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Chapter 1: The Science of Microbiology

Microbiology is derived from the Greek words mikros ("small"), bios ("life"), and logia or
logos ("study of"). It is therefore the study of organisms that are so small they cannot be seen
with the naked eye. These organisms are called microorganisms or microbes and are
categorized into two: (1) cellular, which may either be prokaryotes (bacteria, cyanobacteria, and
archeans) or eukaryotes (fungi, protozoa, and algae); and (2) acellular, which includes viruses.
Microbiology is further classified into different fields of study, namely: (1) bacteriology, the
study of bacteria; (2) virology, the study of viruses; (3) mycology, the study of fungi; (4)
parasitology, the study of protozoa and parasitic worms; (5) phycology, the study of algae; and,
(6) immunology, the study of the immune system and the immune response.

Why study microbiology? The study of microbiology is important for the following reasons:

1. Microbiology has an impact in the daily lives of humans. Microorganisms are everywhere--in
the air one breathes, in the environment, and even in one's body. About a thousand or more
organisms inhabit the human body. These are collectively called normal flora or indigenous
flora which only produce disease in persons with compromised immune systems.

2. Some microorganisms are essential in biotechnology and a wide range of industries which
include food and beverage, pharmaceuticals, mining, genetics, and many more. Much of the
knowledge available in the study of genetics and biochemistry utilize microorganisms as model
organisms.

3. Some microorganisms, especially bacteria and fungi, are important sources of antimicrobial
agents. For example, penicillin was derived from the fungus Penicillium.

4. Some microorganisms act as saprophytes or decomposers of waste products and dead

organisms, making them essential in maintaining a balanced ecosystem.

5. The study of microorganisms has led to a better understanding of how microorganisms

produce disease, paving the way to better disease management and control. This was further
improved through the discovery of vaccines that helped prevent sickness from infections
diseases. By knowing the sources of disease-producing microbes, sanitation practices
improved immensely, leading to better mitigation of infectious diseases.

Certain diseases which were thought to have been eradicated are now re-emerging. Some
have the potential as biological warfare agents. At the same time, there are now a number of
pathogens that are developing resistance to antibiotics. In this context, the study of
microbiology is relevant for better understanding of the negative instances in which science
can be used.

Evolution of Microbiology

Archaeologists and evolutionists have uncovered evidence demonstrating the existence of

primitive microorganisms. In Western Australia, as many as eleven different types of fossils of
primitive microorganisms have been found in ancient rock formations, dating back to as early
as 3.5 billion years ago, long before the existence of animals and humans. Infectious diseases
have existed for thousands of years. In 3180 BC, an epidemic known as the "plague" broke out
in Egypt. In 1122 BC, an outbreak of a smallpox-like disease that originated in China spread
worldwide. The exhumed mummified remains of Rameses V showed skin lesions resembling
smallpox. In the mid-1600s, the microscope was discovered and with the use of this
instrument, Robert Hooke was able to discover the cell- the basic unit of living organisms. His
discovery heralded the cell theory that stated living organisms are made up of cells. Then in the
1670s,

Anton von Leeuwenhoek, a Dutch merchant, created a single-lens microscope that he used
to make observations of microorganisms which he then called animalcules. Through his
observations, he became known as the "Father of Microbiology" and was the one who first
provided accurate descriptions of bacteria, protozoa, and fungi

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In the middle and late 1800s, Louis Pasteur performed countless experiments that led to his
germ theory of disease. He postulated that microorganisms were in the environment and could
cause infectious diseases. He also developed the process of pasteurization, which kills
microorganisms in different types of liquids, and which became the basis for aseptic
techniques. He also introduced the terms aerobes and anaerobes and developed the
fermentation process. Pasteur's attempts to prove his germ theory of disease were
unsuccessful. It took Robert Koch to prove that microorganisms caused certain diseases
through a series of scientific steps which led to his formulation of the Koch's postulates. This
led to an increased effort by other scientists to prove and illustrate further the germ theory that
was initially formulated by Louis Pasteur. Thus, the late 1800s and the first decade of the 1900s
came to be known as the Golden Age of Microbiology. Since then, numerous scientists have
made significant contributions to the field of Microbiology. Edward Jenner discovered the
vaccine for smallpox. Joseph Lister applied the theory to medical procedures paving the way
for the development of aseptic surgery. After World War II, antibiotics were introduced to the
medical world. Paul Ehrlich discovered Salvarsan for the treatment of syphilis. This drug was
heralded the "magic bullet" of chemotherapy, which is treatment of disease by using chemical
substances. Alexander Fleming discovered the antibiotic penicillin from the mold Penicillium
notatum. With the discovery of antibiotics, the incidence of infectious diseases like
tuberculosis, pneumonia, meningitis, and others was significantly reduced. Most of the
experiments conducted in the field of microbiology during the early 20th century involved the
study of bacteria. During this time scientists were not yet equipped with advanced technology
in their study of microorganisms. It was only in the 1930s when the electron microscope was
developed that experimentations in microbiology became more complex. It was also during
that time when viral culture was introduced paving the way for rapid discoveries on viruses.
The vast knowledge gained from the experiments performed by microbiologists together with
the discovery of other vaccines in the 1940s and 1950s have led to better prevention and
control of numerous potentially fatal infectious diseases. Microscopy Microorganisms are
miniscule organisms that cannot be seen with the naked eye. The discovery of the microscope
has led to their close observation, allowing microbiologists and other scientists to study them
further. A microscope is an optical instrument that can magnify organisms a hundredfold or
even a thousand fold. From the time of its initial discovery in the 1600s, the microscope has
undergone steat revolutionary changes. Making it more advanced and complex throughout
time. The following are the different types of microscopes that have evolved from von
Leeuwenhoeks simple prototype.

Compound Microscope
The compound microscope is a type of microscope that contains more than one magnifying
lens. It can magnify objects approximately a thousand times their original size. Visible light is its
main source of illumination. As such, it is also known as the compound light microscope. The
compound microscope utilized today consists of two magnifying lens systems. The eyepiece
(or ocular) contains what is called the ocular lens that has a magnifying power of 10x. The
second lens system is located in the objective that is positioned directly above the

organism to be viewed.

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Component Function

Ocular Lens or Eyepiece Topmost part of the microscope which is the lens
the viewer looks through to see the specimen.
Revolving Nose Piece Located above the stage, it holds the objective
lenses.
Diopter adjustment It is used to change focus on one eyepiece in
order to correct any difference in vision between
the two eyes.
Body tube or head It connects the eyepiece to the objective lenses.

Arm It connects the body tube to the base of the

microscope.
Coarse Adjustment It brings the specimen into general focus.

Fine Adjustment It fine-tunes the focus and increases the details of

the specimen.
Objective Lenses This is held in place above the stage by the
revolving nosepiece and are the lenses that are
closest to the specimen. It contains 3 to 5
objectives ranging in power from 4X to 100X.
Stage Located beneath the revolving nosepiece. It is the
flat platform on which the specimen is placed.
Stage Clips Situated above the stage, these are metal clips
that hold the slide in place.
Stage Control Found beneath the stage, these knobs move the
stage either left or right or forward and backward.
Aperture The hole in the middle of the stage that allows
light from the illuminator to reach the slide
containing the specimen.
On/Off Switch The switch located at the base of the microscope
that turns the illuminator on or off.
Illuminator The light source of the microscope.

Iris Diaphragm Found on the condenser. It is used to adjust the

amount of light coming through the condenser.
Condenser It is found beneath the stage and contains a lens
system that focuses light onto the specimen. It
gathers and focuses light onto the specimen.
Base It supports the microscope and it is where the
illuminator is found.
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Brightfield Microscope
Made up of a series of lenses and utilizing visible light as its source of illumination, the
brightfield microscope can magnify an object 1,000 to 1,500 times. This is used to visualize
bacteria and fungi. Objects less than or thinner than 0.2 um cannot be visualized by this type

of microscope. The term "brightfield" is derived from the fact that the specimen appears dark

against the surrounding bright viewer field of this microscope. However, it has very low
contrast and most of the cells need to be stained to be properly viewed.

Darkfield Microscope
This microscope utilizes reflected light instead of transmitted light, with a special condenser
that has an opaque disc that blocks the light, such that only the specimen is illuminated. The
specimen to be studied appears bright against a dark background. This type of microscope is
ideal for studying specimens that are unstained or transparent and absorb little or no light. It is
also useful in examining the external details of the specimen such as its outline or surface. This
type of microscope is used to view spirochetes.

Phase-contrast Microscope
Phase-contrast microscopy is based on the principle that differences in refractive indices and
light waves passing through transparent objects assume different phases. This type of
microscopy was first introduced by Frits Zernike, a Dutch physicist, in 1934. The phase-
contrast microscope has a contrast-enhancing optical technique in order to produce high-
contrast images of specimens that are transparent which include thin tissue slices, living cells
in culture, and subcellular particles (such as nuclei and organelles).

Differential Interference Contrast Microscope

The differential interference contrast microscope is similar to the phase-contrast microscope
except that it utilizes two beams of light instead of one and therefore has higher resolution. The
resulting contrasting colors of the specimen being studied are due to the prisms that split the
light beam. It was developed by Georges Nomarski in 1952 as an improvement to the phase-
contrast microscope. It is useful in examining living specimens when normal biological
processes might be inhibited by standard staining procedures. However, the three-dimensional
image of the specimen produced may not be accurate since the enhanced areas of light and
shadow may distort the appearance of the image. »»es

Fluorescence Microscope
The fluorescence microscope makes use of ultraviolet light and fluorescent dyes called
fluorochromes. The specimen under study fluoresces or appears to shine against a dark
background. Fluorescence microscopy is based on the principle that certain materials emit
energy that is detectable as visible light when they are irradiated with the light of a given
wavelength. It uses a higher intensity of light source and this in turn excites a fluorescent
species. The fluorescent species then emits a lower energy light of a longer wavelength which
produces the magnified image instead of the original light source. Fluorescence microscopy
can be used to visualize structural components of small specimens such as cells and to detect

the viability of cell populations. It may also be used to visualize the genetic material of the cell
(DNA and RNA).

Confocal Microscope
Also known as the confocal laser scanning microscope (CL-SM). or laser confocal scanning
microscope (LCSM), the confocal microscope uses an optical imaging technique that increases
optical resolution and contrast of the micrograph by using a spatial pin-hole to block our-of-
focus light in image formation. The specimen is stained with a fluorescent dye to make it emit
or return light. The object is scanned with a laser into planes and regions. This is used,
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together with computers, to produce a three-dimensional image. It is also useful in the study of
cell physiology.

Electron Microscope
The electron microscope utilizes a beam of electrons to create an image of the specimen. The
electron beams serve as the source of illumination and magnets are used to focus the beam.
The first prototype of this microscope was built by the German Engineer Ernst Ruska in 1993,
which had a resolution power of up to 50 nm. Modern electron microscopes are capable of
magnifying objects up to 2 million times. It is used to visualize viruses and subcellular
structures of the cell. There are two types of electron microscopes-_-transmission electron
microscope and scanning electron microscope. The transmission electron microscope (TEM) is
the original form of the electron microscope. It produces two-dimensional, black and white
images, and magnifies objects up to 200,000 times. The scanning electron microscope (SEMI)
relies on interactions at the surface rather than transmission. It can magnify bulk samples with
greater depth of view so that the image produced represents the 3-D structure of the sample,
but the image is still only black and white. Generally, it can magnify the object 10,000 times.

Scanning Probe Microscope

The scanning probe microscope was developed in the 1980s by the Swiss scientists Dr. Gerd
Binnig and Dr. Heinrich Rohrer. It is used to study the molecular and atomic shapes of
organisms on a nanoscale. A physical probe is used to scan back and forth over the surface of
a sample. A computer then gathers data that are used to generate an image of the surface. It
can also be used to determine the variations in temperature inside the cell as well as its
chemical properties.

Staining
Most microorganisms besides being very tiny are also devoid of any color and are thus difficult
to see, even with the use of the microscope. To facilitate visualization, staining procedures
have been developed by various scientists. These staining procedures are meant to give color
to the organisms, making them easier to see under the microscope.

Simple Stains
Simple stains make use of a single dye which can either be aqueous (water-based) or alcohol-
based. This method of staining is a quick and easy way to visualize cell shape, size, and
arrangement of bacteria. It uses basic dyes such as safranin, methylene blue, or crystal violet.
These stains give up or accept hydrogen ion, leaving the stain positively charged. Most
bacterial cells and cytoplasm are negatively charged and since the dye is positively charged, it
adheres readily to the cell surface enabling the visualization of bacterial cell morphology.

Differential Stains
Differential stains are used to differentiate one group of bacteria from another. There are two
types of differential staining procedures commonly used, namely:

1. Gram stain - distinguishes gram-positive bacteria from gram-negative bacteria. or pink. As

a general rule, all cocci are gram-positive except Neisseria, Veilonella, and Branhamella. On
the other hand, all bacilli are gram-negative except Corynebacterium, Clostridium, Bacillus,
and Mycobacterium.

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Reagent Function Result if Gram- Result if Gram-

positive negative

Crystal violet Primary stain Purple or blue Purple or blue

Gram’s iodine Mordant Purple or blue Purple or blue

Acetone or 95% Decolorizer Purple or blue Colorless

alcohol
Safranin Counterstain or Purple or blue Red or pink
secondary stain

2. Acid -fast stain - stain used for bacteria with high lipid content in their cell wall hence
cannot be stained using Gram stain. Two methods are used, namely;

a. Ziehl-Neelsen stain - also known as the "hot method" because it requires steam-bathing
the prepared smear after addition of the primary dye. This is because the primary stain used is
aqueous and will not bind to the cell wall of the organism. Acid-fast organisms will appear red
on a blue background.

b. Kinyoun stain - also known as the "cold method" as it does not utilize heat after addition of
the primary stain, which is oil-based. The acid-fast organisms will appear red on a green
background.

Special Stains. These are used to demonstrate specific structures in a bacterial cell. For
instance, metachromatic granules can be visualized using the LAMB (Loeffler Alkaline
Methylene Blue) stain. Other special stains include Hiss stain (capsule or slime layer); Dyer
stain (cell wall), Fischer-Conn stain (flagella), Dorner and Schaeffer-Fulton stain (spores), and
India ink or nigrosine (capsule of the fungus Cryptococcus neoformans).

Culture Media
Staining procedures only give clues as to the probable organism being studied. To identify a
specific organism, culture using specific culture media is the most ideal. Media (sing, medium
are used to grow microorganisms. A culture medium is basically an aqueous solution to which
all the necessary nutrients essential for the growth of organisms are added. These are
classified into three primary levels: physical state, chemical composition, and functional type.

According to Physical State

1. Liquid media - commonly called broths, milk, or infusions, these are water-based solutions

that do not solidify at temperatures above the freezing point. These contain specific amounts
of nutrients but do not contain gelling agents such as gelatin or agar. Liquid media are suited
for the propagation of a large number of organisms, fermentation studies, and other tests.

2. Semi-solid media - exhibit a clot-like consistency at ordinary room temperature and contain
agar at concentrations of 0.5% or less that allows thickening of the media without producing a
firm substance. They have a soft consistency similar to custard and are best suited for culture
of microaerophilic bacteria or for the study of bacterial motility.

3. Solid media - contain a solidifying agent such as 1.5%-2% agar, giving them a firm surface
on which cells can form discrete colonies. They are used for isolation of bacteria and fungi or
for determining the colony characteristics of the organism under study. Solid media come in
two forms: (a) liquefiable (or reversible) solid media and (b) non-liquefiable (or non-reversible)
solid media.

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According to Chemical Composition

1. Synthetic media - contain chemically-defined substances which are pure organic and/ or
inorganic compounds. The precise chemical composition of a synthetic medium is known.
They may be simple or complex, depending on what supplement is added to it.

2. Non-synthetic media - complex media that contain at least one ingredient that is not
chemically defined, which means that it is neither a simple or pure compound. It is not
representable by an exact chemical formula. Most are extracts of animals, plants, or yeasts.
Non-synthetic media can support the growth of more fastidious organisms.

According to Functional Type

1. General Purpose media - are designed for primary isolation of a broad spectrum of
microbes and contain a mixture of nutrients that support the growth of both pathogenic and
non-pathogenic organisms. Examples are peptone water, nutrient broth. and nutrient agar.
2. Enrichment media - contain complex organic substances such as blood, serum, or special
growth factors, and are designed to increase the number of desired microorganisms without
stimulating the rest of the bacterial population. These are used to grow fastidious or
nutritionally exacting bacteria. There are two commonly used enrichment

media, namely:

a. Blood agar - contains general nutrients with 5%-10% (by volume) blood added to a blood
agar base. Certain gram-positive bacteria produce exotoxins that cause hemolysis of red blood
cells contained in the blood agar. Their hemolytic reaction is categorized into three, which is
useful in the classification of these bacteria. The hemolytic patterns are:

i. Beta hemolysis - shows complete lysis of red blood cells resulting in complete

clearing around the colonies.

ii. Alpha hemolysis - shows incomplete lysis of red blood cells, producing a greenish
discoloration of the blood agar around the colonies.

iii. Gamma hemolysis - shows no hemolysis, resulting in no change in the medium.

b Figure 1.6 Three types of hemolytic reactions seen in the culture: (a) beta hemolysis or
complete hemolysis; b) alpha hemolysis or incomplete hemolysis; and © gamma hemolysis or
no hemolysis

b. Chocolate agar - a type of nutrient medium that is used for the culture of fastidious
organisms such as Haemophilus sp. Heat is applied to lyse the red blood cells, causing the
medium to turn brown.

3. Selective media - contain one or more substances that encourage the growth of only a
specific target microorganism and inhibit the growth of others. It is designed to prevent the
growth of unwanted contaminating bacteria or commensals so only the target bacteria will
grow. Examples of approaches that will make the medium selective include changing the pH of
the culture medium or adding substances such as antibiotics, dyes, or other chemicals. These
are usually agar-based solid media that allow isolation of individual bacterial colonies.
Examples of this type of culture medium include the following:

a. Thayer-Martin agar - contains the antibiotics trimethroprim, nystatin, vancomycin, and

colistin. It is used for the isolation of Neisseria.

b. Mannitol Salt agar - contains 10% NaCI and used for the isolation of Staphylococcus
aureus.

c. MacConkey's agar - promotes the growth of gram-negative bacteria, primarily those

belonging to the family Enterobacteriaceae, and inhibits the growth of gram- positive bacteria
through the addition of bile salts. It is both selective and differential.

d. Löwenstein-Jensen medium - a selective medium used to recover Mycobacterium

tuberculosis. It is made selective by the incorporation of malachite green.

e. Saboraud's dextrose agar - used for the isolation of fungi.

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4. Differential media - allow the growth of several types of microorganisms. These are
designed to show visible differences among certain groups of microorganisms. The differences
may be in the form of variations in colony size or color, changes in color of culture media, or
formation of precipitates or gas bubbles. Differential media allow the growth of more than one
target microorganism that demonstrate morphologic variations in colony morphology.
Examples include MacConkey's agar and Triple Sugar Iron agar.

5. Transport media - used for clinical specimens that need to be transported to the laboratory
immediately after collection. These media prevent the drying of specimen and inhibit the
overgrowth of commensals and contaminating organisms. Charcoal is added to neutralize
inhibitory factors. Examples are the Cary Blair transport medium for transport of feces of
suspected cholera patients and Pike's medium which is used to transport throat specimens of
patients with streptococcal infection.

6. Anaerobic media - media used specifically for organisms that cannot survive in the
presence of oxygen and require reduced oxidation-reduction potential and other nutrients.
These are supplemented with nutrients such as vitamin K and hemin. They undergo boiling to
remove dissolved oxygen. To reduce the oxidation-reduction potential, substances such as 1%
glucose, 0.1% ascorbic acid, 0. 1% thioglycolate, or 0.05% cysteine ate added. Methylene
blue or resazurin is added as an indicator of the oxidation- reduction potential. Examples are
chopped cooked meat and thioglycolate broth.

Chapter 1 Summary:
Microbiology is the study of small, living microorganisms or microbes that cannot be seen with
the naked eye. These organisms may be cellular (prokaryotes, eukaryotes, and the like) or
acellular such as viruses. Microbiology is divided into several fields that deal with the study of
bacteria (bacteriology), viruses (virology), fungi (mycology), protozoa and parasitic worms
(parasitology), algae (phycology), and the immune system (immunology). Microorganisms may
be beneficial or harmful. Some microorganisms are used in different industries such as in food
and beverage. Some microorganisms are sources of antibiotics while some are used in the field
of biotechnology and genetic engineering. Microorganisms are also important in maintaining a
balanced ecosystem. While some microorganisms are essential and have beneficial uses, there
are also numerous microorganisms that produce disease in humans, some of which are
potentially fatal. Some microorganisms have the potential to be used as biological warfare
agents. Microorganisms are so miniscule that for them to be visualized, they need to be stained
and studied using the microscope. Several types of microscopes have been developed for this
purpose from the compound microscope to the more sophisticated electron microscopes.

The use of various staining procedures has made visualization of microorganisms easier. These
stains may be classified into simple, differential, and special stains. › Simple stains make use of
a single water- or alcohol-based dye that is used to demonstrate the shape and basic
structures of the organism. Differential stains are used to distinguish one group of bacteria from
another group. These include the Gram stain and the acid-fast stain. Special stains are mainly
used to demonstrate specific bacterial structures such as the spores (Dorner or Schaeffer-
Fulton), flagella (Fischer & Conn), capsule (Hiss stain), or the metachromatic granules (LAMB
stain). Specific culture media are the most ideal in identifying specific organisms. Several
classes of culture media have been developed and these culture media can be classified into
three primary levels: physical state (liquid, semi-solid, solid), chemical composition (synthetic
and non-synthetic), and functional type (general purpose, enrichment, selective, differential,
transport, and anaerobic),

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Chapter 2: Prokaryotic and Eukaryotic Cells

Living Cells can be classified into two general categories- prokaryotes and eukaryotes.
Prokaryotes are organisms that do not possess a true nucleus and membrane-bound
organelles (e.g., bacteria). Eukaryotic organisms are those that possess a true nucleus and
membrane-bound organelles. They are usually multicellular organisms and include plants,
animals, fungi, parasites, and algae. Viruses are acellular organisms that possess only DNA or
RNA. They are dependent on host cells for their replication and are considered as obligate
intracellular parasites.

