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