Genus: Streptococcus
The genus streptococcus contains Gram positive streptococci and diplococcic (pneumococci).
Classification based on hemolytic activity:
When cultured o blood agar, aerobic Streptococcus species can be classified as follows:
 Beta-hemolytic Streptococci:
Colonies are surrounded by a zone of complete hemolysis with decolorization of the haemoglobin.
Beta-hemolysis is marked when the plate has been incubated anaerobically.
 Alpha-hemolytic Streptococci:
Colonies are surrounded by an area of partial hemolysis and a green-brown colour (reduced haemoglobin).
 Non-hemolytic Streptococci:
Colonies show neither typical Alpha nor Beta-hemolysis.
There may be, however, slight discoloration in the medium.
Lancefield grouping of Streptococci:
 Beta-hemolytic Streptococci and some other Streptococci produce group specific carbohydrates (C carbohydrates) group-specific cell wall antigens. This carbohydrate is contained in the cell wall of many Streptococci and forms the basis of serologic grouping (Lancefield groups).
 Extracts of group-specific antigen for grouping Streptococci may be prepared by extraction of centrifugated culture with hot hydrochloric acid, nitrous acid, or formamide; by enzymatic lysis of Streptococcal cells(eg: with pepsin or trypsin);or by autoclavingof cells suspensions at 15Ib pressure for 15 minutes. The serologic specificity of the group-specific carbohydrate is determined by an amino sugar.
 Carbohydrate identification enables a strain to be placed in one of the lancefield groups designated A-H and K-V.
Species: Streptococcus pyogenes (Group A)
Normal habitat:
Streptococci are widely distributed in nature, being found in water, dust,vegetation, milk and milk products.
Streptococcus pyogenes can occur as commensals in the upper respiratory tract.
Pathogenicity:
Streptococcus pyogenes (Group A)
This species causes:
 Acute sore throat (tonsillitis and pharyngitis) and peritonsillar abscess (quinsy).
 Scarlet fever caused by erythrogenic toxin- producing strains (about 20% of S.pyogenes strains). The toxin-producing gene is carried by plasmids.
 Ear infections (otitis media and mastoiditis).
 Puerperal sepsis.
 Skin infection such as cellulitis.
 Septicemia, and occasionally endocarditis.
 Cutaneous & Soft Tissue Infections.
 Pyoderma (Impetigo: contagious pyoderma with superficial yellow weeping lesions),(blistering of the skin).
 Erysipelas: Acute superficial cellulitis of skin with lymphatic involvement; face and lower extremities, skin and subcutaneous tissues.
Post streptococcal diseases:
Serious complication may arise following an immunological response to an acute Group A streptococcal infection. The most serious are:
• Acute glomerulonephritis , usually following a streptococcal infection of the skin. Such infection is common in developing countries; especially rural areas. There is inflammation of the kidney due to the deposition of immune complexes in the glomeruli. Blood and protein are passed in the urine.
• Rheumatic fever usually following a respiratory streptococcal infection. This is the most serious complication because damage to heart valves and muscle can lead to chronic rheumatic heart disease.
Enzymes and toxins produced by S.pyogenes :
• Streptolysin O, that haemolyeze red cells under reduced conditions. It stimulates the production of antistreptolysin O (ASO) antibody that can be measured in patient’s serum.
• Streptolysin S , that haemlyzes red cells (beta-hemolysis on blood agar) but does not stimulate the production of any detectable antibody.
• Streptokinase, that causes fibrinolysis.
• Hyaluronidase, that breaks down hyaluronic acid. Streptococcal hyaluronidase is antigenic.
• DNAse (types A, B, C, D), that breakdown deoxyribonucleic acid (DNA) and stimulate an antibody response, especially against DNAse B.
• DPNAse that kills leucocytes by attacking diphosphopyridine nucleotides (DPN).
• Erythrogenic toxin that is the cause of the rash seen in scarlet fever.
Sensitivity to physical and chemical agents:
 Killed by 54 Co in 30 minutes.
 Culture should be stored at 3 Co -5 Co in blood agar or cooked meet medium (CMM), or else freeze-dried.
 Sensitive to most antiseptics, benzylpencillin.
Laboratory diagnosis:
Specimens:
Throat swab, pus, or blood for culture and serum for serology.
Microscopy:
Streptococci are gram positive cocci, occur in chains of varying lenght, non-motile, non-spore-forming.
*Some strains of group A Streptococcus and other Streptococi are capsulated(in very young cultures).
Culture:
Can grow aerobic and anaerobic, at 22-42 Co with optimum of 35-37 Co, for 24hr.
• On blood agar:
small colonies are usually less than 1mm in diameter, grey-white or colourless, transparent to translucent, low convex, discrete, and with matt or glossy surface, dry or shiny and usually irregular in outline. Beta-hemolysis is shown as large clear zone around the colonies.
*The matt colonies contain M antigen.
*Mucoid colonies may occur when a strain is heavily capsulate.

