Microbial Ecology and Classification In 1968 the Kingdom Procaryotae was

Phylogeny: The Study of Evolutionary accepted by biologists.

Relationships of Living Organisms
• Over 1.5 million different organisms
have been identified to date.
Many similarities among
organisms:
• Made up of cells surrounded by a
plasma membrane.
• Use ATP as energy source.
• Store genetic information as DNA.
• Ribosomes are the site of protein
synthesis.
In 1969 Robert Whittaker proposed a five-
kingdom system of biological classification for
all living organisms.
living
Five-Kingdom System of Biological
Classification
1. Kingdom Prokaryotae (Monera)
- Oldest known cells.
- Lived over 3.5 billion years ago.
- Lack a nucleus and membrane
bound organelles.

Both differences and similarities among The other four kingdoms are eukaryotes.
organisms are caused by natural selection Have a true nucleus and membrane bound
(Darwin, 1858). organelles.

Organisms can be classified into taxonomic 2. Kingdom Protista

categories (taxa), based on the differences and - Mostly unicellular, lack tissue
similarities among them. organization.
- Most have flagella during life.
Systematics is the discipline of identifying and
classifying organisms. 3. Kingdom Fungi
- May be unicellular (yeasts) or
Ancient Greeks classified all living organisms
multicellular (molds). Many are
into two groups:
saprotrophs.
1. Kingdom Plantae
2. Kingdom Animalia
4. Kingdom Plantae
- Multicellular, photosynthetic.
In 1850s bacteria and fungi were incorrectly
placed in the Plant Kingdom.
5. Kingdom Animalia
- Multicellular, heterotrophs that ingest
In 1860s Kingdom Protista was proposed to
include bacteria, fungi, algae, and protozoa, food through a mouth or oral cavity
but many scientists still classified .
bacteria and fungi as plants. Five-Kingdom Classification System
Domains
Intense disagreement over classification of • The highest – largest category, recent
bacteria and fungi persisted over 100 years. addition
• 3 domains:
In 1930s electron microscopy made it clear that 1. Archaea
bacterial cells lacked a nucleus. The term o ancient “bacteria”, unicellular
procaryote was introduced in 1937. like bacteria, also simple cell
structure (prokaryote) but
In 1959 Kingdom Fungi was established. have distinct metabolism
(chemistry) allowing them to
In 1961 the current definition of the term exist in “extreme”
procaryote was established. environments
 2. Bacteria Categories of Classification
o unicellular, prokaryote, found Domain
everywhere (Old kingdom Kingdom Most inclusive
name – Monera) Phylum
Class
3. Eukarya Order
o unicellular to multicellular, Family
complex and organized cells Genus Least inclusive
with nuclei and organelles Species
(e.g. mitochondria)
Did King Philip Came Over For Good
Domain Archaea Spaghetti?
• Archaea are singlecelled organisms that
lack a membrane-bound nucleus Naming and classifying microorganisms
(Prokaryote) • Carolus Linnaeus established the
• Archaea can be found in environments system of scientific nomenclature in
that are too hostile for other life forms. 1739.
• Each organism has two names →
Domain Bacteria Binomial nomenclature: Genus +
• Bacteria are single- celled organisms specific epithet (species)
that lack a membrane-bound nucleus • Italicized (or underlined), genus
(Prokaryote) capitalized, “latinized”, used worldwide.
• Bacteria are found almost everywhere • May be descriptive or honor a scientist.
on the planet Earth.
Examples
Domain Eukarya • Staphylococcus aureus (S. aureus) -
• The cells of all eukaryotes have a Describes the clustered arrangement of
membrane-bound nucleus. Members of the cells (staphylo-) and the golden color
the Domain Eukarya are further of the colonies (aur-).
categorized into one of four Kingdoms.
• Escherichia coli (E. coli)- Honors the
Three Domain Classification discoverer, Theodor Escherich, and
1. Bacteria describes the bacterium’s habitat–the
2. Archaea large intestine or colon.
3. Eukarya
a. Protista Types of Microorganisms
b. Fungi • Bacteria
c. Plantae • Archaea
d. Animalia • Fungi
• Protozoa
Microbiology • Algae
• Older 5 kingdom scheme is still widely used • Viruses
1. Monera – bacteria (Prokaryotic) • Multicellular animal parasites
2. Protista – Protozoans (Eukaryotic) • Prions
3. Fungi - yeast, molds, etc. (Eukaryotic)
4. Plant – photosynthetic producers 1. Bacterium / Bacteria
(Eukaryotic) - Prokaryotic
5. Animals – heterotrophic consumers - Peptidoglycan cell wall
(Eukaryotic) - Reproduction by binary fission
 - Gain energy from use of organic - Helminths are parasitic flatworms
chemicals, inorganic chemicals, or and round worms
photosynthesis - Microscopic stages in life cycles
- Reproduce sexually (individuals can
either be monoecious/hermaphrodite
2. Archaea or dioecious)
- Prokaryotic
- No peptidoglycan 7. Prions
- Live in extreme environments - derived from the initial letters of the
- Reproduce asexually by binary or words proteinaceous and
multiple fission, fragmentation, or infectious, with -on added by
budding analogy to the word virion
- Include: Methanogens, Extreme - Small infectious proteins that cause
halophiles, Extreme fatal neurologic diseases in animals;
thermophiles/psychrophiles contains no nucleic acid, does not
trigger an immune response, and is
3. Fungus/Fungi not destroyed by extreme heat or
- Eukaryotic cold
- Chitin cell walls - examples: Scrapie, Bovine
- Use organic chemicals for energy. Spongiform Encephalopathy (“Mad
- Molds and mushrooms are Cow Disease”) and Creutzfeldt-
multicellular consisting of masses of Jacob disease
mycelia, which are composed of
filaments called hyphae. Microbes and Human Disease
- Yeasts are unicellular.
- Reproduce asexually by Most microbes are either beneficial or harmless
to humans – Normal microbiota (flora) in and
fragmentation, budding, or producing
on the human body
spores
Less than 1% of microbes cause disease.
4. Protozoan/ Protozoa
Pathogens overcome the host’s resistance →
- Eukaryotes
infectious disease
- Absorb or ingest organic chemicals
- May be motile via pseudopods, cilia, In 1962, the surgeon general of the United
or flagella States stated: “The war against infectious
- Reproduce both asexually through diseases has been won”.
mitosis and sexually through meiosis
and gamete fusion Today it is clear that this was overly optimistic:
Emerging diseases: New diseases like AIDS,
5. Viruses hantavirus, Ebola fever, Lyme disease,
- Are acellular Hepatitis C, and others that did not exist a few
- Have either DNA or RNA in core years ago.
- Core is surrounded by a protein coat. Antibiotic and Drug Resistance: Many old
- Coat may be enclosed in a lipid diseases are becoming resistant to traditional
envelope. therapies: Tuberculosis, gonorrhea, malaria,
- Viruses only replicate within a living etc.
host cell.
Today infectious diseases cause 50% of the 52
6. Multicellular Animal Parasites million worldwide deaths per year.
- Eukaryotes
- Multicellular animals 1. Avian influenza A
 • Influenza A virus (H5N1) - Several devastating epidemics
• Primarily in waterfowl and poultry throughout history.
• Sustained human-to-human - High mortality: Up to 80% of those
transmission has not occurred yet infected die.
- 1347-1351: Over 75 million died in
2. West Nile Encephalitis Europe, Asia, and Africa.
• Caused by West Nile virus - Over 25% of population of Europe
• First diagnosed in the West Nile died.
region of Uganda in 1937 - Cause was unknown for over 500
• Appeared in New York City in 1999 years, leading to superstition,
3. MRSA (Methicillin-resistant persecution, and hysteria.
Staphylococcus aureus) - Bacterial disease transmitted by rat
• 1950s: Penicillin resistance
fleas.
developed
- Rare today but still occurs:
• 1980s: Methicillin resistance
o 10-15 cases/year in U.S.
• 1990s: MRSA resistance to
vancomycin reported o Last epidemic occurred in India in
• VISA: Vancomycin-intermediate- 1994.
resistant S. aureus - Left: Swollen lymph nodes in bubonic
• VRSA: Vancomycin-resistant S. plague infection.
aureus - Right: Infected flea bite with eschar and
carbuncle.
4. Bovine Spongiform Encephalopathy
• Caused by a prion 2. Smallpox
• Also causes Creutzfeldt-Jakob - One of deadliest human infectious
disease (CJD). New variant CJD in diseases throughout history.
humans is related to beef - Caused by smallpox virus.
consumption - First known case in 1175 B.C.:
• Escherichia coli O157:H7 – Toxin- Egyptian pharaoh Ramses V died
producing strain of E. coli from smallpox.
• First seen in 1982 - Several hundred million deaths
• Leading cause of diarrhea worldwide through history.
- Up to 90% of Native American
5. Acquired immunodeficiency population was killed by smallpox
syndrome (AIDS) and other diseases (measles and
• Caused by human immunodeficiency plague) introduced during European
virus (HIV) conquests.
• First identified in 1981 - Native population of Central and
• Worldwide epidemic infecting 30 South America dropped from 130
million people; 14,000 new infections million to about 1.6 million over
every day several decades.
• Sexually transmitted infection - Smallpox was used as a biological
affecting males and females weapon by British colonists in North
• HIV/AIDS in the U.S.: 30% are America.
female, and 75% are African - 600,000 deaths/year in Europe from
American
1500-1700.
• Neonatal tetanus kills over 600,000
- Smallpox infection in a small child.
infants every year.
- Disease was eradicated worldwide
Microbes and Disease in Human History by immunization in 1977.
1. Bubonic Plague (Black death) - 75% of survivors were severely
scarred and/or blinded.
 - An effective vaccine was developed
in 1870s by Edward Jenner, using a
related virus (cowpox).
- Smallpox was the first and only viral
disease to be completely eradicated
(1977).
- Worldwide immunization campaign in
1960s.
- Only infects humans.
3. Tuberculosis (TB)
- Caused by a bacterium that mainly
infects lungs but may spread to other
parts of body.
- Leading killer of world’s infectious
diseases:
- 3 million die worldwide every year.
- Over 1 million killed in U.S. between
1930-49.
- One out of three people infected
worldwide.
- In U.S. 10 million people are
presently infected, but only 5% will
develop active disease.
- Most healthy individuals can contain
infection.
- Treatment: Antibiotics for up to one
year.
- After introduction of antibiotics, TB
declined from 1950s to 80s, and then
started to increase again.
- Low patient compliance with
treatment has caused antibiotic
resistant TB.
- AIDS epidemic has caused an
increase in cases.
- Tuberculosis is leading killer among
infectious diseases worldwide.
Patient with lymph node necrosis.
- Extremely common before the
1900s.
- About 1 in 17 women who gave birth
would become infected (fever, chills,
delirium, and death).
- Cause was unknown.
- Austrian doctor Semmelweiss
showed that washing hands and
instruments with a disinfectant
solution greatly reduced case.
- Today common in women who have
illegal abortions, especially in third
world countries.
5. AIDS: Acquired Immune Deficiency
Syndrome.
- First cases reported in 1981 at
UCLA.
- Cause: Human Immunodeficiency
Virus (HIV)
- Transmitted by sexual contact, blood
transfusions, mother-to-child, and
infected needles.
- Destroys an individual’s immune
system, making them susceptible
- to many infectious diseases and
cancer.
- Number of cases has grown rapidly
during the last two decades.
- As of 2001: Over 900,000 infected
individuals in the U.S. Over 40
million deaths worldwide

