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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Primary Targets of Control

• Microorganisms capable of causing infection or
  spoilage
• vegetative bacterial cells
• endospores
• fungal hyphae and spores
• yeast
• protozoan trophozoites and cysts
• worms
• viruses
• prions

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Relative Resistance of Microbes

• Highest resistance
• Bacterial endospores
• 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

• Sterilization
• destroys all viable microbes
• including viruses endospores
• Disinfection
• destroy vegetative pathogens
• not endospores
• Sanitization
• cleansing technique that mechanically removes
  microbes
• Degermation
• reduces the number of microbes
• Microbial agents
• Bactericide
• Sporocide
• Fungicide
• Viricide
• Microbistasis
• Bacteriostatic
• Fungistatic

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Microbial Death

• Involves permanent loss of reproductive
  capability
• even under optimum growth conditions
• Still alive, but cant reproduce!!
• Not viable in environment
• Hard to detect
• Loss of movement??

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Factors That Affect Death Rate

• Number of microbes
• Nature of microbes in the population
• Temperature pH of environment
• Concentration or dosage of agent
• Mode of action of the agent
• 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
• Filtration

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1. Heat

• Moist heat use of hot water or steam
• mode of action
• denaturation of proteins
• destruction of membranes
• destruction of DNA
• Sterilization
• Steam under pressue
• Autoclave
• Intermittent sterilization
• unpressurized steam
• 100oC 30-60 min for 3 days
• Disinfection
• boiling at 100oC for 30 minutes
• destroy non-spore-forming pathogens
• Pasteurization

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Pasteurization

• 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 heat - higher temperatures than moist heat
• incineration
• 600-1200oC
• combusts dehydrates cells
• dry ovens
• 150-180oC
• coagulate proteins
• 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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5. Filtration

• physical removal of microbes by passing a gas or
  liquid through filter
• used to sterilize heat sensitive liquids air in
  hospital isolation units industrial clean rooms

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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

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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 (25)
• 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

• Amonia 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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Antimicrobial Therapy
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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
• 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
• 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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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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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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Targets of Antimicrobial Drugs

• Inhibition of cell wall synthesis
• Inhibition of nucleic acid synthesis, structure
  or function
• Inhibition of protein synthesis
• 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
• Antimicrobics usually have a selective action
  against prokaryotes
• can also damage the eukaryotic mitochondria
• 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
• 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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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

• Identify the microorganism causing the infection
• Specimens should be taken before antimicrobials
  initiated
• Test the microorganisms susceptibility
  (sensitivity) to various drugs in vitro when
  indicated
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• 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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Major Antimicrobial Drug Groups

• Antibacterial drugs
• Antibiotics
• Synthetic drugs
• Antifungal drugs
• Antiparasitic drugs
• Antiviral drugs
• About 260 different antimicrobial drugs are
  classified in 20 drug families

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Antimicrobial Drug Groups

• Act on cell wall
• Penicillin
• Cephalosporin
• Non-beta-lactam
• Damage cell membranes
• Polymixins
• Act on DNA and RNA
• Fluoroquinolones
• Interfere with protein synthesis
• Aminoglycosides
• Tetracycline
• Chloramphenicol
• Block metabolic pathways
• Sulfonamides

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Penicillins

• More economical to obtain natural penicillin
  through microbial fermentation
• Then modify it to semi-synthetic forms
• Penicillium chrysogenum
• major source
• Differ in side chains

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Penicillins

• Penicillins G and V most important natural forms
• Drug of choice for gram-positive cocci
  (streptococci)
• Some gram-negative bacteria (meningococci and
  syphilis spirochete)
• Semisynthetic penicillins
• ampicillin, carbenicillin amoxicillin have
  broader spectra
• gram negative enterics rods
• Penicillinase-resistant
• methicillin, nafcillin, cloxacillin
• Primary problems
• allergies and resistant strains of bacteria

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Cephalosporins

• Account for majority of all antibiotics
  administered
• Isolated from Cephalosporium acremonium mold
• Beta-lactam ring that can be altered
• Relatively broad-spectrum
• resistant to most penicillinases
• cause fewer allergic reactions
• Some are given orally

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Four Generations of Cephalosporins

• First generation
• cephalothin, cefazolin
• most effective against gram-positive cocci
• Second generation
• cefaclor, cefonacid
• more effective against gram-negative bacteria
• Third generation
• cephalexin, cefotaxime
• broad-spectrum activity against enteric bacteria
  with beta-lactamases
• Fourth generation
• Cefepime
• Effective against gram positive and negative

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Polymixins

• Source is Bacillus polymyxa
• Damages cell membrane
• Can treat drug-resistant organisms
• Pseudomonas
• Severe urinary tract infections

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Aminoglycosides

• composed of 2 or more amino sugars
• and an aminocyclitol (6C) ring
• products of various species of soil actinomycetes
  in genera Streptomyces Micromonospora
• Uses
• broad-spectrum
• inhibit protein synthesis
• especially useful against aerobic gram-negative
  rods certain gram-positive bacteria
• Streptomycin bubonic plague, tularemia, TB
• Gentamicin less toxic, used against
  gram-negative rods

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Fluoroquinolones

• Acts on DNA or RNA
• Readily absorbed from intestines
• Binds gyrase
• Quinolones, ciprofloxacin

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Tetracycline Antibiotics

• Broad-spectrum, block protein synthesis
• Doxycycline minocycline
• oral drugs taken for STDs, Rocky Mountain spotted
  fever, Lyme disease, typhus, acne protozoa

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Chloramphenicol

• Isolated from Streptomyces venezuelae
• Blocks peptide bond formation
• No longer derived from natural source
• Very toxic, restricted uses, can cause
  irreversible damage to bone marrow
• Typhoid fever, brain abscesses, rickettsial
  chlamydial infections

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Antifungal Drugs

• Macrolide polyene
• Amphotericin B
• mimic lipids, most versatile effective, topical
  systemic treatments
• Nystatin
• topical treatment
• Griseofulvin
• stubborn cases of dermatophyte infections,
  nephrotoxic
• Synthetic azoles
• broad-spectrum
• ketoconazole, clotrimazole, miconazole
• Flucytosine
• analog of cytosine cutaneous mycoses or in
  combination with amphotericin B for systemic
  mycoses
• Echinocandins
• Damage cell walls

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Antiparasitic Drugs

• Antimalarial drugs
• quinine, chloroquinine, primaquine, mefloquine
• Antiprotozoan drugs
• Metronidazole (Flagyl), quinicrine, sulfonamides,
  tetracyclines
• Antihelminthic drugs
• immobilize, disintegrate, or inhibit metabolism
• mebendazole, thiabendazole
• broad-spectrum
• inhibit function of microtubules
• interfers with glucose utilization disables
  them
• pyrantel, piperazine
• paralyze muscles
• niclosamide
• destroys scolex

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Antiviral Drugs

• Block penetration into host cell
• Block transcription or translation
• Nucleotide analogs
• Acyclovir herpesviruses
• Ribavirin- a guanine analog RSV, hemorrhagic
  fevers
• AZT thymine analog - HIV
• Prevent maturation of viral particles
• Protease inhibitors HIV

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