MICROBIOLOGY LECTURE 5 – Genus Streptococcus There is a clear colorless

Notes from Lecture zone around a streptococcal

USTMED ’07 Sec C – AsM colony in which the RBC have
undergone complete
Generalities destruction
Alpha hemolysis on 5% sheep blood agar
1. Gram positive cocci that grow in pairs or chains
2. Widely distributed in nature an indistinct zone of partial
3. 27 recognized species lysis of red blood cells
4. Some are members of the normal human flora causing a green to greenish
5. Others are associated with important human disease brown discoloration of the
medium immediately
attributable to infection by streptococci and to
surrounding the colony
sensitization to them
6. The most important of the human pathogens:
a. Streptococcus pyogenes (Group A) Alpha hemolysis showing
b. Streptococcus agalactiae (Group B) partially hemolyzed RBC
immediately surrounding the
c. Enterococcus faecalis(Streptococcus faecalis) (Group colonoy
D)
d. Streptococcus pneumoniae
e. some of the oral streptococci
7. Elaborates a variety of extracellular substances and
enzymes
8. Streptococci cause a wide variety of infections: Gamma hemolysis on 5% sheep blood agar

a. Streptococcus pyogenes (Group A) streptococci- Gamma denotes lack of

leading bacterial cause of pharyngitis and cellulitis; hemolysis; the RBC
inciting factor of two important immunologic surrounding the colonies are
diseases: acute rheumatic fever and acute intact
glomerunephritis
b. Streptococcus agalactiae (Group B streptococci)-
leading cause of neonatal sepsis and meningitis
c. Enterococcus faecalis – important cause of hospital
acquired urinary tract infections and endocarditis
Macroscopically there is no
d. Viridans group streptococci – most common cause of apparent hemolytic activity
endocarditis or discoloration produced by
e. Streptococcus bovis – may also cause endocarditis the colony
9. Most species are facultative anaerobes; maybe strictly
anaerobic to capnophilic
10. Have complex nutritional requirements necessitating
blood or serum enriched medium for isolation
11. Carbohydrates are fermented with the production of lactic
acid but not gas
12. Catalase negative CLASSIFICATION OF STREPTOCOCCI
13. Can be classified as to:
1. Group A streptococcus – Streptococcus pyogenes
a. clinical presentation(pyogenic,oral,enteric)
b. serological properties (Lancefield grouping A-H, K-V)
a. contains the group A antigen
b. beta hemolytic
based on the antigenic composition of cell wall
carbohydrates c. main human pathogen associated with local or
• serogroups A,B,C,D and G – most commonly systemic invasion and poststreptococcal
found associated with human disease immunologic disorders
d. PYR positive(hydrolysis of L-pyrrolidonyl-2-
c. Hemolytic patterns in blood agar plates napthylamide)
• Beta hemolysis – clear zone of hemolysis e. usually susceptible to bacitracin
around the colony as a result of complete
lysis of the red blood cells 2. Group B streptococcus – Streptococcus agalactiae
• Alpha hemolysis – zone of partial hemolysis a. contains the group B antigen
with a greenish discoloration of the b. members of the normal flora of the female
medium genital tract
c. an important cause of neonatal sepsis and
• Gamma hemolysis – no color change or
meningitis
lysis of red blood cells ; nonhemolytic d. beta hemolytic
d. Biochemical (physiological) properties e. hydrolyzes sodium hippurate
• Sugar fermentation reactions f. gives a positive response to CAMP test
• Tests for the presence of enzymes
• Tests for susceptibility or resistance to
3. Groups C and G streptococci
a. occur sometimes in the nasopharynx
certain chemical agents
b. may cause sinusitis, bacteremia or endocarditis
e. antigenic specificity of the capsular polysaccharide c. beta hemolytic
d. identified by reactions with specific antisera for
Beta hemolysis on 5% sheep blood agar groups C or G

4. Group D streptococci
a. enterococcal – Enterococcus faecium
There is complete lysis of RBC Enterococcus durans
surrounding the colony that can • Part of the normal enteric flora
be seen macroscopically
• Usually nonhemolytic; occasionally
alpha hemolytic
• PYR positive
• Grow in the presence of bile
 • Hydrolyze esculin (bile esculin c. usually alpha hemolytic; may be
positive) nonhemolytic
• Grow in 6.5% NaCl
• resistant to penicillin G d. part of the normal flora
e. occasionally cause bacteremia or
• Some strains are vancomycin resistant
endocarditis
f. can be found in brain abscesses and other
infections

