Streptococcus

Background
• Chain forming GPC, facultatively anaerobic
• Catalase negative
• Mostly occur as commensal in mouth and throat, may act as
opportunistic pathogen.
• But a few eg. S.pyogens and S.agalactiae act as primary
pathogen.
• Staphylococcal infections have tendency to localize but
Streptococcal infections have tendency to spread.
1. Morphology
• GPC (0.5), arranged in chains or pairs
• Chains of variable length, longer in log phase in liquid media.
Chain length has no role in pathogenicity.
• Capsule: mostly present
group A and C– hyaluronic acid capsule
group B and D- polysaccharide capsule
2. Culture
• Facultatively anaerobic, optimum temp. 37 degree C.
• Pathogenic streptococci more exacting in nutritional
requirements, grows well in blood agar
• 10% CO2 enhances growth and haemolysis.
3. Biochemical test
• Catalase negative
• Oxidase negative
• Ferment glucose
4. Classification
A. On the basis of haemolysis
Brown (1919), alpha haemolytic, beta haemolytic, and
gamma haemolytic.
• Alpha haemolytic Streptococci: exhibit incomplete haemolysis
(1-2mm wide) imparting green discoloration around the
colony. Consists of group referred to as S. viridans (viridis
means green) and S. pneumoniae.
• Beta haemolytic Streptococci: Streptolysin O and S. Complete
lysis (zone 2-4 mm wide), Group A streptococcus
• Gamma/ Non –haemolytic Streptococci: No haemolysis. not
primary pathogens , mostly commensals
B. Lancefield’s classification
• Serological classification
• Rebecca lancefield and co-workers
• On the basis of antigenic difference in cell wall carbohydrate
(group specific polysaccharide antigen in cell wall.
• Most beta haemolytic, few alpha and gamma too are
classified into group A to H and K to V
• Groups determined by precipitaion test with specific antisera
or immunofluoresence.
eg. Group A- S. pyogens, Group B- S. agalactiae, Group D-
Enterococcus spp. (enterorococcal), Streptococcus bovis (non-
enterococcal)
Type specific antigen:
• Strains of Gr A are further classified into different types, based
on M proteins (found on cell wall, virulent, interfering in
phagocytosis)
• Also known as Griffith classification; 80 serotypes known.
• M protein is of –a) Rheumatogenic – causes rheumatic fever
b)Nephritogenic – causes acute glomerulonephrits
Other proteins are T and R proteins, not aiding in pathogenicity.
Pathogenicity of Gr. A Streptococci is determined by hyaluronic acid and M
protein.
5. Normal habitats
Group C, G and viridans---commensals in URT
Enrerococci (Gr. D) –intestinal tract
S. agalactiae (Gr. B)—genital tract of women.
 S. pyogens
• Beta haemolytic causing pharyngitis
• Adhere to pharyngeal epithelial via pili covered with
lipoteichoic acid and M-protein
• Inhibited by bacitracin

1. Morphology
Spherical cocci (0.7-0.9 micron)
Occurring in chains of varying length
Non-motile
Have hyaluronic acid capsule (antiphagocytic)
2. Cultural characheristics
• Facultatively anaerobic
• Fastidious
• Temperature range 22 – 42 °C (optimal 37 °C)
• Colonies: small, 0.5 to 1mm, semi transparent, glossy surface

Selective media:
Crystal violet agar, PNF medium (polymyxin B sulphate,
Neomycin, Fusidic acid)
These media inhibit the growth of throat commensals and
facilitate the detection of small number of S.pyogens.
3. Biochemical characters
Catalase: negative
Bile insoluble
PYRase (pyrrolidonyl aminopeptidase): positive
 4. Pathogenesis

