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Sterilization and Chemotherapy

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Title: Sterilization and Chemotherapy


1
Sterilization and Chemotherapy
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  • ?? 5634
  • Email sswang23_at_mail.ncku.edu.tw

2
Outline
  • Definition of Sterilization and Disinfection
  • Physical and Chemical Methods of Antimicrobial
    Control
  • Antibiotics and Mechanisms of Antimicrobial
    Action
  • References
  • Chapters 8 20 in Medical Microbiology
  • (Murray, P. R. et al 6th edition)

3
  • Early civilizations practiced salting, smoking,
    pickling, drying, and exposure of food and
    clothing to sunlight to control microbial growth.
  • Use of spices in cooking was to mask taste of
    spoiled food. Some spices prevented spoilage.
  • In mid 1800s Semmelweiss and Lister helped
    developed aseptic techniques to prevent
    contamination of surgical wounds. Before then
  • Nosocomial infections caused death in 10 of
    surgeries.
  • Up to 25 mothers delivering in hospitals died
    due to infection

4
Antimicrobial Definitions
  • Sterilization
  • To completely remove all kinds of microbes
    (bacteria, mycobacteria, viruses, fungi) by
    physical or chemical methods
  • Effective to kill bacterium spores
  • Sterilant material or method used to remove or
    kill all microbes

5
Antimicrobial Definitions
  • Disinfection
  • To reduce the number of pathogenic microorganisms
    to the point where they no longer cause diseases
  • Usually involves the removal of vegetative or
    non-endospore forming pathogens
  • May use physical or chemical methods
  • Disinfectant An agent applied to inanimate
    objects.
  • Antiseptic A substance applied to living
    tissue.
  • Degerming Removal of most microbes in a limited
    area. Example Alcohol swab on skin.
  • Sanitization Use of chemical agent on
    food-handling equipment to meet public health
    standards and minimize chances of disease
    transmission. e.g. Hot soap water

6
Antimicrobial Definitions
  • Bacteriostatic
  • prevents growth of bacteria
  • Germicide
  • An agent that kills certain microorganisms.
  • Bactericide An agent that kills bacteria. Most
    do not kill endospores.
  • Viricide An agent that inactivates viruses.
  • Fungicide An agent that kills fungi.
  • Sporicide An agent that kills bacterial
    endospores of fungal spores.

7
Method of Control
  • physical or chemical?
  • physical control includes heat, irradiation,
    filtration and mechanical removal
  • chemical control involves the use of
    antimicrobial chemicals
  • depends on the situation
  • degree of control required

antimicrobial chemicals
air filters
8
Factors influence the effectiveness of
antimicrobial treatment
  • Number of Microbes The more microbes present,
    the more time it takes to eliminate population.
  • Type of Microbes Endospores are very difficult
    to destroy. Vegetative pathogens vary widely in
    susceptibility to different methods of microbial
    control.
  • Environmental influences Presence of organic
    material (blood, feces, saliva, pH etc.) tends to
    inhibit antimicrobials.
  • Time of Exposure Chemical antimicrobials and
    radiation treatments are more effective at longer
    times. In heat treatments, longer exposure
    compensates for lower temperatures.

9
Rate of Microbial Death
  • When bacterial populations are heated or treated
    antimicrobial chemicals, they usually die at a
    constant rate.

10
Physical Methods of Microbial Control
  • heat
  • filtration
  • radiation

11
Physical Methods of Microbial Control
  • Heat
  • Kills microorganisms by denaturing their enzymes
    and other proteins. Heat resistance varies widely
    among microbes.
  • fast, reliable, inexpensive
  • does not introduce potential toxic substances
  • types of heat control include
  • moist heat
  • pasteurization
  • dry heat

12
Physical Methods of Microbial Control
  • Moist Heat Kills microorganisms by coagulating
    their proteins.
  • Boiling Heat to 100oC or more at sea level.
    Kills vegetative forms of bacterial pathogens.
    Most pathogens can be killed within 10 minutes or
    less. Endospores and some viruses are not
    destroyed this quickly.
  • In general, moist heat is much more effective
    than dry heat.