11. Peptostreptococcus
b. Non-enterococcal – Streptococcus bovis
• Part of the enteric flora a. grow only under anaerobic or
• Occasionally cause endocarditis microaerophilic conditions
b. variably produce hemolysis
• Sometimes cause bacteremia in
c. part of the normal flora of the mouth,
patients with colon carcinoma
upper respiratory tract, bowel and female
• Nonhemolytic and PYR negative
genital tract
• Grow in the presence of bile
• Hydrolyze esculin (bile esculin- d. often participate in mixed anaerobic
positive) infections in the abdomen, pelvis, lung or
brain
• Do not grow in 6.5% NaCl
• Sensitive to penicillin Antigenic Structure of Group A Streptococcus

5. Streptococcus anginosus or Streptococcus milleri,

Streptococcus intermedius, Streptococcus
constellatus
a. part of the normal flora
b. may be beta, alpha or nonhemolytic
c. includes:
• Beta hemolytic streptococci that form
minute colonies (<0.5 mm in diameter)
and react with groups A,C or G
antisera
• All beta hemolytic group F
streptococci
• Voges Proskauer test positive
• Those that are group A are PYR 1. Capsule
negative - hyaluronic acid; nonimmunogenic; antiphagocytic
• May be classified as Viridans
streptococci 2. Cell wall

6. Group N streptococci a. group specific carbohydrates of group A – is a dimer

a. rarely found in human disease states of N-acetylglucosamine and rhamnose

b. produce normal coagulation (souring ) of b. type specific protein antigens

milk i. M protein
o a major antigen associated with virulent
7. Groups E,F,G,H and K-U streptococci streptococci
- occur primarily in animals
o located at the end of the hairlike fimbriae
8. Streptococcus pneumoniae that are anchored in the cell wall and
a. alpha hemolytic extending through the capsule
o antiphagocytic
b. growth inhibited by optochin
(ethylhydrocupreine hydrochloride) o anticomplimentary
c. colonies are bile soluble ii. T or trypsin resistant protein
o With M protein – an important
9. Viridans streptococci – Streptococcus mitis, epidemiological marker of group A strains
Streptococcus salivarius, Streptococcus sanguis o no relationship to virulence
(Group H), Streptococcus mutans iii. R protein
a. typically alpha hemolytic ; may be
nonhemolytic c. Other surface antigens
b. growth not inhibited by optochin i. F protein or fibronectin binding protein
o Has a receptor for fibronectin
c. colonies are not soluble bile(deoxycholate)
o Major adhesin for bacterial attachment to
d. most prevalent members of the normal
the epithelial cells of the pharynx and skin
flora of the upper respiratory tract ii. Lipoteichoic acid
e. important for the healthy state of the o Lipid moiety is implicated to binding to
mucous membranes fibronectin.
f. may reach the blood stream due to trauma o Possibly mediates adherence to epithelial
g. a principal cause of endocarditis on cells
abnormal heart valves
Antigenic structure of Group B Streptococcus
h. Streptococcus mutans – synthesizes large
polysaccharides (dextrans and levans) from
sucrose which may lead to dental caries