a. Virulence factors
i. Capsular hyaluronic acid: Present on group A and also on group C
streptococci. Protects the cells against phagocytosis. It is not
immunogenic.
ii. Group specific polysaccharide antigen:
This is present in cell wall . Believed to play role in the non-suppurative
sequelae of streptococcal infections. These are chemically and
structurally similar to human tissue antigens like those of heart, kidneys
and joints, as a result of which immune response directed against
S.pyogens are auto reactive and cause endocarditis, glomerulonephritis
and arthritis.
iii. Type specific protein antigen
a. M protein: located on the surface of the cell wall . Helps in resisting
phagocytosis, promotes adherence. S.pyogens without M protein is
avirulent. May be rheumatogenic or nephritogenic type.
b. T and R proteins: No role in virulence
Iv. Other surface proteins
Protein F (fibronectin binding protein )helps in adhesion to pharyngeal
epithelial cells. Protein G binds IgG and hinders antibody binding.
v. Exotoxins
a. Erythrogenic toxin
Produced by lysogenised strains of group A streptococci. It is antigenic
and is neutralised by convalescent sera. Acts directly on the
hypothalamus and causes pyrogenicity. Also is known to damage the
blood capillaries under the skin and produce a red skin rash
(characteristic of scarlet fever).
• Dick test is a method of determining susceptibility to scarlet fever by
injecting into the skin 0.1 ml of erythrogenic (scarlet fever) toxin. A
reddening of the skin in an area over 10 millimetres (0.4 inch) in
diameter within about 24 hours indicates a lack of immunity
(susceptible) to the disease whereas formation of no skin rash indicates
immunity against the disease or convalescence due to neutralization of
the toxin by antibody. The test was developed in 1924 by the U.S.
physicians George and Gladys Dick.
b. Exotoxin A:
Closely related to erythrogenic toxin and TSST of S.aureus. It is
superantigen and is responsible to streptococcal TSS.
c. Exotoxin B:
Responsible for tissue destruction in patients with necrotizing
fascitis.
d. Cardiohepatic toxin: responsible for heart and liver failure

vi. Haemolysin:
• Produced by most Group A and few group C and G streptococci
• Are of two types S and O.
• Also called streptolysins
• Can act on cells other than RBC too. eg. WBC, platelets, cardiac
tissue (cytolysin)
a. Streptolysin O:
• Oxygen labile. Causes beta haemolysis only when grown
under surface of blood agar.
• Antigenic, antibody to it (ASO) develops after group A
steptococcal infection. ASO blocks haemolysis by blocking
the action of streptolysin O. Streptolysin O lyses RBC by
binding to cholesterol containing cell membrane and
producing holes in it
• Also acts as cytolysin

b. Streptolysin S:
• Oxygen stable, beta haemolysis even when colonies grow on
surface
• Not antigenic, though protein in nature
vii. Spreading factors:
• Most strains of group A and some members of group C and G
streptocci produce streptokinase (fibrinolysin) , Dnase ,
lipoproteinase, phosphatase ,hyaluronidase etc. which help in
spread of infection.

b. Spectra of diseases
• Cause two types of infection:
Pyogenic/suppurative infection
a. Primary infection
Toxin mediated infection