• On blood agar + Bacitracin disk (0.05units):
Show sensitivity of Beta-hemolytic strain to Bacitracin disk, show clear zone of no growth around the disk.
*To demonstrate beta-hemolytic activity, blood agar plates should be prepared from horse, sheep, or goat blood. Human blood should not be used.
• On blood agar containing crystal violet (1 in 50 000) is recommended as a selective medium for isolating Streptococcus pyogenes from throat swabs or from patients with impetigo where Staphylococcus aureus and Streptococcus pyogenes often occur together.
• On MacConkey agar: no growth.
Biochemical reactions:
1. Catalase test:
Streptococcus pyogenes gives negative result.
2. In soluble in bile.
3. PYRase test:
Aim:
To distinguishes Streptococcus pyogenes from non-group A haemolytic streptococci.
Principle:
Group A streptococci and Enterococci hydrolyse L-pyrrolidonyl-B-naphthylamide(PYR).
Method:
1) Prepare a solution of (PYR) by dissolving 25mg PYR in 1ml methanol and add 100ml distilled water.
2) Take up two to four colonies from blood agar on to the tip of a cotton-wool swab and moisten with two drops of PYR solution.
3) Hold he swab in a petridish at room temperature for 10min.
4) Add one drop of Cinnamaldehyde reagent.
Result:
 Development of a red colour within 15min is a positive reaction.
 Negative reactions stay colourless for 30-45 min.
4. Estimation of antistreptolysin O (ASO) titre:
 Used to investigate the post streptococcal diseases.
 There is a rise in ASO antibody level in 80-85% of patients with rheumatic fever. The rise begins early in the course of the disease with highest levels being reached soon after the onset of the disease. In second week, the level usually begins to fall.
 Commercially available tests for the investigation of raised ASO antibody levels are of two types:
 ASO latex slide agglutination test to screen for significantly raised ASO titres and if indicated to semiquantify the antibody.
 ASO microtitration or tube haemolysis test to titrate the antibody.
 ASO latex test:
Aim:
Screens for ASO antibody levels over 200 international units (IU) per ml. levels over 200 IU/ml are generally considered to be abnormal.
Method:
1) Patient’s serum is first incubated with streptolysin O reagent (reduced form) containing 200IU antigen/ml.
2) Add one drop of latex suspension (latex particles coated with streptolysin O antigen).
3) Mixing.
4) Examination for agglutination result.
Result:
• If the patient serum contains more than 200IU ASO antibody, the excess antibody will agglutinate the antigen in the latex reagent.
• If no agglutination occurs the antibody level is below 200IU/ml.
• If the antibody level is greater than 200IU/ml, further testing is required to estimate the a pproximate titre of the antibody.
 ASO titration kits:
1) A constant amount of Streptolysin O antigen reagent is added to a series of dilutions of the patient ’s serum.
2) Incubation.
3) Add Group O washed human or rabbit red cells.
4) The tubes examined for lysis of the Red blood cells.
Result:
• Haemolysis occurs in those tubes in which there is insufficient antibody to neutralize the antigen.
• The highest dilution of serum showing no haemolysis is the ASO titre.
5. Estimation of DNAse B antibody.
6. Streptozyme test.
Antimicrobial sensitivity:
o Antibiotic with activity against Streptococcus pyogenes include penicillin and erythromycin.
o Streptococcus pyogenes is resistant to polymyxin and nalidixic acid.
o Can acquire resistance to sulphonamide, tetracyclines, and less commonly to clindamycin and macrolides.






