MLAB 2434: Microbiology Keri Brophy-Martinez

1. MLAB 2434: MicrobiologyKeri Brophy-Martinez Streptococci, Enterococci and Other Catalase-Negative Gram Positive Cocci

2. Streptococcus and Enterococcus: General Characteristics • Members of the Streptococcaceae family • Facultatively anaerobic • Aerotolerant • Catalase negative

3. Streptococcus and Enterococcus: General Characteristics • Most are typically spherical; some may appear elongated or ovoid • They may appear in chains or pairs

4. Streptococcus and Enterococcus: Habitat and Clinical Infections • Habitat • Normal Flora • Respiratory tract • Gastrointestinal tract • Urogenital tracts • Clinical Infections • Upper and lower respiratory tract infections • Urinary tract infections • Wound infections • Endocarditis

5. Streptococcus and Enterococcus:Cell Wall Structure • Thick peptidoglycan layer • Teichoic acid • Carbohydrate layer present • Used in Lancefield grouping of Streptococcus spp. • Capsule • Virulence factor • S. pneumoniae

6. Classification Overview • Physiologic characteristics • Pyogenic: produce pus • Lactococci: found in dairy products • Enterococci: normal gut flora • Viridans: normal URT flora • Hemolysis • J. H Brown • Alpha, beta, gamma classifications • Serological grouping • Typing of C carbohydrate • Lancefield group • Performed only on β-hemolytic hemolysis • Biochemical • Based on reaction of isolate

7. Classification:Hemolysis • J.H. Brown- 1903 • Grouped streps on ability to lyse RBCS • Alpha • Beta • Gamma • Alpha-prime

8. Hemolysis Patterns • Alpha (α): • Greenish discoloration • Caused by partial lysis of RBCs in media

9. Hemolysis Patterns • Beta (ß): • Complete lysis of RBCs • Produces a clear, colorless zone

10. Hemolysis Patterns • Gamma : • Colonies show no hemolysis or discoloration • Called non-hemolytic

11. Classification:Serological Grouping • Rebecca Lancefield – 1930 • Based on presence of carbohydrates in cell wall • Groups A, B, C, and D most significant • Typing done on beta-hemolytic colonies

12. Classification:Biochemical Identification/Susceptibility • Bacitracin • “A” disk or “Taxo A” disk • 0.04 units • Identifies Group A streptococci (S. pyogenes) • Zone of inhibition is presumptive ID of Grp. A strep Group A streptococcus is susceptible to “A” disk (left)

13. Biochemical Identification/Susceptibility • Optochin • P disk or“Taxo P” disk • Differentiates S. pneumoniae from other alpha-hemolytic streptococci

14. Biochemical Identification • Bile solubility test • Detects amidase enzyme • Under bile salt or detergent lyses cell wall • Clearing through lysis of colonies • Diagnostic for S. pneumoniae

15. Biochemical Identification • PYR hydrolysis • Substrate L-pyrrolidonyl-b-napthlyamide (PYR) is hydrolyzed by the enzyme pyrrolidonyl arlamide • Group A Streptococci and Enterococcus sp. posses the necessary enzyme. • More specific than Bacitracin for Group A streptococci The disk on the right has turned a red color, indicating a indicating a positive reaction. The left disk remains a yellow color indicating a negative result.

16. Biochemical Identification • Hippurate hydrolysis • Differentiates Group B streptococci from other beta hemolytic streptococci • Group B streptococci hydrolyzes sodium hippurate forming a purple color

17. Biochemical Identification • CAMP test • Christie,Atkins, Munch-Petersen • Detects the production of enhanced hemolysis that occurs when b-lysin and the hemolysins of Group B streptococci come in contact with each other Group B streptococci showing the classical “arrow-shaped hemolysis near the staphylococcus streak

18. Biochemical Identification • Bile esculin hydrolysis • Ability to grow in bile and hydrolyze Esculin • Characteristic of streptococci that possess group D antigen and Enterococci Both Group D streptococci and enterococci produce a positive (top) bile Esculin hydrolysis test.

19. Biochemical Identification • Salt Tolerance • Growth in 6.5% NaCl broth • Differentiates Group D streptococci from enterococci • Enterococcus= POSITIVE • Tube on left • Group D Streptococcus= NEGATIVE • Tube on right

20. Non-culture Identification • Slide agglutination kits • Latex beads are coated with group specific anti-serum, which clump when mixed with a small amount of colony from the specific Streptococcus sp. • Nucleic Acid Probes • Detect genes for specific groups

21. Slide Agglutination Tests

22. Slide Agglutination Tests

23. Break Time!

24. Virulence Factors: Streptococcus pyogenes • Fimbrae: Protein F • Attachment and adherence • M protein: • Resistance to phagocytosis • Hyaluronic acid capsule: • Prevents phagocytosis • Lipoteichoic acid: • Adheres to molecules on host epithelial cells

25. Virulence Factors:Streptococcus pyogenes • Hemolysins • Streptolysin O (O2 labile) detected in ASO titers • Streptolysin S (O2 stable) Causes hemolysis on plates • Erythrogenic toxin/Streptococcal pyogenic exotoxin: • Scarlet fever • Enzymes • Streptokinase • DNases • Hyaluronidase – “spreading factor”

