Staphylococci

• Focus in the introduction of MLS in Diagnostic Bacteriology

  • Characteristics
  • Significance
  • Laboratory identification
  • Anti-susceptibility testing
• Workflow in Bacterial Identification

  1. Specimen Collection

  2. Direct Microscopic Examination

  3. Culture

  4. Bacterial Identification

  5. Biochemical Test

  6. Anti-Susceptibility Testing
• Staphylococcaceae


  Family's name
• Staphylococcus spp.


  • Catalase (+)
  • Coagulase (+) and some are Coagulase (-)
  • Gram (+) [purple] cocci in singly, in pairs, and in grape-like clusters
• Micrococcaceae


  Genus Micrococcus spp.
• Micrococcus spp.


  • Catalase (+)
  • Coagulase (-)
  • Gram (+) cocci in pairs, tetrads, and ultimately, irregular clusters
  • Found in environment and human skin
  • Colonies: yellow pigment
• Micrococcaceae and Staphylococcaceae are not in the same family and are in different orders
• Other normal flora that resembles staphylococcus

  • Kocuria spp.
  • Kytococcus spp.

  
• Bacteria that resemble staphylococci in terms of characteristics

  • Rothia mucilaginosa
  • Aerococcus spp.
  • Alloiococcus otitis

  
• Modified oxidase (Microdase)


  Negative (-) for Staphylococci, Positive (+) for Micrococci
• Anaerobic acid production [fermentation] from glucose

  Positive (+) for Staphylococci, Negative (-) for Micrococci (except M. kristinae and M. varians)
• Growth on Furoxone-Tween 80-oil red O agar

  Negative (-) for Staphylococci [no growth; inhibited by furoxone], Positive (+) for Micrococci [not inhibited by furoxone]
• Anaerobic acid production from glycerol in the presence of erythromycin

  Positive (+) for Staphylococci, Negative (-) for Micrococci
• Resistance to bacitracin (0.04 U)


  Resistance+ for Staphylococci, Sensitive for Micrococci
• Resistance to Lysosome (50-mg disk)


  Resistance for Staphylococci, Sensitive for Micrococci
• Resistance to Lysostaphin (200 ug/mL)


  Sensitive+ for Staphylococci, Resistance for Micrococci
• Micrococci


  Obligate Aerobe - Oxidation; requires oxygen to grow and for metabolism; use glucose with the presence of oxygen
• Staphylococci


  Facultative Anaerobe - Fermentation; use organic compounds
• Disk Diffusion Method


  1. Use MHA or Blood Agar Plate

  2. Culture organism entirely to cover the entire media

  3. Place antibiotic disk on top of the culture

  4. Incubate for 18-24 hours

  5. Measure the zone of inhibition to interpret as susceptible/sensitive (>10mm) or resistant (-)
• Not all staphylococcus are Microdase negative. The following bacteria are staphylococcus that are microdase positive: S. sciuri, Macrococcus caseolyticus, S. lentus, S. vitulus

  
• Staphylococcus spp.


  • Nonmotile, non–spore-forming, and aerobic or facultatively anaerobic except for S. saccharolyticus and S. aureus subsp. Anaerobius
  • Normal flora of skin and mucous membranes of humans and animals
  • Colonies: medium-sized (4-to-8-millimeter [mm]) and appear cream-colored, white, or rarely light gold, and "buttery looking" [butyrous]
  • Slime layer: glycocalyx (Staphylococcus aureus and Staphylococcus epidermidis)
  • Fastidious strains requirements: CO2, hemin [x-factor], or menadione with at least 48 hours of incubation

  
• Cutaneous infections caused by Staphylococcus

  • Folliculitis
  • Furuncles
  • Carbuncles
  • Bullous impetigo
• Staphylococcus aureus Virulence Factors

  • Enterotoxins (Groups A-E and G-J)
  • Toxic Shock Syndrome Toxin-1
  • Exfoliative Toxin (Epidermolytic toxin A & B)
  • Cytolytic Toxin or Cytotoxin (Hemolysins and Leukocidins)
  • Enzymes (Coagulase, protease, hyaluronidase, and lipase)
  • Protein A
  • Peptidoglycan
  • Teichoic Acid
• Coagulase (+) and CoNS


  • Act on lipids present on the surface of the skin
• Protein A


  • Found in cell wall of the S. aureus
  • Bind the Fc portion of immunoglobulin G (IgG) and complement
  • Block phagocytosis and inhibit action of IgG
• Peptidoglycan


