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II. Microbial Control

1. Factors Affecting Microbial Growth

Microbial growth is affected by many different environmental factors, including the availability of nutrients and
moisture, temperature, pH, osmotic pressure, barometric pressure, and composition of the atmosphere. These
environmental factors affect microorganisms in their daily lives and play important roles in the control of
microorganisms in laboratory, industrial, and hospital settings. Whether scientists wish to encourage or inhibit
the growth of microorganisms, they must first understand the fundamental needs of microbes.

Microbes are exposed to a wide variety of environmental factors in addition to nutrients. Microbial
ecology focuses on ways that microorganisms deal or adapt to such factors as heat, cold, gases, radiation,
osmotic and hydrostatic pressures, and even other microbes. Adaptation is a complex adjustment in
biochemistry or genetics that enables long-term survival and growth. For most microbes, environmental
factors fundamentally affect the function of metabolic enzymes. Thus, survival in a changing environment is
largely a matter of whether the enzyme systems of microorganisms can adapt to alterations in their habitat
(textbook, pp. 192-193).

1. Availability of Nutrients – all living organisms require nutrients–the various chemical compounds
that organisms use to sustain life. Therefore, to survive in a particular environment, appropriate
nutrients must be available. Many nutrients are energy sources; organisms will obtain energy from
these chemicals by breaking chemical bonds. Nutrients also serve as sources of carbon, oxygen,
hydrogen, nitrogen, phosphorus, and sulphur as well as other elements (e.g., sodium, potassium,
chlorine, magnesium, calcium, and trace elements such as iron, iodine, and zinc) that are usually in
lesser amounts.

2. Moisture – on earth, water is essential for life. Cells consist between 70 to 95% water. All living
organisms require water to carry out their normal metabolic processes, and most will die in
environments containing too little moisture. There are certain microbial stages (e.g., bacterial
endospores and protozoan cysts), however, that can survive the complete drying process
(desiccation). The organisms contained within the spores and cysts are in a dormant resting state; if
they are placed in a moist, nutrient-rich environment, they will grow and reproduce normally.

3. Temperature – most microorganisms grow well at the temperatures favored by humans. However,
certain bacteria are capable of growing at extremes of temperature that would certainly hinder the
survival of almost all eukaryotic organisms.

Microorganisms are classified into three primary groups on the basis of their preferred range of
temperatures: psychrophiles (cold-loving microbes), mesophiles (moderate-temperature-loving
microbes), and thermophiles (heat-loving microbes). Most bacteria grow only within a limited range
of temperatures, and their maximum and minimum growth temperatures are only about 30 oC apart.
They grow poorly at the temperature extremes within their range.

A psychrophile is a microorganism that has an optimum temperature below 15 0C and is capable of

growth at 0°C. It is obligate with respect to cold and generally cannot grow above 20°C. The habitats
of psychrophilic bacteria, fungi and algae are lakes and rivers, snowfields, polar ice, and the deep
ocean. Rarely, if ever, they are pathogenic.

Refrigeration is the most common method of preserving household food supplies. It is based on the
principle that microbial reproductive rates decrease at low temperatures. Although microbes usually
survive even subfreezing temperatures, they gradually decline in number. Psychrotrophs or
facultative psychrophiles grow slowly in cold but have an optimum temperature above 20°C. These
organisms grow best at refrigerator temperature; given time, they are able to slowly degrade food.
Such spoilage might take on the form of a mold mycelium, slime on food surfaces, or off-tastes of off-
colors in foods. The temperature inside a properly set refrigerator will greatly slow the growth of most
spoilage of organisms and will entirely prevent the growth of all but a few pathogenic bacteria. Bacteria
such as Staphylococcus aureus and Listeria monocytogenes are a concern because they can grow in
refrigerated food and cause food-borne illness.

The majority of medically significant microorganisms are mesophiles, organisms that grow at
intermediate temperatures. Although an individual species can grow at the extremes of 10°C or 50°C,
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the optimum growth temperature (optima) of most mesophiles fall into the range of 20°C to 40°C.
Organisms in this group inhabit animals and plants as well as soil and water in temperate, subtropical,
and tropical regions. Most human pathogens have optima somewhere between 30°C and 40°C (human
body temperature is 37°C). Thermoduric microbes, which can survive short exposure to high
temperatures but are normally mesophiles, are common contaminants of heated or pasteurized foods.
Examples include heat-resistant cysts such as Giardia or spore-formers such as Bacillus and
Clostridium.

A thermophile is a microbe that grows optimally at temperatures greater than 45°C. Such heat-loving
microbes live in soil and water associated with volcanic activity, in compost piles, and in habitats
directly exposed to the sun. Thermophiles vary in heat requirements, with a general range of growth of
45C to 80°C. Most eukaryotic forms cannot survive above 60°C, but a few thermophilic bacteria, called
hyperthermophiles, grow between 80°C and 120°C (currently thought to be the temperature limit
established by enzymes and cell structures). Strict thermophiles are so heat tolerant that researchers
may use autoclave to isolate them in cultures.

4. Gases – microorganisms vary with respect to the type of gaseous atmosphere that they require . An
aerobe (aerobic organism) can use gaseous oxygen in its metabolism and possesses the enzymes
needed to process toxic oxygen products.

Examples:
1. Bacillus subtilis
2. Bordetella pertussis
3. Mycobacterium leprae
4. Mycobacterium tuberculosis
5. Neisseria gonorrhoeae
6. Neisseria meningitidis
7. Pseudomonas spp.

An organism that cannot grow without oxygen is an obligate aerobe. Most fungi and protozoa, as
well as many bacteria (genera Micrococcus and Bacillus), have to have oxygen in their metabolism. A
facultative anaerobe is an aerobe that does not require oxygen for its metabolism and is capable of
growth in the absence of it. This type of organism metabolizes by aerobic respiration when oxygen is
present, but in its absence, adopts an anaerobic mode of metabolism such as fermentation. A large
number of bacterial pathogens fall into this group (for example, gram-negative intestinal bacteria and
staphylococci).

Notes: Facultative anaerobe - a microorganism that can live and grow with or without
molecular oxygen.
Obligate anaerobe - an organism that can grow only in the complete absence of molecular oxygen.
Examples:
1. Bacillus anthracis
2. Corynebacterium diphtheriae
3. Escherichia coli
4. Gardnerella vaginalis
5. Hemophilus influenzae
6. Klebsiella spp.
7. Lactobacillus spp. (can also be microaerophilic)
8. Mycoplasma pneumoniae
9. Salmonella spp.
10. Shigella spp.
11. Staphylococcus aureus
12. Streptococcus spp.
13. Vibrio cholerae

A microaerophile does not grow at normal atmospheric concentrations of oxygen but requires a small
amount of it in metabolism. Most organisms in this category live in a habitat (soil, water, or the human
body) that provides small amounts of oxygen but is not directly exposed to the atmosphere.

Examples:
1. Helicobacter pylori
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2. Lactobacillus spp. (can also be a facultative anaerobe)

3. Treponema pallidum (can also be an anaerobe)

An anaerobe (anaerobic microorganism) lacks the metabolic enzyme systems for using oxygen in
respiration. Because strict, or obligate anaerobes also lack enzymes for processing toxic oxygen,
they cannot tolerate any free oxygen in the environment and will die if exposed to it. Strict anaerobes
live in highly reduced habitats such as deep muds, lakes, oceans, and soil. Even though human cells
use oxygen and oxygen is found in the blood and in tissues, some body sites present anaerobic pockets
or microhabitats where colonization or infection can occur. One region that is an important site for
anaerobic infections is the oral cavity. Dental caries are partly due to the complex actions of aerobic
and anaerobic bacteria, and most gingival infections consist of similar mixtures of oral bacteria that
have invaded damaged gum tissues. Another common site for anaerobic infections is the large
intestine, a relatively oxygen-free habitat that harbors a rich assortment of strictly anaerobic bacteria.
Anaerobic infections can occur following abdominal surgery and traumatic injuries (gas gangrene and
tetanus). Bacteria which are incapable of survival in the presence of O 2 are included Clostridium
botulinum and Clostridium tetani.

An aerotolerant anaerobes do not utilize oxygen but can survive and grow to a limited extent in its
presence. These anaerobes are not harmed by oxygen, mainly because they possess alternative
mechanisms for breaking down peroxide and superoxide (lactobacilli and streptococci).

5. pH – most microorganisms prefer a neutral or slightly alkaline growth medium (pH 7.0 to 7.4), but
acidophilic microbes (acidophiles) prefer a pH of 2 to 5. Because many molds and yeasts tolerate
moderate acid, they are the most common spoilage agents of pickled foods. Also, these organisms can
live in the stomach. Fungi prefer acidic environments. Most human pathogens are neutrophiles. An
exception is Vibrio cholerae which is an alkalinophile.

pH range classifications include:

1. acidophile (pH < 5.4)
2. neutrophile (pH 5.4 - 8.5)
3. alkalinophile (pH 7.0 - 11.5)

6. Osmotic pressure – when a microbial cell is in a solution that has a higher concentration of
solutes than in the cell (hypertonic), the cellular water passes out through the plasma membrane to
the high solute concentration. This osmotic loss of water causes plasmolysis, or shrinkage of the cell’s
plasma membrane.

The importance of this phenomenon is that the growth of the cell is inhibited as the plasma membrane
pulls away from the cell wall. Thus, the addition of salts (or other solutes) to a solution, and the
resulting increase in osmotic pressure, can be used to preserve foods. Salted fish, honey, and
sweetened condensed milk are preserved largely by this mechanism; the high salt or sugar
concentrations draw water out of any microbial cells that are present and thus prevent their growth.
These effects of osmotic pressure are roughly related to the number of dissolved molecules and ions in
a volume of solution.

Sugar solutions for jellies and pickling brines (salt solutions) for meats preserve these foods by
inhibiting the growth of most microorganisms. However, some types of molds and bacteria can survive
and even grow in a salty environment.

If a bacterial cell is placed in a hypotonic solution (such as distilled water), the cell may not burst
(because of rigid cell wall), but the fluid pressure within the cell increases greatly. This increased
pressure occurs in cells having rigid cell walls such as plant cells and bacteria. If the pressure becomes
so great that the cell ruptures, the escape of cytoplasm from the cell is referred to as plasmoptysis.
In an isotonic environment, excess water neither leaves nor enters the cell, and thus, no plasmolysis or
plasmoptysis occurs; the cell has normal turgor (fullness).

Most microbes exist under hypotonic or isotonic conditions. The exceptions are organisms adapted to
growth at relatively high and very high salt concentrations, which are referred to as facultative and
extreme halophiles, respectively. Facultative halophiles are remarkably resistant to salt, even
though they do not normally reside in high-salt environments. For example, S. aureus can grow on
NaCl media ranging from 0.1% up to 20%. Although it is common to use high concentrations of salt
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and sugar to preserve food (jellies, syrups, and brines) many bacteria and fungi actually thrive under
these conditions and are common spoilage agents. Molds and yeasts tend to be much more resistant to
high or low osmotic pressures than are bacteria. This is one reason that molds, but not bacteria, tend
to be the spoilers of fruits and grains.

7. Type of microbe – disinfectants and antiseptics have a greater effect on gram–positive bacteria.
8. Physiological state of microbe – growing cells are more susceptible than the older cells.

