Download Chemotherapeutic Agents

Control of Microorganisms by Physical and Chemical
Agents
What is the meaning of sterilization?
 It is the complete destruction or removal of all living organism including viruses,
bacteria, fungi and their spores.
 Cleaning : is a process intended to remove foreign material, like dust, dirt, organic
materials including microorganism.

 Disinfection is the destruction of infectious organism( not including bacterial spores)
by using chemical ( disinfectants and antiseptics. It results in reduction of the
contaminating organism.
 -Disinfectant are toxic and irritant material and are used for control of
microorganism ( on the floors for example)
 - Antiseptic are less irritant and less toxic and used for disinfection of body surfaces (
living tissues).
 Preservation: is the prevention of multiplication of microorganism in the formulated
products including pharmaceuticals and foods.
Figure 8.1-Microbial Control Methods
Definition of Frequently Used Terms in Microbial control methods
• Sterilization
• destruction or removal of all viable organisms from an object or habitat.
• Disinfection
• killing, inhibition, or removal of pathogenic organisms that may cause
disease:substantial reduction of total population.
• Disinfectants
• agents, usually chemical, used for disinfection; not necessary kills viable spores
• Sanitization
• reduction of microbial population to levels deemed safe (based on public health
standards)
• Antisepsis
• prevention of infection or sepsis. of living tissue by microorganisms using
antiseptics.
• antiseptics
• chemical agents that kill or inhibit growth of microorganisms when applied to
tissue-should not be toxic as disinfectants to kill host tissues.
Antimicrobial Agents
• agents that kill microorganisms or inhibit their growth e;g Chemotherapy- chemical
agent to kill or inhibit growth of microorganisms within host tissues.
• -cidal agents to kill
• -static agents to inhibit growth
• cide
• suffix indicating that agent kills
• germicide
• kills pathogens and many nonpathogens but not necessarily endospores
• include bactericides, fungicides, algicides, and viricides
• -static
• suffix indicating that agent inhibits growth
• include bacteriostatic and fungistatic
The Pattern of Microbial Death
• A microbial population is not killed instantly
• population death usually occurs exponentially as growth rate.
• 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.
• Decimal Reduction Time (D):
• D is the time required to kill 90% of the microorganisms or spores in a sample
under specified conditions.
• Microorganisms is defined as dead when they don’t grow or reproduce
when inoculated in culture medium.
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. Bacterial spores are much more resistant to Microbial agents
e.g Mycobacterium tuberculosis.
Concentration or intensity of an antimicrobial agent:
 usually higher concentrations or intensities kill more rapidly
 relationship is not linear
Duration of exposure:
longer exposure to antimicrobial agents more organisms killed.
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. Organic matter in biofilms protects biofilm
microorganisms.
The Use of Physical Methods in Control

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Heat
Low temperatures
Filtration
Radiation
Moist Heat Sterilization
 must be carried out above 100oC which requires
saturated steam under pressure
 Moist heat destroys viruses, bacteria and fungi
 carried out using an autoclave (Fig8.3) also known
as Steam Sterilizer
 effective against all types of microorganisms
including spores
 degrades nucleic acids, denatures proteins, and
disrupts membranes
The Autoclave or Steam Sterilizer
Figure 8.3-Autoclave Steam Sterilizer
Table 8.2
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 .
Pasteurization
 controlled heating at temperatures well below boiling- Louis Pasteur
 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.
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.
Filtration
 reduces microbial population or sterilizes solutions of heat-sensitive
materials by removing microorganisms
 also used to reduce microbial populations in air
Filtering Liquids
Depth filters
 thick fibrous or granular materials bonded in thick layers
that remove microorganisms by physical screening(size),
entrapment, and/or adsorption to the surface of the filter
materials.
 Solution contain microorganisms are sucked under vacuum
 membrane filters
 porous membranes with defined pore sizes that remove
microorganisms primarily by physical screening. This has
replaced Depth Filters.
Figure 8.5-Membrane Filter Sterilisation
Figure 8.6-Membrane Filter
Filtering Air
 surgical masks used in
hospitals and Labs
 cotton plugs on
culture vessels
 high-efficiency
particulate air (HEPA)
filters used in laminar
flow biological safety
cabinets (remove
99.97% of particles)
Figure 8.7 (a)-Laminar Floor
Ultraviolet (UV) Radiation
 UV (260nm) quite lethal is
limited to surface sterilization
because it does not penetrate
glass, dirt films, water, and
other substances.
 UV prevent replication and
transcription of Microbial
DNA.
 has been used for water
treatment
Figure 7.9
Ionizing Radiation
 Excellent sterilization agent e.g Gamma 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
Figure 8.8-Sterilization with Ionization; Radiation machine which uses
Cobalt 60 as a Gamma radiation to sterilize fruits, veg, fish, meat, etc..
Chemical Control Agents -Disinfectants and Antiseptics
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
Alcohols
 bactericidal, fungicidal, but not sporicidal
 inactivate some viruses
 denature proteins and possibly dissolve membrane lipids
Halogens
 any of five elements: fluorine, chlorine, bromine, iodine, and astatine
 iodine and chlorine are important antimicrobial agents
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
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
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
Ammonium Compounds
 These are detergents that have antimicrobial activity and are effective disinfectants
 organic molecules (cleansing agents) with hydrophilic and hydrophobic ends for
food utensils, small instruments and skin antiseptics.
 Because of its positively charged nitrogen, cationic detergents/ammonium
compound, are effective disinfectants.
 They disrupt microbial membrane , may denature protein.
 kill most bacteria, but not Mycobacterium tuberculosis or endospores
 safe and easy to use, but inactivated by hard water and soap
 E.g Benzalkonium chloride and Cetylpyridinium
Aldehydes
 highly reactive molecules
 sporicidal and can be used as chemical sterilants
 combine with and inactivate nucleic acids and proteins
 E.g Formal dehyde and glutaraldehyde
Sterilizing Gases
 used to sterilize heat-sensitive materials such as
disposable petri dishes, syringes, heart lung machine
components, sutures, catheters
 microbicidal and sporicidal
 combine with and inactivate proteins
 E.g Ethylene oxide gas (EtO)
Chemical in common use are :
 1-Alcolhol: kill vegetative bacteria only e.g. 75% ( absolute alcohol is not
effective becuase water is important for the denaturation of bacterial
protein). It is used for skin disinfection.
 2- Phenol 2% used as a disinfectant in microbial laboratories.
 3- ethylene oxide gas ; used for the disinfection f plastic and rubber
articles.
 4-halogenes: kill vegetative bacteria, spores and viruses.
 Iodine: used in alcohol for skin antisepsis ( e.g. betadine).
 5- Glutaraldehyde 2 % used for the decontamination of endoscopes.
 6-hydrogen peroxide used in wound cleaning.
 7-formaldehyde gas used as a disinfectant for fumigation of areas
contaminated by infection agents, for rubber, leather, shoes, books.
 8- Quaternary ammonium salts; cetavlon ( soap)
 9- Heavy metals ( e.g. mercury and silver) they cause denaturation of
enzymes and other essential proteins.`
Figure 8.11-Ethylene Oxide Sterilizer
Chemotherapeutic Agents
 chemicals that can be used internally to kill or
inhibit the growth of microbes within host cells
(covered later in book)
 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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CHEMOTHERAPEUTIC AGENTS
Definitions:

Antimicrobial agents are substances that inhibit or kill the growth of microorganisms. Antimicrobial agents that
are produced by microorganisms are naturally occurring chemotherapeutics and were defined by Waxman as
antibiotics.
Examples of naturally occurring chemotherapeutic agents are : penicillins and cephalosporins,
chloroamphinicol, tetracyclines, macrolids, nystatin and griseofulvin.
Examples of synthetics chemotherapeutic agents are: sulphonamides, nitrofuran, isonictinc acid deriivatives,
diaminopyrimidines, quinolones and imidazoles.
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The chemical substance that used as a chemotherapeutic agent must be a selective toxic. By means toxic for the
microorganism and not toxic to the host.
Antibiotics: An antibiotic is a product produced by a microorganism or a similar substance produced wholly or
partially by chemical synthesis, which in low concentrations, inhibits the growth of other microorganisms.

The minimum inhibitory concentration (MIC): is the lowest concentration of an antibiotic that inhibit the
growth of an organism.

Bactericidal antibiotics: are those antibiotics that destroy bacteria, like penicillins, cephalosporins.

Bacteriostatic antibiotics: are those inhibit the multiplication of the bacterial cells. Like tetracyclines and
chloroamphenicol
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Antibacterial spectrum—Range of activity
of an antimicrobial against bacteria. A
broad-spectrum antibacterial drug can
inhibit a wide variety of gram-positive and
gram-negative bacteria, whereas a
narrow-spectrum drug is active only
against a limited variety of bacteria.
Antibiotic combinations—Combinations of
antibiotics that may be used (1) to broaden
the antibacterial spectrum for empiric
therapy or the treatment of polymicrobial
infections, (2) to prevent the emergence of
resistant organisms during therapy, and (3)
to achieve a synergistic killing effect.
Bacteriostatic activity—The level of
antimicrobial activity that inhibits the
growth of an organism. This is determined
in vitro by testing a standardized
concentration of organisms against a
series of antimicrobial dilutions. The
lowest concentration that inhibits the
growth of the organism is referred to as
the minimum inhibitory concentration
(MIC).
Antibiotic synergism—Combinations of
two antibiotics that have enhanced
bactericidal activity when tested together
compared with the activity of each
antibiotic.
Bactericidal
activity—The
level
of
antimicrobial activity that kills the test
organism. This is determined in vitro by
exposing a standardized concentration of
organisms to a series of antimicrobial
dilutions. The lowest concentration that
kills 99.9% of the population is referred to
as
the
minimum
bactericidal
concentration (MBC).
Antibiotic antagonism—Combination of
antibiotics in which the activity of one
antibiotic interferes With the activity of the
other (e.g., the sum of the activity is less
than the activity of the individual drugs).
Beta-lactamase—An
enzyme
that
hydrolyzes the beta-lactam ring in the
beta-lactam class of antibiotics, thus
inactivating the antibiotic. The enzymes
specific for penicillins and cephalosporins
aret
he
penicillinases
and
cephalosporinases,
respectively.
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Susceptibility Tests
1.
Broth dilution -
MIC test
2.
Agar dilution
-
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MIC test
Minimal Inhibitory Concentration (MIC)
vs.
Minimal Bactericidal Concentration (MBC)
32 ug/ml 16 ug/ml 8 ug/ml
Sub-culture to agar medium
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4 ug/ml
2 ug/ml
1 ug/ml
MIC = 8 ug/ml
MBC = 16 ug/ml
Susceptibility Tests
3. Agar diffusion
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Antibiotic Mechanisms of Action
Alteration of
Cell Membrane
Polymyxins
Bacitracin
Neomycin
Transcription
Translation
Translation
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