4. Childbirth Fever
- Common nosocomial (hospital
acquired) infection.
- Bacterial infection of the uterus as a
result of childbirth or abortion.
- Transmitted by hands and
instruments of physicians and
midwives.
Microbial Cell Structure and Function
Cells of Bacteria and Archaea
Cell Morphology
• Morphology: cell shape
• Major morphologies of prokaryotic cells:
 Coccus (pl. cocci): spherical or ovoid
 Bacillus (pl. bacilli): rodlike
 Spirillum: curved, spiral, corkscrew
shape
 Vibrio: comma shape
 Spirochete: flexible, wavy shape
 Pleomorphic: variety of shapes and
sizes
 Some stay grouped/clustered after cell
division in characteristic arrangements
o Diplococcus: pair of cocci
o Streptococcus: chain of cocci
o Tetracoccus: packets of 4 cocci
o Staphylococcus: grapelike
clusters of cocci
o Streptobacilli: long chains of
bacilli
• Cells with unusual shapes – appendaged
bacteria, and filamentous bacteria
• Many variations on basic morphological
types known
Cell Arrangement
• Morphology typically does not predict
physiology, ecology, phylogeny, or other
properties of a prokaryotic cell.
• May be selective forces involved in
setting the morphology optimization for
nutrient uptake (small cells and high
surface-to-volume ratio, such as
appendaged cells)
• swimming motility in viscous
environments or near surfaces (helical
or spiral-shaped cells)
• gliding motility (filamentous bacteria)
Bacterial cell size
• Size range for prokaryotes: 0.2 µm to
>700 µm in diameter
• Most cultured rod-shaped bacteria are
between 0.5 and 4.0 µm wide and <15
µm long.
• examples of very large prokaryotes:
o Epulopiscium fishelsoni
o Thiomargarita namibiensis
• Size range for eukaryotic cells: 2 to
>600 µm in diameter
• Surface-to-volume ratios, growth rates,
and evolution
• advantages to being small:
o more surface area relative to cell
volume than large cells (i.e., higher
S/V ratio).
o support greater nutrient and waste
product exchange per unit cell
volume
o tend to grow faster than larger cells
o Mutations lead to faster evolution.
o Eukaryotic cells adapt slower.
• Lower limits of cell size
o Cellular organisms <0.15 µm in
diameter are unlikely.
(need volume to house proteins,
nucleic acids, ribosomes, and so on)
o Open oceans tend to contain small
cells (0.2–0.4 µm in diameter)
known as “ultramicrobacteria.”
The Cell Membrane and Wall
The Cell/Plasma Membrane
 Surrounds cytoplasm
 Separates it from environment
 Main function: selective permeability
(nutrients transported in and waste
products out)
1. Bacterial cytoplasmic membrane
- General structure is phospholipid
bilayer containing embedded proteins.
- contain both hydrophobic (water-
repelling) and hydrophilic
(waterattracting) components
 hydrophobic = fatty acids
 hydrophilic = glycerol + phosphate
and another functional group (e.g.,
sugars, ethanolamine, choline)
- Fatty acids point inward to form
hydrophobic environment; hydrophilic
- portions remain exposed to external
environment or the cytoplasm.
- 8–10 nm wide
- some species strengthened by
hopanoids (sterol-like molecules)
- embedded proteins including integral
membrane proteins (significantly
embedded) or peripheral membrane
proteins (loosely attached)
Prokaryote cell membrane
• has respiratory enzymes
• assist DNA replication
• has attachment points for bacterial
flagella
2. Archaeal membranes
- ether linkages in phospholipids of
Archaea in contrast to Bacteria and
Eukarya that have ester linkages in
phospholipids
- Archaeal lipids have isoprenes
instead of fatty acids.
- Major lipids are phosphoglycerol
diethers with phytanyl C20 side chains
and diphosphoglycerol tetraethers with
biphytanyl C40 side chains, which can
form lipid monolayers.
 Cytoplasmic membrane function
o permeability barrier
- Polar and charged molecules
must be transported.
- Transport proteins accumulate
solutes against the concentration
gradient.
o protein anchor
- holds transport proteins in place
o energy conservation and
consumption
- generation of proton motive force
Bacterial Cell Walls: Peptidoglycan
• Species of Bacteria separated into two
groups based on Gram stain
• Gram-positives and gram-negatives have
different cell wall structures.
1. gram-negative cell wall (also called
cell envelope) at least two layers: LPS
and peptidoglycan
2. gram-positive cell wall – thicker,
primarily one layer of peptidoglycan
• Structure of Peptidoglycan
 rigid layer that provides strength
 typically composed of alternating
modified glucose (N-acetylglucosamine
and N-acetylmuramic acid) in β-1,4
linkages amino acids L-alanine, D-
alanine, D-glutamic acid, and either L-
lysine or diaminopimelic acid (DAP)
 cross-linked differently in gram-negative
bacteria and gram-positive
 bacteria (often “interbridges”) can be
destroyed by lysozyme (enzymes that
cleave glycosidic bond between sugars)
 found in human secretions, major
defense against bacterial infection
 100+ distinct peptidoglycans have been
described
• Overview of the gram-positive cell wall
 up to 90 percent peptidoglycan
 common to have teichoic acids (acidic
substances) covalently bound to
peptidoglycan
o bind divalent metal ions (e.g., Ca+2
and Mg+2) prior to transport
 o lipoteichoic acids: teichoic acids - Alcohol dissolves outer
covalently bound to membrane lipids membrane and leaves holes
in peptidoglycan.
• LPS: The Outer Membrane - CV-I washes out
 Small amount of total cell wall
contains peptidoglycan. • Few prokaryotes lack cell walls
 Most of cell wall composed of outer o Mycoplasmas – group of pathogenic
membrane or the bacteria related to gram-positives
lipopolysaccharide (LPS) layer. o Thermoplasma – Archaea
 barrier against antibiotics and other o have tough cytoplasmic membranes
harmful agents (e.g., sterols or lipoglycans)
 LPS consists of core polysaccharide,
O-polysaccharide, and lipid A. Bacteria with No Cell Wall: Mycoplasmas
 LPS replaces most of phospholipids • Instead, have cell membrane which
in outer half of outer membrane. incorporates cholesterol compounds
 Lipid A of LPS acts as endotoxin; O (sterols), similar to eukaryotic cells
polysaccharides are antigens for • Cannot be detected by typical light
typing, e.g., E. coli O157:H7 microscopy
a. Periplasm: space located Acid-fast Cell Walls
between cytoplasmic and outer • Genus Mycobacterium and Nocardia
membranes (periplasmic space) - mycolic acid (waxy lipid) covers thin
- 15 nm wide peptidoglycan layer
- houses many extracellular - Do not stain well with Gram stain →
proteins use acid-fast stain
b. Porins: transmembrane protein Damage to Cell Wall
channels for entrance and exit of • Lysozyme digests disaccharide in
solutes peptidoglycan.
• Penicillin inhibits peptide bridges in
• Gram + Cell Wall peptidoglycan.
 Thick layer of peptidoglycan
 Negatively charged teichoic acid on Archaeal Cell Walls
surface • No peptidoglycan
• Pseudomurein
• Gram – Cell Wall o found in cell walls of certain
 Thin peptidoglycan methanogenic Archaea
 Outer membrane o polysaccharide similar to
 Periplasmic space
peptidoglycan
o composed of N-acetylglucosamine
• Gram Stain Mechanism
(in peptidoglycan) and
 Crystal violet-iodine crystals form in
Nacetyltalosaminuronic acid
cell.
(different, replaces N-acetylmuramic
a. Gram-positive
acid)
- Alcohol dehydrates
o β-1,3 glycosidic bonds instead of β-
peptidoglycan
1,4
- CV-I crystals do not leave
o amino acids all L-stereoisomer
o cannot be destroyed by lysozyme
b. Gram-negative
and penicillin
 • Cell walls of some Archaea lack
pseudomurein.
o contain other polysaccharide
polymers
• S-Layers
o most common cell wall type
o consist of protein or glycoprotein
o paracrystalline structure
o in many organisms, S-layers present
in addition to other cell wall
o components, usually
polysaccharides
o always outermost layer
Inside the Bacterial Cell
Cell cytoplasm
• Dense gelatinous solution of sugars,
amino acids, and salts
• 70-80% water (Why is this important to
know?)
• Serves as solvent for materials used in
all cell functions
Nucleoid