9. Environment - organic matter interferes with the action of the chemical agent. Pus, feces, vomitus,
mucus and blood contain proteins that serve as protective coating to insulate the pathogens.

Terminologies Related to Microbial Control

 Sterilization – the destruction of ALL viable microorganisms, including viruses. Any material that has
been subjected to this process is said to be sterile.
 Disinfection – the destruction or removal of pathogens from nonliving objects by physical or chemical
methods. It destroys most microbial life (except bacterial endospores), reducing contamination on
inanimate surfaces. A disinfectant is a disinfecting chemical agent, normally applied to non-living
material because in the concentrations required to be effective, they can be toxic to living tissue, such
as bedside equipment and operating rooms.
 Asepsis – a condition free of viable pathogenic microorganisms (such as bacteria, viruses, fungi, and
parasites).
The term asepsis refers to any practice (practices used to promote or induce asepsis in an
operative field in surgery or medicine) that prevents the entry of infectious agents into sterile tissues
and thus prevents infection. Ideally, a field is "sterile" — free of all biological contaminants, not just
those that can cause disease, putrefaction, or fermentation — but that is a situation that is difficult to
attain. However, elimination of infection is the goal of asepsis, not sterility.
 Antisepsis – the prevention of infection by inhibiting growth of pathogens. It is the same as
disinfection, except a living surface is involved. It also refers to the use of chemical treatments to kill or
inhibit the growth of all vegetative microorganisms on body surfaces. An antiseptic is a growth-
inhibiting agent used on skin and other living tissues, especially wounds, to prevent infection.
 Decontamination – the mechanical removal of most microbes from an animate or inanimate surface.
 Sanitization – any cleansing technique that mechanically removes microorganisms as well as other
debris to reduce contamination to safe levels. An example is the cleaning of pathogenic
microorganisms from public eating utensils and objects such as that done by the kitchen of a
restaurant. Cooking utensils, dishes, bottles, cans, and used clothing that have been washed and dried
may not be completely free of microbes, but they are considered safe for normal use (sanitary). A
sanitizer is a compound such as soap or detergent used to perform this task.
 Degerming – (or degermation) the removal (or reduction of the numbers) of microorganisms from
the skin, i.e., as in cleaning the skin prior to injections. The concepts of antisepsis and degermation
clearly overlap, because a determining procedure can simultaneously be antiseptic and vice versa.
 Germicide – also known as germicidal, biocidal (biocide) and microbicidal (microbicide) agents, are
disinfectants that kill microbes. A germicide can be used on inanimate materials or on living tissue, but
it cannot kill resistant microbial cells.
 Bacteristatic/bacteriostatic agent – a chemical that specifically inhibits the metabolism/growth
and reproduction of bacteria on tissues or on objects in the environment. Bacteriostasis implies the
inhibition of bacterial growth by means other than killing.
 Bactericide – chemical that destroys bacteria except for those in the endospore stage.
 Bactericidal – substance capable of killing bacteria
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Effects of Antimicrobial Agents on Cellular Structures

An antimicrobial agent’s adverse effect on cells is known as its mode (or mechanism) of action. Agents affect
one or more cellular targets, inflicting damage progressively until the cell is no longer able to survive.
Antimicrobials have a range of cellular targets that are least selective in their targeting tending to be effective
against the widest range of microbes (examples include heat and radiation). More selective agents (drugs, for
example) tend to target only a single cellular component and are much more restricted as to the microbes
they are effective against (textbook, p. 318).

The cellular targets of physical and chemical agents fall into four general categories:
1. the cell wall,
2. the cell membrane,
3. cellular synthetic processes (DNA, RNA), and
4. proteins.

1. Damage to cell wall integrity

The cell wall maintains the structural integrity of bacterial and fungal cells. Several types of chemical
agents damage the cell wall by blocking its synthesis, digesting it, or breaking down its surface. A cell
deprived of a functioning cell wall becomes fragile and is lysed very easily. Detergents and alcohol can also
disrupt cell walls, especially in gram-negative bacteria. Some antimicrobial drugs (penicillins) interfere with
the synthesis of the cell wall in bacteria.

2. Alteration of membrane permeability

A microorganism’s plasma membrane, located just inside the cell wall, is the target of many microbial
control agents. This membrane actively regulates the passage of nutrients into the cell and the elimination
of wastes from the cell. Damage to the lipids or proteins of the plasma membrane by antimicrobial agents
causes cellular contents to leak into the surrounding medium and interferes with the growth of the cell. If
this membrane is disrupted, a cell loses its selective permeability and cannot bar the entry of damaging
chemicals. Loss of such ability leads to cell death.

Detergents called surfactants work as microbicidal agents because they lower the
surface tension of cell membranes. Surfactants are polar molecules with hydrophilic
and hydrophobic regions that can physically bind to the lipid layer and penetrate the
internal hydrophobic region of membranes. In effect, this process “opens up” the
once tight interface, leaving leaky spots that allow injurious chemicals to seep into
the cell and important ions to seep out. Alcohols exert a related effect by dissolving membrane lipids and
stripping membranes away from cells.

3. Damage to protein and nucleic acids

The hydrogen bond of proteins is susceptible to breakage by heat or certain chemicals; breakage results
into the denaturation (or disruption) of the protein. In general, denaturation occurs when the bonds that
maintain the secondary and tertiary structure of the protein are broken. Breaking these bonds will cause
the protein to unfold or create random, irregular loops and coils. One way that proteins can be denatured
is through coagulation by moist heat (the same reaction seen in the irreversible solidification of the white
of an egg when boiled). Chemicals such as strong organic solvents (alcohols, acids) and phenolics also
coagulate proteins. Other antimicrobial agents, such as metallic ions, attach to the active site of the protein
and prevent it from interacting with its correct substrate. Regardless of the exact mechanism, such losses
in normal protein function can promptly arrest metabolism. Most antimicrobials of this type are non-
selective as to the microbe they affect.

The nucleic acids DNA and RNA are the carriers of the cell’s genetic information. Damage to these nucleic
acids by heat, radiation, or chemicals is frequently lethal to the cell; the cell can no longer replicate, nor
can it carry out normal metabolic functions such as the synthesis of enzymes. Some agents bind
irreversibly to DNA, preventing both transcription and translation; others are mutagenic agents. Gamma,
ultraviolet, or X radiation causes mutations that result in permanent activation of DNA. Chemicals such as
formaldehyde and ethylene oxide also interfere with DNA and RNA function.

2. Methods of Microbial Control

The methods of microbial control belong to the general category of decontamination procedures, in
that they destroy or remove contaminants. In microbiology, contaminants are microbes present at a
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given place and time that are undesirable or unwanted. Most decontamination methods employ either
physical agents, such as heat or radiation, or chemical agents, such as disinfectants and antiseptics.

The targets of microbial control processes are microorganisms capable of causing infection or spoilage
that are constantly present in the external environment and on the human body. This targeted
population is rarely simple or uniform; it often contains mixtures of microbes with extreme differences
in resistance and harmfulness.

A. Physical Methods

1. Heat as an Agent of Microbial Control

Heat is the most practical, efficient, and inexpensive method of sterilization of those inanimate objects and
materials that can withstand high temperatures. Because of these advantages, it is the means most frequently
used.
A sudden departure from a microbe’s temperature of adaptation is likely to have a detrimental effect
on it. As a rule, elevated temperatures (exceeding the maximum growth temperature) are microbicidal,
whereas lower temperatures (below the minimum growth temperature) are microbistatic. The two physical
states of heat used in microbial control are moist and dry. Moist heat occurs in the form of hot water, boiling
water, or steam (vaporized water). In practice, the temperature of moist heat usually ranges from 60 0C to
1350C. As we shall see, the temperature of steam can be regulated by adjusting its pressure in a closed
container. The expression of dry heat denotes air with a low moisture content that has been heated by a flame
or electric heating coil. In practice, the temperature of dry heat ranges from 160 0C to several thousand
degrees Celsius.

Heat Resistance and Thermal Death of Spores and Vegetative Cells

Bacterial endospores exhibit the greatest resistance, and vegetative states of bacteria and fungi are the least
resistant to both moist and dry heat. Destruction of spores usually requires temperatures above boiling,
although resistance varies widely.
Vegetative cells also vary in their sensitivity to heat, though not to the same extent as spores. Among
bacteria, the death times with moist heat range from 50°C for 3 minutes ( Neisseria gonorrheae) to 60°C for 60
minutes (S. aureus). It is worth noting that vegetative cells of sporeformers are just as susceptible as
vegetative cells of non-sporeformers and that pathogens are neither more nor less susceptible than
nonpathogens. Other microbes, including fungi, protozoa, and worms, are rather similar in their sensitivity to
heat. Viruses are surprisingly resistant to heat, with a tolerance range extending from 55°C for 2 minutes to 5
minutes (adenoviruses) to 60°C for 600 minutes (hepatitis A virus). For practical purposes, all non-resistant
forms of bacteria, yeasts, molds, protozoa, worms, and viruses are destroyed by exposure to 80°C for 20
minutes.
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A. Common Methods of Moist Heat Control

Heat applied in the presence of moisture, as in boiling or steaming, is faster and more effective than dry heat,
and can be accomplished at a lower temperature; thus, it is less destructive to many materials that otherwise
would be damaged at higher temperatures. Although many cellular structures are damaged by moist heat, its
most microbicidal effect is the coagulation and denaturation of proteins, which quickly and permanently halts
cellular metabolism. And because cellular enzymes are proteins, they are inactivated by moist heat, leading to
cell death.
The vegetative forms of most pathogens are quite easily destroyed by boiling for 30 minutes. Thus,
clean articles made of metal and glass such as syringes, needles, and simple instruments, may be disinfected
by boiling for 30 minutes. Because the temperature at which water boils is lower at higher altitudes, water
should always be boiled for longer times at high altitudes. Boiling is not always effective, however, because
heat-resistant bacterial endospores, mycobacteria, and viruses may be present. The endospores of the
bacteria that can cause anthrax, tetanus, gas gangrene, and botulism as well as hepatitis viruses, are
especially heat resistant and often survive boiling. Also, because thermophiles thrive at high temperatures,
boiling is not an effective means of killing them.
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The four ways that moist heat is employed to control microbes are:
1. Autoclaving – use of steam under pressure to completely destroy all microbial life; a very
effective method of sterilization. The autoclave is used by health and commercial industries for
this purpose, and a comparable home appliance is the pressure cooker. At about 15 psi (pounds
per square inch) of pressure (121°C), all vegetative cells and their endospores are killed in about
15 minutes. Sterilization is achieved when the steam condenses against the objects in the chamber
and gradually raises their temperature. It is used to sterilize culture media, solutions, linens,
utensils, dressings, equipment and other items that can withstand temperature and pressure.
2. Nonpressurized steam

