Sterilization and Chemotherapy

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Sterilization and Chemotherapy

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• Email sswang23_at_mail.ncku.edu.tw

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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)

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• 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

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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

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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

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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.

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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
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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.

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Rate of Microbial Death

• When bacterial populations are heated or treated
  antimicrobial chemicals, they usually die at a
  constant rate.

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Physical Methods of Microbial Control

• heat
• filtration
• radiation

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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

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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.

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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.

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Autoclave Closed Chamber with High Temperature
and Pressure
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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.

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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.

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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.

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Physical Methods of Microbial Control

• Filtration
• used for heat sensitive fluids
• air

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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.

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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.

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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.

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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.

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Forms of Radiation
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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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

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Outline

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

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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.

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Microbial Sources of Antibiotics
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Antibiotic Spectrum of Activity

• No antibiotic is effective against all microbes

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Mechanisms of Antimicrobial Action

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

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Modes of Antimicrobial Action
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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

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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.

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Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis

• The weakness in the cell wall causes the cell to
  lyze.

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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

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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)

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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

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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

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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

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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

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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

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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

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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)

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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

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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

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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

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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

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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

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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

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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.