Gram Positive Cocci-Staphylococcus Light growth of white pin head non-hemolytic colonies

BAP – DIFFERENTIAL MEDIUM

• BAP: creamy, white or light gold, and “buttery

looking”
• Other species have gray colonies
• Some species are beta hemolytic (S. aureus)
• B-hemolysis - Clear and complete zone of
hemolysis

Staphylococcus aureus

• Au – Gold; because of gold-looking appearance

on culture
• Most virulent species; coagulase (+)
• Culture: golden yellow pigment caused by
Stain + Shape + Arrangement
lipochrome
Gram Staining alone cannot differentiate the genus • BAP on culture beta hemolytic
staphyloccocus • On MSA, resembles “old sock/stocking odor”
• Halophilic bacteria (organism that can survive
Both Micrococci and Staphylococci = can demonstrate
in high salt concentration)
gram positive cocci arranged in clusters
• Responsible for various skin, wound and deep
14.1 Study Mahon pg. 308 tissue infections
• Has the most numerous virulence factors
Staphylococcci

- Gram-positive belongs to Staphylococcaceae

- Catalase-producing bacteria
- Are facultatively anaerobic (except S.
saccharolyticus – obligate anaerobe)
- Non-motile and glucose fermenter
Microscopy:
- Spherical cells - singly in pairs, and in clusters
- Staple means “bunches of grapes”

Notice the gold appearance of the colonies

Culture of Staphylococcus aureus

 • Carbuncles
- occur when larger more invasive lesions
develop from multiple furuncles
- Treatment: Incision and Drainage: incision and
drain the pus (slice surgical blade)
• Bullous Impetigo (blister)
- pustules are larger and surrounded by zone of
erythema

Blood Agar Plate with clear zone of hemolysis (beta

hemolytic) folliculitis Furuncles

Mannitol salt agar – S. aureus turns yellow

Associated disease and infections

1. Toxin-induced cases
• Scalded Skin syndrome
- Bullous (blister) exfoliative dermatitis caused Bullous impetigo (blisters surrounded by erythema)
by staphylococcal exfoliative or epidermolytic
toxin
- Usually seen in newborns esp. when the mother
is infected with S. aureus
• Toxic Shock Syndrome (TSS)
- Rare but potentially fatal, multisystem disease
characterized by sudden onset of fevers, chills,
and vomiting diarrhea.
2. Cutaneous infections
- Suppurative in nature – pus (presence of wbc)
• Folliculitis
- inflammation of hair follicle or oil gland
• Furuncles (boils)
Non-bullous impetigo caused by Streptococci
- “pigsa”
pyogenes
- Large, raised superficial abscesses
 Note the bubble formation (due to the release of
oxygen)
Small bubble formation = false positive
Other associated disease and infections:
3. Bacteremia – bacteria in blood
4. Sepsis – severe effect of bacteremia
5. Osteomyelitis – infection of bone
6. Septic arthritis
7. Food poisoning
8. Acute bacterial endocarditis
Enzymes and Toxins Produced (virulence factors):
Virulence factor – structures/substances/enzymes that
make the organism pathogenic
1. Catalase
- heme enzyme that catalyzes the decomposition
of H202 to water and oxygen.
REMEMBER:
Staphylococci (catalase +) HAS CATALASE
Streptococci (catalase -) NO CATALASE
Catalase test
- uses 3% hydrogen peroxide
- Catalase is found in the bacteria
- Indicator: Bubble formation for positive
The catalase found in the bacteria will act on the
hydrogen peroxide and catalase will break H202 down to
become oxygen and water thus the formation of
bubbles.
2. Staphylocoagulase
- most important virulence factor of
Staphylococcus aureus
- Coagulates fibrinogen in plasma as a result, will
form clot or coagulum formation
- Promotes the formation of a fibrin layer
around staphylococcal abscess thereby
protecting the bacteria from phagocytosis
Two types:
o Cell-bound coagulase/ clumping factor
- Coagulase attached/bound in the
cell wall
- Clots human, rabbit, or pig
plasma
o Unbound coagulase/ free coagulase
- Free from the cell wall
- Causes clot to form when bacteria
cells are incubated with plasma
3. Hyaluronidase
- Also called as spreading factor
- Hyarunoric acid is rich in the tissues in human
and the hyraluronidase will degrade this and
increase the passage of the pathogen to the
body
- Enhances invasion and survival in tissue
- Binds cell together and renders the intercellular
spaces passable to pathogen (increase
permeability)
4. Staphylokinase (fibrinolysin)
- It has fibrinolytic activity; dissolves fibrin clot
- Human body defense: Clotting, to trap organism
inside so that WBC can go directly and kill the
organism
- Can dissolve the clot in human to invade the
body
 5. Lipase (fat-splitting enzyme) 10. Cytolytic Toxin
- Essential for survival in sebaceous areas of the (Hemolysin/Cytolysin/Hemolytic enzyme)
body esp. in skin because it has sebaceous
Hemolysin Destruction of NTR
glands
Alpha RBC, Platelets, causes sever
- Important in the formation of furuncles,
hemolysin Macrophages tissue damage
carbuncles and boils
Beta hemolysin sphingomyelin Exhibited in the
6. Deoxyribonuclease (DNAse) and Phosphatase (hot and cold and RBC around CAMP test
- DNase: destroy DNA lysin) nerves (Christie–
- Phosphatase: destroy phosphate group Atkins–Munch-
- Lowers viscosity of exudates (fluid in nature), Peterson test)
giving the pathogen more mobility even S. for group B
aureus is non-motile, it can permit to go in to streptococci
the different parts of the body Gamma RBC injury in Less toxic than
7. Beta-lactamase hemolysin culture and alpha and beta
- responsible for antibiotic resistance producing
- breaks down penicillin and other beta-lactam edematous
drugs as these inhibit cell wall synthesis lesions
- 90% or more of clinical staphylococci isolates Delta RBC Associated with
hemolysin PVL
are resistant to penicillin as a result of enzyme
REMEMBER!
production.
8. Enterotoxins (heat-stable) • ALPHA AND BETA HEMOLYSIN CAN CAUSE
- It is stable at 100 degrees Celsius for 30 minutes SEVERE DESTRUCTION IN HUMANS
- Reheating contaminated food will not prevent • GAMMA AND DELTA HEMOLYSIN ARE LESS
food poisoning TOXIC
- It is resistant to hydrolysis by gastric and
jejunal enzymes WHY BETA HEMOLYSIN IS CALLED THE HOT AND COLD
- Examples: LYSIN?