1-10 um - Typical Prokaryotic Cell

10-100 um - Typical Eukaryotic Cell

Feature Prokaryotic Eukaryotic

Genetic Material Not enclosed within a Enclosed within a membrane;

membrane, not associated with
histones, usually circular
Size Smaller (1-2um by 1-4um or
less)
Cell type Mostly unicellular
associated with histones; usually
linear
Greater than 5um in diameter
Mostly multicellular

Nucleus No true nucleus and nuclear With true nucleus enclosed by

membrane; called nucleoid nuclear membrane
Cell Wall Simple Complex

Cell Division Budding or binary fission Mitosis

Sexual Reproduction No meiosis; transfer of DNA only Meiosis

Cytoskeleton Absent Present

Mesosome Functions as mitochondria and Absent

Golgi complex
Ribosomes 70S; located in cytoplasm 80S; located in membranes such
as in the endoplasmic reticulum

70S; found in organelles such as

mitochondria or chloroplast
Membrane-bound organelles Absent Present

Extrachromosomal plasmid Present Absent

Duration of cell cycle Short (20-60 minutes) Long (12-24 hours)

Medically Important Microorganisms - Organisms that are considered medically important

are those that have the potential or the ability to produce significant clinical disease in humans.
They may be part of the normal flora of the body or are true pathogenic organisms. These may
be categorized into bacteria, viruses, fungi, algae, and parasites (protozoa and helminths).
Viruses are acellular organisms. Their outer surface is called capsid, which is composed of
repeating sub-units called capsomeres. Viruses possess only a single nucleic acid, either DNA

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or RNA, but never both. In addition, viruses lack the necessary cellular parts that can allow
them to replicate independent of the host cell. They also lack the genes and enzymes that are
necessary for energy production. They rely on the cellular machinery of the host cell for protein

and energy production. Hence, viruses are considered obligate intracellular parasites.

Viruses are classified based on the following: (1) type of nucleic acid they possess; (2) shape
of the capsid (icosahedral, helical, polyhedral, or complex); (3) number of capsomeres; (4) size
of the capsid; (5) presence or absence of an envelope; (6) type of host they infect (humans,
plants, or animals); (7) type of disease they produce; (8) target cell or tropism (e.g., T helper
cells for HIV); and (9) immunologic or antigenic properties.

Bacteriophages are a special type of viruses that primarily infect bacteria. They are similar to
other viruses in that: (1) they are obligate intracellular parasites; (2) they are similarly shaped
like other viruses; and (3) they may also be classified based on the type of nucleic acid they
possess. They play a role in the acquisition of virulence factors of certain bacteria (e.g.,
diphtheria toxin of Corynebacterium diphtheriae), as well as in the transfer of genetic material
from one bacterium to another (as in transduction).

Bacteria are prokaryotic cells with majority having an outer covering called the cell wall that is
composed mainly of peptidoglycan. Unlike viruses, they possess both DNA and RNA. Unlike
eukaryotic organisms, bacteria possess a nucleoid instead of a true nucleus, smaller
ribosomes, and lack mitochondria. Based on their physical characteristics, bacteria may be
broadly categorized into (1) gram-negative bacteria with cell wall (e.g., Escherichia coli); (2)
gram-positive bacteria with cell wall (e.g., Staphylococcus aureus); (3) acid-fast bacteria with
lipid-rich cell wall (e.g., Mycobacterium tuberculosis); and, (4) bacteria without cell wall (e.g.,
Mycoplasma).

Fungi are eukaryotic cells with an outer surface composed mainly of chitin. Their cell
membrane is made up mostly of ergosterol. Like bacteria, fungi possess both DNA and RNA.
Unlike bacteria, they possess a true nucleus that is enclosed by a nuclear membrane and
mitochondria that function for ATP production. Fungal ribosomes are also larger than bacterial
ribosomes (80 Svedberg units). Table 2.2 summarizes the major differences between fungi and
bacteria.

Protozoa are the representatives for parasites. Like bacteria and fungi, these are also
eukaryotic cells that have an outer surface called a pellicle. These are unicellular organisms
that usually divide through binary fission, similar to bacteria. Majority exist in two morphologic
forms--cysts and trophozoites. The infective stage is the cyst while the pathogenic stage is the
trophozoite. Protozoa possess both DNA and RNA as well as other cellular features seen in
typical eukaryotic cells.

Features Bacteria Fungi

Cell Type Prokaryotic; unicellular Eukaryotic, unicellular or

multicellular
Role in ecosystem Can be both producers and Mainly decomposers
decomposers
Optimal pH Neutral pH (6.5-7.0) Slightly acidic (4.0-6.0)
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Features Bacteria Fungi

Cell Structure
Main component of cell wall
Sterols in cell membrane
No true nucleus and membrane- Possess true nucleus and
bound organelles membrane-bound organelles
Peptidoglycan, except in Chitin
archaebacteria
Absent except in Mycoplasma Present

Mode of nutrition Heterotrophic, Heterotrophic, majority aerobic

chemoautotrophic, and facultative anaerobic
photoautotrophic, aerobic,
anaerobic, facultative anaerobic
Reproduction Binary fission Sexual and asexual spores.

Algae are eukaryotic organisms whose outer surface consists primarily of cellulose. They are
described as plant-like organisms because most of them have chlorophyll and are thus
capable of photosynthesis. Unlike plants, they do not possess true roots, stems, and leaves.
Table 2.3 summarizes the major differences between algae and plants. Algae vary in size from
the single- celled phytoplanktons to the large seaweeds found in the ocean floor. Algae do not
produce significant disease in humans. Most algae are beneficial in that they are important
sources of food, iodine, and other minerals. They may also be used as fertilizers, emulsifiers for
puddings, and stabilizers for ice cream and salad dressings.

Features Algae Plants

Taxonomic classification Kingdom Protista Kingdom Plantae

Cellular structure Unicellular, multicellular or Multicellular

colony-forming

Photosynthetic Yes Yes

Energy source Carbon Dioxide Carbon Dioxide

Storage form of energy Starch Starch

Vascular system Absent Present

Habitat Mostly water Mostly rooted to the ground

Composed of roots, stems and No Yes

leaves

Method of reproduction Both asexual and sexual Sexual (complex)

Diatoms are unicellular algae that inhabit both fresh- and saltwater. Their cell wall contains
silicone dioxide that may be utilized in filtration systems, insulation, and as abrasives.
Dinoflagellates are also unicellular algae that are important members of the phytoplankton
group. They contribute greatly to the oxygen in the atmosphere and serve as important links in
the food chain. On the other hand, they are also responsible for what is known as "red tide."
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These small organisms produce a powerful neurotoxin which, when ingested in significant
amounts, is responsible for the potentially fatal disease called paralytic shellfish poisoning.

Chapter 2 Summary
Living cells can be classified as either prokaryotic or eukaryotic.

• Prokaryotic cells, as exemplified by bacteria, are usually unicellular, do not possess a true

nucleus and membrane-bound organelles, and multiply by means of binary fission.

• Eukaryotic cells vary from unicellular (e.g., protozoa) to multicellular (e.g., fungi). They

possess a true nucleus surrounded by a nuclear membrane as well as membrane-bound

organelles.

Viruses are not classified as cells since they only possess an outer covering called capsid and
a nucleic acid (either DNA or RNA). As such, they are dependent on the host cell machinery for
their replication and are thus considered as obligate intracellular parasites. Medically important
organisms are those which produce significant disease in humans. These may take the form of
viruses, bacteria, fungi, protozoa, and algae.

Viruses are acellular, obligate intracellular parasites possessing only DNA or RNA and may be
classified based on: (1) type of nucleic acid they possess; (2) shape of the capsid (icosahedral,
helical, polyhedral, or complex); (3) number of capsomeres; (4) size of the capsid; (5) presence
or absence of an envelope; (6) type of host they infect (humans, plants, or animals); (7) type of
disease they produce; (8) target cell or tropism (e.g., T helper cells for HIV); and (9)
immunologic or antigenic properties. Bacteria are prokaryotic organisms that possess both
DNA and RNA. Most possess a cell wall composed predominantly of peptidoglycan. Fungi are
eukaryotic organisms with a cell wall composed mainly of chitin and cell membrane that
contains ergosterol.

Protozoa are mostly unicellular parasites that are eukaryotic. Most divide by binary

fission similar to bacteria.

› Algae are eukaryotic, aquatic, plant-like organisms. Similar to plants, they are photosynthetic
but unlike plants, they do not have true roots, stems, or leaves.

Chapter 3: Bacterial Morphology

Bacteria, which are prokaryotic, have simpler structures compared to eukaryotic organisms. In
terms of morphology, bacteria may be classified into three basic shapes: coccus (pl. cocci),
bacillus (pl. bacilli), and spiral-shaped or curved. Cocci can be described as spherical or round-

shaped organisms (e.g., Staphylococcus, Streptococcus). They may be arranged singly, in

pairs (diplococci), in chains (streptococci), in clusters (staphylococci), in groups of four (tetrad),

or in groups of eight (octad). Rod-shaped organisms are called bacilli (e.g., Escherichia coli,
Salmonella). Some may be very short, resembling elongated cocci called coccobacilli (e.g.,
Haemophilus influenza). Curved and spiral-shaped organisms may show variations in their
morphology. Vibrio cholerae, the organism causing cholera, is described as comma-shaped.
The causative agent of syphilis, Treponema pallium, is spiral in shape while the causative agent

of diphtheria, Corynebacterium diphtheria, is club-shaped.

Envelope Structures
Prokaryotic cells are surrounded by a complex envelope that may vary in composition. The
envelope serves to protect the bacteria from harsh environmental conditions.

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Glycocalyx
This is the outermost covering of some bacteria. It is a gelatinous substance that is located
external to the cell wall, composed of polysaccharide or polypeptide, or both. It is called
capsule if it is strongly attached to the cell wall and slime layer if it is loosely attached. The
presence of the capsule is indicative of the virulence of an organism, aiding the organism in the
evasion of phagocytosis. It can stimulate an antibody response from the immune system. The
capsule serves to protect the organism from dehydration.

Cell Wall
The bacterial cell wall is sometimes called the murein sacculus. Its principal component is
peptidoglycan, which is also called murein or mucopeptide. It is multi-layered in gram-positive
bacteria and single-layered in gram-negative bacteria. The cell wall provides rigid support and
gives shape to the bacteria. It protects the bacteria from osmotic damage and plays an
important role in cell division.

Special components of gram-positive cell walls

1. Teichoic acids - comprise major surface antigens of gram-positive organisms and can elicit
antibody response. In some gram-positive organisms such as Staphylococcus aureus, teichoic
acids function for the attachment of the organism to the host cell. These also provide tensile
strength to gram-positive bacterial cell walls.

2. Polysaccharides - polysaccharide molecules include neutral sugars such as mannose,

arabinose, rhamnose, and glucosamine. It also includes some acidic sugars such as glucuronic
acid and mannuronic acid.

Special components of gram-negative cell walls

1. Outer membrane - a bi-layered structure where the inner leaflet is composed of a

lipopolysaccharide (LPS). It has special protein channels that allow the passage of small or
low-molecular-weight hydrophilic substances such as sugars and amino acids. LPS has a
complex glycolipid called lipid A, responsible for its endotoxin activity. It is located in the outer
leaflet of the outer membrane. The inner core is a polysaccharide made up of repeat units. This
repeat unit is also called the O antigen, which is unique for every species of bacteria.

2. Lipoprotein - functions to anchor the outer membrane to the peptidoglycan layer and
stabilizes the outer membrane.

3. Periplasmic space - a fluid-filled space between the outer membrane and the inner plasma
membrane. It contains enzymes for the breakdown of large non-transportable molecules into
transportable ones and enzymes that serve to detoxify and inactivate antibiotics.

Acid-fast cell wall

Unlike gram-positive and gram-negative bacteria, acid-fast organisms such a Mycobacterium
tuberculosis possess an outer layer that is lipid-rich. The cell wall of acid-fare organisms is
composed of large amounts of waxes that are known as mycolic acids. The inner layer of the
cell wall is also made up of peptidoglycan but because the outermost layer is lipid. rich, cell
walls of acid-fast organisms are hydrophobic. This is the reason why they cannot be stained
using the reagents used in gram staining. The hydrophobic nature of their cell wall protects
them from harsh chemicals such as strong acids and detergents.

Projecting Structures
Flagella
These are thread-like structures made up entirely of molecules of the protein sub-unit flagellin.
They project from the capsule and are organs for motility. Flagella are classified into four types,
namely: (a) monotrichous (single polar flagellum); (b) lophotrichous (a tuft of flagella at one end
of the bacterium); (c) amphitrichous (flagella at both ends of the bacterium); and (d) peritrichous
(flagella all around the bacterium). Bacteria without flagella are called atrichous.

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Pili or Fimbriae
These are rigid surface appendages found on many gram-negative bacteria. They are fine and
short in comparison with flagella. Their structural protein sub-units are called pilins. Pili may
also function for motility. They function for adherence to cell surface (common pili) or
attachment to another bacterium during a form of bacterial gene exchange called conjugation
(sex pili).

Axial Filaments
Axial filaments are also called endoflagella and are found in spirochetes (e.g., Treponema
pallidum causing syphilis). These are composed of bundles of fibrils, the structures of which
are similar to flagella. They arise from the ends of the bacterial cell and spiral around the cell.
The filaments rotate producing movement of the outer sheath of the spirochetes propelling
them forward.

Cytoplasmic Membrane
Also called cell membrane or plasma membrane, the cytoplasmic membrane is located
beneath the cell wall. It is sometimes called the cell sac because it encloses the cytoplasm of
the cell The cytoplasmic membrane is a selectively permeable membrane that allows for
transport of selected solutes. In aerobic organisms, it is the site of the electron transport chain
and serves as the site of ATP production. It therefore serves the function of the mitochondria,
which are not found in prokaryotic cells. The cytoplasmic membrane also contains the
enzymes needed for the biosynthesis of DNA, cell wall components, and membrane lipids.

Internal Structures
Nucleoid
Bacteria have no true nucleus that is surrounded by a nuclear membrane. Its genetic material is
packaged in a structure called the nucleoid. Bacteria possess a single, circular, double-
stranded DNA.

Mesosomes

The mesosome functions for cell division. It is also involved in the secretion of substances
produced by bacteria.

Ribosomes

The ribosomes function for protein synthesis. Unlike eukaryotic ribosomes, bacterial ribosome
is smaller (70S).

Granules or Inclusion Bodies

These are found in certain bacteria and serve for storage of food and energy) (e.g.,
metachromatic granules of Corynebacterium or Much granules of Mycobacterium
tuberculosis).

Endospores
Endospores are structures produced by many bacteria when they are placed in a hostile
environment. It is composed of dipicolinic acid which confers resistance to heat, drying,
chemical agents, and radiation; making it very difficult to destroy. The process of spore
production is called sporulation, and this occurs when the environmental conditions are
detrimental to the bacteria. When environmental conditions become favorable, the endospores
revert to their vegetative state through a process called germination. Some gram-positive, but
never gram-negative, bacteria form spores.

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Chapter 3 Summary
There are three basic shapes of bacteria: (a) spherical or cocci; (b) rod-shaped or bacilli,

and (c) curved or spiral. A typical prokaryotic cell is composed of three major components-an
outer envelope with its projecting structures, the cell membrane, and the internal structures.

The envelope is composed of the following:

The outermost covering is the glycocalyx, also known as the capsule if it is adherent to the cell
wall and slime layer when it is loosely attached to the cell wall.

The cell wall or the murein sacculus provides rigid support and shape to the bacteria. Its main
component is peptidoglycan, which is multilayered in gram-positive bacteria and monolayered
in gram-negative bacteria.

- Gram-positive cell wall contains teichoic acids which may function for the attachment of the
bacterium to the host cell, as well as polysaccharide molecules.

- Gram-negative cell walls contain lipopolysaccharide made of a lipid A molecule and

polysaccharides. The lipid A component is responsible for the endotoxic activity of gram-
negative bacteria. The lipopolysaccharide is an integral part of the outer membrane of gram-
negative bacteria. Gram-negative bacteria also have a periplasmic space where important
enzymes are found.

- Acid-fast organisms possess a cell wall that is also made up of an inner layer of
peptidoglycan and an outer layer rich in waxes composed of mycolic acid and other lipids.
This is responsible for the hydrophobic nature of its cell wall and the main reason why acid-
fast organisms cannot be stained using the reagents for Gram-staining.

- Structures projecting from the bacterial capsule include pili or fimbriae of gram-negative
organisms, flagella, and axial filaments of spirochetes.

- There are two types of pili: common pili which functions for attachment and sex pili which
participates in gene exchange among bacteria in a process called conjugation.

- Flagella may be of four patterns: (1) lophotrichous (a tuft of flagella on one end of the
bacterium), (2) amphitrichous (a single flagellum on each end of the bacterium), (3)
peritrichous (flagella surrounding the bacterium), and (4) monotrichous (only one flagellum at
one end of the bacterium).

- Axial filaments are similar in structure to flagella and help propel the spirocheles forward.

- Bacterial cytoplasmic membrane is the functional analogue of the mitochondria. It is

selectively permeable and is the site of ATP production of aerobic bacteria.

- Bacteria do not have a true nucleus. Its genetic material is packaged in a structure called
nucleoid. Bacterial ribosome is smaller than a typical eukaryotic ribosome.

- Bacteria possess structures that enable them to withstand adverse environmental

conditions. These structures are the endospores which are mainly composed of dipicolinic
acid.

- Other structures found in bacterial cells are the mesosomes, which play a role in cell
division, and inclusion bodies or granules in some bacteria which serve as storage for food.

Chapter 4: Bacterial Growth Requirements

Growth as defined in medical dictionaries involves an orderly and organized increase in the
sum of all components of the organism. The process entails the replication of all cellular
structures, organelles, and components. Microbial growth is concerned with the increase in the

number of cells and not an increase in the size of the organism. A bacterial colony is composed

of thousands of cells; hence, colonies in culture are actually composed of billions of cells. As in
any living organism, bacteria require certain nutrients and physical conditions that will promote
their growth. This chapter discusses the various nutritional and physical requirements of
bacteria for growth.

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Nutritional Requirements
Carbon

Carbon makes up the structural backbone or skeleton of all organic molecules. Based on their
carbon source, microorganisms may be classified into autotrophs (lithotrophs) and
heterotrophs (organotrophs).

Autotrophs are microorganisms that utilize inorganic compounds (e.g, carbon dioxide) and

inorganic salts as their sole carbon source. Organotrophs are organisms that make use of
organic substances like sugars or glucose as their carbon source, For both autotrophs and
heterotrophy, their energy may be derived from either light (photolithotrophs and
photoorganotrophs) or the oxidation of inorganic substances (chemolithotrophs and
chemoorganotrophs). Most medically important bacteria are chemoorganotrophs.

Nitrogen, Sulfur, Phosphorus. These are necessary for the synthesis of cellular materials like
proteins and nucleic acids. Nitrogen and sulfur are required for the synthesis of proteins.
Nitrogen and phosphorus are essential for the synthesis of nucleic acids and ATP.
Approximately 14% of the dry weight of a bacterial cell is nitrogen and about 4% is sulfur and
phosphorus.

Inorganic lons

These include magnesium, potassium, calcium, iron, and trace elements (e.g., manganese,
zinc, copper, cobalt). Magnesium stabilizes ribosomes, cell membranes, and nucleic acids. It
also serves as a co-factor in the activity of many enzymes. Potassium is required for the normal
functioning and integrity of ribosomes and participates in certain enzymatic activities of the
cell.

Calcium is an important component of gram-positive bacterial cell wall and contributes to the
resistance of bacterial endospores against adverse environmental conditions. Iron 1s a
component of cytochrome, a component of the electron transport chain, and functions as a
co-factor for enzymatic activities. Trace elements are components of enzymes and function as
co-factors. Some are necessary for the maintenance of protein structure.

Growth Factors

Growth factors are essential to promote the growth and development of the bacterial cell.
These include vitamin B complex and amino acids.

Physical Requirements
Moisture/Water

The bacterial cell is composed mainly of water. It serves as the medium from which bacteria
acquire their nutrients.

Oxygen is used by aerobic bacteria for cellular respiration and serve as the final electron
acceptor. Microorganisms are classified as either aerobes or anaerobes based on their oxygen
requirements.

Microorganisms that utilize molecular oxygen for energy production are referred to as aerobes.
Strict aerobes are organisms that strictly require oxygen for growth. Microbes that cannot
survive in the presence of oxygen are called obligate anaerobes. These organisms do not have
the enzymes that break down free radicals produced in the body (i.e., catalase and superoxide
dismutase).

There are organisms that can grow and survive under both aerobic and anaerobic conditions.
These are called facultative organisms. Most medically important bacteria are facultative. Some
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organisms are able to grow at low oxygen tension but their rate of growth is diminished. These
are called microaerophiles. There are some organisms though that may require the addition of
carbon dioxide to enhance their growth. These are called capnophiles.

Temperature

Enhanced enzyme activity requires certain temperatures. Microbes are classified into three
groups based on their temperature requirements, namely: (1) thermophiles, which grow best at
temperatures higher than 40°C; (2) mesophiles, which require an optimal temperature of
20°C-40°C; and, (3) psychrophiles, which require an optimum temperature of 10°C-20°C. Most
medically important bacteria are mesophiles.

pH

Another requirement of bacteria is the extent of acidity or alkalinity of their environment, which
is referred to as the pH. Microorganisms that grow best in pH 8.4-9.0 are called alkalophiles.
Those that grow best in pH 6.5-7.5 are called neutrophiles. Most medically important bacteria
are neutrophiles. The pH of most human tissues are 7.0-7.2. Certain bacteria require a pH less
than 6.0. These bacteria are called acidophiles.

Osmotic Conditions

Most organisms grow best under ideal conditions of osmotic pressure, which is determined by
the salt concentration. The normal microbial cytoplasmic salt concentration is approximately
1%. The optimum condition is if the external environment also has the same salt concentration.

of the extracellular salt concentration is increased (e.g., when food is salted), water will flow out
of the microbial cell and the organism will shrink and die. On the other hand, if the external
environment does not contain salt, water will flow into the bacterial cell causing the organism
to swell and rupture. Organisms that require high salt concentrations for growth are called
balophiles (e.g., diatoms and dinoflagellates) and those that require high osmotic pressure for
optimal growth are called osmophiles.

Bacterial Growth Curve

The bacterial growth curve illustrates the phases in the growth of the population of bacteria
when they are grown in a culture of fixed volume. It reflects the different stages in the growth of
the organism and is divided into four phases: lag phase, log phase, stationary phase, and
death or decline phase.

Lag Phase

This is the period of adjustment for the bacteria in the new environment. During this phase,
there is no appreciable increase in the number of microorganisms. The organisms will show
increased metabolic activity in order to synthesize DNA as well as secrete enzymes which
might not be present in their new environment but which are needed by the organism. Bacteria

attain their maximum size toward the end of the lag phase. This phase may last for 1 to 4
hours.

Log/Logarithmic/Exponential Phase

This period is characterized by rapid cell division, resulting in an increase in the number of
bacteria. The organism exhibits high metabolic activity. This is the period when the generation
time or doubling time of the organism (i.e., the time required for the bacterial cells to double in
number) is determined. A generation time of 10 minutes means that the bacteria will double in
number every 10 minutes showing exponential growth. The average duration of this phase is
about 8 hours.

