Streptococci ssp

Gram’s +ve Cocci Clusters

Chains or Pairs
Staphylococci
Streptococci
Catalase +ve
Catalase -ve

Streptococcus General Characteristics 

Classification of Streptococci
Streptococci can be classified according to: Oxygen requirements Anaerobic (Peptostreptococcus) Aerobic or facultative anaerobic (Streptococcus) Serology (Lanciefield Classification) Hemolysis on Blood Agar (BA)

Growth on Blood Agar

Sterptococci are divided into three main groups according to Hemolytic reactions on blood agar α-hemolytic Sterptococci.β-hemolytic Sterptococci.3- Non-hemolytic streptococci Culture : Streptococci can grow well on blood agar ,although enrichment of media with glucose and serum

It causes: 1. Zone of greenish discolouration around the colonies. 2. It is changes oxidation haemoglobin to met-hemoglobin by hydrogen pyroxide.
α-hemolytic Sterptococci: Example: S.pneumonia, viridans streptococci.
-hemolysis


It causes complete hemolysis to RBCs (caused by hemolysins) leading to formation of clear zone around the colonies
β-hemolytic Sterptococci: Example: S. pyogenes (group A β-hemolytic Strept.), S.agalactiae. b-hemolysis


*

Streptococci classified into many groups from A-K & H-VClassification based on C- carbohydrate antigen of cell wall which called Lancefield grouping. and it may be further sub – divided into types based on protein ( M , T , R ) present on the cell surface which called ( Griffth typing ) and more than 65 typing of Griffth of Streptococcus Pyogenes have been recognized.Biochemical reactions are used for species that can not be classified into the Lancefield classification e.g. viridans streptococci Lancefield serological group
Serology

Serology: Lanciefield Classification

Pathogenesis and Virulence Factors
Structural components M protein Lipoteichoic acid & F protein Hyaluronidase spreading factor, Enzymes Streptokinases DNAase Streptolysins A-Streptolysin O lyses red blood cells, white blood cells, and platelets. B- Streptolysin S Erythrogenic toxin/streptococcal pyogenic exotoxin:scarlet fever
spread of streptococci through tissues


Differentiation between -hemolytic streptococci The following tests can be used to differentiate between -hemolytic streptococciLanciefield ClassificationBacitracin susceptibility TestSpecific for S. pyogenes (Group A)CAMP testSpecific for S. agalactiae (Group B)

Bacitracin sensitivity

Principle: Bacitracin test is used for identification of group A To distinguish between S. pyogenes (sensitive to B) & non group A such as S. agalactiae (Resistant to B) Procedure Inoculate Blood agar with heavy suspension of tested organism Bacitracin disk (0.04 U) is applied to inoculated Blood agar. After incubation, any zone of inhibition around the disk is considered as sensitive

CAMP test

Principle: Group B streptococci produce extracellular protein (CAMP factor) Procedure: a strain of Staphylococcus aureus is inoculated down the center of a sheep blood agar plate. A single streak of an isolate to be identified is inoculated vertical to the S. aureus streak to within 3-4 mm of the S. aureus streak. After incubation, a positive result appear as an arrow-head shaped zone of complete hemolysis

4-Christie-Atkins, Munch-Petersen (CAMP test)

-hemolytic streptococci

Some of recognized species viridans Streptococci (oral streptococci)

Species
Group
Str. mutans, serotypes c, e, f
Mutans group
Str. sobrinus, serotypes d, g
Str. cricetus, serotypes a
Str. rattus, serotypes b, and others
Str. salivarius
Salivarius group
Str. vestibularis
Str. constellatus
Anginosus group
Str. intermedius
Str. anginosus
Str. Sangius & Str. oralis
Mitis group



Differentiation between -hemolytic streptococci The following definitive tests used to differentiate between S. pneumoniae & viridans streptococci 1-Optochin Test (ethyl-hydrocupreine hydrochlorid) 2-Bile Solubility Test 3- Qwelling reaction

Optochin Susceptibility Test

Principle: Optochin test is used to identify S. pneumoniae Procedure: Blood agar plate inoculated with organism to be tested Optochin disk is placed on the center of inoculated Blood agar. After incubation at 37oC for 18 hrs, measure the diameter of the inhibition zone S. pneumoniae is positive (S) while S. viridans is negative (R)

