Staphylococcus Spp.

• Staphylococcus


  • Catalase (+)
  • Coagulase (+) & some are Coagulase (-) -CoNS (Coagulase Negative Staphylococcus)
  • Gram (+) cocci in singly, in pairs, and in grape-like clusters

  
• Micrococcus


  • Catalase (+)
  • Coagulase (-)
  • Gram (+) cocci in pairs, tetrads, and ultimately, irregular clusters
  • Found in environment and human skin
  • Colonies: yellow pigment

  
• Gram positive cocci


  • Staphylococcus – Catalase (+)
  • Micrococcus – Catalase (+)
  • Streptococcus

  
• Tests to differentiate Staphylococci and Micrococci

  • Modified oxidase (Microdase)
  • Anaerobic acid production from glucose
  • Anaerobic acid production form glycerol in the presence of erythromycin
  • Growth on Furoxone-Tween 80-oil red O agar
  • Resistance to bacitracin (0.04 U)
  • Resistance lysosome (50-mg disk)
  • Resistance to lysostaphin (200 ug/mL)
• Staphylococcus colonies


  • Medium sized (4 to 8mm) and appear cream-colored, white, or rarely light gold, and "buttery-looking"

  
• Small Colony Variants (SCV)

  Fastidious strains of staphylococci that are able to grow on media containing blood within 48 hrs of incubation
• Tests to differentiate Staphylococci, Micrococci, Rothia, Aerococcus, and Enterococcus

  • Catalase
  • Modified oxidase
  • Aerotolerance
  • Resistance to bacitracin (0.04 U)
  • Resistance to furazolidone (100 ug)
  • Resistance to lysostaphin (200 ug/mL)

  
• Staphylococcus aureus


  • Virulence factors: Enterotoxins, Toxic Shock Syndrome Toxin-1, Exfoliative Toxin, Cytolytic Toxin/Cytotoxin, Enzymes (Coagulase, protease, hyaluronidase & lipase), Protein A, Peptidoglycan, Teichoic acid

  
• Staphylococcus aureus virulence factors

  • Enterotoxins
  • Toxic Shock Syndrome Toxin-1
  • Exfoliative Toxin
  • Cytolytic Toxin/Cytotoxin
  • Enzymes (Coagulase, protease, hyaluronidase & lipase)
  • Protein A
  • Peptidoglycan
  • Teichoic acid

  
• Enterotoxins


  Resistant to hydrolysis by the gastric and intestinal enzymes, Groups A-E and G-J, Cause staphylococcal food poisoning (A-E) and Toxic Shock Syndrome (B, C, G, I), Heat stable exotoxins (100°C for 30 mins)
• Toxic Shock Syndrome Toxin-1


  Cause systemic effects (fever, hypotension, desquamation) – lead to shock and death
• Exfoliative Toxin


  Epidermolytic toxin A & B, Cause Staphylococcal Scalded Skin Syndrome (SSS) and Bullous impetigo, Able to cause epidermal layer of skin to slough off
• α-hemolysin


  Disrupts the smooth muscle in blood vessels and is toxic to erythrocytes, leukocytes, hepatocytes, and platelets, Able to lyse RBC, Can damage the platelet and macrophages, Cause severe tissue damage
• β-hemolysin


  Also known as Sphingomyelinase C or "hot-cold lysin", Acts on sphingomyelin in the plasma membrane of RBCs, Has enhanced hemolytic activity on incubation at 37'C and subsequent exposure to cold (4'C)
• δ-hemolysin


  Less toxic than α-hemolysin or β-hemolysin, Found in CoNS (S. aureus, S. epidermidis, & S. haemolyticus)
• γ-hemolysin


  Functions with (PVL) Panton-Valentine leukocidin
• Enzymes produced by Staphylococcus