Stationary Phase

This is considered as the period of equilibrium. During this period, the rate of growth slow
down, nutrients start to deplete, and toxic wastes begin to accumulate. As a consequence,
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some bacterial cells may die. However, since there are still bacterial cells undergoing cell
division, the number of living cells equals the number of dead cells. Gram-positive organisms
may become gram-negative organisms in this phase. Sporulation occurs towards the end of
this phase, or in the case of spore-forming organisms, during the beginning of this phase.

Death or Decline Phase

This is the period of rapid cell death where the number of dead cells is greater than the number
of living cells. This is due to the continuous depletion of nutrients and accumulation of waste
materials. Sporulation continues to occur during this stage. The duration of this phase varies
from a few hours to a few days.

Chapter 4 Summary
Bacteria require optimum nutrient and physical conditions for their growth.

Nutritional requirements of bacteria include adequate supply of carbon, nitrogen, sulfur,

phosphorus, inorganic ions, and growth factors.

Bacteria are classified into two groups based on their carbon source: autotrophs/lithotrophs
and heterotrophs/organotrophs.

Autotrophs utilize inorganic compounds

for their carbon source while organic compounds such as glucose serve as the carbon source
of heterotrophs. Bacteria derive energy by two means: from sunlight or from oxidation of
inorganic substances.

Physical requirements of bacteria include moisture, oxygen, temperature, pH, and osmotic
conditions.

Bacterial cell is made up mostly of water, which serves as the medium from which bacteria
derive their nutrients.

» Organisms that require oxygen for optimal growth are called aerobes while those that cannot
survive in the presence of oxygen are called anaerobes.

» Facultative organisms are those which can grow in the presence or absence of oxygen.

- Bacteria may be grouped into three based on their temperature requirements: (1) those that
require high temperature (thermophiles); (2) those that require temperature of 20 C-40°C
(mesophiles); and (3) those that require temperature of 10°C-20°C

(psychrophiles).

- Acidophiles are organisms that grow best in pH < 6.0. Neutrophiles grow best at pH of
7.0-7.2 while alkalophiles are those that grow best at pHI of 8.4-9.0.

- Organisms that require salt for growth are called halophiles. Osmophiles are those that need
high osmotic pressure for maximal growth.

Based on their nutritional and physical requirements, most medically important bacteria are
chemoorganotrophs, facultative, mesophiles, and neutrophiles.

The bacterial growth curve illustrates the phases of growth of a bacterial population grown in
culture of fixed volume. It is divided into a lag phase, log phase, stationary phase, and death or
decline phase.

Chapter 5: Normal Flora of the Human Body

Microbial Ecology is the study of the relationships between microorganisms and their
environment. Among these relationships is the relationship of microbes with humans, and such

include the normal flora (or indigenous flora) of the human body. Normal flora consists of the
group of organisms that inhabit the body of a normal healthy individual in the community.
These indigenous flora may be non-pathogenic or pathogenic and may at times behave as
opportunistic pathogens.

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There are two types of flora, namely: (1) resident flora and (2) transient flora. Resident flora are
organisms that are relatively of fixed types and are regularly found in a given area of the body
at a given age. Transient flora are those that inhabit the skin and mucous membrane
temporarily for hours, days, or weeks and are derived from the environment. Normal flora are
beneficial to the human body because they can inhibit the growth of pathogenic organisms by
priming the immune system of newborns. At the same time, normal flora protects the body's
organs and systems that are in direct contact with the external environment and are therefore
subject to the attack of invasive organisms. Normal flora do this by either competing with
invasive organisms for nutrients essential for their growth or by producing substances that can
kill them. Normal flora synthesize important vitamins that are essential to humans.

Normal Intestinal Flora secrete vitamin K that is needed for the activity of some clotting factors.
Other beneficial effects of normal flora include the following:

1. Normal fora can prevent pathogenic organisms from attaching to and penetrating the sin and
other tissues by producing mucin which make it difficult for the pathogenic organisms to attach
to the tissues to produce disease.

2. Normal flora in the intestines aid in the digestion of food by producing enzymes such as
cellulase, galactosidase, and glucosidase.

3. Intestinal flora also help in the metabolism of steroids. The healthy fetus is normally sterile
until birth, following the rupture of the bag of water. Once born, the newborn normal flora is
derived from the mother's genital tract during delivery, from the skin and respiratory tract of
individuals who handled the newborn, and from the environment.

There are certain body tissues and fluids that are normally sterile. Body fluids that are sterile
include the cerebrospinal fluid (CSF), synovial fluid, and blood. In the blood, there may be low
transient bacteremia brought about by physiologic trauma. The sterile tissues include the
urinary bladder, uterus, fallopian tubes, middle ear, and paranasal sinuses. Presence of bacteria

in these tissues and body fluids may lead to serious infections in these areas. For example,
bacteria in the CF can gain entry into the central nervous system, leading to a potentially fatal
encephalitis.

Normal Flora on Different Sites of the Body

Skin
The skin is the part of the human body that is in constant contact with the environment, making
it the most exposed to microorganisms. There are certain factors that eliminate non-resident
flora from the skin, namely: (1) lysozyme in the skin; (2) acidic pH of the shin due to sweat; (3)
free fatty acids in sebaceous secretions; and (4) the constant sloughing off of the skin.

The normal flora of the skin consists mainly of bacteria and fungi. The microorganisms vary
depending on the region of the skin. The skin may be divided into three regions: (1) axilla
perineum, and toe webs; (2) hand, face, and trunk; and (3) upper arms and legs. Skin of ti
axilla, perineum, and toe webs is characterized by having higher moisture levels, higher bod
temperature, and higher levels of surface lipids. These regions have more microorganisms
compared to the others and are predominantly inhabited by gram -negative bacilli. Dry side
(e:B, hands, forearms, feet, legs) have diverse flora because of their exposure to the
environment. Predominant flora in these areas include Staphylococus epidermidis and
Staphylococus hominis.

Most microorganisms in the skin are found in its superficial layers (stratum corneum) and hair
follicles. Anaerobes inhabit the deeper structures and layers of the skin, such as hair follicles,
sebaceous glands, and sweat glands. Table 5.1 summarizes the various microorganisms that
inhabit the skin.

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Organism Remarks

Staphylococcus epidermidis Major skin inhabitant, comprimising

Staphylococcus aureus
Micrococci (Micrococcus luteus)
approximately 90% of resident aerobic flora
Most commonly found in nose and perineum; in
the nose, number varies with age (greater in
newborns than in adults)
Accounts for 20%-80% of micrococci in the skin

Diphtheroids (Coryneforms) Classified into: lipophilic (common in axilla) or

non-lipophilic (more common on glabrous or
hairless skin such as palms of hands)

Anaerobic diphtheroids (Propionibacterium acnes)

Gram-negative Bacilli (Enterobacter, Klebsiella,
Escherichia coli, and Proteus spp.)
Nail Flora
areas rich in sebaceous glands
Seen in moist intertriginous areas such as toe
webs and axilla
Similar to that of the skin. Fungi may also be
present (Aspergillus, Penicillum, Cladosporium,
Mucor)

Mouth and Respiratory Tract

The tongue and buccal mucosa are inhabited mostly by Streptococcus viridans group, which
includes S. mutans, S. milleri, S. salivarius, and S. sanguis. Although they are part of the
normal fora of the mouth, the viridans streptococci have been implicated in the pathogenesis
of dental caries. The gingival crevices and the tonsillar crypts are primarily inhabited by
anaerobic flora. The normal flora of the pharynx and trachea are similar to those found in the
oral cavity. However, there may be transient carriage in the pharynx of potentially pathogenic
organisms. These include Haemophilus influenzae, Streptococcus pneumoniae, Neisseria
meningitidis, and Mycoplasma.

In the upper respiratory tract, initial colonization by path@schle organiems may be sen. These
include Neiseria meningitidis, Corynebacterium diphiberiae and Bordetellà pertunt, The lower
respiratory tract is usually sterile and organisms that reach this region are wall, destroyed by
the defense mechanisms of the body such as the alveolar macrophages.

Conjunctiva

The normal flora in the conjunctivae are very scanty because they are held in check by the flow
of tears that contain lysozyme. The lysozyme may interfere with the cell wall synthesis of
organisms. However, some bacteria may transiently colonize the conjunctiva including
Neisseria, Moraxella, and Corynebacterium. Staphylococci and streptococci may also be
present.

Digestive Tract

The esophagus contains transient mouth flora. Minimal bacteria may be found in the stomach
due to the relatively hostile environment in the stomach. Bacteria that may be found in the
stomach are those that may be swallowed with the food or those that are dislodged from the
mouth. The acidity in the environment of the stomach is further increased after meals because
of the release of gastric acid. However, there are certain bacteria that are able to survive in the
acidic environment of the stomach. One of these is Helicobacter pylori, the most common
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cause of duodenal ulcer. This organism produces urease that causes alkalinization of gastric
acid thereby enabling it to colonize the stomach.

The number of bacterial flora differs between the small intestine and large intestine. In the small
intestine, scanty flora may be found due to the constant peristaltic movement of the intestines.
Most of the bacteria cultured in the small intestine include streptococci, lactobacilli, and
Bacteroides which are all transient.

The number of bacterial flora in the large intestine is far greater than' in the small intestine. The
colon is inhabited predominantly by anaerobes (95%-99%) which includes Bacteroides fragilis
(most common), Bifidobacterium/Lactobacillus bifidum (predominant in breast-fed infants),
Eubacterium, Peptostreptococcus, and Clostridium. In bottle-fed infants, the predominant
intestinal flora is Lactobacillus acidophilus. About 1%-4% of the flora of the colon are
facultative aerobes, predominantly Escherichia coli and other Enterobacteriaceae. Intestinal
flora play important roles in the body, namely: (1) synthesis of vitamin B complet and vitamin K;
(2) conversion of bile into bile acids; (3) competition with transient flora lof nutrients; (4)
prevention of colonization of the intestines by transient flora; and (5) production of potentially
pathogenic end-products of metabolism that are toxic to transient flora.

Genitourinary Tract

The urinary tract is sterile above the distal 1 cm of the urethra. In the anterior urethra, the
predominant flora isolated are S. epidermidis, enterococci, and diphtheroids. In both males and
females, Mycobacterium smegmatis may be found as normal commensals in their secretions.
In addition, Gardnerella vaginalis, bacteroides, and alpha streptococci may be found in penile
urethra. The female urethra is either sterile or contains Staphylococcus epidermidis.

Vaginal flora varies depending on the age, hormonal levels, and vaginal pHI of the host. In
female infants, the predominant vaginal flora is Lactobacillus spp. From 1 month of age until
puberty, there is cessation of glycogen secretion making the vaginal pH higher (around 7.0).
The microorganisms that may inhabit the vagina at this time include Staphylococcus
epidermidis, Streptococci, diphtheroids, and Escherichia coli. At the onset of puberty, there is
resumption of glycogen secretion making the vaginal pH acidic. Predominant flora include
Lactobacillus acidophilus, corynebacteria, peptostreptococci, streptococci, Bacteroides, and
staphylococci. Lactobacillus plays a crucial role in preventing gonococcal infection by
producing lactic acid that adds to the acidity of the vagina. Young girls are more prone to the
development of gonococcal infection compared to adult women because the normal acidic pt
of the vagina as Well as the normal vaginal flora are not yet fully developed.

After menopause, the vaginal pH increases once more due to the lessened production of
glycogen. Normal flora that predominate during this period are similar to those found during
pre-puberty; Most of these flora are derived from the skin and from the colon. Pungi such as
Torulopsis and Candida may also be found (10%-30%). Conditions that will allow the
overgrowth of these fungi (eg, intake of antibiotics) can lead to vaginal infections such as
vaginitis.

Normal or indigenous flora refers to organisms that inhabit the body of a normal healthy
individual. Resident flora, also known as normal flora, refers to microorganisms that are
regularly found in a given area at a given age. Transient flora are those organisms that inhabit
the skin and mucous membrane temporarily for a few hours, days, or weeks. They do not
establish themselves permanently in the body tissues.

Normal flora have important roles in the body which can be beneficial or harmful.

Advantages of normal flora include:

1. Inhibition of growth of pathogenic organisms by priming of the immune system

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2. Synthesis of vitamin B12 and vitamin K in the intestines.

3. Synthesis of substances that may inhibit growth of pathogenic organisms

(e.g., enzymes, fatty acids, bacteriocins).

Disadvantages of normal flora include:

1. Production of disease if the individual becomes immunocompromised or if they change

their usual anatomic location.

2. Production of disease since most of them are pathogens or opportunistic pathogens.

3. Most of the normal flora in the skin are found in moist, intertriginous areas. Diphtheroids
and Staphylococcus epidermidis are the predominant flora of the skin. The tongue and
buccal mucosa are inhabited mostly by Streptococcus viridans group, which includes S.
mutans, S. milleri, S. salivarius, and S. sanguis. The gingival crevices and the tonsillar
crypts are primarily inhabited by anaerobic flora. There may be transient carriage in the
pharynx of potentially pathogenic organisms. These include Haemophilus influenza,
Streptococcus pneumonia, Neisseria meningitidis, and Mycoplasma. In the upper
respiratory tract, initial colonization by pathogenic organisms may be seen. These include
Neisseria meningitidis, Corynebacterium diphtheria, and Bordetella pertussis. Most of the
bacteria cultured in the small intestine include streptococci, lactobacilli, and Bacteroides
which are all transient.

The colon is inhabited predominantly by anaerobes (95%-99%) which includes Bacteroides

fragilis (most common), Bifidobacterium/Lactobacillus bifidum (predominant in breastfed
infants), Eubacterium, Peptostreptococcus, and Clostridium. Vaginal flora varies depending on
the age, hormonal levels, and vaginal pH of the host.

» In female infants, the predominant vaginal flora is Lactobacillus spp. From 1 month of age
until puberty, microorganisms which may inhabit the vagina include Staphylococcus
epidermidis, Streptococci, diphtheroids, and Escherichia coli.

» At puberty the predominant flora include Lactobacillus acidophilus, corynebacteria,

peprostreptococci, streptococci, Bacteroides, and staphylococci.

» Fungi such as Torulopsis and Candida may also be found (10%-30%).

Chapter 6: Medical and Surgical Asepsis

Infection Control is one of the major concerns that healthcare workers in healthcare facilities
and hospitals constantly address. There are certain terminologies associated with infection
control that a healthcare worker must be familiar with. These terminologies are often related to
the chain of infection, how the organisms are transmitted, asepsis, the specific types of
infection, and personal protective equipment (PPE). These include the following:

1. Chain of infection - how an individual acquires the infectious agents and includes the
infectious agent, the source of infection or its reservoir, how the organism is transmitted, and
the organism's portal of entry into the susceptible host

2. Mode of transmission - the manner in which the infectious organism is acquired by the
host.

3. Standard precautions - the specific measures used to prevent the spread of infection
among all patients and healthcare workers, including measures to protect them from
contaminated blood and other body fluids.

4. Contamination - denotes contact of a sterile or aseptic item with microorganisms. Medically

aseptic items become contaminated if they get in contact with disease-producing organisms.
Sterile items become contaminated if they get in contact with items that are not sterile.

5. Decontamination - the process where physical or chemical means are used to remove,
inactivate, or destroy pathogens on a surface or item making them safe for handling or use and
incapable of transmitting infectious agents.

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6. Disinfection - the process of using physical or chemical means to destroy pathogens,

excluding the spores.

7. Sterilization - the process by which all pathogens are destroyed, including the spores. The
various methods of physical and chemical sterilization will be discussed in the succeeding
chapters.

8. Antiseptic - a chemical solution that inhibits the growth of some microorganisms. Most
antiseptics can be used directly on the skin (e.g., alcohol and iodine).

9. Healthcare-associated infection - any infection that is acquired during the time a patient is
admitted in a healthcare facility. The most common healthcare-associated infection is the
urinary tract infection (UTI).

10. Iatrogenic infection - infection that is acquired in the course of undergoing diagnostic
tests or therapeutic procedures.

11. Occupational exposure - the acquisition or exposure to an infectious agent of a

healthcare worker during the course of his/her work.

12. Personal protective equipment (PPE) - specialized equipment and attire used by
healthcare workers to protect them from infections. These include gloves, masks, gowns, and
goggles.

Asepsis refers to a condition in which the individual and his/her surrounding environment are
free of any microorganisms. Sepsis, the opposite of asepsis, refers to the clinical condition
where an individual develops a systemic reaction to a bacterial infection that starts from a
localized infection in one part of the body. The goals of asepsis are to protect the patient from
hospital-acquired or nosocomial infections and to prevent the spread of pathogenic
microorganisms.

All patients in healthcare facilities are vulnerable to pathogenic organisms. Some of the factors
that play a role in the occurrence of infection among patients include: (1) suppression of the
immune system; (2) prolonged duration of illness; and (3) procedures that patients undergo in
the healthcare facility such as insertion of in-dwelling catheters, use of antibiotics, and insertion
of intravenous lines or endotracheal tubes. The most commonly occurring pathogenic
microorganisms that lead to nosocomial infections are Escherichia coli, Staphylococus aureus,
Pseudomonas aeruginosa, Candida albicans, and Enterococcus. The primary locations of
infections from these organisms are surgical wounds, the urinary tract, the respiratory tract,
and the bloodstream.

Pathogens may be introduced to the patient through contact with hospital personnel, the
hospital environment, or hospital equipment such as respiratory machines, catheters, and
intravenous lines or needles. Situations that require aseptic measures are surgery and the
insertion of intravenous lines, urinary catheters, and drains. All personnel must constantly
monitor not only their own movements and practices but those of others as well.

Asepsis may be categorized into medical asepsis and surgical asepsis. Medical or clean
asepsis refers to the absence of disease-producing microorganisms. It is the infection control
process that aims to reduce the spread of infection. It involves certain procedures aimed to
decrease the number of organisms and prevent their spread in the general clinical setting.
Proper hand hygiene, the administration of all medications except those that are given
intravenously, and the preparation of the patient's skin before administration of subcutaneous
medication are instances when medical asepsis is applied.

Surgical or sterile asepsis is defined as the absence of all microorganisms. It involves

procedures that aim to eliminate microorganisms from an area in the body where surgical
procedures will be performed as well as the location where the surgical procedure will be
carried out. There are some procedures and treatment modalities that necessitate surgical
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asepsis and there are others that would only require medical asepsis. The principles of surgical
asepsis are applied when the skin is not intact and when internal areas of the body are involved
in procedures, whether for diagnostic or treatment purposes. Applications of surgical asepsis
include wound care, during invasive procedures (e.g., endoscopy), administration of
intravenous drugs, and during insertion of urinary catheter and other internally placed tubes.

General aseptic procedures that help to preserve and maintain a clean medical environment
include: (1) frequent handwashing of hospital personnel (doctors, nurses, medical
technologists, and orderlies); (2) prompt and safe disposal of contaminated materials like
bandages and needles; (3) regular checking and emptying of containers for surgical drains; (4)
prompt cleaning of soiled or moist areas; and (5) proper labeling of containers regarding the
date and time of disposal.

Handwashing - The most frequent source of microorganisms leading to outbreaks of infection

in health institutions is the hands of the healthcare workers. This is the reason why proper
handwashing is one of the most basic means of preventing the spread of pathogenic
organisms. It is essential in the healthcare environment for the following reasons: (1) to reduce
the flora on the healthcare worker's skin; (2) to proteer the healthcare worker in the event that
there is a breale in his or her skin; (3) to reduce risk of contact with infectious agents if gloves
worn ate punctured; and (4) to reduce the chances of disease transmission. Healthcare
workers must be aware that the healthcare environment is highly susceptible to a number of
healthcare-acquired infections. These include infections with methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (RE), and penicillin-
resistant Streptococcus pneumoniae.

When should handwashing be done? The United States Center for Disease Control
recommend routine handwashing for at least 15 seconds with a 10-second rinse. For
healthcare workers a longer period of time for handwashing that entails thorough washing of
the hands, lathering at least twice, and careful cleaning of the fingernails is recommended in
the following situations: (1) at the beginning and end of each shift; (2), when the hands are
visibly soiled, (3) after contact with a possible source of microorganisms such as blood or body
fluids, mucous membrane, non-intact skin, or contaminated objects; (4) before and after
performing invasive procedures; or (5) before removing gloves if they are visibly soiled and
each time after removing gloves.

Proper handwashing can be done with friction and regular soap and water. Hands must be
thoroughly washed with vigorous scrubbing, paying special attention to the areas around the
nailbeds and between the fingers. These are areas that usually have high bacterial load. The
fingernails should be kept clean and short. Patients, as well as their relatives, must also be
taught the proper way of handwashing. Remember that the best way to prevent the spread of
communicable diseases is health education!

Alcohol-based sanitizing antimicrobial solutions or hand cleansers must not be used as

substitute for proper handwashing. However, if running water and soap are not available, one
may use alcohol-based hand cleansers to decontaminate the hands. The alcohol-based hand
cleansers must be liberally applied to the entire hand after which the hands are rubbed until the
entire hand is completely dried.

Personal Protective Equipment (PPE) Personal protective equipment (PPE) are specialized
equipment and attire used in healthcare facilities to protect not only the healthcare workers but
also the patients and visitors against infections. These include masks, gowns, and goggles.

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Gloves

Among the various PPEs in use, gloves are the most commonly used. Gloves used during
medical procedures are disposable and the most commonly used are of tho types: (2),
examination gloves, which may be sterile or non-sterile, and (2) surgical gloves which are
sterile. They serve as a protective barrier when handling or touching open wounds, blood, or
body fluids. Gloves provide protection from microorganisms and help prevent the spread of
infectious agents from one person to another. Sterile, disposable gloves must be provided to all
personnel in healthcare facilities, particularly those who have direct contact with patients. The
gloves must be disposed of immediately after use. Hands must be washed thoroughly after
using gloves since the wearing of gloves can also promote multiplication of microorganisms
because of the moist environment that it provides.

The World Health Organization (WHO) has come up with guidelines for the proper use of gloves
in healthcare facilities. Some of the recommendations listed in the guidelines include the
following:

1. Gloves are not meant to replace observance of proper hand hygiene. The practice of hand
hygiene must still be observed before and after wearing of gloves.

2. Gloves must be worn if contact with blood or body fluids, mucous membranes, open
wounds, or potentially infectious material is anticipated.

3. Gloves must be removed and disposed of after caring for a patient. Healthcare workers must
not wear the same gloves if caring for more than one patient.

4. Gloves must be removed or changed if moving from a contaminated body site to another
body site in the course of caring for a patient.

