BACT211: CLINICAL BACTERIOLOGY

TOPIC: STREPTOCOCCI AND ENTEROCOCCI: ISOLATION AND IDENTIFICATION

2ND SEMESTER | S.Y 2023-2024
LECTURER: Ma’am Pamela Sevilla, RMT, MLS (ASCPi)
TOPIC • Alpha – greenish
SUBTOPIC • Gamma – growth of colony, but no lysis, no change in
SUB SUBTOPIC the agar.

BETA-HEMOLYTIC GROUPS
STREPTOCOCCI • Streptococcus pyogenes
- Will produce clear zones [2-4x
• Differs from staphylococci in 2 significant larger than the diameter of the
characteristics: colony]
- Known for having long chains - Morphology: spherical in shape
- Lack the enzyme catalase [negative in [cocci], arranged in short chains
catalase test] in clinical specimens, longer
• Gram-positive cocci when grown in broth
• Facultative anaerobes and generally considered non- • In order to differentiate S. pyogenes
motile from other streptococci and
GROUP A enterococci, isolates are tested for
• Occur singly or in pairs, however, they are best known
STREPTOCOCCI resistance to bacitracin.
for their characteristic formation of long chains
• If a bacterial isolate is beta-
• Some species are capnophilic hemolytic and sensitive to
• Pyogenic causing bacteria bacitracin, it is presumed to be S.
pyogenes.
• There are some organisms from the
Group D that are beta-hemolytic,
but they will be discussed under the
alpha hemolytic section because
they are variably hemolytic.

• Streptococcus agalactiae
- This pathogen may be found in
the pharynx, skin, and rectum;
however, it is more likely to be
• 1930s – Rebecca Lnacefield
found in the genital and
HEMOLYTIC PATTERNS intestinal tracts of healthy
adults and infants.
- S. agalactiae colonies are large,
GROUP B with a narrow zone of beta-
STREPTOCOCCI hemolysis
- Preliminary identification of this
species relies heavily on a
positive CAMP reaction
- Important cause of serious
neonatal infection: sepsis,
meningitis; adult population:
• Beta – clear [the organism was able to lyse the blood
abscesses, endocarditis,
present in the ager] septicemia.
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 • Uncommon human pathogens but
may be involved in zoonoses
• Streptococcus dysgalactiae
produce large colonies with a large
zone of beta-hemolysis on blood
agar
- Pharyngitis, endocarditis,
GROUP C
meningitis [occur in patients
STREPTOCOCCI
with other illnesses]
• Presumptive differentiation of S.
dysgalactiae from other beta-
hemolytic streptococci (S. pyogenes
and S. agalactiae) is based primarily
on resistance to bacitracin and a
negative CAMP test.
ALPHA-HEMOLYTIC GROUPS
• A significant human pathogen
- Bacterial pneumonia
- Meningitis
- Otitis media [ear infection
commonly found in
children]
- Colonizes the pharynx, and
in some cases, in lungs,
sinuses, middle ear
- Virulence: covered with
polysaccharide capsule
STREPTOCOCCUS
• Colonies appear smooth,
PNEUMONIAE
mucoid, and surrounded by a
zone of greenish discoloration
(alpha-hemolysis)
• In culture, these cells usually
grow as diplococci, but they can
also occur in singly or in short
chains
• Presumptive identification of S.
pneumoniae can be made with
a positive optochin
susceptibility test.
• Constituent of the normal flora
or introduced in the tissue via
dental or surgical means [can
cause infection]
VIRIDANS
- Most serious infection:
STREPTOCOCCI
subacute endocarditis
GROUP
• Blood Agar: very small, gray to
whitish gray, and opaque
• Culture: rod-like and grow in
chains
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• The Viridans group can be
differentiated from the
pneumococci and enterococci
by a negative result in the bile
esculin hydrolysis test, the salt-
tolerance test, and the
optochin susceptibility test
• Can be isolated from the
genitourinary tract and the oral
cavity
• Do not possess many virulence
factors; they are important
pathogens in hospitalized
patients where they can cause
UTI, bacteremia, and
endocarditis
GROUP D • Blood Agar: large colonies that
ENTEROCOCCI can appear nonhemolytic,
alpha-hemolytic, or rarely beta-
hemolytic
• Culture: diplococci in short
chains
• E. faecalis appear either
nonhemolytic or beta-
hemolytic
• E. facium: alpha-hemolytic
• S. bovis
- Human pathogen and is
known to be causative
agent of endocarditis and
meningitis
- Associated with
malignancies in the GITs
GROUP D - Large, mucoid (many
NONENTEROCOCCI strains have a capsule), and
either nonhemolytic or
alpha-hemolytic
- Culture: in pairs and short
chains
• Key reactions for this group are
a positive bile esculin test and
negative salt broth test
Transcribed by: JAMARA
 LABORATORY DIAGNOSIS BIOCHEMICAL TESTS - ALPHA-HEMOLYSIS
• GRAM STAIN – Gram positive cocci in pairs or in chains OPTOCHIN DISK [TAXO P]
• GROWTH ON BAP AND CAP
- GRP A – Grayish white, Transparent to translucent,
matte or glossy, large zone of bet hemolysis
- GRP B – Larger than Grp A, Translucent to opaque,
flat or glossy, narrow zone of beta hemolysis
- GRP C – Grayish white, glistening, widezone of
hemolysis
• CATALASE TEST – NEGATIVE

