Cocci 2011


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Cocci 2011

  1. 1. Laboratory Diagnosis• Colonial Morphology (BAP) • S. aureus  Golden yellow colonies, smooth, entirely raised, - hemolytic (>24 hrs of incubation) • S. epidermidis  translucent, gray-white colonies, non- hemolytic • S. saprophyticus  (same as S. epidermidis)
  2. 2. Laboratory DiagnosisGram stain: Gram-positive cocciDifferentiatesStaphylococcus and Catalase testStreptococcusStaphylococcus Streptococcussp. sp.
  3. 3. Catalase test (Slide)Negative Positive
  4. 4. Medically important Staphylococci• Staphylococcus aureus• Staphylococcus epidermidis• Staphylococcus saprophyticus
  5. 5. Laboratory Diagnosis Catalase test Coagulase test/ MSAStaphylococcussp. S. aureus S. saprophyticus S. epidermidis
  6. 6. Coagulase Test• 2 types1.) Bound/Clumping factor  Slide test  (+) Result: agglutination of organism when mixed w/ plasma  not all strains of S. aureus produced clumping factor2.) Free  Tube test  (+) Clot formationAnticoagulant: EDTA
  7. 7. Coagulase test (Tube) Positive Negative
  8. 8. Mannitol Salt Agar• pH indicator: phenol red• Sugar: Mannitol• Salt conc.: 10%• (+) Result: Yellow Halo
  9. 9. Laboratory DiagnosisCoagulase test/ Novobiocin test MSA (S) (R)S. saprophyticusS. epidermidis S. epidermidis S. saprophyticus
  10. 10. Novobiocin disc testS. epidermidis S. saprophyticus
  11. 11. GENUS: Staphylococcus• Gram-positive• (0.5 – 1 m)• Non-motile, non-sporeforming• Catalase (+)• Facultative anaerobes
  12. 12. Medically important Staphylococci• Staphylococcus aureus• Staphylococcus epidermidis• Staphylococcus saprophyticus
  13. 13. Staphylococcus aureus• Catalase-positive, Coagulase-positive• Salt tolerant (7.5% NaCl)• Ferments mannitol on Mannitol salt agar
  14. 14. Reservoir• Normal flora on nasal mucosa and skin
  15. 15. Transmission• Spread via the hands and sneezing• Fomites• Surgical wounds• Lungs of cystic fibrosis patients• Foods associated with food poisoning (Ham/Canned meats, Custard pastries and potato salad)
  16. 16. Predisposing Factors for Infections• Any break in skin (sx)• Any foreign body (sx packing, sutures, tampons)• Ventilators• WBC <500/ L• Dse: CF, CGD• IV drug abuse
  17. 17. Virulence factors:A. Cell associated factorsB. Extracellular factors
  18. 18. A) CELL ASSOCIATED FACTORS:a) CELL ASSOCIATED POLYMERS 1. Cell wall polysaccharide (Peptidoglycan) 2. Teichoic acid 3. Capsular polysaccharide (some strains)b) CELL SURFACE PROTEINS: 1. Protein A (S. aureus) 2. Clumping factor (bound coagulase)- S. aureus
  19. 19. Staphylococcal cell wall
  20. 20. B) EXTRACELLULAR FACTORSa) Enzymes:1. Catalase – all Staph.2. Free coagulase (S. aureus)3. Lipase4. Hyaluronidase (Spreading factor)5. β-lactamases6. Staphylokinase (Fibrinolysin)7. Proteinases
  21. 21. B) EXTRACELLULAR FACTORSb) Toxins:1. Cytolytic toxins i) Hemolysins (α,β,γ,δ) ii) Leucocidin (Panton-Valentine toxin)2. Enterotoxin A-F (heat stable 60°C, 10 mins)3. Toxic shock syndrome toxin-1 (TSST-1)4. Exfoliative (epidermolytic toxin) .
  22. 22. Staphylococcal Diseases Infections Intoxications
  23. 23. 1) Skin and soft tissue infections:• Folliculitis, furuncle (boil), carbuncle, styes, abscess, wound infections, impetigo, paronychia and less often cellulitis.• Coagulase
  24. 24. 2) Musculoskeletal• Osteomyelitis, arthritis, bursitis, pyomyositis.