  • Coagulase
  • Protease
  • Hyaluronidase
  • Lipase
• Staphylocoagualase


  Responsible for positive tube coagulase test, Able to produce coagulation or clump/clot formation, Present for S. intermedius, S. pseudointermedius, S. hycius, S. delphini, S. lutrae, S. agnetis, and some S. schleiferi
• Hyaluronidase


  Hydrolyzes hyaluronic acid present in the intracellular ground substance that makes up connective tissues, Permits bacteria to spread to through connective tissues
• Lipase


  Act on lipids present on the surface of the skin, Degrades lipids on the skin making it more susceptible to bacterial entry into epidermal layers
• Protein A


  Found in cell wall, 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 (polymorphonuclear leukocytes), Interleukin 1 – endogenous pyrogen (Induces fever)
• Teichoic acid


  Important in cell wall metabolism, Mediate binding to fibronectin
• Specimen collection


  1. From the site of infection with aseptic technique

  2. No special procedures – usually cutaneous (within the surface of the skin)

  3. Aspirates: ideal sample

  4. Swabs: less satisfactory for both culture and smear results
• Direct Microscopic Examination


  1. Perform gram staining

  2. Gram (+) cocci with PMN cells
• Culture


  1. BAP, CAP, MSA, CNA, PEA, CHROMagar Staph aureus

  2. 18 to 24 hours of incubation at 35°C to 37°C

  3. For general culture media: BAP, CAP, or NA

  4. For selective media: MSA, CNA, PEA, CHROMagar
• MSA


  High concentration of salt – S. aureus are halophilic; 7.5 to 10% of salt, If mannitol fermenter – yellow colonies, If non-mannitol fermenter – pink colonies
• CNA & PEA


  Gram (+) organism; reduce contamination from gram (-); especially if from stool
• CHROMagar


  For identification of Methicillin-Resistant Staphylococcus aureus
• Macroscopic examination – growth of colony

  Colonies: S. aureus - round, smooth, white, creamy colonies on BAP, S. epidermidis - small to medium sized, nonhemolytic (gamma hemolytic), gray-to-white colonies
• Microscopic examination – gram stain

  Gram (+) cocci in singly, in pairs, and in grape like clusters
• Catalase test


  Principle: catalase mediates the breakdown of hydrogen peroxide (30% H2O2) into oxygen and water ---> produce BUBBLES, Result: (+) bubble formation (Staphylococcus spp. & Micrococcus spp.), (-) no or few bubble formation (Streptococcus spp.), Causes of False Positive Reaction: Peroxidase (enzymes), Sample contaminated from blood agar
• Microdase Test/Modified Oxidase Test


  To differentiate Staphylococcus from Micrococcus

  
• Staphylococcus species and their infections

  • S. aureus - Cutaneous infections, Food poisoning, Scalded skin syndrome, Toxic shock syndrome, Toxic epidermal necrolysis
  • S. epidermidis - Nosocomial infections
  • S. haemolyticus - Septicemia, UTIs, Native valve infections
  • S. lugdunensis - Catheter-related bacteremia and endocarditis
  • S. saprophyticus - UTI
  • Micrococcus spp. - Rarely implicated in infections
  • Kocuria spp., Kytococcus spp.

  
• Catalase test


  1. Get a colony on a glass slide

  2. Add 1 drop of 30% H2O2 or 3% H2O2

  3. Observe bubble formation
• Catalase positive


  Bubble formation (Staphylococcus spp. & Micrococcus spp.)
• Peroxidase


  Some organisms as Enterococci (under Streptococci) produces peroxidase which slowly catalyzes the breakdown of hydrogen peroxide to water and oxygen, producing a weak positive result - small number of bubbles or late reaction
• Sample contaminated from blood agar

  Pseudoperoxidase activity- formation of bubbles
• Microdase test/Modified oxidase test result

  Positive (blue to purple-blue color) for Micrococcus spp., Negative (no color change) for Staphylococcus spp.

  
• Bacitracin test


  Incubate for 18 to 24 hours and observe the zone of inhibition (measure the diameter)