26. Clinical Conditions: Streptococcus pyogenes(Group A)

27. Clinical Conditions: Streptococcus pyogenes(Group A) • Pyodermal infections • Impetigo: weeping lesion • Erysipelas • Cellulitis • Wound Infections Erysipelas due to Streptococcus pyogenes

28. Clinical Conditions: Streptococcus pyogenes(Group A) • Scarlet Fever • Starts with pharyngitis and causes rash on trunk and extremities • Due to untreated Group A infections

29. Invasive Group A Streptococcal Infections • Streptococcal toxic shock syndrome • Multi-organ system failure similar to staphylococcal toxic shock • Initial infection may have been pharyngitis, cellulitis, peritonitis, or other wound infections

30. Invasive Group A Streptococcal Infections • Cellulitis/Necrotizing Fasciitis • Severe form of infection that is life-threatening • Bacteremia and sepsis may occur • In patients necrotizing fasciitis, edema, erythema, and pain in the affected area may develop • Streptococcal myositis resembles clostridial gangrene

31. Post–Group-A Streptococcal Infections • Rheumatic fever • Fever • Inflammation of the heart, joints, blood vessels, and subcutaneous tissues • Chronic, progressive damage to the heart valves (most evidence favors cross-reactivity between Strep. antigens and heart tissue) • ASO titer will be elevated

32. Post–Group-A Streptococcal Infections • Acute glomerulonephritis (AGN) • Follows either cutaneous or pharyngeal infections • More common in children than adults • Antigen-antibody complexes deposit in the glomerulus • Inflammatory response causes damage to the glomerulus and impairs the kidneys

33. Laboratory Diagnosis: Group A Streptococcus Grams stained wound smear showing gram-positive cocci in chains with numerous “polys” (PMNs)

34. Laboratory Diagnosis: Group A Streptococcus • Colony morphology • Transparent, smooth, and well-defined zone of complete or b- hemolysis

35. Laboratory Diagnosis: Group A Streptococcus • Identification • Catalase-negative • Bacitracin-susceptible • PYR-positive • Hippurate hydrolysis- negative • Slide agglutination Group A streptococci is susceptible to Bacitracin disk (left); The right shows resistance

36. Group B b-Hemolytic Streptococcus (Streptococcus agalactiae) • Colonize the urogenital tract of pregnant women (10-30% rate – can cause OB complications such as premature rupture of membranes and premature delivery) • Mother fails to pass protective antibodies to fetus • Cause invasive diseases in newborns • Early-onset infection • Late-onset disease

37. Invasive Disease in the Newborn

38. Invasive Streptococcus agalactiae Infections • In adults • Occurs in immunosuppressed patients or those with underlying diseases • Often found in a previously healthy adult who just experienced childbirth

39. Laboratory Diagnosis: Streptococcus agalactiae • Colony morphology • Small • Grayish-white • Mucoid, creamy • Narrow zone of b-hemolysis

41. Laboratory Diagnosis: Streptococcus agalactiae • Presumptive Identification tests • Gram stain- GPC in chains • Catalase-negative • Bacitracin-resistant • Bile esculin- negative • Does not grow well in 6.5% NaCl. • CAMP- positive • Slide agglutination S. agalactiae shows the arrow-shaped hemolysis near the staphylococcus streak, showing a positive test for CAMP factor

42. Streptococcus pneumoniae • General characteristics • Inhabits the nasopharyngeal areas of healthy individuals • Typical opportunist • Possess C substance • Virulence factors • Polysaccharide capsule

43. Clinical Conditions:Streptococcus pneumoniae • Pneumonia • Most common cause of bacterial pneumonia • Meningitis • Bacteremia • Sinusitis/otitis media • Most common cause of otitis media in children < 3 years

44. Laboratory Diagnosis:Streptococcus pneumoniae • Microscopic morphology • Gram-positive cocci in pairs; lancet-shaped (somewhat oval in shape)

45. Laboratory Diagnosis:Streptococcus pneumoniae • Colony morphology • Smooth, glistening, wet-looking, mucoid • a-Hemolytic • CO2enhances growth • As colony ages, autolytic collapse causes “checker shape”

46. Laboratory Diagnosis: Streptococcus pneumoniae • Identification • Catalase negative • Optochin-susceptibility-test–susceptible • Bile-solubility-test–positive

47. Identification Schema

48. EnterococcusSpecies • Clinically Significant Isolates • E. faecalis • E. faecium • Opportunistic pathogens • In the GI tract, genitourinary tract and oral cavity • Associatedinfections • Bacteremia • Urinary tract infections • Wound infections • Endocarditis • Hospital-acquired Infections

49. Laboratory Diagnosis: EnterococcusSpecies • Microscopic morphology • Cells tend to elongate • Colony morphology • Small, grey • Most are non-hemolytic, although some may show a- or, rarely, b-hemolysis • Possess Group D antigen

50. Laboratory Diagnosis:EnterococcusSpecies • Identification tests • Catalase: may produce a weak catalase reaction • Hydrolyze bile esculin • Differentiate Group D from Enterococcus sp. with 6.5% NaCl or PYR test • Important to identify Enterococcus from non-Enterococcus, because Enterococcus must be treated more aggressively.