Selected substances that cannot withstand the high temperature of the autoclave can be subjected
to intermittent (or fractional) sterilization or tyndallization. This technique requires a chamber to
hold the materials and a reservoir for boiling water. Items in the chamber are exposed to free-
flowing steam for 30 to 60 minutes. This temperature is not sufficient to reliably kill spores, so a
single exposure will not suffice. On the assumption that surviving spores will germinate into less
resistant vegetative cells, the items are incubated at appropriate temperatures for 23 to 24 hours,
and then again subjected to steam treatment. This cycle is repeated for 3 days in a row. Because
the temperature never gets above 100°C, highly resistant spores that do not germinate may
survive even after 3 days of this treatment.
Intermittent sterilization is used most often to process heat-sensitive culture media, such as those
containing sera, egg or carbohydrates (which can break down at higher temperatures) and some
canned foods. It is probably not effective in sterilizing items such as instruments and dressings that
provide no environment for spore germination, but it can certainly disinfect them.
3. Pasteurization: Disinfection of Beverages
Fresh beverages such as milk, yogurt, ice cream, fruit juices, beer and wine are easily
contaminated during collection and processing. Because microbes have the potential for spoiling
these foods or causing illness, heat is frequently used to reduce the microbial load and destroy
pathogens. Pasteurization is a technique in which heat is applied to liquids to kill potential agents
of infection and spoilage, while at the same time retaining the liquid’s flavor and food value.
Ordinary pasteurization techniques require special heat exchangers that expose liquid to 71.6°C
for 15 seconds (flash method) or to 63°C to 66°C for 30 minutes (batch method). The first method
is preferable because it is less likely to change flavor and nutrient content, and it is more effective
against certain resistant pathogens such as Coxiella and Mycobacterium.
Although these treatments inactivate most viruses and destroy the vegetative stages of 97 to 99%
of bacteria and fungi, they do not kill endospores or thermoduric microbes (mostly nonpathogenic
lactobacilli, micrococci, and yeasts). Milk is not sterile after regular pasteurization. In fact, it can
contain 20,000 microbes per milliliter or more, which explains why even an unopened carton of milk
will eventually spoil. Newer techniques can also produce sterile milk that has a storage life of 3
months. This milk is processed with ultrahigh temperature (UHT) – 134°C – for 1 to 2 seconds.
One important aim in pasteurization is to prevent the transmission of milk-borne diseases from
infected cows or milk handlers. The primary targets of pasteurization are non-spore-forming
pathogens: Salmonella spp. (a common cause of food infection), Campylobacter jejuni (acute
intestinal infection), Mycobacterium bovis, M. tuberculosis and several enteric viruses.
Pasteurization also has the advantage of extending milk storage time, and it can also be used by
some wineries and breweries to stop fermentation and destroy contaminants.

4. Boiling water or flowing steam – kills vegetative bacterial and fungal pathogens and many
viruses within 10 minutes; less effective on endospores. Because a single processing at 100°C will
not kill all resistant cells, this method can be relied on only for disinfection and not for sterilization.
Exposing materials to boiling water for 30 minutes will kill most non-spore-forming pathogens,
including resistant species such as tubercle bacillus and staphylococci. It is usually used in dishes,
basins, pitchers and various equipment. The use of boiling to sanitize and disinfect baby bottles is a
familiar example. Boiling is also a recommended method of disinfecting unsafe drinking water.

B. Dry Heat

Dry heat dehydrates the cell, removing the water necessary for metabolic reactions, and it also denatures the
proteins. However, the lack of water actually increases the stability of some protein conformations,
necessitating the use of higher temperatures when dry heat is employed as a use of microbial control. At very
high temperatures, dry heat oxidizes cells, burning them to ashes.
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a. Direct flaming – very effective method of sterilization; for use in inoculating loops. The flame
of a Bunsen burner reaches 1,870°C at its hottest point. This method is fast and effective, but
is also limited to metals and heat-resistant glass materials.
b. Incineration – destruction of microbes by subjecting them to extremes of dry heat. Microbes
are reduced to ashes and gases by this process. Large incinerators are regularly employed in
hospitals and research labs for complete destruction and disposal of infectious materials such as
syringes, needles, cultural materials, contaminated dressings, bedding, animal carcasses and
pathology samples.
c. Hot-air sterilization – very effective method of sterilization, but requires temperature of 180°
C for about 2 to 4 hours to ensure thorough heating of the objects and destruction of spores.
Items to be sterilized are placed in a dry oven, usually electric and occasionally gas. The dry
oven is used in laboratories and clinics for heating resistant items that do not sterilize well with
moist heat. Substances appropriate for dry ovens are glassware, metallic instruments, powders,
and oils that steam does not penetrate well. This method is not suitable for plastics, cotton, and
paper, which may burn at high temperature, or for liquids, which will evaporate. Another
limitation is the time required for it to work.

2. Cold

The principal benefit of cold treatment is to slow growth of cultures and microbes in food during processing
and storage. It must be emphasized that cold merely retards the activities of most microbes .

1. Refrigeration – has a bacteriostatic effect; it merely slows the growth of most microorganisms, but
does not completely inhibit growth. Useful on food, drug, and culture preservation.
2. Deep-freezing – an effective method of preserving microbial cultures, in which cultures are quick-
frozen between –50° and – 95°C. For food, drug, and culture preservation.
Although it is true that some microbes are killed by cold temperatures, most are not adversely
affected by gradual cooling, long-term refrigeration, or deep-freezing temperatures. Freezing
temperatures ranging from – 70°C to –135°C, provide an environment that can preserve cultures of
bacteria, viruses and fungi for long periods. Some psychrophiles grow very slowly even at freezing
temperatures and can continue to secrete toxic products. Ignorance of these facts is probably
responsible for numerous cases of food poisoning from frozen foods that have been defrosted at room
temperature and then inadequately cooked. Pathogens able to survive several months in the
refrigerator are S. aureus, Clostridium spp. (sporeformers), Streptococcus spp., and several types of
yeasts, molds, and viruses. Outbreaks of Salmonella food infection traced backed to refrigerated foods
such as ice cream, eggs, and Tiramisu are testimony to the inability of freezing temperatures to reliably
kill pathogens.
3. Lyophilization/Freeze-Drying – a process that combines dehydration (drying) and freezing. Most
effective method for long term preservation of microbial cultures; water removed by high vacuum at
low temperature. Lyophilized materials are frozen in a vacuum; the container is then sealed to maintain
the inactive state. This freeze-drying method avoids the formation of ice crystals that would damage
the cells. It is widely used to preserve foods, antibiotics, antisera, microorganisms, and other biologic
materials such as pure cultures. It should be remembered that lyophilization cannot be used to kill
microorganisms, but, rather, is used to prevent them from reproducing and to store them for future
use.

3. Desiccation – or dehydration, involves removing water from microbes; in the absence of water,
microorganisms cannot grow or reproduce but can remain viable for years. Then, when water is made
available to them, they can resume their growth and division. This ability is used in the laboratory when
microbes are preserved by lyophilization, or freeze-drying, as discussed earlier. Certain foods are freeze-dried
to preserve them (e.g., coffee, and some fruit and vegetable additives).
The resistance of vegetative cells to desiccation varies with the species and the organism’s
environment. For example, the gonorrhea bacterium can withstand dryness for only about an hour, but the
tuberculosis bacterium can remain viable for months. Viruses are generally resistant to desiccation, but they
are not as resistant as bacterial endospores, some of which have survived for centuries. Endospores of Bacillus
and Clostridium are viable for millions of years under extremely arid conditions. This ability of certain dried
microbes and endospores to remain viable is important in hospital setting. Dust, clothing, bedding, and
dressings might contain infectious microbes, in dried mucus, urine, pus, and feces
. Desiccation can be a valuable way to preserve foods because it greatly reduces the amount of water
available to support microbial growth.
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4. Osmotic Pressure – plasmolysis; results in loss of water from microbial cells. The use of high
concentrations of salts and sugars to preserve food is based on the effects of osmotic pressure. High
concentrations of these substances create a hypertonic environment that causes water to leave the microbial
cell. This process resembles preservation by desiccation, in that both methods deny the cell the moisture it
needs for growth. The principle of osmotic pressure is used in the preservation of foods. For example,
concentrated salt solutions are used to preserve fruits.
As a general rule, molds and yeasts are much more capable than bacteria of growing in materials with
low moisture or high osmotic pressures. This property of molds, sometimes combined with their ability to grow
under acidic conditions, is the reason fruits and grains are spoiled by molds rather than by bacteria. It is also
part of the reason molds are able to form mildew on a damp wall or shower curtain.

5. Radiation

Another way in which energy can serve as antimicrobial is through the use of radiation. Radiation is defined
as energy emitted from atomic activities and dispersed at high velocity through matter or space. Radiation has
various effects on cells, depending on its wavelength, intensity, and duration. Radiation that kills
microorganisms (sterilizing radiation) is of two types:

a. Ionizing – when a cell is bombarded (bombardment or irradiation) by certain waves or

particles, its molecules absorb some of the available energy, leading to the ejection of orbital
electrons from an atom to form an ion. Its effect is the destruction of proteins by gamma rays
and high energy electron beams. X rays, which are produced in by machines in a manner
similar to the production of electron beams, are similar in nature to gamma rays. The principal
effect of ionizing radiation is the ionization of water, which forms highly reactive hydroxyl
radicals. These radicals react with organic cellular components, especially DNA and other
proteins. Secondary lethal effects appear to be chemical changes in organelles and the
production of toxic substances.
It is a highly effective alternative for sterilizing materials that are sensitive to heat such
as pharmaceuticals (drugs & vaccines), and disposable medical and dental supplies, such as
plastic syringes, surgical gloves, suturing materials, and catheters. Its main advantages include
speed, high penetrating power (it can sterilize materials through outer packages and
wrappings), and the absence of heat.
b. Nonionizing – damage to DNA by UV light. Irradiation excites atoms by raising them to a
higher energy state, but does not ionize them. This atomic excitation, in turn leads to the
formation of abnormal bonds within molecules such as DNA and is thus a source of mutations.
A UV or “germicidal” lamp is commonly found in hospital rooms, nurseries, operating
rooms, schools, food preparation areas, cafeterias and dental offices. Germicidal lamps can cut
down on the concentration of airborne microbes by as much as 99%. Ultraviolet disinfection of
air has proved effective in reducing postoperative infections, preventing the transmission of
infections by respiratory droplets, and curtailing the growth of microbes in food-processing
plants and slaughter houses.
UV light is also used to disinfect vaccines and other medical products. A major
disadvantage of UV light as a disinfectant that the radiation is not very penetrating (especially
through solid materials such as glass, metal, cloth, plastic and even paper), so the organisms
killed must be directly exposed to the rays. Another potential problem is that UV light can
damage human eyes (retinal damage), and prolonged exposure can cause burns, wrinkles and
skin cancer in humans.

