Staphylococci 09 10 Med

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Staphylococci 09 10 Med

  1. 1. Gram Positive Cocci
  2. 2. Gram positive Staphylococci <ul><li>Objective </li></ul><ul><li>To know the general characters of the genus Staphylococcus </li></ul><ul><li>To know how to differentiate between staphylococci and other gram-positive cocci </li></ul><ul><li>To know the virulence factors associated with staphylococci </li></ul><ul><li>To know the clinical infections associated with staphylococci </li></ul><ul><li>To know the differential tests that used to identify the clinically relevant staphylococcus species </li></ul><ul><li>To know the methicillin resistance in a serious of clinical problem </li></ul>
  3. 3. STAPHYLOCOCCI Staphyle (Greek) Bunch of grapes or Berries Cocci (Rounded) <ul><li>General Characteristics </li></ul><ul><li>Gram positive cocci </li></ul><ul><li>Arranged in grape like clusters </li></ul><ul><li>Facultative anaerobic </li></ul><ul><li>Catalase-positive </li></ul><ul><li>Grow in ordinary culture media </li></ul><ul><li>(Nutrient agar and broth) </li></ul>
  4. 4. Species of Staphylococci <ul><li>33 species known </li></ul><ul><li>Three are medically important: </li></ul><ul><li>S. aureus </li></ul><ul><li>S. epidermidis </li></ul><ul><li>S. saprophyticus </li></ul>
  5. 5. STAPHYLOCOCCUS AUREUS <ul><li>Morphology & Cultural Characters </li></ul><ul><li>Staphyle - bunch of grapes </li></ul><ul><li>Aureus - golden color colonies </li></ul><ul><li>On blood agar - beta-hemolytic colonies </li></ul><ul><li>In contrast to other Staphylococci </li></ul><ul><li>Can grow in 7.5% NaCl & basis of mannitol </li></ul><ul><li>ferment mannitol salt agar as selective medium for S.aureus </li></ul>
  6. 6. Staphylococcal cell wall structure Capsule or polysaccharide slime layer Phospholipid PBP PBP Transport protein Cytoplasmic membrane Peptidoglycan layer Capsule Antigenic & Antiphagocytic <ul><li>Teichoic acid </li></ul><ul><li>Binds to fibronectins </li></ul><ul><li>on surface of host cells </li></ul>Protein A Has great affinity to Fc portion of IgG and makes it unavailable to its receptors on phagocyte Protein A Techoic acid
  7. 7. Coagulase EXTRACELLULAR ENZYMES The clot coats the organisms and inhibit their phagocytosis 1. Coagulase Fibrin Clot Fibrinogen
  8. 8. 2. Catalase May prevent killing of S. aureus by PMNLs cells EXTRACELLULAR ENZYMES Catalase H 2 O 2 Water O 2 +
  9. 9. EXTRACELLULAR ENZYMES <ul><li>3. Lipase </li></ul><ul><li>4. Hyaluronidase </li></ul><ul><li>5. Protease </li></ul><ul><li>6. DNAase </li></ul><ul><li>7. Penicillinase </li></ul>
  10. 10. TOXINS OF S. AUREUS <ul><li>Cytolytic toxins (Haemolysins & Leukocidins) </li></ul><ul><li>A- Haemolysins (hemolyzing RBCs) </li></ul><ul><li> , hemolysin: hemolyzing RBCs and destroying platelets </li></ul><ul><li> , hemolysin: als known as hot-cold lysin, enhances haemolytic activity when incubated at 37 0 C and 4 0 C </li></ul><ul><li>δ hemolysin: less lethal than  and  tHaemolysins. </li></ul><ul><li>B-Leukocidins </li></ul><ul><li>Is an exotoxin lethal to PMNs also it has been implicated to as contributing to invasiveness of the organisms by suppressing phagocytosis </li></ul><ul><li>2. Exfoliatins (epidermolytic ) </li></ul><ul><li>Cause nerosis of epidermis (locally or at other sites) </li></ul>
