Mod 6

• Staphylococcaceae


  Family of Gram-positive cocci
• Micrococcaceae


  Family of Gram-positive cocci
• Most Important Human Pathogens

  • Staphylococcus aureus (skin and soft tissue infections, bacteremia, toxic shock syndrome)

  Streptococcus pyogenes (pharyngitis, skin and soft tissue infections)

  Streptococcus agalactiae (neonatal bacteremia and meningitis)

  Streptococcus pneumoniae (CAP, meningitis)

  Enterococcus faecalis and E. faecium (nosocomial UTIs and bacteremia)
• Family Staphyllococcaceae


  • Staphylococcus

  Macrococcus

  Gemella

  Salinicoccus
• Staphylococcus


  Greek words: staphule (bunches of grapes) and kokkos (berries)

  General Characteristics:

  1. Catalase-producing / Catalase (+)

  2. Aerobic or facultative anaerobic

  3. Nonmotile, non-spore-forming glucose fermenters

  Normal inhabitants of the skin, mucous membranes, and intestines
• Staphylococcus aureus


  • Most virulent species of staphylococci

  Halophilic

  Causes infection when it enters a normally sterile site
• Clinical Manifestations of S. aureus

  • Skin infections (impetigo, boils, folliculitis, mastitis, and infection of surgical wounds)

  2. Most common cause of septic arthritis in prepubertal children

  3. Among the leading causes of bacteremia in hospitalized patients (may cause endocarditis)

  4. Most common cause of spinal epidural abscess and suppurative intracranial phlebitis

  5. Toxin-mediated diseases (e.g. scalded skin syndrome and toxic shock syndrome)

  6. Others: food poisoning, UTI, osteomyelitis
• Pathogenic Determinants of S. aureus

  • Staphylokinase (enables infection once fibrin clot is dissolved)

  Coagulase (converts fibrinogen to fibrin; coats PMN with fibrin to inhibit phagocytosis)

  Hyaluronidase (spreading factor: dissolves hyaluronic acid in connective tissues)

  Lipase (hydrolyze lipids in plasma and skin; responsible for skin infections)

  Exfoliatin (aka epidermolytic toxin; responsible for scalded skin syndrome)

  Protein A (binds to Fc portion of IgG; inhibits phagocytosis; involved in coagglutination)

  Toxic shock syndrome toxin (TSST-1) (aka Enterotoxin F; causes toxic shock syndrome)

  Enterotoxins A–E (food poisoning)

  Hemolysin (lyse RBCs; causes anemia and makes iron available for bacteria)

  Leukocidin (aka Panton-Valentine leukocidin (PVL); lyse neutrophils and macrophages)
• Methicillin-resistant S. aureus (MRSA)

  S. aureus strain resistant to antibiotics such as methicillin, nafcillin, and oxacillin

  Acquired after prolonged stay in the hospital, close contact with carriers, broad-spectrum antibiotics

  In the community, mostly skin infections

  In healthcare settings usually more severe and life-threatening (bloodstream infections, surgical site infections, or pneumonia)
• CHROM agar test: changes in the color of MRSA colonies within 24-48 hours against colorless colonies of non-MRSA
• Novobiocin Susceptible Coagulase-Negative Staphylococci (CoNS)

  • S. epidermidis, S. capitis, S. haemolyticus, S. hominis subsp. hominis, S. lugdunensis, S. saccharolyticus, S. warneri, etc.
• Novobiocin Resistant Coagulase-Negative Staphylococci (CoNS)

  • S. cohnii, S. kloosii, S. saprophyticus, and S. xylosus
• Staphylococcus epidermidis


  Resistant flora of the skin/mucous membrane

  Blood culture contaminant

  Opportunistic pathogen

  Distinct predilection for foreign bodies (artificial heart valves, CNS shunts, IV catheters)
• Staphylococcus saprophyticus


  Normal flora of skin and urethra

  Common cause of UTI in young sexually active women

  2nd most common cause of UTI in young women

  Low urine colony count is considered significant (<100,00 Urine CFU/mL)

  
• Micrococcaceae


  Family includes Micrococcus, Arthrobacteria, Kocuria

  Occur worldwide and are ubiquitous

  Indigenous skin biota, often recovered with staphylococci

  Rarely infectious
• Micrococcus spp.


