.g. wound S. zooepidemicus Nasopharyngeal commensal Opportunistic e.g. 2° respiratory infections; wound; metritis; etc. Infects various host species: cattle, sheep, dogs, etc. S. equi [carrier animals?] Pharyngitis Lymphadenitis Fever, swollen lymph nodes, discharge - STRANGLES Septicaemia (fatal) - BASTARD STRANGLES Highly contagious: Quarantine & antibiotic treatment

40. Horse with strangles: swollen lymph nodes at neck

41. Head of horse with strangles split to show gutteral pouch empyaema

42. Two fibronectin binding proteins- Adhesion Two M-like proteins-SeM, SzPSe Anti-phagocytic, anti-opsonic. SzPSe present in S.zooepidemicus SeM more important Hyaluronic acid capsule Anti-phagocytic Non-immunogenic Toxins Streptolysin S (SLS)-like toxin, anti-phagocytic Virulence factors of S.equi

43. Strangles vaccine Intervet Equilis StrepE. Live genetically modified S.equi vaccine, S.equi aro mutant Administer inside upper lip.

44. S.canis: toxic-shock syndrome Canine toxic-shock syndrome. An emerging disease? Symptoms of toxic shock syndrome vary depending on the underlying cause. Typically characterized by high fever, accompanied by low blood pressure, malaise and confusion, which can rapidly progress to coma, and multi-organ failure.

45. Strep suis china

46. Enterococci In Veterinary Medicine • Commensals in gut of humans and animals • Referred as group D Streptococci • Resistant to Bile Salts • Grow on MacConkey agar • Grow on 6.5% NaCl • Opportunist pathogens in all species • Bacteraemias in immunosuppressed animals and people • Often very resistant to antibiotics