Optochin Susceptibility Test

Optochin susceptible S. pneumoniae
Optochin resistant S. viridans

Bile Solubility test

Principle: S. pneumoniae produce a self-lysing enzyme to inhibit the growth The presence of bile salt accelerate this process Procedure: Add ten parts (10 ml) of the broth culture of the organism to be tested to one part (1 ml) of 2% Na deoxycholate (bile) into the test tube Incubate at 37oC for 15 min Record the result after 15 min

Bile Solubility test

Results: Positive test appears as clearing in the presence of bile while negative test appears as turbid S. pneumoniae soluble in bile whereas S. viridans insoluble

This test useful for rapidly Identification of the organism .Procedure :Mix a loopful of broth culture with loopful of diagnostic antiserum and drop of methylene blue or safranine stain on the slide.Cover slip applied over the mixture.After 10 – 12 min examine under oil immersion objective lenses . Qwelling reaction (capsular swelling reaction)


Result : + ve capsule appear swollen - ve capsule quite invisible

Part of Streptococcus until 1984. Gram-positive cocci arranged singly, in pairs, and in short chains. Catalase-negative Most strains react with Lancefield group D antisera. Enterococcus faecalis is the most important species. Effect on blood agar: It has no effect on RBCs (Non hemolytic)

Definitive test for Enterococcus faecalis

Growth on MacConkey’s agar: Principle:
MacConkey’s agar is a selective medium for Gram’s –ve bacteria.It contains bile salts and crystal violet to inhibit the growth of Gram’s +ve bacteria.Enterococcus faecalis is the only gram-positive cocci which can grow on MacConkey’s agar giving pink colonies.

1-Growth on MacConkey’s agar: Procedure:

1. Inoculate MacConkey’s agar plate with the test organism by streaking. 2. Incubate the plate at 37oC for 24 hrs.
Flam & Cool
Flam & Cool
Flam & Cool

Results:

Enterococcus faecalis
Growth of pink colonies
No Growth
Sterptococci

2-Bile esculin test :

Enterococci hydrolyze the glycoside esculin to esculetin and dextrose. Esculetin reacts with an iron salt to form a dark brown or black complex. Ferric citrate is incorporated into the medium as an indicator of esculin hydrolysis and resulting esculetin formation.

Bile esculin test :

Positive result
Negative result

Bile esculin test :

Positive result
Negative result

3- grow on 6.5% NaCl medium

Enterococcus spp. Grow well in 6.5% NaCl medium(6.5%NaCl in brain heart infusion broth ) but S.bovis will not.
Negative after 24hrs S.bovis
Positive after 24hrs Enterococcus spp.

Identification of Sterptococci

β-hemolytic Sterptococci α-hemolytic Sterptococci Enterococci
Gram’s Stain Catalase test
Growth on blood agar
Gram’s +ve Cocci arranged in chains - ve
- ve
- ve
Complete hemolysis
Greenish discoloration
Non hemolytic

Identification of Sterptococci

β-hemolytic Sterptococci α-hemolytic Sterptococci Enterococci
CAMP test
Arrow head shaped zone
-
-
Inhibition zone
Bacitracin sensitive
S.pyogenes
No zone
Bacitracin resistant
Non group B β-hemolytic Strept. -
-
Bacitracin sensitivity
S.agalactiae
Non group A β-hemolytic Strept.


Identification of Sterptococci
β-hemolytic Sterptococci α-hemolytic Sterptococci Enterococci
Qwelling reaction
(+) (-)
Inhibition zone
Optochin sensitive
S.pneumoniae
No zone or
Optochin resistant
Viridans Streptococci
Optochin sensitivity
Bile Solubility
Visibile clearance
Remain turbid
-
-
-
-
(+ve)
(-ve)

Identification of Sterptococci

β-hemolytic Sterptococci α-hemolytic Sterptococci Enterococci
Esculin test Grow in 6.5%Nacl
(+) (+)

Growth on MacConkey’s Agar No Growth

No Growth
Growth of pink colonies
Enterococcus faecalis