b. Secondary infection
A. Primary infection

Pyogenic infection
Suppurative, induced locally at the site of infection.
Examples are
• Sore throat : Acute tonsilitis or pharyngitis. The bacteria adhere
to the epithelium by means of lipoteichoic acid and M protein
• Skin infection: Impetigo, Erysipelas, Cellulitis
• Others: sepsis, endocarditis, pneumonia , otitis media, sinusitis,
bacteremia, lymphadenitis etc.
Toxin mediated infection
• Non-suppurative, toxin is directly involved. Causes wide-
spread systemic symptoms in areas of body where there
are no organisms.
• Eg.
• Scarlet fever: It occurs as a complication of sore throat
when the infecting strain produces erythrogenic toxin and
the patient has got no antitoxic immunity (usually a child).
The disease consists of a combination of sore throat and a
generalized erythematous rash.
• TSS due to exotoxin A.
• Erythema nodusom: due to hypersensitivity to PG portion
of cell wall. Characterized by red nodules under the
surface of skin.
B. Secondary infection/ post streptococcal disease
• Complication on following primary infection, followed weeks
later after a latent period.
• Non pyogenic. immunologic in nature
• Occurs when antibody against cell wall polysaccharide of
organism cross-reacts with normal tissue and forms immune
complex that damages normal tissue.
• Examples are acute rheumatic fever (often preceeded by
streptococcal respiratory infection) and acute
glomerulonephritis (often preceeded by streptococcal
infection of skin).
• ASO level increases in both the cases.
5. Laboratory diagnosis
a. Specimen: blood, swab from infectd site, pus
b. Direct smear/Gram stain : It is of no use in streptococcal
pharyngitis as viridans are member of normal flora and cannot
be visually distinguished from S.pyogens. It is useful in skin
lesions or wounds.
c. Culture:
• Specimen (blood, exudate, swabs) inoculated immediately or
sent to lab in pikes transport medium
• Inoculated into sheep/horse blood agar plate, overnight at
37°C, hemolysis better in 5-10% CO2.
• Small , translucent, glossy, beta-haemolytic colonies
• Sensitive to 1 µg benzyl penicillin or 0.04 unit bacitracin and
resistant to cotrimoxazle (1.25pg/23.75µg). So these are put in
well of primary culture.
d. Serological tests
• Though antibodies are produced against most enzymes and
toxins, they are not useful in diagnosis due to their slow
appearance in blood.
• Culture provides proof of continuing presence of S.pyogens,
while serological test proves recent infection. Following
serological tests are in use:
ASO titre
Anti Dnase test
Anti streptokinase
Anti hyaluronidase..etc

Serological grouping and typing is done when needed for

definite classification and epidemiological study.
 S. agalactiae
1. Habitat:
• Commensal in oral cavity, intestinal tract and vagina.
• Important pathogen in neonates, infants, post partum women and
debilated persons

2. Serotypes
• Five distinct serotypes known, type III is the most common

3. Biochemical test:
i. Hippurate hydrolysis
ii. Bacitracin resistance
iii. CAMP test
 Hippurate hydrolysis test
• Hippurate is the glycine conjugate of benzoic acid. When hippurate is
hydrolysed by an organism glycine and benzoic acid are formed.
• In the classical test (48 hour), the benzoic acid produced was detected
using ferric chloride indicator, by its precipitation in an excess of ferric
chloride. Gas Liquid Chromatography (GLC) method for detection of
benzoic acid is also available nowadays.
• In the rapid test (2 hour) Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is
used as the indicator to detect Glycine, the second byproduct of hippurate
hydrolysis. Ninhydrin reacts with glycine to form a deep blue or purple
color (Ruhemann’s purple).
• Principle:
• Hippuric acid is hydrolyzed to benzoic acid and glycine by the enzymatic
action of hippuricase. The glycine end product is detected by the addition
of ninhydrin reagent.
Uses:
Aids in the differentiation of β-hemolytic Streptococcus agalactiae from other β-
hemolytic streptococci.

Hippurate hydrolysis test is also used in identification of Campylobacter

jejuni (hydrolyzes hippurate) and Campylobacter coli (negative) strains.

