Physical and Chemical Control of Microorganisms

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Physical and Chemical Control of Microorganisms
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Controlling Microorganisms

• Reduce or destroy undesirable microbes in a given
  area
• Physical
• Chemical
• Mechanical

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Relative Resistance of Microbes

• Highest resistance
• Bacterial endospores prions
• Moderate resistance
• Pseudomonas sp.
• Mycobacterium tuberculosis
• Staphylococcus aureus
• Protozoan cysts
• Least resistance
• most vegetative cells
• fungal spores
• enveloped viruses
• yeast
• protozoan trophozoites

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Terms for Microbial Control

• Sterile
• Inanimate objects free of all life
• Sterilization
• destroys all viable microbes
• including viruses endospores
• Disinfection
• destroy vegetative pathogens
• not endospores
• Sanitization
• cleansing technique that mechanically removes
  microbes to safe levels
• Degerming
• removing organisms from an objects surface

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Terms for Microbial Control

• Microbicidal agents
• Causes microbial death
• Bactericide
• Sporocide
• Fungicide
• Viricide
• Microbistasis
• Prevents microbial growth
• Bacteriostatic
• Fungistatic

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Factors That Affect Death Rate

1. Number of microbes
2. Nature of microbes in the population
3. Temperature pH of environment
4. Concentration or dosage of agent
5. Mode of action of the agent
6. Presence of solvents, organic matter, or
   inhibitors

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Cellular Targets of Control

• Mode of action of antimicrobials
• Cell wall
• Cell membrane
• Cellular synthetic processes (DNA, RNA)
• Proteins

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Practical Concerns for Microbial Control

• Does the application require sterilization?
• Is the item to be reused?
• Can the item withstand heat, pressure, radiation,
  or chemicals?
• Is the method suitable?
• Will the agent penetrate to the necessary extent?
• Is the method cost- and labor-efficient is it
  safe?

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Methods of Physical Control

• Heat Moist verse Dry
• Cold temperatures
• Desiccation
• Radiation

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1. Heat (Moist)

• Moist heat use of hot water or steam
• mode of action
• denaturation of proteins
• destruction of membranes
• destruction of DNA
• Sterilization
• Steam under pressure
• Autoclave
• 15psi/121oC/10-40 min
• Steam must reach surface of item being sterilized!

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Autoclave Tape
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1. Heat (Moist)

• Intermittent sterilization
• unpressurized steam
• 100oC 30-60 min for 3 days
• spores, which are unaffected, germinate during
  the intervening periods and are subsequently
  destroyed
• Disinfection
• boiling at 100oC for 30 minutes
• destroy non-spore-forming pathogens
• Pasteurization

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1. Heat (Moist)

• Pasteurization
• heat applied to kill potential agents of
  infection and spoilage
• without destroying the food flavor or value
• 63C - 66C for 30 minutes
• batch method
• 71.6C for 15 seconds
• flash method
• Not sterilization
• kills non-spore-forming pathogens and lowers
  overall microbe count
• does not kill endospores or many nonpathogenic
  microbes

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1. Heat (Dry)

• Dry heat - higher temperatures than moist heat
• incineration
• 600-1200oC
• combusts dehydrates cells
• dry ovens
• 150-180oC
• coagulate proteins
• Bunsen burner
• 1870oC
• Dehydrates cells and removes water
• Can also sterilize

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Thermal Death Measurements

• Thermal death time (TDT)
• shortest length of time required to kill all
  microbes at a specified temperature
• Thermal death point (TDP)
• lowest temperature required to kill all microbes
  in a sample in 10 minutes

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2. Cold Temperatures

• Microbistatic
• slows the growth of microbes
• refrigeration 0-15oC
• freezing lt0oC
• used to preserve food, media and cultures

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3. Desiccation

• gradual removal of water from cells
• leads to metabolic inhibition
• not effective microbial control
• many cells retain ability to grow when water is
  reintroduced

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4. Radiation

• Ionizing radiation
• deep penetrating power breaks DNA
• gamma rays, X-rays, cathode rays
• used to sterilize medical supplies food
  products
• Nonionizing radiation
• little penetrating power to sterilize air, water
  solid surfaces
• UV light creates thymine pyrmidines
• interfere with replication

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Chemical Agents in Microbial Control

• Chemicals that sterilize
• Disinfectants, antiseptics, sterilants
• Chemicals that inhibit deterioration
• Degermers, and preservatives
• Desirable qualities of chemicals
• rapid action in low concentration
• solubility in water or alcohol, stable
• broad spectrum, low toxicity
• penetrating
• noncorrosive and nonstaining
• affordable and readily available