Enterotoxin Associations Beta hemolysin has an enhanced hemolytic activity on

Enterotoxin A Food poisoning incubation at 35 C (heat-labile) and subsequent
Enterotoxin B exposure to 4 C
Enterotoxin D
11. Exfoliative toxin A and B
Enterotoxin B Toxic shock syndrome
- Causes the epidermal layer of the skin to slough
Enterotoxin C
off – stratum granulosum (surface layers of the
Enterotoxin G
Enterotoxin I skin) is destroyed
Enterotoxin B Pseudomembranous - Causes calded skin syndrome (SSS)
enterocolitis - Severe SSS is called Ritter’s disease
WITH “ENTEROTOXIN” HA COMPLETE DAPAT ‘TO SA 12. Toxic Shock syndrome Toxin-1 (TSST-
QUIZ NAKO 1)/Enterotoxin F/Pyogenic exotoxin
- Chromosomal-mediated toxin
9. Panton-Valentine Leukocidin - Causes almost all cases of menstruating-
- PVL: Targets the WBC (kill them) (PMN, associated TSS (ex. Tampons) Infects female
macrophage, monocytes) reproductive organ
- Often with community-acquired or hospital- 13. Protein A
acquired staphylococcal infections - not an enzyme
- immunologically active substance found in the
cell wall (prevents phagocytosis)
 - Is antiphagocytic by competing with
neutrophils for the Fc portion of specific
2. Mannitol Fermentation Test
opsonins
- Differential (used to differentiate pathogenic
What is A in Protein A? from nonpathogenic staphylococci) and
selective (MSA – only promotes halophilic
SELF STUDY: Mahon TABLE 14.3 Virulence factors of
organism)
Staphylococcus aureus
- MSA: 1% mannitol + 7.5% NaCl BOTH SELECTIVE
1. Coagulase test ANSD DIFFERENTIAL
- best single criterion of pathogenicity of Differential
Staphylococcus aureus Positive result (+):
- Reagent: Rabbit plasma, pig plasma, human - Pathogenic: yellow-colored S. aureus colonies
plasma (colonies surrounded by yellow halo) “old sock
odor”
METHOD Slide method Tube method - Non-pathogenic: pink
DETECTS clumping extracellular/unbound/free
factor/ bound coagulase
coagulase
INDICATOR clot/coagulum clot/coagulum formation
formation after 1-4 hours of
within 30 incubation
seconds
COAGULASE For slide For tube coagulase (-):
coagulase (+) Staphylococcus hyicus,
but tube Staphylococcus
coagulase (-): indermedius,
Staphylococcus Staphylococcus delphini,
lugdunensis Staphylococcus scheliferi
subsp. coagulans,
Staphylococcus Staphylococcus aureus
schleiferi

NTR This test is This test is sensitive and Why do you think S. aureus yellow in MSA?
insensitive definitive test The growth of Staphylococci will ferment the mannitol
component of the MSA producing acid. Hence, this
fermentation of the manitol will give the colonies and
the medium a yellow color

3. Voges-Proskauer test
- It differentiates S.aureus (+) from S.
intermedius (-)
- (+) result: pink color acetoin (acethylmethyl
carbinol) production
- Staphylococcus aureus and Staphylococcus
indermedius are both positive in tube-
coagulase test
- But, only Staphylococcus aureus is positive to
this test thus producing acetoin
Methicillin-resistant Staphylococcus aureus (MRSA)

- Penicillin resistant strains require treatment

with penicillinase-resistant penicillin
- Betalactam drug = useless (antibiotic resistant)
- Nafcillin or oxacillin: drug of choice
- if the organisms are resistant to Nafcillin or
oxallin, they will be termed as MRSA.
- Acquired after prolonged hospital stay (ICU and
burn patients); proximity to patietns with
MRSA; after receiving broad spectrum
antibiotics; nasal carriage
- found in nasal carriage
- controlled by: proper isolation of organism,
hand hygiene, rapid identification of the
bacteria and control and treatment of sources
- identification:
o specimen for testing: nasal swab
o chromogenic test: (+) mauve-colored colonies
within 24hrs and confirmed within 48 hrs
o Rapid test: IDI-MRSA test and BD Gene Ohm
assay (2 hours)
o Manual method: Oxacillin – generally used for
detection; CLSI 100 – recommends Cefoxitin
(latest)
Placing the antibiotic disc either oxacillin and
cefoxitin. If cefoxitin is resistant, then it is
MRSA