1. Chromosome
• Single, circular, double-stranded DNA
molecule that contains all the genetic
information required by a cell
2. Plasmids
• Free small circular, double-stranded
DNA
• Not essential to bacterial growth and
metabolism
• Used in genetic engineering - readily
manipulated and transferred from cell to
cell
Bacterial Ribosome
1. Ribosomes
• Made of 60% ribosomal RNA and 40%
protein
• Consist of two subunits: large and
small
• Prokaryotic differ from eukaryotic
ribosomes in size and number of
proteins
• Site of protein synthesis
Bacterial Internal Structures
1. Cytoskeleton
• Many bacteria possess an internal
network of protein polymers that is
closely associated with the cell wall
(Actin filaments)
Cell Surface Structures and Inclusions
Cell Surface Structures
1. Glycocalyx
• thick, gummy material; reservoir for
nutrients and protects from changes in
the environment
• layer of polysaccharides and proteins
• Capsules and slime layers
o not considered part of cell wall
because these do not confer
significant structural strength
o polysaccharide layers (may be
thick or thin, rigid or flexible)
a. capsule: if tightly attached,
tight matrix; visible if treated
with India ink
b. slime layer: loosely attached,
easily deformed (e.g.,
Leuconostoc)
o assist in attachment to surfaces
o role in development and
maintenance of biofilms
o virulence factors: protect against
phagocytosis
o prevent dehydration/desiccation
2. Fimbriae and pili
• filamentous protein structures; 2–10 nm
wide
• Fimbriae enable organisms to stick to
surfaces or form pellicles (thin sheets of
cells on a liquid surface).
• Pili are typically longer, and fewer (1 or
a few) found per cell than fimbriae; for
attachments to surfaces
a. Conjugative/sex pili facilitate
genetic exchange between cells
(conjugation).
b. Type IV pili adhere to host tissues
and support twitching motility (e.g.,
Pseudomonas and Moraxella).
3. Hamus/hami
• Archaeal “grappling hooks” assist in
surface attachment, forming biofilms.
• structurally resemble type IV pili except
for barbed terminus, which attaches
cells to surfaces or each other
Cell Inclusions
• Inclusions function as energy reserves,
carbon reservoirs, and/or have special
functions.
• Enclosed by thin membrane
• Reduces osmotic stress
• Carbon storage polymers
 poly-β-hydroxybutyric acid (PHB): lipid
polymer
 glycogen: glucose polymer
1. Polyphosphate granules: inorganic
phosphate
2. Sulfur globules: elemental sulfur
found in periplasm, oxidized to sulfate
(SO42–)
3. Carbonate minerals:
biomineralization of barium, strontium,
and magnesium
4. Magnetosomes: magnetic iron
oxides; allow cell to undergo
o magnetotaxis: migration along
magnetic field lines
Endospores
• Formed during endosporulation or
sporulation
• Highly differentiated cells resistant to
heat, harsh chemicals, and radiation
• Survival structures to endure
unfavorable growth conditions
• “Dormant” stage of bacterial life cycle
• Ideal for dispersal via wind, water, or
animal gut
• Present only in some gram-positive
bacteria, (e.g., Bacillus and Clostridium)
Formation and germination
 vegetative cell converted to nongrowing,
heat-resistant, light-refractive structure
 only occurs when growth ceases due to
lack of essential nutrient such as carbon
or nitrogen
• can remain dormant for years but
converts rapidly back to being
vegetative
Three Steps:
1. activation: heated for several
minutes at elevated but sub-lethal
temperature
2. germination: rapid (minutes), loss of
refractility and loss of resistance to
heat and chemicals
3. outgrowth: swelling from water
uptake and synthesis of RNA,
proteins, and DNA
Structure and Features
• many layers: exosporium
(outermost), spore coats, cortex,
core
• contains dipicolinicacid
• enriched in Ca2+
• Core contains small acid-soluble
spore proteins (SASP), which
bind and protect DNA and
function as carbon and energy
source for outgrowth.
Cell Locomotion
Flagella, Archaella, and Swimming Motility
1. Flagella/archaella: structure that assists
in swimming in Bacteria and Archaea,
respectively
• tiny rotating machines
Flagellar and flagellation
• long, thin appendages
• different arrangements
Flagellar Arrangements
1. Monotrichous – single flagellum at
one end
2. Lophotrichous – small bunches
emerging from the same site
3. Amphitrichous – flagella at both
ends of cell
4. Peritrichous – flagella dispersed
over surface of cell
Chemotaxis and Other Taxes
• Taxis: directed movement in response
to chemical or physical gradients
 chemotaxis: response to
chemicals
 phototaxis: response to light
 aerotaxis: response to oxygen
 osmotaxis: response to ionic
strength
 hydrotaxis: response to water
 • Chemotaxis • results in four haploid (one
- best studied in E. coli (peritrichous) copy of each chromosome)
- “run and tumble” behavior gametes
o run: smooth forward motion,
flagellar motor rotates Mitochondria and Chloroplasts
counterclockwise - All specialize in energy metabolism.
o tumble: stops and jiggles,
flagellar motor rotates clockwise, 2. Mitochondria
flagellar bundle comes apar - respiration and oxidative
- move by rotation phosphorylation for aerobic
- Bacteria respond to temporal, not eukaryotes
spatial, difference in chemical - few—1000+ per cell
concentration. - surrounded by two membranes
- monitor/sample environment with  cristae: folded internal
chemoreceptors that sense membranes – contain enzymes
attractants and repellents needed for respiration and ATP
production
Eukaryotic Microbial Cells  matrix: innermost area of
The Nucleus and Cell Division mitochondrion – contains citric
Eukaryotes acid enzymes
• contain a membrane-enclosed nucleus
• Other organelles include mitochondria, 3. Chloroplasts
Golgi complex, lysosomes, endoplasmic - chlorophyll-containing organelle
reticula, microtubules, and found in phototrophic eukaryotes
microfilaments. - relatively large; number of
• Some have motility (flagella or cilia). chloroplasts vary
• Some have cell walls. - double membrane
• Membranes contain sterols that lend - Inner membrane surrounds stroma,
structural strength. which contains large amounts of
RubisCO (key for Calvin cycle that
1. Nucleus converts CO2 to organics).
- contains the chromosomes - thylakoids: flattened membrane
- DNA is wound around histones. discs contain chlorophyll and ATP
- enclosed by two membranes that synthetic components, form proton
interact with nucleoplasm (inner motive force
membrane) and cytoplasm (outer
membrane) 4. Organelles and Endosymbiosis
- Within the nucleus is the nucleolus - Endosymbiotic hypothesis:
(site of ribosomal RNA synthesis) Mitochondria and chloroplasts
descended from respiratory and
Cell division phototrophic bacterial cells,
a. mitosis respectively, associating with
• normal form of nuclear nonphototrophic eukaryal hosts.
division in eukaryotic cells - Free-living symbionts became part of
• results in two diploid (two eukaryotic cell.
copies of each chromosome) - Evidence: mitochondria,
daughter cells hydrogenosomes, chloroplasts
contain circular DNA genomes and
b. meiosis ribosomes.
• specialized form of nuclear
division
Other Eukaryotic Cell Structures
1. Endoplasmic reticulum (ER)
• network of membranes continuous
with nuclear membrane
• two types (rough and smooth)
• Rough contains attached ribosomes;
smooth does not.
 Smooth ER participates in the
synthesis of lipids and
carbohydrate metabolism.
 Rough ER is a major producer of
glycoproteins and new
membrane material.
2. Golgi complex: stacks of cisternae
(membrane-bound sacs) functioning in
concert with the ER
 modifies products of the ER destined
for secretion
5. Flagella and cilia
• organelles of motility that allow cells
to move by swimming
• Cilia are short flagella.
• structurally distinct from prokaryotic
flagella and do not rotate; instead
whip (flagella) or beat in synchrony
(cilia)
• bundle of nine pairs of microtubules
surrounding a central pair of
microtubules
• Dynein is attached to the
microtubules and uses ATP to drive
motility

