1. Streptococcaceae
Chapter 15: Streptococcus, Enterococcus, and Other Catalase-Negative Gram-Positive Cocci

2. Streptococcaceae: Genera
Streptococcus – major cause of human infection
Enterococcus – frequent cause of human infection
Aerococcus – occasionally endocarditis, meningitis, urinary tract infection
Lactococcus – rare human pathogen
Pediococcus – NF of lower GI tract, occasional isolate from abscess
Leuconostoc – infrequently isolated from blood, wound, abscess
Gemella – rare isolate of upper RT infection

3. Streptococcaceae: Classification
Medical importance: Streptococcus, Enterococcus
Differentiation on CBA – partial, complete, nonhemolytic (, , or none)
Serologic classification developed 1930’s by Lancefield
CHO (C substance) extracted from cell wall
Group A through U
Some Strep don’t have this antigen, not ID by this method
Lancefield typing used today primarily for beta hemolytic Group A, B

4. Classification
Physiologic or biochemical characteristics used to differentiate species within Streptococci and Enterococci
In future, DNA probes and hybridization, PCR DNA amplification tests will be used (greater sensitivity and specificity)

5. Gram Stain
Gram (+) cocci, in pairs or chains (most often seen in broth culture); cell division occurs in 1 plane
Strep pneumoniae is lancet shaped diplococcus
Strep quickly lose ability to retain crystal violet in Gram stain; appear G (-) in old cultures

6. Strep Characteristics
Form capsule
More fastidious than Staphylococci, require enriched media such as CBA
Hemolytic on CBA and source of blood (sheep vs. rabbits) may affect hemolysis, clinical labs use 5% sheep RBCs in CBA
Group A Strep two hemolysins:
Streptolysin O - oxygen labile; stabbing CBA after inoculation provides lower oxygen tension to identify strains that only produce streptolysin O
Streptolysin S – oxygen stable

7. Characteristics
Addition of sulfamethoxazole & trimethoprim (SXT) to CBA may be used to suppress growth of NF and allow better recovery of beta hemolytic Strep
Microaerophilic, good growth require reduced oxygen (candle jar or CO2 incubator)
Nutritionally deficient Strep may require vitamin B6 or pyridoxal for growth and may be found satelliting Staph aureus
Strep pneumoniae is sensitive to cold and may fail to grow if refrigerated

8. Strep Differentiation From Other Genera
Other Gram(+) cocci:
Strep = catalase(-)
Staph & Micrococci are catalase(+)
Other cocci:
Neisseria are Gram(-)
May be confused with Strep that lose G(+) as they age
However, Neisseria = oxidase(+)

9. Differentiation Within Strep Genus
Respiratory Tract by CBA hemolysis:
Viridans group (Strep mitis, Strep salivarius, Strep sanguis) - partial (α) hemolysis, NF
Strep pneumoniae – partial (α) hemolysis
Strep pyogenes – complete (β) hemolysis, may be pathogen
Differential diagnosis by clinical syndrome:
Group A – sore throat
Group B – neonatal meningitis

10. Strep pyogenes (Group A)
Sensitive to Bacitracin (0.04 units, Taxo A disk)
Resistant to SXT disk (both Gp. A & B)
PYR hydrolysis: Production of pyroglutamyl aminopeptidase; both Gp. A & Enterococcus = (+)

11. Strep agalactae (Group B)
CAMP (Christie, Atkins, Munch-Peterson) test – extracellular protein that enhances Staph aureus hemolysin
Hippurate hydrolysis (hippuricase) – hippurc acid to Na benzoate + glycine (amino acid)
Growth in 6.5% NaCl (Gp. B & Enterococcus)
Resistance to SXT disk (both Gp. A & B)

12. Group D: Enterococcus, Streptococcus bovis
CBA - may be partial, complete, nonhemolytic (α, β, none)
Bile esculin agar - all Group D grow in bile, hydrolyze esculin = growth + blackening on agar
6.5% NaCl – Enterococcuss = (+) growth
PYR hydrolysis – Enterococcus = (+)

