Infective Organisms

Type

  • Staphylococcus epidermis & Staphylococcus aureus 50-70%
  • Other Gram positive 25%
    • Streptococcus viridans
  • Gram negative & facultative bugs 10-20%
    • E Coli, Proteus
    • Pseudomonas, Salmonella
  • Single Organism 75%
    • Mixed 25%
  • Increased incidence of resistant organisms
    • MRSA, MRSE & VREC
  • Gram negative becoming more common

Cell Structure

  • Cell Wall
    • Gram positive & Gram negative different
      • Gram positive
        • 3 layers
    • Gram negative
      • Complex multi-layered structure
    • Function
      • Maintains cell shape
      • Corsets cell as high internal osmotic pressure
      • Cell wall injury » Lysis
  • Layers
    • Cytoplasmic Membrane
      • Innermost layer
      • Present in both Gram positive & Gram negative
      • Functions
        • Permeability barrier
        • Transport system
    • Cell Wall
      • Intervening layer
      • Gives osmotic protection
      • Is difference between Gram positive & Gram negative
        • In Gram positive
          • Thick Peptidoglycan Layer
        • In Gram negative
          • Thin Peptidoglycan Layer
          • Lipoprotein
          • Outer membrane
          • Lipopolysaccharide
            • Hence Gram negative
    • Capsule & Glycocalyx
      • Extracellular Polymer
      • If forms condensed, well-defined layer, called Capsule
      • If forms loose meshwork of fibrils, called Glycocalyx (Slime)
  • Slime
    • Exo-polysaccharide Glycocalyx
    • Envelopes bugs infecting a prosthesis
      • Staphylococcus aureus & epidermis
      • Pseudomonas
    • Protects from host-defense factors
      • Complement fixation
      • Neutrophil ingestion
    • Worst infections
    • 500x more resistant
    • Enables the bacteria
      • Adhere to surfaces
      • Survival on synthetic surfaces
      • Avoid destruction

Concepts

“Race for Surface”

  • Protective host cells race to beat bugs to coat prosthesis on implantation

“Window of Opportunity”

  • Theoretical period when can kill bugs before biofilm forms
  • Basis behind Acute Early TJR infection regimes
  • Time frame unknown ? 2/52

Spores

  • Gram positive rods, Bacillus & Clostridium
  • Occurs in unfavourable conditions
  • Resistant to drying/ antiseptic
  • Only killed by Betadine
  • Killing spores (Sterilization) different from Disinfection

Staining

Gram stain

  • Difference is in cell wall
  • Reason unclear
  • Sequence
    • Crystal Violet & Iodine – 30s » All cells purple
    • Alcohol & Acetone – 30s » G pos purple & G neg colourless
    • Dilute Carbol-Fuschin – 30s » G pos Purple & G neg Pink
  • Process
    • Crystal Violet & then Iodine applied & each washed off
      • All bugs are purple at this point
    • Then treatment with alcohol & acetone
      • Gram positive retain crystal Violet-Iodine complex (purple)
      • Gram negative cells completely decolorized
    • Saffron counterstain applied
      • Gram negative cells take on contrasting Red
      • Gram positive cells Purple

Acid-Fast Stain

  • Ziehl-Neilsen
    • Acid-Fast bacteria retain Carbol-Fuschin stain
      • Related to lipids in cell wall
    • Even when decolorized with acid-alcohol
    • Most commonly Mycobacterium
    • Process
      • Red Carbol-Fuschin applied
      • Heated on steam bath
      • Decolorized with hydrochloric acid in alcohol
      • Contrasting blue counterstain applied
      • Acid-fast bacteria appear red
      • Others are blue

Antimicrobial Treatment

  • Four Basic MOA
    • Inhibition of Cell wall Synthesis
    • Inhibition of Cell membrane Function
    • Inhibition of Protein Synthesis
    • Inhibition of Nucleic acid Synthesis

Inhibition of Cell Wall Synthesis

  • ß lactam drugs
    • Penicillin
    • Cephalosporins
  • Vancomycin

Inhibition of Cell Membrane Function

  • Polymyxins (Gram negative cells)
  • Amphoterecin B (Fungi)

Inhibition of Protein Synthesis

  • Gentamicin
  • Erythromycin
  • Tetracyclines

Inhibition of Nucleic Acid Synthesis

  • Rifampicin*
  • Ciprofloxacin**
  • Sulfonamides
  • Trimethoprim
  • Quinolones (Ciprofloxacin)
    • *Rifampicin
      • Binds to DNA-dependent RNA polymerase
      • Thus inhibits RNA synthesis
    • **Ciprofloxacin
      • Inhibits DNA Gyrase

Antibiotic Resistance

  • Transmitted by
    • Genetic Exchange
      • Plasmids
      • Entire chromosomes
    • Mutation
  • Genes encode proteins which
    • Degrade antibiotics
      • Lactams
    • Modify receptor sites or target
      • Some Gram negatives alter 30s binding site of ribosome
        • Decrease bacteria’s permeability to the antibiotic
    • Protective glycocalyx
      • Variety of polysaccharides
      • Synthesized by bacteria
      • As well as a range of host molecules
      • Biofilm of glycocalyx 500x resistant to AB
      • Resistant to complement & neutrophils
    • Produce cell membrane antibiotic pumps

Antimicrobial Activity

  • Factors
    • Metabolic Activity
      • Dormant (No biosynthetic activity)
      • Resistant
    • Distribution
      • Drug unequally distributed in body
      • Poor penetration into
        • BBB
        • Abscesses
    • Location
      • Bacteria may be intracellular
      • Drug may not penetrate cell well
        • No true Blood-Bone Barrier
      • Biomaterial Susceptibility
        • PMMA > Polyethylene
        • Cobolt chrome > Titanium by 20x
        • Surface Area
          • Porous > Polished Stem
    • Interfering substances
      • Acid pH
      • Slime
    • Plasma Level
      • At least MIC x4