  11. 11. TOXINS BY S. AUREUS <ul><li>3. Toxic shock syndrome toxin (TSST-1 ) </li></ul><ul><li>Stimulates macrophages to produce IL-1 & TNF-alpha </li></ul><ul><li>Multiple effects  toxic shock </li></ul><ul><li>4. Enterotoxins </li></ul><ul><li>Resist boiling for 30 min. </li></ul><ul><li>Resist gastrointestinal enzymes. </li></ul><ul><li>Act on neuronal receptors in upper GIT </li></ul><ul><li>Cause gastroenteritis in 1-5 hours after ingestion </li></ul>
  12. 12. Diseases by S. aureus Pyogenic Due to toxins Superficial Deep
  13. 13. S. AUREUS DISEASES <ul><li>I: PYOGENIC INFECTIONS </li></ul><ul><li>A. Superficial </li></ul><ul><li>1. Furuncle (boil) </li></ul><ul><li>Infection of hair follicle, sweat gland, sebaceous glands </li></ul><ul><li>Blockage of ducts predispose to infections like </li></ul><ul><li>acne vulgaris & stye </li></ul><ul><li>2. Carbuncle </li></ul><ul><li>(Furuncle + Inflammation of subcutaneous tissue) </li></ul><ul><li>Can lead to abscess formation and bacteremia </li></ul>
  14. 14. Furuncle Folliculitis
  15. 15. <ul><li>3. Paroncyhia </li></ul><ul><li>Infection of nail bed </li></ul><ul><li>Can be autoinfection OR from an external source </li></ul><ul><li>4. Postoperative wound infections </li></ul><ul><li>Autoinfection or from carriers like doctors and nurses </li></ul><ul><li>5. Nosocomial (hospital acquired) infections </li></ul><ul><li>A common cause </li></ul>S. AUREUS DISEASES
  16. 16. <ul><li>I: PYOGENIC INFECTIONS </li></ul><ul><li>B. Deep Infections </li></ul><ul><li>Usually caused by bacteremic spread </li></ul><ul><li>1. Osteomyelitis and arthritis </li></ul><ul><li>3. Bronchopneumonia </li></ul><ul><li>4. Empyema thoracis </li></ul><ul><li>5. Meningitis </li></ul><ul><li>6. UTI </li></ul><ul><li>7. Endocarditis: in drug abusers using injections </li></ul>S. AUREUS DISEASES
  17. 17. S. AUREUS DISEASES II: DISEASES DUE TO TOXINS Exfoliatin in Staph lesion Staph not isolated from lesions 1. Scalded Skin Syndrome (SSS) Toxemia Necrosis of epidermis Loose skin (vesicles) Vesicles breakup
  18. 18. Staphylococcus Scalded Skin Syndrome (SSSS)
  19. 19. <ul><li>2. Impetigo </li></ul><ul><li>Localized SSS </li></ul><ul><li>Produce blisters that contain pus and Staph </li></ul><ul><li>3. Scarlet Fever </li></ul><ul><li>Mild form of SSS </li></ul><ul><li>Characterized by erythema without vesicles </li></ul>S. AUREUS DISEASES II: DISEASES DUE TO TOXINS
  20. 20. <ul><li>4. Toxic Shock Syndrome (TSS) </li></ul><ul><li>Occur in young women during and immediately after menstruation </li></ul><ul><li>Due to intravaginal use of tampons infected with S. aureus  produce TSS </li></ul><ul><li>Fever, vomiting, diarrhea, body pain within 48 hours  severe shock </li></ul>S. AUREUS DISEASES II: DISEASES DUE TO TOXINS
  21. 21. Acute vomiting & diarrhoea (1-5 hrs) 5. Staph Food Intoxication Staph Carrier S. AUREUS DISEASES II: DISEASES DUE TO TOXINS Staph multiplication Enterotoxin in food Resists reheating Food ingested Poor referigeration Salads, potato creamy dishes Contaminates
