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Lesson 5

⦿ The streptococci are gram-positive spherical bacteria that characteristically form pairs or chains during

⦿ They are widely distributed in nature.

⦿ Some are members of the normal human flora;

⦿ Catalase negative

⦿ Non-sporeformer and generally non-motile;

⦿ Non-encapsulated except, Streptococcus pneumoniae;

⦿ Facultative anaerobe; some strains require CO2 for initial isolation;

⦿ Colonies are transluscent to milky, circular and pinpoint with shiny surface on agar plate.

Classification of Streptococci
⦿ Academic or Bergey’s Classification
› Based on physiologic properties of Streptococci

⦿ Smith and Brown Classification
› Based on colony morphology and hemolytic reactions on Blood agar plate

⦿ Lancefield Classification
› Based on serologic specificity of the cell wall group-specific substance and other cell wall or
capsular antigens


Group 100C 370C 450C 6.5% NaCl

Pyogenic Group
- + - -
S. pyogenes

Enterococcal Group
+ + + +
E. faecalis

Viridans Group
- + + -
S. viridans

Lactic Group
+ + - -
S. lactis

Types of Hemolysis
Type Type of Hemolysis Organisms

Alpha Incomplete/partial Greenish zone of S. pneumoniae

hemolysis of RBC Hemolysis S. viridans
Beta Complete Clear zone of S. pyogenes

hemolysis of RBC Hemolysis S. agalactiae

Gamma Non-hemolytic No hemolysis S. faecalis

Lesson 5


Group A rhamnose-N- S. pyogenes Scarlet fever

acetylglucosamine AGN & Rheumatic fever

Group B rhamnose-glucosamine S. agalactiae Neonatal meningitis

polysaccharide Sepsis

Group C rhamnose-N- S. equi / equimilis
Pneumonia, abscess,
acetylgalactosamine dysagalactiae

Group D glycerol teichoic acid E. faecalis, faecium, UTI and wound infections
enterococci containing D-alanine and durans, avium

Group D glycerol teichoic acid S. bovis,
non-enterococci containing D-alanine and S. equimus


Group F glucopyranosyl-N- E. anginosus Bacteremia, sepsis


Streptococcus pyogenes
✦ Group A, β-hemolytic

✦ facultative anaerobes

✦ the main human pathogen associated with local or systemic
invasion and poststreptococcal immunologic disorders

✦ produces large (1 cm in diameter) zones of hemolysis

✦ PYR-positive (hydrolysis of L-pyrrolidonyl-2-naphthylamide)

✦ Bacitracin susceptible

✦ colonies are transparent to transluscent, convex or domed,
entire, circular, shiny, surrounded by a wide-zone of beta


⦿ M-protein – the major virulence factor of Group A Streptococci by agglutination or precipitation
reaction with M-type specific sera.

⦿ Streptokinase (Fibrinolysin) – produced by beta-hemolytic Streptococci which transforms

plasminogen to plasmin, and active proteolytic enzyme that digest fibrin and other proteins
⦿ Streptodornase (Streptococcal deoxynuclease) – it polymerizes DNA
⦿ Hyaluronidase (Spreading Factor) – splits hyaluronic acid, an important component of the ground

substance of connective tissue

⦿ Erythrogenic Toxin – an antigenic substance responsible for the rash that occurs in scarlet fever.
⦿ Hemolysins – causes the lysis of red blood cells
› Streptolysin O (SLO) – a protein (MW 60,000) that is hemolytically active in the reduced state
but rapidly inactivated in the presence of oxygen (oxygen labile) responsible for sub-surface

Lesson 5
› Streptolysin S (SLS) – non-antigenic, oxygen stable responsible for surface hemolysis around
streptococcal colonies on blood agar plate.

̣ Erysipelas – a skin infection with edema due to the entry of S. pyogenes on the skin

̣ Puerperal fever (sepis) – septicemia resulting from the entry of S. pyogenes in the uterus
(endometritis) after delivery.