3. Lysosomes
• membrane-enclosed compartments
• contain digestive enzymes used for
hydrolysis of food
• degrade and recycle damaged cell
components
• separate lytic activity from cytoplasm

4. Cytoskeleton
• internal structural support
a. microtubules
- 25 nm in diameter; composed
of α- and β-tubulin
- maintain cell shape and
motility; move chromosomes
and organelles

b. microfilaments
- seven nm in diameter;
polymers of actin
- maintain cell shape; involved
in amoeboid motility and cell
division

c. intermediate filaments
- 8–12 nm in diameter; keratin
proteins
- maintain cell shape and
position organelles
Microbial Diversity
 Algae
• Characteristics and Classification
- Algae are photosynthetic, eucaryotic
organisms.
- All algal cells consist of cytoplasm, a
cell wall (usually), a cell membrane,
a nucleus, plastids, ribosomes,
mitochondria, and Golgi bodies.
o Some have a pellicle, a stigma,
and/or flagella
- Algae range in size from unicellular
microorganisms (e.g., diatoms) to
large, multi-cellular organisms (e.g.,
seaweeds or kelp).
- Algae produce energy by
photosynthesis.
o Some may use organic
nutrients.
- Algae may be arranged in colonies
or strands and are found in fresh and
salt water, in wet soil, and on wet
rock
- Most algal cell walls contain
cellulose.
- Depending on their photosynthetic
pigments, algae are classified as
green, golden, brown, or red
algae.
- Algae include: diatoms,
dinoflagellates, desmids, Spirogyra,
Chlamydomonas, Volvox, and
Euglena.
- Algae are an important source of
food, iodine, fertilizers, emulsifiers,
and stabilizers and gelling agents for
jams and culture media.
• Common Pond Water Algae and
Protozoa
 Amoeba sp. (protozoa)
 Euglena sp. (algae)
 Stentor sp. (protozoa)
 Vorticellasp. (protozoa)
 Volvox sp. (algae)
 Paramecium sp. (protozoa)
• Algae: Medical Significance
1. One genus of algae, Prototheca, is
a very rare cause of human
infections
2. Causes protothecosis
3. Algae in several other genera
secrete toxic substances called
phycotoxins
o Poisonous to humans, fish, and
other animals
o If ingested by humans, the
phycotoxins produced by the
dinoflagellates that cause “red tides”
can lead to a disease called
paralytic shellfish poisoning
 Protozoa
• Characteristics
- Protozoa are nonphotosynthetic,
eukaryotic organisms.
- Most protozoa are unicellular and
free-living; found in soil and water.
 Most protozoa are more animal-
like than plant-like.
 All protozoal cells possess a
variety of eukaryotic
structures/organelles.
 Protozoa cannot make their own
food; they ingest whole algae,
yeasts, bacteria, and smaller
protozoa for nutrients.
- Protozoa do not have cell walls, but
some possess a thickened cell
membrane called a “pellicle,” which
serves the same purpose –
protection.
- A typical protozoan life cycle has 2
stages – a trophozoite and a cyst.
o The trophozoite is the motile,
feeding, dividing stage.
o The cyst is the nonmotile,
dormant, survival stage.
- Some protozoa are parasites.
 Parasitic protozoa cause many
human diseases, such as
malaria, giardiasis, and
trypanosomiasis.
- Protozoa are divided into groups,
based on their method of
locomotion:
1. Amoebae move by means of
pseudopodia (“false feet”) –
 example: Entamoeba histolytica,
the cause of amebic dystentery.
2. Ciliates move by means of
hairlike cilia – example:
Balantidium coli, the cause of
balantidiasis.
3. Flagellates move by means of
whiplike flagella – example:
Giardia lamblia, the cause of
giardiasis.
4. Sporozoa have no visible means
of locomotion – example:
Plasmodium spp., which cause
malaria.
• Protozoa That Cause Human
Diseases
 B. coli trophozoite
 Giardia lamblia trophozoite.
 Fungi
• Characteristics
- The study of fungi is called
mycology; scientists who study
fungi are called mycologists.
- Fungi are found virtually everywhere.
- Some fungi are harmful, some are
beneficial.
- Fungi represent a diverse group of
eukaryotic organisms that include
yeasts, moulds, and fleshy fungi
(e.g., mushrooms).
- Fungi are the “garbage disposers” of
nature.
- Fungi are not plants – they are not
photosynthetic.
- Fungal cell walls contain a
polysaccharide called chitin.
- Some fungi are unicellular, while
others grow as filaments called
hyphae.
o Hyphae intertwine to form a
mass called a mycelium.
o Some fungi have septate
hyphae (the hyphae are
divided into cells by cross
walls or septa).
o Some fungi have aseptate
hyphae (the hyphae do not
have septa).
- Whether or not a fungus has
aseptate or septate hyphae is an
important clue to its identification.
• Reproduction
- Depending on the species, fungal
cells can reproduce by budding,
hyphal extension, or the formation of
spores.
- There are 2 general categories of
spores:
1. Sexual spores
2. Asexual spores (also called
conidia)
- Some fungi produce both asexual
and sexual spores.
- Fungal spores are very resistant
structures.
• Fungi Classification
- Classification of fungi is based
primarily on their mode of sexual
reproduction and the type of sexual
spore they produce.
- The 5 phyla of fungi are:
Zygomycotina, Chytridiomycotina,
Ascomycotina, Basidiomycotina, and
Deuteromycotina.
- Deuteromycotina or Deuteromycetes
include the medically important
moulds such as Aspergillus and
Penicillium.
o Fungi in this phylum have no
mode of sexual reproduction or
the mode of sexual reproduction
is not known.
a. Fungi: Yeasts
• Yeasts are Eukaryotic, unicellular
organisms that lack mycelia.
• Individual yeast cells, also referred to
as blastospores or blastoconidia, can
only be observed using a
microscope.
• Yeasts usually reproduce by
budding, but occasionally by a type
of spore formation.
• A string of elongated buds is known
as a pseudohypha (not Really a
hypha).
• Some yeasts produce thick-walled,
spore-like structures called
chlamydospores (or
chlamydoconidia).
 • Yeasts are found in soil and water
and on the skins of many fruits and
vegetables.
o Yeasts have been used for centuries
to make wine and beer.
o Saccharomyces cerevisiae is a yeast
used in baking.
o Candida albicans is the yeast most
frequently isolated from human
clinical specimens, and is also the
fungus most frequently isolated from
human clinical specimens.
• Yeast colonies may be difficult to
distinguish from bacterial colonies.
o A simple wet mount can be used
to differentiate yeast colonies
from bacterial colonies.
 Yeasts are larger than
bacteria and are usually oval-
shaped.
 Yeasts are often observed in
the process of budding.
 Bacteria do not bud.
b. Fungi: Moulds
- Often spelled “molds.”
- Moulds are often seen in water and
soil and growing on food.
- Moulds produce cytoplasmic
filaments called hyphae.
 Aerial hyphae extend above the
surface of whatever the mould is
growing on.
 Vegetative hyphae grow beneath
the surface.
- Reproduction is by spore formation,
either sexually or asexually, on the
aerial hyphae (also known as
reproductive hyphae).
- Moulds have great commercial
importance.
o Some produce antibiotics.
Examples: Penicillium and
Cephalosporium
o Some moulds are used to
produce large quantities of
enzymes that are used
commercially.
o The flavor of cheeses like bleu
cheese, Roquefort, camembert,
and limburger are due to moulds
that grow in them.
c. Fungi: Fleshy Fungi
- Include mushrooms, toadstools,
puffballs and bracket fungi
- Consist of a network of filaments or
strands (the mycelium) that grows in
soil or on rotting logs
- The fruiting body that grows above
the ground forms and releases
spores
- Some mushrooms are edible; some
are extremely toxic
Fungi: Medical Significance
• A variety of fungi including yeasts,
moulds, and some fleshy fungi, are of
medical, veterinary and agricultural
importance because of the diseases
they cause in humans, animals, and
plants.
• The infectious diseases of humans and
animals that are caused by moulds are
called mycoses.
• Fungal infections of humans are
categorized as superficial, cutaneous,
subcutaneous, and systemic mycoses.
1. Superficial and Cutaneous Mycoses
- Superficial mycoses are fungal
infections of the outermost areas of
the human body – hair, nails and
epidermis.
- Cutaneous mycoses are fungal
infections of the living layer of the
skin, the dermis.
o A group of moulds collectively
referred to as dermatophytes
cause tinea (“ringworm”)
infections.
“ringworm” infections have
nothing to do with worms.
o The yeast, Candida albicans,
can also cause cutaneous,
oral, and vaginal infections.
2. Subcutaneous and Systemic
Mycoses
 - Subcutaneous and systemic  In vivo, dimorphic fungi exist as
mycoses are more severe types of yeasts.
fungal infections.
- Subcutaneous mycoses are fungal - Dimorphic fungi that cause human
infections of the dermis and diseases include:
underlying tissues.  Histoplasma
o Example: Madura foot. capsulatum(histoplasmosis)
- Systemic mycoses are fungal  Sporothrix
infections of the internal organs of schenckii(sporotrichosis)
the body.  Coccidioides immitis
o Spores of some pathogenic (coccidioidomycosis)
fungi may be inhaled with dust  Blastomyces dermatitidis
from contaminated soil or (blastomycosis)
dried bird or bat feces. They
may also enter through 2. Lichens and Slime Moulds
wounds of the hands and feet. - Lichens are observed as colored,
often circular patches on tree trunks
 Examples of deep-seated pulmonary and rocks.
infections include blastomycosis, o Lichens are composed of an alga
coccidioidomycosis, cryptococcosis, and and a fungus living in a
histoplasmosis. mutualistic relationship.
 Inhalation of common bread moulds like o Lichens are classified as
Rhizopus and Mucor spp. can cause protists.
disease and even death in
immunosuppressed patients. - Slime moulds are found in soil and
 Diagnosis of mycoses is accomplished on rotting logs.
by culture techniques and o Slime moulds have both fungal
immunodiagnostic procedures. and protozoal characteristics.
o Yeasts are identified using a o Slime moulds are classified as
series of biochemical tests. protists
o Moulds are identified using a
combination of macroscopic and
microscopic observations.

1. Dimorphic Fungi
- A few fungi, including some
pathogens, can live as either yeasts
or moulds, depending on growth
conditions.
- This phenomenon is known as
dimorphism and the fungi are called
dimorphic fungi.
 When grown in vitro at body
temperature (37oC), dimorphic
fungi grow as yeasts and produce
yeast colonies.
 When grown in vitro at room
temperature (25oC), dimorphic
fungi exist as moulds, producing
mould colonies.
Microbial Nutrition
Microbial Physiology
Introduction
• Physiology is the study of the vital life
processes of organisms.
• Microbial physiology concerns the vital
life processes of microorganisms.
• Scientists can learn about human cells
by studying the nutritional needs of
bacteria, their metabolic pathways, and
why they live, grow, multiply, or die
under certain conditions.
• Bacteria, fungi, and viruses are used
extensively in genetic studies because
they produce generation after
generation so rapidly.
Growth requirements for microorganisms
• a characteristic of microorganisms is
their ability to grow and form a
population of organisms
• one of the results of microbial
metabolism is an increase in the size of
the cell
• the many requirements for successful
growth include those both chemical and
physical
 Chemical requirements - supply of
water as well as numerous other
substances including mineral elements,
growth factors, and gas, such as oxygen
 Physical requirements – certain
physical conditions that affect the type
and amount of microbial growth
Nutritional Requirements (Chemical)
• All living protoplasm contains 6 major
chemical elements: carbon, hydrogen,
oxygen, nitrogen, phosphorus, and
sulfur.
• Combinations of these and other
elements make up vital macromolecules
of life, including carbohydrates, lipids,
proteins, and nucleic acids.
• Materials that organisms are unable to
synthesize, but are required for building
macromolecules and sustaining life, are
termed essential nutrients (e.g.,
certain essential amino acids and
essential fatty acids).
• Nutrition – process by which chemical
substances (nutrients) are acquired from
the environment and used in cellular
activities
• Nutrients
- supply of monomers (or precursors
of) required by cells for growth
- essential nutrients – must be
provided to an organism
Essential Nutrients
1. Macronutrients
• nutrients required in large amounts
• play principal roles in cell structure
and metabolism
• proteins, carbohydrates
2. Micronutrients or trace elements
• nutrients required in minute amounts
• trace metals and growth factors
• involved in enzyme function and
maintenance of
• protein structure
• manganese, zinc, nickel
Nutrients
1. Organic nutrients – contain C and H
atoms and are usually the products of
living things
o methane (CH4), carbohydrates,
lipids, proteins, and nucleic acids
2. Inorganic nutrients – atom or molecule
that contains a combination of atoms other
than C and H
o metals and their salts (magnesium
sulfate, ferric nitrate, sodium
phosphate), gases (oxygen, carbon
dioxide) and water
Carbon, nitrogen, and other macronutrients
• required by ALL cells
I. carbon (C): major element in ALL
classes of macromolecules
o Typical bacterial cell is ~50% carbon
(by dry weight).
 o Most microbes (heterotrophs) use
organic carbon (sugars, proteins,
lipids, and their monomers).
o Autotrophs use carbon dioxide
(CO2).
II. nitrogen (N): for synthesis of amino
acids, proteins, nucleic acids
o Bulk of nitrogen in nature is
ammonia (NH3), nitrate (NO3-), or
nitrogen gas (N2).
o Nearly all microbes can use NH3.
o Bacteria that obtain nitrogen directly
from the atmosphere are called
nitrogen-fixing bacteria (include
species of Rhizobium and
Azotobacter, both found in the soil)
a. Obligate anaerobe – lacks the
enzymes to detoxify oxygen so
cannot survive in an oxygen
environment
b. Aerotolerant anaerobes – do not
utilize oxygen but can survive and
grow in its presence
 Capnophile – grows best at higher CO2
tensions than normally present in the
atmosphere
O2 requirements vary greatly:

III. oxygen (O) and hydrogen (H): from

water
IV. phosphorus (P)
o used by aerobic bacteria during the
o for synthesis of nucleic acids and
process of cellular respiration as a
phospholipids
final electron acceptor
o an absolute requirement for their
V. sulfur (S)
energy-yielding properties
o for synthesis of sulfur-containing
amino acids (cysteine and
Categories of Microbes: Gas Requirements
methionine)
Oxygen
o vitamins (e.g., thiamine, biotin, lipoic
• as oxygen is utilized it is transformed into
several toxic products: singlet oxygen acid)
(1O2), superoxide ion (O2-), peroxide
(H2O2), and hydroxyl radicals (OH-) VI. potassium (K)
• most cells have developed enzymes that o required by enzymes for activity in
neutralize these chemicals: superoxide protein synthesis
dismutase, catalase o for membrane function
• if a microbe is not capable of dealing with
toxic oxygen, it is forced to live in oxygen- VII. magnesium (Mg)
free habitats o stabilizes ribosomes, membranes,
and nucleic acids
 Aerobe – utilizes oxygen and can o also required by many enzymes
detoxify it
a. Obligate aerobe – cannot grow VIII. calcium (Ca)
without oxygen o stabilizer of cell walls and
b. Facultative anaerobe – utilizes endospores
oxygen but can also grow in its
absence IX. sodium (Na)
o certain types of cell transport
 Microaerophilic – requires only a small
amount of oxygen Micronutrients
I. iron (Fe), copper (Cu), zinc (Zn)
 Anaerobe – does not utilize oxygen
 o often used for the synthesis of
enzymes
o (Fe) cellular respiration (important
component of cytochrome proteins)
o trace metals – enzyme cofactors
Growth factors
• organic compounds that cannot be
synthesized by an organism because they
lack the genetic and metabolic mechanisms
to synthesize them
• organic compounds required in small
amounts by certain organisms; must be
provided as a nutrient
o examples: vitamins, essential amino
acids, purines, pyrimidines
• vitamins
o most frequently required growth factors
o most function as coenzymes
Categorizing Microorganisms According to
Their Energy and Carbon Sources
Energy source
• Phototrophs – use light
• Chemotrophs – use either inorganic or
organic chemicals
 Chemoorganotrophs – use organic
chemicals
 Chemolithotrophs – use inorganic
chemicals
o Methanogens (Archaea) - use
hydrogen gas to produce
methane and to fix carbon dioxide
o Nitrifying bacteria - produce
nitrite/nitrate from ammonia to fix
carbon dioxide
Carbon source
• Autotrophs – use carbon dioxide (CO2) as
their sole source of C
• Heterotrophs – use organic compounds
other than CO2
Energy and Carbon source
• Photoautotrophs – use light as an energy
source and CO2 as a C source
• Chemoautotrophs – use chemicals as an
energy source and CO2 as a C source
• Chemoheterotrophs use chemicals as an
energy source and organic compounds
other than CO2 as a C source
Physical requirements
1. Temperature
• affects enzyme activity and growth
• 3 cardinal temperatures
 Minimum temperature – lowest
temperature that permits a
microbe’s growth and metabolism
 Maximum temperature – highest
temperature that permits a
microbe’s growth and metabolism
 Optimum temperature –
promotes the fastest rate of
growth and metabolism
• microorganisms are classified in three
groups according to their temperature
preferences:
 psychrophilic organisms
(psychrophiles) prefer cold
temperatures of about 0°C to 20°C
 mesophilic organisms
(mesophiles) prefer temperatures
at 20°C to 40°C
 thermophilic organisms
(thermophiles) prefer
temperatures higher than 40°C
2. pH
• the extent of acidity or alkalinity of a
solution
• for most bacteria, the optimum pH is
between 6.5 and 7.5
• Categories of Bacteria
 neutrophiles – usually grow well in
the body since the pH of most
human tissue is 7.0 to 7.2
 acidophiles – certain bacteria (in
sauerkraut and yogurt), yeast and
molds prefer acidic environments of
6.0 or below
 alkalinophiles – grow at extreme
alkaline pH
3. Osmotic pressure should be ideal for
microbial growth to proceed best
• Normally, the salt concentration of
microbial cytoplasm is about 1 percent.
When the external environment also
 has a 1 percent salt concentration,
then the osmotic pressure is optimum.
• Most microbes exist under hypotonic or
isotonic conditions
 Halophiles – require a high
concentration of salt
 Osmotolerant – do not require
high concentration of solute but can
tolerate it when it occurs
 Barophiles – can survive under
extreme pressure and will rupture if
exposed to normal atmospheric
pressure
Metabolic Enzymes
• Metabolism refers to all the chemical
reactions that occur in a cell. The chemical
reactions are referred to as metabolic
reactions.
o Metabolic reactions are enhanced and
regulated by enzymes known as
metabolic enzymes.
• Biologic Catalysts
o Enzymes are biologic catalysts; they are
proteins that either cause a particular
chemical reaction to occur or accelerate
it.
o Enzymes are specific in that they only
catalyze one particular chemical
reaction.
o A particular enzyme can only exert its
effect on one particular substance,
known as the substrate for that enzyme.
o The unique 3-dimensional shape of an
enzyme enables it to fit the combining
site of the substrate like a key fit into a
lock.
o An enzyme does not become altered
during the chemical reaction it catalyzes.
(They don’t last forever, however!)
a. Endoenzymes are enzymes produced
within a cell that remain within the cell to
catalyze reactions.
Example: digestive enzymes within
phagocytes
b. Exoenzymes are produced within a cell
and then released outside of the cell to
catalyze extracellular reactions.
Examples: cellulase and pectinase,
which are secreted by saprophytic fungi
to break down cellulose and pectin,
respectively
o Hydrolases and polymerases are
examples of metabolic enzymes.
Factors That Affect the Efficiency of
Enzymes
• Many factors affect the efficiency or
effectiveness of enzymes; enzymes
function best under optimum conditions.
 pH - extreme acidity for example
 Temperature - heat can denature
enzymes by breaking bonds
 Concentration of enzyme and/or
substrate – may be too high or too low
 Inhibitors, for example heavy metals like
lead, zinc, mercury and arsenic
Metabolism
• again, metabolism refers to all of the
chemical reactions within a cell - reactions
known as metabolic reactions.
 A metabolite is any molecule that is a
nutrient, an intermediary product, or an
end product in a metabolic reaction.
Metabolic reactions fall into 2 categories:
catabolism and anabolism.
 Catabolism refers to all catabolic
reactions in a cell.
 Anabolism refers to all anabolic
reactions in a cell.
• Catabolic reactions involve the breaking
down of larger molecules into smaller ones.
o Whenever chemical bonds are broken,
energy is released. Catabolic reactions
are a cell’s major source of energy.
• Anabolic reactions involve the assembly
of smaller molecules into larger molecules,
requiring the formation of bonds. Once
formed, the bonds represent stored energy.
• Much of the energy released during - Think of nutrients as energy sources for
catabolic reactions is used to drive anabolic organisms and think of chemical bonds as
reactions. stored energy.
- Glucose, for example, can be catabolized
• Energy can be temporarily stored in high- by one of 2 common biochemical pathways:
energy bonds in special molecules, usually aerobic respiration and fermentation.
adenosine triphosphate (ATP).
o ATP molecules are the major energy- Aerobic Respiration of Glucose
storing or energy-carrying molecules in – Catabolism of glucose by aerobic
a cell. respiration occurs in 3 phases (each is a
• ATP molecules are found in all cells biochemical pathway):
because they are used to transfer 1. Glycolysis
energy from energy-yielding molecules 2. The Krebs cycle
like glucose, to energy-requiring 3. The electron transport chain
reactions.
– The 1st phase (glycolysis) is usually
• When ATP is used as an energy source, anaerobic, but the other 2 phases are
it is hydrolyzed to adenosine aerobic.
diphosphate (ADP).
1. Glycolysis (also called the glycolytic
• ADP can be used as an energy source pathway, the EmbdenMeyerhof
by hydrolysis to adenosine pathway and the Meyerhof-Parnas
monophosphate (AMP). pathway) is a 9-step biochemical
pathway. Each step requires a specific
• Energy is required not only for metabolic enzyme.
pathways, but also for growth,
reproduction, sporulation, and 2. The Krebs Cycle (also known as the
movement of the organism, as well as citric acid cycle, the tricarboxylic acid
active transport of substances across cycle (TCA cycle)):
membranes. o A biochemical pathway consisting of
8 separate reactions, each controlled
• Some organisms (e.g., marine by a different enzyme.
dinoflagellates) use energy for o Only 2 ATP molecules are produced,
bioluminescence. but a number of products (e.g.,
NADH, H+, FADH2) are formed,
• Cellular mechanisms that release small which enter the electron transport
amounts of energy as the cell needs it chain.
usually involve a sequence of catabolic o In eucaryotes, the TCA cycle and the
and anabolic reactions. electron transport chain occur in
mitochondria.
Catabolism o In procaryotes, both occur at the
- Catabolic reactions release energy (by inner surface of the cell
breaking bonds) and are a cell’s major membrane.
source of energy.
o Some energy is lost as heat in catabolic 3. The electron transport chain (also
reactions. referred to as the electron transport
- Biochemical pathways are a series of linked system or respiratory chain):
biochemical reactions occurring in a o A series of oxidation-reduction
stepwise manner, from a starting material to reactions, whereby energy is
an end product. released as electrons which are
 transferred from one compound to
another.
o Many enzymes are involved in the
electron transport chain, including
cytochrome oxidase, which transfers
electrons to oxygen (the final
acceptor).
o A large number of ATP molecules
are produced by oxidative
phosphorylation.
o Aerobic respiration is very efficient!
Fermentation of Glucose
• Fermentation reactions do not involve
oxygen. They take place in anaerobic
environments.
• There are many industrial applications of
fermentation reactions.
o First step is glycolysis (anaerobic).
o The next step is conversion of pyruvic
acid into an end product. The end
product varies from one organism to
another. Example: yeasts are used to
make wine and beer; the end product is
ethanol.
o Fermentation reactions produce very
little energy (~ 2 ATP molecules).
Oxidation-Reducton (Redox) Reactions
• Oxidation-reduction reactions are paired
reactions in which electrons are transferred
from one compound to another.
• Oxidation occurs whenever an atom, ion, or
molecule loses one or more electrons in a
reaction; in which case, the molecule is said
to be oxidized.
• The gain of one or more electrons by a
molecule is called reduction and the
molecule is said to be reduced.
• Within a cell, an oxidation reaction is always
paired with a reduction reaction; hence the
term, oxidation-reduction reaction.
• In a redox reaction, the electron donor
(compound A) is the reducing agent, and
the electron acceptor (compound B) is the
oxidizing agent.
• Many biologic oxidations are referred to as
dehydrogenation reactions because
hydrogen ions, as well as electrons, are
removed.
Anabolism
• Anabolic reactions require energy because
chemical bonds are being formed. The
energy that is required comes from
catabolic reactions, which are occurring
simultaneously.
• Anabolic reactions are also called
biosynthetic reactions.
• Biosynthesis of organic compounds
requires energy. The energy may be
obtained through photosynthesis (from light)
or chemosynthesis (from chemicals).
o Photosynthetic reactions trap the radiant
energy of light and convert it into
chemical bond energy in ATP and
carbohydrates (e.g., glucose).
Transport: Movement of Chemicals Across
the Cell Membrane
1. Passive transport – does not require
energy; substances exist in a gradient and
move from areas of higher concentration
toward areas of lower concentration
• Diffusion
• Osmosis – diffusion of water
• Facilitated diffusion – requires a
carrier
2. Active transport – requires energy and
carrier proteins; gradient independent
• Carrier-mediated active transport
• Group translocation – transported
molecule chemically altered
• Bulk transport – endocytosis,
exocytosis, pinocytosis
Diffusion – Net Movement of Molecules
Down Their Concentration Gradient
(Passive Transport)

