Safely Using Pathogens and Determining Drug Resistance

Sterilization

• Destroy or remove all viable microorganisms

• Heat + pressure (autocalves)

• Sterilants (chemical agents that destroy endospores) 

Disinfection

• Physical/chmical agent destroy vegetative pathogens

• NOT bacterial endospores 

• Removes harmful toxins from microorganisms

• Bleach

• Iodine

• Heat (boiling)

decontamination/sanitation

• Cleaning technique

• Removes microorganisms mechanically

• Soaps

• Detergents

• Dishwashers 

antisepsis/degermination

• Reduce # of microbes on human skin (living tissues)

• Scrubbing skin/immersing in chemicals

• Alcohol

• Surgical hand scrubs 

Autoclaves vs pasteurization vs radiation 

• Autoclaves

  • steam 121ºC + pressure (18atm)

  • Kills endospores (sterilization) 

• Pasturization

  • Short burst of heat

  • 70ºC for short period of time

  • NOT sterilize

  • Don’t kill every microbe 

• Radiation

  • Uses UV light

Chemiclave

• Chemicals for sterilization

• NOT for solutions

• ONLY for instruments

Filters

•  Want small pores like 0.2µm (bacteria 1µm)

• Microplasmids, not viruses 

Bacteriostatic vs vs Bactericidal vs Bacteriolytic

• Bacteriostatic

  • Total cell count same as viable cell count

  • Not killing cells

    • Hoping to stop their growth 

• Bactericidal

  • Total cell count higher than viable cell count

  • Kills bacteria directly, structure still intact 

• Bacteriolytic 

  • Kills bacteria by lysing (breaking open) cell walls/membranes 

Test susceptibility to antimicrobials

• Minimum inhibitory concentration (MIC) (test tubes) 

• Etest (strip) 

• Kirby Bauer test (disc)

• Micro dilution (wells) 

Minimum inhibitory concentration (MIC)

• Tests resistant strains of bacteria

• Control = no growth

• Least concentration of AB = growth occurs

• Minimum concentration with no growth = highest antibiotic concentration

Vancomycin

• Resistant b/c binds of ala - ala of peptidoglycan

• VRSA binds to ala- something else (lac) = vancomycin cannot bind 

Etest

• Filter strip to measure MIC and ZOI (zone of inhibition)

• Higher concentration = larger ZOI

• Quantify how much AB needed to inhibit growth 

Kirby Bauer test

• Disc

• To test

  • Same antibiotic, different concentrations

  • Different antibiotics as growth inhibitors

• Measure the ZIO

• To discover which antibiotic or concentration needed

Micro dilution

• Like titring

• Positive control = media + bacteria, NO AB

• negative control = media + AB, NO BACTERIA

• Add media + bacteria + AB to wells

Doxyclyclin

• Later gen of tetracycline

• Treat infections/acne

• Decrease risk of schizophrenia in adolescents

  • anti-inflammatories

• Doxy = decrease in bacterial infection = decrease inflammation = decrease in schizophrenia

Tetracycline

• Prescribed during 1st trimester of pregnancy

• Tetra + Ca2+ for bone and tooth formation

  • Tetra gets stuck = brown colour of teeth

  • Can pass through placenta 

• Can inhibit birth control pill

  • Lowers gut microbe that metabolises est

  • Decreased efficacy of birth control pill

Cell wall synthesis antibiotics

• Vancomycin (binds to ala - ala)

• Penicillins

• Cycloserine

DNA gyrase antibiotics

Ciprofloxacin

Cytoplasmic membrane structure and function

Daptomycin

Protein synthesis (30S inhibitors) 

• Tetracyclines

• Streptomycin 

• Can affect binding affinity to proteins

Tetracycline - dose and target

• Target 70S ribosomes (30S + 50S)

• Produced by streptomyces 

• Broad spectrum = both gram -/+

• Tetracycline analog

  • Increase efficacy

  • Bypass resistance

  • Help deliver and target 

Why AB are Resistance

• Lack target

• Impermeable (capsule, thick cell wall) 

• Alternative pathways present

• Enzymatic alteration of antibiotics 

  • how beta lactamase inhibits penicillin

• Modify the target (mutation)

  • PBP2’

• Membrane transport

  • Multi drug therapy (MDR)

  • Transport proteins on membranes export toxins from cell

  • More toxin = greater expression of MDR protein

    • Cocaine addiction

    • Chemo for cancer (some)

Antibiotics

• 1st blockbuster

  • Early 1900s

  • Salvarsan - syphilis and gonorrhea

  • Social issues - HIV

• Supha drugs

  • Early 1930s

  • Part of red dye = AB qualities

  • PABA in bacteria, enzyme converts to folic acid

  • DHF (di, 2) → THF (tetra, 4) (methyl donor)

    • Enzyme facilitator = DHFR)

    • Donator CH3 = U → T

    • THF → DHF

Folic acid

• Humans get from diet

• CH# (methyl) on Uracil to make Thymine 

Analogs

• Derivatives

• Phenylalanine → p=f;urophenylalamine (therapeutic version)  

• Uracil → 5-fluorouracil (chemotherapy) 

Penicillin analogs 

• Natural penicillin = only gram+, b-lactamase sensitive

• Semisynthetic penicillin (block resistance, delivery, broad spectrum) 

  • Methicillin and Oxacilllin

    • Acid stable

    • B-lac resistant

  • Ampicillin

    • Broad spectrum = gram -/+

    • Acid stable

    • B-lac resistant

  • Cabenicillin 

    • Broad spectrum = gram -/+

    • Acid stable

    • B-lac resistant

    • Ineffective orally 

• Why CAN Amp kill E. coli but pen CAN’T

  • Amp can b/c can cross LPS layer of gram - E.coli 

• Penicillin as anaphylactic = allergies

Drug design 

• HIV protease important b/c of life-cycle

• Drug with peptide bond = block action = limit secondary effects

  • Ex Aspirin

• Do not CURE HIV but limit recurrent infections

Why would bacteria make AB

• Competitive advantage