13
Physical Methods of Microbial Control
  • Moist Heat (Continued)
  • Reliable sterilization with moist heat requires
    temperatures above that of boiling water.
  • Autoclave Chamber which is filled with hot steam
    under pressure. Preferred method of
    sterilization, unless material is damaged by
    heat, moisture, or high pressure.
  • Temperature of steam reaches 121oC at twice
    atmospheric pressure.
  • All organisms and endospores are killed within
    15 minutes.

14
Autoclave Closed Chamber with High Temperature
and Pressure
15
Physical Methods of Microbial Control
  • Moist Heat (Continued)
  • Pasteurization Developed by Louis Pasteur to
    prevent the spoilage of beverages. Used to
    reduce microbes responsible for spoilage of beer,
    milk, wine, juices, etc.
  • Classic Method of Pasteurization Milk was
    exposed to 65oC for 30 minutes.
  • High Temperature Short Time Pasteurization
    (HTST) Used today. Milk is exposed to 72oC for
    15 seconds.

16
Physical Methods of Microbial Control
  • Dry Heat
  • Direct Flaming Used to sterilize inoculating
    loops and needles. Heat metal until it has a red
    glow.
  • Incineration Effective way to sterilize
    disposable items (paper cups, dressings) and
    biological waste.
  • Hot Air Sterilization Place objects in an oven.
    Require 2 hours at 170oC for sterilization. Dry
    heat is transfers heat less effectively to a cool
    body, than moist heat.

17
Physical Methods of Microbial Control
  • Filtration Removal of microbes by passage of a
    liquid or gas through a screen like material with
    small pores. Used to sterilize heat sensitive
    materials like vaccines, enzymes, antibiotics,
    and some culture media.
  • Membrane Filters Uniform pore size. Used in
    industry and research. Different sizes
  • 0.22 and 0.45um Pores Used to filter most
    bacteria. Dont retain spirochetes, mycoplasmas
    and viruses.
  • 0.01 um Pores Retain all viruses and some large
    proteins.
  • High Efficiency Particulate Air Filters (HEPA)
    Used in operating rooms to remove bacteria from
    air.

18
Physical Methods of Microbial Control
  • Filtration
  • used for heat sensitive fluids
  • air

19
Physical Methods of Microbial Control
  • Low Temperature Effect depends on microbe and
    treatment applied.
  • Refrigeration Temperatures from 0 to 7oC.
    Bacteriostatic effect. Reduces metabolic rate of
    most microbes so they cannot reproduce or produce
    toxins.
  • Freezing Temperatures below 0oC.

20
Physical Methods of Microbial Control
  • Desiccation In the absence of water, microbes
    cannot grow or reproduce, but some may remain
    viable for years. After water becomes available,
    they start growing again.
  • Susceptibility to desiccation varies widely
  • Neisseria gonnorrhea Only survives about one
    hour.
  • Mycobacterium tuberculosis May survive several
    months.
  • Viruses are fairly resistant to desiccation.
  • Clostridium spp. and Bacillus spp. May survive
    decades.

21
Physical Methods of Microbial Control
  • Osmotic Pressure The use of high concentrations
    of salts and sugars in foods is used to increase
    the osmotic pressure and create a hypertonic
    environment.
  • Plasmolysis As water leaves the cell, plasma
    membrane shrinks away from cell wall.
  • Yeasts and molds More resistant to high osmotic
    pressures.
  • Staphylococci spp. that live on skin are fairly
    resistant to high osmotic pressure.

22
Physical Methods of Microbial Control
  • Radiation Three types of radiation kill
    microbes
  • 1. Ionizing Radiation Gamma rays, X rays,
    electron beams, or higher energy rays. Have
    short wavelengths (less than 1 nanometer).
  • Used to sterilize pharmaceuticals, disposable
    medical supplies and food.
  • Disadvantages Penetrates human tissues. May
    cause genetic mutations in humans.

23
Forms of Radiation
24
Physical Methods of Microbial Control
  • Radiation Three types of radiation kill
    microbes
  • 2. Ultraviolet light (Nonionizing Radiation)
    Wavelength is longer than 1 nanometer. Damages
    DNA by producing thymine dimers, which cause
    mutations.
  • Used to disinfect operating rooms, nurseries,
    cafeterias.
  • Disadvantages Damages skin, eyes. Doesnt
    penetrate paper, glass, and cloth.

