CLS03 - Gram Positive Cocci

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  1. Four main genera of gram-positive cocci w/ basic gram stain morphology?
    • Staphylococci: bunches of grapes
    • Micrococci: tetrads
    • Streptococci: string of beads
    • Enterococci: short chains
  2. Ingredients in sheep's blood agar?  What is it used for?
    • Trypticase soy agar w/ 5% sheep's blood
    • Can detect hemolytic activity (streptococcus and staphylococcus)
    • Non-selective nutrient medium
  3. Describe the various forms of hemolysis seen on a BAP w/ examples
    • Beta: complete hemolysis (clear)
    • S. pyogenes (group A Strep) - wide zone
    • S. agalactiae (group B Strep) - narrow zone
    • Alpha: partial hemolysis (green)
    • S. pneumoniae, Viridans, Enterococcus, group D strep
    • Gamma: no hemolysis
  4. Ingredients in Mannitol Salt Agar and their purpose?
    • Peptones/beef extract: growth factors
    • 7.5% NaCl: selective agent for gram +
    • Mannitol: differential agent for fermentation
    • Phenol Red: pH indicator
    • Agar: solidifying agent
  5. What is Mannitol Salt agar used for?  How?
    • Selective/differential medium
    • used for selective isolation of staphylococci
    • S. aureus causes a color change (from red to yellow)
    • Only halophiles can grow (high NaCl)
  6. Ingredients in phenylethyl alcohol agar and their purpose?
    • 5% sheep's blood
    • Peptones (nitrogen, carbon, sulfur, trace nutrients): nutrition
    • NaCl: maintain osmotic equilibrum
    • β-phenylethyl alcohol: selective agent - bacteriostatic for gram-negative!
  7. What is Phenylethyl alcohol used for?  How?  When should it be used?
    • Selective medium
    • supports growth of gram positive bacteria (bacteriostatic for gram negative)
    • Use where large number of gram negatives may be present (eg. a wound culture)
  8. Ingredients in Bile Esculin agar and their purpose?
    • Peptone/beef extract (nitrogen, carbon, sulfure, trace nutrients): nutrition
    • Oxgall (synthetic bile salts): inhibits non-enterococci, gram pos
    • Ferric citrate: reacts with esculetin creating dk brown/black complex
    • Esculin: hydrolyzed by enterococci -> esculetin + dextrose
    • Agar: solidifying agent
  9. What is Bile Esculin agar used for?  How?
    • Differentiates Enterococci from other streptococci
    • Enterococci hydrolyze esculin creating a reaction that causes the medium to blacken
  10. How do you differentiate Staphylococci from Streptococci?
    • The catalase test
    • Catalase positive: Staphylococci (Staphylococcus, Micrococcus)
    • Catalse negative: Streptococcus, Enterococcus)
  11. Describe the catalase test procedure and its results w/ pos and neg control spp.  What is its purpose?
    • Place drop of 3% hydrogen peroxide on small amount of colony and look for immediate bubbles
    • Bubbles: positive for catalase (Staph. spp)
    • No bubbles: negative for catalase (Strep spp)
    • *NOTE - false pos can occur if taken from blood agar
  12. What tests are used to ID spp of Staphylococcus?
    • Coagulase
    • Staphyloside agglutination
    • Antibiotic resistance (novobiocin, bacitracin)
  13. Describe the coagulase tube test theory, procedure, and results.  What is its purpose?  What other methods are there?
    • Theory: Staphylocoagulase is an extracellular enzyme
    • Coagulase-reacting factor (CRF) is thermostable and thombin-like
    • enzyme rxn w/ CRF  converts fibrinogen to fibrin creating visible clot
    • Procedure: Emulsify colonies in .5mL rabbit plasma w/ EDTA (milky)
    • Incubate @ 35C for 4 hours
    • Check for clot formation
    • Staph aureus is the only spp that will be positive
    • New methods: use plasma-coated carrier particles, much more common (eg. staphaurex)
  14. Staph aureus microscopic appearance
    gram positive cocci in grape-like clusters
  15. Staph aureus colony morphology incl. hemolysis, resistance, and ___robic
    • Large colony
    • Creamy to yellow ("buttery")
    • β hemolytic w/ narrow zone
    • non-motile
    • resistant to bacitracin
    • *NOTE - facultative anaerobes (hearty)
  16. Staph aureus diseases caused by infection and by toxins
    • Caused by infection: Wounds
    • Furuncles and carbuncles
    • Folliculitis
    • Impetigo
    • Disseminated disease - bacteremia, endocarditis, pneumonia, osteomyelitis, septic arthritis
    • Caused by toxins: Staphylococcus Scalded Skin Syndrome
    • Food Poisoning (salted meats, custard, potato salad, ice cream)
    • Toxic shock syndrome
  17. Describe all antibiotic resistant S. aureus
    • β lactamase: gives many isolates resistance to beta lactam drugs (penicillin, oxacillin, amoxicillin, cephalosporins)
    • MRSA: IDd by PCR for mecA gene or latex assay for penicllin-binding protein (the gene's product)
    • Vancomycin is drug of choice
    • VISA (vancomycin intermediate S. aureus): reduced resistance to vancomycin 
    • often needs higher dose or multiple drugs
    • VRSA (vanc resistant)
