Streptococci

Presentation on streptococci

1. Streptococcus Dr. Amirul Huda Bhuiyan Lecturer Department of Microbiology Shaheed Syed Nazrul Islam Medical College, kishorganj

2. Streptococci General criteria Gram positive, cocci, arrange in pairs or chains Nutritionally fastidious, require complex media supplemented with blood or serum Most are facultative anaerobe, but do not use O2 metabolically Some are capnophilic and others are anaerobic Ferment carbohydrate and produce lactic acid. No gas production C atalase negative, Non motile

3. Streptococcus pneumoniae Streptococcus pyogens

4. Staphylococcus Arrange in cluster Grow in ordinary media Colony – pin head, large Selective media – Mannitol salt agar Catalase – positive Produce thick pus Penicillin – usually resistant Streptococcus Arrange in chain Require enriched media Colony - small, pin point Selective media - Crystal Violet Blood Agar Catalase – negative Produce thin pus Penicillin – usually sensitive

5. Lancefield Grouping Based on C carbohydrate Ag on bacterial cell wall. 21 Lancefield group A-W

6. Hemolysis Trait Alpha hemolysis Beta Hemolysis Gamma Hemolysis Type incomplete Complete No Color Greenish discoloration Clear zone No change Margin Indefinite Sharp define Area of zone 1-2 mm wide 2-4 mm wide Mechanism H 2 O 2 produce by bacteria it o xidizes HB and produce biliverdin (green) Hemolysin Completely lyse RBC example Viridans streptococci ( viridis = green) S. Pneumonae S. Pyogen S. agalactiae Enteroccocci

7. S. Pyogens Gram positive, spherical / oval cocci Non-motile, non sporing 0.5 to 1 µm in diameter They divide in a plane perpendicular to the long axis of the chain Form long chain, when recovered from liquid culture May arrange in short chain, clusters or pairs in gram stains of samples from infected tissue

8. Antigenic structure A. Capsule Many strains produce hyaluronic acid capsule such strains grow as mucoid colony on Blood Agar, T hese are highly virulent A nti-phagocytic effect Non-immunogenic (chemically Identical to hyaluronic acid of host so, not recognize as foreign)

9. B. Cell wall composition – Outer layer of protein and lipoteichoic acid Middle layer of group specific C carbohydrate ( rhamnose & N acetylglucosamine ) Inner layer of peptidoglycan M protein Most important virulence factor determines the type of S. pyogenes Appears as hair-like projections on Streptococcal cell wall

10. M Protein A ntiphagocytic activity S. pyogenes is not infectious in the absence of M Protein It is antigenic and antibody to M Protein provides type specific immunity More than 100 different types of M Proteins have been identified by serological means I ndividual may suffer from recurrent infections with strains expressing different versions of M Protein

11. M protein Depending on M protein there are Rheumatogenic strain – Causes primarily rheumatic fever Nephritogenic strain- Causes primarily AGN

12. Enzymes Streptokinase ( Fibrinolysin ) Two types – Streptokinase A & B Catalyses conversion of plasminogen to plasmin it then cleaves fibrin and fibrinogen Result in lyses of clots and fibrin deposits, thrombi and emboli Facilitate rapid spread of S. pyogenes in infected tissues by breaking the fibrin barrier In contrast, S. aureus aims at hiding behind a wall of fibrin and produce a localized abscess It is used for treatment of pulmonary emboli and of coronary artery and venous thrombi A nti-streptokinase antibody are useful marker of infection

13. B. Deoxyribonucleases / Streptodornase / DNAase Four types ( Dnase ) are produced – DNAase A, B, C, D DNase B is the most common form They liqufy DNA (thick) which was released from dead neutrophil and make the pus thin Thus act as ‘spreading factor’ by liquefying purulent exudates Antibiotics thus gain better access and infection recover quickly

14. B. Deoxyribonucleases / Streptodornase / DNAa Use Mixture of streptokinase and streptodornase are used to liquefy exudate and facilitate removal of pus & necrotic tissue Anti Dnase B is of great value in diagnosing AGN following skin infection, not sore throat ( streptolysin O is irreversibly inhibited by skin cholesterol; so S. pyogenes fail to produce ASO in cutaneous infections.)

15. C. Hyaluronidase Spreading factor. Streptococcus causes spreading type of skin infection because this enzymes breaks hyaluronic acid, the ground substance of connective tissue . Strains produce large amount of hyaluronidase (M4, M22) are non-capsulated.

16. Toxins Hemolysin / Streptolysin Two types- Streptolysin O and Streptolysin S Streptolysin O Oxygen labile, heat labile, Immunogenic . Hemolytic in reduce state. Responsible for complete hemolysis around the colony. Inhibited by cholesterol

17. Mechanism Lyse RBC = Binds with cholesterol containing cell membrane leads to production of holes Degranulation and lysis of PMNs Inhibit phagocytosis by macrophage Impairs respons of lymphocyte to mitogen Cytotoxic for platelet and cardiac tissues

18. Antisterptolysin O Antibody titre of Streptolysin O, (ASO) >160-200 units use for diagnosis of recent streptococcal infection (pharyngeal) ASO block hemolysis by sterptolysin O Skin infection does not induce ASO. Why ASO response following streptococcal skin infection is low Inactivation of SLO by cholesterol present in skin.

