Streptococci- Overview of Detection, Identification and Differentiation Techniques

 

Overview:

Streptococci are Gram-positive cocci, non-motile, non-spore-forming bacteria arranged in pairs or chains in structure. Most of the streptococci are facultative anaerobes and some are obligative anaerobe it requires enriched media (Blood agar).

Streptococci are catalase-negative and oxidase negative and the sole product of glucose fermentation is lactic acid with no gas formation is called homofermentative.

Streptococcal species may be serologically classified on the basis of cell surface carbohydrate antigens (either cell wall polysaccharides (group A, B, C, F and G streptococci) or (are cell wall lipoteichoic acid (group D Enterococcus sp.). other streptococci are viridans group.

Virulence factor:

Group A Streptococci

M- protein are acid and head stable, tripsin- labile, fibrillar proteins are associated with the outer surface of the cell wall.

Hyaluronic acid capsule function to help the organism resist the complement dependent killing by phagocytic cell.

Pyrogenic toxin (erythrogenic), which causes the rash of scarlet fever.

Streptokinases produced by group A streptococci hydrolyze fibrin clot and may function virulence by preventing the formation of fibrin barriers at the periphery of spreading streptococci lesions.

Group B Streptococci

The virulence factors are including capsule, Lipoteichoic acid, CAMP factor, Beta hemolysin, superoxide dismutase, C5a peptidase, extracellular matrixp intractions hyluronate lyase, C- protein and induction of apoptosis.

Culture identification:

Colonies are small, translucent, and circular, generally >0.5 mm of diameter, Streptococci are round to ovoid and appear as minute beads of moisture. The different species of streptococci produce hemolysis in blood agar as follows:

          β- hemolysis (clear zone of complete hemolysis)

                      Streptococcus pyogenes

                      Streptococcus agalactiae

   α -hemolysis (Greenish discoloration around the colonies with partial hemolysis)

                      Streptococcus viridians

                      Streptococcus pneumoniae

Tests for Streptococcus pyogenes:

Bacitracin sensitivity: Disc containing 0.04 IU of bacitracin is placed on a lawn culture of the bacterium and incubated for 18 -24 hours. Any zone of inhibition around the disc should be reported as Streptococcus pyogenes.

Serological confirmation is done by using latex kits:

1. In a sterile test tube take 400ul of extraction enzyme (Dilute hydrochloric or nitrous acid), add 3-4 beta hemolytic colonies from BA plate and emulsify it.

2.  Take 20µl of above suspension in each circle of reaction card.

3.  Add one drop of test latex reagent- A, B, C, D, F and G respectively.

4.   Mix and rotate the card for 2 min and observe for agglutination.

5.   Positive indicated by agglutination.

Trimethoprim/Sulfamethoxazole Susceptibility Test:

The combination trimethoprim-sulfamethoxazloe (SXT) provides an easy and inexpensive method for the presumptive identification of both group A and group B β hemolytic streptococci. Group A streptococci are susceptible to relatively low concentrations of bacitracin and are resistant to SXT.

Group B streptococci are resistant to both antibiotics, other β-hemolytic streptococci show varying susceptibility to bacitracin, but these organisms are usually susceptible to SXT. Therefore, the performance of the SXT test along with the bacitracin test increases the sensitivity and predictive value of the bacitracin test.

Tests for Streptococcus agalactiae:

CAMP test:  On a blood agar plate Staphylococcus is streaked horizontally. Perpendicular to this the isolate of streptococci to be tested is streaked care taking to avoid touching the staphylococcal growth. The plate is incubated and after 18-24 hours.  An arrow headed zone of hemolysis at the junction of both the colonies is taken as CAMP positive. This is due to a synergistic action of the β toxin of staphylococcus and toxin of Streptococcus agalactiae.

 

CAMP Test

Tests for Streptococcus viridans:

      They produce α hemolysis on blood agar and occur in long chains and are optochin resistant.

Differentiation of β hemolytic streptococci

Species	S. pyogenes	S. agalactiae	E. faecalis	others
Hemolysis	β	β	β	β
Bacitracin (0.04 IU)	S	R	R	R
Bile Esculin	-	-	+	-
SXT Sensitivity	R	R	R	S

 Test for Streptococcus pneumoniae:

Microscopy:

Gram staining: They occur as gram positive diplococci lanceolate shaped and surrounded by a capsule.

India Ink Preparation: The capsule can be demonstrated.

Culture identification:

Chocolate Agar: They grow well on chocolate agar enriched with 5-10% CO₂ and produce draughtsman colonies.

Blood Agar: They are α hemolytic on blood agar. S. pneumoniae is differentiated from S.viridans by being optochin sensitive. 

Optochin Susceptibility test:

Ethylthydrocupreine hydrochloride (Optochin), a quinine derivative, selectively inhibits the growth of Streptococcus pneumoniae at very low concentrations (5µg/mL or less) Optochin may also inhibit other viridians streptococci, but only at much higher concentrations. The test has a sensitivity of more than 95%, is simple to perform, and is inexpensive. Optochin is water-soluble and diffuses readily into agar medium.

Therefore, filter paper disks impregnated with Optochin can be used in a disk diffusion test format to determine susceptibility of suspected pneumococci, thereby, confirm their identity as such. S. Pneumoniae cells surrounding the disk are lysed owing to changes in the surface tension, and a zone of inhibition is produced.

A viridians streptococcus can be presumptively identified as S. Pneumoniae if it shows a zone of inhibition of 14 mm or more around a 6-mm disk, Organisms showing zones smaller than these should be tested for bile solubility.

References

1. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Sixth edition by Washington et al., 2006.

 

 

 

 Read Also:

Colistin Broth Disk elution method