Download Integumentary System – Catalase, Mannitol Salt Agar

Lab 5: INTEGUMENTARY SYSTEM
BACTERIOLOGY AND
IDENTIFICATION
THE INTEGUMENTARY SYSTEM:
A PROTECTIVE BARRIER
USUAL MICROBIOTA OF THE
INTEGUMENTARY SYSTEM
Trillions of bacteria live on our skin
So then why aren’t we sick all of the time?
Because the bacteria are mostly harmless and some even beneficial.
INTEGUMENTARY SYSTEM
WHEN THE LAYERS OF THE SKIN ARE
INTACT, THE BACTERIA REMAIN ON THE
OUTSIDE.
INFECTIONS CAN ARISE WHEN THE SKIN IS
DAMAGED BY:
• EXTERNAL TRAUMA – SURGERY, BITES,
BURNS, ABRASIONS, TRAUMATIC
IMPLANTATION
INTEGUMENTARY SYSTEM
INFECTIONS CAN ALSO ARISE FROM
SYSTEMIC DISEASES OR CHRONIC
INFECTIONS SUCH AS:
• DIABETES
• VASCULAR
• METABOLIC
• IMMUNE (HIV) OR AUTOIMMUNE
USUAL MICROBIOTA OF THE
INTEGUMENTARY SYSTEM
• Bacteria most commonly found on skin include:
– Staphylococcus species (Gram-positive cocci in
clusters)
– Streptococcus species (Gram-positive cocci in
pairs and chains
– Corynebacterium species (Diphtheriods) (Grampositive rods)
– Anaerobic bacteria
– Fungi
– Pathogens in low numbers
PATHOGENS OF THE
INTEGUMENTARY SYSTEM
• Staphylococcus aureus
• Gram-negative rods (Pseudomonas spp. or
enteric)
• Streptococcus spp. (S. pyrogenes (Group A
strep or S. agglactica (Group B strep)
• Enterococci (E. faecalis or E. faecium)
• Fungi (yeast [Candida spp.] and filamentous)
Staphylococcus aureus
• Most pathogenic of the staphylococci
– Produce and secrete several toxins and enzymes that
act on host immune system to mediate cell destruction
• Normal microbiota: Skin surface, upper respiratory tract,
nares, axilla, groin, and perineum; can colonize various
epithelial and mucosal membranes
• Cause a wide range of infections:
– Superficial skin infections; wound infections
– Deep infections—can cause bacteremia and involve
bones, joints, deep organs, lungs and respiratory
– Scalded skin syndrome; toxic shock syndrome; food
poisoning
Staphylococcus epidermidis
• Ubiquitous member of normal microbiota and often
encountered in the laboratory as a contaminant
• Normal microbiota: skin and mucous membranes;
distributed widely over body surface
• Can cause infections in immunocompromised
patients.
– Important nosocomial pathogen
– Associated with indwelling prosthetic devices—
intravascular catheters, cardiac valves, joints,
cerebral shunts; bacteremia; UTIs; wounds
Staphylococcus saprophyticus
• Second most common cause of uncomplicated UTIs in
women
• Normal microbiota: skin and mucosa of the urogenital tract
• Cause urinary tract infections:
– Sexually active, young females
– Prostatitis in men
– Infections in sites outside of urinary tract are
uncommon
TYPES OF INFECTION
Folliculitis
Necrotizing fascitis
Furuncle
Cellulitis
Surgical wounds
Impetigo
Bug bites
THE INTEGUMENTARY SYSTEM:
SPECIMEN COLLECTION FOR
DIAGNOSIS
• THE SAMPLE (TISSUE OR FLUID) IS COLLECTED WITH A SYRINGE
OR SPECIAL SWAB USING ASEPTIC TECHNIQUE.
• UPON RECEIPT, THE MICROBIOLOGY
LABORATORY WILL PERFORM A:
– GRAM STAIN
• LOOKING FOR WHITE BLOOD CELLS AND PRESENCE OF
MICROORGANISMS
– CULTURE
• TO ISOLATE, IDENTIFY AND PERFORM SUSCEPTIBILITY TESTING ON
PATHOGENIC MICROORGANISMS.
