1. MLAB 2434: Microbiology Keri 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
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
Early Onset
Late-Onset
Age of Onset
< 7 days
7 – 30 days
Median age of onset
1 hour
27 days
Maternal complications of labor
Common
Less common
Incidence of prematurity
25%
Source of Organism
Maternal genital tract
Maternal genital tract; nosocomial; community
Clinical presentation
Nonspecific (35-55 %)
Meningitis 5-10 %
Respiratory diseases %
Focal
Meningitis %
Types
I, II III, V
III (75%)
Mortality Rate
5-15 %
2-10 %

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
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. Enterococcus Species Clinically Significant Isolates
E. faecalis
E. faecium
Opportunistic pathogens
In the GI tract, genitourinary tract and oral cavity
Associated infections
Bacteremia
Urinary tract infections
Wound infections
Endocarditis
Hospital-acquired Infections

49. Laboratory Diagnosis: Enterococcus Species
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: Enterococcus Species
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.

51. Identification Schema
Or PYR disk

52. Other Streptococcal Species
Viridans group (Viridans means “green”)
Members of the normal oral, nasopharyngeal flora, GI tract and female genital tract
Most are  hemolytic but also includes nonhemolytic species
The most common cause of subacute bacterial endocarditis (SBE)
Also involved with gingivitis and dental carries
PYR= negative
Optochin= negative
Bile solubility= negative

53. Viridans 5 groups Anginosus
S. anginosus, S. intermedius, S. constellatus
Mitis
S. sanguig, S. parasanguis, S. gordonii, S. crista, S. infantis, S. mitis, S. oralis, S. oralis, S. peroris
Mutans
S. criceti, S. downei, S. macacae, S. mutans, S. rattus, S. sobrinus
Salivarius
S. salivarius, S. thermophilus, S. vestibularis
Bovis
S. equinus, S. gallolyticus,S. infantarius, S. alactolyticus

54. Abiotrophia & Granulicatella
Once referred to as Nutritionally variant streptococci (NVS)
Causes endocarditis and otitis media
Normal flora of oral cavity
Requires pyridoxal to grow (can satellite around Staph, E. coli, Klebsiella, Enterobacter and yeasts)

55. Streptococcus and Enterococcus

56. Streptococcus and Enterococcus
Treatment
Generally, streps are not routinely tested for susceptibility since penicillin drug of choice. If the patient is allergic to pen use erythromycin.
Antibiotic resistance seen with Enterococcus, use vancomycin

57. References
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Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.