13. S. pneumo & Viridans Group
CBA - both partial (α) hemolysis
Taxo P disk- Optochin (ethyl hydrocupreine hydrochloride) susceptibility, detergent that activates autolytic enzyme, lyse cell wall of Strep pneumoniae
Bile solubility – bile salt (Na desoxycholate) activate autolytic enzyme of Strep pneumoniae (test - 37°, 30 min.) dissolve isolated colonies
Most labs do not speciate the Viridans group (Strep mitis, Strep salivarius, Strep sanguis) except for blood isolates or epidemiology

14. Serology Tests Serogroups – Lancefield typing
Group A-H, K-V
Group A Serotypes - >80; based on M protein of cell wall
Quellung test - for Strep pneumoniae
84 serotypes based on specific soluble substance in capsule
specific antisera binds to substance, under microscope capsule appears swollen

15. Strep pyogenes Virulence: Extracellular products
Streptolysin S – alters membrane permeability; lyses RBCs, leukocytes, and other cells containing sterols in membrane
Streptolysin O – forms membrane penetrating channels (porin) leading to membrane defects and cell lysis
Cytotoxic to RBCs and many tissue cells, particularly cardiotoxic
Antigenic - for strep throat infections (but not skin), anti-streptolysin O (ASO) antibody used to diagnose recent infection

16. Strep pyogenes Virulence: Extracellular products
Erythrogenic toxin
Act as superantigen - fever, cardiotoxic, enhanced susceptibility to endotoxin shock, suppress antibody response
Rash seen in scarlet fever; due to delayed hypersensitivity reaction to other streptococcal antigens enhanced by toxin
Toxin involved in toxic shock syndrome similar to Staph. aureus

17. Strep pyogenes Virulence: Extracellular products
Streptokinase – cleaves plasminogen to plasmin, then acts as protease to lyse fibrin clots
Hyaluronidase – spreading factor
DNAse – degrades accumulated inflammatory exudate DNA from leukocyte disintegration; antigenic and antibodies used to diagnose recent Strep infection (including skin infections)

18. Strep pyogenes Virulence: Cell Associated Factors
M protein
Fibrous protein on cell surface
MAJOR virulence factor for group A Strep
>80 different types
Protects MO from opsonization and phagocytosis by binding to fibrinogen
Also play role in adherence
Some types of M protein act as super antigen causing shock in patient
Poorly immunogenic; antibodies, if made, are protective, long lasting, type specific

19. Strep pyogenes Virulence: Cell Associated Factors
Lipoteichoic acid
mediates attachment to epithelial cells
Capsule
hyaluronic acid (note organism also produces a hyaluronidase)
antibodies to capsule are not protective against infection

20. Strep agalactiae Virulence: Extracellular Products
Hemolysin - increase vascular permeability in lungs, leading to respiratory distress
Neuraminidase – cleaves sialic acid residues, involved in hyaline membrane disease of lung
Hyaluronidase – spreading factor
DNAse – spreading factor
Protease – spreading factor

21. Strep agalactiae Virulence: Cell associated Factors
Capsule – antibodies are type specific and protective
Lipoteichoic acid – for attachment

22. Strep pneumoniae Virulence: Extracellular Products
Pneumolysin O
cytolytic for RBCs and other tissue cells
Cytoplasmic rather than extracellular protein, released when bacteria lyse
Acts as porin to disrupt host cell membranes
Leukocidan
Neuraminidase
Hyaluronidase
IgA protease

23. Strep pneumoniae Virulence: Cell Surface Structures
Capsule – most important virulence factor
> 80 different types capsular polysaccharide
Antibodies against capsule type specific and protective (used for vaccine)

24. Streptococcus Clinical Significance
A wide variety of diseases:
Suppurative - pus producing lesions
Toxemia - Clinical symptoms suggesting toxin
Infants - more susceptible, with prolonged, low grade infections; rarely nephritis and rheumatic fever follow
Older children and adults - more acute and self-limited infections; occasional complication of nephritis and rheumatic fever (with no pus or MO present)