̣ Streptococcal pharyngitis – a classic strep throat which is characterized by swollen red tonsils
and pharynx with purulent exudates on tonsils, increased temperature and swollen lymph nodes

which will last for 5 days.
̣ Streptococcal pyoderma – local infection of the superficial layers of skin especially in children
(commonly called impetigo). This is characterized by superficial blisters that break down and
eroded areas whose denuded surface is covered with pus or crusts. Highly communicable.
̣ Scarlet fever – characterized by a skin rashes which begin from the trunk and neck, then spread
to extremities sparing only the face. Skin may peel off in fine scales during healing.
̣ Poststreptococcal Glomerulonephritis – also Acute Glomerulonephritis; this maybe initiated by
Ag-Ab complexes on the glomerular basement membrane of the kidney.
̣ Rheumatic fever – one of the most serious sequelae of untreated recurrent strep throat
̣ Other infections: Endocarditis and Subacute Bacterial Endocarditis

Puerperal fever Pharyngitis

Pyoderma Scarlet Fever
Lesson 5
1. M-protein — causes the streptococcal cell to resist phagocytosis and plays a role in bacterial
adherence to mucosal cells

2. Streptokinase — digests blood clotting factor (fibrinolysin)

3. Streptodornase — polymerizes DNA

4. Erythrogenic toxins —SpeA, SpeB, SpeC and SpeF functions as super antigens

5. Hyaluronidase — spreading factor

6. Hemolysin — lysis of RBC

• Streptolysin O

- responsible for subsurface hemolysis

- responsible for hemolysis on SBA plates incubated anaerobically

- O - being oxygen labile

- active only in reduced form; achieve in an anaerobic environment

- highly immunogenic; infected indivoduals readily form antibodies to hemolysin

- antigenic

• Streptolysin

- oxygen stable

- non-immunogenic

- incubated aerobically

- achieved in an aerobic environment

- non-antigenic

- surface hemolysis around streptococcal colonies on BAP


✦ Bacitracin Disk Test (Taxo A)
› a presumptive test which differentiates group A, beta-hemolytic Streptococci (+) from other

beta-hemolytic Streptococci (-)
✦ PYR Test
› hydrolysis of L-pyrrolidonyl-2-naphthylamide

✦ Dick’s Test
› susceptibility test for scarlet fever which demonstrates the person’s susceptibility to
erythrogenic toxin produced by S. pyogenes

✦ Schultz Charlton Reaction (Blanching Phenomenon)
› based on the neutralization of erythrogenic toxin

Bacitracin Disk Test
✦ Also “Taxo A”

› The growth of Group A Streptococci is inhibited by a paper disk containing 0.02 – 0.04 units of

✦ Positive Result:

› any zone of inhibition regardless of the diameter.

Lesson 5
PYR Test
✦ Principle:

› Based on the presence of L-pyroglutamyl aminopeptidase which hydrolyzes an aminosubstrate
to form the free beta-naphthylamine, which when reacted with a cinnamaldehyde reagent (PYR
reagent) forming a brilliant red end product

✦ Other than S. pyogenes, Enterococcus species also posses the aminopeptidase enzyme.

Dick's Test
✦ susceptibility test for scarlet fever which demonstrates the person’s susceptibility to erythrogenic toxin
produced by S. pyogenes

✦ Procedure:

› Inject 0.1 ml of standardized erythrogenic toxin (broth culture filtrate) intradermally to the

› Observe for the appearance of erythema/edema (reddening and swelling) on the test site
measuring more than 10mm in diameter within 8-24 hours from injection time.

Schultz Charlton Reaction
✦ Blanching Phenomenon

✦ based on the neutralization of erythrogenic toxin where 1 ml of antitoxin is injected into the skin of the
patient showing rashes

✦ If rashes fade away (blanch), then it is of Strep origin.

- Benzathine penicillin as single dose – intramuscular
- Oral penicillin for 10 days

- Erythromycin, clindamycin or cefalexin for penicillin allergic patients

Streptococcus agalactiae
✦ Group B, β-hemolytic

✦ typically are β-hemolytic and produce zones of hemolysis that are only slightly larger than the colonies
(1–2 mm in diameter).

