Sterilization, Disinfection and
Antibacterial Agents
王淑鶯
微生物免疫學所
國立成功大學醫學院
分機: 5634
Email: sswang23@mail.ncku.edu.tw
Outline
Definition of Sterilization and Disinfection
Physical and Chemical Methods of Antimicrobial Control
Antibiotics and Mechanisms of Antimicrobial Action
Reference:
Chapters 8 & 20 in Medical Microbiology
(Murray, P. R. et al; 6th edition)
Joseph Lister (5 April 1827 – 10
February 1912) was an
English surgeon and a pioneer
of antiseptic surgery, who
promoted the idea of sterile
surgery. Lister successfully
introduced carbolic acid
to sterilize surgical instruments
and to clean wounds, which led
to reduced post-operative
infections and made surgery
safer for patients.
http://en.wikipedia.org/wiki/Joseph_Lister,_1st_Baron_Lister
While he was a professor of surgery at the University of
Glasgow, Lister became aware of a paper published by
the French chemist Louis Pasteur showing that rotting
and fermentation could occur under anaerobic conditions
if micro-organisms were present. Pasteur suggested
three methods to eliminate the microorganisms
responsible for gangrene: filtration, exposure to heat, or
exposure to chemical solutions. Lister confirmed
Pasteur's conclusions with his own experiments and
decided to use his findings to develop antiseptic
techniques for wounds. As the first two methods
suggested by Pasteur were inappropriate for the
treatment of human tissue, Lister experimented with the
third.
http://en.wikipedia.org/wiki/Joseph_Lister,_1st_Baron_Lister
Carbolic acid (phenol) had been in use as a means of
deodorising sewage, so Lister tested the results of
spraying instruments, the surgical incisions, and
dressings with a solution of it. Lister found that carbolic
acid solution swabbed on wounds remarkably reduced
the incidence of gangrene.
http://en.wikipedia.org/wiki/Joseph_Lister,_1st_Baron_Lister
Lister also noticed that midwife-delivered babies had a
lower mortality rate than surgeon-delivered babies,
correctly attributing this difference to the fact that
midwives tended to wash their hands more often than
surgeons, and that surgeons often would go directly from
one surgery, such as draining an abscess, to delivering a
baby. He instructed surgeons under his responsibility to
wear clean gloves and wash their hands before and after
operations with 5% carbolic acid solutions.
http://en.wikipedia.org/wiki/Joseph_Lister,_1st_Baron_Lister
Before then:
• Nosocomial infections caused death in
10% of surgeries.
• Up to 25% mothers delivering in hospitals
died due to infection.
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
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.
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
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.
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
air filters
antimicrobial
chemicals
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.
Rate of Microbial Death
When bacterial populations are heated or treated
antimicrobial chemicals, they usually die at a constant
rate.
Physical Methods of Microbial Control
heat
filtration
radiation
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
Physical Methods of Microbial Control
Moist Heat: Kills microorganisms by coagulating
their proteins.
u 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.
u In general, moist heat is much more effective than
dry heat.
Physical Methods of Microbial Control
Moist Heat (Continued):
Reliable sterilization with moist heat requires
temperatures above that of boiling water.
u Autoclave: Chamber which is filled with hot steam under
pressure. Preferred method of sterilization, unless material
is damaged by heat, moisture, or high pressure.
u Temperature of steam reaches 121oC at twice
atmospheric pressure.
u All organisms and endospores are killed within 15
minutes.
Autoclave: Closed Chamber with High
Temperature and Pressure
Physical Methods of Microbial Control
Moist Heat (Continued):
u Pasteurization: Developed by Louis Pasteur to prevent
the spoilage of beverages. Used to reduce microbes
responsible for spoilage of beer, milk, wine, juices, etc.
u Classic Method of Pasteurization: Milk was
exposed to 65oC for 30 minutes.
u High Temperature Short Time Pasteurization
(HTST): Used today. Milk is exposed to 72oC for 15
seconds.
Physical Methods of Microbial Control
Dry Heat:
u Direct Flaming: Used to sterilize inoculating loops and
needles. Heat metal until it has a red glow.
u Incineration: Effective way to sterilize disposable items
(paper cups, dressings) and biological waste.
u 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.
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.
u Membrane Filters: Uniform pore size. Used in industry and
research. Different sizes:
u 0.22 and 0.45um Pores: Used to filter most bacteria. Don’t
retain spirochetes, mycoplasmas and viruses.
u 0.01 um Pores: Retain all viruses and some large proteins.
u High Efficiency Particulate Air Filters (HEPA): Used in operating
rooms to remove bacteria from air.
Physical Methods of Microbial Control
Filtration
used for heat sensitive
fluids
air
Physical Methods of Microbial Control
Low Temperature: Effect depends on microbe and
treatment applied.
u Refrigeration: Temperatures from 0 to 7oC. Bacteriostatic effect.
Reduces metabolic rate of most microbes so they cannot
reproduce or produce toxins.
u Freezing: Temperatures below 0oC.
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:
u Neisseria gonnorrhea: Only survives about one hour.
u Mycobacterium tuberculosis: May survive several months.
u Viruses are fairly resistant to desiccation.
u Clostridium spp. and Bacillus spp.: May survive decades.
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.
u Yeasts and molds: More resistant to high osmotic pressures.
u Staphylococci spp. that live on skin are fairly resistant to high osmotic
pressure.
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.
