1. Medically Important Bacteria Gram Positive Cocci

2. Group B b-Hemolytic Streptococcus (Streptococcus agalactiae)
Has been known to cause mastitis in cattle
Colonize the of urogenital tract pregnant women
Cause invasive diseases in newborns
Early-onset infection
Late-onset disease

3. Streptococcus agalactiae: Invasive Infections
Early-onset infection
Occurs in neonates who are less than 7 days old neonates
Vertical transmission of the organism from the mother
Manifests in the form of pneumonia or meningitis with bacteremia
Associated with a high mortality rate

4. Streptococcus agalactiae: Invasive Infections
Late-onset infection
Occurs between 1 week and 3 months after birth
Usually occurs in the meningitis form
Mortality rate is not as high as early-onset
In adults
Occurs in immunosuppressed patients or those with underlying diseases
Often found in a previously healthy adult who just experienced childbirth

5. Laboratory Diagnosis: Group B b-Hemolytic Streptococcus
Presumptive identification tests
Bile-esculin-hydrolysis–negative
Does not grow in 6.5% NaCl
CAMP-test–positive
Hippurase positive
S. agalactiae shows the arrow-shaped hemolysis near the staphylococcus streak, showing a positive test for CAMP factor

6. Bile Esculin test
Bile-esculin test is based on the ability of certain bacteria to hydrolyze esculin in the presence of bile (4% bile salts or 40% bile).
Bacteria that are bile-esculin positive are, first of all, able to grow in the presence of bile salts. Hydrolysis of the esculin in the medium results in the formation of glucose and a compound called esculetin.
Esculetin, in turn, reacts with ferric ions (supplied by the inorganic medium component ferric citrate) to form a black diffusible complex.

7. Hippurate test
Hippurate hydrolysis test is used to detect the ability of bacteria to hydrolyse hippurate into glycine and benzoic acid by action of hippuricase enzyme present in bacteria.
an oxidizing agent ninhydrin is used as an indicator. Ninhydrin reacts with glycine to form a deep blue or purple color (purple).

8. Mode of control Penicillin G is the drug of choice
A combination from penicillin and aminoglycoside is used in patients with serious infection.
Vancomycin is used for patients allergic to penicillin.

9. Identification Schema
Schema to differentiate Group A and B from other b-hemolytic streptococci

10. Streptococcus Group D and Enterococcus Species
Members of the gut flora
Associated infections
Bacteremia
Urinary tract infections
Wound infections
Endocarditis

11. Group D Streptococcus Enterococcus – 2 imp. species E. fecalis1
Enterococcus – 2 imp. species
E. fecalis
E. faecium
Normal flora in GIT, lower genital tract
Nosocomial / opportunistic pathogen
Resistance to cephalosporins, even vancomycin

12. Laboratory Diagnosis: Streptococcus Group D and Enterococcus Species
Microscopic morphology
Cells tend to elongate
Colony morphology
Most are non-hemolytic, although some may show a- or, rarely, b-hemolysis
Possess Group D antigen

13. Laboratory Diagnosis: Streptococcus Group D and Enterococcus Species
Identification tests
Catalase: may produce a weak catalase reaction
Hydrolyze bile esculin
Differentiate Group D from Enterococcus sp. with 6.5% NaCl
Penicillin resistance

14. Identification Schema
Schema to differentiate Enterococcus and Group D streptococci from other nonhemolytic streptococci

15. Other Streptococcal Species
Viridans group
Members of the normal oral and nasopharyngeal flora
Includes those that lack the Lancefield group antigen
Most are  hemolytic but also includes nonhemolytic species
The most common cause of subacute bacterial endocarditis (SBE)

16. Streptococcus pneumoniae
General characteristics
Inhabits the nasopharyngeal areas of healthy individuals
Typical opportunist
Possess C substance
Virulence factors
Polysaccharide capsule
Clinical infections
Pneumonia - meningitis
Bacteremia - sinusitis/otitis media

17. Laboratory Diagnosis: Streptococcus pneumoniae
Microscopic morphology
Gram-positive cocci in pairs; lancet-shaped

18. Laboratory Diagnosis: Streptococcus pneumoniae
Colony morphology
Smooth, glistening, wet-looking, mucoid
a-Hemolytic
CO2enhances growth

19. Laboratory Diagnosis: Streptococcus pneumoniae
Identification
Catalase negative
Optochin-susceptibility-test–susceptible
Bile-solubility-test–positive

20. Identification Schema
Schema to differentiate S. pneumoniae from other a-hemolytic streptococci

21. Differentiation between -hemolytic streptococci
The following definitive tests used to differentiate between S. pneumoniae & viridans streptococci
Optochin Test
Bile Solubility Test
Inulin Fermentation

22. Optochin Susceptibility Test
Principle:
Optochin (OP) test is presumptive test that is used to identify S. pneumoniae
S. pneumoniae is inhibited by Optochin reagent (<5 µg/ml) giving a inhibition zone ≥14 mm in diameter.
Procedure:
BAP inoculated with organism to be tested
OP disk is placed on the center of inoculated BAP
After incubation at 37oC for 18 hrs, accurately measure the diameter of the inhibition zone by the ruler
≥14 mm zone of inhibition around the disk is considered as positive and ≤13 mm is considered negative
S. pneumoniae is positive (S) while S. viridans is negative (R)

23. Optochin Susceptibility Test
Optochin resistant
S. viridans
Optochin susceptible
S. pneumoniae

24. Bile Solubility test Principle: Procedure:
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
Negative control is made by adding saline instead of bile to the culture
Incubate at 37oC for 15 min
Record the result after 15 min

25. 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

26. Differentiation between -hemolytic streptococci
CAMP test
Bacitracin
sensitivity
Hemolysis
Negative
Susceptible 
S. pyogenes
Positive
Resistant
S. agalactiae
Differentiation between -hemolytic streptococci
Inulin Fermentation
Bile solubility
Optochin sensitivity
Hemolysis
Not ferment
Soluble
Sensitive (≥ 14 mm) 
S. pneumoniae
Ferment
Insoluble
Resistant
(≤13 mm)
Viridans strep

28. Points to Remember General characteristics and hemolytic patterns of
streptococcal and enterococcal species
Infections produced by pathogenic species
Microscopic and colony morphology
Tests used to identify these species
Emergence of resistant strains