6. Filtration – an effective method to remove microbes from air and liquids. In practice, it involves the
passage of liquid or gas through a screen-like material (filter) with pores small enough to trap and retain
microorganisms. Most filters in use consist of cellulose acetate and nitrocellulose, polycarbonate, and a variety
of plastic materials (Teflon, nylon) whose pore size can be carefully controlled and standardized. Ordinary
substances such as charcoal, diatomaceous earth, or unglazed porcelain are also used in some applications.
Filtration is used to prepare liquids that cannot withstand heat, including serum and other blood products,
vaccines, drugs (antibiotic solutions), IV fluids, enzymes, and media. Filtration has been employed as an
alternative method for decontaminating milk and beer without altering their flavor. It is also an important step
in water purification. It has the disadvantage of not removing soluble molecules (toxins) that can cause
disease.
Filtration is also an efficient means of removing airborne contaminants that are a common source of
infection and spoilage. High-efficiency particulate air (HEPA) filters are widely used to provide a flow of
decontaminated air to hospital rooms (operating & rooms occupied by burn patients) and sterile rooms.
 P a g e | 12

B. Chemical Methods

Chemical disinfection means the use of chemical agents to inhibit the growth of microorganisms,
either temporarily or permanently. Chemical agents are used to control the growth of microbes on both
living tissue and inanimate objects. Unfortunately, very few chemical agents achieve sterility; most of them
merely reduce microbial populations to safe levels or remove vegetative forms of pathogens from objects. A
common problem is the selection of an agent. No single disinfectant is appropriate for all circumstances.
Antimicrobial chemicals occur in the liquid, gaseous, or even solid state. They serve as disinfectants,
antiseptics, sterilants (chemicals that sterilize), degermers, or preservatives (chemicals that inhibit the
deterioration of substances). For the sake of convenience, (and sometimes safety), many solid or gaseous
antimicrobial chemicals are dissolved in water, alcohol, or a mixture of the two to produce a liquid solution.
Solutions containing pure water as the solvent are termed aqueous, whereas those dissolved in pure alcohol or
water-alcohol mixtures are termed tinctures.

Characteristics of a Good Antimicrobial Chemical Agents

1. rapid action in low concentrations
2. solubility in water or alcohol and long term stability
3. broad-spectrum microbicidal action and nontoxic to human tissues
4. penetration of inanimate surfaces to sustain a cumulative or persistent action
5. resistance to becoming inactivated by organic matter
6. noncorrosive or nonstaining properties
7. sanitizing and deodorizing properties
8. affordability and ready availability
9. nonoffensive odor

As of yet, no chemical can completely fulfil all of those requirements, but glutaraldehyde and hydrogen
peroxide approach this ideal.
Germicides are evaluated in terms of their effectiveness in destroying microbes in medical and dental
settings. The three levels of chemical decontamination procedures are high, intermediate, and low.
High-level germicides kill endospores and, if properly used, are sterilants. Materials that necessitate high-
level control are medical devices – for example, catheters, heart-lung equipment, and implants – that are not
heat-sterilizable and are intended to enter body tissues during medical procedures. Intermediate-level
germicides kill fungal (but not bacterial) spores, resistant pathogens such as the tubercle bacillus, and
viruses. They are used to disinfect items (respiratory equipment, thermometers) that come into intimate
contact with the mucous membranes but are noninvasive. Low levels of disinfection eliminate only vegetative
bacteria, vegetative fungal cells, and some viruses. They are used to clean materials such as electrodes,
straps, and pieces of furniture that touch the skin surfaces but not the mucous membranes.

Factors Affecting the Effectiveness of Chemical Methods

1. nature of microorganisms being treated
2. nature of material being treated – smooth, solid, objects are more reliably disinfected than are
those with pores or pockets that can trap soil
3. degree of contamination
4. concentration (strength) of the chemical
5. chemical action on cellular targets (proteins, nucleic acids, the cell wall, and the cell membrane)
6. length of exposure – time allowed for the chemical to work
7. pH or acidity of solution
8. temperature
9. presence of organic material such as proteins, blood, pus, feces, mucus secretions and vomitus –
can hinder the penetration of a disinfectant and, in some cases, can form bonds that reduce its
activity.
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Limitation of Disinfectants
1. Many disinfectants that are effective against pathogens in the controlled conditions of the
laboratory become ineffective in the actual hospital or clinic environment.
2. The stronger and more effective antimicrobial chemical agents have limited use because of their
destructiveness to human tissues and to other substances.

Microorganisms Susceptible to Chemicals

1. almost all bacteria in their vegetative state
2. fungi
3. protozoa
4. viruses

Microorganisms Resistant to Chemicals

1. Mycobacteria of TB and Leprosy – w/ thick cell walls and waxy layers that can block the
entrance of many disinfectants, and have moderate resistance
2. bacterial endospores - Bacillus and Clostridium have highest resistance
3. fungal spores – moderate resistance
4. hepatitis B and poliovirus – moderate resistance
5. Pseudomonas, Acinetobacter, and other gram-negative rods – with moderate resistance due to
their cell walls & outer membrane that prevent penetration of chemical agents
6. Staphylococcus species – with moderate resistance due to their thick, protective peptidoglycan
cell walls
Note: Prions (proteinaceous infectious particles) – highest resistance

Categories of Chemical Agents

1. Halogens – are fluorine, bromine, chlorine, and iodine, a group of non-metallic

elements with similar properties. Because fluorine and bromine are difficult and dangerous to
handle and no more effective than chlorine and iodine, only the latter two are used routinely in
germicidal preparations. These elements are highly effective components of disinfectants and
antiseptics because they are microbicidal and not just microbistatic, and they are sporicidal with
longer exposure. For these reasons, halogens are the active ingredients in nearly one-third of
all antimicrobial chemicals currently marketed.
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Chlorine compounds combine with water to interfere with the structure of various enzymes.
The resulting denaturation of enzymes is permanent and suspends metabolic reactions. Chlorine
kills not only bacteria and endospores but also fungi and viruses. Death of almost all
microorganisms usually occurs within 30 minutes, although endospores may require several
hours. Chlorine compounds are less effective if exposed to light, alkaline pH, and excess organic
matter.
a. Gaseous and liquid chlorine are used almost exclusively for large-scale disinfection of
drinking water, sewage, and wastewater. Chlorination to a concentration of 0.6 to 1.0 parts of
chlorine per million parts of water will usually ensure that water is safe to drink.
b. Hypochlorite bleach used extensively in sanitization and disinfection of food equipment,
treatment of swimming pools, spas, drinking water, and fresh foods; for wound antisepsis and
routine medical and household disinfection, deodorizing, and stain removal. Common household
bleach is a 5% solution of sodium hypochlorite.

Iodine is a pungent chemical that forms brown-colored solutions when dissolved in water or
alcohol. Iodine rapidly penetrates the cells of microorganisms, where it apparently disturbs a
variety of metabolic functions by interfering with the hydrogen and disulfide bonding of proteins
(a mode of action similar to chlorine). All classes of microorganisms are killed by iodine if proper
concentrations and exposure times are used. Iodine activity is not as adversely affected by
organic matter and pH as chlorine is.
a. Iodophors – most common iodine for skin and mucous membranes; antiseptic prep for
surgery and injections; for surgical handscrubs; to disinfect equipment and surfaces; possibly
for burn; and may be an alternative preventive for eye infections in newborn. Common products
are betadine and povidone which contain 2% to 10% of available iodine.
b. Aqueous or Tinctures – topical antiseptic prior to surgery; sometimes for burned or injured
skin (weak solutions of 1% to 3% in water or in alcohol tinctures. Medium-level disinfection for
plastic instruments, thermometers (aqueous solutions or tinctures of 5% to 10%; somewhat
limited by their toxicity and tendency to stain).

2. Phenol (carbolic acid) – an acrid, poisonous compound derived from

distillation of coal tar. Solutions of phenol are now used only in certain
limited cases. In high concentrations, they are cellular poisons, rapidly disrupting cell walls and
membranes and precipitating proteins; in lower concentrations, they inactivate certain critical
enzyme systems. The phenolics are strongly microbicidal and will destroy vegetative bacteria
(including tuberculosis bacterium), fungi and most viruses (not hepatitis B), but they are reliably
not sporicidal. Their continued activity in the presence of organic matter and their detergent actions
contribute to their usefulness. Unfortunately, the toxicity of the phenolics makes them too
dangerous to use as antiseptics.
a. Cresols – simple phenolic derivatives that are common hospital and
household disinfectants, bearing the brand name Lysol.

For low or intermediate levels of disinfection in the hospital. 1% to 3% emulsions that are combined w/ soap;
can be too toxic for antisepsis; tend to be absorbed by membranes into the blood.
b. Bisphenols – widely employed commercially, clinically, and in the home; Lysol spray often used in hospital
and laboratory disinfection. One type, orthophenyl phenol is the major ingredient in disinfectant aerosol spray.
C. Hexachlorophene – was once a widespread additive of cleansing soaps (pHisoHex) for hospital and home
use; now, occasionally used to control outbreaks of skin infections. When hexachlorophene was found to be
absorbed through the skin and a cause of neurological damage, it was no longer available without
prescription.
d. Triclosan – widely used antibacterial compound added to soaps, cosmetics, and many other household
products. It acts as both disinfectant and antiseptic and is broad-spectrum in its effects.

3. Chlorhexidine (compound containing chlorine and phenol)

Its mode of action targets both cell membranes (lowering surface tension until selective
permeability is lost) and protein structure (causing denaturation). At moderate to high
concentrations, it is bactericidal for both gram-positive and gram-negative bacteria but inactive
against spores. Its effects on viruses and fungi vary. It is not absorbed into deeper tissues to
any extent. Chlorhexidine solution serves as an obstetric antiseptic (GynePro), a neonatal wash,
a wound degermer, a mucous membrane irrigant, as preservative for eye solutions and also as
an active ingredient in mouthwash (Orahex, Astring-o-sol Specialist) designed to reduce dental
plaque and oral bacteria.
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4. Alcohols – are colorless hydrocarbons. Its mechanism of action depends

upon its concentration. Concentrations of 50% and higher dissolve membrane lipids, disrupt cell
surface tension, and compromise membrane integrity. Alcohol that has entered the protoplasm
denatures proteins through coagulation but only in alcohol-water solutions of 50% to 95%.
Alcohol is the exception to the rule that higher concentrations of an antimicrobial chemical have
greater microbicidal activity. Because water is needed for proteins to coagulate, alcohol shows a
greater microbicidal activity at 70% concentration (that is, 30% water) than at 100% (0%
water). Absolute alcohol (100%) dehydrates cells and inhibits their growth but is generally not a
protein coagulant.
a. Ethyl alcohol, also called ethanol or grain alcohol, is known for being germicidal,
nonirritating, and inexpensive. Solutions of 70 to 95% are routinely used as skin degerming
agents because the surfactant action removes skin oil, soil, and some microbes sheltered in
deeper skin layers. One limitation to its effectiveness is the rate at which it evaporates. Ethyl
alcohol is occasionally used to disinfect electrodes, face masks, and thermometers, which are
first cleaned and then soaked in alcohol for 15 to 20 minutes.
b. Isopropyl alcohol, sold as rubbing alcohol, is even more microbicidal and less expensive
than ethanol, but these benefits must be used with caution in disinfection or skin cleansing,
because inhalation of its vapors can adversely affect the nervous system.
5. Oxidizing agents
a. Hydrogen peroxide – colorless, caustic liquid that decomposes in the
presence of light, metals, or catalase into water and oxygen gas. The
antimicrobial activity stems from their oxidation of cellular components. The
germicidal effects of hydrogen peroxide are due to the direct and indirect
actions of oxygen. Oxygen forms hydroxyl free radicals which is highly toxic
and reactive to cells. Although most microbial cells produce catalase to
inactivate the metabolic hydrogen peroxide, it cannot neutralize the amount
of hydrogen peroxide entering the cell during disinfection and antisepsis.
Although hydrogen peroxide is bactericidal, virucidal, and fungicidal, and in
higher concentrations, sporicidal; it is not a very effective open-wound
antiseptic because the host catalase freed in the wound quickly inactivates
hydrogen peroxide. It is useful in treating infections by anaerobic bacteria
because of the lethal effects of the oxygen released.
b. Ozone (O3) is a gas for disinfection of air, water, industrial air conditioners and cooling towers.