10. Nutritionally variant streptococci or pyridoxal-

dependent streptococci – Streptococcus defectives,
Streptococcus adjacens
a. require pyridoxal or cysteine for growth on
blood agar
b. grow as satellite colonies around colonies
of staphylococci and other bacteria
 fever patient blanches
the rash of a patient
PATHOGENESIS with scarlet fever
Group A streptococci (Streptococcus pyogenes) cause disease
by three mechanisms:
- pyogenic inflammation – induced locally at the site
of the organisms in tissue
- exotoxin production – can cause widespread b. Exotoxin B
systemic symptoms in areas of the body where there
are no organisms  a cysteine protease that rapidly
destroys tissue and is produced
- immunologic – occurs when antibody against a by strains that cause necrotizing
component of the organism cross-reacts with normal fasciitis
tissue or forms immune complexes that damage c. Exotoxin A
normal tissue  May cause streptococcal toxic
shock syndrome
Inflammation-related enzymes produced by Group A
Streptococcus (Streptococcus pyogenes) 2. Hemolysins
1. hyaluronidase(spreading factor)
a. Streptolysin S
- degrades hyaluronic acid which is the ground
substance of the connective tissue  oxygen stable, nonimmunogenic
cell bound hemolysin capable of
- Facilitates spreading of the microorganisms
lysing eryhtrocytes, leukocytes
- Antigenic – specific antibodies are found in the serum and platelets
after infection with hyaluronidase producing  stimulate release of lysosomal
organisms
contents after engulfment
2. streptokinase(fibrinolysin)  responsible for the hemolytic
- Transforms plasminogen of human plasma into zones around streptococcal
colonies growing on the surface
plasmin, an active proteolytic enzyme that digests
of blood agar
fibrin and other proteins
 not antigenic
- Given intravenously for treatment of pulmonary
emboli and of coronary artery and venous thromboses
b. Streptolysin O
3. streptodornase (streptococcal deoxyribonuclease)
 a protein that is hemolytically
- Depolymerizes DNA in exudates or necrotic tissue active in the reduced state
- With streptokinase – used in enzymatic debridement;  responsible for the hemolysis
Helps liquefy exudates and facilitates removal of pus seen when growth is in cuts deep
and necrotic tissue into the medium in blood agar
- Antibody to Dnase develops after streptococcal skin  antigenic – antibodies are formed
infection (normal limit –100 units) against streptolysin O following
infection with streptococci that
Toxins and hemolysins produced by Group A streptococci produce streptolysin O
(Streptococcus pyogenes)  ASO serum titer in excess of 160-
200 units – suggests:
1. Streptococcal pyrogenic toxins • Recent infection with
streptococci
- Three antigenically distinct toxins
• Persistently high
antibody levels dueto
a. Exotoxin C– classic erythrogenic toxin an exaggerated immune
 causes the rash in scarlet fever response to an earlier
exposure in a
 Produced only by strains hypersensitive person
lysogenized by a bacteriophage
carrying the gene for the toxin Pathogenesis of Streptococcus agalactiae (Group B
 Dick test – The injection of a skin streptococci)
test dose of erythrogenic toxin a. is based on the ability of the organism to induce
gives a positive result (an an inflammatory response
erythematous reaction in the skin b. no cytotoxic exotoxins are produced
of nonimmune persons who lack c. role of enzymes in the pathogenesis of infection is
antitoxins)
unknown – deoxyribonucleases, hyaluronidase,
 Schultz Charlton reaction – neuraminidase, proteases, hiuppurase and
antitoxin injected into the skin of hemolysins
a patient with scarlet fever d. no evidence for any immunologically induced
causes localized blanching as a disease
result of neutralization of e. has a polysaccharide capsule – antiphagocytic
erythrogenic toxin f. anticapsular antibody is protective
Dick test – positive – CLINICAL FINDINGS
erythema surrounding
the injection site Types of diseases produced by Streptococcus pyogenes
1. Pyogenic diseases
a. pharyngitis
b. cellulitis and erysipelas
c. impetigo(pyoderma)
2. Toxigenic diseases
a. scarlet fever
b. toxic shock syndrome
3. Immunologic diseases
Schultz Charlton a. rheumatic fever
reaction. Convalescent b. acute glomerunephritis
serum from a scarlet
1a. Pharyngitis 1. The primary site of the infection is usually the
1. Streptococcus pyogenes is the major cause of pharynx, with the distinctive rash resulting from an
bacterial pharyngitis erythrogenic toxin produced by the streptococcus.
2. A disease of children 5-15 years 2. The rash appears within 2 days after the onset of the
3. Spread by person to person by respiratory droplets sore throat and disappears in 6-10 days.