Species: Streptococcus agalactiae (Group B)
Normal habitat:
S. agalactiae is a member of the gastrointestinal normal flora in some humans and can spread to secondary sites - including the vagina in 10-30% of women. This is of clinical importance: S. agalactiae can be transferred to a neonate passing through the birth canal and can cause serious group B streptococcal infection.
Pathogenicity:
Streptococcus agalactiae (Group B)
This species causes:
 Neonatal septicaemia.
 Pneumonia.
 Meningitis.
 Septic abortion and puerperal sepsis.
 Endocarditis.
 Cellulitis, arthritis in adults particularly in compromised and debilitated subjects.
Laboratory diagnosis:
Specimens:
Blood, sputum, CSF.
Microscopy:
Streptococci are gram positive, non-motile, non-sporing, non-capsulated.
Culture:
Can grow aerobic and anaerobic, at 22-42 Co with optimum of 35-37 Co, for 24hr.
• On blood agar: Coloniesare usually less than 1mm in diameter, grey-white or colourless, transparent to translucent, dry or shiny and usually irregular in outline. Beta-hemolysis are shown as double zone of haemolysis around the colonies, when incubated anaerobically.
• On Kanamycin blood agar:
Used of this medium is recommended as a selective medium for isolating Group B streptococcus from urogenital specimens.
• On Serum starch agar:
Produce an orange pigment.
• On MacConkey agar:
Some strains can grow on this medium.
• PNF medium:
o Selective medium for B-haemolytic streptococci, inhibit the growth of staphylococci and coli form bacteria.
o Method: nutrient agar cooled to 50 Co then add sterile horse blood, polymyxin B sulphate, neomycin sulphate, fusidic acid.
• Other selective medium:
Prepare a solution of 15ug/ml nalidixic acid and 8ug/ml gentamicin, and add 0.5ml of it and 0.25ml sterile defibrinated sheep blood to 4.75ml Todd Hewitt broth.
Biochemical reactions:
1) Catalase test: negative result.
2) CAMP (Christie, Atkins, and Munch Peterson) test:
Aim:
To differentiate Streptococcus agalactiae from other B-haemolytic streptococcus.


Principle:
Streptococcus agalactiae produces an extracellular diffusible protein referred to as CAMP, CAMP factor which interact with the Staphylococcal haemolysin as shown by an arrow-head shaped area of haemolysis.
Method:
 Streak a known Staphylococcus culture across a 10% sheep blood agar plate.
 Inoculate the test organism at right angles to it, the test organism must not touch the staphylococcal inoculum.
 An Enterococcus culture is also inoculated as a negative control.
 Incubation overnight at 37 Co.
 Read the result.
Result:
Arrow shape, positive result (Streptococcus agalactiae).
3) Other CAMP method:
1) Add 1-2 drops of staphylococcal B toxin (10 units/ml) to an overnight blood agar plate culture of the test organism.
2) After 2hour incubation.
3) Development of an area of haemolysis around the colonies, indicates that the organism is Streptococcus agalactiae (group B).
4) Hippurate hydrolysis test:
Aim:
To differentiate Streptococcus agalactiae from other B-haemolytic streptococcus.
Principle:
Streptococcus agalactiae have the ability to hydrolysis hippurate and produce glycine.
Method:
1) Prepare a 1% solution of sodium hippurate and dispense it in 0.4ml volumes in capped tubes.
2) Store at -20Co until used.
3) Thaw a tube, inoculate a large loopful of solid growth from a rich blood-containing medium, and emulsify throughly.
4) Incubate for 2 hour at 37 Co .
5) Add 0.2ml ninhydrin solution (3.5g ninhydrin in 100ml of a mixture of equal parts of acetone and butanol).
6) Incubate for a further 10min at 37 Co.
Result:
Look for purple colouration showing that glycine has been produced on hydrolysis of the hippurate.
5) Pigment test:
 Culture strains anaerobically at 37 Co for 18hours on cloumbia agar, starch serum agar, or best, Islam’s medium.
 Many group B strains, but not other Streptococcus, form orange-coloured colonies.
Antimicrobial sensitivity:
o Antibiotic with activity against Streptococcus agalactiae include penicillin and erythromycin.