  25. 25. Osteomyelitis
  26. 26. 1. Respiratory: Tonsillitis, pharyngitis, sinusitis, otitis, bronchopneumonia, lung abscess, empyema, rarely pneumonia.2. Central nervous system: Abscess, meningitis, intracranial thrombophlebitis.3. Endovascular: Bacteremia, septicemia, pyemia, endocarditis.4. Urinary: Urinary tract infection.
  27. 27. Infective endocarditis (Acute)• Fever, malaise, leukocytosis, heart murmur (may be absent initially)• # 1 cause  S. aureus• Fibrin platelet mesh, cytolytic toxins
  28. 28. B) INTOXICATIOINS:The disease is caused by the bacterial exotoxins,which are produced either in the infected hostor preformed in vitro.There are 3 types-1. Food poisoning2. Toxic shock syndrome3. Staphylococcal scalded skin syndrome
  29. 29. Gastroenteritis (food poisoning)• 1-8 hours after ingesting toxin • Nausea • abdominal pain • Vomiting • followed by diarrhea • No fever• Enterotoxins A-F preformed in food (heat-stable)
  30. 30. Toxic Shock Syndrome (TSS) • High fever (abrupt) • Vomiting • Diarrhea • Myalgias • Scarlatiniform rash • Hypotension • Cardiac/Renal failure (severe cases) • TSST-1
  31. 31. Staphylococcal Scalded Skin Syndrome(SSSS)• Exfoliative toxin
  32. 32. Laboratory Diagnosis:Specimens collected: Depends on the type of infection.• Suppurative lesion- Pus,• Respiratory infection- Sputum,• Bacteremia & septicemia- Blood,• Food poisoning- Feces, vomit & the remains of suspected food,• For the detection of carriers- Nasal swab.
  33. 33. Treatment• Methicillin/ Nafcillin/ Oxacillin/ Cloxacillin• Methicillin-resistant S. aureus (MRSA) (due to changes in major penicillin-binding proteins) is commonly resistant to all antibiotics EXCEPT Vancomycin and Fusidic acid.• Topical mupirocin reduces nasal colonization.
  34. 34. Prevention• Basic hospital infection control
  35. 35. Staphylococcus epidermidis• Reservoir: skin and mucous membrane• Neonatal Sepsis• Peritonitis in patients with renal failure who are undergoing peritoneal dialysis through an indwelling catheter• Most common  CSF shunt infection• Infxn related to intravenous catheters and prosthetic implants (e.g., heart valves, vascular grafts, and joints)• Coagulase (-); Novobiocin (S)
  36. 36. Staphylococcus saprophyticus• Causes U.T.I., particularly in sexually active young women.• 2nd cause community acquired U.T.I. young women (Most common cause E. coli)Coagulase (-); Novobiocin (R)
  37. 37. Characteristics S.aureus S.epidermidis S.saprophyticusCoagulase + - -Novobiocin Sensitive Sensitive ResistantsensitivityAcid from + - -mannitolfermentationanaerobicallyHemolysis beta - (most) -
  38. 38. Laboratory DiagnosisGram stain: Gram-positive cocciDifferentiatesStaphylococcus and Catalase testStreptococcusStaphylococcus Streptococcussp. sp.