6. Aldehydes - disrupt the activity of enzymes within the cell and inactivate proteins to kill microorganisms.

a. Formaldehyde is a sharp, irritating gas that readily dissolves in water to form an aqueous
solution called formalin. Pure formalin is a 37% solution of formaldehyde gas dissolved in
water. The chemical is microbicidal through its attachment to nucleic acids and functional
groups of amino acids. Formalin is an intermediate– to high-level disinfectant, although it acts
more slowly than glutaraldehyde. Formaldehyde’s extreme toxicity (it is classified as a
carcinogen) and irritating effects on the skin and mucous membranes greatly limit its clinical
usefulness. Limited uses as a disinfectant for surgical instruments; used in aquaculture to kill
fish parasites and control growth of algae and fungi;    preserving dead tissues, as it is one of
the active ingredients of embalming fluid;   use in vaccine to inactivate viruses and to detoxify
bacterial toxins (Formaldehyde is diluted during the vaccine manufacturing process).

b. Glutaraldehyde – milder chemical for sterilizing materials that are damaged by heat. It is
rapid and broad-spectrum, and is one of the few chemicals officially accepted as sterilant and
high-level disinfectant. It kills spores in 3 hours and fungi and vegetative bacteria (even
Mycobacterium and Pseudomonas) in a few minutes. Viruses, including the most resistant
forms, appear to be inactivated after relative short exposure times. Commercial products
(Cidex, Sporicidin) diluted to 2% are used to sterilize respiratory therapy equipment, hemostats,
fiberoptic endoscopes (laparoscopes, arthroscopes) and kidney dialysis equipment.

7. Gaseous Sterilants and Disinfectants

Processing inanimate substances with chemical vapors, gases, and aerosols provides a versatile
alternative to heat or liquid chemicals. Gaseous chemosterilizers are very penetrating; like the
aldehydes, these gases are strong alkylating agents and react vigorously with functional groups
of DNA and proteins. Through these actions, it blocks both DNA replication and enzymatic
actions. They are effective against vegetative bacteria, fungi, viruses, and endospores.
 P a g e | 16

a. Ethylene Oxide (ETO) is an effective way to sterilize and disinfect plastic materials and
delicate instruments in hospitals and industries. It can safely sterilize prepackaged medical
devices, surgical supplies, syringes, and disposable Petri dishes. It has also been used
extensively to disinfect sugar, spices, dried fruits, and drugs. For all its effectiveness, It has
some unfortunate features. Its explosiveness makes it dangerous to handle; it can damage the
lungs, eyes, and mucous membranes if contacted directly; and it is rated as a carcinogen.
b. Propylene Oxide – strerilization of foods such as nuts, powders, starches and spices. It is
safer to use because it breaks down into a relatively harmless substance.
c. Chlorine Dioxide – air and surface sterilant; for treatment of water, food processing
equipment, and medical waste; decontamination of whole rooms and space probes

8. Surface-Active Agents/Surfactants – can decrease surface tension among

molecules of a liquid
a. Detergents are polar molecules that act as surfactants. The positively charged end of the
detergent molecule binds well with the predominantly negatively charged bacterial surface
proteins while the long, uncharged hydrocarbon chain allows the detergent to disrupt the cell
membrane. Eventually, the cell membrane loses selective permeability, leading to cell death.
Quaternary ammonium compounds (quats) are cationic detergents. When used at high
concentrations they are effective against some gram-positive bacteria, viruses, fungi, and algae.
In low concentrations, they exhibit only microbistatic effects. Drawbacks to the quats include
their ineffectiveness against tuberculosis bacterium, hepatitis virus, Pseudomonas, and spores
at any concentration. Quats are mixed w/ cleaning agents to disinfect and sanitize floors,
equipment surfaces, and restrooms; for sanitizing restaurant utensils, food-processing
equipment, and clothing; common preservatives in ophthalmic solutions and cosmetics. Two
popular quats are Zephiran, a brand name of benzalkonium chloride and cetylpyridinium
chloride (Ceepryn – brand name).
b. Soaps are alkaline compounds made by combining the fatty acids in oils with sodium or
potassium salts. In usual practice, soaps are only weak microbicides, and they destroy only high
sensitivity forms such as the agents of gonorrhea, meningitis, and syphilis. The common
hospital pathogen Pseudomonas is so resistant to soap that various species grow abundantly in
soap dishes.
Soaps function primarily as cleansing agents and sanitizers in industry and the home.
The superior sudsing and wetting properties of soaps help to mechanically remove large
amounts of surface oil, greases, and other debris that contains microorganisms. Soaps gain
greater germicidal value when mixed with agents such as chlorhexidine or iodine. They can be
used for cleaning instruments before heat sterilization, degerming patient’s skin, routine hand
washing by medical and dental personnel, and preoperative hand scrubbing. Vigorously
brushing the hands with germicidal soap over a 15-second period is an effective way to remove
dirt, oil, and surface contaminants as well as some resident microbes, but it will never sterilize
the skin.

9. Heavy metal compounds

Various forms of the metallic elements mercury, silver, gold, copper, arsenic, and zinc, have
been applied in microbial control over several centuries. These are often referred to as heavy
metals because of their relatively high atomic weight. However, from this list, only preparations
containing mercury and silver still have any significance as germicides.
Mercury, silver, and most other metals exert microbicidal effects by binding onto functional
groups of proteins and inactivating them, rapidly bringing metabolism to a standstill. This mode
of action can destroy many types of microbes, including vegetative bacteria, fungal cells and
spores, algae, protozoa, and viruses (but not endospores).
a. organic mercury - tinctures such as thimerosal (Merthiolate) and nitromersol (Metaphen)
are weak antiseptics and infection preventive, but should never be used on broken skin because
of its toxic effect and can delay healing.
b. Silver nitrate is a preparation used for preventing gonococcal infections in the eyes of
newborn infants who had been exposed to an infected birth canal (Crede’s prophylaxis). This is
not used as often now because many pathogens are resistant to it. It has been replaced by
antibiotics in most instances. Silver sulfadiazine ointment (Silvadene, Flammazine), when added
to dressings, effectively prevents infection in second and third-degree burn patients, and pure
silver is now incorporated into catheters to prevent urinary tract infections in the hospital.

.
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10. Dyes (Crystal violet or gentian violet)

Gentian violet is a bactericide and an antifungal agent, the primary agent used in the Gram
stain test, perhaps the single most important bacterial identification test in use today, and it is
also used by hospitals for the treatment of serious heat burns and other injuries to the skin and
gums. Typically prepared as a weak (e.g. 1%) solution in water, it is painted on skin or gums to
treat or prevent fungal infections. It is thought to work by binding to the DNA of target
organisms and causing disruption, mutation or inhibition of DNA replication.
For the most part, dyes will continue to have limited applications because they stain and
have a narrow spectrum of activity.

11. Acids and Alkalis

Conditions of very low or high pH can destroy or inhibit microbial cells, but they are limited in
applications due to their corrosive, caustic, and hazardous nature.
Organic acids are used in food preservation because they prevent spore germination, bacterial
and fungal growth and because they are generally regarded as safe to eat. The key basic
principle on the mode of action of organic acids on bacteria is that non-dissociated (non-
ionized) organic acids can penetrate the bacteria cell wall and disrupt the normal physiology of
certain types of bacteria that we call “pH-sensitive” meaning that they cannot tolerate a wide
internal and external pH gradient. Among those bacteria are Escherichia coli, Salmonella spp.,
C. perfringens, Listeria monocytogenes, and Campylobacter spp.
Acetic acid in the form of vinegar is a pickling agent that inhibits bacterial growth; propionic
acid is commonly incorporated into breads and cakes to retard molds; lactic acid is added to
sauerkraut and olives and also meat and poultry products such as ham and sausages to prevent
growth of anaerobic bacteria (especially the clostridia); and benzoic and sorbic acids are added
to beverages, syrups, and margarine to inhibit yeasts.

Notes:
 Chemical food preservatives are frequently added to foods to retard spoilage. A
preservative is a substance or preparation added to a product to destroy or inhibit the
multiplication of microorganisms. Food additive is any of a large variety of substances added to
foods to prevent spoilage, improve appearance, enhance flavor or texture, or increase
nutritional value. Common additives: sodium benzoate, sorbic acids, potassium sorbate,
calcium propionate. Sodium nitrate and sodium nitrite are added to meat products, such as
ham, bacon, hot dogs, and sausage. The nitrite has two main functions: to preserve the
pleasing red color of the meat by reacting with blood components in the meat, and to prevent
the germination and growth of any botulism endospores that might be present. There has been
some concern that the reaction of nitrites with amino acids can form certain carcinogenic
products known as nitrosamines, and the amount of nitrites added to foods has generally
been reduced recently for this reason. Sulfur dioxide has long been used as a disinfectant,
especially in wine-making.
 Prions (infectious particles and responsible for progressive brain diseases) are extraordinarily
resistant to heat and chemicals. Instruments or other objects contaminated must be discarded
as biohazards, incinerated, or decontaminated with combination of chemicals and heat.
 P a g e | 18

C. Antimicrobial Drugs

Antimicrobial chemotherapy involves the use of chemicals to prevent and treat infectious diseases. The goal of
antimicrobial therapy is deceptively simple: administer a drug to an infected person, which destroys the
infective agent without harming the host’s cells. In actuality, this goal is rather difficult to achieve, because
many (often contradictory) factors must be taken into account. The ideal drug should be easily administered
yet be able to reach the infectious agent anywhere in the body, be absolutely toxic to the infectious agent
while simultaneously being non-toxic to the host, and remain active in the body as long as needed yet be
safely and easily broken down and excreted. In short, the perfect drug does not exist; but by balancing drug
characteristics against one another, a satisfactory compromise can be achieved.
Chemotherapeutic agents are described with regard to their origin, range of effectiveness, and whether
they are naturally produced or chemically synthesized.

Terminologies

Chemotherapy – treatment of disease with chemical agents (drugs).

* Note: The word “chemotherapy” is most commonly associated with the treatment of cancer. Its
official meaning is broader and can also be applied to antimicrobial treatment.
Chemotherapeutic drug – any chemical used in the treatment, relief or prophylaxis of a disease
Prophylaxis – use of a drug to prevent imminent infection of a person at risk
Antimicrobial chemotherapy – the use of chemotherapeutic drugs to control infection.
Antimicrobials –chemical substances that destroys disease-causing microorganisms with minimal damage to
host tissues. It is an all-inclusive term for any antimicrobial drug, regardless of its origin.
Antibiotics – substances produced by microorganisms through natural metabolic processes that are effective
in killing or inhibiting the growth of another microorganism; being produced by organisms like bacteria in
the genera Streptomyces and Bacillus and from molds in the genera Penicillium and Cephalosporium.
Semisynthetic drugs – drugs that are chemically modified in the laboratory after being isolated from natural
resources. Semisynthetic antibiotics include semisynthetic peniclllins, such as amoxicillin and ampicillin.
Synthetic drugs – the use of chemical reactions to synthesize antimicrobial compounds in the laboratory.
Narrow Spectrum (Limited Spectrum) – antimicrobials effective against a limited array of microbial types;
for example, a drug effective mainly on gram-positive bacteria. Examples are vancomycin which destroys
only gram-positive bacteria; and colistin and nalidixic acid which destroy only gram-negative bacteria.
Broad spectrum (Extended Spectrum) – antimicrobials effective against a wide variety of microbial types; for
example, a drug effective against both gram-positive and gram-negative bacteria. Examples of broad-
spectrum antibiotics are ampicillin, chloramphenicol, and tetracycline.