25
Physical Methods of Microbial Control
  • Radiation Three types of radiation kill
    microbes
  • 3. Microwave Radiation Wavelength ranges from
    1 millimeter to 1 meter.
  • Heat is absorbed by water molecules.
  • May kill vegetative cells in moist foods.
  • Bacterial endospores, which do not contain
    water, are not damaged by microwave radiation.
  • Solid foods are unevenly penetrated by
    microwaves.

26
Chemical Methods of Microbial ControlTypes of
Disinfectants
  • 1. Phenols and Phenolics
  • Phenol (carbolic acid) was first used by Lister
    as a disinfectant.
  • Rarely used today because it is a skin irritant
    and has strong odor.
  • Phenolics are chemical derivatives of phenol
  • Cresols (Lysol) Derived from coal tar.
  • Biphenols Effective against gram-positive
    staphylococci and streptococci. Excessive use in
    infants may cause neurological damage.
  • Destroy plasma membranes and denature proteins.
  • Advantages Stable, persist for long times after
    applied, and remain active in the presence of
    organic compounds.

27
Chemical Methods of Microbial ControlTypes of
Disinfectants
  • 2. Halogens Effective alone or in compounds.
  • A. Iodine
  • Iodine tincture (alcohol solution) was one of
    first antiseptics used.
  • B. Chlorine
  • When mixed in water forms hypochlorous acid
  • Cl2 H2O ------gt H Cl- HOCl
  • Hypochlorous acid
  • Used to disinfect drinking water, pools, and
    sewage.

28
Chemical Methods of Microbial ControlTypes of
Disinfectants
  • 3. Alcohols
  • Kill bacteria, fungi, but not endospores or
    naked viruses.
  • Act by denaturing proteins and disrupting cell
    membranes.
  • Used to mechanically wipe microbes off skin
    before injections or blood drawing.
  • Not good for open wounds, because cause proteins
    to coagulate.
  • Ethanol Drinking alcohol. Optimum
    concentration is 70.
  • Isopropanol Rubbing alcohol. Better
    disinfectant than ethanol. Also cheaper and less
    volatile.

29
Chemical Methods of Microbial ControlTypes of
Disinfectants
  • 4. Heavy Metals
  • Include copper, selenium, mercury, silver, and
    zinc.
  • Very tiny amounts are effective.
  • A. Silver
  • 1 silver nitrate used to protect infants against
    gonorrheal eye infections, now has been replaced
    by erythromycin.
  • B. Mercury
  • Organic mercury compounds like merthiolate and
    mercurochrome are used to disinfect skin wounds.
  • C. Copper
  • Copper sulfate is used to kill algae in pools
    and fish tanks.

30
Chemical Methods of Microbial ControlTypes of
Disinfectants
  • 5. Quaternary Ammonium Compounds (Quats)
  • Cationic (positively charge) detergents.
  • Effective against gram positive bacteria, less
    effective against gram-negative bacteria.

31
Chemical Methods of Microbial ControlTypes of
Disinfectants
  • 6. Aldehydes
  • Include some of the most effective
    antimicrobials.
  • Inactivate proteins by forming covalent
    crosslinks with several functional groups.
  • A. Formaldehyde
  • Excellent disinfectant, 2 aqueous solution.
  • Commonly used as formalin, a 37 aqueous
    solution.
  • Formalin was used extensively to preserve
    biological specimens and inactivate viruses and
    bacteria in vaccines.
  • Irritates mucous membranes, strong odor.

32
Chemical Methods of Microbial ControlTypes of
Disinfectants
  • 6. Aldehydes
  • B. Glutaraldehyde
  • Less irritating and more effective than
    formaldehyde.
  • Commonly used to disinfect hospital instruments.
  • 7. Gaseous Sterilizers
  • Chemicals that sterilize in a chamber similar to
    an autoclave.
  • Denature proteins, by replacing functional groups
    with alkyl groups.
  • Ethylene Oxide
  • Kills all microbes and endospores, but requires
    exposure of 4 to 18 hours.