19. Streptolysin S: Oxygen stable. Cell-bound, not antigenic (low molecular wt˂20,000 D). Produced in the presence of serum. S indicate serum stable Responsible for hemolysis in media. Mechanism Interact with membrane phospholipid in exerting toxic effect Can lyse erythrocytes, leukocytes and platelets . Kills phagocytes by releasing the lysosomal contents after engulfment .

20. Pyrogenic ( Erythrogenic ) Exotoxins Produced by both the scarlet fever strains and new invasive S. pyogenes strains . Four serologically distinct toxins ( Spe A, B, C and F ). Spe A and C called erythrogenic toxin. Responsible for scarlet fever; extensive rash. Antibody prevents rash . Associated with streptococcal toxic shock syndrome, necrotizing fasciitis (exotoxin B) rash of scarlet fever .

21. Pyrogenic ( Erythrogenic ) Exotoxins They are superantigens (except for Spe B, which is a cysteine protease) and may exhibit the following biological activities: Enhances release of pro-inflammatory cytokines, e.g. IL-1,2, TNF- α , INF- γ ( pyrogenicity ) causes skin rash Inflammation Hypotensive shock Organ failure Immuno-suppression

22. Diseases by S. pyogens Fig- rash of scarlet fever Fig- Necrotizing fascitis

23. Infection deep in subcutaneous tissues that spreads along fascial planes, destroying muscle and fat Initially cellulitis followed by bullae (fluid filled blisters; bulla is singular), gangrene, systemic toxicity, multi-organ failure. Prompt medical intervention is essential . Necrotizing fasciitis: (“flesh-eating bacteria”)

24. Scarlet fever If streptococci produce erytrogenic toxin Frequently develop scarletina rash on upper chest spreading to extremities within 1 to 2 days after the initial clinical symptoms of pharyngitis develops. The rash disappears over the next 5 to 7 days, followed by desquamation. strawberry tongue a characteristic lesion

25. Streptococcal Toxic Shock Syndrome (TSS) Occurs in invasive and bactaraemic infection, particularly in necrotizing fasciitis. Multisystem toxicity following soft tissue infection progressing to shock and organ failure. This disease results from release of pyrogenic exotoxin A and B. Superantigen , stimulate T cell and release cytokines that mediate shock and tissue injury.

26. Acute rheumatic fever (ARF ) Develops within 2-3 weeks following pharyngitis. Streptococcal skin infection do not cause RF. Autoimmune disease Pathogenesis : cross-reactivity of heart and joint tissues with antibody formed against M protein (Type II Hypersensitivity). Prevention- if treated with penicillin within first 10 days following initiation of pharyngitis.

27. Commonly involved M type 1, 3, 5, 6, 18. Inflammatory reaction characterized by fever, arthritis, carditis , chorea, erythema marginatum or subcutaneous nodules. ASO titre and ESR elevated Morbidity & mortality linked to subsequent disease of heart valve (Rheumatic Heart Disease) Recurrences common, lifelong penicillin therapy needed. Acute rheumatic fever (ARF)

28. Acute Glomerulonephritis Acute inflammation of renal glomeruli Occur after either pharyngeal or skin infection (most common) Following 2-3 weeks of respiratory (pharyngitis) or 1-2 weeks of cutaneous (pyoderma) streptococcal infection. Associated with M types 2, 4, 12 & 49 (most frequent) .

29. Granular accumulations of immunoglobulin due to deposition of immune complexes on the basement membrane of kidney (Type III Hypersensitivity) Rheumatic fever can be reactivated by recurrent streptococcal infections, whereas nephritis does not Acute Glomerulonephritis

30. Lab diagnosis A. Specimen : Can be obtained from throat or skin Swab, pus, sputum, blood . Serum obtained for antibody detection. B. Gram staining and microscopy Gram positive cocci arranged in chains or pairs with pus cells .

31. C. Culture ( Definitive diagnosis) small pinpoint, dome shaped colonies surrounded by a large zone of beta haemolysis in blood agar . Encapsulated cells produce mucoid colonies

32. D. Biochemical test Catalase negative. D ifferentiates from Staphylococcus

33. Bacitracin test: To distinguishing between Group A beta-hemolytic streptococci (bacitracin POS) and other beta-hemolytic streptococci that are isolated from pharyngeal swabs 95 % sensitivity for Grp A strep When grown on blood agar, Group A streptococci are sensitive to (killed by) the antibiotic bacitracin Bacitracin Sensitive - S.pyogenes Bacitracin Resistant - S.agalactiae

34. E. rapid test Antibody detection Detect S. pyogenes directly in throat swab without culture. Diagnostic kits use specific antibody to detect group specific Abs to detect group A CHO Ag in the swab material. Anti DnaseB Positive following streptococcal skin infection. Important in diagnosing AGN. ASO titre Titre high soon after Group A streptococcal infection-within 3 to 4 wks Indicate previous infection, as the time of development of RF culture is usually negative. Unreliable in AGN.