LABORATORY TESTING
So how do we identify any staphylococci
that is growing and whether or not it
could be causing an infection?
To Rule Out Staphylococci:
– GRAM STAIN
– BIOCHEMICAL TESTS:
• CATALASE
• MANNITOL SALT AGAR
• COAGULASE TEST
GRAM STAIN
Staphylococci are:
• Gram positive
• Cocci
• Arrangement in pairs or clusters
CATALASE TEST
• Hydrogen peroxide is toxic to cells
• The catalase test detects the presence of the enzyme
catalase
• Catalase breaks down hydrogen peroxide into water and
oxygen
• Used on the bench-top to differentiate two common groups
of microorganisms that can cause skin infections
– Staphylococci produce catalase
– Streptococci do not produce catalase
CATALASE TEST PROCEDURE
SPECIALIZED MEDIA REVIEW
Selective
Differential
MANNITOL SALT AGAR (MSA)
• Mannitol Salt Agar is a growth medium containing the sugar
mannitol and high concentrations of salt.
• Mannitol salt agar is both a selective and differential medium used
to detect and differentiate Staphylococcus species
• Selective because it contains a high concentration of salt (7.5% NaCl)
which allows for growth of only salt-tolerant (halophiles) staphylococci
to grow.
• Differential because it contains mannitol and the pH indicator phenol
red. Fermentation of mannitol by pathogenic staphylococci produces
acid, which decreases the pH and causes the agar to change from red to
yellow.
Mannitol Salt Agar Lab Procedure
COAGULASE TEST
Bacteria that produce coagulase use it as a defense
mechanism by clotting the areas of plasma around them,
thereby enabling themselves to resist phagocytosis by the
host's immune system
1.Mix a drop of plasma and
the catalase positive
organism on a glass slide
2. Look for the presence or
absence of agglutination
CATALASE TEST
Streptococci are
Gram-positive,
catalase negative (no
bubbles)
Staphylococci are
Gram-positive,
catalase positive
(bubbles)
Mannitol Salt Agar Results
MANNITOL SALT AGAR
NEGATIVE MSA TEST
POSITIVE MSA TEST
POSITIVE TEST IS GROWTH AND
FERMENTATION OF MANNITOL (YELLOW)
Mannitol Salt Agar Lab: RESULTS
COAGULASE TEST
NEGATIVE
(Coagulase - negative
staphylococci)
POSITIVE
(Coagulase - positive
staphylococci)
(Staphylococcus aureus)
THE USE OF MSA / COAGULASE TEST
IN DIAGNOSIS
• Staphylococcus aureus – COAGUALASE POSITIVE
STAPHYLOCOCCI
• The most pathogenic of the staphylococci
• Produce toxins and enzymes that lead to harmful effects on
the body
• Cause of a variety of skin, respiratory, and blood infections
(MRSA and VRSA)
• COAGULASE NEGATIVE STAPHYLOCOCCI
(Staphylococcus epidermidis and other species)
• Staphylococci that are not as pathogenic as S. aureus, but
can cause infection in compromised hosts, BSI (catheterrelated)
DICHOTOMOUS KEY FOR IDENTIFICATION OF STAPHYLOCOCCI
GRAM POSITIVE
COCCI IN PAIRS
OR CLUSTERS
STREPTOCOCCI
CATALASE
NEGATIVE
Staphylococcus
aureus
CATALASE TEST
MSA PLATE
POSITIVE
Staphylococci, not
S. aureus
CATALASE POSITIVE=
Staphylococci
MSA
PLATE
MSA PLATE
NEGATIVE
DICHOTOMOUS KEY FOR IDENTIFICATION OF STAPHYLOCOCCI
GRAM POSITIVE
COCCI IN PAIRS
OR CLUSTERS
STREPTOCOCCI
CATALASE
NEGATIVE
Staphylococcus
aureus
CATALASE TEST
POSITIVE
Coagulase – negative
Staphylococci
CATALASE POSITIVE=
Staphylococci
COAGULASE
TEST
NEGATIVE