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) (Gram-positive 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 Furuncle Cellulitis Impetigo

Necrotizing fascitis Surgical wounds 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 TESTINGSo 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 STAINStaphylococci are:

•Gram positive

•Cocci

•Arrangement in pairs or clusters

• 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

CATALASE TEST PROCEDURE

Selective Differential

SPECIALIZED MEDIA REVIEW

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

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

COAGULASE TEST

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

NEGATIVE(Coagulase - negative

staphylococci)

POSITIVE(Coagulase - positive

staphylococci)(Staphylococcus aureus)

COAGULASE TEST

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 (catheter-related)

DICHOTOMOUS KEY FOR IDENTIFICATION OF STAPHYLOCOCCI

GRAM POSITIVECOCCI IN PAIRS OR CLUSTERS

CATALASE TEST CATALASE NEGATIVE

CATALASE POSITIVE=Staphylococci

MSAPLATE

MSA PLATEPOSITIVE

MSA PLATENEGATIVE

Staphylococcusaureus

Staphylococci, not S. aureus

STREPTOCOCCI

DICHOTOMOUS KEY FOR IDENTIFICATION OF STAPHYLOCOCCI

GRAM POSITIVECOCCI IN PAIRS OR CLUSTERS

CATALASE TEST CATALASE NEGATIVE

CATALASE POSITIVE=Staphylococci

COAGULASE TEST

POSITIVE

NEGATIVE

Staphylococcusaureus

Coagulase – negative Staphylococci

STREPTOCOCCI