Medically Important Bacteria Gram Positive Cocci

1. Medically Important BacteriaGram Positive Cocci Phase I/ Module VII Dr Ekta Chourasia

2. Learning Objectives At the end of the session, you should be able to: • Enlist medically important GPCs • Compare & contrast the features of staphylococci & streptococci • List important virulence factors, toxins & enzymes, produced by them • Explain the role of virulence factors in the development of infection by GPCs • List pathogenicity of staphylococci & streptococci • Explain the basis of isolation & identification of GPCs in clinical specimens in laboratory. Learning resources: • Lecture & practical notes • Ananthanarayan Ch 22, 23, 24 Phase I/ Module VII Dr Ekta

3. GPC Staphylococcus Streptococcus Enterococcus GNC Neisseria Moraxella Branhamella Medically Important Cocci Phase I/ Module VII Dr Ekta

4. Gram Positive Cocci (GPC) Staphylococcus(GPCs in clusters) • Staphylococcus aureus • S. epidermidis • S. saprophyticus Streptococcus(GPCs in chains) • Streptococcus pyogenes (Group A ) • Streptococcus agalactiae (Group B) • Streptococcus pneumoniae (Pneumococcus) • Streptococcus viridans • Enterococcus (Group D) Phase I/ Module VII Dr Ekta

5. In clusters (grapes like) or short chains Catalase + ve Facultative anaerobes Grows on ordinary medium Major components of normal flora – skin, nose & mucosa Produces localized infections Resistant to drying, Penicillin In chains or in pairs Catalase -ve Obligate & facultative anaerobes, grows in 5-10% CO2 Fastidious – requires enriched medium like BA Normal flora in throat, colon, female genital tract Infections tend to spread Delicate organism, sensitive to penicillin Staphylococci v/s Streptococci Phase I/ Module VII Dr Ekta

6. Staphylococcus 1 Phase I/ Module VII Dr Ekta

7. Clinically Important Species • Many species are medically important • S. aureus – Coagulase + ve • Most virulent species • Most common cause of bacterial infections, food poisoning & toxic shock syndrome • Coagulase –ve Staphylococci • S. epidermidis – important cause of prosthetic implant infections • S. saprophyticus – Urinary Tract Infection in young women Phase I/ Module VII Dr Ekta

8. Staphylococcus aureus – General features • Coagulase positive • Beta hemolytic colonies on BA • Produces golden yellow pigment • Highly resistant bacteria • Can grow in the presence of 10 – 15% NaCl Phase I/ Module VII Dr Ekta

9. Virulence Factors • Toxins – cytolytic & superantigen exotoxins • Enzymes & • Cell associated polymers and surface proteins Phase I/ Module VII Dr Ekta

10. 1. Toxins • Haemolysins- Cytolytic, lyse RBCs of various animal species • Leucocidins - Kills leucocytes • Enterotoxin A to E - Food poisoning • Toxic shock syndrome toxin (TSST/ Enterotoxin F) – Toxic Shock Syndrome: rash, desquamation, multi organ failure • Epidermolytic ( Exfoliative ) toxin A & B – SSSS: Staphylococcal scalded skin syndrome (epidermal splitting & exfoliation) Phase I/ Module VII Dr Ekta

11. 2. Extracellular enzymes • Catalase – enhance their survival in phagocytes by inactivating toxic H2O2 & free radicals released after the ingestion of staphylococci. • Coagulase - Clots plasma, responsible for ‘tube coagulase test’, confirmatory test for S.aureus • Hyaluronidase - Breaks down hyaluronic acid (connective tissue): initiation & spread of infection • Fibrinolysin (Staphylokinase) - Lyses fibrin clots: spread of infection • Nuclease - hydrolyses DNA • Lipase – Lipolytic: infection of skin & subcutaneous tissue Phase I/ Module VII Dr Ekta

12. 3. Surface proteins • Protein A - Antiphagocytic • Clumping factor - bound coagulase, responsible for ‘slide coagulase test’, screening test for S.aureus Phase I/ Module VII Dr Ekta

13. Pathogenicity (Staphylococcal diseases) Phase I/ Module VII Dr Ekta

14. Pathogenicity of S. aureus • Cutaneous infections – • Folliculitis (boils), furuncle, burns and wounds • Deep infections – • Osteomyelitis, abscesses, pneumonia, endocarditis, septicemia • Toxin mediated infections – • Staphylococcal scalded skin syndrome (SSSS), • Toxic Shock Syndrome (TSS), • Food poisoning (in 1-8hr, vomiting ,diarrhea, nausea, self limited ) Phase I/ Module VII Dr Ekta

15. Folliculitis Abscess Toxic shock syndrome SSSS Phase I/ Module VII Dr Ekta

16. Lab diagnosis – S. aureus • Specimens: wound swab, pus, blood, feces • Microscopy: Gram stain - GPC in clusters • Culture • BA : beta hemolysis • NA : golden yellow pigment • Catalase positive • Coagulase positive Phase I/ Module VII Dr Ekta

17. Coagulase Test Phase I/ Module VII Dr Ekta

18. Staphylococcus epidermidis Major component of skin flora Nosocomial infections: device/ implant associated infections - shunts, catheters, artificial heart valves / joints, pacemaker BA: Non - hemolytic Identification Coagulase negative Prosthetic valve endocarditis Phase I/ Module VII Dr Ekta

19. Drug Resistance in Staphylococci & MRSA • First developed resistance against Penicillin • Resistance to penicillin is mainly attributed to the production of enzyme, penicillinase (beta-lactamase) • To combat resistance due to penicillinase, Methicillin was developed & now methicillin resistant strains have evolved – MRSA Phase I/ Module VII Dr Ekta

