Microbiology

Prokaryotes

• No membrane-bound nucleus or organelles
• Bacteria
• Rickettsia
• Chlamydia

Eukaryotes

• Nucleus enclosed in membranes (eg. plants)
• Fungi
• Animal cells

Bacterial structure

• Prokaryotic cells
• Most have cell walls which are inert
• The cell membrane however is an osmotic barrier
• Contain DNA and RNA
• Nuclear DNA lies free in the cytoplasm
• Plasmids contain DNA fragments
• No other organelles as such
• Mesosomes are sacs containing enzymes
• Flagella for motility
• Reproduction by binary fission

Bacterial metabolism

• Bacteria need CO2 to grow
• Energy comes from
• Respiration (primarily used by aerobes): producing CO2 and H2O
• Fermentation (primarily used by anaerobes): producing lactic or pyruvic acid

• Note: aerobes and anaerobes MAY use the other method too (eg. aerobes that also use fermentation are termed facultative anaerobes. Strict anaerobes rely only on fermentation)

Gram staining

• Gentian/crystal violet stains the peptidoglycans in Gram positive cells walls
• Lugol’s iodine is applied which fixes the crystal violet to the cells
• A black-purple complex is seen
• Treated with acetone or alcohol
• Some bacteria retain the complex: Gram positive
• Others lose it and become colourless: Gram negative
• These are stained pink with safranin for contrast (counterstain)

Note: methylene blue is used as a counterstain when producing a Ziehl-Neelsen stain for acid-fast bacilli

Fungal stains

• Haematoxylin and eosin (pink)
• Gomori methanamine/Grocott hexamine silver (black)
• Periodic acid-Schiff (purple)

Antibody detection

• Complement fixation
• Haemagglutination inhibition
• Indirect immunofluorescence

Bacterial virulence

• Invasiveness
• Adherence to target cells
• Local tissue damage

• Hyaluronidase (dissolves collagen), streptokinase and collagenase promote bacterial spread

• Haemolysins: kill host phagocytes
• Coagulases precipitate fibrin clots to wall the bacteria off from defences
• Toxigenicity 
• Defence strategies
• Interference with complement activation

• Eg. Streptococci possess a surface M-factor which facilitates C3b breakdown and binds fibrinogen

• E. coli (and other Gram negatives) can divert complement activation
• Pseudomonas releases elastase which inactivates C3a and C5a
• Neisseria gonorrhoeae binds complement
• Some bacterial capsules are antiphagocytic
• Intracellular bacteria evade the immune system by living within macrophages
• Eg. mycobacterium tuberculosis
• Listeria monocytogenes

Staphylococcus aureus

• Flora of many normal people therefore infection often endogenous
• Transmission: air, direct, indirect
• Virulence factors:
• Lysozymes
• Coagulase
• Hyaluronidase
• Adherence to cell surfaces
• Capsule resistant to opsonisation
• Protein A: decreases phagocytosis and inhibits complement
• Epidermolytic toxin (“scalded skin”)
• Enterotoxin (food poisoning)
• Produces coagulase (which staphylococcus epidermidis does not)
• MRSA: 
• Coagulase positive
• May also be a normal commensal
• Actually NOT more virulent or resistant to sterilisation than normal staph aureus
• Resistant to conventional antimicrobials
• Found in the anterior nares

Disinfection vs sterilisation

• Disinfectants (removal of most microorganisms)
• UV radiation

• Pasteurisation: steam generated at 80 degrees at half an atmosphere of pressure kills vegetative organisms but not spores

• Examples
• Chlorhexidine
• Ethyl alcohol with povidone-iodine
• Sterilisation (removal of all microorganisms)
• Gamma radiation
• Dry heat: requires temperatures of 160 degrees for several hours

• Moist heat: requires temperatures of 134 degrees for 3 minutes, or 121 degrees for 120 minutes

• Ethylene oxide

Normal ocular flora

• No viruses exist as normal flora in the eye

• Staphylococcus epidermidis (40-45%) is the most common (coagulase negative, catalase positive)

• Staphylococcus aureus (25%), coagulase positive
• Diphtheroids (25-40%)
• Streptococcus viridans (2-3%)
• Propionibacterium acne (most common anaerobe)
• Demodex folliculorum (protozoa)
• Corynebacteria
• Micrococcus 
• Up to 100 fungi/yeasts live on the lashes and lids

Toll-like receptors

• Single transmembrane receptors
• Recognise intact microbial products

• Activation of TLRs results in proinflammatory and chemotactic cytokine release (recruiting neutrophils, macrophages and lymphocytes)

• TLR4/MD-2 recognises the lipid A moiety of lipopolysaccharide (LPS)

NOD-like receptors

• Intracellular family of PRRs (pattern recognition receptors)

• Recognise components of degraded bacterial cell wall peptidoglycan therefore respond to invading Gram-negative and Gram-positive bacteria

• NLRC4 recognises the flagellin of Gram-negative bacteria including Pseudomonas

• Activated NLRs form a large multi-protein complex called an inflammasome which activates caspase-1 and cleaves multiple interleukins to their active forms with proinflammatory and chemotactic effects

• Prolonged inflammasome activation can lead to caspase-1 mediated cell death (pyroptosis)

C-type lectins

• Fungal cell wall components are recognised via activation of C-type lectins on cell surfaces

Neutrophils

• Most common leucocytes in blood (25% of total WBCs)
• Recruited from capillaries to infected tissues

• Pro-inflammatory cytokines at site of infection induce expression of adhesion molecules on vascular endothelium (selectins) which bind to integrins on neutrophils

• They then transmigrate across endothelium (mediated by chemokines)
• Chemokine (eg IL-8) gradient then dictates their migration to site of infection

Contact lenses

• Most common risk factor for corneal infections

• Long-term wear inhibits epithelial proliferation, migration and suppresses limbal stem cell production of basal epithelial cells

• Reduce flow and effectiveness of tears: trap microbes on cell surfaces

Prions

• Small proteinaceous infective elements
• No genome
• Not antigenic: do not produce an immune response
• Highly resistant: to formaldehyde, ionizing radiation and other methods
• Autoclaving at 15 psi for 1 hour destroys them

Prion protein may be detected in a biopsy sample using Western blot