Medically Important Bacteria
Gram Positive Cocci
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
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
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
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
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.
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).
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.
Identification Schema
Schema to differentiate Group A and B from
other b-hemolytic streptococci
Streptococcus Group D
and Enterococcus Species
Members of the gut flora
Associated infections
– Bacteremia
– Urinary tract infections
– Wound infections
– Endocarditis
Group D Streptococcus
1
Enterococcus – 2 imp. species
E. fecalis
E. faecium
Normal flora in GIT, lower genital tract
Nosocomial / opportunistic pathogen
Resistance to cephalosporins, even vancomycin
Laboratory Diagnosis: Streptococcus
Group D and Enterococcus Species
Microscopic morphology
– Cells tend to elongate
Colony morphology
– Most are non-hemolytic,
although some may show aor, rarely, b-hemolysis
– Possess Group D antigen
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
Identification Schema
Schema to differentiate Enterococcus and Group D
streptococci from other nonhemolytic streptococci
Other Streptococcal Species
Viridans group
– Members of the normal oral and
nasopharyngeal flora
– Includes those that lack the Lancefield
group antigen
– Most are a hemolytic but also includes
nonhemolytic species
– The most common cause of subacute
bacterial endocarditis
(SBE)
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
Laboratory Diagnosis:
Streptococcus pneumoniae
– Microscopic
morphology
– Gram-positive
cocci in pairs;
lancet-shaped
Laboratory Diagnosis:
Streptococcus pneumoniae
Colony morphology
– Smooth, glistening,
wet-looking, mucoid
 a-Hemolytic
– CO2enhances growth
Laboratory Diagnosis:
Streptococcus pneumoniae
Identification
– Catalase negative
– Optochin-susceptibilitytest–susceptible
– Bile-solubility-test–
positive
Identification Schema
Schema to differentiate S.
pneumoniae from other ahemolytic streptococci
Differentiation between a-hemolytic
streptococci
The following definitive tests used to differentiate
between S. pneumoniae & viridans streptococci
– Optochin Test
– Bile Solubility Test
– Inulin Fermentation
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)
Optochin Susceptibility Test
Optochin resistant
S. viridans
Optochin susceptible
S. pneumoniae
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
– 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
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
Differentiation between b-hemolytic streptococci
Hemolysis
Bacitracin
sensitivity
CAMP test
S. pyogenes
b
Susceptible
Negative
S. agalactiae
b
Resistant
Positive
Differentiation between a-hemolytic streptococci
Hemolysis Optochin
Bile
sensitivity solubility
Inulin
Fermentation
S. pneumoniae
a
Sensitive
(≥ 14 mm)
Soluble
Not ferment
Viridans strep
a
Resistant
(≤13 mm)
Insoluble
Ferment
27
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