4. Characterized by sore throat, fever, malaise, Left – scarlet fever

headache and nausea Right – measles
5. Posterior pharynx erythematous with an exudate;
cervical lymphadenopathy present
6. Can result to complications (tonsillar abscesses,
mastoiditis, septicemia, osteomyelitis, rheumatic
fever)

2b. Streptococcal toxic shock syndrome (also called toxic

shock like syndrome)
1. Characterized by hypotension, diffuse erythroderma,
hypoalbuminemia and multiorgan failure(kidney,
lungs, liver, heart)
2. serotypes implicated – M1,M3 or M18
3. due to the production of pyrogenic exotoxins –
exotoxin A
1b. Cellulitis
1. Cardinal features – desquamation of skin
erythema, swelling, heat occurs 10-14 days
and pain after infection at sites
2. Erythema may be pink or that were
red but lacks the erythematous during
intense, fiery red or the initial phase
salmon colored
appearance of
erysipelas. 3a. Rheumatic fever
3. Initiated by infection 1. most serious sequelae of hemolytic streptococcal
through a small break in infection because it results in damage to heart
the skin muscle and valves
4. Can invade the 2. occurs 2 weeks after a group A streptococcal
subcutaneous tissue and infection usually a pharyngitis
advance rapidly through 3. results in a systemic inflammatory process involving
lymphatics --à the connective tissue, heart, joints, and CNS
septicemia 4. Characterized by fever, migratory polyarthritis, and
1b. Erysipelas carditis
1. Characteristic appearance – bright red or salmon red 5. Due to an immunologic reaction between cross-
painful confluent erythema in a “butterfly” reacting antibodies to certain streptococcal M
distribution involving the nasal eminence, cheeks, proteins and antigens of joint and heart tissue
and nose with abrupt borders along the nasolabial 6. Treat promptly with penicillin which is continued
folds
prophylactically to prevent recurrence and increased
2. Erythema increases over a course of 3-6 days and damage
usually resolves in 7-10 days
Revised Jones Criteria for the Diagnosis of Rheumatic Fever

The diagnosis of rheumatic fever is highly likely if supported by

evidence of a preceding group A streptococcal infection and the
presence of two major manifestations or one major and two
minor manifestations.

Supporting evidence of antecedent group A streptococcal

infection

Positive throat culture

Positive streptococcal antigen test
3. Erysipelas usually occur on the face, although any Elevated or rising streptococcal antibody titer
skin surface such as the leg, can be affected.
Major Manifestations
4. Note the sharp line of demarcation and bright red
color, features that distinguish it from cellulitis Carditis
Polyarthritis
1c. Impetigo Chorea
1. A superficial infection Erythema marginatum
that usually begins as Subcutaneous nodules
small vesicles
progressing to weeping Minor manifestations
lesions with amber crust
and slightly cloudy Clinical findings: arthalgia, fever
purulent exudate. Laboratory findings
2. Serotypes implicated – M Elevated acute phase reactants (erythrocyte
types 2,49, 55 & 57 sedimentation rate, C-reactive protein)
3. May result to nephritis Prolonged PR interval on elevtrocardiography
as a complication
3b. Acute glomerulonephritis

2a. Scarlet fever

1. Typically occurs 2-3 weeks after streptococcal skin
infections with M types 2,4,12 or 49 (most frequent)
2. More frequent after skin infections than after
pharyngitis
 3. Characterized by hypertension, edema of the An alternative to PYR test for the presumptive identification of
Group A beta hemolytic streptococci 0.04 units of bacitracin
face(especially periorbital edema) and ankles, &
disk is placed on an inoculum of the microorganism on sheep
“smoky” urine
blood agar
4. Complete recovery; reinfection with streptococci
rarely leads to recurrence.
5. Initiated by deposition of soluble streptococcal
antigen-antibody complexes and complement on the
glomerular basement membrane – lumpy-bumpy
pattern on immunofluorescence
6. Can be prevented by early eradication of
nephritogenic streptococci from skin colonization
sites Positive test – zone of inhibition

Diseases produced by Streptococcus agalactiae

II. Streptococcus agalactiae - Group B beta hemolytic
streptococcus

1. Microscopy
- Gram stain of vaginal
secretions
Gram positive cocci in
pairs,suggestive of
Streptococcus
agalactiae which colonize
the genitourinary tract of
women
2. culture

Colonies of Group B
streptococcus on 5% sheep
Neonatal Group B streptococcal disease blood agar
Colonies are larger than other
LABORATORY DIAGNOSIS beta hemolytic streptococci.
Hemolytic zone surrounding the
I. Streptococcus pyogenes colony is smaller.
Tests
1. Microscopy – Gram
stain 1. Hippurate hydrolysis
test
Gram stain of streptococci in
a positive broth culture Incubate a suspension of the
Gram positive microorganism for 2 hours at
cocci in chains 35 C in a hippurate solution.
Add ninhydrin(indicator)