Osmosis - Diffusion of Water (Passive

Transport)

Inset shows a close-up of As the H2O diffuses Even as the solution

the osmotic process. The into the sac, the becomes diluted,
gradient goes from the volume increases and
outer container (higher
there will still be
concentration of H2O) to forces the excess osmosis into the sac.
the sac (lower solution into the tube, Equilibrium will not
concentration of H2O). which will rise occur because the
Some water will diffuse continually. solutions can never
the opposite direction but become equal.
the net gradient favors
osmosis into the sac.
Response to solutions of different osmotic
content

Summary of Transport Processes

Facilitated Diffusion (Passive Transport)

Carrier Mediated Active Transport

Group Translocation

Endocytosis: Eating and Drinking by Cells

Endocytosis: bringing substances into the cell
through a vesicle or phagosome
o Phagocytosis ingests substances or cells
o Pinocytosis ingests liquids
Bacterial Genetics - The phage DNA integrates into the
 Genetics = the study of heredity. bacterial chromosome but does not
 An organism’s genotype is its complete cause the lytic cycle to occur – this
collection of genes. is known as lysogeny.
 An organism’s phenotype refers to its - A phage is called a prophage when
physical traits (e.g., includes hair and all that remains of it is its DNA.
eye color in humans). - The bacterial cell containing the
 An organism’s phenotype is the prophage is referred to as a
manifestation of that organism’s lysogenic cell.
genotype. - The bacterial cell exhibits new
 Genes direct all functions of the cell. properties, directed by the viral
 A particular segment of the chromosome genes – lysogenic conversion.
constitutes a gene.

 Mutations
 A change in a DNA molecule (genetic
alteration) that is transmissible to
offspring is called a mutation.
o Categories of Mutations:
1. Beneficial mutations
2. Harmful mutations (some are
lethal mutations)
3. Silent mutations
 Mutation rate (the rate at which
mutations occur) can be increased by
exposing cells to physical or chemical
agents called mutagens.
 The organism containing the mutation is
called a mutant.

 Ways in Which Bacteria Acquire New

Genetic Information  Transduction (“to carry across”):
 (i.e.,) acquire new genes: - Also involves bacteriophages.
o Lysogenic Conversion - In transduction, bacterial genetic
o Transduction material is “carried across” from one
o Transformation bacterial cell to another by a
bacterial virus; thus, in transduction,
o Conjugation
bacteria acquire new bacterial
genes.
 An extrachromosomal DNA molecule is
- differs from lysogenic conversion,
called a plasmid.
wherein bacteria acquire new genetic
o An organism that acquires a
information in the form of viral genes.
plasmid acquires new genes. - Only small amounts of genetic
o A plasmid that can either exist by material are transferred by
itself or can integrate into the transduction.
chromosome is called an
episome.

 Lysogenic Conversion
- Temperate phages (or lysogenic
phages) inject their DNA into a
bacterial cell.
  Conjugation
- Involves a specialized type of pilus
called a sex pilus.
- A bacterial cell with a sex pilus
(called the donor cell) attaches by
means of the sex pilus to another
bacterial cell (called the recipient
cell).
- Some genetic material (usually a
plasmid) is transferred through the
hollow sex pilus from the donor cell
to the recipient cell.
- A plasmid that contains multiple
genes for antibiotic resistance is
known as a resistance factor or R-
factor.
- A bacterial cell that receives a R-
factor becomes a “superbug.”

 Transformation
- A bacterial cell becomes genetically
transformed following the uptake of
DNA fragments (“naked DNA”) from
its environment.
- The ability to absorb naked DNA into
the cell is called competence and
bacteria capable of absorbing naked
DNA are said to be competent
bacteria.
- Transformation is probably not
widespread in nature.
 Genetic Engineering
 Genetic engineering or recombinant
DNA technology involves techniques to
transfer eukaryotic genes (particularly
human genes) into easily cultured cells
to manufacture important gene products
(mostly proteins).
 Plasmids are frequently used as
vehicles for inserting genes into cells.
 There are many industrial and medical
benefits from genetic engineering.
 Examples: synthesis of antibodies,
antibiotics, drugs and vaccines; also, for
synthesis of important enzymes and
hormones for treatment of diseases.

 Microbial Ecology
 Ecology is the study of the interactions
between living organisms and the world
around them.
 Ecosystem refers to the interactions
between living organisms and their
nonliving environment.
 Interrelationships among the different
nutritional types are of prime importance
in the functioning of the ecosystem.
 Example: Phototrophs, such as algae
and plants, are the producers of food
and oxygen for chemoheterotrophs,
such as animals.

 Gene Therapy
 Gene therapy of human diseases
involves the insertion of a normal gene
into cells to correct a specific genetic
disorder caused by a defective gene.
 Viral delivery is the most common
method for inserting genes into cells;
specific viruses are selected to target
the DNA of specific cells.
 Genes may someday be regularly
prescribed as “drugs” in the treatment of
diseases (e.g., autoimmune diseases,
sickle cell anemia, cancer, cystic
fibrosis, heart disease, etc.)
Control of Microbial Growth (In Vitro)
Factors that Affect Microbial Growth
The Study of Microbial Growth
 Microbial growth occurs at two levels:
1. growth at a cellular level with increase in
size
2. increase in population
• Division of bacterial cells occurs
mainly through binary fission
(transverse)
- Parent cell enlarges, duplicates its
chromosome, and forms a central
transverse septum dividing the cell into
two daughter cells
 Rate of Population Growth
• Time required for a complete fission
cycle = generation, or doubling time
• Each new fission cycle increases the
population by a factor of 2 –
exponential growth (1, 2, 4, 8, 16, 32,
64, etc)
• As long as the environment is
favorable, the doubling effect
continues at increasing rate
(number/time)
• Length of the generation time - a
measure of the growth rate of an
organism
• Average generation time - 30 to 60
minutes under optimum conditions
• Can be as short as 10 to 12 minutes
→growth pattern = exponential
Equation for calculating population size
over time: Nƒ = (Ni)2n
• Nƒ is total number of cells in the
population Ni is starting number of cells
• Exponent n denotes generation time
• 2n is the number of cells in that
generation
Bottom line – “can grow at an exponential rate”
means real fast!!
 The Population Growth Curve
• A population of bacteria does not
maintain its potential growth rate and
doubles endlessly ...
• Rate slows as generation time gets
longer, because of • reduced resources
o increased density (number/volume)
o increased waste
- K= (Kmax [S] / (Ks + [S])
• A population displays a predictable
growth pattern called “growth curve”,
where the cell # is a function of time
o N = No x e[k x t]
In laboratory studies, populations typically
display a predictable pattern over time – growth
curve.
Stages in the Normal Growth Curve
1. Lag phase – “flat” period of adjustment,
enlargement; little growth.