  39. 39. Important Streptococci• Streptococcus pyogenes• Streptococcus agalactiae• Enterococcus faecalis• Streptococcus bovis• Streptococcus pneumoniae• Viridans group
  40. 40. Streptococcus• Hemolysis varies by species:•••
  41. 41. Laboratory Diagnosis  OPTOCHIN BACITRACIN 6.5% NaCl (S)S. pyogenes (R)S. agalactiae(S)S.pneumoniae (+)Enterococcus(R)Viridans group ( - )S. bovis
  42. 42. Laboratory Diagnosis• Optochin “Taxo P” Disc test
  43. 43. Laboratory Diagnosis• Bacitracin “Taxo A” Disc test (S)
  44. 44. GENUS: Streptococcus • Gram-positive • Non-motile, non-sporeforming • Catalase (-) • Facultative anaerobes
  45. 45. Streptococci• Are serogrouped using known antibodies to the cell wall carbohydrates (Lancefield’s Group A-H, K-U)• Group A- Rhamnose-N-acetylglucosamine• Group B-Rhamnose-glucosamine polysaccharide• Group C-Rhamnose-N-acetylgalactosamine• Group D- Glycerol teichoic acid• Group F- Glucopyranosyl-N-acetylgalactosamine
  46. 46. Laboratory Diagnosis• Specimen Collection & Processing: • No special consideration; site• Antigen Detection • S. pyogenes  (throat) latex agglutination, Coagglutination, ELISA• Gram Stain
  47. 47. Laboratory Diagnosis• Cultivation • MOC: 5% Sheep’s Blood Agar (BAP)
  48. 48. Laboratory Diagnosis• Colonial appearance - Grayish white - Hemolysis
  49. 49. Streptococcus pyogenes (GABS)Distinguishing Characteristics Beta-hemolytic Group A Colonies inhibited by Bacitracin on BA Gram-positive cocci in chains Catalase-negative PYR (+)
  50. 50. Reservoir• Human throat• Skin
  51. 51. Transmission• Spread by respiratory droplets• Direct contact
  52. 52. Group A Streptococcal cell wall
  53. 53. Cell wall components• Hyaluronic acid capsule (a polysaccharide) is non- immunogenic; inhibits phagocytic uptake• M-protein: major virulence factor, hair-like projections; antiphagocytic, used to type group A Strep
  54. 54. Toxins• Hemolysins • Streptolysin O: immunogenic, hemolysin/cytolysin • Streptolysin S: non-immunogenic, hemolysin/cytolysin
  55. 55. Exotoxins A-C(pyrogenic/erythrogenic)• Phage-coded (e.g., the cells are lysogenized by a phage)• Cause fever and the rash of Scarlet fever• Inhibit liver clearance of endotoxin (from normal flora), creating shock-like conditions• Superantigens: activate many helper T cells by bridging T cell receptors and MHC class II markers without processed antigen
  56. 56. Spreading factors:• Streptokinase (fibrinolysin): breaks down fibrin clot• Streptococcal Dnase (Streptodornase): liquefies pus, extension of lesion• Hyaluronidase: hydrolyzes the ground substances of the connective tissues; important to spread in cellulitis
  57. 57. Diseases• Streptococcus pyogenes causes a wide variety of acute infections; some have immunologic sequelae
  58. 58. Acute (Suppurative) S.pyogenes Infxn • Pharyngitis (most common) • Scarlet fever • Pyoderma/ Impetigo (Also, cellulitis,necrotizing fasciitis (flesh-eating bacteria!), puerperal fever, lymphangitis, pneumonia, a toxic shock- like syndrome, etc.
  59. 59. Pharyngitis• Abrupt onset of sore throat• Fever• Malaise• Headache• Tonsillar abscesses• Tender anterior cervical lymph nodes• Lab: For Strep throat: Rapid antigen test (misses 25% of the strep throat); culture all “negatives”
  60. 60. Pyoderma/ Impetigo• Pus-producing skin infection (honey-crusted lesions)
  61. 61. Erysipelas• Brawny edema, advancing margin of infection
  62. 62. Necrotizing fasciitis• S. pyogenes  “flesh-eating bacteria”
  63. 63. Scarlet fever• Initial: S/Sx’s of pharyngitis• Followed by blanching, “sandpaper” rash
  64. 64. Scarlet fever• Pastia lines
  65. 65. Scarlet fever• Strawberry tongue
  66. 66. Non-suppurative Sequelae to GroupA Streptococcal Infections• Rheumatic fever• Acute glomerulonephritis (M12 serotype)
  67. 67. Rheumatic fever• Sequelae to : Pharyngitis with group A Strep (not group C)• Mechanism: in genetically susceptible individuals, the infection results in production of antibodies that cross- react with cardiac antigens
  68. 68. Rheumatic fever• Symptoms occurs 2-3 weeks after a pharyngeal infection• Lab: elevated ASO titers (>200)• Jones Criteria
  69. 69. Major Jones Criteria• “J NES”• J- Joints (Migratory arthritis)• -Carditis• N- Subcutaneous Nodules• E- Erythema marginatum• S- Sydenham chorea
  70. 70. Minor Jones Criteria• Fever• Arthralgias• Elevated acute phase reactants
  71. 71. Rheumatic fever• DIAGNOSIS: • 2 major or • 1 major & 2 minor
