CASE NO.

5
A CASE OF Staphylococcus aureus
INFECTION
EVANGELISTA | FABROS | FARILLAS
GASTARDO | ILAGAN
CASE PRESENTATION
An 18 month old female infant was rushed to the
emergency room because of difficulty of breathing .
1 week ago, she was noted to have multiple
abscess formation on her scalp. 3 days prior to
consult she had fever and cough. Consult was made
in a nearby health center where carbocisteine and
amoxicillin were prescribed. However cough and
fever persisted until day of consult when she was
noted to have difficulty of breathing manifested as
chest indrawing with flaring of the alae nasi. Chest
xray done in the emergency room revealed
pneumatocoele formation.

Staphyloccocus aureus
1. What is the most likely etiologic agent
involved?
Characteristics
Gram-positve cocci occuring in clusters
Non-motile, non-sporeforming, facultative
anaerobe
Fermentation of glucose produces mainly lactic acid
Ferments Mannitol (distinguishes from S.
epidermidis)
Catalase positive
Coagulase positive

Characteristics
Golden yellow colony on agar
Normal flora of humans found on nasal passages,
skin and mucous membranes
Pathogen of humans
Causes a wide range of suppurative infections, as
well as food poisoning and toxic shock syndrome

Morphology
Kayser, Medical Microbiology 2005 Thieme, p. 231

2. What biochemical tests can be requested to
assist you in differentiating this microorganism
from the rest of its relative species?
Biochemical tests that may be requested
include:
Catalase Test
Coagulase Test
DNAse Test

Catalase Test
This is performed to differentiate between
genus Staphylococcus and Streptococcus:
Place a drop of H2O2 on a clean glass slide.
Take a small portion of test organism using a
sterile wooden stick or plastic loop and mix with
the drop of H2O2.
Observe for immediate bubbling (gas production).

Catalase Test
Negative (no bubbles)
Positive (bubbles)
Coagulase Test
Coagulase
An extracellular protein which binds to
prothrombin in the host, forming
staphylothrombin
Clots blood plasma surrounding the cocci;
enabling bacteria to resist phagocytosis by the
hosts immune system
Good indicator of pathogenic potential of S.
Aureus

Two forms of Coagulase
Bound Coagulase/Clumping Factor
Bount to the cell wall
Adsorbs fibrinogen from the plasma and alters it
so it precipitates on the staphylocci, causing them
to clump (cell agglutination)
Detected by the slide coagulase test

Two forms of Coagulase
Free Coagulase
Liberated by the cell wall
Reacts with a substance in plasma to form a fibrin
clot
Detected by tube coagulase test (together with
bound coagulase)

Two Types of Coagulase Test
Coagulase Slide Method
Emulsify a colony of Staph. sp. From the media on
a drop of saline solution on a glass slide
Mix a loopful of fresh plasma with the emulsified
specimen
Observe in the next 5 seconds for the appearance
of white clumps on the slide, indicating a positive
result


Two Types of Coagulase Test
Coagulase Tube Method
Place 0.5 ml diluted plasma into a sterile test tube
Inoculate a loopful of bacteria into the dilute plasma
Incubate at 35
o
C
Observe for clotting at 1, 2 and 24 hour

*False Negatives can occur when clots were only
observed at the 24
th
hour because some strains that
produce coagulase also produce fibrinolysin, which
dissolves clot.


DNAse Test
This is done to detect production of DNAse enzyme
Inoculate a single colony of test organism on DNAse
agar plate.
Incubate the plate overnight at 37
o
C.
Cover the surface of the agar plate with HCl
(Hydrochloric acid)
Look for clear zones around the colonies within 5
minutes after adding the acid.
If clear zones seen around the colonies the test is
DNAse +ve

DNAse negative DNAse positive
3. Cite the characteristics of the clinically significant
species in this genus in terms of hemolysis and tests
used for presumptive diagnosis.

Test(s) S. aureus S. epidermidis S.
saprophyticus
Pigment Gold-white White-yellow White-yellow
Hemolysis + - -
Catalase + + +
Coagulase + - -
Novobiocin Susceptible Susceptible Resistant
Mannitol + - -
Mannose + + -
Trehalose + - +
3. Cite the characteristics of the clinically significant
species in this genus in terms of hemolysis and tests
used for presumptive diagnosis.