5. Reusing of gloves after decontamination is not recommended.

Furthermore, WHO recommends the use of gloves in the following situations:

1. Before performing a sterile procedure.

2. When in contact with a patient and his or her surroundings in conditions where

contact precautions are warranted.

3. When contact with blood or body fluids, non-intact skin, and mucous membranes is

anticipated.

The removal of gloves is indicated in the following:

1. When hand hygiene is indicated.

2. After contact with a single patient and his or her surroundings is ended or when contact with
a contaminated body site is ended.

3. As soon as the gloves are damaged or there is loss of integrity of the gloves.

4. After contact with blood or body fluids, non-intact skin, and mucous membrane.

Masks

The mask must cover the mouth and nose. It must be tied in a way that there should be
minimal gaps between the face and the mask. The healthcare worker must also avoid touching

the mask while it is worn. The moment it becomes damp, it should be replaced with a clean
and dry one. Remember that masks are supposed to be single-use items. Therefore, it must be

discarded and disposed of as clinical waste the moment the procedure which necessitated its
wearing is completed. It is also recommended that hands are decontaminated by washing with

soap and water or by using alcohol-based hand sanitizers after the mask is disposed.

Sterile Gowns

Healthcare workers are recommended to wear gowns or aprons when there is probability of
contact with blood, body secretions excluding sweat, or other body substances. Likewise,
wearing of gowns is recommended if the healthcare worker has close contact with patients,
equipment, or materials that can introduce infectious agents to the healthcare worker's skin,
uniform, or other clothing. The type of apron or gown to wear depends on the degree of risk
with the infectious agents and the potential for body substances and blood to penetrate
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through the clothes or skin of the healthcare worker. The protective wear can either be in the
form of an apron or gown.

If there is a risk for body substances, blood, or body secretions to contaminate the clothing or
skin of the healthcare worker, a fluid-resistant apron or gown is recommended. Clean, non-
sterile gowns or aprons are generally sufficient to protect the skin and prevent soiling of
clothing during procedures or other in-patient activities that may lead to splashing or spraying
of blood and body substances. Fluid-resistant gowns or aprons are always worn with gloves
and other personal protective equipment. Healthcare workers must make sure that they
change gowns or aprons in between treating different patients.

Disposable, single-use gowns are usually used to protect the healthcare worker during
procedures and other activities related to patient care where there is likelihood of generating
splashing or sprays of blood or body substances. The length of the sleeves will depend on the
specific procedure being performed or how much is the risk of exposure of the healthcare
worker's arms.

Fluid-resistant, single-use, long-sleeved, full body gowns are usually worn (1) when there is a
risk of contact of the healthcare worker's skin with a patient who has broken skin, (2) if there is
extensive skin to skin contact between the healthcare worker and the patient, and (3) if the risk
of contact with body substances or fluids cannot be contained such as when the patient has
diarrhea or is vomiting incessantly.

In cases of surgical procedures and other invasive procedures, care must be taken to prevent
the invasion of microorganisms into the surgical site. Sterility parameters have been developed

to maintain the sterile field. These parameters are as follows:

1. The front of a sterile gown is considered sterile from the chest down to the level of the sterile
field. The reason for this is because most scrubbed personnel work next to a sterile table and/
or bed.

2. The gown sleeves are sterile from two inches above the elbow to the cuff, circumferentially.

3. The back of the gown is not considered sterile because it cannot be constantly monitored.

4. The neck, sleeve cuffs, and underarms of the gloves are not considered sterile and are not
considered as effective microbial barriers.

If contamination of the surgical gown occurs at any point during the procedure, the gown as
well as the gloves must be changed. The circulating nurse needs to obtain sterile gloves and

gown for the scrubbed person who needs to change his or her gown. The individual concerned

must step away from the sterile field while the circulating nurse wears sterile gloves and unties

the scrubbed person's gown at the neck and waist. The scrubbed person in turn grasps the
front of the gown at the shoulders below the neckline, pulls the gown off inside out and rolls it
away from the body. The circulating nurse then turns to face the scrubbed person, grasps the
gown at the shoulders and pulls it off. The gloves are then removed next. The moment the
gloves are removed, the scrubbed person is now ready to re-glove and re-gown.

Isolation Precautions

Isolation is the process of separating an individual with an infectious disease from the rest of
the healthy population to prevent the spread of the infection to other individuals. The Center for
Disease Control (CDC) in the United States has come up with guidelines to follow towards this
end. These recommendations, which they termed universal precautions, are geared towards
handling of patients with an infection from an unknown pathogen to decrease the risk of
transmission. These precautions apply to all body fluids including blood, skin, and mucous
membranes. These include (1) proper handwashing; (2) the use of personal protective
equipment such as gloves, aprons, gowns, masks or face shields; (3) proper handling and
disposal of secretions and excretions excluding sweat; (4) proper handling and disposal of
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soiled linen and equipment; (5) environmental control; (6) prevention of injury from sharp
devices such as needles; and (7) patient placement.

Transmission-based Precautions

Transmission-based precautions have been developed to further prevent the prey of infectious
agents. These precautions are based on the mode of transmission of the infectious agents and
are classified into (1) contact precautions; (2) droplet precautions an (3) airborne precautions.

Contact Precautions

Contact precautions are used to prevent the spread of infections or infectious agents that are
transmitted through touching of patients or items in the room where the infectious agents may
be deposited (called fomites). These include infectious agents such as methicillin-resistant
Staphylococcus aureus (MRSA), viruses such as respiratory syncytial virus, agents that cause
diarrhea whether viral or bacterial, and open wounds. All individuals whether healthcare
personnel or non-healthcare personnel, must wear gowns and gloves.

Droplet Precautions

These precautions are used for diseases or infectious agents that are spread in tiny droplets
caused by coughing and sneezing. These are used to prevent contact with secretions from the
respiratory tract. Examples of such disease are influenza, mumps, or pertussis (whooping
cough). These droplets that are spread when the individual coughs or sneezes can travel a
distance of approximately 3 feet (or 90 centimeters). All persons entering the rooms of these
patients are required to wear a surgical mask.

Airborne Precautions

These are measures geared towards preventing the spread of diseases or infectious agents
that are spread through the air from one person to another. These microorganisms are so tiny
that they can float in the air and travel long distances. These include infectious agents that
cause chickenpox, measles, and tuberculosis. Patients who are admitted to the hospital with
the said infections must be placed in a room with negative air pressure where the air is gently
sucked out and not allowed to flow into the hallway thereby preventing contact with the
outside environment. The door must remain closed at all times and all individuals entering the
room must wear a protective mask. This is also called reverse isolation.

Aseptic Measures in the Operating Room

To prevent post-operative infection, asepsis must be strictly observed in the operating room.
Thorough cleaning of the operating room with detergent or detergent germicides, soap and
water must be done. In addition, all equipment that would be directly in contact with the
patient must be properly sterilized. Surgical instruments can be sterilized in the autoclave or by
using chemical agents. Radiation is seldom used because of its toxic effects to body cells.
Personnel must ensure sterility by making sure that sterile packages are dry and intact. Sterile
surgical clothing and operating room gowns and other protective devices (e.g., surgical gloves,
face masks, goggles, eye/face shields) serve as barriers against microorganisms and must be
used to maintain asepsis in the operating room. Gowns used by the surgical team are
considered sterile in front from the chest to the level of the sterile surgical field and two inches
above the elbows to the cuff of the sleeves. This must be put on with extreme care to avoid
contact between its external, sterile surfaces and non-sterile objects, including the skin. The
operating room nurse is usually the one who assists the surgeons in donning the gloves and
gowns. The nurse also prepares and arranges the surgical instruments to minimize the risk of
contamination.

Sterile drapes are sterilized linens placed around the field to delineate sterile areas. These are
used to create a sterile field. Only the top surface of the draped area is considered sterile. The
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drapes must be used to cover the patient, furniture, and equipment to be included in the sterile
field so that only the incisional site is exposed. Only the scrubbed personnel must handle the
sterile drapes. Once positioned, the sterile drapes should not be removed or rearranged.

Wrapped kits of instruments and/or equipment are opened in such a way that the contents do
not touch non-sterile items or surfaces. This is ensured by opening the farthest areas of a
package first. Sterile packages are opened as close as possible to the time of actual use.
Leaning over the contents of packages must be avoided and opened flaps must be prevented
from falling back onto contents. All sterile objects that will touch open wounds or enter body
cavities must be handled only with sterile forceps or with sterile, gloved hands.

To reduce the chances of introducing endogenous flora of the skin to the deeper tissues, the
patient must be prepared prior to surgery. Preparations include shaving of body hair on the
surgical site and thorough cleansing with a disinfectant. The most commonly used and most
effective antiseptic is iodine because it can destroy all forms of microorganisms. Surgical scrub
must be performed by all members of the surgical team and all others who will perform the
surgical procedure or will have access to and contact with the patient, including equipment
and instruments inside the operating room. Surgical scrubbing requires the use of long-acting
powerful antimicrobial soap on the hands and forearms and must be done for a longer period
of time (at least 2-5 minutes) than that of typical handwashing.

The interdigital areas of the hands must also be thoroughly washed. The hands must be held
below the elbows during the surgical scrub and above the elbows following the surgical sen;
Contact with the faucet or other potential contaminants must be avoided; Thorough drying with
a sterile towel is essential, since moist surfaces invite the presence of pathogens. The fauce
can be turned off through use of a foot pedal.

During the operation, only properly scrubbed personnel should be allowed at the vicinity of the
sterile field. The hands and arms of the scrubbed personnel must remain within the sterile site.
Personnel should not turn their backs from the sterile field. Only those areas that can be seen
by the surgeon are considered sterile. Items that are not sterile should not pass over the sterile
field. Talking, laughing, coughing, or sneezing are not allowed across a sterile field.

Preventing Infection in the Community

Controlling the spread of communicable diseases is best achieved at the community level,
before the occurrence of disease. Healthcare personnel must not only be involved in infection
control in the hospital but, more importantly, in the community. It is the duty of healthcare
professionals to educate the public on infectious diseases, particularly their modes of
transmission, because if the people have a clear understanding of the disease process, they
would know what steps to take to prevent its spread.

Infection control in the community includes sanitation techniques, improvement of health

practices, and vaccination. Sanitation techniques include water purification, improvement of
health practices, proper sewage disposal, and other measures that will ensure a clean
environment. Improvement of health practices involves educating the members of the
community on the proper handling, storage, and preparation of food. The members of the
community must be made aware that infectious and parasitic diseases can be obtained from
contaminated and improperly cooked food as well as contaminated water. Lastly, people
should be made aware of the value of immunization. Information about individual vaccines and
vaccine schedules should be made available to the people.

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Chapter Summary
- Sepsis is a clinical condition where infectious agents are spread throughout the body of an
individual from a localized site of infection and manifest with symptoms or organ damage.

- Asepsis is the absence of disease-producing organisms and is divided into medical asepsis
and surgical asepsis.

- Medical asepsis is aimed at reducing the number of disease-producing organisms to prevent

its spread from healthcare workers to the patients and vice versa.

- Surgical asepsis is aimed at total elimination of disease-producing organisms particularly in

areas in the body where surgical procedures will be performed as well as the location where
the surgical procedure will be carried out.

- Handwashing is the most basic and universally accepted measure used to prevent the
spread of infection. Routine handwashing for at least 15 seconds with a 10-second rinse is
recommended.

- Personal protective equipment (PPE) are specialized equipment and attire used in healthcare
facilities to protect not only the healthcare workers but also the patients and visitors against
infections. These include masks, gowns, and goggles. Guidelines have been set in the
proper use of these personal protective equipment.

- Universal precautions are specific measures geared towards handling of patients with an
infection from an unknown pathogen to decrease the risk of transmission. These precautions
apply to all body fluids including blood, skin, and mucous membranes.

- Transmission-based precautions have been developed to further prevent the spread of

infectious agents. These precautions are based on the mode of transmission of the
infectious agents and are classified into (1) contact precautions; (2) droplet precautions; and
(3) airborne precautions.

- The operating room is one of the most sterile areas in the hospital. Strict measures must be
followed to ensure sterility not only of the operating room but also of the instruments and
materials to be used in a surgical procedure.

All healthcare personnel entering the operating room must observe strict precautions to
maintain its sterility. The best way to prevent the spread of infection is at the community level.
Proper health education on the sources of infection as well as the transmission of disease-
producing microorganisms is essential. Preventive measures such as vaccination must also be
emphasized.

Definition of Terms

1. Sterilization - the process of killing or removing all microbial forms, including spores.

2. Disinfection - the process by which most microbial forms on inanimate objects are killed

without necessarily destroying saprophytes and bacterial endospores which leads to

a reduction in the number of organisms to a level that they cannot produce infection.

3. Antisepsis - use of chemical agents on living tissue (e.g., skin) to prevent the spread of
microorganisms either by inhibiting their growth or destroying them.

A. Bactericidal or germicidal agent - agent, physical or chemical, that kills bacteria.

5. Bacteriostatic agent - agent, physical or chemical, capable of inhibiting the growth of

bacteria without necessarily killing them.

6. Sporicidal, fungicidal, viricidal - agents capable of destroying spores, fungi, and viruses,
respectively.

Physical Methods of Sterilization

Heating

Heating is the most common physical method of sterilization. the rate of killing in expressed in
thermal death time, i.e., the minimum time required to kill a suspension of a organism at a
predetermined temperature and environment. The mechanisms of action f heating include: (1)
formation of single-strand breaks in the bacterial DNA; (2) coagulation and denaturation of
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proteins; (3) accumulation of toxic levels of electrolytes; and (4) alteration of cell membrane
structure. Several factors can affect the process of sterilization through heating. These include:

1. Nature of the heat - moist heat has greater killing action than dry heat.

2. Temperature and time - as temperature increases, the time taken to sterilize decreases. In
other words, there is an inverse relationship between time and temperature.

3. Number of microorganisms - the more microorganisms there are, the higher the temperature
and the longer the duration of the process required to destroy all of them.

4. Nature of microorganisms - spore-forming microorganisms are more difficult to destroy than

non-spore-forming ones.

5. Type of material - the temperature required to sterilize materials depend on the sensitivity of
the material to heat. Heat-sensitive materials will require lower temperature than heat-resistant
materials.

6. Presence of organic material - the presence of organic materials such as fats, proteins, and
sugars may necessitate higher temperatures.

Types of Heat

1. Moist heat - preferred over dry heat because of its more rapid killing action. Its main
mechanism of action is to cause coagulation and denaturation of proteins. The various
methods of moist heat may be classified according to the temperature used.

These include:

a. Temperature below 100 °C

Pasteurization

This is the method of destroying disease-producing organisms in milk and milk products as
well as other beverages. There are several variations of this method based on the temperature
utilized. One method is called the conventional method where the milk is heated at 60°C-65°C
followed by rapid cooling.

The flash method involves heating at 72°C for 15 seconds followed by quick cooling to 13°C. A
newer pasteurization method developed is what they call ultra-high temperature (UHT) method
where heating is done at 140°C for a period of 15 seconds and 149°C for 0.5 seconds.

Vaccine bath - This is used to destroy contaminating bacteria in vaccine preparations. The
vaccine preparation is heated in a water bath at 60°C for one hour. This procedure is not
sporicidal. Only the vegetative forms of the bacteria are destroyed.

Serum bath

This is used to inactivate bacteria contaminating serum preparations and is done by heating at
56°C for several successive days. Similar to vaccine bath, only the vegetative forms are
destroyed since higher temperatures will cause coagulation of proteins present in the serum.

Inspissation

This technique is used to solidify and disinfect egg-containing and serum- containing media.
The culture medium is placed in the slopes of a device called an inspissator and is heated at
80°C-85°C for 30 minutes for three successive days. The basis for the method is that on the
first day, vegetative forms will die and the spores that will germinate the following day will also
die.

b. Temperature of 100 °C
Boiling

This method involves utilizing water at boiling temperature of 100°C. It is not sporicidal and will
destroy only the vegetative forms. The killing action can be enhanced by the addition of 2%
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sodium bicarbonate. Certain metal articles and glasswares can be disinfected using this
method for 10-20 minutes without opening the lid of the boiler.

Fractional sterilization (Tyndallization)

This method is also known as intermittent sterilization and involves exposing the material to be
sterilized to live steam at 100°C for 30-90 minutes for three consecutive days, depending on
the material to be sterilized. This sterilization method can be used to sterilize culture media
such as CBS and selenite broth. The vegetative forms are killed on the first day and the spores
that will germinate will be destroyed on the next successive days. Only vegetative forms of the
bacteria are destroyed with this method.

Temperature above 100°C

Autoclave (Steam under pressure)

This is the most efficient method of sterilization because it can destroy all microbial forms. The
temperature for sterilizing is dependent on the pressure of the steam. When the pressure
reaches 15 pounds per square inch (psi), the temperature inside the vessel reaches 121 C.
Because of the high temperature and pressure, it would take only 15-20 minutes to sterilize the
material. This method is used to sterilize instruments, surgical bandages, culture media, and
other contaminated materials that can withstand high temperature and high pressure.

Dry heat - the effectiveness of dry heat depends on the penetration of heat through the
material to be sterilized. It is used to sterilize materials in enclosed tubes, oils, jellies, powders,
and glasswares such as test tubes and Petri dishes.

a. Red flame

This method is used to sterilize articles like bacteriological wire loops, straight wires, tips of
forceps, and searing spatulas. The materials are held over the flame of a Bunsen burner until
they become red hot. It is limited only to articles that can be heated to redness in flame.

b. Open flame (Flaming)

This method also makes use of the Bunsen burner or alcohol lamp. The material to be sterilized
is passed over the flame several times but is not heated to redness. It is aimed at burning the
organism into ashes and is used to sterilize such articles as mouths of test tubes, scalpels,
glass slides, and cover slips. Only vegetative forms are destroyed. In addition, cracking of the
glassware may occur.

C. Incineration

This method is aimed at burning the organism into ashes. The contaminated material is burned
using an incinerator. Articles that must be incinerated include soiled dressings and beddings,
animal carcasses, and pathological material. This will result in loss of the article and hence
must be used only for articles that have to be disposed. Some materials such as polystyrene
emit dense smoke and must not be incinerated.

d. Hot air oven

The use of the hot air oven was first introduced by Louis, Pasteur. Ariel, to be sterilized are
placed in the oven with a temperature of 160 C for a peric, of one hour. This can be used to
sterilize metallic instruments such as forcep, scalpels, and scissors. It can also be used to
sterilize certain glasswares (eg, pet; dishes, pipettes, flasks) and it is the only method used to
sterilize powders and ointments. The disadvantage of using this method is that because air is a
pay conductor of heat, then hot air will have poor penetration of the materials to be sterilized.
In addition, cotton wool and paper may get slightly charred and glasses, can become smoky.

C. Infrared rays

In this method, the articles to be sterilized are placed in a conveyor belt and passed through a
tunnel that is heated by infrared radiators. The temperature to which the materials are
32 of 63

subjected to is 180°C for a period of 7.5 minutes. It can be used to sterilize metallic equipment
and glassware.

Dessication

This method is based on the principle of depriving the microorganism of moisture. It is used
mainly for food preservation, such as in the preparation of dried fish and fruits. It may

destroy vegetative forms. Endospores are resistant to drying.

Freezing

Freezing is not a reliable method of sterilization because most pathogenic organisms are
resistant to low temperatures. Its main use in the laboratory is for the preservation of
microorganisms in a process called lyophilization or freeze-drying where the organism is rapidly
frozen then dehydrated in high vacuum and stored in a vacuum-sealed container.

Filtration

This is a form of mechanical sieving that does not kill microorganisms but merel separates
them from the fluid. A cellulose ester filter with a pore size of 0.22 um-0.45 um s used which
can filter all microorganisms except viruses and the three smallest bacteria - Mycoplasma,
Rickettsia, and Chlamydia. It is used for liquid solutions that will be destroyed by heat or
freezing such as serum, antibiotic solutions, sugar solutions, or urea solution. This method can
be used to remove bacteria from culture media or to prepare suspensions viruses and phages.

1. Ultraviolet Light (UVL)/Non-ionizing radiation - the effective UVL wavelength is in the range
of 200 nm-280 nm, with 260 nm as the most effective. This corresponds with the maximum
absorption of bacterial DNA. UVL acts by inducing formation of thymine- thymine dimers
resulting in lethal frameshift mutations. Microorganisms such as bacteria, viruses, and yeasts
can be inactivated within seconds. However, UVL is not sporicidal and is more frequently used
for surface disinfection. It is used to disinfect hospital wards, operating rooms, laboratories,
and other rooms in the hospital that need to be sterilized. The disadvantage of UV ray is that it
has low penetrance. It is also limited by the lifespan of the UV bulb. In addition, there are some
bacteria that have DNA repair systems that can counteract the damage done by UV rays. Care
should also be observed by the handler because UV rays can be harmful to the skin and eyes.

2. Ionizing radiation - ionizing rays have greater penetrance than UV rays. It exerts its effect by
causing formation of free radicals that chemically interact with proteins and nuclei acids,
resulting in cell death. It is not routinely used because of its potential to harm human tissues.
There are two types of ionizing radiation used for sterilization purposes: electron beams and
electromagnetic rays.

a. Electron beams

Electron beams are particulate in nature. A linear accelerator from a heated cathode is used to
generate high speed electrons. It can be used to sterilize syringes, gloves, dressing packs,
food, and some pharmaceuticals. It has lower penetrance and requires sophisticated
instruments.

b. Electromagnetic rays (Gamma rays) Electromagnetic rays are produced from nuclear
disintegration of selected radioactive isotopes. They have greater penetrance than electron
beams but require longer exposure time. The high energy radiation produced cause damage to
the microorganism's nucleic acid. It is bactericidal, fungicidal, viricidal, and sporicidal. It is
used commercially to sterilize disposable Petri dishes, plastic syringes, vitamins, antibiotics,
hormones, fabrics and glassware.

Sonic and Ultrasonic Vibrations

Some bacteria can be killed after exposure to certain frequency of sound waves. Exposure to
sound waves at a frequency of approximately 20,000 cycles/second for one hour can kill some
33 of 63

bacteria and viruses. High frequency sound waves act by disrupting cells. They are used to
disinfect and clean instruments and to reduce microbial load.

Osmotic Pressure

This method is based on the principle of osmosis so that concentration of the fluid surrounding
the organism is altered. this will cause the bacterial cell to collapse, This used for preservation
of fruits in syrup and meats in brine.

Chemical Methods of Sterilization

Chemicals can inhibit the growth of pathogenic organisms, either temporarily or permanently.
Several factors can affect the efficacy of a chemical agent. These include:

1. Concentration and potency of the chemical agent. In general, a higher concentration is

bactericidal whereas a lower concentration may only be bacteriostatic. This is not true for
alcohol. For alcohol, the effective bactericidal concentration is at 50% to 80%.