Pneumococci
Species:
The species of medical importance is:
Streptococcus pneumoniae (formerly Diplococcus pneumoniae)
Normal habitat:
Streptococcus pneumoniae can found as a commensal in the upper respiratory tract (naso and oropharyngeal flora in many healthy persons).
Pathogenicity:
Streptococcus pneumoniae
This species causes:
 Lobar pneumonia, bronchopneumonia, and bronchitis.
 Bacteraemia and meningitis.
 Endocarditis and pericarditis.
 Middle ear infections, sinusitis, and conjunctivitis.
 In tropical countries, Streptococcus pneumoniae is a common cause of serious infections in patients with sickle cell disease and nephrotic syndrome.
Enzymes and toxins:
1. Leucocidin.
2. Hyaluronidase.
3. Haemolysin.



Laboratory diagnosis:
Specimens:
Blood, sputum, CSF.
Microscopy:
Streptococcus pneumoniae are gram positive, non-motile, non-sporing, elongated (lancet-shaped) diplococci occur in pairs.
Culture:
 Can grow aerobic and facultative anaerobe, at 25-40 Co with optimum of 35-37 Co, for 24hr.
 Grow best in air on hydrogen with 5-10% CO2.
 Can grow on ordinary medium, but better on media with 5-10% serum, blood or heated blood, which supplies nutrients, PH buffers and catalase.
• On blood agar:
Colonies are small, smooth, and transparent, low covex while tiny, they become flattened or depressed centrally, showing the (draughtman form).
A partial clearing of blood and greenish discolouration (alpha-haemolytic).
• On selective medium:
- Used to facilitate the isolation of Streptococcus pneumoniae from sputum, which is heavily contaminated with commensal bacteria.
- Nutrient agar (Columbia agar), horse blood10%, crystal violet, and nalidixic acid and gentamicin (inhibit the many respiratory tract bacteria other than Pneumococcus, Viridans streptococci and Enterococci).



Biochemical reactions:
1). Catalase test:
negative result.
2). Oxidase test:
negative result.
3). Form acid but not gas from glucose, lactose, and sucrose.

4). Optochin sensitivity test:
Aim:
To distinguish Streptococcus pneumoniae from Viridans streptococci.
Method:
1. Prepare a blood agar plate with suspected colonies (pneumococcus-like colonies).
2. Place a paper disc containing 5ug of optochin (ethylhydrocuprein) on an area of heavy inoculum (area A).
3. Incubate at 37CO in air with 5-10% CO2.
Result:
 The growth of pneumococcus will be inhibited in a zone extending radially for at least 5mm from the margin of the disc.
 Viridans streptococci will grow right up to the disc.