✦ part of the normal vaginal flora in 5–25% of women

✦ hydrolyze sodium hippurate and give a positive response in the so-called CAMP test (Christie, Atkins,

✦ colonies are large to mucoid, more transluscent to opaque, whitish gray, soft, smooth surrounded by a
small zone of beta-hemolysis


⦿ Neonatal meningitis and sepsis – most common
⦿ Skin infections

⦿ Endocarditis

⦿ Puerperal infection
⦿ Osteomyelitis

Lesson 5

Hippurate Hydrolysis Test
✦ Group B Streptococci and some Enterococci can hydrolyze sodium hippurate
resulting in the formation of glycine and sodium benzoate.
Positive Negative
✦ A suspension of microorganism is incubated for 2 hours at 35°C in a hippurate
solution and then the indicator, ninhydrin is added.

✦ Deamination of glycine is detected by the development of purple color within 10

✦ A negative reaction remains colorless.

CAMP (Christie, Atkins and Munch-Peterson)
✦ Certain organisms (including group B Streptococci) produce a diffusible
extracellular protein (CAMP factor) that acts synergistically with the beta-
lysin of Staphylococcus aureus to cause enhanced lysis of red blood

✦ arrowhead zone of hemolysis at the juncture between Streptoccus
agalactiae and Staphylococcus aureus streak.

- Penicillin G – antibiotic of choice

- Erythromycin,
- Clindamycin,
- Chloramphenicol,
- Vancomycin and
- Impenem

Group C Streptococci
✦ Members of this group are primarily animal pathogen that may infect humans as well

✦ S. equi, S. equimilis, S. zooepidemicus,

› Streptococcus equi – can cause disease in horses

› Streptococcus equimilis – may cause pharyngitis, puerperal sepsis, bacteremia, osteomyelitis,
brain abscess, post-operative wound infection and pneumonia in humans.

✦ All are beta-hemolytic, except S. dysagalactiae which maybe alpha-hemolytic or non-hemolytic

✦ They can be differentiated by CHO fermentation

✦ Treatment: Similar to Group A Streptococci

Group D Streptococci
✦ Normal flora of the skin, upper respiratory tract, GIT and GUT.

✦ Bacterial endocarditis in elderly, urinary and biliary tract infections, septicemia, wound infections and
intra-abdominal abscess.

› Group D Enterococci – grow in 6.5 % NaCl and 40 % bile and able to hydrolyze esculin; Penicillin

● Organism: Enterococcus faecalis

Entrococcus faecium

Enterococcus durans

Lesson 5
› Group D non-enterococci – grow in 40 % bile and able to hydrolyze esculin, inhibited by 6.5 %
NaCl; Penicillin susceptible

● Organism: Streptococcus equinus

Streptococcus bovis


Bile esculin slant and 6.5% NaCl broth
The bile esculin slant (left) is black (positive), indicating that the microorganism
can grow in the presence of bile and hydrolyze esculin. Growth in a broth
contains a salt concentration of 6.5% (tube on right) is indicated by turbidity and
a change in the indicator from pink to yellow following overnight incubation. The
reactions shown here confirm the identification of Enterococcuus spp. A positive
PYR test may be also used to confirm the identification of suspected
Enterococcus spp. Nonenterococcal group D (S. bovid) are unable to grow in the
presence of 6.5% NaCl broth.

✦ For enterococcal strains
› Ampicillin

› Penicillin in combination with aminoglycosides (gentamycin or streptomycin)

› Vancomycin – for penicillin allergic patients
✦ For nonenterococcal strains
› Penicillin G

Viridans Streptococci
✦ Normal flora of the nasopharynx, mouth, gingival crevices, GIT, female genital tract

✦ They are opportunistic pathogens thought to be of low virulence

› Streptococcus sanguis – bacterial endocarditis

› Streptococcus mutans – dental caries (plaque)

Cellulitis and wound infection, meningitis, sinusitis, biliary and intra-abdominal infections
✦ Treatment:

› Penicillin G + gentamycin

› Vancomycin – for penicillin allergic patients

Lesson 5


Test Group A Group B Group D Group D
Enterococci Nonenterococci

Bacitracin Inhibition + - - -
Hippurate - + - -
Bile Esculin - - + +
6.5 % NaCl - - + -

Streptococcus pneumoniae
✦ gram-positive diplococci, often lancet-shaped or arranged in chains
possessing a capsule of polysaccharide

✦ Facultative anaerobe requiring an increased CO2 tension (can be obtained
using candle jar)

✦ Has an absolute nutritional requirement for choline

✦ Most energy is obtained from fermentation of glucose; this is accompanied
by the rapid production of lactic acid, which limits growth.