2. Duration of exposure. The longer the time of exposure to the chemical agent, the better the
killing action.

3. Temperature. A higher temperature speeds up the rate of a chemical reaction and thus
accelerates killing action. However, there are also certain chemical agents that exert optimal
effect at lower temperatures.

4. Nature of the surrounding medium. The pHI of the medium and the presence of extraneous
materials like pus or blood decreases the efficiency of the chemical agent. These materials may
inactivate or lower the concentration of the chemical agent or may bind the chemical agent to
its surface.

5. Nature of the organism. This refers to the innate resistance of the microorganism to
disinfectants. Microorganisms vary in their resistance to disinfectants. Bacteria that produce
endospores may be resistant to most chemical agents. Mycobacterial cell wall is lipid-rich that
makes it difficult for the chemicals to penetrate it. Gram-negative bacteria have an outer
membrane that confers resistance to disinfectants.

6. Number of organisms/Size of inoculum. The larger the number of microorganism present, the
more time needed for a disinfectant to destroy all of them.

A chemical agent, to be effective as a disinfectant or antiseptic, must be chosen carefuly

based on the specific purpose, pathogen, and environment. A good chemical agent must
possess the following characteristics:

1. It should be broad spectrum, able to destroy a wide variety of microorganisms.

2. It should be fast-acting, able to destroy microbes within a short period of time.

3. It should be active in the presence of organic matter.

4. It should be active in any pH.

5. It should be stable.

6. It should be non-toxic, non-allergenic, non-irritative, and non-corrosive.

7. It should be soluble in water and easy to apply.

8. It should leave a residual antimicrobial film on the treated surface.

9. It should have high penetrating power.

10. It should not be expensive and must be easily available.

11. It should be safe under storage and shipping for reasonable periods of time.

12. It should not have a bad odor.

Classification of Chemical Disinfectants

Chemical disinfectants may be classified based on the following: (1) consistency (liquid or
gaseous); (2) spectrum of activity (high level, intermediate level, low level); or (3) mechanism of
action.

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Mechanism of Action

Damage to the cell membrane

Damage to the cell membrane can cause smaller molecules to leak out of the bacterial cell and
interfere with the active transport and energy metabolism within the cell. Chemicals under this
include the following:

1. Surface active agents - compounds have long chain hydrocarbons that are fat-soluble and
charged ions that are water-soluble. They concentrate on the surface of membranes and
disrupt membrane resulting in leakage of cell components. These agents are active against
vegetative microbial forms including Mycobacteria as well as enveloped viruses. They are
widely used as disinfectants in homes and hospitals but their activity is reduced in the
presence of hard water and organic matter.

a. Cationic agents

These are detergents where the fat-soluble portion is positively charged due to combination
with a quaternary nitrogen atom. These are called quaternary ammonium compounds and are
effective at alkaline pH. Examples are cetrimide and benzalkonium chloride.

b. Anionic agents

These are negatively charged agents that contain long chain hydrocarbons. Examples are
soaps and bile salts. They remove dirt through the process of emulsification and are most
effective at acidic pH.

2. Phenolic compounds - these act by disrupting cell membranes as well as causing

precipitation of proteins and inactivation of enzymes. These are coal-tar derivatives that act as
disinfectants at high concentration and as antiseptic at low concentrations. Phenols are
bactericidal and fungicidal with good activity against Mycobacteria but have poor activity
against spores and most viruses.

a. Phenol is no longer used as a disinfectant because it is toxic to human cells. It is used as a

gold standard in the chemical evaluation of new chemical agents using the phenol coefficient
test.

b. Cresols are phenol derivatives more potent and safer than phenol. An example is Lysol®.

c. Chlorhexidine is used as a skin disinfectant if in isopropanol solution. The aqueous

preparation is used for wound irrigation. Its main use is as antiseptic hand wash.

d. Chloroxylenols are used for topical purposes. They are effective against gram-positive
bacteria.

e. Hexachlorophene is a chlorinated diphenyl which has greater activity against gram-positive

bacteria similar to chloroxylenols.

f. Triclosan, an organic phenyl ether, has good activity against gram-positive bacteria and a
number of gram-negative bacteria including Pseudomonas. It has some activity on fungi and
viruses.

3. Alcohols - disorganize the lipid structure of the cell membrane, dehydrate cells, and cause
denaturation and coagulation of cellular proteins. The microbial killing property of alcohol is
seen better in a 70% aqueous solution compared to absolute alcohol. The disadvantage of
using alcohols is that they are skin irritants and are also flammable.

a. Ethyl alcohol - used as skin antiseptic, it is bactericidal and removes lipids from skin
surfaces.

b. Isopropyl alcohol - it has greater bactericidal activity than ethyl alcohol and is less volatile. It
can be used to disinfect surfaces. Inhalation of its fumes can cause narcosis.

c. Benzyl alcohol - it is used mainly as a preservative.

d. Methyl alcohol - it is fungicidal and sporicidal used in disinfecting inoculation hoods.

Denaturation of cellular proteins

Substances that cause denaturation or loss of the normal structure of proteins pave die way for
the eventual destruction of the bacterial cell. Denaturing agents include: (1) acids.and alkalis,
(2) alcohol and acetone, and (3) phenol and cresol.

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Modification of the functional groups of proteins and nuclei acids

1. Heavy metals - cause damage to the enzyme activity of bacteria. They also cause
bacteriostatic than bactericidal. I precipitation of proteins and oxidation of sulthydryl groups.
Heavy metals are mostly bacteriostatic than bactericidal.

a. Mercurials (e.g., mercurochrome and merthiolate) are biocidal and are used as antiseptics.
These are active against viruses at dilution of 1:500 to 1:1000.

b. Silver compounds (e.g., silver nitrate) are bactericidal. 1% silver nitrate solution is used
clinically as treatment for ophthalmia neonatorum (Crede's prophylaxis). Silver sulfadiazine is
used topically in the treatment of burn wounds.

2. Halogens - bactericidal oxidizing agents that cause oxidation of essential sulfhydry| groups
of enzymes causing inactivation of the enzymes.

a. Iodine (tincture of iodine, iodophors) is considered the best antiseptic because it is

sporicidal, bactericidal, fungicidal, viricidal, and amoebicidal. It can be combined with neutral
carrier polymers to produce iodophores (e.g., povidone-iodine). A 10% solution of povidone-
iodine is used for pre-operative and post-operative skin disinfection.

b. Chlorine is mainly used in the treatment of water (chlorine gas). Hypochlorites are used for
sanitizing dairy and tood processing equipment. It is also common household disinfectant. At
higher concentrations, it is used to disinfect swimming pools.

c. Hydrogen peroxide is a weak antiseptic and used only for cleaning wounds and in the
disinfection of surgical devices and soft plastic contact lenses.

3. Alkylating agents

a. Aldehydes damage nucleic acids by alkylation of amino-, carboxyl-, or hydroxyl groups. It

kills all microorganisms including spores.

- Formaldehyde (formalin) is used for surface disinfection. It can be used to sterilize bedding
and furniture. It is also used to kill Mycobacterium tuberculosis in sputum and fungi in athlete's
foot.

- Glutaraldehyde is sporicidal and used as a cold sterilant in sterilizing medical equipment such
as respiratory therapy machines and other equipment that can be damaged by heat. It is more
potent than aldehyde. It requires alkaline pH for Its action and exposure time of at least 3 hours
to be effective.

b. Ethylene oxide is also sporicidal and is used in the gaseous sterilization of heat-sensitive
materials or equipment like heart-lung machine, respiratory and dental equipment, and
polyethylene tubes in anesthesia machines. It is more potent than glutaraldehyde but slower-
acting. It is highly flammable and is usually combined with 10% CO,. It causes eye irritation
and is mutagenic and carcinogenic.

Chapter Summary:

Sterilization is the process of removing all microbial forms, including spores. Disinfection is the
proces of removing most of the microbial forms. It is meant to redue the microbial load to
prevent the development of infection.

Factors that affect the effectivity of physical methods of sterilization include:

1. Nature of the heat

2. Temperature and time

3. Number of microorganisms

4. Nature of microorganisms

5. Type of material

6. Presence of organic material

Heating is the most common physical method of sterilization. It acts by the following

mechanisms:

1. formation of single-strand breaks in bacterial DNA

2. coagulation and denaturation of proteins

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3. accumulation of toxic levels of electrolytes

4. alteration of cell membrane structure

- An effective chemical disinfectant must have a broad spectrum of activity, be fast-acting,

inexpensive, easy to apply, odorless, and non-toxic to human tissues.

- Chemical agents used for disinfection and sterilization are classified based on their main
mechanisms of action.

- Agents that cause damage to the cell membrane include surface active agents, phenolic
compounds, and alcohols.

» Acids and alkalis, alcohol and acetone, phenols and cresols all cause denaturation of

» Modification of functional protein groups is the mechanism of action of heavy metals.

halogens, and alkylating agents.

Chapter 8: Antimicrobial Agents

Antibiotics or antimicrobials are substances produced from microorganisms or synthetically
that are capable of inhibiting or destroying microorganisms even at low concentrations. Natural
sources include fungi and bacteria. The antibiotic penicillin, for example, was derived from the
fungus Penicillium. Polymixin and bacitracin were developed from the bacterium Bacillus sp.
while Actinomyces was the source for the drugs tetracycline, chloramphenicol, and
streptomycin.

Antibiotics are mainly used in the treatment of infectious diseases. An ideal antimicrobial agent
must possess the following characteristics:

1. It should be able to kill the microbial agent or inhibit its growth.

2. It must have a broad spectrum of activity.

3. It should not cause any damage or adverse effect to the patient.

4. It should remain stable when stored in either a solid or a liquid form.

5. It should be able to remain in specific body tissues long enough for it to be effective.

6. It should be able to kill the organism or inhibit its growth before it has had a chance to
mutate and develop resistance.

7. It must exhibit selective toxicity. It must be toxic to the microbial cell but not to the host’s
cells.

Antibiotics may be classified in several ways. Based on spectrum of activity, they may be
classified as broad spectrum or narrow spectrum antibiotics. Broad spectrum antibiotics are
those with a wide coverage of activity against a wide spectrum of microorganisms while narrow
spectrum antibiotics are those with a limited coverage of activity, effective only against a
limited number of microorganisms.

Antibiotics may also be classified based on their antimicrobial activity, An antibiotic is said to
be bactericidal if it is capable of killing the microorganism, An antibiotic is bacteriostatic if it
can only inhibit the growth of the organism. In the choice of antibiotics, bactericidal agents are

more preferred than bacteriostatic drugs.

Another way of classifying antibiotics is based on their absorbability from the site of
administration. A locally-acting antibiotic is one that limits its action at the site where it is
administered. Examples are topical agents such as topical ointments or eye drops. A
systemically-acting antibiotic is one that affects several body systems. Examples are
antibiotics that are administered intramuscularly or intravenously.

37 of 63

Classification of Antibiotics According to Mechanism of Action

Agents that Interfere with the synthesis of bacterial cell wall:

These agents act by inhibiting the different stages of peptidoglycan synthesis of, b) destroying
an already formed peptidoglycan by activating autolytic enzymes. The most common used are
the b-lactam antibiotics as exemplified by penicillins and cephalosporin. Also called penicillin-
binding proteins (PBPs), these act by inhibiting the conversion of immature peptidoglyean to
mature peptidoglycan by directly inhibiting bacterial transpeptidases. The peptidoglycan
produced is weakly cross-linked making the organism susceptible to cell lysis and death.

Agents that Alter the Function or Permeability of the Cell Membrane

The microbial cell membrane is essential to the survival of the organism because not only does
it serve as a barrier by its selective permeability but more importantly it is the site of bacterial
ATP production. Agents that target the cell membrane can be classified into cationic, anionic,
and neutral agents. The most well-known are polymyxin B and colistemethate (polymyxin E)
which are cationic agents. These agents initially act by disrupting the outer membrane
structure enabling them to enter the cell and inhibit metabolic processes in the bacterial cell.
Among the damaging effects of polymyxin B are (1) disturbance of the surface charge and lipid
composition of the cell membrane, (2) disruption of the potassium gradient on the cell
membrane, and (3) depolarization of the cell membrane.

Antifungal drugs such as polyenes (nystatin, amphotericin B) alter the permeability of the cell
membrane. Azoles (clotrimazole, ketoconazole, miconazole, fluconazole), another group of
anti-fungal drugs, intertere with the synthesis of ergosterol, a major component of the fungal
cell membrane.

Agents that Inhibit Protein Synthesis

These agents bind with the ribosomes, either the 30S or the 50S ribosomal sub-units or both.
Binding with the ribosome results in failure to initiate the synthesis of proteins, interference with
protein elongation or misreading resulting in deformed proteins. Inhibitors of the 30S ribosomal
subunit interfere primarily with the initiation process. The representative drugs are the
aminoglycosides and tetracycline. Aminoglycosides cause formation of non-functional
complexes and misreading. Spectinomycin is an antimicrobial agent related to the
aminoglycosides that binds to a protein in the 30S of ribosomes different from the target of
aminoglycosides. Similarly, tetracycline also targets bacterial 30S ribosomal subunit.
Spectinomycin and tetracycline are only bacteriostatic but inhibit a wide variety of bacteria
including Chlamydia and Mycoplasma.

Agents that bind to the 50S ribosomal sub-unit are inhibitors of the elongation process of
protein synthesis. There are three classes of drugs under this--chloramphenicol, macrolides,
and lincinoids. Chloramphenicol acts by binding to a peptidy) transterase enzyme thereby
inhibiting peptide bond formation. It is a bacteriostatic agent that is eftective against a number
of gram-positive and gram-negative organisms. Macrolides also act on peptidyl transferase
enzyme by interfering with its reaction or translocation. The most popular macrolide is
erythromycin which can effectively inhibit certain gram-positive and gram-negative bacteria
including Haemophilus, Mycoplasma, Chlamydia, and Legionella. Newer classes of macrolides
are azithromycin and clarithromycin which have broader spectrums of activity than
erythromycin;

Agents that Alter the Function or Permeability

of the Cell Membrane

The microbial cell membrane is essential to the survival of the organism because not only does
it serve as a barfier by its selective permeability but more importantly it is the site of bacterial
ATP production. Agents that target the cell membrane can be classified into cationic, anionic,
and neutral agents. The most well-known are polymyxin B and colisterethate (polymyxin E)
38 of 63

which are cationic agents. These agents initially act by disrupting the outer membrane
structure enabling them to enter the cell and inhibit metabolic processes in the bacterial cell.
Among the damaging effects of polymyxin B are (1) disturbance of the surface charge and lipid
composition of the cell membrane, (2) disruption of the potassium gradient on the cell
membrane, and (3) depolarization of the cell membrane. Antifungal drugs such as polyenes
(nystatin, amphotericin B) alter the permeability of the cell membrane. Azoles (clotrimazole,
ketoconazole, miconazole, fluconazole), another group of anti-fungal drugs, intertere with the
synthesis of ergosterol, a major component of the fungal cell membrane.

Agents that Inhibit Protein Synthesis

These agents bind with the ribosomes, either the 30S or the 50S ribosomal sub-units or both.
Binding with the ribosome results in failure to initiate the synthesis of proteins, interference with
protein elongation or misreading resulting in deformed proteins. Inhibitors of the 30S ribosomal
subunit interfere primarily with the initiation process. The representative drugs are the
aminoglycosides and tetracycline. Aminoglycosides cause formation of non-functional
complexes and misreading. Spectinomycin is an antimicrobial agent related to the
aminoglycosides that binds to a protein in the 30S of ribosomes different from the target of
aminoglycosides.

Similarly, tetracycline also targets bacterial 30S ribosomal subunit. Spectinomycin and
tetracycline are only bacteriostatic but inhibit a wide variety of bacteria including Chlamydia
and Mycoplasma. Agents that bind to the 50S ribosomal sub-unit are inhibitors of the
elongation process of protein synthesis. There are three classes of drugs under this--
chloramphenicol, macrolides, and lincinoids. Chloramphenicol acts by binding to a peptidy)
transterase enzyme thereby inhibiting peptide bond formation. It is a bacteriostatic agent that
is eftective against a number of gram-positive and gram-negative organisms. Macrolides also
act on peptidyl transferase enzyme by interfering with its reaction or translocation. The most
popular macrolide is erythromycin which can effectively inhibit certain gram-positive and gram-
negative bacteria including Haemophilus, Mycoplasma, Chlamydia, and Legionella. Newer
classes of macrolides are azithromycin and clarithromycin which have broader spectrums of
activity than erythromycin;

There are several factors that contribute to the development of antimicrobial resistance of
microorganisms. The most common is the overuse of broad-spectrum antibiotics due to over-
prescription. Other factors include incorrect diagnosis, unnecessary prescription of antibiotics,
indiscriminate or Improper use of antibiotics by the patient, and the use of antibiotics as
additives to livestock teeds to improve the growth of the animals.

Agents that Act on the Nucleic Acid

1. Agents char inhibit DNA topoisomerases - topoisomerase enzymes (types I and type II) are
essential to DNA synthesis and are critical enzymes involved in protein translation and cell
replication. DNA Gyrase, a type II topoisomerase is found only in prokaryotic organisms and is
essential for their survival. Among the different antimicrobial agents quinoles have been found
to be most effective against DNA gyrase. First generation quinolones include nalidixic acid and
oxolinic acid. Newer generation of quinolones include the fluoroquinolones, which have wider
spectrum of activity than the quinolones.

1. 2. Agents that inhibit RNA synthesis - agents that act by interfering with the B-subunit of an

RÑA polymerase that is needed for RNA synthesis. Rifampicin is a first-line drug used for the
treatment of tuberculosis that specifically inhibits bacterial RNA synthesis.

Agents that Inhibit Microbial Metabolic Pathways

These agents interfere with metabolic pathways crucial for the survival of the microorganism.
Trimethoprim and sulfonamides are antibiotics that interfere with folic acid metabolism. They
act as competitive inhibitors of tetrahydrofolic acid which is important in the synthesis of DNA,
39 of 63

RNA, and bacterial cell wall proteins. Bacteria cannot utilize preformed folic acid from the
environment and thus must synthesize their own. Sulfonamides act specifically by inhibiting
formation of dihydrofolic acid. Trimethoprim inhibits formation of tetrahydrofolic acid by
inhibiting the enzyme dihydrofolate reductase.

Mechanisms of Drug Resistance

Drug resistance is a growing concern in the field of infection control. An organism is said to
have developed resistance to an antibiotic if it is not affected anymore by that particular
antibiotic. Development of resistance may either be innate (intrinsic) or acquired. Intrinsic
resistance is a stable genetic property that is encoded in the chromosome of the organism and
shared by, all strains of the species. Acquired resistance is resistance arising from the ability of
an organism to resist an antimicrobial drug to which the species, as a whole, is naturally
susceptible. It is not normally encoded in the chromosome of the organism but developed in
the course of time due to constant exposure to the antimicrobial agent involved. It can be due
to chromosomal mutation or the result of genetic exchange between organisms.

Resistance acquired through genetic exchange can occur through any of three ways-
transformation, transduction, and conjugation. Transformation is the simplest and the earliest
form of genetic exchange studied. In transformation, naked or free microbial DNA inserts itself
into the DNA of the same species. Transduction is the transfer of genetic material by a
bacteriophage. Conjugation is the transfer of genetic material through the sex pillus. In
conjugation, what is transferred to another bacterium is an extrachromosomal DNA called
plasmid. The resistance gene is carried by the plasmid.

Certain resistance genes may affect the activity of an antibiotic in two ways. A resistance gene
may code for enzymes that can alter its chemical structure leading to the inactivation of the
antibiotic, or the products of the resistance genes may cause hydrolysis of the antibiotic
thereby destroying the antibiotic. For example, certain bacteria produce beta-lactamases
which can hydrolyze the beta-lactam bonds in the chemical structure of the antimicrobial
agent. This is the most common mechanism of beta-lactam resistance and is the mechanism
involved in the resistance of certain microorganisms to penicillin and cephalosporin.

Prevention of cellular uptake or efflux pump

Gram-negative bacteria have developed the ability to change the lipid composition of their
outer membrane thereby preventing the antibiotic from reaching its cellular target. This
prevents their accumulation in the bacterial cell. In addition, there are gram-positive and gram-
negative bacteria that have developed an efflux pump that can prevent the antibiotic to
accumulate within the bacterial cell. This is true in the case of bacterial resistance to
tetracyclines and fluoroquinolones. Efflux pumps are effective against a wide range of
antimicrobials in multiple classes.

Modification of target sites

Antimicrobials have specific targets in the bacterial cell. Any change in the structure of these
target structures will lead to the inability of the antibiotic to exert its action on the target
bacteria. Certain bacteria have developed the ability to alter the normal target

Target site modified Antibiotic involved

Peptide sub-units of peptidoglycan Glycopeptides

Ribosome sub-units Macrolides, tetracyclines, aminoglycosides

Metabolic enzymes Sulfa drugs, sulfones, trimethroprim

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Target site modified Antibiotic involved

Lipopolysaccharide structure Polymyxins

DNA gyrase Fluoroquinolones

RNA polymerase Rifampicin

Overproduction or bypass of target enzyme

One of the mechanisms developed by bacteria is targeting specific enzymes that are essential
to the metabolism of the organism. This is true in the case of antimicrobials that function as
anti-metabolites. One way by which this is achieved is by over-production of the target enzyme
of the bacteria. By overproducing the target enzyme of the antibiotic, there will still be enough
amount of the enzyme that is free from the antibiotic allowing the organism to still carry out the
essential enzymatic reaction. Some bacteria have developed alternative or bypass
mechanisms that can serve as alternative for the target enzyme. Both these mechanisms are
involved in bacterial resistance to sulfonamides.

Target mimicry is a new mechanism of antimicrobial resistance that has been discovered. It
involves bacteria producing proteins that are similar in structure to the target sites of the
antibioties. Due to the similarity in structure of the new proteins and the target proteins, the
antimicrobial binds the new proteins and not the target protein. For instance, the organism
Mycobacterium tuberculosis produces a protein that can be mistaken for the structure of DNA

The protein selectively binds Ruoroquinolones preventing its binding to the organisms DNA

making the organism resistant to the drug.

Definition of Terms

Immunology - the study of the immune system and the immune response.

Immunogen - any substance capable of inducing an immune response, whether humoral or

cell-mediated or both.

Antigen - a substance recognized by the immune system, whether by the B cell or the T cell,

that serves as the target of the immune response but may not necessarily lead to an immune

response.

Epitope - the structure in the antigen that is recognized by the B cell or the T cell.

Hapten - a substance that is of low molecular weight that can only induce an immune response

if bound to another substance that is already immunogenic (carrier molecule).

Chapter Summary:

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Chapter 9: Host Response to Infection

1. Immunology - the study of the immune system and the immune response.
2. Immunogen - any substance capable of inducing an immune response, whether humoral
or cell mediated or both.