  72. 72. Acute Glomerulonephritis• Sequelae to: Pharyngitis or Cutaneous strep infxn• Mechanism: Immune complexes bound to glomeruli
  73. 73. Laboratory Diagnosis• PYR test• Principle:hydrolysis of L- pyrrolidonyl-- naphthylamide (PYR)
  74. 74. Treatment• Penicillin G  DOC• Beta-lactam drugs• Erythromycin
  75. 75. Prevention• Penicillin in RF px to prevent recurrent S. pyogenes pharyngitis
  76. 76. Streptococcus agalactiae = Group BStreptococci (GBS)• Distinguishing Characteristics • Beta-hemolytic • Bacitracin-resistant on BAP • Gram-positive cocci in chains • Group B • Catalase-negative, hydrolyzes hippurate • CAMP test-positive: CAMP(Christie-Atkins-Munch-Peterson) factor is a polypeptide that “compliments” a Staph aureus sphingomyelinase to make an area of new complete beta- hemolysis
  77. 77. Reservoir• Colonizes human vagina (15 – 20% of women)
  78. 78. Transmission• Newborn infected during birth• Increased risk with PROM
  79. 79. Pathogenesis• Beta-hemolysin
  80. 80. Diseases• Neonatal septicemia• Neonatal meningitis (Neonate – 2 mths)• Most common causative agent ( GEL) # 1 – S. agalactiae (GBS) 2 – E. coli Rare: L. monocytogenes
  81. 81. Laboratory Diagnosis• 0.04 U Bacitracin disk – Resistant• CAMP (Christie, Atkins, Munch- Peterson) Test  detects production of a diffusible, extracellular protein that enhaces hemolysis of sheep erythrocytes by S. aureus • (+) Arrowhead shape at the juncture of S. agalactiae & S. aureus
  82. 82. Treatment• Ampicillin with Cefotaxime or Gentamicin
  83. 83. Prevention• Treat mother prior to delivery if she had a previous baby with GBS, has documented GBS colonization, or prolonged rupture of membranes
  84. 84. Streptococcus pneumoniae(Pneumococcus)• Distinguishing Characteristics - Alpha-hemolytic - Colonies inhibited by optochin on BAP - Gram-positive, lancet-shaped diplococci (or short chains) - Lyse by bile- Quellung (+)
  85. 85. Reservoir• Human Upper Respiratory Tract
  86. 86. Transmission• Respiratory droplets; not considered highly communicable• Often colonizes without causing disease
  87. 87. Pathogenesis• IgA protease: colonization
  88. 88. Pathogenesis• Teichoic acids: attachment• Polysaccharide capsule: major virulence factor• Pneumolysin O: hemolysin/cytolysin • Damages respiratory epithelium (hemolysin similar to streptolysin O, which damages eukaryotic cells) • (Inhibits leukocyte respiratory burst and inhibits classical complement fixation.)
  89. 89. Pathogenesis• Pneumococcus in alveoli stimulate release of fluid and red and white cells producing “rusty sputum”• Peptidoglycan/ teichoic acids highly inflammatory in CNS
  90. 90. Diseases• Bacterial Pneumonia• Adult Meningitis• Otitis Media and Sinusitis in children• Septicemia
  91. 91. Bacterial Pneumonia• Most common bacterial cause, especially after 65 years but also in infants• Sx: • “big” shaking chills • Sharp pleural pain • High fever • Lobar with productive blood-tinged sputum (rusty-colored)
  92. 92. Predisposing Conditions forPneumonia• Antecedent influenza or measles infection: damage to mucociliary elevator• Chronic obstructive pulmonary disorders• Congestive heart failure• Alcoholism• Asplenia predisposes to septicemia
  93. 93. Adult Meningitis• Most common cause (> 40 y/o)• CSF: WBC (PMN) Glucose Protein Pressure
  94. 94. Otitis Media and Sinusitis in Children • Most common cause
  95. 95. Septicemia• In splenectomized patients
  96. 96. Treatment• Penicillin G  DOC• Resistance (both low level and high level) is chromosomal (altered penicillin-binding proteins); major concern in meningitis (Vancomycin  Rifampin used)
  97. 97. Prevention• Vaccine 23 serotypes of capsule
  98. 98. Viridans Streptococci (S. sanguis, S.mutans, etc.)• Distinguishing Characteristics • Alpha-hemolytic, resistant to optochin • Gram-positive cocci in chains • NOT bile soluble
  99. 99. Reservoir• Human oropharynx (normal flora)
  100. 100. Diseases• Dental carries• Infective Endocarditis (Subacute)
  101. 101. Dental carries• S. mutans dextran-mediated adherence glues oral flora onto teeth, forming plaque and causing caries
  102. 102. Infective Endocarditis• Sx: • Malaise • Fatigue • Anorexia • Night sweats • Weight loss• Predisposing factors: • Damage (or prosthetic) heart valve • Dental work w/o prophylactic antibiotics • Extremely poor oral hygiene
  103. 103. Pathogenesis• Dextran (biofilm)-mediated adherence onto tooth enamel or damaged heart valve and to each other (vegetation). Growth in vegetation protects organism from immune system.
