Chapters 7 and 34

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Chapters 7 and 34

• Control of Microorganisms by Physical and
  Chemical Agents
• and
• Antimicrobial Chemotherapy

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Figure 7.1
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Definition of Frequently Used Terms

• sterilization
• destruction or removal of all viable organisms
• disinfection
• killing, inhibition, or removal of pathogenic
  organisms
• disinfectants
• agents, usually chemical, used for disinfection
• usually used on inanimate objects

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More definitions

• sanitization
• reduction of microbial population to levels
  deemed safe (based on public health standards)
• antisepsis
• prevention of infection of living tissue by
  microorganisms
• antiseptics
• chemical agents that kill or inhibit growth of
  microorganisms when applied to tissue

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Antimicrobial agents

• agents that kill microorganisms or inhibit their
  growth
• -cidal agents kill
• -static agents inhibit growth

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-cidal agents

• -cide
• suffix indicating that agent kills
• germicide
• kills pathogens and many nonpathogens but not
  necessarily endospores
• include bactericides, fungicides, algicides, and
  viricides

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-static agents

• -static
• suffix indicating that agent inhibits growth
• include bacteriostatic and fungistatic

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The Pattern of Microbial Death

• microorganisms are not killed instantly
• population death usually occurs exponentially
• microorganisms were previously considered to be
  dead when they did not reproduce in conditions
  that normally supported their reproduction
• however we now know that organisms can be in a
  viable but nonculturable (VBNC) condition
• once they recover they may regain the ability to
  reproduce and cause infection

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Table 7.1
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Conditions Influencing the Effectiveness of
Antimicrobial Agent Activity

• population size
• larger populations take longer to kill than
  smaller populations
• population composition
• microorganisms differ markedly in their
  sensitivity to antimicrobial agents

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More conditions

• concentration or intensity of an antimicrobial
  agent
• usually higher concentrations or intensities kill
  more rapidly
• relationship is not linear
• duration of exposure
• longer exposure ? more organisms killed

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More Conditions

• temperature
• higher temperatures usually increase amount of
  killing
• local environment
• many factors (e.g., pH, viscosity and
  concentration of organic matter) can profoundly
  impact effectiveness
• organisms in biofilms are physiologically altered
  and less susceptible to many antimicrobial agents

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The Use of Physical Methods in Control

• heat
• low temperatures
• filtration
• radiation

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Moist Heat Sterilization

• must be carried out above 100oC which requires
  saturated steam under pressure
• carried out using an autoclave
• effective against all types of microorganisms
  including spores
• degrades nucleic acids, denatures proteins, and
  disrupts membranes

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Table 7.2
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The Autoclave or Steam Sterilizer
Figure 7.3
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Pasteurization

• controlled heating at temperatures well below
  boiling
• used for milk, beer and other beverages
• process does not sterilize but does kill
  pathogens present and slow spoilage by reducing
  the total load of organisms present

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Pasteurization

• Batch or holding method 63 C for 30 min.
• Flash or HTST (High Temp/ Short Time) method
  72 C for 15 sec.
• UHT (Ultra High Temp) method 140 - 150 C for
  1- 3 sec.
• This is a type of sterilization used so that
  milk products can be left at room temp.

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Dry Heat Sterilization

• less effective than moist heat sterilization,
  requiring higher temperatures and longer exposure
  times
• items subjected to 160-170oC for 2 to 3 hours
• oxidizes cell constituents and denatures proteins

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Dry Heat Incineration

• bench top incinerators are used to sterilize
  inoculating loops used in microbiology
  laboratories

Figure 7.4
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Measuring Heat-Killing Efficiency

• thermal death time (TDT)
• shortest time needed to kill all microorganisms
  in a suspension at a specific temperature and
  under defined conditions
• decimal reduction time (D or D value)
• time required to kill 90 of microorganisms or
  spores in a sample at a specific temperature

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• Z value
• increase in temperature required to reduce D by
  1/10

Figure 7.5
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Table 7.3
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Low Temperatures

• freezing
• stops microbial reproduction due to lack of
  liquid water
• some microorganisms killed by ice crystal
  disruption of cell membranes
• refrigeration
• slows microbial growth and reproduction