25. Streptococcus: Clinical Significance
Complete (ß) hemolytic:
Gp. A, B, and C most virulent
Gp. A greatest number infections
Partial (α) hemolytic & non hemolytic:
Viridans group found as NF in RT and GI tract, most common cause of subacute bacterial endocarditis
Step pneumoniae leading cause of death due to bacterial infection throughout the world, particularly in elderly and very young

26. Strep pyogenes Infections: Pyoderma (Impetigo)
Superficial skin infection characterized by vesicular rash, becomes pustular, in later stages thick crust
Staph aureus may be secondary invader
Endemic in tropical areas caused by M serotypes different from those that cause pharyngitis
Spread by close, direct contact
Disease starts with skin colonization, followed by invasion through abrasions
Type specific antibodies to M are made, but antibodies to Streptolysin O are uncommon
Most serious complication is acute glomerulonephritis, occur 2-3 weeks post infection

27. Strep pyogenes Infections: Erysipelas
Diffuse, erythematous skin infection
Most often on face, occurs following pharyngitis

28. Strep pyogenes Infections
Wound infections - occasionally following trauma, due to simple contamination of wound
Cellulitis – subcutaneous tissue, accompanied by lymphangitis and abscess; typically exhibit chills, fever, marked signs of toxicity
Necrotizing fasciitis (”flesh eating bacteria”) – fibrous tissue deep in skin; toxin destruction of sheath that covers muscle

29. Strep pyogenes Infections: Myositis
Invasion of muscle tissue
Extensive muscle necrosis, overwhelming sepsis
Usually fatal

30. Strep pyogenes Infections: Pharyngitis (Strep Sore Throat)
Most common disease caused by Strep pyogenes
Spread by droplets of respiratory secretions or via food or water
Abrupt onset of sore throat, malaise, headache, fever
Throat erythematous with grayish-white exudate
Tonsils and lymph nodes are enlarged; tonsils may become infected chronically
May spread to sinuses and middle ear
Usually self-limited, penicillin used to prevent secondary complications such as rheumatic fever

31. Strep pyogenes Infections: Scarlet Fever
From URT infection
Strep lysogenized by phage with gene for erythrogenic toxin
Symptoms similar to pharyngitis - erythematous rash upper chest, spreads to rest of body, ~1 week; followed by weeks of extensive desquamation of skin
“strawberry tongue” - red spots on yellowish-white tongue
“raspberry tongue” - followed by peeling, red beefy tongue
Immunity to toxin is demonstrated by Dick test:
Inject toxin in skin, produces erythema in absence of antibody
If no erythema, indicates antibodies present, patient infected
Erythrogenic toxin may also be involved in causing a toxic-shock like syndrome

32. Non-suppurative Complications Strep pyogenes: Rheumatic Fever
Inflammatory disease; involve heart, joints, subcutaneous tissue, CNS
Primarily in children 6-15 yrs.
1-5 weeks after Strep throat
Major manifestations - polyarthritis, carditis, chorea, erythema marginatum
Carditis can involve all layers of heart, can lead to rheumatic heart disease - chronic, progressive damage to heart, possibly death

33. Rheumatic Fever
By autoimmune, cross-reacting antibodies made against Strep that react with self tissues
Attracts host reactive cells, Complement gets activated and leads to cellular damage
Diagnosis by serum antibody test - ASO assay
Prevent by treating Strep throat infection with penicillin

34. Non-suppurative Complications Strep pyogenes: Acute Glomerulonephritis
Occur 2-3 weeks following pharyngitis or pyoderma
Edema, hypertension, headache, malaise, circulatory congestion, hematuria
Only associated with specific nephritogenic M serotypes of Gp. A Strep
Immune complex disease - deposition of circulating Strep antigens and antibodies complexes in glomeruli of kidney
Damage due to host reactive cells and subsequent Complement activation