33
Chemical Methods of Microbial ControlTypes of
Disinfectants
  • 8. Oxidizing Agents
  • Oxidize cellular components of treated microbes.
  • Disrupt membranes and proteins.
  • A. Ozone
  • Used along with chlorine to disinfect water.
  • Helps neutralize unpleasant tastes and odors.
  • More effective killing agent than chlorine, but
    less stable and more expensive.
  • Highly reactive form of oxygen.
  • Made by exposing oxygen to electricity or UV
    light
  • B. Hydrogen Peroxide
  • Not good for open wounds because quickly broken
    down by catalase present in human cells.
  • Effective in disinfection of inanimate objects

34
Outline
  • Definition of Sterilization and Disinfection
  • Physical and Chemical Methods of antimicrobial
    control
  • Antibiotics and Mechanisms of Antimicrobial
    Action

35
Definition of an Antibiotic
  • Substance produced by a microorganism or a
    similar product produced wholly (synthetic) or
    partially (semi-synthetic) by chemical synthesis
    and in low concentrations inhibits the growth of
    or kills microorganisms.

36
Microbial Sources of Antibiotics
37
Antibiotic Spectrum of Activity
  • No antibiotic is effective against all microbes

38
Mechanisms of Antimicrobial Action
  • Bacteria have their own enzymes for
  • Cell wall formation
  • Protein synthesis
  • DNA replication
  • RNA synthesis
  • Synthesis of essential metabolites

39
Modes of Antimicrobial Action
40
Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis
  • Bacteria cell wall contains peptidoglycan
  • Antimicrobials that interfere with the synthesis
    of cell wall do not interfere with eukaryotic
    cell
  • Antimicrobials of this class include
  • ß- lactam drugs
  • Vancomycin
  • Daptomycin
  • Bacitracin

41
Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis
  • Penicillins and Cephalosporins
  • Part of group of drugs called ß lactams
  • Have shared chemical structure called ß-lactam
    ring
  • Competitively inhibits function of
    penicillin-binding proteins (involved in the
    final stages of the synthesis of peptidoglycan)
  • Inhibits peptide bridge formation between glycan
    molecules
  • This causes the cell wall to develop weak points
    at the growth sites and become fragile.

42
Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis
  • The weakness in the cell wall causes the cell to
    lyze.

43
Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis
  • Natural penicillins
  • Narrow range of action
  • Susceptible to penicillinase (b- lactamase)
  • Semisynthetic Penicillins
  • Penicilinase-resistant penicillins
  • Carbapenems very broad spectrum
  • Monobactam Gram negative
  • Extended-spectrum penicillins

44
Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis
  • Cephalosporins
  • chemical structures make them resistant to
    inactivation by certain ß-lactamases
  • most effective against Gram bacteria.
  • chemically modified to produce family of related
    compounds
  • 2nd, 3rd, and 4th generations more effective
    against gram-negatives (4th generation against
    almost Enterobacteriaceae and Pseudomonas
    aeruginosa)

45
Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis
  • Bacitracin
  • Interferes with transport of peptidoglycan
    precursors across cytoplasmic membrane
  • Toxicity limits use to topical applications
  • Common ingredient in non-prescription first-aid
    ointments

46
Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis
  • Vancomycin
  • Inhibits formation of glycan chains
  • Important in treating infections caused by
    penicillin resistant Gram organisms
  • Acquired resistance most often due to alterations
    in side chain of NAM molecule
  • Prevents binding of vancomycin to NAM component
    of glycan
  • Important "last line" against antibiotic
    resistant S. aureus

47
Antibacterial Antibiotics Inhibitors of Protein
Synthesis
  • Inhibition of protein synthesis
  • Structure of prokaryotic ribosome acts as target
    for many antimicrobials of this class
  • Differences in prokaryotic and eukaryotic
    ribosomes responsible for selective toxicity
  • Drugs of this class include
  • Aminoglycosides
  • Tetracyclins
  • Macrolids
  • Chloramphenicol