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Levels of Chemical Decontamination

• High-level germicides
• kill endospores
• devices that are not heat sterilizable and
  intended to be used in sterile environments (body
  tissue)
• Intermediate-level
• kill fungal spores (not endospores), tubercle
  bacillus, and viruses
• used to disinfect devices that will come in
  contact with mucous membranes but are not
  invasive
• Low-level
• eliminate only vegetative bacteria, vegetative
  fungal cells, and some viruses
• clean surfaces that touch skin but not mucous
  membranes

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Factors that Affect Germicidal Activity of
Chemicals

• Nature of the material being treated
• Degree of contamination
• Time of exposure
• Strength and chemical action of the germicide
• Dilutions
• Volume of liquid chemical diluted in larger
  volume of solvent (water)

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Chemical Control Of Microbial Agents

• Halogens
• Phenolics
• Chlorhexidine
• Alcohols
• Hydrogen peroxide
• Detergents soaps
• Heavy metals
• Aldehydes
• Gases and aerosols

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1. Halogens

• Chlorine
• Cl2, hypochlorites (chlorine bleach), chloramines
• Denaturation of proteins by disrupting disulfide
  bonds
• Can be sporicidal
• Iodine
• I2, iodophors (betadine)
• Denature proteins
• Can be sporicidal
• Milder medical dental degerming agents,
  disinfectants, ointments

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2. Phenolics

• Disrupt cell membranes precipitating proteins
• bactericidal, fungicidal, virucidal, not
  sporicidal
• Lysol
• Triclosan
• antibacterial additive to soaps

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3. Chlorhexidine

• A surfactant protein denaturant with broad
  microbicidal properties
• Not sporicidal
• Used as skin degerming agents for preoperative
  scrubs, skin cleaning burns

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4. Alcohols

• Ethyl, isopropyl in solutions of 50-90
• Act as surfactants
• dissolve membrane lipids and coagulating proteins
  of vegetative bacterial cells and fungi
• Not sporicidal
• Good for enveloped viruses

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5. Hydrogen Peroxide

• Weak (3) to strong (35)
• Produce highly reactive hydroxyl-free radicals
  that damage protein DNA while also decomposing
  to O2 gas
• toxic to anaerobes
• Strong solutions are sporicidal
• in increasing concentrations
  

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6. Detergents Soaps

• Ammonia compounds act as surfactants
• alter membrane permeability of some bacteria
  fungi
• Not sporicidal
• Soaps
• mechanically remove soil and grease containing
  microbes
• Low concentrations
• Only have microbistatic effects

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7. Heavy Metals

• Solutions of silver mercury
• kill vegetative cells in low concentrations by
  inactivating proteins
• Oligodynamic action
• Not sporicidal

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8. Aldehydes

• Glutaraldehyde formaldehyde kill by alkylating
  protein DNA
• glutaraldehyde in 2 solution (Cidex) used as
  sterilant for heat sensitive instruments
• formaldehyde
• disinfectant, preservative, toxicity limits use

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9. Gases Aerosols

• Ethylene oxide, propylene oxide,
  betapropiolactone chlorine dioxide
• Strong alkylating agents, sporicidal

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Mechanical Control

• Filtration
• physical removal of microbes
• passing a gas or liquid through filter
• organisms above a certain size trapped in the
  pores
• used to sterilize heat sensitive liquids air in
  hospital isolation units industrial clean rooms
• Air can be filtered using a high-efficiency
  particulate air (HEPA) filter

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Antimicrobial Therapy
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Origins of Antimicrobial Drugs

• Antibiotics
• Common metabolic products of aerobic
  spore-forming bacteria fungi
• bacteria in genera Streptomyces Bacillus
• molds in genera Penicillium Cephalosporium
• Inhibiting other microbes in the same habitat
• antibiotic producers have less competition for
  nutrients space

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Ideal Antimicrobial Drug..

• Selectively toxic to microbe
• Not host cells
• Microbicidal, not microbistatic
• Soluble
• Potent
• No antimicrobial resistance
• Remains active
• Readily delivered to site of infection
• Expense
• Not allergen

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Chemotherapy

• Antimicrobial
• Control infection
• Antibiotic
• Produced by the natural metabolic processes of
  microorganisms
• Can inhibit or destroy other microorganisms
• Semisynthetic
• Chemically modified drugs in lab
• Synthetic
• Synthesized compounds in lab

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Chemotherapy

• Narrow spectrum
• Effective against limited microbial types
• Target a specific cell component that is found
  only in certain microbes
• Broad spectrum
• Effective against wide variety microbial types
• Target cell components common to most pathogens