2. Exponential growth phase – a period of  Viable Plate Count

maximum growth will continue as long as
cells have adequate nutrients and a
favorable environment.
3. Stationary phase – rate of cell growth
equals rate of cell death caused by
depleted nutrients and O2, excretion of
organic acids and pollutants
4. Death phase – as limiting factors intensify,
cells die exponentially
 Additional Direct Measurements
1. Filtration method of choice for low
counts
2. Direct microscopic count: Counting
chambers (slides) for microscope
3. Use of the hemocytometer or Petroff-
Hausser counting chamber (direct
microscopic count)

 Direct Measurements of Microbial

 Estimating Bacterial Numbers by

Growth
Indirect Methods
• Turbidometry
Viable cell counts: Plate counts: Serial dilutions - most simple
put on plates→ CFUs form colonies - Degree of cloudiness, turbidity,
reflects the relative population
size (estimate)

 Measuring Microbial Growth

• Direct Methods
1. Plate counts
2. Filtration
3. MPN
4. Direct microscopic count

• Indirect Methods
1. Turbidity
2. Metabolic activity
3. Dry weight

Factors that Affect Microbial Growth

• Environmental Factors
• Temperature
• Gas (oxygen and CO2) •pH
• Osmotic pressure
• Other factors
• Microbial association – Temperate, subtropical,
and tropical regions
• Availability of Nutrients – Most pathogens of
- All living organisms require nutrients mammals have growth
to sustain life. optima between 30°C and
- Nutrients are energy sources. 40°C; grow best at
Organisms obtain energy by moderate temperatures
breaking chemical bonds. (e.g., 37o C)

 Moisture o Thermophile - 45 to 80 °C
- Water is essential for life. It is – A microbe that grows
needed to carry out normal metabolic optimally at temperatures
processes. greater than 45°C
- Certain microbial stages (e.g., – Vary in heat requirements
bacterial endospores and protozoal – General range of growth
cysts) can survive a drying process of 45°C to 80°C
(dessication). – Hyperthermophiles grow
between 80°C and 120°C
 Temperature
- For optimal growth and metabolism o Hyperthermophile >80 °C
o Psychrophile - 0 to 15 °C
– A microorganism that has - Pasteurization (72 °C, 15 seconds)
an optimum temperature
below 15°C and is capable • Gas
of growth at 0°C; prefer - Two gases that most affect microbial
cold temperatures (like growth:
deep ocean water) o Oxygen
– True psychrophiles are – Oxidizing agent
obligate with respect to – Respiration
cold and cannot grow o Carbon dioxide (carbon source)
above 20°C
– Psychrotrophs or  Oxidizing agent (electron
facultative psychrophiles acceptor)
grow slowly in cold but - Oxygen metabolites are toxic
have an optimum - These toxic metabolites must be
temperature above 20°C; neutralized for growth
particular group of
psychrophiles, prefer  Categories of Bacteria
refrigerator temperature 1. Obligate aerobe
(4oC) - Requires oxygen gas for metabolism
– Psychroduric organisms - prefer the same atmosphere that
prefer warm temperatures, humans do (~20-21% O2 and 78-
but can endure very cold 79% N2, other gases < 1%)
or even freezing - Possesses enzymes that can
temperatures. neutralize the toxic (active) oxygen
metabolites
o Mesophile - 20 to 40 °C - Superoxide dismutase and catalase
– An organism that grows at - Effectively sequesters Iron
intermediate temperatures - Ex. Most fungi, protozoa, and
– Optimum growth bacteria
temperature of most: 20°C
to 40°C
 2. Microaerophiles
3. Facultative anaerobe
- Does not require oxygen for
metabolism, but grows better in its
presence
- Usually possess superoxide
dismutase and catalase
- In the absence of oxygen, cells
utilize other oxidants to dispose
electrons (anaerobic respiration or
fermentation occurs)
- Ex. Gram-negative pathogens
4. Obligate (strict) anaerobe
- Does not use oxygen for metabolism
- Do not possess superoxide
dismutase and catalase
- The presence of oxygen is toxic to
the cell (H2O2, O2-, •OH)
• Carbon Dioxide
- All microbes require some carbon
dioxide in their metabolism, some
need it as their sole source of
carbon
- Capnophiles grow best at a higher
CO2 tension (5-10% CO2) than is
normally present in the atmosphere
and they REQUIRE CO2
- Capnotrophs tolerate higher CO2
concentrations
• pH
- Cells grow best in the “temperate
range” between pH 6-8
- Most microorganisms prefer a
neutral or slightly alkaline growth
medium (pH 7.0 - 7.4)
- Acidophiles prefer a pH of 2 to 5 •
Alkali[no]philes prefer a pH > 8.5
• Water availability – Osmotic pressure
and salinity
Osmotic pressure – pressure that is
exerted on a cell membrane by solutions
both inside and outside the cell
o Osmosis
o Hypertonic solution
- Plasmolysis – condition in
which the cell membrane and
cytoplasm of a cell shrink
away from the cell wall;
occurs when bacteria with
rigid cell walls are placed into
a hypertonic solution
o Hypotonic solution
- If a bacterial cell is placed into
a hypotonic solution, it may
not burst (because of the rigid
cell wall); if it does burst, the
cytoplasm escapes –
plasmoptysis
o Isotonic solution
 Changes in Osmotic Pressure
• Halophiles (salt)
- Requires high salt concentrations
- Osmophiles/halophiles withstand
hypertonic conditions
- Ex. Halobacterium
• Facultative halophiles or haloduric
organisms
- do not prefer to live in salty
environments, but which are capable of
surviving there
- do not require salt
- Ex. Staphylococcus aureus
 Other factors
• (Hydrostatic) Pressure - Barophiles
withstand high pressures
• Piezophiles – microbes that can survive
in high atmospheric pressure (> 14.7
psi)
• Radiation - pigmented or fortified walls
withstand high energy (UV), heat
(infrared)
Spores and cysts can survive dry
habitats.
 Ecological Association • The time it takes for one cell to become
• Influence microorganisms have on other two cells is called the generation time
microbes (e.g., E. coli = 20 minutes).

1. Symbiotic relationship  Culture Media

- Organisms that live in close • Media (sing., medium) are used in
nutritional relationship (syntrophy) microbiology labs to culture (i.e., grow)
bacteria; media prepared in the lab are
 Types: referred to as artificial media or synthetic
• Mutualism – both organism media.
benefit • Chemically defined medium –
• Commensalism – one ingredients are known
organisms benefits without • Culture media can be liquid or solid
disadvantage to the other
• Parasitism (host/microbe 1. Enriched medium – a broth or solid
relationship) – one organism containing a rich supply of special
benefits on the expense of the nutrients that promote the growth of
other fastidious organisms; ex. chocolate
agar
An example of commensalism, where 2. Selective medium – has added
Staphylococcus aureus provides inhibitors that discourage growth of
vitamins and amino acids to certain organisms while allowing the
Haemophilus influenzae. growth of a desired organism; ex. PEA
(phenylethyl alcohol) agar
2. Non-symbiotic relationship 3. Differential medium – permits the
- Organisms are free-living, and do not differentiation of organisms that grow
rely on each other for survival on the medium; ex. MacConkey agar

 Types: • The various categories of media are not

• Synergism – shared mutually exclusive; e.g., blood agar is
metabolism, not required enriched and differential
• Antagonism - competition
between microorganisms (to Thioglycollate broth (THIO)
each other’s detriment) - a popular liquid medium in bacteriology
labs; it supports the growth of all
Encouraging the Growth of Microbes in categories of bacteria from obligate
Vitro aerobes to obligate anaerobes.
Culturing Bacteria in the Laboratory - There is a concentration gradient of
 Bacterial Growth dissolved oxygen in the tube; organisms
• increase in the number of organisms grow only in that part of the broth where
rather than an increase in their size the oxygen concentration meets their
• Bacteria divide by binary fission (one needs.
cell divides to become two cells) when
they reach their optimum size
• Binary fission continues through many
generations until a colony is produced
on solid culture medium
• Binary fission continues for as long as
there is a sufficient supply of nutrients,
water, and space
 Inoculation of Culture Media
• Culture media are inoculated with
clinical specimens (i.e., specimens
collected from patients with a suspected
infectious disease).
• Inoculation involves adding a portion of
a specimen to the medium.
• Inoculation is accomplished using a
sterile inoculating loop.
• A spectrophotometer can be used to
determine growth by measuring the
turbidity of the medium.
• A viable plate count is used to determine
the number of viable bacteria in a liquid
sample by making serial dilutions of the
liquid and inoculating onto nutrient agar;
after overnight incubation, the number of
colonies is counted.

 Importance of Using “Aseptic  Bacterial Population Growth Curve

Technique”
• Aseptic technique is practiced when it is
necessary to exclude microbes from a
particular area (e.g., when inoculating
culture media).
• Unwanted organisms are referred to as
contaminants; the growth medium or
plate is said to be contaminated.
• The sterility of the media must be
maintained before inoculation.
o Avoid touching the surface of the
agar!
• Inoculating media within a biologic
safety cabinet minimizes contamination
and protects the laboratorian.
• A population growth curve for any
particular species of bacterium may be
determined by growing a pure culture of
the organism in a liquid medium at a
constant temperature.
o Samples of the culture are collected
at fixed intervals to determine the
number of viable organisms.
o A graph is prepared by plotting the
logarithmic number of viable
organisms (on the vertical or Y- axis)
against the incubation time (on the
horizontal or X-axis).

 Incubation
• After media are inoculated, they must be
placed into an incubator which will
maintain the appropriate atmosphere,
temperature, and moisture level;
(process = incubation)
• 3 types of incubators are used in clinical  A Chemostat is used for continuous
microbiology laboratories:
o A CO2 incubator (contains 5-10%
CO2)
o A non-CO2 incubator (contains
room air)
o An anaerobic incubator (the
atmosphere is devoid of O2)

 Bacterial Population Counts

• Microbiologists sometimes need to know
how many bacteria are present in a
particular liquid at a given time (e.g., to
determine bacterial contamination of cultures
drinking water).
o Can determine either the total
number of bacterial cells or the
number of viable (living) cells
 Culturing Obligate Intracellular
Pathogens in the Laboratory
• Obligate intracellular pathogens
- microbes that can only survive and
multiply within living cells
- called host cells
- include viruses and 2 groups of
Gram-negative bacteria – rickettsias
and chlamydias
- Culturing these organisms in the
laboratory is a challenge; they must
be grown in embryonated chicken
eggs, lab animals, or cell cultures

 Culturing Fungi in the Laboratory

• Fungi (including yeasts, moulds and
dimorphic fungi) grow on and in a
variety of solid and liquid culture media.
• There is no single medium that is best
for all medically important fungi.
• Examples of culture media for fungi
include brain heart infusion (BHI) agar,
BHI with blood, and Sabouraud
dextrose agar (SDA); due to its low pH,
SDA is selective for fungi.
• Caution must be exercised when
culturing fungi – some are highly
infectious!