48
Antibacterial Antibiotics Inhibitors of Protein
Synthesis
  • Aminoglycosides
  • binds to ribosomal subunits
  • Examples of aminoglycosides include
  • Gentamicin, streptomycin and neomycin
  • Often used in synergistic combination with
    ß-lactam drugs
  • Allows aminoglycosides to enter cells that are
    often resistant
  • Side effects
  • Nephrotoxicity

49
Antibacterial Antibiotics Inhibitors of Protein
Synthesis
  • Tetracyclins
  • Reversibly bind 30S ribosomal subunit
  • Blocks attachment of tRNA to ribosome
  • Effective against certain Gram and Gram
  • Can cause discoloration of teeth if taken as
    young child

50
Antibacterial Antibiotics Inhibitors of Protein
Synthesis
  • Macrolids
  • Reversibly binds to 50S ribosome
  • Prevents continuation of protein synthesis
  • Effective against variety of Gram organisms and
    those responsible for atypical pneumonia
  • Often drug of choice for patients allergic to
    penicillin
  • Macrolids include
  • Erythromycin, clarithromycin and azithromycin

51
Antibacterial Antibiotics Inhibitors of Protein
Synthesis
  • Chloramphenicol
  • Binds to 50S ribosomal subunit
  • Prevents peptide bonds from forming and blocking
    proteins synthesis
  • Effective against a wide variety of organisms
  • Generally used as drug of last resort for
    life-threatening infections
  • Rare but lethal side effect is aplastic anemia
    (because it disrupts protein synthesis in human
    bone marrow cells)

52
Antibacterial Antibiotics Inhibitors of Nucleic
Acid Synthesis
  • Fluoroquinolones
  • Inhibit action of topoisomerase DNA gyrase
  • Examples include
  • Ciprofloxacin and ofloxacin
  • Urinary tract infections
  • Rifamycins
  • Block prokaryotic RNA polymerase
  • Primarily used to treat tuberculosis and
    preventing meningitis after exposure to N.
    meningitidis

53
Antibacterial Antibiotics Inhibitors of
Metabolic Pathway
  • Sulfonamides (sulfa drugs)
  • Inhibit folic acid synthesis
  • Structurally similar to para-aminobenzoic acid
  • Substrate in folic acid pathway
  • Through competitive inhibition of enzyme that
    aids in production of folic acid
  • Inhibit growth of Gram and Gram - organisms

54
Antibacterial Antibiotics Disruption of Plasma
Membrane
  • Polymyxin B
  • Binds membrane of Gram - cells
  • Alters permeability
  • Leads to leakage of cell and cell death
  • Also bind eukaryotic cells but to lesser extent
  • Limits use to topical application
  • Common ingredient in first-aid skin ointments

55
Mechanisms of Antibiotic Resistance
  • Enzymatic destruction of drug
  • Some organisms produce enzymes that chemically
    modify drug
  • Penicillinase breaks ß-lactam ring of penicillin
    antibiotics
  • Alteration of drug's target site
  • Minor structural changes in antibiotic target can
    prevent binding
  • Changes in ribosomal RNA prevent macrolids from
    binding to ribosomal subunits

56
Mechanisms of Antibiotic Resistance
  • Prevention of penetration of drug
  • Alterations in porin proteins decrease
    permeability of cells
  • Prevents certain drugs from entering
  • Rapid ejection of the drug
  • Some organisms produce efflux pumps
  • Increases overall capacity of organism to
    eliminate drug
  • Enables organism to resist higher concentrations
    of drug
  • Tetracycline resistance

57
EFFECTS OF COMBINATIONS OF DRUGS
  • Synergism
  • the chemotherapeutic effects of two drugs given
    simultaneously is greater than the effect of
    either given alone
  • For example, penicillin and streptomycin in the
    treatment of bacterial endocarditis. Damage to
    bacterial cell walls by penicillin makes it
    easier for streptomycin to enter

58
EFFECTS OF COMBINATIONS OF DRUGS
  • Antagonism
  • the chemotherapeutic effects of two drugs given
    simultaneously reduce the effect of either given
    alone
  • For example, the simultaneous use of penicillin
    and tetracycline is often less effective than
    when wither drugs is used alone. By stopping the
    growth of the bacteria, the bacteriostatic drug
    tetracycline interferes with the action of
    penicillin, which requires bacterial growth.
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