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Filtration

• reduces microbial population or sterilizes
  solutions of heat-sensitive materials by removing
  microorganisms
• also used to reduce microbial populations in air

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Filtering liquids

• depth filters
• thick fibrous or granular filters that remove
  microorganisms by physical screening, entrapment,
  and/or adsorption
• membrane filters
• porous membranes with defined pore sizes that
  remove microorganisms primarily by physical
  screening

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Figure 7.6
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Figure 7.7
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Filtering air

• surgical masks
• cotton plugs on culture vessels
• high-efficiency particulate air (HEPA) filters
• used in laminar flow biological safety cabinets

Figure 7.8 (a)
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Figure 7.8 (b)
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Ultraviolet (UV) Radiation

• limited to surface sterilization because it does
  not penetrate glass, dirt films, water, and other
  substances
• has been used for water treatment

Figure 7.9
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Ionizing Radiation

• penetrates deep into objects
• destroys bacterial endospores not always
  effective against viruses
• used for sterilization and pasteurization of
  antibiotics, hormones, sutures, plastic
  disposable supplies, and food

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Figure 7.10
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• Gamma radiation from Cobalt 60 "Cold
  Sterilization"
• Radicidation Disinfection, pathogens destroyed
• Radurization Sanitization, reduce numbers,
  increase shelf life
• Has been approved to be used in the food industry
• Possible problems associated with radiolytic
  products - epoxides (more in high fat foods)
• Rad appertization Sterilize (500,000 RADS, LD
  for humans 500 RADS)
• UV Radiation - 2650 Angstroms lethal to bacteria
  but does not penetrate

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Characteristics of an Ideal Antimicrobial
Chemical Agent

• 1. Antimicrobial activity
• 2. Selective toxicity
• 3. Stable
• 4. Soluable in water and lipids
• 5. Homogenous
• 6. Not affected by extraneous organic material
• 7. Toxic at room or body temp
• 8. Capacity to penetrate
• 9. Noncorroding
• 10. Nonstaining
• 11. Deodorizing ability
• 12. Available
• 13. Inexpensive

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Table 7.4
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Table 7.5
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Chemical Control Agents -Disinfectants and
Antiseptics
Figure 7.11
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Phenolics

• commonly used as laboratory and hospital
  disinfectants
• act by denaturing proteins and disrupting cell
  membranes
• tuberculocidal, effective in presence of organic
  material, and long lasting
• disagreeable odor and can cause skin irritation

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Alcohols

• bactericidal, fungicidal, but not sporicidal
• inactivate some viruses
• denature proteins and possibly dissolve membrane
  lipids

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Halogens

• any of five elements fluorine, chlorine,
  bromine, iodine, and astatine
• iodine and chlorine are important antimicrobial
  agents

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Halogens - Iodine

• skin antiseptic
• oxidizes cell constituents and iodinates proteins
• at high concentrations may kill spores
• skin damage, staining, and allergies can be a
  problem
• iodophore
• iodine complexed with organic carrier

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Halogens - Chlorine

• oxidizes cell constituents
• important in disinfection of water supplies and
  swimming pools, used in dairy and food
  industries, effective household disinfectant
• destroys vegetative bacteria and fungi, but not
  spores
• can react with organic matter to form
  carcinogenic compounds

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Heavy Metals

• e.g., ions of mercury, silver, arsenic, zinc, and
  copper
• effective but usually toxic
• combine with and inactivate proteins may also
  precipitate proteins

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Quaternary Ammonium Compounds

• detergents that have antimicrobial activity and
  are effective disinfectants
• organic molecules with hydrophilic and
  hydrophobic ends
• act as wetting agents and emulsifiers
• cationic detergents are effective disinfectants
• kill most bacteria, but not Mycobacterium
  tuberculosis or endospores
• safe and easy to use, but inactivated by hard
  water and soap

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Aldehydes

• highly reactive molecules
• sporicidal and can be used as chemical sterilants
• combine with and inactivate nucleic acids and
  proteins

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Sterilizing Gases

• used to sterilize heat-sensitive materials
• microbicidal and sporicidal
• combine with and inactivate proteins

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Figure 7.13
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Evaluation of Antimicrobial Agent Effectiveness