Historical Overview
 Salvarsan – the first chemotherapeutic agent discovered by Paul Ehrlich–The Father of Chemotherapy–
for treatment of syphilis in the late 1800s.
 Sulfa drugs – came into prominence by late 1930s.
 Antibiotic (Penicillin) – discovered by Alexander Fleming from a mold, Penicillium notatum, that had
contaminated a Petri plate of Staphylococcus aureus.

Characteristics of the Ideal Antimicrobial Drug (textbook, Table 12.1, page 344)
1. Selective toxicity to the microbe, but nontoxic to host cells
2. Microbicidal rather than microbistatic
3. Relatively soluble; functions even when highly diluted in body fluids
4. Remains potent long enough to act and is not broken down or excreted prematurely
5. Doesn’t lead to development of antimicrobial resistance
6. Complements or assists the activities of the host’s defenses
7. Remains active in tissues and body fluids
8. Readily delivered to the site of infection
9. Reasonably priced
10. Does not disrupt the host’s health by causing allergies or predisposing the host to other infections;
should not produce hypersensitivity to most patients

Mechanisms of Drug Action

If the goal of chemotherapy is to disrupt the structure or function of an organism to the point where it can no
longer survive, then the first step toward this goal is to identify the structural and metabolic needs of a living
cell. Once the requirements of a living cell have been determined, methods of removing, disrupting, or
 P a g e | 19

interfering with these requirements can be employed as potential chemotherapeutic strategies. The
metabolism of an actively dividing cell is marked by the production of a new cell wall components (in most
cells), DNA, RNA, proteins, and cell membrane. Consequently, antimicrobial drugs are divided into categories
based on which of these metabolic targets they affect. These categories include:

1. inhibition of cell wall synthesis

2. inhibition of nucleic acid (RNA and DNA) structure and function,
3. inhibition of protein synthesis,
4. interference (damage) with cell membrane structure or function, and
5. inhibition of enzyme activity – particularly that of folic acid synthesis.

Major Antimicrobial Drug Groups

1. Antibacterial Agents

Antimicrobial agents work well against bacterial pathogens because the bacteria (being prokaryotic) have
different cellular structures and metabolic pathways that can be disrupted or destroyed by drugs that do not
damage the eukaryotic host cells. As mentioned earlier, bactericidal agents kill bacteria, whereas bacteriostatic
agents stop them from growing and dividing. Bacteriostatic agents should only be used in patients whose host
defense mechanisms are functioning properly (i.e., only in patients whose bodies are capable of killing the
pathogen once its multiplication is stopped). Bacteriostatic agents should not be used in immunosuppressed or
leukopenic patients (patients having an abnormally low number of white blood cells).

1.1. Antimicrobial Drugs that Affect the Bacterial Cell Wall

The cell walls of most bacteria contain a rigid girdle of peptidoglycan, which protects the cell against rupture
from hypotonic environments. Active cells must constantly synthesize new peptidoglycan and transport it to its
proper place in the cell envelope. Drugs such as penicillins and cephalosphorins (a group known as beta
lactam antibiotics) react with one or more of the enzymes required to complete the process, causing the
cell to develop weak points at growth sites and to becomes osmotically fragile. Antibiotics that produce this
effect are considered bactericidal, because the weakened cell is subject to lysis. It is essential to note that
most of these antibiotics are active only in young, growing cells, because old, inactive or dormant cells do not
synthesize peptidoglycan. (One exception is a new class of antibiotics called the “-penems.”)
Cycloserine inhibits the formation of the basic peptidoglycan subunits, and vancomycin hinders the
elongation of the peptidoglycan. Penicillins and cephalosphorins bind and block peptidases that cross-link the
glycan molecules, thereby interrupting the completion of the cell wall. Penicillins that do not penetrate the
outer membrane are less effective against gram-negative bacteria, but broad-spectrum penicillins and
cephalosporins, such as carbenicllin or ceftriaxone, can access the cell walls of gram-negative species.
Note that penicillin is more active against Gram-positive cell walls due to the lack of an outer membrane
in these bacteria.   However, there exist derivatives of penicillin that display significant activity against Gram-
negative organisms (e.g., amoxicillin, ampicillin, ticarcillin, piperacillin, carbenicillin) including, particularly,
Pseudomonas spp.
Cell wall synthesis inhibitors include:
o Penicillins G and V – most important natural forms.
o Semisynthetic penicllins such as: amoxicillin, ampicillin, methicillin, oxacillin
Note: Clavulanic acid is often added to semisynthetic penicllins to augment their effectiveness.
Clavulanic acid is a chemical that inhibits beta-lactamase enzymes, thereby increasing the longevity
of beta-lactam antibiotics in the presence of penicillinase-producing bacteria. For example,
clavamox is a combination of amoxicillin and clavulanic acid and is marketed under the trade name
Augmentin. Zosyn is a similar combination of tazobactam, a beta-lactamase inhibitor, and
piperacillin that is used for a wide variety of systemic infections.
o Bacitracin – belongs to the class of Polypeptides; produced by a strain of the
bacterium, B. subtilis
o Cephalosporins – structurally related to penicillins but isolated from a different
organism (Cephalosporium rather than Penicillium)
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o Carbapenems – other beta-lactam antibiotics, structurally related to penicillins and

cephalosphorins
o Vancomycin – belongs to the class of Glycopeptides; used against methicillin-
resistant Gram-positive cocci, particularly Staphylococcus as well as Streptococcus and
Enterococcus
o Isoniazid (INH) – bactericidal to Mycobacterium tuberculosis, but only against
growing cells.

1.2. Antimicrobial Drugs that Affect Nucleic Acid Synthesis

The metabolic pathway that generates DNA and RNA is a long, enzyme-catalyzed series of reactions. Like any
complicated process, it is subject to breakdown at many different points along the way, and inhibition at any
point in the sequence can block subsequent events. Antimicrobial drugs interfere with nucleic acid synthesis by
blocking synthesis of nucleotides, inhibiting replication, or stopping transcription. Because functioning DNA and
RNA are required for proper translation as well, the effect on protein metabolism can be far-reaching.
Other antimicrobials inhibit DNA synthesis. Chloroquine (an antimalarial drug) binds and cross-links the
double helix. The newer broad-spectrum quinolones inhibit DNA unwinding enzymes or helicases, thereby
stopping DNA transcription. Antiviral drugs that are analogs of purines and pyrimidines, including
azidothymidine (AZT) and acyclovir, insert in the viral nucleic acid and block further replication.
Antibacterial drugs targeting DNA or RNA include:
o Fluoroquinolones – e.g. Nalidixic acid, norfloxacibn, ciprofloxacin and others with
the suffix “–xacin”
o Rifamycin – product of the genus Streptomyces, chemically altered into rifampin;
most prominent in treating mycobacterial infections, especially tuberculosis and leprosy.

1.3. Antimicrobial Drugs that Block Protein Synthesis

Most inhibitors of translation, or protein synthesis, react with the ribosome mRNA complex. Although human
cells also have ribosomes, the ribosomes of eukaryotes are different in size and structure from those of
prokaryotes, so these antimicrobials usually have a selective action against bacteria. One potential therapeutic
consequence of drugs that bind to the prokaryotic ribosome is the damage they can do to eukaryotic
mitochondria, which contain a prokaryotic type of ribosome. This gives antibiotics that inhibit protein synthesis
a potential for toxicity. Two possible targets of ribosomal inhibition are the 30S subunit and the 50S subunit.
Aminoglycosides (streptomycin, gentamicin, for example) insert on sites on the 30S subunit and cause the
misreading of the mRNA, leading to abnormal proteins. Tetracyclines block the attachment of tRNA on the A
acceptor site and effectively stop further protein synthesis. Other antibiotics attach to sites on the 50S subunit
in a way that prevents the formation of peptide bonds (chloramphenicol) or inhibits translocation of the
subunit during translation (erythromycin).
Antibacterial drugs targeting DNA or RNA include:
o Aminoglycosides – exclusively the products of the soil fungus, actinomycetes such as Streptomyces;
include amikacin, gentamicin, neomycin; with possible side effects of hearing loss, vertigo, and
kidney damage
o Streptomycin – antituberculosis; not given orally, but through intramuscular injections; may cause
ototoxicity and neurotoxicity
o Macrolide anti infectives – azithromycin, clarithromycin, erythromycin
o Lincosamides – clindamycin used topically for acne
o Tetracyclines – doxycyline, tetracycline; potential toxicity to mother and fetus during pregnancy
o Chloramphenicol

1.4. Antimicrobial Drugs that Disrupt Cell Membrane Function

A cell with a damaged membrane invariably dies from disruption in metabolism or lysis and does not even
have to be actively dividing to be destroyed. The antibiotic classes that damage cell membranes have
specificity for particular microbial groups, based on differences in the types of lipids in their cell membranes.
Polymyxins, belonging to the class of Polypeptides (same group as that of Bacitracin, but different
mechanism of action) are antibiotics that act by disrupting the Gram-negative outer membrane (they
additionally serve to inhibit the toxic effects of endotoxin though this comes at costs associated with their low
chemotherapeutic indices). Polymyxins interact with membrane phospholipids, distort the cell surface, and
cause leakage of proteins and nitrogen bases, particularly in gram-negative bacteria. The polyene antifungal
antibiotics (amphotericin B and nystatin) form complexes with the sterols on fungal membranes; these
complexes cause abnormal openings and seepage of small ions. Unfortunately, this selectivity is not exact, and
 P a g e | 21

the universal presence of membranes in microbial and animal cells alike means that most of these antibiotics
can be quite toxic to humans.
Bacillus polymyxa is the source of the Polymyxins, narrow-spectrum peptide antibiotics with a unique
fatty acid component that contributes to their detergent activity. Only two polymyxins–B and E–have any
routine applications, and even these are limited to their toxicity to the kidney. Either drug can be indicated to
treat drug-resistant Pseudomonas aeruginosa and severe urinary tract infections caused by other gram-
negative rods.