3. Antigen - a substance recognized by the immune system, whether by the B cell or the T
cell, that serves as the target of the immune response but may not necessarily lead to an
immune response.

4. Epitope - the structure in the antigen that is recognized by the B cell or the T cell.

5. Hapten - a substance that is of low molecular weight that can only induce an immune
response if bound to another substance that is already immunogenic (carrier molecule).

Properties of Antigens

There are several properties that an antigen must possess to make it immunogenic. These
include (1) foreignness and genetic composition, (2) chemical composition and complexity, 13)
molecular size and stability, and (4) mode of entry of the antigen. Antigens are genetically
foreign to the host or recognized by the body as non-self. For example, if a pig's heart is
transplanted into a human being, the chances of the human body developing an immune
reaction leading to the rejection of the transplanted heart will be very high because it is
genetically different from humans. In the same manner, most humans are exposed to similar
environmental components (e.g., dust) but not all will have similar reactions. One person may

manifest a hypersensitivity reaction to a substance that will provoke no reaction in another. This
is because each individual has a different genetic composition from another. The chemical
composition and complexity of an antigen may also affect its immunogenicity. Most organic
substances can be antigenic except for pure lipids and nucleic acids. Of the different chemical
groupings, proteins are the most immunogenic. This is because proteins are larger molecules
than others that have more complex structures. Likewise, between a pure protein and a
glycoprotein, a glycoprotein will be more antigenic because its structure is more complex.

The molecular size of an antigen is another property that can affect its immunogenicity, As a
rule, molecules with molecular weights below 10,000 daltons are weakly immunogenic or nor
immunogenic at all. Those with molecular weights greater than 10,000 daltons are very potent
immunogens. However, one needs to consider the stability of the molecule. There are some
substances that have high molecular weights that break up into smaller molecules once they
enter the body, in which case they lose their immunogenicity. Finally, immunogenicity also
depends on how the antigen is administered. The dose of the antigen as well as the mode of
administration should be taken into consideration. For instance, one might need a small
amount of antigen to induce an immune response if the antigen introduced is a protein as
compared to a larger amount if the antigen were a polysaccharide. In the same manner,
antigens may not elicit a reaction intramuscularly but may provoke a good response when
given subcutaneously.

The Immune System

The immune system is composed of molecular and cellular components that are derid firm the
central primary) and peripheral (secondary) lyraphoid organs. The central (uphil DEw a bite he
primary sites for diferentiation and maturation of the important cells th 8:% b him port role in
dapise innuniy which die ta 7 1 m pho oytes (or T cell) ot (ha to vraphoores for B cell, These
consit of the bone at OWanda the thymus. The botf naton/seditedic fem where blood cells
orginate. "he pretWor celli for lymphogyts:

found in the adult bone marrow and this is where they differentiate into B cells and T cells.

Once differentiated, B cells remain in the bone marrow and undergo maturation in the bone

marrow. On the other hand, the T cells will go out of the bone marrow as immature and

42 of 63

incompetent forms then go to the thymus where they mature and become competent. After

maturation, the mature B cells and T cells proceed to the peripheral lymphoid organs to await

any antigen that may enter the body.

The peripheral lymphoid organs consist of the lymph nodes, spleen, and the mucosa-
associated lymphoid tissues (MALT), which include the tonsils, adenoids, Peyer's patches in
the ileum, and the appendix. These organs are the site of reactivity of lymphoid cells. These are

where antigens are trapped and subsequently encounter the T and B cells. Antigens are
brought to these peripheral lymphoid organs where the cells needed for their destruction are
located. Both mature T cells and B cells are found in the peripheral lymphoid organs.

Cells of the Immune System

The various responses of the human immune system are mediated by specific cells and the
substances they produce. These cells include the white blood cells which include (1)
granulocytes (e.g., neutrophil) which are 50%-80% of white blood cells; (2) lymphocytes,
approximately 20%-45% of total white blood cells; and (3) monocytes and macrophages,
3%-8% of white blood cells. Neutrophils play a major role in acute inflammation as well as in
bacterial infections. Lymphocytes and macrophages are mainly involved in chronic
inflammation. Lymphocytes are the predominant inflammatory cells in viral infections.
Macrophages are also predominant in chronic inflammation. In addition, cells that belong to the
mononuclear- phagocyte system play crucial roles as antigen presenting cells.

As the name implies, antigen presenting cells are cells that are involved in the processing and
presentation of antigens to the T cells. These include the macrophages, B cells, dendritic cells,
Langerhans cells in the skin, Kupffer cells in the liver, and glial cells in the central nervous
system. B cells, macrophages, and dendritic cells are the professional antigen presenting cells,

the most important of which are the dendritic cells. Some of these cells are found in
association with lymphoid follicles in the lymph nodes and are thus called follicular dendritic
cells. Langerhans cells in the skin also bring antigens to the paracortical zone of the lymph
node where they are called interdigitating dendritic cells. Dendritic cells are considered as the
true link between innate and adaptive immunity.

Other white blood cells that are part of the innate arm of the immune system include
eosinophils, basophils and platelets. Eosinophils posses eosinophilic granules that play a role
in Type I Hypersensitivity reaction or allergy. In addition, eosinophils also secrete a substance
that is called minor basic protein that is toxic to parasites, especially helminths or worms Like
eosinophils, basophils also play a role in allergies. The granules of both eosinophils and
basophils contain histamine which when released is responsible for the changes seen during
the initial phase of an allergic reaction. Finally, platelets are membrane-bound cell fragments
that are derived from large cells called megakaryocytes. Platelets are mainly involved in blond
coagulation, however, they secrete substances that play a role in inflammation. Natural killer
cells are large granular lymphocytes that are also called NK cells or Null cells They were
originally classified as cytotoxic T cells because they had the same manner of killing target
antigens. However, studies conducted on their structures showed that all T cells had a T-Cell
Receptor (TCR) on their surface that was not present in NK cells, hence NK cells are not
classified anymore as T cells. NK cells are classified as large granular lymphocytes and are part
of the body's innate immune system.

1. Neutrophil - multi lobed nucleus, pale red and blue cytoplasmic granules

2. Eosinophil - Bilobed nucleus, red cytoplasmic granules

3. Basophil - Bilobed nucleus purplish-black cytoplasmic granules

4. Lymphocyte (small) - Large spherical nucleus , thin rim of pale blue cytoplasm

5. Monocyte - Kidney-shaped nucleus, abundant pale blue cytoplasm

43 of 63

The more important cell of the immune system that play pivotal roles in adaptive immunity are
the T lymphocytes and the B lymphocytes. As mentioned, both cells originate from the bone
marrow. B cells mature in the fetal liver and in the adult bone marrow, which is the equivalent of
the bursa of Fabricius in birds. In the peripheral lymphoid organs, they are the appropriate
antigen, the B cells differentiate into antibody-producing plasma cells as well as memory B
cells. They are involved in the body’s humoral immunity. At the same time, the B cells also
function as a professional antigen presenting cell.

T cells are located mainly in the paracortical and interfollicular areas of the lymph node and
spleen. They are involved in the body's cell-mediated immunity. The T cells further differentiate
into CD4+ T cells and CD&+ T cells (cytotoxic or cytolytic). The CD4+ T call consist of the
helper T cells and the regulatory T cells (CD4+CD25+ T cells). The helper T cells do not have
the direct capacity to destroy an antigen. Instead, it activates the cytotoxic T cells and
stimulates differentiation of B cells into antibody-producing plasma cells. The regulatory T cells
play an important role in the maintenance of self-tolerance or the ability of the immune system
to recognize self from non-self. The T cells, most especially the CD4+T cells are the
predominant lymphocytes in the circulation and constitute part of the body’s immune
surveillance. Some T cells also differentiate into memory T cells.

Innate Immunitv

Innate immunitv is also known as natural immunity. This immunity is already active from the
time of birth, prior to exposure to an antigen. Innate immunity is non-specific. It includes host
barriers that prevent entry of microorganisms such as the skin and mucous membranes (first
line of defense), and processes such as phagocytosis and inflammation (second line of
defense) which prevents the multiplication of organisms that gain entry to the body preventing
them from multiplying before they have a chance to produce disease. It is activated within
minutes following exposure to the antigen. However, it does not improve after exposure to the
antigen and does not possess memory and thus provides only short-term protection.

The innate arm of the immune system performs two major functions: killing invading
microorganisms and activating adaptive immune responses. As mentioned, it consists of the
body's first and second lines of defense. The first lines of defense serve to prevent entry of the
organism to the body and limit microbial survival. Physical and chemical barriers Prevent
attachment and entry of the. organisms. These include the skin, fatty acids in sebaceous
secretions, and sweat. The low pH of the fates acids and evert inhibit the growth of
microorganisms. In addition, the normal flora of the skin and other parts of the body form!,
biological barrier that inhibits the colonization and multiplication of pathogenic organisms
competing with the pathogenic organisms for nutrients and by priming the immune system

Microorganisms that penetrate the first line of defense are prevented from multiply in inside the
body by the body's second line of defense. If the primary barriers are breached, inflammation
is activated as well as the natural killer cells. The microorganisms are recognized by innate
immune cells and soluble mediators because of their molecular patterns called pathogen-
associated molecular patterns (PAMP). Inflammatory cells possess pattern recognition
receptors which allow them to act on these pathogenic organisms. Soluble host proteins
specifically those that are part of the complement system also possess such pattern
recognition receptors (e.g., mannose-binding lectin). Recognition of these patterns in turn
activate the inflammatory cells and the complement system. Activation of inflammatory cells
lead to phagocytosis of the antigen while activation of the complement system results in the
production of membrane attack complex which help degrade the antigen.

44 of 63

Factors that limit the entry of microorganisms Mode of Action

Keratin layer of intact skin Acts as a mechanical barrier

Lysozome in tears and other secretions Degrades bacterial cell wall

Respiratory cilia Directs organisms trapped in mucus out of the

respiratory passages

Low pH of stomach and vagina; fatty acids in Inhibits the growth of microorganisms
skin

Surface phagocytes Ingest and destroy microbes

Normal flora Prevent colonization by pathogens

Factors that limit growth of the Mode of Action

microorganisms within the body

Natural killer cells Kills virus-infected cells

Neutrophils Ingest and destroy microbes

Macrophages and dendritic cells Ingest and destroy microbes present antigens to T
cells

Interferons Inhibit viral replication; produce anti viral state

Complement system Membrane attack complex creates holes in

bacterial cell membrane components activate
inflammation

Fever

Inflammatory response

Inhibits bacterial growth

Limits spread of microbes by destroying them

Microbes able to escape the second line of defense are acted upon by the final line of defense
which is the immune response. This involves the B cells and the T cells, cells that are involved
in adaptive immunity.

Adaptive immunity is specific. It is activated after exposure to a particular antigen. Unlike

innate immunity, it is an acquired response to an antigen that is initiated by recognition of
specific epitopes of the foreign invaders. It involves production of antibodies by the B cells and

activation of the cytotoxic T cells. The response is delayed compared to innate immunity since

it takes about 7-10 days before sufficient levels of antibodies are produced by the body. At the

same time, antigens need to be processed first before they can be acted upon by the cytotoxic

T cells. However, unlike innate immunity, the protection given by adaptive immunity is longer
and, in most cases, lasts throughout the lifetime of the individual.

45 of 63

An important distinction between innate and adaptive immunity is the fact that adaptive
immunity possesses memory; Once the B or T cells are activated, some of the B and I cell af
converted to memory cell. The presence of these memory cells ensures a higher response
once there is re-exposure to the same antigen, making the response amplifiable.

Immune Response

First exposure with an antigen leads to the activation of a specific set of helper I cells called the
Th1 cells. Activation of Th1 cells leads to activation of the inflammatory response and delayed
type hypersensitivity as well as stimulation of B cells to produce IgN and IgG.THe antibodies
become detectable in the serum after about 7-10 days but can be longer depending on the
nature of the antigen and the dose of the antigen. The serum Level of antibodies continues to
rise for several weeks and then declines and may drop to very low levels.

Secondary immune response occurs after re-exposure to the same antigen. A second
encounter with the same antigen or a closely-related one occurring months or vears after the
primary response will activate another set of helper T cell called the 722 cell. This will lead to
further production of antibodies (except IgM). This response leads to a rapid antibody response
of a much higher intensity than the primary response. This is explained by the persistence of
antigen-specific memory cells. The predominant antibody involved is IgG and the levels tend to
persist much longer than the primary response. However, if there is a need for other antibodies.
some of the IgG in the circulation can undergo modifications in their structure to become
converted to another antibody (e.g., IgA or IgE). This process is called class switching.

Humoral Immunity

Innate and adaptive immunity can be humoral or cell-mediated. Innate humoral immunity
involves cytokines and the complement system. Adaptive humoral immunity involves the action
of antibodies. Antibody-mediated immunity is directed primarily against (1) extracellular
Pathogens, (2) toxin-induced diseases, (3) certain viral infections, and (4) infections caused by
encapsulated pathogens (e.g., pneumococci and Haemophilus infuenzae).

Antibodies

Antibodies are globulin proteins (immunoglobulins) that react specifically with the antigens chat
stimulate their production. The most important functions of antibodies are (1) to neutralize
toxins and viruses; (2) to opsonize microbes so that they will be readily recognized and more
easily phagocytosed; (3) to activate complement system; and (4) to prevent the attachment of
microbes to mucosal surfaces.

Antibody Structure

A typical immunoglobulin is shaped like a letter "Y" and consists of polypeptide chain linked br
disulfide bonds. An immunoglobulin is made up of two identical heavy chains (50- 70 WDI and
two identical light chains (23 kD). The heavy chains consist of polypeptide chains of 440-550
amino acid residues in length. Each immunoglobulin class has its own structurally distinct
heavy chain- gamma (y) for IgG, mu (p) for IgM, alpha (a) for IgA, delta (8) for ID, and epsilon (E)
for IgE. The light chains are approximately 220 amino acid residues long and are either kappa
(k) chains or lambda (1) chains. Each chain is composed of a variable region and a constant
region. The variable region contains the hypervariable region that represents the antigen
binding site of the antibody. The antigen-binding site is therefore composed of the variable
regions of both the heavy and light chains. Interchain disulfide bonds hold together two heavy
chains. Intrachain disulfide bonds are found within each of the polypeptide chains. The region
at which the arms of the antibody molecule form a letter Y is a flexible region called the hinge
region. Digestion of this region with either papain or pepsin will yield two which binds to
effector cells. identical antigen binding fragments (called Fab) and one crystallizable fragment
(called Fo)

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Classes of Immunoglobulins

There are five classes of immunoglobulins found in all species and all individuals. Each

immunoglobulin class is defined by its component heavy chain.

1. IgG - a monomer and is the predominant antibody in the secondary immune response
(anamnestic response) and is a major defense against bacteria and viruses. It comprises
approximately 73% of the immunoglobulins in the serum. It consists of four subclasses or
isotypes: IgG, (most common), IgG2, IgGs, and IgG. It is the only antibody to cross the
placenta (except IgG.). It is therefore the most abundant antibody in newborns. Together with
IgM, it can fix or activate complement system (except IgG.). It also functions as an opsonin,
thus enhancing phagocytosis. It is the main immunoglobulin in chronic infections.

2. IgM - the largest among the immunoglobulins and is a pentamer. It has a J chair Veining
Chain) that holds the lgM pentamer together. It is the main immunoglobulin produced early in
the primary response and is the predominant antibody in acute infections. Together with IgG, it
can activate the complement system. It is the more efficient activator of complement owing to
its large size. It is also present on the surface of B cells where it acts as an antigen receptor.

3. IgA - called the secretory immunoglobulin and is the main immunoglobulin in secretions
such as colostrum, saliva, and tears, as well as respiratory, gastrointestinal and genitourinary
tract secretions. It exists as a monomer in serum and as a dimer in secretions where the two
monomeric units are held together by a ] chain. It is an important component of mucosal
immunity.

4. IgE - also called the reaginic antibody. It is medically important for two reasons: (1) it
mediates immediate or anaphylactic hypersensitivity reaction, and (2) it provides defense
against parasites such as helminths or worms. It binds to the surface of mast cells and
basophils where it serves as antigen receptor for the allergen. It exists in monomeric form.

5. IgD - a monomer that has no known antibody function. It is found on the surface of many B
cells and serves as the surface marker for B cells but may also function as an antigen receptor.
It is present in small amounts in serum (approx. 1%).

Cell-mediated immunity has four basic functions, namely: (1) provide resistance and aid in
recovery from infections due to intracellular organisms (e.B, viruses): (2) important defense
against fungi, parasites, and bacteria; (3) involved in transplant and graft rejection; and (4) main

defense against tumor cells.

The components of the cell-mediated immune system include several cell types. These are the
macrophages, natural killer cells, helper T cells, and cytotoxic T cells. Macrophages, together
with B cells and dendritic cells present antigens to T cells. In addition, macrophages pre
phagocytic cells that ingest and destroy microbes.

Helper T cells (CD4+ T cells) are of several sub-types. The most important are Th,, Thy, and Th,
In general, the helper T cells function to stimulate differentiation of B cells to antibody-
producing plasma cells as well as to activate the cytotoxic T cells. Th, cells are activated on
first encounter with an antigen and is responsible for triggering inflammation, delayed type
hypersensitivity and synthesis of IgM and IgG. Th response is seen on re-encounter with the
same antigen leading to further synthesis of antibodies as well as class switching. Thy cells are
chemotactic for neutrophils and play a role when further inflammation is required.

Cytotoxic T cells or the CD8+ T cells destroy antigens primarily through the perforin-granzyme
mechanism. Once the antigen is presented to the CD8+ T cells, these release perforin which
will cause formation of pores on the wall or membrane of the antigen. Afterwards, granzyme is
released by the CD8+ T cells which will cause destruction of the antigen while at the same time
activate caspases leading to activation of apoptosis or programmed cell death. CDS+ T cells
are the body's main defense against intracellular pathogens. It is also involved in transplant
and graft rejection as well as destruction of tumor cells.

47 of 63

Complement System

The complement system consists of a group of soluble proteins (C1 - C9) which are proteases
that cleave and activate one another in a sequential manner. They are secreted as inactive
enzymes which are enzymatically activated by other complement proteins. This pathway is
mediated by a single molecule of IgM or two molecules of IgG (IgG1, IgG, or IgG3). There are
three main effects of activation of the complement system, namely (1) lysis of cells; (2)
generation of inflammatory mediators; and (8) opsonization leading to enhanced phagocytosis.
It involves four basic steps: (1) initiation, (2) formation of C3 convertase, (3) formation of C5
convertase, and (4) formation of membrane attack complex (MAC). C3 convertase is required
to cleave C3 to form C convertase, which is essential to cleave C5 to form the terminal product
which is MAC. There are three pathways that act synergistically with each other- alternative or
properdin pathway, classical pathway, and mannose binding lectin or MBL pathway.

The alternative or properdin pathway is activated by bacterial products such as endotoxin or

complexes of immunoglobulins. It is part of innate immunity and is the first to be activated on
initial exposure to the antigen. It bypasses C1, C2, and C4. Upon exposure to the antigen, the
C3 component undergoes spontaneous hydrolysis and binds to activated factor B forming
C3bBb (C3 convertase). The C3 convertase cleaves C3 to form C3bBb3b (C5 convertase)
which cleaves C5 to form C5b6789 (MAC). The C3 convertase is stabilized by properdin P. C3b
produced by the other pathways can be utilized by the pathway in what is known as an
amplification loop.

The classical pathway of complement system is activated by antigen-antibody complexes.

These complexes bind to C1qrs (recognition unit) which will lead to spontaneous activation and
cleavage of complement proteins C2 and C4. The resulting C4b and C2b combine to form
C4b2b (C3 convertase) which will cleave C3 to form C4b2b3b (C5 convertase). This pathway
functions in both innate and adaptive immunity. It is the last to be activated since it requires

some time for specific antibodies to be produced.

The mannose binding lectin or MBL pathway is activated by specific patterns of sugars found
on the bacterial cell wall. Lectin, the first to be discovered, is a pattern recognition receptor that
recognizes the pattern of mannose-containing sugars. Other sugar patterns that do not contain
mannose are recognized by another set of pattern recognition receptors called ficolins. Upon
binding of lectin with the sugar on the bacterial cell wall, there is simultaneous activation of C4
and C2 as in the classical pathway. The sequence of activation of the complement proteins in
both classical and MBL pathways are the same except that the MBL pathway does not utilize
complement protein C1 and the C3 convertase produced is C4b2a. C5 convertase in this
pathway is therefore composed of C4b2a3b.

It should be noted that all three pathways lead to the production of C3b, called the central
molecule of the complement system. C3b functions as an opsonin. At the same time, B cells
have a receptor for C3b and binding of C3b to the receptor on the B cells will stimulate
differentiation of B cells into antibody-producing plasma cells, promoting further production of
antibodies. In addition to C3b, C1q in the classical pathway also functions as an opsonin.

Other

important products of complement activation include the following:

1. C3a and C5a - chemotactic for neutrophils; chemical mediators in inflammation causing
vascular leakage or increased vascular permeability

C3a C42 ,and C5a- function as anaphylatoxins, causing degranulation of mast els

and release of histamine

3. Membrane attack complex (MAC) - cause lysis of the bacterial cell

48 of 63

Hvpersensitivity Reactions

Hypersensitivity reactions are exaggerated and inappropriate immune responses that lead to
tissue injury resulting in harm to the host. It occurs when an already sensitized person is e-
exposed to the same foreign antigen. The injury may be brought about by the various
substances and chemical mediators activated during inflammation as well as the activation of

free complement system. Hypersensitivity reactions may be categorized into four -types I, Il, TI,
and iV. Types I, Il, and Ill are mediated by antibodies while type IV is mediated by T cells.

Type I: Immediate (Anaphylactic) Hypersensitivity

This is what is commonly known as an allergic reaction and is mediated by IgE. It occurs in
response to environmental or administered antigens. Common allergens include pollens,
animal fur, foods, and various drugs. The process begins when an allergen is presented by
dendritic cells to naive CD4+ T cells which differentiate into Th, cells. The Th, cells in turn
release interleukin-4 (IL-4) which stimulate class switching to IgE. The allergen binds to the IgE
and the complex formed binds to and sensitizes mast cells. Subsequent exposure to the
allergen leads to cross-linking among the IgE-allergen complexes stimulating degranulation of
mast cells and subsequent release of histamine.

Type I hypersensitivity can be divided into two phases: immediate phase and late phase. The
immediate phase represents the vascular events of inflammation which include vasodilation
and increased vascular permeability. The major chemical mediator during this phase is
histamine. The reaction occurs within minutes of re-exposure to the allergen and will eventually
subside. About 2-24 hours after the initial phase, there is a recurrence of the symptoms of the
patient. The late phase represents the cellular events of inflammation where the tissues will
show infiltration by inflammatory cells which include neutrophils and eosinophils. Eosinophils
recruit release enzymes that further cause damage to the mucosa. Main chemical mediators
during this phase include slow-reacting substances of anaphylaxis or SRS-A consisting mainly
of leukotrienes C4, D4, and E4 (LTC4, LTD4, and LTE4, respectively). Prostaglandins also play a
role during this phase.