• Test: Optochin Test

- This test is exclusive for S. pneumoniae
• Media: 5% Sheep Blood Agar
• Antibiotic: Optochin/Taxo P/ethylhydrocupreine
hydrochloride [interferes the ATPase (walang maggrow
sa palibot ni optochin)]
• Incubation Period: 18-24 hrs.
• Result:
- Positive/Susceptible: Streptococcus pneumoniae
[Zone of inhibition]
- Negative/Resistant: Other Alpha-hemolytic
streptococci [further test the specimen (Bile Esculin
Test)]

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 BILE SOLUBILITY TEST
• Exclusively for S. pneumoniae
• Media: 5% Sheep Blood Agar
• Reagent:
- Plate: 10% Sodium deoxycholate
- Tube: 2% Sodium deoxycholate
• Incubation Period: 35 degrees Celsius to 37 degrees
Celsius for 30 minutes
• Lysis – presence of autolytic enzymes (amidase)
BILE ESCULIN TEST
• Used to identify the enterococci
• Media: Bile Esculin Agar (slant)
• Grows of a 4% bile – they will be able to hydrolyze
esculin to esculetin
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• Esculin + Ferric = dark brown to black precipitate
• Incubate for 48 hours and look for the presence of black
agar
• Results:
- Positive: Growth and Blackening of the agar slant
- Negative: Growth and no blackening
SALT TOLERANCE TEST
• Enterococci – halophilic (salt-loving) [positive]
• Media: Heart infusion broth with 6.5% NaCl
• Indicator for acid production: Glucose and bromocresol
purple
• Method:
1. Inoculate one or two colonies from an 18 to 24 hour
culture into 6.5% NaCl broth
2. Incubate the tube at 35 degrees Celsius to 37
degrees Celsius in ambient air for 48 hours.
3. Check daily for growth
• Expected Results:
- Positive: Visible turbidity in the broth, with or
without a color change from purple to yellow
o Turbidity alone is indicative of a positive
test
- Negative: No turbidity and no color change after 72
hours of incubation
Transcribed by: JAMARA
 organisms. After incubation, the inoculated plates
are examined for zones of inhibition surrounding
the disks.
• Method
1. Using an inoculating loop, streak two or three
suspected colonies of a pure culture onto a
blood agar plate
2. Using heated forceps, place a bacitracin disk in
the first quadrant (area of heaviest growth).
SCHEMATIC DIAGRAM FOR DIFFERENTIATION OF Gently tap the disk to ensure adequate contact
GAS FROM GBS with the agar surface.
3. Incubate the plate for 18 to 24 hours at 35
degrees Celsius to 37 degrees Celsius in ambient
air for staphylococci and in 5% to 10% carbon
dioxide (CO2) for streptococci differentiation.
4. Look for a zone of inhibition around the disk
• Expected Results
- Positive: Any zone of inhibition greater than 10 mm;
susceptible
- Negative: No zone of inhibition; resistant
• Antibiotic: bacitracin (0.04) Taxo A
• Media: 5% Blood Agar

CAMP TEST

BIOCHEMICAL TEST – B-HEMOLYSIS

BACITRACIN SUSCEPTIBILITY
• Purpose
- The test is used for presumptive identification and
differentiation of beta-hemolytic group A
streptococci [Streptococcus pyogenes – susceptible)
from other beta-hemolytic streptococci.
- It is also used to distinguish staphylococci species
[resistant] from micrococci [susceptible].
• Principle
- The antibiotic bacitracin inhibits the synthesis of
bacterial cell walls by interfering the peptidoglycan
synthesis of bacteria. A disk impregnated with a
small amount of bacitracin (0.04 units) is placed on
an agar plate, allowing the antibiotic to diffuse into
the medium and inhibit the growth of susceptible

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 4. Cap and incubate the tube for 2 hours at 35 degrees
Celsius; use of water bath is preferred
5. Ad 0.2 mL ninhydrin reagent and re-incubate for an
additional 15 to 30 minutes. Observe the solution
for the development of a deep purple color
• Expected Results:
- Positive: A positive test is indicated by the
appearance of a deep blue or violet color in 30
minutes
- Negative: Colorless or slightly yellow pink color
• Limitations:
- A false-positive result may occur if incubation with
ninhydrin exceeds 30 minutes

• S. agalactiae
• CAMP stands for Christie, Atkins, and Munch-Peterson
• Media: 5% Sheep’s Blood Agar
• CAMP Factor + beta – lysin S. aureus = enhanced lysis of
RBC

HIPPURATE HYDROLYSIS

• Hippuricase – glycine and benzoic acid

• Glycine – OXIDIZE – Ninhydrin = purple color
• Reagent: 0.2 mL n=Ninhydrin Reagent
• S. agalactiae
• Method:
1. Add 0.1 mL of sterile water to a 12x75 mm plastic
test tube
2. Make a heavy suspension of the organism to be
tested
3. Using heated forceps, place a rapid Hippurate disk
in the mixture

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