1.5. Antimicrobial Drugs that Inhibit Folic Acid Synthesis

Sulfonamides and trimethoprim are drugs that act by mimicking the normal substrate of an enzyme in a
process called competitive inhibition. They are supplied to the cell in high concentrations to ensure that a
needed enzyme is constantly occupied with the metabolic analog rather than the true substrate of the
enzyme. As the enzyme is no longer able to produce a needed product, cellular metabolism slows or stops.
Sulfonamides and trimethoprim interfere with folate metabolism by blocking enzymes required for the
synthesis of tetrahydrofolate, which is needed by bacterial cells for the synthesis of folic acid and the eventual
production of DNA and RNA and amino acids. Sulfonamides and trimethoprim are often given simultaneously
to achieve a synergistic effect, which, in pharmacological terms, refers to an effect that is more than additive
achieved by multiple drugs working together, thus requiring a lower dose of each.
The sulfonamide drug sulfanilamide inhibits production of folic acid (a vitamin) in those bacteria that
require p-aminobenzoic acid (PABA) to synthesize folic acid. Because the sulfonamide molecule is similar in
shape to the PABA molecule, bacteria attempt to metabolize sulfanilamide to produce folic acid. However, the
enzymes that convert PABA to folic acid cannot produce folic acid form the sulfonamide molecule. Without folic
acid, bacteria cannot produce certain essential proteins and finally die.
Sulfa drugs are bacteriostatic as opposed to being bactericidal. The selective toxicity of these
compounds is explained by the fact that mammals derive folic acid from their diet and do not possess this
enzyme system. Therefore, inhibition of bacterial and protozoan parasites, which must synthesize folic acid, is
easily accomplished while leaving the human unaffected.
Antibacterial drugs targeting folic acid synthesis include:
o Sulfonamides or sulfa drugs – sulfisoxazole, sulfadiazine (in combination with silver, used as a
topical burn cream on second- and third-degree burns), sulfamethoxazole
o Trimethoprim – usually given in combination with Sulfamethoxazole, known as Co-trimoxazole
(Bactrim, Septra)
o Sulfanilamide is an antimetabolite that inhibits bacterial folic acid synthesis (a B vitamin) from
PABA (p-aminobenzoic acid, another B vitamin)

2. Antifungal Agents

Antifungal agents are being used with greater frequency because of the emergence of resistant strains and an
increase in the number of immunosuppressed patients, especially those with AIDS. Because the cells of fungi
are eukaryotic, they present special problems in chemotherapy. For one, the great majority of
chemotherapeutic drugs are designed to act on bacteria and are generally ineffective in combating fungal
infections. For another, the similarities between fungal and human cells often mean that drugs toxic to fungal
cells are also capable of harming human tissues. A few agents with special antifungal properties have been
developed for treating systemic and superficial fungal infections.
Most antifungal agents work in one of three ways:
o By binding with cell membrane sterols (amphotericin B and nystatin)
o By interfering with sterol synthesis (clotrimazole and miconazole)
o By blocking mitosis (mitotic inhibitors) or nucleic acid synthesis (griseofulvin and flucytosine)

Four main drugs groups currently in use are the macrolide polyene antibiotics, griseofulvin, synthetic azoles,
and flucytosine.
Polyenes bind to fungal membranes and cause loss of selective permeability. They are specific for
fungal membranes because these membranes contain a particular sterol component called ergosterol, while
human membranes do not. The toxicity of polyenes is not completely selective, however, because mammalian
cell membranes contain compounds similar to ergosterol that bind polyenes to a small extent.
Macrolide polyenes, represented by amphotericin B (named for its acidic and basic–amphoteric–
properties) and nystatin (for New York State, where it was discovered) have a structure that mimics the lipids
in some cell membranes. Amphotericin B is by far the most versatile and effective of all antifungals. Not only
does it work on most fungal infections, including skin and mucous membrane lesions caused by Candida
 P a g e | 22

albicans, but it is one of the few drugs that can be injected to treat systemic fungal infections such as
histoplasmosis and cryptococcus meningitis. Nystatin is used only topically or orally to treat candidiasis of the
skin and mucous membranes, but it is not useful for subcutaneous or systemic fungal infections or for
ringworm.
Griseofulvin (Grisovin), derived from the mold Penicillium griseofulvum, is an antifungal product
especially active in certain dermatophyte infections such as athlete’s foot. The drug binds to tubulin,
interfering with microtubule function, thus inhibiting mitosis. It binds to keratin and is deposited in the
epidermis, nails, and hair, where it inhibits fungal growth. Because complete eradication requires several
months and griseofulvin is relatively nephrotoxic, this therapy is given only for the most stubborn cases.
The azoles are broad-spectrum antifungal agents with a complex ringed structure. The most effective
drugs are ketoconazole, fluconazole, clotrimazole, and miconazole. Ketoconazole (Nizoral) is used orally and
topically for cutaneous mycoses, vaginal and oral candidiasis, and some systemic mycoses. Fluconazole
(Diflucan) can be used in selected patients for AIDS-related mycoses such as aspergilosis and cryptococcus
meningitis. Clotrimazole (Canesten) and miconazole (Daktarin, Monistat) are used mainly as topical ointments
for infections in the skin, mouth, and vagina.
Flucytosine is an analog (synthetic) of the nucleotide cytosine that has antifungal properties. It is
rapidly absorbed after oral therapy, and it is readily dissolved in the blood and cerebrospinal fluid. Alone, it can
be used to treat certain cutaneous mycoses. Many fungi are resistant to flucytosine, so it is usually combined
with amphotericin B to effectively treat systemic mycoses.

3. Antiparasitic Chemotherapy

3.1. Antiprotozoal Agents

Antiprotozoal drugs are usually quite toxic to the host and work by (1) interfering with DNA and RNA synthesis
(e.g. chloroquine, pentamidine and quinacrine), or (2) interfering with protozoal metabolism (e.g.
metronidazole; brand name Flagyl).

3.1.1. Antimalarial Drugs: Quinine and Its Relatives

Quinine, extracted from the bark of the cinchona tree, was the principal treatment for malaria for hundreds of
years, but it has been replaced by the synthesized quinolones, mainly chloroquine and primaquine, which have
less toxicity to humans. Because there are several species of Plasmodium (the malaria parasite) and many
stages of its life cycle, no single drug is universally effective for every species and stage, and each drug is
restricted in application. For instance, primaquine eliminates the liver phase of infection, and chloroquine
suppresses acute attacks associated with infection of red blood cells. Chloroquine is taken alone for
prophylaxis and suppression of acute forms of malaria. Primaquine is administered to patients with relapsing
cases of malira. Mefloquine is a semisynthetic analog of quinine used to treat infections caused by
chloroquine-resistant strains of Plasmodium.

3.1.2. Chemotherapy for Other Protozoan Infections

A widely used amoebicide, metronidazole (Flagyl), is effective in treating mild and svere intestinal infections
and hepatic disease caused by Entamoeba histolytica. Given orally, it also has applications for infections by
Giardia lamblia and Trichomonas vaginalis. Other drugs with antiprotozoan activities are quinicrine (a quinine-
based drug), sulfonamides, and tetracyclines.

3.1.3. Antihelminthic (Anthelminthic) Drug Therapy

Anthelmintics or antihelminthics are drugs that expel parasitic worms (helminths) from the body, by
either stunning or killing them. They may also be called vermifuges (stunning) or vermicides (killing).
Treating helminthic infections has been one of the most difficult and challenging of all
chemotherapeutic tasks. Flukes, tapeworms, and roundworms are much larger parasites than other
microorganisms and, being animals, have greater similarities to human physiology. Also, the usual strategy of
using drugs to block their reproduction is usually not successful in eradicating the adult worms. The most
effective drugs immobilize, disintegrate, or inhibit the metabolism of all stages of the life cycle.
Mebendazole and thiabendazole are broad-spectrum Antiparasitic drugs used in several roundworm
intestinal infestations. These drugs work locally in the intestine to inhibit the function of the microtubules of
worms, eggs, and larvae, which interferes with their glucose utilization and disables them. The compounds
pyrantel and piperazine paralyze the muscles of intestinal roundworms. Consequently, the worms are unable
to maintain their grip on the intestinal wall and are expelled along with the feces by the normal peristaltic
action of the bowel. Two newer antihelminthic drugs are praziquantel, a treatment for various tapeworm and
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fluke infections, and ivermectin, a veterinary drug now used in humans in the treatment of oncocercosis, but is
also effective against other worm infestations (such as strongyloidiasis, ascariasis, trichuriasis, filariasis and
enterobiasis).

4. Antiviral Chemotherapeutic Agents

The chemotherapeutic treatment of viral infections presents unique problems. With viruses, we are dealing
with an infectious agent that relies upon the host cell for the vast majority of its metabolic functions.
Disrupting viral metabolism requires that we disrupt the metabolism of the host cell to a much greater extent
than is desirable. Put another way, selective toxicity with regard to viral infection is almost impossible to
achieve because a single metabolic system is responsible for both the well-being of both the virus and host.
Although viral disease such as measles, mumps, and hepatitis are routinely prevented by the use of effective
vaccinations, epidemics of AIDS, influenza, and even the common cold attest to the need for more effective
medications for the treatment of viral pathogens.
Over the last few years, several antiviral drugs have been developed that target specific points in the
infectious cycle of viruses. Although antiviral drugs are certainly a welcome discovery, the chemotherapeutic
treatment of viruses is still in its infancy and most antiviral compounds are fairly limited in their usefulness.

Most compounds have their effects on some stage of the viral replication/multiplication cycle. Three
major modes of action are:

1. barring penetration of the virus into the host cell,

2. blocking the transcription and translation of viral genetic material, and
3. preventing the normal maturation of viral particles.

Although antiviral drugs protect uninfected cells by keeping viruses from being synthesized and
released, most are unable to destroy extracellular viruses or those in a latent state.

4.1. Drugs for Treating Influenza

a. Amantadine & Rimantidine – to treat infection by influenza A virus; prevent fusion of the virus w/
cell membranes and consequently interfere w/ entry, uncoating and release of virus
b. Tamiflu & Relenza – broader-spectrum antiviral drugs because they block neuraminidase enzymes in
both influenza A and influenza B thus preventing the entry of virus into the cell. Both types of drugs may be
used as prophylaxis and must be given early in an infection to be most effective
4. 2. Antiherpes Drugs
a. Acyclovir (Zovirax) and related compounds valacyclovir (Valtrex), famciclovir (Famvir), and
peniciclovir (Denavir) – inhibit nucleic acid synthesis; for treatment of herpesvirus infections, chickenpox, and
shingles
4. 3. Drugs for Treating HIV Infection and AIDS
- Nucleoside reverse transcriptase inhibitors (Zidovudine brand names retrovir & AZT) – stop the action
of reverse transcriptase in HIV, and blocking viral DNA production
- Non-nucleoside reverse transcriptase inhibitors (Nevirapine) – attach to HIV reverse transcriptase
binding site and stop its action
- Protease inhibitors (indinavir, crixivan, and saquinavir) – blocking protease HIV and causing the
viruses to be defective and not infective
- Integrase inhibitor (raltegravir) – interracts w/ integrase and blocks its action; HIV DNA is not being
incorporated into the host’s chromosomes
- Maravoc – keeps HIV out of cells by covering up the CD4 receptors on WBC that HIV usually infects
thus preventing the viruses from adhering to host cells
- Fuzeon – stops the envelope of the virus from fusing w/ the host cell after it has attached

Note: Interferons – a sensible alternative to artificial drugs has been a human-based substance. It is
a glycoprotein produced primarily by fibroblasts and leukocytes in response to various immune stimuli. It has a
numerous biological activities, including antiviral and anticancer properties.