5). Bile solubility test:
Aim:
Rapid presumptive test made on the primary culture plate to differentiate between Streptococcus pneumoniae and other streptococcus species.
Method(1):
1. Touch a suspected pneumococcal colony with a loopful of 2% sodium deoxycholate solution at PH 7.
2. Incubate the plate for 30min at 37CO.
Result:
Colonies of pneumococcus disappear, leaving an area of alpha-haemolysis on the blood agar.
Control:
• Standard cultures of Streptococcus pneumoniae as positive control.
• Standard cultures of Streptococcus faecalis as negative control.
Method(2):
1. Grow the isolate to be tested for 18hr at 37CO in 5ml serum, digest broth or infusion broth.
2. While still warm, add 0.5ml of 10% sodium deoxycholate solution.
3. Reincubate at 37CO.
Result:
Pneumococci are lysed within 15 min, and the initially turbid culture becomes clear and transparent.

6). Swelling test:
• Pneumococci have about 83 capsule serotypes.
• Antipneumococcal rabbit sera, a dye such as methylene blue is often added to typing sera so that the bodies of cocci may be stained and thus made easier to see and distinguishable from the unstained capsules.
Method:
• Suitable suspension of cocci can be prepared from pure culture in saline (0.85%Nacl).
• Then separated from saline and dissolved by centrifugation.
• Resuspended to a low density in fresh saline.
• Place a loopful of the culture suspension on a microscope slide.
• Add a loopful of antiserum.
• Mix.
• Apply a thin cover slip, and examine by x100.
Result:
• If the serum contains antibodies homologous for cocci, the margins of the capsules will become refractile and visible, separated from the coccal bodies by the width of capsules.
• With heterologous serum the capsules remain invisible and only the coccal bodies can be detected.

7). Test of pneumococcal antigen:
 Coagglutination test on CSF (COA).
 Latex agglutination test (LA).
 Counter-current immunoelectrophoresis (CIE).
 ELISA to detect capsular antigens.

Animal pathogenicity:
 Most, but not all strains from infective conditions in man are virulent for the mouse.
 Intraperitoneal injection of a small dose of pneumococci generally causes peritonitis, septicaemia and death of the mouse within 1-3 days.
 Rabbits also are highly susceptible to the pneumococcus.

Antimicrobial sensitivity:
o Antibiotic with activity against Streptococcus pneumoniae include penicillin, erythromycin and cotrimoxazole.
o Some strains of Streptococcus pneumoniae that are resistant to penicillin.













Species: Streptococcus viridans
Normal habitat:
• Streptococcus viridans can occur as a commensals in upper respiratory tract.
• Streptococcus viridans are present in large numbers in the mouth, but under certain conditions are associated with oral infections and the production of dental caries.
Pathogenicity:
• Infective endocarditis (especially in patients with artificial heart valves).
 Streptococcus viridans account for about 40% of cases of infective endocarditis.
• Dental caries.
• Occasionally abdominal and brain abscesses.
Laboratory diagnosis:
Specimens:
Blood, sputum, CSF.
Microscopy:
Streptococcus viridans are gram positive, non-motile, non-sporing.
Culture:
• On blood agar:
Show alpha-haemolytic.
• On blood agar + optochin disc (5ug disc):
Resistant to optochin.

• On chocolate blood agar:
See alpha-haemolysis as green colouration around colonies.
• On MacConkey bile salt agar:
Fail to grow.
Biochemical reactions:
1). Catalase test:
negative result.
2). Oxidase test:
negative result.
3). Not dissolved in bile salts.
 Differential characters of pneumococci and viridans streptococci:
Character pneumococci viridans streptococci
Morphology Ovoid or lanceo-diplococci, some short chains Short or long chains of rounded cocci
Capsule Present Usually absent
Colonies Become flattened or draughtman appearance Convex
Effect on blood agar Narrow zone of alpha-haemolysis Wide or narrow zone of alpha-haemolysis
Optochin sensitivity Sensitive Resistant
Bile solubility Soluble Not soluble
Inulin fermentation Ferment Not ferment
Virulence in mice Virulent Non virulent