• complex process regulated by US federal agencies
• Environmental Protection Agency
• Food and Drug Administration

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Phenol coefficient test

• potency of a disinfectant is compared to that of
  phenol

Table 7.6
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Other evaluation methods

• use dilution test
• determines rate at which selected bacteria are
  destroyed by various chemical agents
• in-use testing
• testing done using conditions that approximate
  normal use of disinfectant

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Chemotherapeutic Agents

• chemicals that can be used internally to kill or
  inhibit the growth of microbes within host cells
• their selective toxicity allows them to target
  the microbe without harming the host
• most are antibiotics, chemicals synthesized by
  microbes that are effective in controlling the
  growth of bacteria

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Definitions

• Chemotherapy The treatment or prevention of a
  disease with a chemical substance
• Chemotherapeutic Agent the chemical substance
  used in chemotherapy
• may be derived from
• A living organism bacteria, fungi, plants
• Totally synthesized
• Partially synthesized synthetic alterations of
  an agent derived from a living organism
• Produced by genetic engineering
• Antibiotic A chemical substance produced by a
  living organism to kill or inhibit the growth of
  other living organisms, is more broadly used to
  refer to all substances used in chemotherapeutic
  treatment of microbial infections

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• Therapeutic Index Used to rate the
  chemotherapeutic agents a ratio between -
• The minimum toxic dose to the host and the
  minimum effective microbial lethal dose
• Kirby-Bauer Antimicrobial Sensitivity Testing
  Used to measure the zone of inhibition of
  antimicrobial agents on growing cultures to
  determine effectiveness
• Broad-spectrum drugs Effective against a broad
  range of organisms
• Narrrow-spectrum drugs Effective against a
  limited variety of organisms
• MIC Minimum Inhibitory Concentration The lowest
  dose that can be used to inhibit the growth of a
  bacterium
• MBC Minimum Bacteriocidal Concentration The
  lowest lethal dose

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Properties of an Ideal Chemotherapeutic Agent

• 1. Relatively nontoxic to the host - selective
  toxicity able to destroy or prevent the activity
  of a parasite without injuring the cells of the
  host or with only minor injury to its cells (some
  toxicity may be acceptable)
• 2. Ability to come in contact with the infectious
  agent - penetration of tissue and cells,
  solubility in fluids
• 3. Exhibit antimicrobial activity at a low
  concentration
• 4. Leave the host's defenses unaltered
• 5. Not produce hypersensitivity
• 6. Remain stable (shelf life)
• 7. Reasonable cost

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Classification of Antibiotics

• 1. By mechanism of action
• 2. Bacteriocidal or bacteriostatic
• 3. Spectrum of activity broad or narrow

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Antibiotic Resistance

• 1. Resistant bacteria occur by and then are
  selected for
• 2. May be resistant due to the inability of the
  drug to enter the organism (ex. capsule or
  multilayer Gram -)
• 3. The cell membrane may have transport proteins
  that "pump" the drug out of the cell (referred to
  as "multi-drug resistant pumps")
• 4. The organisms may release substances which
  inactive the drug by cleaving or binding (ex.
  penicillinase hydrolyzes the beta-lactam ring of
  penicillin)
• 5. The organism may use an alternate pathway and
  bypass the drug
• 6. Resistance may occur by mutation or be
  transmitted by way of R plasmids

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GENERAL MECHANISMS OF ACTION FORANTIMICROBIAL
AGENTS
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Inhibition of Cell Wall Synthesis or Damage to
Existing Cell Wall

• These have the highest therapeutic index because
  they are the most selective -
• (no other animal cells have a cell wall)
• Penicillins - penicillin G, ampicillin
• Cephalosporins
• Bacitracin
• Vancomycin

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Damage to Cell Membrane

• Polymyxins
• Polyenes - Amphotericin B

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Inhibition of Protein Synthesis

• Fairly high therapeutic index because they bind
  with the procaryotic ribosomes
• Streptomycin
• Gentamiciun
• Tetracycline
• Chloramphenicol
• Erythromycin
• Clindamycin

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Inhibition of Nucleic Acid Synthesis

• Rifampin
• Ciprofloxacin

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Inhibition of Metabolic Activity

• Sulfonamides