Effects of Combinations of Drugs

Combinations of drugs may be used to minimize the development of resistant strains, to employ a synergistic
effect, to provide therapy prior to diagnosis, and to use small concentrations of each drug to lessen toxicity.
 P a g e | 24

The use of two antimicrobial agents to treat an infectious disease sometimes produces a degree of
pathogen killing that is far greater achieved by either drug than alone. This is known as synergism. Some
combinations of drugs are synergistic – they are more effective when taken together. The chemotherapeutic
effect of two drugs given simultaneously is sometimes greater than the effect of either given alone. Many
urinary, respiratory, and gastrointestinal infections respond particularly well to a combination of trimethoprim
and sulfamethoxazole, a combination referred to as co-trimoxazole (Bactrim & Septra).
There are situations, however, when two drugs are prescribed (perhaps by two different clinicians who
are treating the patient’s infection) that actually work against each other. This is known as antagonism.
Some combinations of drugs are antagonistic – when taken together, both drugs become less effective than
when taken alone.

Multidrug Therapy

In some cases, a single antimicrobial agent is not sufficient to destroy all the pathogens that develop during
the course of a disease; thus, two or more drugs may be used simultaneously to kill all the pathogens and to
prevent resistant mutant pathogens from emerging. In tuberculosis, for example, in which multidrug-resistant
strains of M. tuberculosis are frequently encountered, four drugs (isoniazid, rifampin, pyrazinamide, and either
ethambutol or streptomycin) are routinely prescribed.

Interactions between Drugs and Microbes

The goal of antimicrobial drugs is either to disrupt the cell processes or structures of bacteria, fungi, and
protozoa or to inhibit viral replication. Most of the drugs used in chemotherapy interfere with the function of
enzymes required to synthesize or assemble macromolecules, or they destroy structures already formed in the
cell. Above all, drugs should be selectively toxic, which means they should kill or inhibit microbial cells
without simultaneously damaging host tissues. This concept of selective toxicity is central to chemotherapy,
and the best drugs are those that block the actions or synthesis of molecules in microorganisms but not in
vertebrate cells. Examples of drugs with excellent selective toxicity are those that block the actions or
synthesis of cell wall in bacteria (penicillins). They have low toxicity and few direct effects on human cells
because human cells lack the chemical peptidoglycan and are thus unaffected by this action.

A. Drug Resistance

One unfortunate outcome of the use of antimicrobials is the development of drug resistance, an adaptive
response in which microorganisms begin to tolerate an amount of drug that would ordinarily be inhibitory.
Drug resistance refers to the ability of microorganisms to resist the antimicrobial effects of a chemotherapeutic
agent. The development of mechanisms for circumventing or inactivating antimicrobial drugs is largely due to
the genetic versatility and adaptability of microbial populations. The property of drug resistance can best be
exemplified by the fact that bacteria, must, of course, be resistant to any antibiotic they produce. This type of
resistance is limited, however, to a small group of organisms and is generally not a problem with regard to
antimicrobial chemotherapy.
Microbes become newly resistant to a drug after one of the following events occurs: (1) spontaneous
mutations in critical chromosomal genes, or (2) acquisition of entire new genes or sets of genes via transfer
from another species. Chromosomal drug resistance usually results from spontaneous random mutations in
bacterial populations. The chance that such a mutation will be advantageous is minimal, and the chance it will
confer resistance to a specific drug is still lower. Nevertheless, given the huge numbers of microorganisms in
any population and the constant rate of mutation, such mutations do occur. The end result varies from slight
changes in microbial sensitivity, which can be overcome by larger doses of the drug, to complete loss of
sensitivity.
Resistance occurring through intermicrobial transfer originates from plasmids called resistance (R)
factors that are transferred through conjugation, transformation, or transduction. Studies have shown that
plasmids encoded with drug resistance are naturally present in microorganisms before they have been
exposed to the drug. Such traits are “lying in wait” for an opportunity to be expressed and to confer
adaptability on the species. Many bacteria also maintain transposable drug resistance sequences (transposons)
that are duplicated and inserted from one plasmid to another or from a plasmid to the chromosome.
Chromosomal genes and plasmids containing codes for drug resistance are faithfully replicated and inherited
by all subsequent progeny. This sharing of resistance genes accounts for the rapid proliferation of drug-
resistant species. A growing body of evidence points to the ease and frequency of gene transfers in nature,
from totally unrelated bacteria living in the body’s normal biota and the environment.

Specific Mechanisms of Drug Resistance

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Inside a bacterial cell, the net effect of the two events described earlier is one of the following, which actually
causes the bacterium to be resistant.

1. New enzymes are synthesized; these inactivate the drug (only occurs when new genes are
acquired).
Microbes inactivate drugs by producing enzymes that permanently alter drug structure. One
example, bacterial exoenzymes called beta-lactamases, hydrolyze the beta-lactam rings of some
penicillins (penicllinase) and cephalosporins (cephalosporinase) rendering the drugs inactive.

2. Permeability or uptake of drug into bacterium is decreased or eliminated (can occur via mutation of
acquisition of new genes) – (1) prevention of penetration to the target site within the microbe and
(2) rapid efflux (ejection), which pumps the drug out of the cell before it can become effective.
The resistance of some bacteria can be due to a mechanism that prevents the drug from
entering the cell and acting on its target. For example, the outer membrane of the cell wall of
certain gram-negative bacteria is a natural blockade for some of the penicillin drugs. Many bacteria
possess multidrug-resistant (MDR) pumps that actively transport drugs and other chemicals out of
cells. These pumps are proteins encoded by plasmids or chromosomes. They are stationed in the
cell membrane and expel molecules by a proton-motive force similar to ATP synthesis.

3. Binding sites for drug are decreased in number or affinity – alteration of the drug’s target sites (can
occur via mutation or acquisition of new genes)
Because most drugs act on specific targets such as protein, RNA, DNA, or membrane structure,
microbes can circumvent drugs by altering the nature of this target.

4. An affected metabolic pathway is shut down or an alternate pathway is used (occurs due to the
mutation of original enzymes).
The action of antimetabolites can be circumvented if a microbe develops an alternative
metabolic pathway or enzymes.

Some bacteria can become resistant by lapsing into dormancy or, in the case of penicillin, by converting to
a cell-wall-deficient form (L form) that penicillin cannot affect.
.Some bacteria are naturally resistant to a particular antimicrobial agent because they lack the specific
target site for that drug (e.g., mycoplasmas have no cell walls and are, therefore, resistant to any drugs that
interfere with cell wall synthesis). Other bacteria are naturally resistant because that drugs i unable to cross
the organisms’ cell wall or cell membrane and thus, cannot reach its site of action (e.g., ribosomes). Such
resistance is known as intrinsic resistance. It is also possible for bacteria that were once susceptible to a
particular drug to become resistant to it; this is called acquired resistance. Hereditary drug resistance is
carried by plasmids called resistance (R) factor. Resistance can be minimized by the discriminate use of drugs
in appropriate concentrations and dosages.

Misuse of antibiotics selects for resistance mutants.

Includes:
◦ Using outdated, weakened antibiotics
◦ Using antibiotics for the common cold and other inappropriate conditions
◦ Use of antibiotics in animal feed
◦ Failure to complete the prescribed regimen
◦ Using someone else's leftover prescription

Adverse Effects of Antimicrobial Drugs

a. Toxicity to Organs
Drugs can adversely affect the following organs: the liver (hepatotoxic), kidneys (nephrotoxic), GIT,
cardiovascular system, and blood-forming tissue (hemotoxic), respiratory tract, nervous system
(neurotoxic), skin, bones, and teeth. Because the liver is responsible for metabolizing and detoxifying
foreign chemicals in the blood, it can be damaged by a drug or its metabolic products. Injury to liver
cells can result in enzymatic abnormalities, fatty liver deposits, hepatitis, and liver failure. The kidney is
involved in excreting drugs and their metabolites. Some drugs irritate the nephron tubules, creating
changes that interfere with their filtration abilities. Drugs such as sulfonamides can crystallize in the
kidney and form stones that can obstruct the flow of urine.

The most common complaint associated with oral antimicrobial therapy is diarrhea, which can progress
to severe intestinal irritation or colitis. Although some drugs directly irritate the intestinal lining, the
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usual GIT complaints are caused by disruption of the intestinal microflora . Many drugs given for
parasitic infections are toxic to the heart, causing irregular heartbeat and even cardiac arrest in
extreme cases. Chloramphenicol can severely depress blood-forming cells in the bone marrow,
resulting in either a reversible or a permanent (fatal) anemia. Some drugs hemolyze the RBC, others
reduce WBC counts, and still others damage platelets or interfere with their formation, thereby
inhibiting blood clotting.

Certain antimicrobials act directly on the brain and cause seizures. Aminoglycosides (streptomycin)
damage nerves (very common 8th cranial nerve), leading to dizziness, deafness, motor and sensory
disturbances. The skin is a frequent target of drug-induced side effects. The skin response can be a
symptom of drug allergy or a direct toxic effect. Tetracyclines are contraindicated for children from
birth to 8 years of age because they bind to the teeth enamel, creating a permanent gray to brown
discoloration. Pregnant women should avoid tetracyclines because they cross the placenta and can be
deposited in the developing fetal bones and teeth.

b. Allergic Responses

One of the most frequent drug reactions is heightened sensitivity, or allergy. This reaction occurs
because the drug acts as an antigen and stimulates an allergic response. This response can be
provoked by the intact drug molecule or by substances that develop from the body’s metabolic
alteration of the drug. In the case of penicillin, it is not the penicillin molecule itself that causes the
allergic response but a product benzylpenicilloyl. Allergic reactions have been reported for every major
type of antimicrobial drug, but the penicillins account for the greatest number of antimicrobial allergies,
followed by the sulfonamides.

People who are allergic to a drug become sensitized to it during the first contact, usually without
symptoms. Once the immune system is sensitized, a second exposure to the drug can lead to a
reaction such as skin rash (hives); respiratory inflammation; and, rarely, anaphylaxis, an acute,
overwhelming allergic response that develops rapidly and can be fatal.

c. Suppression and Alteration of the Microflora

With prolonged use, broad-spectrum antibiotics may destroy the normal flora of the mouth, intestine,
or vagina. The person no longer has the protection of the indigenous microflora and thus becomes
much more susceptible to infections caused by opportunists or secondary invaders. The resultant
overgrowth by such organisms is referred to as a superinfection. A superinfection can be thought of
as a “population explosion” of organisms that are usually present in small numbers. For example, the
prolonged use of oral antibiotics can result in a superinfection of Clostridium difficile in the colon, which
can lead to such disease as antibiotic-associated diarrhea (AAD) and pseudomembranous colitis (PMC).
Yeast vaginitis often follows antibacterial therapy because many bacteria of the vaginal flora were
destroyed, leading to a superinfection of the indigenous yeast, C. albicans.

Other Approaches of Controlling Infections

a. Probiotics are preparations of live microorganisms that are fed to animals and humans to improve
the intestinal flora. This can serve to replace microbes lost during antimicrobial therapy or simply to
augment the biota that is already there. For example, people consume yogurt because of the beneficial
microbes it contains. A probiotic approach is also sometimes used in female patients who have
recurring urinary tract infections. These patients receive vaginal inserts or perineal swabs of
Lactobacillus in an attempt to restore a healthy acidic environment to the region and to displace
disease-causing microorganisms. Probiotics also seem to inhibit the development of food allergies.

b. Prebiotics are nutrients that encourage the growth of beneficial microbes in the intestine. For
instance, certain sugars such as fructans are thought to encourage the growth of Bifidobacterium in
the large intestine and to discourage the growth of potential pathogens.