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