Hippurate hydrolysis test: left positive, right negative

Bacitracin resistance test
• Bacitracin is a bactericidal drug useful in the treatment of superficial skin
infections but too toxic for systemic use. Bacitracin is a polypeptide antibiotic
produced by Bacillus subtilis. This drug interferes in the peptidoglycan synthesis
of Bacteria. The presumptive identification of group A streptococci (GAS) is
usually done by testing for sensitivity to bacitracin.
• Principle: Bacitracin test is used to determine the effect of a small amount of
bacitracin (0.04 U) on an organism. Streptococcus pyogenes (group A
streptococci) is inhibited by the small amount of bacitracin in the disk; other
beta-hemolytic streptococci usually are not. Staphylococci are also bacitracin
sensitive.
• Procedure of Bacitracin test
• Using an inoculating loop, streak two or three suspected colonies of a pure
culture onto a blood agar plate
• Using heated forceps, place a bacitracin disk in the first quadrant (area of
heaviest growth). Gently tap the disk to ensure adequate contact with the agar
surface.
• Incubate the plate for 18 to 24 hours at 35oC in ambient air.
• Look for zone of inhibition around disk.
• S. agalactiae is resistance to bacitracin
whereas S.pyogenes are sensitive.
CAMP test
• CAMP test was first identified by Christie, Atkins, and Munch-Peterson in 1944
and CAMP test is an acronym of three researchers.
• CAMP (Christie, Atkins, and Munch-Peterson) test is used for the presumptive
identification of Group B Streptococci (Streptococcus agalactiae). It is the only
beta-hemolytic Streptococcus which secrete a protein called CAMP factor or
“protein B”. CAMP test rarely give false positive with other Streptococci.
• Variety of methods are currently available to identify Group B Streptococci (GBS)
isolated from clinical specimens.
Principle
• CAMP test detects the production of diffusible, thermostable, extracellular
protein known as CAMP factor, produced by Group B Streptococcus. The CAMP
factor acts synergistically with the beta lysin produced by Staphylococcus aureus
to produce a zone of enhanced lysis of sheep or bovine erythrocytes. The
standard CAMP test depends on the elaboration of two toxins during growth to
form a typical arrowhead or flame-shaped clearing at the junction of the two
organisms when they are placed perpendicular to each other.
• This test is useful in the identification of S. agalactiae and gram-positive rods,
like Listeria monocytogenes.
Procedure
1. Using an inoculating loop, streak a beta-lysin-producing Staphylococcus aureus
(ATCC25923) in a straight line across the center of a sheep blood agar plate.
2. Streak test organism in a straight line perpendicular to the S. aureus leaving 1cm
space between the two streaks. (Multiple organisms can be tested on a single plate
if they are 3 to 4mm apart).
3. Incubate the plate at 37 degree Celsius for 18-24 hours.

Result
4. Pathogenicity
i. Neonates: Meningitis, septicemia, pneumonia. Predisposing factors are:
>18 hr membrane rupture in women colonised with organism, children
born prior to 37 week gestation, children of mother lacking Ab to Gr B
streptococci and those born without transplacentally acquired IgG
ii. Adults: endocarditis, pneumonia, arthritis, osteomyelitis, postpartum
endometritis. Predisposing factors are debilation, immunosuppression
(like pregnancy, chemotherapy, HIV, diabetes mellitus etc.

5. Laboratory diagnosis
iii. Direct gram stain
iv. Culture : On BAP- beta haemolysis, zone narrower, sometimes alpha or
gamma type, colonies gray, mucoid, larger (2mm) than other
streptococci
v. Biochemical tests: CAMP test, Hippurate hydrolysis test, Bacitracin
resistance test.
vi. Definitive diagnosis done by lancefield grouping.
 Group D Streptococci
• Heterogenous group of streptococci, possessing Lancefield group D
antigen
• Enterococci: E. faecalis, E.faecium
Streptocci/ Non-enterococcal: S.bovis, S.equinis

• Enterococci are Gram Positive Cocci, occurring in short chain or pairs

1. Habitat: commensal of colon. Can cause urinary, biliary and
cardiovascular infection.
2. Cultural characteristics:
• Very hardy organisms, bile resistant, grows on 6.5% NaCl, pH 9.6 and
temperature 10 -45°C, withstand 60°C for 30 min.
• Resistant to beta lactam antibiotics (penicillin),many to vancomycin too.
• Mostly non-hemolytic, few beta hemolytic
3. Pathogenicity:
• Diseases caused mostly by E.faecalis (90%) and E.faecium (5-10%).
• Following are the diseases caused:
i. Endocarditis: third major cause of endocarditis after streptococcus and
staphylococcus. Mostly in old patients
ii. Genitourinary tract infection: Mostly in hospitalized patients
iii. Others: Abdomenal lesion, Biliary tract infection, neonatal meningitis
4. Laboratory Diagnosis