  Activates complement, iL-1, chemotactic to PMNs
• Teichoic Acid


  Mediate binding to fibronectin
• STAPHYLOCOCCUS AUREUS WORKFLOW


  1. SPECIMEN COLLECTION

  2. DIRECT MICROSCOPIC EXAMINATION

  3. CULTURE

  4. MACROSCOPIC EXAMINATION

  5. MICROSCOPIC EXAMINATION

  6. BIOCHEMICAL TESTS

  
• Catalase


  • Principle: catalase mediates the breakdown of hydrogen peroxide (30% H2O2) into oxygen and water
  • Result: (+) bubble formation [Staphylococcus spp. and Micrococcus spp.], (-) no or few bubble formation [Streptococcus spp. & Enterococcus spp.]
• Microdase Test [Modified Oxidase Test]

  • Principle: oxidase enzyme reacts with the oxidase reagent and cytochrome C to form the colored compound, indophenol after 2 minutes of exposure or incubation
  • Result: (+) development of blue to purple-blue color [Micrococcus spp.], (-) no color change [Staphylococcus spp.]
• Bacitracin Test


  • Principle: determine the effect of a small amount of bacitracin (0.04U) on an organism
  • Result: (+) presence of zone of inhibition around the disk [Staphylococcus spp.], (-) no zone of inhibition
• Oxidation-Fermentation (O/F) Reactions


  • Principle: determine whether an organism uses carbohydrate substrates to produce acid byproducts using OF glucose medium
  • Result: (+) Staphylococcus spp. [+/+], (-) Micrococcus spp. except M. kristinae & M. [-/-]
  • Fermentation: GLUCOSE → [glycolysis] PYRUVIC ACID; end product: single acid (homolactic acid fermenters) or mixed acids (lactic acid, propionic acid and succinic acid)
  • Oxidation: GLUCOSE → [glycolysis] PYRUVIC ACID → CO2, Requires oxygen (aerobic respiration) or Inorganic molecule (anaerobic respiration)
• Coagulase Test


  • Principle: used to differentiate Staphylococci aureus from CoNS by detecting enzyme coagulase
  • Result: (+) fibrin clot [staphylococcus], (-) no fibrin clot [CoNS]
  • 2 Forms of Coagulase Enzyme: Slide Coagulase Test [bound coagulase/clumping factor], Free Coagulase or Staphylocoagulase [confirmatory]
• Pyrrolidonyl Arylamidase (PYR) Test


  • Principle: differentiate S. aureus from other CoNS by the presence of the enzyme L-pyrroglutamylaminopeptidase
  • Result: (+) Bright red color [S. lugdunensis, S. intermedius, S. schleiferi], (-) No color change or an orange color [S. aureus]
• Vogue-Proskauer (VP) Test


  • Principle: determine the ability of some organisms to produce neutral end products from glucose fermentation
  • Result: (+) Red color [S. aureus], (-) Yellow color [S. intermedius, S. lugunensis, S. haemolyticus, S. schleiferi]
• DNA Hydrolysis


  • Principle: determine the ability of an organism to hydrolyze DNA based on the production of deoxyribonuclease
  • Result: (+) clear/colorless [S. aureus], (-) green
• Novobiocin Susceptibility Test


  • Principle: determine the effect of a 5-g novobiocin disk on an organism
  • Result: (+) presence of zone of inhibition around the disk [susceptible: S. epidermidis, S. capitis, S. haemolyticus, S. hominis subsp. Hominis, S. lugdunensis, S. saccharolyticus, S. warneri], (-) no zone of inhibition [resistant: S. cohnii, S. kloosii, S. saprophyticus, S. xylosus]
• MRSA [METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS]

  • Methicillin – group of antibiotic, example: penicillin
  • Penicillin-resistant (-lactam) strains of S. aureus
  • Requires penicillin-resistant penicillin, such as nafcillin, oxacillin, or cefoxitin
  • Types: CA-MRSA, HA-MRSA, HACO-MRSA, MRSE
  • Treatment of Choice: Vancomycin
  • Gene: MecA gene, better inducer is cefoxitin
• MACROLIDE RESISTANCE


  • Resistance to clindamycin
  • Modified double disk diffusion test (D-zone test): use erythromycin [15 ug] to induce the resistance to clindamycin [2 ug], 15 mm apart
  • Gene: erm gene
• GENERAL CHARACTERISTICS OF STAPHYLOCOCCUS

  1 Gram positive arranged in tetrads or clusters

  2 Facultative anaerobes

  3 Medium-sized, raised creamy colonies on Blood Agar

  4 Catalase positive

  5 Oxidase negative

  6 Non-motile

  7 Non-spore forming

  8 Reduces nitrates to nitrites

  9 Grows in 7.5 to 10% NaCl

  10 Bacitracin resistant