35. Strep agalactiae Infections
NF of adult vagina, urethra, rectum, occasionally pharynx
Sexually transmitted disease (STD)
Adults - rare infections; URT, meningitis, bacteremia, endocarditis
Newborn - most serious disease cases
Use to be major cause of puerperal fever (postpartum sepsis, child-bed fever)

36. Strep agalactiae Infection: Newborn
Early onset disease
Infected in utero or at birth
MO gain access via RT, symptoms of respiratory disease
Septicemia, meningitis may occur
High mortality rate
Late onset disease
7-10 days after birth, MO rarely found in mother prior to birth
MO by direct contact with newborn mucosal surfaces
Meningitis or osteomyelitis, possibly bacteremia
Mortality rate high, but lower than early-onset disease

37. Streptococcal Group C, D Infections
Group C – similar to Gp. A, but without non-suppurative sequelae complications
Group D – NF of GI tract
Common cause of UT infections, subacute bacterial endocarditis, abdominal abscesses, wound infections
Strep bovis in blood suggest surface of colon compromised; associated with carcinoma of colon; proposed as early screen for colon cancer

38. Viridans Group Infections
NF of RT, GI tract, genital tract
Most common cause of subacute bacterial endocarditis, colonize damaged heart valves
Strep mutans plays a role in dental decay

39. Strep pneumoniae Infections
Found as NF in URT in 15% of children and 5% of adults; carrier state is sporadic
Probable pathogen when most predominant MO isolated
Leading cause of bacterial pneumonia, particularly in very young, elderly, those with immunological deficiencies
Mortality rate is 5-7%.
Bacteremia is common, meningitis may occur

40. Strep pneumoniae Infections
Meningitis – not as common as other causes of meningitis except in children under 1 year of age where it is second most common cause of meningitis; mortality rate is high ~40%
Otitis media – most common cause of middle ear infection in children (1/3 of cases)
Also cause purulent sinusitis and peritonitis

41. Antimicrobial Susceptibility
Penicillin for groups A, B, C; erythromycin for individuals allergic to penicillin. Susceptibility testing is not necessary.
Enterococcus and other group D – must do sensitivity testing; now strains of Enterococcus faecalis resistant to all antibiotics
Viridans group Strep – do sensitivity testing
Strep pneumoniae – use penicillin, but now resistant strains, so sensitivity testing is necessary

42. Pneumococcal Vaccine Use of purified polysaccharide
Children: heptavalent pneumococcal vaccine (PCV7) – vaccine of 7 serotypes S. pneumoniae
Adults: 23 capsular polysaccharides (PS23) - vaccine for 23 strains most likely to cause pneumonia
Given to most likely infected - young, old, chronic respiratory problems

43. Class Assignment
Textbook Reading: Chapter 15 Streptococcus, Enterococcus, and Other Catalase-Negative Gram-Positive Cocci
Key Terms
Learning Assessment Questions

44. Case Study 2 - Streptococcus
A 62-year-old man with a history of chronic obstructive pulmonary disease (COPD) came to the emergency department because of a fever of 40°C, chills, nausea, vomiting, and hypotension.
The patient also produced tenacious, yellowish sputum that had increased in quantity over the preceding 3 days.
His respiratory rate was 18 breaths/min, and his blood pressure was 94/52 mmHg.

45. Case Study 2
Chest radiographic examination showed extensive infiltrates in the left lower lung that involved both the lower lobe and the lingula.
Multiple blood cultures and culture of the sputum yielded S. pneumoniae.
The isolate was susceptible to cefazolin, vancomycin, and erythromycin but resistant to penicillin.

46. Case Study 2 - Questions
1. What predisposing condition made this patient more susceptible to pneumonia and bacteremia caused by S. pneumoniae? What other populations of patients are susceptible to these infections? What other infections does this organism cause, and what populations are most susceptible?
2. What infections are caused by S. pyogenes, S. agalactiae, and viridans streptococci?

47. Case Study 2 –Questions
3. S. pyogenes can cause streptococcal toxic shock syndrome. How does this disease differ from the disease produced by staphylococci?
4. What two non-suppurative diseases can develop after localized S. pyogenes disease?