  22. 22. TREATMENT OF STAPH INFECTIONS Drainage of pus in superficial and chronic lesions C/S to select proper antibiotics Severe infections Amoxycillin-clavulinic acid (Augumentin) Chronic infections MRSA (methicillin-resistant S. aureus) Vancomycin VRSA (Vancomycin Resistant S.aureus ) & VISA (Vancomycin Intermediate S.aureus ) Linezolid & Quinupristin-Dalfopristin
  23. 23. <ul><li>1940s : all S.aureus were sensitive to penicillin </li></ul><ul><li>Shortly after use : penicillin resistant strains appeared which produced beta-lactamase - rapidly spread </li></ul><ul><li>In late 1950s : beta-lactamase - resistant penicillin </li></ul><ul><li>(methicillin) was introduced </li></ul><ul><li>In 1961 methicillin-resistant S. aureus (MRSA) </li></ul><ul><li>was discovered (presently a major problem) </li></ul><ul><li>In 1996 Vancomycin Intermediate S. aureus (VISA) </li></ul><ul><li>In 1997 Vancomycin Resistant S. aureus (VRSA) </li></ul>ANTIBIOTICS RESISTANCE Historical aspect
  24. 24. STAPHYLOCOCCAL NASAL CARRIER <ul><li>Anterior nose of 20-40% of adults are carriers </li></ul><ul><li>Physicians & nurses = 50-70% </li></ul><ul><li>Also skin of axillae & perineum </li></ul><ul><li>In hospital - high carrier rate due to environmental load </li></ul><ul><li>MRSA </li></ul><ul><li>Low carriage rate in community </li></ul><ul><li>High in tertiary care hospitals </li></ul><ul><li>Mode of Transmission </li></ul><ul><li>Fomites </li></ul><ul><li>Direct from hospital staff or attendants : contaminated hands </li></ul>
  25. 25. Control of Carrier and Re-infection <ul><li>Wash clothes in hot water (>70 o C) </li></ul><ul><li>Hand washing with antiseptic soap (Dettol soap) </li></ul><ul><li>Antimicrobial nasal cream (Gentamicin/Mupirocin) </li></ul><ul><li>Oral antibiotics that are concentrated in nasal secretions (ciprofloxacin and rifampicin) </li></ul><ul><li>Chemoprophylaxis </li></ul><ul><li>Antibiotics before and at time of surgical operation </li></ul>
  26. 26. COAGULASE-NEGATIVE STAPHYLOCOCCI <ul><li>Medically important species: </li></ul><ul><li>1. S. epidermidis </li></ul><ul><li>2. S. saprophyticus </li></ul>
  27. 27. STAPHYLOCOCCUS EPIDERMIDIS <ul><li>Normal flora in </li></ul><ul><ul><li>Skin </li></ul></ul><ul><ul><li>Anterior nose & </li></ul></ul><ul><ul><li>External ear canal </li></ul></ul><ul><li>White, non-haemolytic colonies on blood agar </li></ul><ul><li>Sensitive to novobiocin; ( S. saprophyticus is resistant) </li></ul>
  28. 28. DISEASES BY S. EPIDERMIDIS <ul><li>Most infections are hospital acquired </li></ul><ul><li>Opportunistic pathogen in immuno-suppressed </li></ul><ul><li>Strongly associated with presence of foreign bodies </li></ul><ul><ul><li>Prosthetic heart valves (endocarditis) </li></ul></ul><ul><ul><li>IV catheters (bacteremia) </li></ul></ul><ul><ul><li>Urinary catheter (UTI in elderly) </li></ul></ul><ul><ul><li>CSF shunts (meningitis) </li></ul></ul><ul><ul><li>Peritoneal dialysis catheter (peritonitis) </li></ul></ul>
  29. 30. STAPHYLOCOCUS SAPROPHYTICUS <ul><li>Saprophytic life </li></ul><ul><li>Resistant to novobiocin </li></ul><ul><li>Most infections are community-acquired </li></ul><ul><li>Primary UTI in 10-20% of young adult women – hormonal factors may be involved. </li></ul><ul><li>Resistant to antibiotics – penicillins & cephalosporins </li></ul>
