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Lab No (3 ): Genus : Streptococci
Characteristics:
• are non-motile, non- spore forming , gram positive and facultative aerobes anaerobes.( which can growth is enhanced with 10% CO2).
• Spherical or oval cells(cocci) characteristically forming pairs or chains during growth.
• Grow well on ordinary solid media enriched with blood, serum, or glucose.
• Most streptococci grow in solid media as discoid colonies.
• catalase negative.
• Distribution of bacteria :
• Streptococci are widely distributed in nature , and are found in upper respiratory tract, gastrointestinal tract and genitourinary tract as normal microbial flora.
• Samples:
• Ear discharge , throat swab, blood , pus
•
• Classification properties :
• the current classification system used in classification of streptococci based on :
• 1.colony growth characteristics ( morphological culture )
• 2.pattern growth on blood agar.( hemolytic reaction )
• 3. Antigenic composition of group specific cell wall substance
• biochemical reaction and resistance to physical and chemical factors.
• Ecologic features.
Table 1: hemolytic reaction of streptococci

hemolytic appearance designation example
complete complete colorless, clear, Beta(? S. pyogenes
sharply defined zone
partial streptococci greenish discoloration Alpha(?) S. Viridans
(reduced hemoglobin)

None none No change Gamma(§) Enterococci



****** Lance field grouping of streptococci :
Streptococci produce group specific carbohydrates (C carbohydrates) identified using group specific antiserum.
It is designated A-H and K-V .( excluding I and J)

The clinically, important streptococci are grouped under A, B,C,D,F and G.
The main species and groups of medical importance.
S. pyogenes …………….. Lancefield group A
S. agalactiae ……………… Lancefield group B
Enterococci …………………. Lancefield group D
NB : Viridians streptococci and anaerobic are not grouped under Lancefield classification
Pathogenicity
It produces different types of enzymes and exotoxins are :
1. Streptokinase : Fibrinolysin .
It is an active proteolytic enzyme which lyses fibrin by catalytic conversion of plasminogen to plasmin.
2. Streptodornase : Streptococcal deoxyribonuclease Mixtures of streptokinase and streptodornase are used in “enzymatic debridement “
3. NADase : Nicotinamide adenine dinucleotidase
4. Hyaluronidase : Spreading factor
It degrades the ground substance of connective tissue (hyaluronic acid) and aids in spreading infectious microorganism
5. Hemolysins : Two types
Streptolysin O and Streptolysin S
Antistreptolysin O antibody titer > 1:200 this supportive evidence for Acute rheumatic fever
Table2: Comparison of the streptolysins

Hemolysin stability of oxygen antigenic

Streptolysin O No Yes

Streptolysin S yes No



6. Erythogenic toxin : pyrogenic exotoxins
It is responsible for the erythematous rash in scarlet fever .
Clinical features
Skin infection, Impetigo, cellulitis, erysipelas, scarlet fever, Acute streptococcal sore throat, ear infection: acute otitis media and mastoiditis, puerperal fever : septicemia originating in the infected uterus, post-streptococcal diseases: immunological diseases:
1. Acute rheumatic fever
Immunological damage to the heart valves and muscle following.
Streptococcal upper respiratory tract infection
It clinically presents with fever, malaise, migratory non- sppurative polyarthritis, carditis, erythema marginatum and subcutaneous nodules.
2. Post streptococcal acute glomerulonephritis .
Immunological damage to the kidney following infection of skin with streptococci .
It clinically manifests with generalized body edema, elevated blood pressure, protein and blood in the urine, blood urea nitrogen retention and low complement level,
Necrotizing fasciitis ( streptococcal gangrene ): extensive and rapidly spreading necrosis of skin and subcutaneous tissue.
S. agalactiae
Clinical features
Neonatal sepsis, pneumonia, and meningitis.
Septic abortion
Puerperal sepsis

Enterococci
Clinical features
. Frequent cause of nosocomial infection
.Abdominal abscess
. Sub-acute bacterial endocarditis
Viridans streptococci
Such as :
Streptococcus mitis
Streptococcus mutans
Streptococcus salivarius
Streptococcus sanguis
Clinical features
Sub-acute bacterial endocarditis
Laboratory Diagnosis.
Specimen :
S. pyogenes . throat swab, pus, blood
S. agalactiae. high vaginal swab of women, blood and cerebrospinal fluid of new born
Enterococci. Blood,pus
Viridans streptococci. Blood
Smear ( microscopic properties ): non- motile gram-positive cocci in chains


Fig .Streptococci
Culture properties :
Grow in aerobic and anaerobic environment at a temperature of 35-37 0c
Grow in ordinary solid media with shiny or dry colonies with grey-white or colorless appearance
S. pyogenes –shows clear zone of hemolysis in blood agar plate.
Dose not grow in mac Conkey agar plate.
Bacitracin sensitive . PYR positive
S. agalactiae : shows clear zone of hemolysis in blood agar plate.
May show double zone of hemolysis when incubated anaerobically ,
Active in hippurate hydrolysis.
CAMP test is used to identify the bacteria.
Viridans streptococci : show greenish discoloration of blood agar plate
Optochin resistant .
Not soluble in bile salts
Do not ferment inulin .
Enterococci : non –hemolytic or ? hemolytic changes in blood agar plate.
Grow in the presence of 6.5 % NaCl .
Grows in in mac Conkey agar.
Identified by litmus milk reduction test.
PYR- positive
Bile esculin –positive
Treatment
Penicillin /Erythromycin and Gentamicin
Streptococcus pneumonia e
• Fastidious, lancet-shaped gram positive diplococcic.
• Possess a capsule of polysaccharide that permits typing with specific antisera.
• Found as a normal flora in the upper respiratory tract.


Antigenic structure :
Capsular poly saccharides: pathogenicity determinant with anti- phagocytic property. There are more than 80 serotypes of the bacteria based on capsular typing.


Identified by capsular swelling test ( quelling reaction)
C substance: cell wall associated antigen .
Protein M antigen .
IgA, protease: enzyme which cleaved IgA.

Clinical features
.labor pneumonia, meningitis, endocarditis,
. otitis media , septic arthritis, Sinusitis, bacteremia

Laboratory Diagnosis:
Specimen : sputum, blood, cerebrospinal fluid, ear discharge and sinus drainage.
Smear: lancet-shaped gram positive diplococcic.
Culture: grow best in chocolate agar media in CO2 enriched atmosphere.
• Shows ? –hemolytic, draughts man colony. Appearance: Sunken centre colony due to spontaneous autolysis of older bacteria.
• Young colonies resemble dew-drops due to capsule.
• Bile soluble, ferment inulin.
• Optochin sensitive.
Serological test :
Quelling reaction:
Procedure :
Mix specific serotype of S. pneumonia ( fresh specimen) with specific anti- polysaccharides serum of the same serotype or with polyvalent anti-serum on a slide.
. look for the appearance of capsule swelling under the 100x objective microscope.
Treatment :
Amoxicillin , Chloramphenicol, and third generation Cephalosporins
Prevention and control : pneumococcal conjugate vaccine : immunization of individuals with type specific polysaccharides vaccine.
Biochemical reaction to diagnose streptococci
. Bile solubility test.
. Litmus milk reduction test .
. CAMP test
. Bacitracin test.
Optochin test.