  Grows on 5% NaCl but not on 7.5% NaCl

  Opportunistic pathogens

  Bacteremia, continuous ambulatory peritoneal dialysis peritonitis, and infections associated with ventricular shunts and CVC
• Lab Diagnosis for Staphylococcus

  Gram stain

  Culture (specimen, incubation conditions, culture media)

  Catalase test

  Coagulase test (slide, tube)

  DNAse test

  Novobiocin susceptibility test

  Polymyxin B susceptibility test

  Modified Oxidase test
• Schematic Diagram for ID of Staphylococci
• Incubation


  1. Incubate for 4 hours @ 35°C

  2. If (-) for 4 hours incubate for another 24 hours @RT
• Coagulase-positive Staphylococcus species


  • S. hyicus
  • S. intermedius
  • S. lutrae
  • S. delphini
  • S. schleiferi subsp. coagulans
• DNAse Test


  Determines if an organism can produce DNAse (hydrolyzes DNA)
• DNAse Test Reagents


  • DNAse agar with methyl green/ toluidine blue as indicator
• Positive DNAse Test


  Clearing of media (methyl green)/ blue into rose pink (toluidine blue)
• Organisms with positive DNAse test


  • S. aureus
  • Moraxella
  • Serratia
• Novobiocin Susceptibility Test


  Differentiates S. epidermidis (susceptible) from S. saprophyticus (resistant)
• Novobiocin Susceptibility Test Reagents

  • 5 ug Novobiocin and sheep BAP
• Positive Novobiocin Susceptibility Test


  ZOI >16mm
• Polymyxin B Susceptibility Test

  S. aureus: resistant, other staph: sensitive
• Staphylococcus saprophyticus (top) is resistant to novobiocin, indicated by the lack of a zone of inhibition around the disk

  
• Modified Oxidase Test


  Rapid method to differentiate Staphylococcus (-) from Micrococcus (+)

  
• Modified Oxidase Test Procedure


  • 18- to 24-hour-old culture is smeared on the disk
  • Reagent: tetramethyl-p-phenylenediamine dihydrochloride in dimethyl sulfoxide
• Positive Modified Oxidase Test


  Blue color
• Oxidation Fermentation Test


  Determines if an organism is an oxidizer or fermenter
• Oxidation Fermentation Test Procedure


  • Glucose in OF tubes
  • Open tube
  • Closed tube sealed with vaspar, vasellin and paraffin
  • Indicator is bromthymol blue
• Fermenter


  Ferments glucose with or without O2<|>Glucose is utilized in open and closed tubes<|>Media turns yellow
• Oxidizer


  Requires O2 to ferment sugar<|>Ferments glucose in open tube only<|>Media remains green in closed tube
• Tests to Differentiate Staphylococcus from Micrococcus

  • Growth (Facultative anaerobe vs Obligate aerobe)
  • Lysostaphin (S vs R)
  • Furazolidone (S vs R)
  • Bacitracin (R vs S)
  • Modified oxidase (- vs +)
  • Glucose utilization (Fermenter vs Oxidizer)

  
• Characteristics of S. aureus, S. epidermidis, and S. saprophyticus

  • Catalase test (+)
  • Coagulase test (+, -, -)
  • Colonies on BAP (Gold-yellow, White, White to yellow)
  • Hemolysis on BA (Beta, None, None)
  • Novobiocin (Sensitive, Sensitive, Resistant)
  • Mannitol fermentation (+, -, +)
• Streptococcaceae


  Family to which Streptococcus belongs

  
• Streptococcaceae


  • Not highly pathogenic
  • Frequent cause of nosocomial infections
  • Can cause UTI, endocarditis, wound infection