  30. 31. LAB IDENTIFICATION OF S. AUREUS <ul><li>Specimens </li></ul><ul><li>Pus, sputum </li></ul><ul><li>Blood, CSF </li></ul><ul><li>Feces, vomit and left over food – in food poisoning </li></ul><ul><li>Anterior nasal swab for carriers </li></ul>
  31. 32. LAB IDENTIFICATION OF S. AUREUS <ul><li>Microscopy </li></ul><ul><li>G+ve cocci in clusters </li></ul><ul><li>Culture </li></ul><ul><li>Blood agar - golden yellow colonies with </li></ul><ul><li>beta-haemolysis </li></ul><ul><li>Mannitol salt agar - yellow colonies (selective and differential for isolation from faeces) </li></ul>
  32. 33. Sputum smear  Staphylococcus aureus Staphylococcus & Streptococcus pneumoniae
  33. 34. LAB IDENTIFICATION OF S. AUREUS <ul><li>Biochemical Tests </li></ul><ul><li>Catalase Test </li></ul><ul><li>Differentiates between Staphylococci (positive) & Streptococci (negative) </li></ul><ul><ul><ul><li>H 2 O 2 Water + Oxygen </li></ul></ul></ul><ul><li>Pour 2-3 ml of H 2 O 2 in a test tube </li></ul><ul><li>With a sterile wooden stick pick a good growth of the </li></ul><ul><li>organism (from blood free medium) and immerse in H 2 O 2 </li></ul><ul><li>Immediate active bubbles – positive test </li></ul>Catalase
  34. 35. LAB IDENTIFICATION OF S. AUREUS <ul><li>Biochemical Tests </li></ul><ul><li>Coagulase Test (positive) </li></ul><ul><li>Differentiates between S. aureus (positive) and other staphylococci (negative) </li></ul><ul><li>Slide Method (for bound coagulase) </li></ul><ul><li>Place a drop of saline on two separate slides. </li></ul><ul><li>Emulsify a colony of test organism in each drop to make thick suspension. </li></ul><ul><li>Add a drop of plasma to one suspension and mix gently. </li></ul><ul><li>Look for clumping within 10 sec for positive test. </li></ul>
  35. 36. EXTRACELLULAR ENZYMES 1. Coagulase a ) Free coagulase (soluble ): <ul><li>Converts fibrinogen to fibrin -clot formation </li></ul><ul><li>The clot coats the organisms and inhibit their phagocytosis </li></ul><ul><li>Detected by tube coagulase test (positive in 3-4 hours) </li></ul>Coagulase Activates CRF* (Prothrombin) in plasma *CRF : Coagulase Reacting Factor Clot Fibrinogen Fibrin Activated CRF
  36. 37. Coagulase EXTRACELLULAR ENZYMES <ul><li>Converts fibrinogen to fibrin -clot formation </li></ul><ul><li>The clot coats the organisms and inhibit their phagocytosis </li></ul><ul><li>Detected by slide coagulase test (positive in few min) </li></ul>2. Bound c oagulase Fibrin Clot Fibrinogen
  37. 38. LAB IDENTIFICATION OF S. AUREUS <ul><li>Biochemical Tests </li></ul><ul><li>Tube Method (for free coagulase) </li></ul><ul><li>1. Mix 0.2 ml of plasma with 1.8 ml of saline (1:10 dil) </li></ul><ul><li>2. Take three small test tubes and label </li></ul><ul><li>T (Test organism) = (18-24 hr broth culture) </li></ul><ul><li>Pos (Positive control) = (18-24 hr broth culture) </li></ul><ul><li>Neg (Negative control) = (Sterile broth) </li></ul>