2. Cultures Hydrolysis of sodium hippurate

leads to the formation of
Colonies of group A glycine and sodium benzoate.
streptococci on 5% sheep
blood agar Deamination of glycine –purple
small colonies with a color
wide zone of beta hemolysis
Group A Streptococcus
Streptococcus selective agar – contains sulfamethoxazole 2. CAMP test (Christie,Atkins, Munich-Peterson) - An
And trimethoprim which inhibits the growth of nongroup alternative to hippurate hydrolysis
A betahemolytic streptococci, staphylococci, viridans
Streptococci and gram negative bacilli Demonstrates the
arrowhead shaped
enhancement of beta
hemolysis that occurs
when the hemolytic beta
toxin producedby
Staphylococcus aureus
acts synergistically with
the CAMP factor

III. Group D streptococci

Left – blood agar plate; right – streptococcus selective agar
1. enterococcus
PYR Test
The presence of an aminopeptidase enzyme that degrades Colonies of Enterococcus spp.
the substrate is a 10 minute presumptive test for Group A on 5% sheep blood agar
streptococci (beta hemolytic) and Aerococcus, Enterococcus Colonies are raised, white to
and Gemella(alpha or nonhemolytic) gray white ranging from 0.5
to 1.5 mm. In size and are
usually nonhemolytic

Tests
1. Bile esculin and 6.5% NaCl
- Bile esculin slant(left) –
indicates that the
left(+) colorless; right (+) red color microorganism can grow in
the presence of bile and
hydrolyze esculin.
Bacitracin susceptibility test
 2. 6.5% NaCl broth(right)- 4. Vancomycin resistant enterococci – linezolid (Zyvox)
growth is indicated by and quinupristin/dalforpristin(Synercid)
turbidity and change in the 5. Nonterococcal Streptococcus bovis:penicillin G
indicator from pink to
yellow 6. Group B streptococcal infections- penicillin G or
ampicillin in combination with an aminoglycoside
3. PYR Test 7. Peptostreptococci – penicillin G

PREVENTION
- The presence of an aminopeptidase enzyme that
degrades the substrate PYR(L-pyrrolidonyl-B
napthylamide) is a 10 minute presumptive test for
group A streptococci(beta Hemolytic) and Aerococcus,
Enterococcus and Gemella(alpha or nonhemolytic)
IV. Viridans streptococci
- Gram stain of a viridans streptococcus species in
Blood culture broth appear in long chains especially
when recovered from a blood culture broth
1. Rheumatic fever can be prevented by prompt
treatment of Group A streptococcal pharyngitis with
penicillin.
2. Penicillin prophylaxis for acute rheumatic fever
patients to prevent recurrence of the disease; not
needed in acute glomerulonephritis
3. In patients with damaged heart valves who undergo
invasive dental procedures, endocarditis can be
prevented by using amoxicillin perioperatively.
4. In patients with damaged heart valves who undergo
gastrointestinal or urinary tract procedures,
endocarditis caused by enterococcus can be
prevented by using ampicillin and gentamicin
perioperatively.
5. Neonatal sepsis caused by group B streptococci can
be prevented by administration of parenteral
ampicillin perinatally to women who experience
prolonged (longer than 18 hours) rupture of
membranes, whose labor begins before 37 weeks
gestation or who have a fever at the time of labor.

- fin -

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- Optochin susceptibility test. A paper disk containing

optochin (ethylhydrocupreine Hydrochloride) is
applied to the surface of an inoculated 5% sheep
blood agar plate.

No zone of inhibition
- Identification of viridans streptococci with
conventional biochemical reactions. Definitive
identification requires several substrates including
Bile esculin, arginine decarboxylase, 6.5% NaCl,
lactose, Mannitol, raffinose, sorbitol, arabinose,
inulin, sucrose and Esculin.

TREATMENT
1. All group A streptococci are susceptible to penicillin
G.
- mild - oral penicillin V
- if allergic: erythromycin or its derivatives
(azithromycin)
2. Endocarditis caused by Viridans streptococci is
curable by prolonged penicillin treatment
3. Enterococcal endocarditis – eradicated only by a
penicillin or vancomycin combined with an
aminoglycoside