I. Colonies are small (0.5 -1 mm), magenta coloured on Mac Conkey Agar
II. Bile esculin hydrolysis: positive
III. Growth on 6.5% NaCl
IV. VP test: positive
V. Growth at 10 to 45 degree C.
VI. Potassium Tellurite reduction: positive
VII. PYRase test : Positive
VIII. Mannitol fermentor
IX. H2S variable
X. Can be non-haemlytic or haemolytic, (alpha or beta)
 Streptococcus viridans
• Heterogenous group of streptococci, mostly alpha haemolytic,
some gamma haemolytic
• Do not react with Lancefield grouping
1. Habitat : Normal flora of oropharynx, can reach blood stream
and cause endocarditis intermittently
2. Types: Seven types known: S anginosus, S.bovis, S.mitis,
S.mutans, S.salivarius, S.sanguis and S.vestibularis.
3. Pathogenicity: These are opportunistic pathogens and can
cause:
i. sub-acute bacterial endocarditis: preceeded by dental
extraction, oral surgery that displaces alpha hemolytic species
from oral cavity to blood stream.
ii. S.mutans and others synthesize polysaccharide (dextran),
which are found in dental plaque and cause dental caries.
4. Laboratory Diagnosis
i. On blood agar: mostly alpha haemolytic, sometimes gamma haemolytic ,
distinguished from S.pneumoniae by :
a. Bile insolubility
b. Optochin resistance

ii. Gram stain: more ovoid than S.pyogens, GPC in short chains
iii. Penicillin sensitive and gentamicin resistant.
 Streptococcus pneumoniae

• Also known as pneumococcus

• Do not come under Lancefield grouping
• Formerly known as diplococcus pneumoniae
• One of the most important pathogenic streptococci

1. Habitat
-Commensals of upper respiratory tract
-Can cause infection of upper respiratory tract as otitis media, sinusitis,
pneumoniae and septicemia

2. Morphology
-Gram Positive Cocci occurring in pair, short chain, ovoid or lanceolate shaped.
- Non-motile, non-sporing, capsulate (freshly isolated colonies)
3. Cultural characteristics
• Facultatively anaerobic, tempr 25- 40 degree C. optimun 37 degree C.
• Fastiduous and needs meat extracts, blood, serum, etc
• Capnophilic
• In blood agar: alpha haemolysis, resembling viridans streptococci, small, smooth,
transparent, 0.5-1mm, while young tiny, low convex, but as the colonies grow, they
become flattened or depressed centrally known as draughtsman form.
• In nutrient broth: turbid after 6-12 hours and cleared by autolysis within 24 hours.
The autolysin is N-acetylmuramyl-L- alanine amidase.
• Hiss ‘ serum broth is used to study sugar fermentation of S.pneumoniae. It
ferments inulin in addition to other common sugars.
• They do not grow well in sugar rich media because fermentation of sugars
produces excess lactic acid which inhibit the bacteria itself.
4. Biochemical tests
i. Catalase: negative
ii. Oxidase: negative
iii. Bile soluble
iv. Optochin (5µg): sensitive
v. Inulin fermentation
5. Antigenicity:
i. Capsular polysaccharide:
• Type specific and immunologically distinct for each of 90 serotypes
• Ellicits B cell response, soluble in tissue and culture and also termed as soluble specific substance
(SSS)
• Serological typing can be done for epidemiological purpose on the basis of capsular polysaccharides
using following reactions
a. Agglutination test (slide test) with type specific antisera
b. Precipitation test
c. Quellung’s reaction (only of historical interest)
ii. Somatic M protein:
• Possessed by both capsulated and uncapsulated penumococci
• Physiochemically similar, but unrelated to M protein of Group A Streptococci.
iii. Cell wall carbohydrate
• The cell wall of S.pneumoniae contains an antigen referred to as C substance (exposed teichoic acid),
which is antigenically unrelated to the C-carbohydrate of various Lancefield groups.
• C substance is of medical importance as it reacts with normal serum protein made by liver called C-
reactive protein (CRP).
• CRP is a beta –globulin in human serum. It is an acute phase protein which increases in acute
inflammation. It is not an antibody. So it acts as a non-specific indicator of inflammation which is
elevated in response to presence of many organisms, and also cancer, not just S.pneumoniae. Its role
is to bind to phosphocholine expressed on the surface of bacteria in order to activate the
complement system vis C1Q complex. It rises within two hours of infection..
6. Virulence factors:
i. Capsule polysaccharide:
• Interferes with phagocytosis, and favours invasiveness
• Only capsulated strains are virulent
ii. IgA proteases: inactivates secretory IgA Ab
iii. Adhesin proteins: mediate initial colonization of oropharynx
iv. Cell wall constituent: (teichoic acid and peptidoglycan): cause tissue destruction by
activating alternate complement pathway.

