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Physical and Chemical Control of
Microorganisms
Controlling Microorganisms Reduce or destroy undesirable microbes in a given area
Physical Chemical Mechanical
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
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 object’s surface
Terms for Microbial Control Microbicidal agents
Causes microbial death Bactericide Sporocide Fungicide Viricide
Microbistasis Prevents microbial growth Bacteriostatic Fungistatic
Factors That Affect Death Rate1. Number of microbes2. Nature of microbes in the population3. Temperature & pH of environment4. Concentration or dosage of agent5. Mode of action of the agent6. Presence of solvents, organic matter, or
inhibitors
Cellular Targets of Control
Mode of action of antimicrobials:
Cell wall Cell membrane Cellular synthetic
processes (DNA, RNA) Proteins
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?
Methods of Physical Control1. Heat – Moist verse Dry2. Cold temperatures3. Desiccation4. Radiation
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!
Autoclave Tape
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
1. Heat (Moist)
Pasteurization heat applied to kill potential agents of
infection and spoilage without destroying the food flavor or value 63°C - 66°C for 30 minutes
batch method 71.6°C 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
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
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
2. Cold Temperatures Microbistatic
slows the growth of microbes
refrigeration 0-15oC freezing <0oC used to preserve food,
media and cultures
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
4. Radiation1. Ionizing radiation
deep penetrating power breaks DNA gamma rays, X-rays, cathode rays used to sterilize medical supplies & food
products
2. Nonionizing radiation little penetrating power to sterilize air, water &
solid surfaces UV light creates thymine pyrmidines
interfere with replication
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
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
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)
Chemical Control Of Microbial Agents
1. Halogens2. Phenolics3. Chlorhexidine4. Alcohols5. Hydrogen peroxide6. Detergents & soaps7. Heavy metals8. Aldehydes9. Gases and aerosols
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
2. Phenolics Disrupt cell
membranes & precipitating proteins bactericidal, fungicidal,
virucidal, not sporicidal Lysol Triclosan
antibacterial additive to soaps
3. Chlorhexidine A surfactant & protein denaturant with
broad microbicidal properties Not sporicidal Used as skin degerming agents for
preoperative scrubs, skin cleaning & burns
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
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
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
7. Heavy Metals Solutions of silver &
mercury kill vegetative cells in
low concentrations by inactivating proteins
Oligodynamic action Not sporicidal
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
9. Gases & Aerosols Ethylene oxide, propylene oxide,
betapropiolactone & chlorine dioxide Strong alkylating agents, sporicidal
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
Antimicrobial Therapy
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
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
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
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
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
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
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
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
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
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
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
Drug Resistance Microorganisms begin to tolerate an
amount of drug that would ordinarily be inhibitory due to genetic versatility or variation intrinsic and acquired
Intrinsic verse Acquired Intrinsic resistance
Microbe must be resistant to antibiotic that they produce
Acquired resistance:1. spontaneous mutations in critical
chromosomal genes2. acquisition of new genes or sets of genes via
transfer from another species originates from resistance factors (plasmids) encoded
with drug resistance, transposons
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
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
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
Considerations in Selecting an Antimicrobial Drug
1. Identify the microorganism causing the infection Specimens should be taken before
antimicrobials initiated
2. Test the microorganism’s susceptibility (sensitivity) to various drugs in vitro when indicated (Next slide)
3. Overall medical condition of the patient
Testing for Drug Susceptibility Essential for groups of
bacteria commonly showing resistance
Kirby-Bauer disk diffusion test
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
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