HEMOLYSIS
TEST FOR PRESUMPTIVE
DIAGNOSIS
S. aureus -hemolysis (+) Coagulase Test
S. epidermidis hemolysis (-) Trehalose fermentation
S. saprophyticus hemolysis
(-) Mannose fermentation
(-) Novobiocin
4. Name the culture media to be used for
definitive diagnosis and describe its colony
characteristics
Gram staining
PRELIMINARY IDENTIFICATION
Definitive Diagnosis
Inoculation in culture media
4. Name the culture media...
4. Name the culture media...
MANNITOL-SALT AGAR (MSA)
pH of 7.4
Contains the following:
Casein digest
Animal tissue digest
Beef extract
D-mannitol
NaCl
Phenol Red - indicator
Agar
4. Name the culture media...
MANNITOL-SALT AGAR (MSA)
Selectively favors growth of S. aureus because of
the 7.5% NaCl
S. aureus can ferment mannitol while other
Staphylococcal organisms can not.
Color change from red to yellow indicates
fermentation of mannitol.
4. Name the culture media...
MANNITOL-SALT AGAR (MSA)


4. Name the culture media...
Blood Agar and Chocolate Agar
Enriched bacterial growth medium.
Includes:
Base with protein source
Soybean protein digest
NaCl
5% sheep blood
Casein
dH
2
O
Agar
4. Name the culture media...
Blood Agar and Chocolate Agar
Enzymes like hemolysin diffuse outward and
cause complete or partial destruction of RBCs in
the medium and complete Hgb denaturation
within cell products
Chocolate agar has similar contents except it
contains lysed RBCs instead of whole RBCs
4. Name the culture media...
Blood Agar and Chocolate Agar
Most staphylococci grow on agar media
containing peptone
Growth within 24 hours at 35 to 37 degrees
Celsius
S. aureus colonies appear YELLOW and exhibit -
HEMOLYSIS on sheep blood agar, but these may
also occur with other Staphylococcal species.
4. Name the culture media...
Blood Agar and Chocolate Agar
Other Diseases
Bacteremia
Empyema
Endocarditis
Meningitis
Osteomyelitis
Pneumonia
Toxic Shock Syndrome
5. Name other diseases that are associated with this
organism; Include toxin-related diseases.

Functions of Virulence Factors
Evade phagocytosis by macrophages
Mediate adherence to host tissues
Promote tissue destruction Production of
toxins and hydrolytic enzymes
6. Name virulence factors associated with
this organism.
Structural Constituents in the Bacteria
BACTERIAL
CAPSULE
Inhibits chemotaxis and phagocytosis;
Prevents proliferation of polymorphonuclear leukocytes
PEPTIDOGLYCAN
Elicits the production of endogenous pyrogen IL-1 and
opsonic antibodies by monocytes
Also a chemoattractant to leukocytes promoting
abscess formation
TEICHOIC ACIDS
Peptidoglycan component that bind to fibronectin to
allow adherence to host tissues
PROTEIN A
Binds to IgG and effectively prevents antibody-
mediated clearance of the bacteria
VIRULENCE FACTORS OF S. aureus
Toxins
CYTOTOXINS
-hemolysin heterogeneous protein that acts on a broad
spectrum of eukaryotic cell membranes
-toxin degrades sphingomyelin and can affect RBCs
-toxin disrupts biological membranes and may be involve
in diarrheal diseases
-hemolysin can interact with Panton-Valentine Leukocidin
to lyse WBCs
EXFOLIATIVE
TOXINS (ETA and ETB)
Epidermolytic toxins that contain serine proteases to split
intercellular bridges of stratum granulosum yielding to
desquamation on staphylococcal scalded skin syndrome
ENTEROTOXINS
(from A to R)
Superantigens that stimulate T cell proliferation and cytokine
release
Stimulate release of inflammatory mediators
Increase intestinal peristalsis and fluid loss leading to nausea
and vomiting
TOXIC SHOCK
SYNDROME TOXIN -1
Prototypical superantigen that stimulates proliferation of T
cells and release of cytokines, leading to leakage and
endothelial cell destruction
Hydrolytic Enzymes
COAGULASE
Enzymatically activate and initiate fibrin
polymerization
HYALURONIDASE
Spreading factor hydrolyzes hyaluronic acids in
connective tissue
FIBRINOLYSIN Dissolves fibrin clots
LIPASES Hydrolyze membrane lipids
NUCLEASES Hydrolyze DNA
VIRULENCE FACTORS OF S. aureus
7. Create a schematic diagram for the
laboratory identification of this organism.
7. Create a schematic diagram for the
laboratory identification of this organism.
END. THANK YOU!