GRAM POSITIVE

• Gram-Positive Cocci


  Bacteria that are purple in Gram stain and have a spherical shape

  
• Learning objectives


  • Review basic concepts on bacterial staining and morphology
  • Review Gram-positive bacteria
  • Active recall on bacterial characteristics

  
• Staphylococcus species


  • Facultative anaerobes except S. saccharolyticus
  • Non-motile
  • Non-spore forming
  • Normal flora on human skin & mucosal surface
  • Purple in color
  • Forms usually in clusters
  • Grows on most bacteriologic media

  
• Blood Agar Plate (BAP)


  • Beef heart infusion with 5% sheep blood
  • Cultivation of fastidious organism
  • Determines hemolytic pattern- lysis of red blood cells
• Beta hemolysis


  Complete clearing of RBCs around bacterial colony

  
• Alpha hemolysis

  Partial lysis of cells which gives a greenish discoloration around bacterial colony

  
• Gamma hemolysis


  No discoloration or halo around colony
• Staphylococcus aureus


  • Gram Positive bacteria in clusters
  • Normal flora on skin & mucosal surface
  • Catalase positive
  • True coagulase positive

  
• Staphylococcus aureus


  • Gold/ light gold colonies due to "Staphyloxanthin" on BAP
  • Beta-hemolytic due to "Alpha-hemolysin"

  
• Virulence Factors of S. aureus

  • Coagulase
  • Protein A
  • Alpha toxin
  • Beta toxin
  • Delta toxin
  • Gamma toxin
  • Pyrogenic exotoxin C
  • Leukocidin
  • Exfoliatin toxin
  • Polysaccharide microcapsule
• Disease Association of S. aureus

  • Boil, abscesses, impetigo, wound infection, Scalded Skin Syndrome
  • Necrotizing fasciitis, cellulitis
  • Osteomyelitis
  • Septic arthritis
  • Bacteremia, septic thrombophlebitis
  • Toxic shock syndrome
  • Pneumonia
  • Brain abscess, meningitis
  • UTI
  • Food poisoning
• Staphylococcus epidermidis


  • Gram positive cocci in clusters
  • Less virulent than S. aureus
  • Opportunistic pathogens
  • Normal flora on skin & mucosal surface
  • Transmission through direct contact & autoinfection
  • Creamy or white; non-hemolytic on BAP
  • Slime-producing due to glycocalyx
  • Novobiocin- Susceptible

  
• Exopolysaccharide (Glycocalyx)


  Forms biofilms that helps bacteria to adhere to surfaces and to each other which also serves as a protective mechanism
• Disease Association of S. epidermidis

  • Nosocomial infection
  • Prosthetic valve endocarditis
  • Septic arthritis in prosthetic joints
  • Ventriculoperitoneal shunt infections
• Staphylococcus saprophyticus


  • Less virulent than S. aureus
  • Opportunistic pathogens
  • Normal flora on skin & mucosal surface
  • White, non-hemolytic on BAP
  • Novobiocin -Resistant
  • 2nd most common cause of UTI in sexually active women
• Novobiocin Test

  • Principle: Novobiocin interferes with DNA synthesis process which inhibits bacterial replication and colony formation
  • Interpretation: Resistant - Zone of inhibition less than 16mm (S. saprophyticus), Susceptible - Zone of inhibition greater than 16mm
• Virulence Factors of S. saprophyticus

  • Exopolysaccharide (Glycocalyx)
  • Surface proteins
  • Enzymes
• Other Staphylococcus spp. (S. haemolyticus, S. lugdunensis) are less virulent than S. aureus and are opportunistic pathogens causing skin, wound, and deep tissue infections

  
• 1. zone Test is used to primarily detect inducible clindamycin resistance in Staphylococcus aureus and Streptococcus spp.

  
• Micrococcus spp.


  • Normal flora on human skin & mucosal surface
  • Extremely low virulence
  • Usual contaminant in clinical specimen
  • Catalase positive, Coagulase negative
  • Appear as tetrads

  
• Differences between Staphylococcus spp. and Micrococcus spp.