The clinical manifestations are typical in a given individual. These are classified into local
anaphylaxis and systemic anaphylaxis. Examples of local anaphvlaxis include food allergy,
urticaria (hives), eczema, allergic rhinitis or hay fever, and asthma. Systemic anaphylaxis is a
severe allergic response where patients manifest symptoms of circulatory collapse such as
hypotension, severe bronchoconstriction, and laryngeal edema. This is a serious reaction that
is potentially fatal and can be induced by foods such as peanuts and seafoods, bee venom,
and certain drugs (e.g., aspirin and penicillin). Individuals with type I hypersensitivity are said *o
be atopic and will have IgE levels higher than the general population. Diagnosis of type I
hypersensitivity involves accurate history taking. The condition is usually familial and good
history taking techniques will licit information of other family members suffering from the same
condition. Other modalities are available aimed at identifying the specific antigen to which an
individual is allergic to. These include the skin prick test and the scratch test. In the skin prick
test, known allergens are administered subcutaneously like doing a skin test. After one hour,
the resulting wheal-and-flare reaction is measured where a size of > 10 mm is considered
positive. In the scratch test, superficial scratches spaced equally are created on the ventral
aspect of the forearm after which varying solutions of known food allergens are applied. The
size of the wheal-and-flare reaction is again measured with ≥ 10 mm considered as positive.

Feather

Cat

Histamine

Negative

Control

Plane

49 of 63

Pollen

Birch

Pollen

Sheep Wool

Dog

Type II: Antibody-mediated Hypersensitivity

Type II hypersensitivity, formerly known as cytotoxic or cytolytic hypersensitivity, involves three

sub-types, two of which involve destruction of cells. However, a third sub-type does not involve
cytotoxicity but instead involve cellular dysfunction which is the reason why the name was
changed. This hypersensitivity reaction is mediated by IgG or IgM. The classification of the
sub-types will be based on the mechanism involved.

The first sub-type involves the processes of opsonization and phagocytosis. Opsonization is
the process where an antigen is coated by molecules that facilitate recognition by phagocytic
cells resulting in enhanced phagocytosis. An antigen stimulates production of antibodies,
usually IgG, and the complex formed stimulates the classical pathway of complement system.
This leads to the generation of C1q and C3b which both function as opsonin thereby
enhancing phagocytosis. Note that IgG is also an opsonin. Opsonization of cells by IgG
antibody can also cause activation of natural killer cells which can destroy the target

The second sub-type involves the process of complement and Fc receptor-mediated

inflammation. This is initiated when antibodies (IgG or IgM) deposit in fixed tissues such as
basement membrane or extracellular matrix. This will again lead to activation of classical
pathway of complement system leading to the generation of C3a and C5a which are both
chemotactic for neutrophils thus promoting inflammation. Acute rheumatic fever is an example
of a condition under this mechanism.

The third subset under type I hypersensitivity reaction involves formation of autoantibodies

directed against specific cellular receptors (called antibody-mediated cellular dysfunction). In

this mechanism, there is no destruction or lysis of target cells. The autoantibodies produced
may act as competitive inhibitors or may mimic the action of the normal ligand for the receptor.
There are two conditions that illustrate this mechanism.

The first example is myasthenia gravis. It is a neuromuscular disease that is characterized by

progressive muscle paralysis. L he pathogenesis involves formation of antibodies directed
against acetylcholine receptors. In normal conditions, acetylcholine is released by vesicles
found at the terminal portion of the nerves. The acetylcholine binds to the receptor found on
muscle cells. Binding of the acetylcholine with its receptor will initiate muscle contraction. In
myasthenia gravis, autoantibodies against acetylcholine receptors bind to the receptors
preventing the binding of acetylcholine to the receptors. In this manner, the autoantibodies act
as competitive inhibitors for acetylcholine. Because acetylcholine cannot bind to the receptor,
there is no stimulation of muscle contraction. The muscles that are involved are initially those
that are frequently used such as the eyelid muscles. The earliest manifestation of the condition
is inability to open the eyelids causing drooping of the eyelids (ptosis).

A similar process is seen in Graves disease. In this condition, antibodies against receptors for
churoid stimulating hormone ('TSH) are produced. TSH normally stimulates the thyroid gland to
produce thyroid hormone in cases when the hormone level is reduced. The autoantibodies
produced bind to the TSH receptors found in the thyroid gland and mimic the action of TSF.
The net result is continuous stimulation of the thyroid gland to produce hormones so the
patient manifests symptoms of hyperthvroidism.

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Type III: Immune Complex-mediated Hypersensitivity

Immune complex-mediated hypersensitivity reaction is initiated by the formation of immune

complexes in the circulation. The immune complexes form deposit in various tissues, usually in
the kidneys and on the endothelium of blood vessels. Like type Il hypersensitivity reaction,
classical pathway of complement system is activated leading to recruitment of inflammatory
cells and inflammation at the site of deposition. As a result, not only is the target antigen
destroyed but the underlying tissues where the immune complexes are deposited as well,
leading to damage to the tissues. In blood vessels, this leads to a condition known as fibrinoid
necrosis. Infectious processes that may involve this type of hypersensitivity reaction include
involvement of the kidneys in malaria ("black water fever"), dengue, and hepatitis infection
(polyarteritis nodosa).

Type Ill hypersensitivity is of two forms--local immune complex disease or systemic immune
complex disease. Local immune complex disease is exemplified by Arthus reaction. This is
seen as a complication of immunization especially with vaccines that are given with multiple
doses (e.g., DPT). These vaccines are usually given at prescribed intervals. If the vaccine is
given before the next schedule is due and is injected at the same site as the previous dose,
immune complexes form and precipitate in the walls of blood vessels leading to fibrinoid
necrosis. The result is the formation of a localized area of tissue necrosis at the injection site.

The second form of type III hypersensitivity is systemic immune complex disease, evemplified
by acute serum sickness. This is triggered by the administration of large amounts of Foreign
serum (e.g., anti-tetanus serum) or after receiving antibodies from another person or species.
Some drugs like penicillin may also induce this reaction. The manifestations are seen around
one week after receipt of the foreign serum, drug, or antibodies and include fever, urticaria, and
joint pains. Enlargement of lymph nodes (lymphadenopathy) and the spleen (splenomegaly) are
also noted.

Type IV: T Cell-mediated Hypersensitivity

This type of hypersensitivity reaction formerly known as delayed type of hypersensitivity. It

involves T lymphocytes (either CD4+ or CD&+ I lymphocytes), not antibodies. The tissue
destruction is due to either inflammation brought about by cytokines produced by the CD4+ T
cells (CD4+ T cell-mediated inflammation) or direct killing of target cells by the cytotoxic I cells
(CD8+ T cell-mediated cvtotoxicity). Reaction involving the helper I cells (CD4+ T cells) is now
what is specifically referred to as delayed type hypersensitivity. It can be induced by
intracellular pathogens like Mycobacterium tuberculosis as well as certain viruses and fungi,
including their skin tests. For instance, in the tuberculin skin test for tuberculosis, the antigen
(PPD) is administered into the skin of a previously immunized individual or someone who was
exposed to a known case of tuberculosis. The detectable skin reaction (called induration) is
seen within 24-48 hours (sometimes up to 72 hours) after administration. A positive tuberculin
test is seen as induration measuring ¿ 10 mm and does not necessarily mean that the tested
person has tuberculosis but may mean. Positive exposure to an active case. Other examples
are contact dermatitis and rheumatoid arthritis. The helper T cells involved in this form are the
Th, cells which activate macrophages, and the Thy, cells which recruit neutrophils leading to
the inflammatory reaction seen.

In CD&+ T cell-mediated cytotoxicity, the cytotoxic T cells destroy cells bearing specific
antigens on its surface leading to tissue destruction. This mechanism has been implicated in
type I diabetes mellitus and plays an important role in the destruction of virus-infected cells as
well as graft rejection and destruction of tumor cells. The principal mechanism of destruction
involves perforins and granzymes that are secreted by the cytotoxic T cells. Once the target
cells are recognized by the cytotoxic I cells, they release perforin which perforates the wall of
the antigen. They also facilitate the release of granzymes which in turn causes activation of
caspases thereby leading to apoptosis of the target cell.

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Vaccines

A great number of infectious diseases can be prevented by administering vaccines that induce
either active or passive immunity. It contains a weakened (also called attenuated) or inactivated
form of the organism. It may contain the entire organism or a specific portion of the microbe
(also known as sub-unit). It may also be derived from toxins produced by the microorganism
(toxoids). Whatever the component, vaccines are designed to stimulate the body's immune
system to produce the antibodies specific to the organism or its components so that these are
recognized as foreign and will be destroyed immediately upon entry of the organism into the
body. The immunization of a population stops the spread of an infectious agent by reducing
the number of susceptible hosts (herd immunity). Immunization programs have achieved the
following goals:

1. Protection of population groups from the development of common infectious diseases

such as pertussis, diphtheria, tetanus, and rabies

2. Control of the spread of measles, mumps, and rubella

3. Elimination of smallpox in the world

Types of Immunization

Passive immunization involves the administration of purified antibody in preparations called

immune globulins or antibody-containing serum. It is given for rapid, temporary protection
(usually 3-4 months) or treatment of a person (e.g., in the treatment of rabies). The protection
given is short-lived. It is used with the following goals in mind: (1) to prevent goals disease after
a known exposure; (2) to reduce the symptoms of an ongoing disease; (3) to protect
immunosuppressed patients; or (4) to block the action of bacterial toxins and prevent the
diseases that they cause. Immune serum globulin preparations are derived from infected
humans or animals and are available as prophylaxis for several bacterial and viral diseases.
Examples are human rabies immune globulin (FRIG) and immune globulins against hepatitis

A or B, measles, chickenpox, and diphtheria.

Active immunization involves the injection of vaccines prepared from organisms or their
products. This stimulates the body's immune system to produce the specific antibodies
against the component organism of the vaccine. The response takes days to weeks to develop
but the protection given is long-term or even lifelong. Active immunization is preferred over
passive immunization. Examples include TDaP, MMR, and BCG.

Type of Vaccines

There are four major groups of vaccines--toxoid, live attenuated, subunit or killed inactivated.
Live attenuated vaccines are prepared using organisms with limited ability to cause disease.
These are especially useful for protection against infections caused by enveloped viruses.
Immunization with a live attenuated vaccine resembles the natural infection leading to
development of humoral, cell-mediated, and memory responses. Immunity acquired is usually
long-lived and, depending on the route of administration, can mimic the normal immune
response to the infecting agent. The first vaccine was developed by Edward Jenner for
smallpox. Albert and Sabin developed the first live oral polio vaccine. Other examples are the
Bacille-Calmette-Guarin (BCG) vaccine for tuberculosis and vaccines against measles, mumps,
rubella (German measles), and chickenpox. There are two problems with the use of live
vaccines. First, the organism may still revert to its original virulent form once it enters the body;

Second, the vaccine may be dangerous to immunocompromised patients and pregnant

women.

Toxoid vaccines were developed based on the principle that certain diseases are caused by
exotoxins produced by the causative agents. Examples are tetanus, botulism, pertussis,
diphtheria, and cholera. The toxoids were produced from the exotoxins. Because the sourcels
52 of 63

C0 olin, they are not as immunogenic and large or multiple doses are needed which may lead
longer lasting immune response.

Toxoid vaccines are advantageous because: (1) they are safe without possibility of reverting to
a virulent form; (2) the component antigens are non-replicating; and (3) they are more stable
compared to live vaccines. The disadvantages include the need for adjuvant and multiple
doses, and the possibility of developing a type III Arthus reaction. This is because of the
presence of excess antibodies forming complexes with the toxoid molecules and activation of
the classical pathway of complement.

Killed vaccines in general refer to vaccines derived from bacterial sources while inactivated
vaccines are derived from viruses. The first killed vaccine to be produced was the typhoid
vaccine during the latter part of the 19th century. Examples of inactivated vaccines that are
popularly used are the polio vaccine and hepatitis A vaccine. Immune response to the killed/
inactivated vaccine is similar to the response to the toxoid vaccine but with a wider range of
target antigens. Several disadvantages are associated with killed/inactivated vaccines. Same
as toxoids, multiple doses are needed to elicit a strong immune response. In addition, because
of the adjuvant used, local reactions may be seen at the site of injection. Also, the immunity
induced may only be humoral and not cell-mediated. Lastly, they do not elicit a local IgA
response. The last vaccine type is the subunit vaccine. This type of vaccine is produced the
same way as the killed/inactivated vaccine but instead of using the entire organism as the
antigen to stimulate antibody production, only a specific antigen or structure on the organism is
used. Its effectivity relies on accurate selection of the sub-unit to be used. Examples are the
hepatitis B vaccine where the surface antigen of the virus was used in its development. Other
examples are the vaccines against Haemophilus influenza and Streptococcus pneumonia
where the capsules of both organisms were used.

The benefits of using subunit vaccines are similar to toxoid vaccine. An additional advantage is
that with subunit vaccines, one can identify or distinguish the infected individuals from the
vaccinated individuals. For example, in hepatitis B, immunized individuals will only have the
antibody to the surface antigen (anti-HBs) and will be the only ones detected in their blood
while infected patients will have additional antibodies present against the core antigen (anti-
HBe) and the envelope (anti-HBe) of the hepatitis B virus. The disadvantages of using of
subunit vaccines are comparable to toxoid vaccines. Such vaccines also requires multiple
doses and addition of an adjuvant as well as local reactions at the site of infection similar to an

Arthus reaction.

Problems with vaccine use

Vaccines have been instrumental in the prevention of a great number of infectious diseases.
However, the use of vaccines also entails certain problems. As mentioned earlier, live vaccines
can occasionally revert to virulent forms and may be life-threatening when given to
immunosuppressed individuals or pregnant women. Adverse side effects have been reported in
association with certain vaccines. These include hypersensitivity and allergic reactions to the
antigen in the vaccine, to non microbial marerial in the watcly thand fo contaminan, the airt.
Fever is a corn mon complaint of parents after having their children vacant) the varcistances,
depending on the susceptibility of the child, the fever may lead to bet;, febrile seizures.

There are also instances of vaccine failures. Certain organisms such as viruses may have more
than one serotype that may be difficult for a vaccine to control. For example, Rhinovirus, The
most common Cause of the common colds has more than 100 serotypes. The existence d
several strains of the infuenza virus led to reduced and limited effectivity of the fu vaccine
Lastly, vaccines do not 100% guarantee that the disease will not develop. A child who receive,

53 of 63

a vaccine against chickenpox may still develop the disease, especially if there is an
outbreaking the community. The advantage of giving the vaccine is that it can prevent the
development of complications.

The immune system is composed of cells and soluble proteins that are designed to defend

the body against any invading organism.

. Cells involved in the immune system are derived from the bone marrow. These are the white
blood cells, the most important of which are the lymphocytes, the major cells involved in
adaptive immunity.

- Antigen-presenting cells are derived from the mononuclear phagocyte lineage. They function
to process and present antigens to the T cells. The professional antigen presenting cells are the
macrophage, B cells, and dendritic cells. The most important among the three are dendritic
cells.

• The bone marrow and the thymus are the central or primary lymphoid organs where the
immune cells undergo differentiation and maturation. For example, B lymphocytes differentiate
and mature in the bone marrow. T lymphocytes differentiate in the bone marrow and undergo
maturation in the thymus.

• Mature B cells and T cells enter the circulation to go to the secondary or peripheral lymphoid
organs. These include the lymph nodes, spleen, and the mucosa-associated lymphoid tissue
sites where antigens encounter the immune cells.

• Innate immunity is immunity that is already present and active at birth. It is non-specific, acts
immediately upon encounter with the antigen but gives short-term protection. It does not
possess memory. It includes the body's first and second lines of defense. The first line of
defense functions to prevent the entry of the organism into the body. These include the skin
and mucous membranes as well as the normal flora found in different parts of the body. The
second line of defense aims to destroy the invading organism before it has a chance to multiply
and cause disease. Included are the natural killer cells, inflammation, and the body's normal
resident flora. Adaptive immunity is activated by certain antigens which makes the response
more specific. It has a more delayed reaction because it takes time for antibodies to be
produced and for cytotoxic T cells to be activated. The protection obtained is long-term and, in
most instances, lifelong. The most important property is memory which allows recognition of
the antigen on re-exposure making the immune response amplifiable. It constitutes the body's
third line of defense.

• Innate and adaptive immunity may both be humoral or cell-mediated.

• - Innate humoral immmunity involves the action of specific proteins and molecules that act to
destroy antigens. This includes cytokines and the complement system.

• Innate, cell/ mediated immunity involves the action of natural killer cell my

phagocytic cells.

• Synthesis of specific antibodies by activated B cells is what constitutes adaptive humoral

immunity.

* IgG is the major immunoglobulin in the circulation and is predominant in the secondary
immune response. It is the only immunoglobulin that truly functions »s an opsonin and the only
immunoglobulin that can cross the placenta. It is involve? in chronic inflammation and is able
to fix complement.

› IgM is predominant in acute inflammation and the primary immune response, ltis the largest
and more effective fixer of complement: It also functions as an antigen receptor on the surface
of B cells.

› IgA is also known as the secretory immunoglobulin and acts to prevent adhesion of microbes
to mucosal surfaces. It provides protection to the gastrointestinal tract, digestive tract, and
genitourinary tract.

› IgE is the reaginic antibody and is the predominant immunoglobulin in type I hypersensitivity
and parasitic infections.

54 of 63

• ID functions as a surface marker for B cells and has no biologic activity. Adaptive humoral
immunity is the body's defense against extracellular organisms. Antibodies produced can help
neutralize viruses. These antibodies also serve as the body’s defense against encapsulated
organisms as well as toxin-producing microbes.

• Adaptive cell-mediated immunity involves both the helper T cells and the cytotoxic I cells.

» Helper T cells are CD4+ and have several important subsets.

Thy acts on first exposure to an antigen and is responsible for stimulating inflammation,
delayed type hypersensitivity and synthesis of IgG and IgM. Thy is activated on re-exposure to
the same antigen and is involved in stimulating class switching to form other antibodies. Thy
function to recruit neutrophils leading to inflammation.

CD4+ T cells act to stimulate differentiation of B cells to antibody-producing plasma cells and
activate cytotoxic T cells.

• CDA-CD25+ I cells are the regulatory cells which forms part of the body's immune
surveillance: It recognizes self from non-self and is therefore important in the maintenance of
immunologic tolerance (ability of the body to recognize self for response of the immune cells.

Tester?, These cells regulate the action of the immune cells preventing exaggerated

Cytotoxic T cells are CD&+ and are the body's main defense against intracellular organisms
such as viruses and tumor cells. They are involved in graft and transplant rejection. The major
mechanism by which they destroy antigens is through the perforin-granzyme mechanism.

• Complement system is composed of various proteins that are activated or cleaved in a

sequential manner. It consists of three pathways.

» The alternative or properdin pathway is the first to be activated on exposure to an antigen. Its
main activator is bacterial products such as endotoxins.

» The mannose binding lectin pathway is activated upon recognition of specific patterns of
sugar found on the cell wall of bacteria. There are two pattern recognition receptors involved in
the pathway-lectin which recognizes mannose containing sugars and ficolin which recognizes
other patterns of sugar. It is the second to be activated on first exposure to an antigen.

» The last to be activated upon antigen exposure is the classical pathway. It is triggered by
antigen-antibody complexes.

» All three pathways lead to formation of C3b which is required in the formation of C5
convertase, the enzyme needed to form the terminal product of complement which is
membrane attack complex. Hypersensitivity reactions are exaggerated immune responses that
lead to tissue damage and injury.

» Type I hypersensitivity is mediated by IgE and is commonly known as allergy. The effector
cells are mast cells and the predominant inflammatory cells are the cosinophils. Type I
hypersensitivity is called antibody-mediated hypersensitivity. It is triggered by antigen-antibody
complexes that causes activation of the classical pathway of complement system.

» Type III hypersensitivity is immune complex-mediated hypersensitivity. It is also triggered by

antigen-antibody complexes. Like type II, the antibody involved is IgG or IgM and similar to
type Il also involves activation of classical pathway of complement. The main difference is the
site of formation of immune complexes and the resulting deposition of these complexes in
tissues and blood vessels leading to fibrinoid necrosis. It has two forms--acute serum sickness
(systemic form) and the Arthus reaction

(localized form).

» Type IV hypersensitivity reaction is the T cell-mediated hypersensitivity reaction which

involves either CD4+ T cells (delayed type hypersensitivity) and CD&+ T cells (cytotoxic T cell-
mediated).

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Chapter 10: Bacteria and Disease

Definition of Terms

1. Disease - result of an undesirable relationship between the host and the pathogen, marked
by interruption in the normal functioning of a body part or parts.

2. Infection - invasion of the body by pathogenic microorganisms. The term is not synonymous

with disease.

3. Symbiosis - prolonged and close interaction between organisms of different species.

4. Mutualism - a form of symbiosis in which both organisms benefit from the relationship.

5.Commensalism - a form of symbiosis in which one organism benefits from another organism
without causing harm to it.

6. Parasitism - a form of symbiosis where one organism benefits from another organism and at

the same time causes harm to the other.

7. Pathogenicity - ability of an organism to produce disease. An organism that can produce

disease in humans is said to be pathogenic.

8. Virulence - describes the degree of pathogenicity of an organism or the degree to which a

organism can produce disease.

9. Contamination - presence of unwanted materials (chemical, biological or radiological)

where they should not be or at concentrations above the normal. The presence of these
substances may not necessarily lead to harm.

10. Pollution - presence of contaminants that can cause adverse biological effects to humans
and communities. All pollutants are contaminants but not all contaminants are pollutants,

11. Bacteremia - presence of bacteria in the blood.

12. Septicemia - presence of actively multiplying bacteria in the blood, usually from a source r

infection. The condition is called sepsis.

13. Pyemia - presence of pus-producing bacteria in the bloodstream.

14. Viremia - presence of viruses in the blood.

15. Toxemia - presence of toxins in the blood.

Koch's Postulates

Robert Koch was a German physician who made significant contributions to the field of
microbiology. One of his greatest and most well-known contribution was proving that certain
microorganisms caused specific diseases. Together with some of his colleagues, he developed
a scientific experimental procedure to prove this relationship. This experimental procedure was
published in 1884 and came to be known as Kock's postulates. These postulates are as
follows;

1. The suspected organism must be absent in healthy individuals but present in those with

the disease.

2. The suspected organism must be isolated from the infected host and grown in pure

culture.