  104. 104. Treatment• Penicillin G with Aminoglycoside for endocarditis
  105. 105. Prevention• For individuals with damage heart valve• Prophylactic penicillin prior to dental work
  106. 106. GENUS: Enterococcus• Catalase negative• PYR +• Hydrolyzes Esculin in 40% bile• (+) growth 6.5% NaCl
  107. 107. Enterococcus faecalis =Streptococcus faecalis• Distinguishing Characteristics • Group D Gram-positive cocci in chains • PYR test + • Catalase-negative, varied hemolysis • Hydrolyzes esculin in 40% bile (bile esculin agar turns black) • (+) growth 6.5% NaCl
  108. 108. Reservoir• Human colon• Urethra • Female genital tract
  109. 109. Pathogenesis/ PredisposingConditions • Bile/ Salt tolerance allows survival in bowel and gall bladder • During medical procedures on GI or GU tract: E. faecalis  bloodstream  previously damaged valves  ENDOCARDITIS (SBE)
  110. 110. Diseases• Urinary, biliary tract Infections• Infective endocarditis (SBE)
  111. 111. Treatment• All strains carry some drug resistance• Some vancomycin-resistant strains of Enterococcus faecium or E. faecalis: no reliably effective treatment
  112. 112. Prevention• Prophylactic use of penicillin and gentamicin in patients with damaged heart valves prior to intestinal or urinary tract manipulation
  113. 113. Gram-negative cocci
  114. 114. GENUS: Neisseria• Gram-negative• Diplococci with flattened sides• Oxidase positive
  115. 115. Important Genera• Neisseria meningitidis• Neisseria gonorrhoeae
  116. 116. NeisseriaSpecies N. meningitidis N. gonorrhoeaeCapsulePiliVaccinePortal of entry Respiratory GenitalGlucose UtilizationMaltose FermentationOxidase testBeta-lactamase prdxn Rare
  117. 117. Neisseria meningitidis(meningococcus)• Distinguishing Characteristics • Gram-negative kidney bean-shaped diplococci • Large capsule • Grows on chocolate (not blood) agar in 5-10% CO2 • Ferments maltose • Oxidase positive • 13 Serogroups: A, B, C, D,29E, H, I, K,L,X,Y,Z & W-135
  118. 118. Reservoir• Human nasopharyngeal area• ≥ 5% carriers (asymptomatic)
  119. 119. Transmission• Respiratory droplets• Oropharyngeal colonization• Spread to the meninges via the bloodstream• Disease occurs in only small percent of colonized
  120. 120. Pathogenesis• Important Virulence Factors • Polysaccharide capsule (most impt) • IgA protease allows oropharynx colonization • Endotoxin (LPS): fever, septic shock in meningococcemia, overproduction of outer membrane • Pili and outer membrane proteins important in ability to colonize and invade • Deficiency in late complement components (C5-8) predisposes to bacteremia
  121. 121. Diseases• Meningitis• Meningococcemia
  122. 122. Waterhouse-Friderichsen Syndrome• Most severe form of meningococcemia• High fever• Shock• DIC• Ecchymoses• Adrenal insufficiency• Coma• Death
  123. 123. Laboratory Diagnosis• Specimen: • Blood  culture only • CSF  smear, culture (tube #2), chemical determination • Petechial aspirate  smear/culture (?)