  • Bacitracin/ Taxo A Disk Test on BAP & MHA: Staphylococcus spp. Resistant, Micrococcus Susceptible
  • Furazolidone susceptibility Test: Staphylococcus spp. Susceptible, Micrococcus Resistant
  • Lysostaphin sensitivity Test: Staphylococcus spp. Susceptible, Micrococcus Resistant
  • Microdase/ Modified oxidase test: Staphylococcus spp. (+) blue color, Micrococcus (-)

  
• Streptococcus spp.


  • Facultative anaerobes
  • Non-motile
  • Non-spore forming
  • Normal flora on human skin & mucosal surface
  • Purple in color
  • Forms usually in chains or pairs
  • Usually a normal flora unless point of entry of sterile site is accessed

  
• Streptococcus spp. on BAP

  Grayish, pinpoint, translucent to slightly opaque with others having mucoid colonies

  
• Streptococcus spp. Biochemical test


  (-) catalase, (-) oxidase, (-) gas production

  
• Streptococcus spp. Classification


  • Beta Hemolytic: Group A (S. pyogenes), Group B (S. agalactiae), Group C (S. dysagalactiae subsp. equisimilis, S. equi)
  • Alpha or Gamma Hemolytic: Group D (S. gallolyticus), Enterococci (formerly streptococci)
  • Alpha, Beta, or Gamma Hemolytic: Viridans streptococci
  • Alpha Hemolytic: S. pnuemoniae

  
• Streptococcus pyogenes


  • Group A Streptococci
  • Not considered a normal flora; pathogenic to humans
  • Primary site of infection: Upper respiratory tract & skin lesions
  • Beta hemolytic on BAP (large zone of hemolysis)
  • Culture: small, translucent, smooth colonies
  • Susceptible to Bacitracin

  
• Streptococcus pyogenes is resistant to drying and may be recovered from swabs several hours after drying. It may be present in the skin, upper respiratory tract of humans, or carried on nasal, pharyngeal, and or anal mucosa.

  
• Virulence Factors of S. pyogenes

  • M protein
  • Streptolysin O
  • Streptolysin S
  • Pyrogenic exotoxins
  • Streptokinase
  • Hyaluronidase
  • DNase
  • C5a peptidase
• Disease Association of S. pyogenes

  • Pharyngitis/ Tonsilitis (Strep Throat)
  • Scarlet Fever
  • Rheumatic Fever
  • Impetigo
  • Bright's Disease
  • Streptococcal Toxic Shock Syndrome (Strep TSS)
• Pyrrolidonyl arylamidase test (PYR) Test


  Detects the presence of PYR enzyme in bacteria, only S. pyogenes in beta hemolytic Streptococcus will be positive

  
• S. pyogenes


  • Disease association
• Diseases associated with S. pyogenes

  • Impetigo
  • Bright's Disease
  • Streptococcal Toxic Shock Syndrome (Strep TSS)
• Impetigo


  May lead to necrotizing fasciitis, "Galloping gangrene", "Flesh-eating bacteria"
• Bright's Disease


  Acute glomerulonephritis, mainly due to deposition of immune complexes in kidney which triggers an immune response leading to activation of immune complexes
• Streptococcal Toxic Shock Syndrome (Strep TSS)


  Due to excessive cytokine release, which lead to systemic inflammatory response, causing widespread tissue damage & organ dysfunction
• S. pyogenes - Tests


  • Pyrrolidonyl arylamidase test (PYR) Test: Detects the presence of PYR enzyme in bacteria, only S. pyogenes in beta hemolytic Streptococcus spp. is (+) (Cherry red color)
  • Bacitracin Disk Test: Differentiates S. pyogenes from other beta hemolytic streptococcus, (+) = any zone of inhibition

  
• Streptococcus agalactiae


  • Group B Streptococci, Normal flora in female genital tract & lower GIT, Causes infection of fetus during passage in labor, Beta hemolytic on BAP (small zone of hemolysis), Culture BAP: grayish-white & mucoid, Resistant to Bacitracin
• CAMP factor


  Produced by S. agalactiae, enhances hemolytic activity of beta hemolysin produced by S. aureus
• Diseases caused by S. agalactiae

  • Neonatal meningitis, Post-partum infection, UTI, endocarditis, pneumonia, osteomyelitis
• S. agalactiae - Tests

  • CAMP Test, Hippurate hydrolysis test, Carrot broth (Swab from pregnant; orange color), Granada agar (orange colonies), Lim broth (enrichment broth for anovaginal specimen)