35. Treatment Oral penicillin V or amoxicillin for streptococcal pharyngitis. For penicillin-allergic patients, oral cephalosporin or macrolides can be used. For severe systemic infection- combined use of I/V penicillin and aminoglycosides recommended. Patient with a history of rheumatic fever require long term penicillin therapy as prophylaxis to prevent recurrence of the disease. Resistance found in – tetracyclines , sulphonamides , erythromycin and newer macrolids (e.g. azithromycin, clarithromycin)

36. Pneumococci Formerly called Diplococcus pneumonia. This was one of the first pathogenic bacteria observed by Gram stain.

37. Morphology Gram-positive cocci, non-motile , encapsulated (polysaccharide) Lancet shaped (oval with somewhat pointed ends) A rranged in pair or short chains in specimen. It has fastidious nutritional requirements and can grow only on enriched media supplemented with blood products . Produce alpha hemolysis on blood agar Ferment carbohydrates, producing lactic acid as the primary metabolic byproduct.

38. Capsular polysaccharide (SSS) Virulent strains are covered with a complex polysaccharide capsule. currently , 94 serotypes are recognized . Encapsulated strains are pathogenic whereas N oncapsulated strains are avirulent . It has antiphagocytic and antigenic activity. Provide type specific immunity. Structure

39. Capsular swelling ( Quellung ) reaction Pneumococcal capsule reacts with the type specific antiserum and becomes apparently swollen and become more easily visible under the microscope.

40. Cell wall carbohydrate The peptidoglycan layer is typical of gram-positive cocci. ‘ C ’ polysaccharide or C substance exposed on cell surface - It precipitates a serum globulin fraction [CRP ]. Structure

41. T oxin Pneumolysin It is a cytosolin similar to streptolysin O. It retain within the cytosol of intact pneumococci. It is released by autolysin from interior of the bacterium. Binds with cholesterol in mammalian host cell membrane and creates pore. It may contribute to pathogenesis by destroying ciliated epithelial cells and phagocytic cells.

42. Autolysin Hydrolase/ amidase present in inactivated form in cell wall. Activated by surface active substances, β- lactam antibiotics, bile or aging and results in cell lysis . Autolysis of aged cells produce colonies with depressed center (Draughtsman colony). Toxin

43. Enzymes IgA proteases It enhances the ability of the organism to colonize the mucosa of the upper respiratory tract by inactivating secretory IgA Ab Hyaluronidase Facilitate spread of the organism

44. Pneumonia Bronchopneumonia – secondary infection following viral infection Meningitis – pneumococcus spread from the pharynx to meninges via blood because of coexistent bacteremia Bacteremia and sepsis Sinusitis and otitis media Other suppurative infection- empyema, pericarditis, suppurative arthritis. Diseases

45. S . pneumoniae S . aureus K . pneumonia S . pyogens H . influenzae Fusobacterium species L . pneumophilia Bacteroids melaninogenicus Anaerobic cocci ( Peptostreptococcus , peptococcus ) Causes of pneumonia

46. Specimen According to site of lesion Blood , Sputum, nasopharyngeal swab, CSF, Pus, aspirate from sinus Smear preparation and microscopy Gram staining - Demonstration of gram positive lancet shaped diplococci surrounded by an unstained halo of capsule Lab diagnosis

47. Culture Streptococcus grows in enriched media like Blood agar media with 5-10% CO2 at 37 C Young culture of encapsulated bacteria produce circular, glistening, dome-shaped colonies. Lab diagnosis

48. Biochemical reaction Bile solubility test- undergo rapid autolysis when exposed to bile on an isolated colony Optochin sensitivity- sensitive. A zone of inhibition is seen around the colony Ferments glucose, lactose, sucrose and inulin giving acid only. Lab diagnosis

49. Penicillin is the drug of choice for susceptible strain For resistant strains and penicillin-allergic patients, cephalosporin, erythromycin, chloramphenicol or vancomycin can be used. Serious pneumococcal diseases should be treated with combination of antibiotics. Vancomycin with ceftriaxone commonly used as empiric treatment. Treatment

50. Pneumococcal conjugate vaccine (PCV13) R ecommended for all children younger than 5 years old, all adults 65 years or older, and people 6 years or older with certain risk factors. Pneumococcal polysaccharide vaccine (PPSV23 ) Recommended for all adults 65 years or older. People 2 to 64 years of age who are at high risk of pneumococcal disease should also receive PPSV23. immunization