3. The organisms grown from pure culture must produce the same disease as that of the

infected source when inoculated to a susceptible animal.

4. The same organism must be isolated from pure culture from the experimentally-infected
Once all the above conditions are fulfilled, it can now be concluded that the organism isolated
is indeed the cause of the disease under study. The validity of Koch's postulates lies in the
ability of the pathogen to grow in the laboratory) using atificial culture media. However, there
are certain organisms that cannot be grown!h artificial culture media. Viruses are obligate
intracellular parasites that need to be grona” ivin3 cells Likewise, Myrobaderium leprae, the
causative agent of leprosy neds to be grown foot pads of mice and armadillo.

56 of 63

Another limitation of Koch's postulates is that not all people who acquire an infection

develop overt disease. Most of the time, infections are sub-clinical. Also, the reaction of
humans to specific pathogens may differ given a specific microorganism. This is because
human beings have different genetic compositions that may modulate their responses to the
same organism. One individual might develop minor illness from a particular pathogen but the
same pathogen may produce fatal infection in another host.

An issue involving Koch's postulates is the requirement that the cultured organism must be
inoculated into a susceptible animal. However, there are certain organisms that are species
specific. There are organisms that produce disease only in animals in the same manner that
there are infectious agents that produce disease only in humans. Therefore, organisms that
produce disease only in humans cannot be tested using laboratory animals and vice versa.
One also needs to consider the ethical issues involved in such testing procedures. Finally, there
ate certain pathogens that become altered when grown in artificial media. Some become less
Pathogenic while others may lose their pathogenicity, in which case Koch's postulates cannot

be applied.

Factors that Influence the Occurrence

of Infection: The Chain of Infection

The development of an infectious disease is a consequence of the interaction among three

components the etiologic agent, the host, and the environment. Transmission starts when the
pathogenic organism leaves its host or a reservoir through a portal of exit. A susceptible
organism acquires the infection through a given mode of transmission, entering the body of the
susceptible host through a portal of entry. Once inside the body, the organism starts to multiply
and produce disease. This is called the chain of infection.

Reservoir

Reservoirs serve as the continual source of disease-producing microorganisms. It is the

site where an infectious agent normally resides and multiplies. It provides the organisms with

conditions that enable them to survive and multiply and provide opportunity for transmission

to a susceptible host. Reservoirs include animals and humans as well as the environment.

Animal Reservoirs

Certain infectious diseases can be transmitted from an animal to humans. These are called
zoonotic infections. In most instances, humans serve only as an incidental host and dead-end
host and thus the disease will not be transmitted to another human. Examples of zoonotic
infections include anthrax, plague, and rabies.

Human Reservoirs

A number of pathogenic organisms have humans as their reservoir. These organisms may be
directly transmitted from one individual to another. Examples are respiratory pathogens and
sexually transmitted infections. The human reservoir may not necessarily manifest with the
disease. There are certain infected humans who may harbor the organism but only develop
sub-clinical disease. There are those who developed the disease, got well but still harbor the
organism thereby transmitting them to others. These are what are known as carriers and
comes in several forms. Those who are infected but do not manifest symptoms are known?
asymptomatic or healthy carriers. Carriers who transmit the causative agent during the
incubation.

Period of the illness are called incubatory carriers. Chronic carriers are those who harbor life
organism for months or longer after the patient developed the initial infection. Individual who
developed the disease, recovered but remain capable of transmitting the causative agent are
known as convalescent carriers. Carriers are individuals who are not aware that they#
transmitting the infectious agent which makes them public health hazards.

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Environmental Reservoirs

Water, soil, and plants can harbor infectious organisms. For instance, the fungus Histoplasma
capsulatum is associated with soil. Water serves as a reservoir for Entamoeba histolytica, a
protozoan parasite that cause amebiasis. Aquatic vegetation such as watercress and
"Kangkong" harbor Fasciola hepatica larvae which causes damage to the liver.

Portal of Exit

The portal of exit is the route by which an infectious agent exits its host. It is usually the site
where the infectious agent is commonly located or localized. For example, the blood fluke
Schistosoma haematobium which preferentially infects the urinary bladder exits the host via
urine. Infectious agents causing respiratory tract infection will leave the host via droplets or
aerosols from the respiratory tract. Sexually transmitted infectious agents exit via vaginal or
urethral secretions. There are also organisms that exit the host through blood-sucking
arthropods such as Plasmodium spp., the causative agent of malaria.

Mode of Transmission

Infectious agents may be transmitted from the source to a susceptible host in several ways,
These can be broadly categorized as direct or indirect contact.

Direct Contact

Most infectious agents are transmitted through direct contact, Contact with environmental
sources harboring infectious agents are also considered direct contact For example, the blood
Fluke Schistosoma spp. can be transmitted when one wades in fresh water containing snails
that harbor the larvae of the parasite. The larvae in turn enter the human host through fie
penetration. The most important methods though of direct contact are the person-to-person
contact and droplet spread.

1. Person- to person contact- involves transmission through skin- to skin contact kissing or
serval transmission. Warts can be transmitted through direct contact with the lesion on the skin
of infected persons. Infectious mononucleosis caused by Epstein Barr Virus is transmitted
through saliva, hence the name ‘ "Kissing Disease." Syphilis, gonorrhea, and other sexually
transmitted infections are spread through vaginal and urethral secretions of infected persons.

2. Droplet spread - patients with respiratory tract infection such as the common colds or
influenza can transmit the causative agents during coughing and sneezing. Droplets are
differentiated from aerosols by its larger size (> 5 microns in size). It is considered as direct
contact because the droplets are sprayed over a few feet before they fall to the ground. Close
proximity with the source is necessary for droplets to be transmitted.

Indirect Contact
1. Airborne transmission - infectious agents may be transferred from an infected person to a
susceptible host through dust or aerosols. Aerosols are droplets with nuclei less than 5 microns
in size. Due to their small size, they may remain suspended in air for a longer time and may
cover farther distance than droplets. There are also organisms that can be cartied with dust.
For example, the fungus Cryptococeus neoformans can be transmitted through aerosolized
pigeon or fowl droppings and inhaled by a susceptible host. Measles a common childhood
illness, can be transmitted through aerosols.

2. Vehicle transmission - refers to transmission of organisms through media such as flour,

water, milks, or biologic substances such as blood and body secretions. Fomies” inanimate
objects such as beddings and clothing may also serve as vehicles. Vehicle Cary an infectious
agent passively or may provide an environment that promote; growth and multiplication of an
58 of 63

infectious agent. The most common vehicles are food and water. Gastrointestinal infections
such as cholera and typhoid are transmitted through contaminated water. In food-borne
transmission, the causative agent is transmitted through ingestion of raw or improperly cooked,
poorly refrigerated food that is contaminated by the causative agent. The food ingested may be
contaminated by feces of the infected patient (fecal-oral transmission). Examples are food
poisoning and gastroenteritis.

3. Vector transmission - vectors are usually insects that can transmit an infectious agent. These
spread the infectious agent by two general methods: mechanical and biological. Mechanical
transmission refers to the passive transport of the organism on the insect’s feet or other body
parts. For example, cockroaches and flies can transfer the organisms from the feces of
infected persons to food, which is later swallowed by the host. Biological transmission is the
active transport of the organism. Here, the organism enters the insect vector after the insect
vector bites an infected person. The organism then multiplies within the insect vector and is
transmitted by the insect vector to another person through bites. For example, malaria is
transmitted to a susceptible host through the bite of the female Anopheles mosquito. Dengue
virus, chikungunya virus, and zika virus are also transmitted through bites of mosquito vectors.
Bite of the rat flea is the mode of transmission of Yersinia pestis, the causative agent of the
plague.

Portal of Entry

How the infectious agent enters a susceptible host is referred to as the portal of entry. It
provides access to tissues where the infectious agent can multiply. More commonly, the portal
of exit of an infectious agent is also the portal of entry into another host. For example,
organisms that leave the respiratory tract will also enter another host through the respiratory
tract via inhalation. Organisms that are transmitted through food and water enter the host
through the mouth but exit through the feces. In infection with the blood fluke Schistosoma
haematobium, the organism leaves the body of the host through urine but enters through skin
penetration by the infective larva. Hepatitis B virus and HIV enter the susceptible host through
blood and blood products.

Host

The final link in the chain of infection is the susceptible host. The host's susceptibility is
affected by several factors such as constitutional or genetic factors and immune status of the
host. Susceptibility to infection may be increased or decreased in certain individuals with
specific genetic make-up. For example, patients born with the gene that codes for the sickle
cell trait, an abnormality in morphology of red blood cells, are less prone to develop malaria
than those who were not born with the trait.

The immune status of the host is probably the most important factor that can affect
development of a disease process. Humans have natural barriers that prevent entry of potential

pathogenic organisms. Besides these, they are also equipped with a highly functional immune

system that can mount adequate defenses to fight and destroy any invading pathogen. Once
there is a breakdown in all these defenses, microorganisms can easily gain entrance into the
body, multiply, and produce disease. Factors that may impact the human immune system
include poor nutritional status, chronic intake of alcoholic beverages, or any condition that
dampens the immune response.

How Organisms Produce Disease

Mechanical: Invasiveness

Organisms can produce disease by directly damaging tissues or body surfaces. This involves
invasion of the epithelial surface and penetration into deeper tissues. Invasiveness
encompasses three important steps--colonization, ability to evade host immune defenses, and

production of extracellular substances that can promote invasion. Colonization involves the
ability of the invading organism to enter the susceptible host and establish itself in the portal of
59 of 63

entry. This can be facilitated by substances produced by the organism that facilitate adhesion
of the organism to specific target cells. These substances are collectively called adhesins.
Gram-negative bacteria possess pili or fimbriae that promote adherence of the organism to

susceptible cells. By promoting adhesion, the organism can easily invade the surfaces and
enter the body of the host. Once the organism enters the body, the immune system of the host
immediately mounts an immune response that will lead to the destruction of the invading
pathogen. However, there are certain factors that allow the organism to evade these immune
defenses. For instance, bacteria that possess a capsule enables the organism to evade
phagocytosis. Staphylococcus aureus secretes the enzyme coagulase that promotes formation
of a coagulum within which the organism may hide to escape detection by the immune
surveillance cells. Mycobacterium tuberculosis can survive and multiply inside macrophages by
inhibiting phagosome-lysosome fusion.

Finally, some microorganisms produce substances or have developed mechanisms that Can
promote invasion. Neisseria gonorrheae can enter and multiply within host cells and alter
multiplication is extruded from the host cell allowing it to infect other host cells. The process of
extrusion from the host cell causes direct destruction of the host cells. Some bacteri irclice
entymes that aids them in invading target cells. Collagenase is an enzyme product. 34
Clostridium perfringens that causes breakdown of collagen, a major component of connectis
issue of muscles thereby contributing to the development of the disease called gas gangrene.

Chemical: Toxin Production

Toxins are poisonous substances and are often the primary factors that contribute to disease
production. There are two major types of toxins--exotoxins and endotoxins. Endotoxins are
integral components of the outer membrane of gram-negative bacteria such as Salmonella,
Shigella, and Escherichia coli. The specific component responsible for the endotoxin activity of
these bacteria is the lipopolysaccharide (LPS), which is further composed of a lipid A moiety
and a polysaccharide moiety. The lipid A component is associated with its toxic activity while
the polysaccharide component is antigenic. Endotoxins exert their effects when the gram-
negative bacteria die and their cell walls undergo lysis, thereby releasing the endotoxin. All
endotoxins produce similar signs and symptoms, although not to the same degree. Exotoxins
are intracellular products of some bacteria as part of their growth and metabolism and are
released into the surrounding medium. These are mainly proteins and many of them are
enzymes. Most of them are produced by gram-positive bacteria but may also be produced by
some gram-negative bacteria.

Exotoxins are soluble in body fluids and are thus easily diffused into the blood and rapidly
transported throughout the body. There are three principal types of exotoxins: (1) cytotoxins
which kill host cells or affect their function; (2) neurotoxins which interfere with normal nerve
impulse transmission; and (3) enterotoxins which affect the cells lining the gastrointestinal tract.
Diseases produced by exotoxin-producing bacteria are often due to the effects of the exotoxin
and not of the bacteria themselves. Exotoxins are therefore disease-specific. Important
examples are the diphtheria toxin, botulinum toxin, and the tetanus toxin.

Property Exotoxin Endotoxin

Bacterial Source Mostly gram-positive bacteria; Gram-negative bacteria only

some gram-negative bacteria

Relation to microorganism Metabolic product of growing

cell; secreted outside the cell
Toxicity Extremely toxic; sometimes fatal
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Property Exotoxin Endotoxin

Bacterial Source Mostly gram-positive bacteria; Gram-negative bacteria only

some gram-negative bacteria

Chemistry Protein or short peptides

Pharmacology (effect on the Specific for a particular cell
body) structure or a function in the
host (mainly affects cell
functions, nerves and GIT)
Antigenicity Extremely antigenic
General such as fever, malaise
and shock; all produce the same
effects.
Less antigenic

Enzymatic Activity Yes No

Heat Stability Heat labile (except Heat stable

staphylococcal enterotoxin)
Fever Production No Yes

Specificity High degree Low degree

Relation to antibodies Can be convicted to toxoids; Cannot be converted to toxoids;

neutralized by anti toxins not neutralized by anti-toxins
Denaturation on boiling Yes No

Location of genes Present on plasmids or Bacterial chromosome

Representative diseases
bacteriophages
Gas gangrene, botulism,
diphtheria, tetanus, scarlet fever
Typhoid fever, urinary tract
infections, meningococcal
meningitis, and
meningococcemia

Immunologic

Some organisms produce disease not as a consequence of mechanical invasion or tonin

production but as a consequence of the immune response of the host to the microorganism or
its product. In hepatitis caused by the hepatitis viruses, the damage to the liver is not a direct
effect of the virus but of the response of the immune system to the virus. Antibodies are
produced against the virus and cytotoxic I cells are activated leading to the destruction of
hepatocytes. In childhood measles and German measles, the rashes seen are due to the
specific immune response of the body to the measles virus

Classification of Infectious Diseases

Based on how they behave within a host and within a given population. A disease that is
spread from one host to another, either directly or indirectly, is called? communicable disease.
Examples are measles, tuberculosis, and typhoid fever. If a disease is easily and rapidly spread
from one person to another, then it is classified as a contagious, disease (ef, measles and
chickenpox). If the infection results in the death of the patient or!, short period of time, it is
called a fulminant infection. An example is meningococcemia a patient may die hours after
confinement in the hospital.

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A non-communicable disease is one that is not spread from one person to another. It is usually
caused by organisms that normally inhabit the body and produce disease only occasionally or
by organisms that produce disease only when introduced into the body such as Clostridium
tetani, the agent that causes tetanus. It produces disease only when it enters the body through
breaks in the skin.

Based on the source of the microorganism.

An infection is said to be exogenous if the source of the infectious agent is from outside the
body. For example, cholera is an exogenous infection because the causative agent enters the
body through ingestion of contaminated water. Hospital-acquired infections or nosocomial
infections can also be considered as exogenous infections where the offending organism was
obtained from the hospital environment during the period of confinement of the patient in the
hospital. On the other hand, an endogenous infection is one where the source of the causative
organism is from inside the body. Escherichia coli is part of the normal flora of the colon that
can enter the urinary tract via the urethra, especially in women, due to its close proximity to the
anal orifice. This can lead to the development of urinary tract infection which can ascend to
involve the kidney and can lead to sepsis in immunocompromised patients.

Based on the occurrence of a disease.

A disease that occurs occasionally is called a sporadic disease. If the disease is constantly
present in a population at low levels, then it is called an endemic disease. Malaria is said to be
endemic in Palawan while schistosomiasis is considered endemic in Leyte. If a great number of
people in a given locality develop an infectious disease in a relatively short period of time, it is
called an epidemic. Influenza is an example of a disease that can cause epidemics. If a disease
has a worldwide occurrence or involves at least three regions in the world, it is said to be a
pandemic. Influenza, especially due to influenza A, SARS, and AIDS are examples of diseases
that can cause pandemics.

Based on the severity or duration of a disease. An acute disease is one that develops rapidly
but lasts for only a short period of time. An example is the common cold. If the disease
develops more slowly and occurs for long periods of time, it is called a chronic disease. A good
example of this is tuberculosis. Hepatitis B infection is an example of a disease that can have
either an acute form or a chronic form. A latent disease is one in which the causative organism
remains inactive for a time but can become active again and produce symptoms of the
disease. Viruses that belong to the family Herpesviridae are characterized by latency,. An
example is shingles, a reactivation of a latent chickenpox infection which occurs years after the
initial infection.

Based on the extent of host involvement

Infections can be classified according to the extent to which the body of the host is affected. A
localized infection is one in which the invading organisms are limited to a relatively small area
of the body. Boils and abscesses are examples of localized infections. A systemic or
generalized infection is one where the causative organisms or their products are spread
throughout the body through blood or lymph. In some cases, the causative agents of a
localized infection may enter a blood or lymphatic vessel, spread to specific parts of the body
and become confined to specific areas. This is called a focal infection. Focal infections can
arise from infections in areas such as the teeth, tonsils, or sinuses.

A primary infection is an acute infection that causes the initial illness, while a secondary
infection is one which is caused by opportunistic pathogens after the primary infection has
weakened the body's defenses. Secondary infections are common in patients whose immune
system have been compromised by another disease process (e.g., AlDS) and can prove to be
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more devastating than the primary infection. A subclinical or inapparent infection is one that
does not cause noticeable illness. Hepatitis due to hepatitis A virus can, for instance, occur in

certain individuals who do not necessarily develop the typical signs and symptoms associated

with the disease.

Stages of an Infectious Disease

Once a microorganism invades a susceptible host, disease follows. The development of the

disease follows a sequence of events that tends to follow a similar pattern whether the disease
is chronic or acute. These periods are the following:

1. Incubation period - the time interval between entry of the offending agent and the
appearance of the initial signs and symptoms of the disease. In most cases, this period is
variable and is usually stated in the form of a range (e.g., 6-12 days). The length of this period
can be affected by the virulence of the organisms as well as the number of infecting
microorganisms. It also depends on the resistance of the host. An organism that is considered
virulent can produce disease within a short incubation period. As for the number of infecting
microorganisms, in general, the greater the number of microorganisms that invade the body,
the shorter the incubation period. However, if the organism is highly virulent, it may take only a
small number of organisms produce disease, hence a shorter incubation period.

2. Prodromal period - a relatively short period, is characterized by early, mild symptoms; of

disease which are generally non-specific. In measles infection, the prodromal. Period is
characterized by non-specific constitutional symptoms such as fever, cough colds, general
aches, and malaise- symptoms which can also be seen in other disease processes and are
thus not specific to measles.

3. Period of illness - corresponds to the period of maximal invasion. It is during this period
that the disease is most acute. During this period, the patient manifests signs and symptoms
distinctive of the disease. For example, the period of illness in measles is marked by the
appearance of the typical rashes seen in measles. Examination of the patient's complete blood
count (CBC) will generally show elevation of the white blood cells although in some infections
there may be a reduction in the total WBC count. As a rule, most bacterial infections will
usually show increased neutrophil count while most viral infections will have a high lymphocyte
count. Several outcomes can arise during this period. The infection may remain acute where
the body's defense mechanisms successfully destroy the invading microorganism leading to
resolution of the infection and recovery of the patient. When the patient does not successfully
overcome the disease-producing agents, he or she may develop severe disease that can lead
to a fulminant infection. The infection may also progress from an acute form into a chronic form
(e.g., hepatitis B infection). Finally, the infection can progress to a carrier state where the
patient is asymptomatic but continues to transmit the infecting microorganism.

4. Period of decline - corresponds to what is known as the period of defervescence. During

this period, the signs and symptoms of the patient start to subside. Body temperature may
return to normal and the feeling of weakness may diminish. However, it is also during this
period that the patient becomes vulnerable to secondary infections.

5. Period of convalescence - this period is marked by recovery of the patient from the
disease. The patient regains strength and the body returns to its pre-diseased, normal
condition.

Robert Koch was the first to scientifically prove that a specific infectious disease is caused by
a specific infectious agent. Together with some colleagues, they experimentally provided
evidence for this in what is now known as Koch's postulates.

- The chain of infection involves the following elements--reservoir, portal of exit, mode of

transmission, and host.

- The reservoir is the site where the organism resides and multiplies. It provides an environment
conducive to the growth and replication of the organism. Reservoirs provide continual source
of the infectious agent and may be humans, animals, or the environment.

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- Portal of exit refers to where the organism exits from its reservoir. For example, organisms
that have the gastrointestinal tract as their reservoir will exit through the feces.

• Mode of transmission refers to how the organism is spread. It is generally classified into direct
contact transmission and indirect contact transmission.

The major routes of direct contact transmission are through contact and through droplet
transmission. person-to-person

- Indirect transmission includes airborne transmission, vector transmission, and vehicle

transmission.

- The final link in the chain of infection is a susceptible host. Development of infection in the
host is affected by several factors such as genetic constitution of the host, the nature of the
organism, and the immune status of the host. The most important among these factors is the
defensive powers of the host.

- Bacteria produce disease in three ways--mechanical (invasion), chemical (toxin production),

and immunological. Invasion involves adhesion to mucosal surfaces and penetration into
deeper tissues.

It is facilitated by structures found on the external surface of certain bacteria (e.g., pili in gram-
negative bacteria), as well as extracellular substances secreted by the bacteria to promote
adhesion (adhesins).

Bacteria produce toxins that may cause damage to the cells or affect the normal physiologic
function of the susceptible host. These are generally exotoxins and endotoxins. classified into

Endotoxins are components of the outer membrane of gram-negative bacteria. These are
mainly lipopolysaccharides composed of a lipid. A component and polysaccharides. The
endotoxin activity is attributed to the lipid A component.

Exotoxins are substances which are secreted by bacteria. These are mainly produced by gram-
positive bacteria but may also be produced by some gram-negative bacteria.

some infections caused by microorganisms, the damage to the host tissues is not a direct
effect of the infecting agent but is a consequence of the body's immune response to the
organism. An example is damage to the liver seen in patients with hepatitis due to infection
with the hepatitis viruses.

• Infectious diseases are classified based on the following: (1) how they behave within a host
and within a given population; (2) the source of the microorganism; (3) the occurrence of a
disease; (4) the severity or duration of a disease; and (5) the extent of host involvement. An
infectious disease may be divided into five stages: (1) incubation period, (2) prodromal period,
(3) period of illness, (4) period of decline, and (5) period of convalescence.

Incubation period corresponds to the time from initial entry of the infectious agent until the time
the patient first manifests signs and symptoms. Prodromal period corresponds to the initial
manifestations of the patient. These manifestations are usually non-specific constitutional
symptoms such as fever, body, malaise cough and cold.