7. Pathogenicity :
• Most frequent bacterial cause of lobar and bronchopneumonia
• Also associated with acute bronchtis, acute exacerbation of chronic bronchitis,
sinusitis, otitis media, meningitis, conjunctivitis, arthritis
• Can cause septicemia
• Generally remains harmless in carriers, unless provoked by viral infection as common
cold, influenza to spread to lower respiratory tract, middle ear, act as secondary
pathogen
• In immuno-compromised persons as those with radio/chemotherapy, with chronic
renal disease, elderly people etc, can act as primary pathogen causing pneumonia.
Pneumonia:
In health, various barriers such as cough reflex, ciliated epithelium,
macrophages etc prevent establishment of infection.
Encapsulated pneumococci get trapped in bronchioles, proliferate and
infect alveoli. Inflammatory response of host leads to accumulation of
alveolar exudate containing WBC and RBC. Sputum also resembles this.

Long term infection may cause fibrous and inelastic alveoli.

8. Laboratory diagnosis:
Samples: sputum, CSF, lung aspirate, pleural fluid, blood, urine, laryngeal
swab, trans-tracheal aspiration
i. Direct Gram staining of sputum: lanceolate shaped Gram Positive
Diplococci.
ii. Culture: On blood agar: alpha haemolytic, optochin sensitive, small,
transparent, low convex initially and flatten later (draughtman’s colony)
iii. Optochin sensitivity test (ethyl hydrocuprein dihydrochloride):
• Distinguishes pneumococci from other viridans sreptococci
• Test is done in well of primary culture
• S.pneumoniae is optochin sensitive

iv. Bile solubility test:

• Distinguishes pneumococci from other viridans streptococci. S.pneumoniae is bile
soluble
• Two methods- 1. using 10%sodium deoxycholate
2. using 2% sodium deoxycholate

5. Quellung’s test
• Pneumococci are mixed with polyvalent anticapsular antibodies and mixture is
examined microscopically. There is increased refractiveness (swelling) around the
bacteria in positive cases. Methylene blue addition stains cocci to distinguish them fro
unsatined capsules.
• Wet films can be prepared from either culture suspension or sample directly.
• Individual type sera can be used for serotyping of 90 types

6. Antigen detection: immunoassays as latex agglutination, ELISA etc have been devised.
• Difference between pneumococci and viridans streptococci

Pneumococci Viridans Streptococci

1. Morphology Ovoid/ Lanceolate Round
2. Capsule Present Absent
3. Colonies Draughtman colonies Convex
4. Alpha hemolysis Narrow Wide/narrow
5. Optochin (5µg) Sensititive Resistant
6. Bile Soluble Insoluble
7. Inulin Fermentation No fermentation