1. Lab 5: INTEGUMENTARY SYSTEM BACTERIOLOGY AND IDENTIFICATION

2. THE INTEGUMENTARY SYSTEM: A PROTECTIVE BARRIER

3. 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.

4. 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

5. INTEGUMENTARY SYSTEM INFECTIONS CAN ALSO ARISE FROM SYSTEMIC DISEASES OR CHRONIC INFECTIONS SUCH AS: DIABETES VASCULAR METABOLIC IMMUNE (HIV) OR AUTOIMMUNE

6. 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

7. 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)

8. 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

9. 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

10. 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

11. TYPES OF INFECTION FolliculitisFuruncle Cellulitis Impetigo Necrotizing fascitis Surgical woundsBug bites

12. 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.

13. 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

14. GRAM STAIN Staphylococci are: Gram positive Cocci Arrangement in pairs or clusters

15. 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

16. CATALASE TEST PROCEDURE

17. Selective Differential SPECIALIZED MEDIA REVIEW

18. 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.

19. Mannitol Salt Agar Lab Procedure

20. 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

22. CATALASE TEST Streptococci are Gram-positive, catalase negative (no bubbles) Staphylococci are Gram-positive, catalase positive (bubbles)

23. Mannitol Salt Agar Results

24. MANNITOL SALT AGAR NEGATIVE MSA TEST POSITIVE MSA TEST POSITIVE TEST IS GROWTH AND FERMENTATION OF MANNITOL (YELLOW)

25. Mannitol Salt Agar Lab: RESULTS

26. NEGATIVE (Coagulase - negative staphylococci) POSITIVE (Coagulase - positive staphylococci) (Staphylococcus aureus) COAGULASE TEST

27. 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)

28. DICHOTOMOUS KEY FOR IDENTIFICATION OF STAPHYLOCOCCI GRAM POSITIVE COCCI IN PAIRS OR CLUSTERS CATALASE TEST CATALASE NEGATIVE CATALASE POSITIVE= Staphylococci MSA PLATE MSA PLATE POSITIVE MSA PLATE NEGATIVE Staphylococcus aureus Staphylococci, not S. aureus STREPTOCOCCI

29. DICHOTOMOUS KEY FOR IDENTIFICATION OF STAPHYLOCOCCI GRAM POSITIVE COCCI IN PAIRS OR CLUSTERS CATALASE TEST CATALASE NEGATIVE CATALASE POSITIVE= Staphylococci COAGULASE TEST POSITIVE NEGATIVE Staphylococcus aureus Coagulase – negative Staphylococci STREPTOCOCCI