  38. 39. LAB IDENTIFICATION OF S. AUREUS <ul><li>3. Pipette 0.5 ml of diluted plasma into each tube </li></ul><ul><li>4. Add 5 drops of test organism in tube “T” </li></ul><ul><li>5. Add 5 drops of S. aureus culture to tube “Pos”. </li></ul><ul><li>6. Add 5 drops of sterile broth to tube ‘Neg’ </li></ul><ul><li>7.Mix gently and incubate at 37 o C for 1 hr. </li></ul><ul><li>8. Examine for clotting each hr : upto 6 hours. </li></ul>
  39. 40. LAB IDENTIFICATION OF S. AUREUS <ul><li>DNASE TEST </li></ul><ul><li>Differentiates S. aureus (positive)) from other Stpahylococci (negative) </li></ul><ul><li>Culture test organism DNA agar ( S. aureus will hydrolyse DNA around the colonies) </li></ul><ul><li>After 24 hrs incubation pour weak HCl on surface of plate </li></ul><ul><li>The acid will ppt. unhydrolyzed DNA and DNAse producing colonies are surrounded by clear areas within 5 min. </li></ul><ul><li>Clear zones: S. aureus – DNAse +ve </li></ul><ul><li>No clearing around colonies – DNAse -ve </li></ul>
  40. 41. Case study #1 <ul><li>An elderly male was admitted to CCU with myocardial infarction. He was helped with IV lines and urinary catheter. On 5th day of his stay, he was found to have : </li></ul><ul><li>High grade temperature. </li></ul><ul><li>The physician noticed redness around IV line. </li></ul><ul><li>Chest X-ray showed normal lung fields. </li></ul><ul><li>Blood culture showed gram-positive cocci in clusters </li></ul><ul><li>Growth on blood agar had coagulase-negative bacteria </li></ul>
  41. 42. Questions <ul><li>1. What is the identity of the organism ? </li></ul><ul><li>2. What type of infection it is ? </li></ul><ul><li>3. What is the source of organism ? </li></ul><ul><li>4. What is antibiotic treatment of the patient ? </li></ul><ul><li>5. What will you do if the organism is resistant to vancomycin ? </li></ul>
  42. 43. Case study #2 <ul><li>An elderly male was admitted to CCU with myocardial infarction. On 5th day of his stay, he was found to have : </li></ul><ul><li>1. High temperature with cough and purulent sputum </li></ul><ul><li>2. Chest X-ray showed multiple abscesses </li></ul><ul><li>3. Sputum Gram-smear showed Gram-positive cocci in clusters </li></ul><ul><li>4. Growth on blood agar had coagulase-positive bacteria </li></ul>
  43. 44. Questions <ul><li>1. What is the identity of the organism ? </li></ul><ul><li>2. What type of infection it is ? </li></ul><ul><li>3. What is the source of organism ? </li></ul><ul><li>4. What is antibiotic treatment of the patient ? </li></ul><ul><li>5. What you will do if the organism is MRSA ? </li></ul>
  44. 45. Case study #3 <ul><li>A19-year-old women complained of fever and flank pain, dysuria, urgency to urinate, and blood-tinged urine were also noted. A urine analysis revealed many WBCs and WBCs cast. A urine culture grew white nonhemolytic colonies on blood agar. The colony count was 45,000 CFU/ml. No growth appeared on MacConkey’s agar. The organism was catalase positive and slide-and tube coagulase negative and produced a 21-mm zone of inhibition in the presence of novnbiocin disc </li></ul>
  45. 46. Questions <ul><li>What is the identity of the isolate ? </li></ul><ul><li>In what patient population does this organism normally cause infection? </li></ul>

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