  124. 124. Laboratory Diagnosis• G/S• Presumptive dx: (+) gram-negative cocci on CSF smear• Presumptive Dx: (+) Oxidase test (tetramethyl-para- phenylenediamine- dihydrochloride)
  125. 125. Laboratory Diagnosis• Culture CAP 5-10% CO2 (candle jar) Incubate at 36-37 C at least 5 days before discarding as negative• Confirmatory test: Carbohydrate Fermentation test• (+) Glucose• (+) Maltose
  126. 126. Treatment• Penicillin G – DOC• Ceftriaxone• β- lactamase production (rare)
  127. 127. Prevention• Vaccine: capsular polysaccharide of strains Y, W-135, C, A
  128. 128. Prophylaxis• Rifampicin• Ciprofloxacin
  129. 129. Neisseria gonorrhoeae• Distinguishing Characteristics • Gram-negative kidney bean-shaped diplococci • Intracellular Gram-negative diplococci in PMNs from urethral smear is suggestive of N.g. • Sensitive to drying and cold
  130. 130. Reservoir• Human genital tract
  131. 131. Transmission• Sexual contact• Birth
  132. 132. Pathogenesis• Pili • Attachment to mucosal surfaces • Inhibit phagocytic uptake • Antigenic (immunogenic) variation • Most impt
  133. 133. Pathogenesis• Outer membrane Proteins • OMP I: Structural, antigen used in serotyping • OPA proteins (opacity): antigenic variation, adherence • IgA protease: aids in colonization and cellular uptake
  134. 134. Disease• Gonorrhea
  135. 135. Laboratory Diagnosis• Specimen • Discharge from the GUT • Discharge from the rectal mucosa • Discharge from the throat/ oropharynx • Skin lesions • Eye/ Conjuntival Discharge • Synovial Fluid
  136. 136. Laboratory Diagnosis• Collection: • Use Non-toxic cotton swabs (treated with charcoal to absorb toxic fatty acid present in the cotton fiber) • Swabs should be plated immediately (best method) or within 6 hours • Specimen from sterile sites requires no special method in transport like synovial fluids in the syringes, they should be transpotred immediately to the laboratory • Blood culture is an exception, N. gonorrheae and N. meningitidis are sensitive to SPS (Sodium Polyanetholsulfate) which is present in vacutainer tubes, if present should < 0.025% • Transport media: • Amie’s charcoal transport medium • Transgrow medium • New York City medium • JEMBEC
  137. 137. Laboratory Diagnosis• G/S & C/S of d/c• Presumptive test – (+) gram-negative intracellular diplococci• Presumptive test – Oxidase test
  138. 138. Laboratory Diagnosis• Culture • Gold-standard in diagnosis • Colonies: translucent, grayish, convex, shiny colonies with entire margin, non-hemolytic • TYPES: • T1 & T2  Small, bright reflective colonies, typical of fresh isolates from gonorrheae (+) fimbrae/pili • T3, T4 & T5  Larger, flatter, non-reflecting (-) fimbrae/pili
  139. 139. Media Used:• Chocolate agar plate (CAP) • Sterile sites• Thayer-Martin Chocolate (T M) medium • Modified medium of CAP • Non-sterile sites • Vancomycin, Colistin, Nystatin• Modified Thayer-Martin (MTM) • T-M + trimetroprim to (-) swarming Proteus• M-Lewis Agar • Same as T-M but instead of Nystatin, Anisomycin is use
  140. 140. Treatment• Ceftriaxone – DOC• Test for Chlamydia trachomatis or treat with tetracycline• Penicillin-binding protein mutations led to gradual increases in penicillin resistance from the 50s to the 70s• Plasmid mediated β lactamase produces high level penicillin resistance
  141. 141. PreventionAdult forms: A B C• Abstinence• Be faithful/careful• Condom
  142. 142. Prevention• Neonatal: • 0.5 % erythromycin ointment • 1.0 % Tetracycline ointment • 1.0 % Silver nitrate drops (Crede’s prophylaxis)
  143. 143. Moraxella catarrhalis• Gram-negative diplococcus (close relative of neisseriae)• Normal upper respiratory flora• Otitis media• Cause bronchitis and bronchopneumonia in elderly with COPD• Drug resistance a problem; most strains produce a β lactamase