Forms of Radiation
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. Doesn’t
penetrate paper, glass, and cloth.
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.
Chemical Methods of Microbial Control
Types of Disinfectants
1. Phenols and Phenolics:
u
u
u
u
Phenol (carbolic acid) was first used by Lister as a
disinfectant.
u Rarely used today because it is a skin irritant and has
strong odor.
Phenolics are chemical derivatives of phenol
u Cresols (Lysol): Derived from coal tar.
u Bisphenols: Effective against gram-positive
staphylococci and streptococci. Excessive use in infants
may cause neurological damage. E.g. hexachlorophene
Destroy plasma membranes and denature proteins.
Advantages: Stable, persist for long times after applied,
and remain active in the presence of organic compounds.
Chemical Methods of Microbial Control
Types of Disinfectants
2. Halogens: Effective alone or in compounds.
A. Iodine:
u Iodine tincture (alcohol solution) was one of first
antiseptics used.
u Precipitates proteins and oxidizes essential enzymes
B. Chlorine:
u When mixed in water forms hypochlorous acid:
Cl2 + H2O ------>
H+ + Cl- + HOCl
Hypochlorous acid
u
Used to disinfect drinking water, pools, and sewage.
Chemical Methods of Microbial Control
Types of Disinfectants
3. Alcohols:
u
u
u
u
Kill bacteria, fungi, but not endospores or 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.
u Ethanol: Drinking alcohol. Optimum concentration is
70%.
u
Isopropanol: Rubbing alcohol. Better disinfectant than
ethanol. Also cheaper and less volatile.
Chemical Methods of Microbial Control
Types of Disinfectants
4. Quaternary Ammonium Compounds (Quats):
u
u
u
u
Cationic (positively charge) compound acts like
detergents.
Denatures cell membranes
Effective against gram positive bacteria, less effective
against gram-negative bacteria.
Benzalkonium chloride, cetylpyridinium chloride
Chemical Methods of Microbial Control
Types of Disinfectants
5. Aldehydes:
Include some of the most effective antimicrobials.
u Inactivate proteins by forming covalent crosslinks with
several functional groups.
A. Formaldehyde:
u Excellent disinfectant, 2% aqueous solution.
u Commonly used as formalin, a 37% aqueous solution.
u Formalin was used extensively to preserve biological
specimens and inactivate viruses and bacteria in
vaccines.
u Irritates mucous membranes, strong odor.
u
Chemical Methods of Microbial Control
Types of Disinfectants
5. Aldehydes:
B. Glutaraldehyde:
u Less irritating and more effective than formaldehyde.
6. Gaseous Sterilizers:
Chemicals that sterilize in a chamber similar to an autoclave.
u Denature proteins, by replacing functional groups with alkyl
groups.
Ethylene Oxide:
u Kills all microbes and endospores, but requires exposure of
4 to 18 hours.
u Commonly used to disinfect hospital instruments
u
Chemical Methods of Microbial Control
Types of Disinfectants
7. Oxidizing Agents:
Oxidize cellular components of treated microbes.
u
Disrupt membranes and proteins.
A. Ozone:
u Used along with chlorine to disinfect water.
u Helps neutralize unpleasant tastes and odors.
u More effective killing agent than chlorine, but less stable and more
expensive.
u Highly reactive form of oxygen.
u Made by exposing oxygen to electricity or UV light
B. Hydrogen Peroxide:
u Not good for open wounds because quickly broken down by catalase
present in human cells.
u
Effective in disinfection of inanimate objects
u
Outline
Definition of Sterilization and Disinfection
Physical and Chemical Methods of Antimicrobial Control
Antibiotics and Mechanisms of Antimicrobial Action
Definition of an Antibiotic
Substance produced by a microorganism or a similar
product produced wholly (synthetic) or partially (semisynthetic) by chemical synthesis and in low
concentrations inhibits the growth of or kills
microorganisms.
Microbial
Sources of
Antibiotics
Antibiotic Spectrum of Activity
No antibiotic is effective against all microbes
Mechanisms of Antimicrobial Action
Bacteria have their own enzymes for
– Cell wall formation
– Protein synthesis
– DNA replication
– RNA synthesis
– Synthesis of essential metabolites
Modes of Antimicrobial Action
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
β- lactam drugs
Vancomycin
Bacitracin
Antibacterial Antibiotics
Inhibitors of Cell Wall Synthesis
Penicillins and Cephalosporins
– Part of group of drugs called β –
lactams
Have shared chemical structure
called β-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.
Antibacterial Antibiotics
Inhibitors of Cell Wall Synthesis
The weakness in the cell wall
causes the cell to lyze.
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
Antibacterial Antibiotics
Inhibitors of Cell Wall Synthesis
Cephalosporins
– chemical structures make them resistant to
inactivation by certain β-lactamases
– most effective against Gram – bacteria.
– chemically modified to produce family of related
compounds
2nd, 3rd, and 4th generations more effective against
gram-negatives
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
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
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
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 β-lactam drugs
Allows aminoglycosides to enter cells that are often
resistant
– Side effects
Nephrotoxicity
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
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
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
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
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
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
Mechanisms of Antibiotic Resistance
Enzymatic destruction of drug
– Some organisms produce
enzymes that chemically modify
drug
Penicillinase breaks β-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
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
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
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.
Summary
Antimicrobial control
– physical methods
– chemical methods
Antibiotics
– mechanisms of action