20. MRSA(Methicillin Resistant S. aureus) • Important cause of Nosocomial infections • post surgical wound infections • blood stream infections • ventilator associated pneumonia • Patients with open wounds, invasive devices and weakened immune systems are at greater risk for infection • Person to person spread – mainly from carriers (hospital staff, visitors), 25-30% carry in their nose. By contact with • colonized or infected patients • colonized or infected body sites of the personnel themselves, • devices, items, or environmental surfaces contaminated with body fluids containing MRSA. Phase I/ Module VII Dr Ekta

21. MRSA(Methicillin Resistant S. aureus) • Difficult to treat • Prevention and infection-control strategies • Screening of staphylococcal carriers among hospital staff • Treatment of carriers with mupirocin, hexachlorophene • Proper sanitary procedures – surface sanitation, hand washing (alcohol gels), personal protective measures • Isolation of patients with open staphylococcal lesions * However carrier status prevents complete control • Treatment of MRSA infection - Glycopeptides • Vancomycin • teicoplanin Phase I/ Module VII Dr Ekta

22. Streptococcus 1 Phase I/ Module VII Dr Ekta

23. Classification Based on O2 Anaerobes Aerobes Peptostreptococci Growth on BA γhemolysis α hemolysis β hemolysis Incomplete hemolysis (green color) Complete hemolysis α / β / no hemolysis Lancefield groupingspecific C carbohydrate Ag on cell wall Enterococcus fecalis Strep. viridans Strep. pneumoniae Group A – U (21 groups) Griffith typing of Group A on MTR proteins into > 100 types Phase I/ Module VII Dr Ekta

24. Beta hemolytic Group A Streptococcus(Streptococcus pyogenes) 1 Phase I/ Module VII Dr Ekta

25. Streptococcus pyogenes – virulence factors Streptolysin O (SLO) Oxygen labile Damage cardiac cells Antigenic – produce ASLO Streptolysin S (SLS) Oxygen stable , non-antigenic Exotoxins Streptococcal Pyrogenic Exotoxin (SPEs) Manifestation of scarlet fever Exoenzymes Streptokinase (fibrinolysin) / Streptodornase (DNAase) / Hyalarunidase Phase I/ Module VII Dr Ekta

26. Pathogenicity of Strep. pyogenes • Respiratory infections – • pharyngitis (sore throat), tonsillitis • otitis media, sinusitis • Skin & subcutaneous infections – • pyoderma, cellulitis • necrotising fasciitis (flesh eating bacteria) • Non suppurative complications – • Acute rheumatic fever – usually follows streptococcal pharyngitis • Acute glomerulonephritis – usually follows pyoderma Phase I/ Module VII Dr Ekta

27. Lab diagnosis – Strep. pyogenes • Specimens: throat swab, pus, blood • Microscopy :Gram stain - GPC in chains • Culture: BA - beta hemolytic colonies • Identification tests - • Catalase Negative • Bacitracin sensitive • Penicillin sensitive • ASO titre / DNAase B test B B Phase I/ Module VII Dr Ekta

28. Beta hemolytic Group B Streptococcus • Normal flora in lower GIT, female genital tract • Pathogenicity • Neonatal meningitis • Puerperal sepsis • Pneumonia 1 Phase I/ Module VII Dr Ekta

29. Lab diagnosis – Group B Streptococci • Specimens: CSF, blood, vaginal smears, urine • Microscopy :Gram stain - GPC in chains • Culture: BA - beta hemolytic colonies • Identification tests • Catalase negative • Bacitracin resistance • CAMP Test + • Penicillin sensitive B P Phase I/ Module VII Dr Ekta

30. Group D Streptococcus 1 Enterococcus – 2 imp. species E. fecalis E. faecium Normal flora in GIT, lower genital tract Nosocomial / opportunistic pathogen UTI, wound infection, endocarditis Resistance to cephalosporins, even vancomycin Phase I/ Module VII Dr Ekta

31. Lab diagnosis - Enterococcus Specimens: urine, pus, blood Microscopy: Gram stain - GPC in pairs or short chains Culture: BA - alpha / beta / no hemolysis Identification tests - Catalase Negative Bile esculin positive Growth in 6.5% Nacl Penicillin resistance Phase I/ Module VII Dr Ekta

32. Alpha hemolytic streptococciStreptococcus pneumoniae 1 Phase I/ Module VII Dr Ekta

33. Features of Pneumococci • Virulence factor – capsule • Pathogenicity • Otitis media, sinusitis - commonest • Pneumonia • Meningitis • Other suppurative lesions - Pericarditis, conjunctivitis, arthritis, peritonitis • Vaccine available for prevention – polyvalent polysaccharide vaccine Phase I/ Module VII Dr Ekta

34. Lab diagnosis • Specimen: CSF, blood, sputum, pus, swabs • Microscopy: Gram stain – GPC in pairs, capsulated, lanceolate shaped • Culture • BA/ CA – alpha hemolytic colonies • Identification tests • Catalase –ve • Optochin sensitive • Bile solubility Phase I/ Module VII Dr Ekta

35. Alpha hemolytic streptococciStreptococcus viridans 1 • Normally present on teeth, throat, colon & female genital tract • Pathogenicity – • Endocarditis & Dental caries Phase I/ Module VII Dr Ekta

36. Differences between Viridans Gp & Pneumococci Phase I/ Module VII Dr Ekta

37. Phase I/ Module VII Dr Ekta

38. Overview of the Medically Important Gram Positive Cocci Phase I/ Module VII Dr Ekta