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Selectively Toxic

• Should kill or inhibit microbial cells without
  simultaneously damaging host tissues
• Complete selective toxicity
• Difficult to achieve
• Characteristics of the infectious agent become
  more similar to the vertebrate host cell
• More side effects are seen

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Selective toxicity

• toxic dose of a drug
• The concentration causing harm to the host
• therapeutic dose
• the concentration eliminating pathogens in the
  host
• Together, the toxic and therapeutic doses are
  used to formulate the chemotherapeutic index

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Targets of Antimicrobial Drugs

1. Inhibition of cell wall synthesis
2. Inhibition of nucleic acid synthesis, structure
   or function
3. Inhibition of protein synthesis
4. Disruption of cell membrane structure or function

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1. Bacterial Cell Wall

• Most bacterial cell walls contain peptidoglycan
• Penicillin and cephalosporin block synthesis of
  peptidoglycan
• Causes the cell wall to lyse
• Penicillins do not penetrate the outer membrane
• less effective against gram-negative bacteria
• Broad spectrum penicillins and cephalosporins
• cross the cell walls of gram-negative bacteria

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2. Inhibit Nucleic Acid Synthesis

• May block synthesis of nucleotides, inhibit
  replication, or stop transcription
• sulfonamides and trimethoprim
• block enzymes required for tetrahydrofolate
  synthesis
• needed for DNA RNA synthesis

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3. Drugs that Block Protein Synthesis

• Ribosomes
• eukaryotes differ in size and structure from
  prokaryotes
• Aminoglycosides (streptomycin, gentamicin)
• insert on sites cause misreading of mRNA
• Tetracyclines
• block attachment of tRNA and stop further
  synthesis

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4. Disrupt Cell Membrane Function

• Damaged membrane
• dies from disruption in metabolism or lysis
• These drugs have specificity for a particular
  microbial group
• based on differences in types of lipids in their
  cell membranes
• Polymyxins
• interact with phospholipids
• cause leakage, particularly in gram-negative
  bacteria
• Amphotericin B and nystatin
• form complexes with sterols on fungal membranes
• causes leakage

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Drug Resistance

• Microorganisms begin to tolerate an amount of
  drug that would ordinarily be inhibitory
• due to genetic versatility or variation
• intrinsic and acquired

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Intrinsic verse Acquired

• Intrinsic resistance
• Microbe must be resistant to antibiotic that they
  produce
• Acquired resistance
• 1. spontaneous mutations in critical chromosomal
  genes
• 2. acquisition of new genes or sets of genes via
  transfer from another species
• originates from resistance factors (plasmids)
  encoded with drug resistance, transposons

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Mechanisms of Drug Resistance

• Drug inactivation by acquired enzymatic activity
• penicillinases
• Decreased permeability to drug or increased
  elimination of drug from cell
• acquired or mutation
• Change in drug receptors
• mutation or acquisition
• Change in metabolic patterns
• mutation of original enzyme

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Antibiotic Resistance in Medical Community

• Improper or excessive use of antibiotics causes
  antibiotic resistance
• Unnecessarily large antibiotic doses
• Allow resistant strains to overgrow susceptible
  ones
• If resistant strains spread to other patients, a
  superinfection occurs
• Antibiotics are available over the counter in
  developing countries
• allows for overuse and incorrect use
• Antibiotic use is widespread in livestock feeds
• can be transmitted to humans through meat
  consumption

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Side Effects of Drugs

• 5 of all persons taking antimicrobials will
  experience a serious adverse reaction to the drug
  
• Toxicity to organs
• Allergic responses
• Suppression and alteration of microflora

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Considerations in Selecting an Antimicrobial Drug

• 1. Identify the microorganism causing the
  infection
• Specimens should be taken before antimicrobials
  initiated
• 2. Test the microorganisms susceptibility
  (sensitivity) to various drugs in vitro when
  indicated
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• 3. Overall medical condition of the patient

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Testing for Drug Susceptibility

• Essential for groups of bacteria commonly showing
  resistance
• Kirby-Bauer disk diffusion test

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Dilution tests

• Minimum inhibitory concentration (MIC)
• smallest concentration of drug that visibly
  inhibits growth
• In vitro activity of a drug is not always
  correlated with in vivo effect
• If therapy fails, a different drug, combination
  of drugs, or different administration must be
  considered
• Best to chose a drug with highest level of
  selectivity but lowest level toxicity

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What about viruses?!?!?

• Do not destroy their target pathogen
• Instead they inhibit their development
• Inhibit virus before enters cell
• Viral-associated proteins
• Stop it from entering the cell
• Stop it from reproducing
• Prevent from exiting the cell