✦ On BAP – young colonies are circular, glistening, dome-shaped while some
are larger and more mucoid.

✦ Described as umbilicated or doughnut appearance or nail-head colonies as it becomes older because of
some autolytic changes which may result in collapse of the center.

✦ Produces a zone of alpha-hemolysis anaerobically due to pneumolysin O (an oxygen-labile toxin)

- Polysaccharide capsule
- Neuraminidase – enzyme that degrades surface structures of host tissue

- Protease – enzyme that facilitates bacterial colonization on mucosal surface by eliminating IgA,
IgG and IgM

- Pneumolysin O – an oxygen-sensitive toxin that is cytolytic for cells

- Autolysin – facilitates the release of Pneumolysin O and other proteins inflammatory for cells

- C-substance – a component of cell wall which is a teichoic acid that reacts with C-Reactive
Protein (CRP) resulting in the activation of some non-specific host immune responses.

✦ Most common cause of bacterial (lobar) pneumonia
✦ Second most common cause of bacterial meningitis
✦ Otitis media and purulent sinusitis

✦ Gram Stain

✦ Culture - CAP

✦ Optochin Sensitivity Test (Taxo P)

✦ Bile Solubility Test

✦ Mouse Virulence Test

✦ Neufeld Quellung Reaction (Capsular Swelling Test)

✦ Francis Test (Skin Test)

Lesson 5
Optochin Sensitivity Test
✦ Widely used presumptive test for differentiating pneumococci from other alpha-hemolytic Streptococci
(e.g. Viridans Streptococci)

✦ Procedure:

Taxo P or Optochin disk (ethylenehydrocupreine hydrochloride) is placed on BAP heavily inoculated
with the suspended organisms and incubated overnight

14-16mm zone of inhibition depending on the size of the optochin disk (6-10mm)

Bile Solubility test
✦ based on the presence of the enzyme autolytic amidase which when activated by bile, results in the lysis
of the organisms

✦ Principle:

This test differentiates Streptococcus pneumoniae (positive) from alpha-hemolytic Streptococci
(negative). Bile or a solution of bile salt, such as sodium deoxycholate rapidly lyses pneumococcal
colonies. Lysis depends on the presence of an intracellular autolytic enzyme. Bile salts lower the
surface tension between the bacterial cell membrane and the medium, thus accelerating the
organism’s natural autolytic process.

✦ Quality control

› Positive :Streptococcus pneumoniae

› Negative : Enterococcus faecalis

Mouse Virulence Test
✦ the mouse is particularly sensitive to even small inoculum of pneumococci

✦ Procedure:

Sputum containing pneumococci is injected intraperitoneally to a mouse which will eventually die
within 16-48 hours.

✦ The heart and blood of the dead mouse harbor a pure culture of pneumococci.

Neufeld Quellung Reaction
✦ Capsular Precipitation Reaction or Capsular Swelling Test

the most useful, specific and rapid method for the identification of pneumococci.
✦ Procedure:
The test is performed by mixing on a slide a loopful of anti-pneumococcal serum and methylene blue.

Positive Result:
Capsule appears swollen due to change in the refractive index which in turns due to serological

Lesson 5
Francis Test
✦ skin test for determining the presence of antibodies against pneumococci

Penicillin – drug of choice; Cephalosporin or erythromycin for pneumonia
Chloramphenicol for meningitis

Pneumococci vs. Streptococci

Test Pneumococci Streptococci
Bile Solubility Test Soluble Insoluble

Inulin Fermentation Test Fermenter Non-fermenter

Capsular Swelling Test Swelling of capsule No swelling

Quinidine Test Sensitive Resistant

Optochin Test Sensitive Resistant

Mouse Virulence Test Mouse dies 16-48 hours Mouse will not die