  18. Micrococcus microscopic appearance
    Gram positive cocci in tetrads
  19. Micrococcus colony morphology incl. resistance and ___robic.  Disease?
    • Very small
    • Yellow
    • Smooth colony
    • Susceptible to bacitracin
    • Strict aerobe
    • Normal flora of skin/mucosa/oral pharynx
    • Opportunistic infection
  20. Staph saprophyticus microscopic appearance
    Gram positive cocci in clusters
  21. Staph saprophyticus colony morphology incl. resisistance, disease
    • Large, opaque, creamy/yellow
    • Novobiocin resistance (distinguishes it from S. epidermidis)
    • Normal flora in urogenital tract
    • Causes UTI
    • **NOTE - picture looks like small/white round/raised colonies!!!
  22. Staph epidermidis microscopic appearance
    Gram positive cocci in clusters
  23. Staph epidermidis colony morpholgy incl resistance, disease
    • Small, white, round, raised, opaque
    • Non hemolytic
    • Novobiocin susceptible (distinguishes it from S. saprophyticus)
    • Normal flora of the skin
    • Associated w/ endocarditis, biofilm formation on prosthetics and shunts
  24. Is Staphylococcus lugdunensis an emerging pathogen?
    • According to CDC yes
    • Often misdiagnosed as S. aureus
    • Mixed populations are common and often found as small-colony variants
    • Was found in blood culture after pacemaker battery replacement
    • Pacemaker removed after recurring symptoms could not be stopped
  25. What tests are used to ID spp of Streptococcus?
    • Colony morphology
    • Type of hemolytic patterns on BAP
    • Resistance: to bacitracin (b disc - a disc?) or optochin (p disc)
    • Biochemical characteristics: Bile esculin hydrolysis, PYR test
    • Serology: C carbohydrates (Lancefield class.)
    • Groups (modern class)
    • Capsular polysaccharide (natural resis. to phago)
    • Surface proteins (Strep pyogenes M protein)
    • CAMP test
  26. Describe the PYR test - how does it work, pos/neg controls?
    • Tests for enzyme L-pyrroglutamyl-aminopeptidase which hydrolyzes L-pyrrolidonyl-B-napthylmid (PYR) to make B-napthylamine
    • B-napthylamine is detected in the presence of N,N-methyl-mainocinnamaldehyde reagent
    • PRODUCES A PINK (not orange) COLOR
    • pos control: Enterococcus or S. pyogenes
    • neg control: most other Streptococci
  27. Bile solubility test - used for? principle? pos/neg control? procedure? results?
    • differentiates S. pneumoniae from other α-hemolytic streptococci
    • principle: bile lyses pnemococcal colonies by lower surface tension, accelerating autolytic process
    • pos control: S. pneumoniae
    • neg control: E. faecalis
    • Procedure: culture grown on SBA
    • place 1-2 drops of 10% sodium desoxycholate beside an isolated colony
    • gently wash liquid over colony
    • incubate 30min @ 35C
    • pos - colonies disintegrate
    • neg - colonies remain intact
  28. Bile esculin hydrolysis test - used for? principle? results?
    • Used to test for enterococci
    • Principle: enterococci possess enzyme that hydrolyzes bile
    • procedure: streak 1-2 isolated colonies onto BE slant or BE agar
    • incubate @ 35C up to 45h
    • pos - blackening of agar
    • neg - no color change
  29. CAMP test - used for? principle? controls? procedure? results?
    • Christie Atkins Munch Peterson
    • used to ID group B strep
    • principle: enhanced hemolysis due to interaction between B-lysis of S. aureus and diffusible extracellular protein (CAMP factor) of Group B strep
    • They act synergistically casuing enhanced RBC lysis (arrow)
    • pos control: S. agalactiae
    • neg control: S. pyogenes
    • procedure: inoculate a single streak of B-hemolytic S. aureas on a SBA plate
    • Inoculate a single streak of organism at right angles to S. aureus (NOT TOUCHING)
    • Incubate @ 36C
    • pos - arrow-head shaped zone of B-hemolysis
    • neg - no enhanced zone of hemolysis
  30. Streptococcus pyogenes - alt name, normal location, microscopic appearance, colony morphology
    • Group A strep
    • normal flora of oropharynx
    • micro: Gram pos cocci in chains
    • colony: small, translucent colonies
    • streptolysin S gives large zone B hemolysis
    • streptolysin O gives enhanced hemolysis around stabs into agar (inhibited by O2)
  31. Streptococcus pyogenes - virulence factors
    • Ability to adhere to surface of host cells (M-protein)
    • Invade epithelial cells
    • Avoid opsonization and phagocytosis and produce toxions and enzymes (hyaluronic capsule)
  32. Streptococcus pyogenes - diseases
    • Suppurative: pharyngitis
    • scarlet fever (toxin production)
    • pyoderma
    • cellulitis
    • Erysipelas
    • Necrotizing fasciitis
    • Bactermia
    • Food poisoning (toxin production)
    • Secondary infection in mumps, measles
    • Non-suppurative: rheumatic fever (chronic progressive dmg to heart valve)
    • acute glomerulonephritis
    • **NOTE - diseases result from anti-streptococcal antibodies cross-reacting w/ host tissue (goodpasture's syndrome)
  33. Streptococcus agalaciae - alt name, normal location, microscopic appearance, colony morphology,
    • Group B strep
    • Normal flora of GI and UroGen tract, other mucosa
    • Micro: gram positive cocci in chains
    • Morphology: small, translucent colonies
    • Small zone of B-hemolysis
  34. Streptococcus agalactiae - diseases
    • Neonates: very serious (innoc. during birth)
    • meningitis
    • pneumonia
    • *NOTE - vag swabs collected from preggos to monitor group B strep, treated w/ antibiotics if pos
    • Adults: less serious
    • UTI
    • wound infections
    • soft-tissue infections
    • pneumonia
    • Bacteremia (most common B-hemolytic strep)
  35. Streptococcus pneumoniae - alt name, normal location, microscopic appearance, colony morphology
    • No alt name?
    • Normal flora of the nasopharynx
    • micro: lancet-shaped diplococci
    • colony: a-hemolytic
    • un-encapsulated strains have small colonies, autolysis after 45 hours
    • encapsulated strains have larger colonies, are mucoid and more round
  36. Streptococcus pneumoniae - diseases, vir. factors
    • Primary cause of bacteria pneumonia, meningitis, otitis media
    • bacteremia
    • capsule is primary virulence factor
    • 30% are penicillin resistant
  37. Enterococcus faecalis - alt name, normal location, microscopic appearance, colony morphology
    • Group D (but NOT the only member!!!)
    • normal flora of GI and vaginal tract
    • micro: gram pos cocci in short chains
    • morph: large, greyish w/ sheen
    • non-hemolytic on BAP (gamma)
    • *NOTE - non-enterococcus group D have varying hemolysis
  38. Enterococcus faecalis - diseases
    • UTI
    • Bacteremia
    • Endocarditis
    • Intra-abdominal abscesses (polymicrobial infection)
    • *NOTE - non enterococcus group D cause septicemia
  39. Enterococcus faecalis - antibiotic resistant strains
    • VRE (vancomycin resistant enterococci): first GPC to exhibit vanc resisitance
    • ~20% can be vanc resis
    • Require strong combinations w/ vanc
    • Intriniscally more resisitant to antibiotics than other GPC
    • resistant to cephalosporins/aminoglycoisdes
  40. Viridans strep - microscopic, normal location, morphology, diseases
    • Gram pos cocci in pairs and chains
    • Normal flora of the respiratory, GI, and urogen tracts
    • Small, a-hemolytic colonies ("think green")
    • diseases: dental carries, intra-abdominal abscesses, subacute endocarditis
  41. Abiotrophia - alt name, colony morphology, diseases
    • Previously viridans strep
    • difficult to culture
    • grows as satellite colonies around others
    • associated w/ speticemia, endocarditis
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CLS03 - Gram Positive Cocci
2016-03-14 06:51:03
CLS03 Gram Positive Cocci
CLS03 - Gram Positive Cocci
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