 Culturing Protozoa in the Laboratory

• Most microbiology laboratories do not
culture protozoa (only some research
and reference labs)
• Examples of protozoa that can be
cultured in vitro are amebae, Giardia
lamblia, Leishmania spp., Toxoplasma
gondii, Trichomonas vaginalis and
Trypanosoma cruzi.
• Due to the severity of diseases that
they cause, it is of greatest importance
to culture amebae: Acanthamoeba spp.,
Balamuthia spp. and Naegleria fowleri.
Control of Microorganisms by Physical and
Chemical Agents • –static agents
- temporarily preventing the growth
 Definition of Terms of microbes
• Sterilization - Stasis and static: to stand still
- the complete destruction of all o Microbistatic agent – drug or
microbes, including cells, spores, chemical that inhibits growth
and viruses and reproduction of microbes
- accomplished by dry heat, o Bacteriostatic agent –
autoclaving (steam under pressure), specifically inhibits the
gas, various chemicals, and certain metabolism and reproduction of
types of radiation bacteria
o Fungistatic agent – inhibit
• Inanimate objects fungal growth
- Surgical instruments
- commercially packaged foods • Resistance (against agents)
- Highest resistance - bacterial spores
• Disinfection and prions
- the destruction or removal of - Moderate resistance - some bacteria,
vegetative pathogens from nonliving protozoan cysts, fungal sexual spores,
objects by physical or chemical naked viruses
methods; e.g., pasteurization - Least resistance - most bacteria,
- Also removes toxins fungal nonsexual spores and hyphae,
- Does not eliminate bacterial enveloped viruses, yeast, protozoan
endospores trophozoites
o Disinfectants – chemical
substances that eliminate
pathogens on inanimate objects
o Antiseptics – solutions used to
disinfect skin and other living
tissues

• –cide or –cidal
- “killing” or destroying
microorganisms
o Germicidal agents, biocidal
agents, and microbicidal
agents – chemicals that kill
microbes • Antisepsis
o Bactericidal agents – - when chemical agents (antiseptics)
chemicals that specifically kill destroy or inhibit vegetative pathogens
bacteria, but not necessarily from animate surfaces
bacterial endospores - Skin and mucous membranes
o Sporicidal agents – kill o Sepsis: the growth of
bacterial endospores microorganisms in the blood and
o Fungicidal agents – kill fungi, other tissues
including fungal spores o Asepsis: any practice that
o Algicidal agents – kill algae prevents the entry of infectious
o Viricidal agents – destroy agents into sterile tissues
viruses
 • Decontamination • Mode of action.
- Used when actual sterilization isn’t  Mode of Actions of Microbial Control
needed but need to decrease the risk Agents
of infection or spoilage (ex. food
industry) 1. Cell wall
• Agents:
o Sanitation - Penicillin (Cephalosporin),
- Any cleansing technique that Vancomycin, Bacitracin
mechanically removes - detergents, alcohols
microorganisms (or substratum
that could support their growth) to • Bacteria and Fungi
reduce contamination to safe - block synthesis
levels - degrade cellular components
- Sanitizer: compound such as - destroy or reduce stability
soap or detergent that sanitizes
- Sanitary: may not be free from 2. Cell membrane
microbes but are safe for normal • Agents:
use - Nisin, Gramicidin, Polymyxin
- Physical and chemical agents - Surfactants

o De-germination • All microbes and enveloped viruses

- Reduces the numbers of - disruption of membrane function
microbes on the human skin (ex. (pores)
alcohol wipes) - bind and penetrate lipids
- Physical and chemical agents - lose selective permeability
(leakage)
 What is Microbial Death?
- when various cell structures become
dysfunctional and the entire cell sustains
irreversible damage
- if a cell can no longer reproduce under
ideal environmental conditions
- Death begins when a certain threshold
of microbicidal agent is met, and
continues in a logarithmic manner.

 Effectiveness of Antimicrobial Treatment

depends on:
• Number of microorganisms. Time it
takes to kill a microbial population is 3. Nucleic acid synthesis
proportional to number of microbes. • Agent:
• Targets. Microbial species and life cycle - Chemical agent
phases (e.g.: bacteria, viruses, o formaldehyde; Trimethoprim,
endospores) have different Novobiocin, Nalidixic Acid,
susceptibilities to physical and chemical Rifamp(ic)in
controls. - Physical agent
• Interfering agents. Organic matter may o radiation
interfere with heat treatments and
chemical control agents. • Action:
• Exposure time: Longer exposure to - Mutations: Irreversible injury to
lower heat produces same effect as DNA
shorter time at higher heat.
 - Inhibition of nucleotide  An Overview of the Microbial Control
biosynthesis Methods
- Inhibition of DNA-gyrase
- Inhibition of DNA-polymerase
- Inhibition of RNA-polymerase

• Consequence:
- Stop of replication
- transcription and(eventually)
- translation

4. Protein synthesis
• Agents:
- Chloramphenicol, Tetracyclines*,  Physical Methods of Microbial
Aminoglycosides; Macrolides, Control
Puromycin 1. Heat – moist and dry
2. Cold temperatures
• Action: 3. Desiccation
- bind to ribosomes (30S subunit, 4. Radiation
50S subunit) 5. Filtration
- stops initiation, elongation* or
termination  Physical Methods of Microbial
- prevents peptide bond formation Control
• Heat is very effective (fast and
5. Protein function cheap).
• Agent: 1. Thermal death point (TDP):
- Physical – Heat, pH change Lowest temperature at which all
- Chemical – alcohols, acids, cells in a culture are killed in 10
phenolics, metallic ions min.
2. Thermal death time (TDT): Time
• Action: to kill all cells in a culture
- block protein active sites 3. Decimal Reduction Time (DRT):
- prevent binding to substrate Minutes to kill 90% of a population
- denature protein at a given temperature

• The effects of heat, pH, and blocking  Moist Heat Sterilization

agents on the function of proteins. • Denatures proteins, coagulates
proteins, destroys membranes and
DNA
• Autoclave: Steam under pressure
• Most dependable sterilization method
• Steam must directly contact material
to be sterilized.
• Pressurized steam reaches
higher temperatures (above 100oC).
• Normal autoclave conditions: 121.5C
for 15 min.
• Prion destruction: 132C for 4.5 hours
• Effectively destroys spores,
vegetative microorganisms, and
viruses
• Sterilizes inanimate objects
(glassware)
 • Limitations of the autoclave
o Clostidium perfringens and C.
botulinum (food poisoning) survive  Tyndallization
hours of boiling • Non-pressurized steam
o Very resistant large numbers of • Intermittent sterilization for
hepatitis A virus, fungal spores substances that cannot withstand
and protozoal cyst autoclaving
• Used for heat-sensitive media, some
 Pasteurization canned foods
• heat is applied to kill potential agents of • Will not destroy spores
infection and spoilage without • Items exposed to free-flowing steam
destroying the food flavor or value for 30–60 minutes, incubated for 23–
• significant number reduction (esp. 24 hours and then subjected to
spoilage and pathogenic organisms) steam again (repeat cycle for 3 days)
• not sterilization – kills non-spore- • Disinfection
forming pathogens and lowers overall
microbe count; does not kill endospores  Boiling Water
or many nonpathogenic microbes. • Decontaminates at 100 °C for 30 min
• Historical goal: destruction of M. • Kills most non-spore forming
tuberculosis pathogens
• Disinfection
• Classic holding method (batch method): • Examples: home sanitizing and
63C for 30 min disinfecting, disinfecting unsafe
• Flash pasteurization (HTST): 71.6C for water
15 sec. Most common in US.
• Thermoduric organisms survive  Dry heat sterilization
• Ultra-High Temperature (UHT): 140C • Dehydration, denaturation, oxidation
for < 1 sec. (burning to ashes)
- Technically not pasteurization • Flaming of loop
because it sterilizes. • Incineration of carcasses
o UHT-pasteurized milk that is o Anthrax
packaged aseptically results in a o Foot and mouth disease
"shelf stable" product that does o Bird flu
not require refrigeration until
opened • Hot-air sterilization (oven)
- Glassware is heated for 2-3
• Disinfection of beverages hours at 320°F-360°F (160°C
- Exposes beverages to 71.6 °C for -180°C)
15 seconds - Coagulates proteins
- Stops fermentation - Also used for powder, water
free oils, inanimate objects
• Prevents the transmission of milk-borne - Advantage: no dulling and
diseases corrosion
- Salmonella, Campylobacter, Listeria,
Mycobacteria Hot-air Autoclave
Equivalent
• Milk industry, wineries, breweries 170 ̊C, 2 hr 121 ̊C, 15 min
treatments

 Incineration
• Destroys microbes to ashes or gas
 •
Flame - 1870°C
•
Furnace - 800°C to 6500°C
•
An infrared incinerator uses flame
to burn or oxidize materials into
ashes.
 Comparing Moist Heat to Dry Heat
 Effects of cold and desiccation
• Cold
- reduce the activity - slows the
growth of some microbes (micro
biostatic), but NOT psychrophiles
- not a disinfection or sterilization
method
- refrigeration 0–15°C and freezing
<0°C
- used to preserve food, media, and
cultures
• Desiccation or dehydration
- kill some microorganisms
- gradual removal of water from cells,
leads to metabolic inhibition
- not effective microbial control – many
cells retain ability to grow when
water is reintroduced
- Lyophilization – freeze drying
(process that combines dehydration
(drying) and freezing); widely used in
industry to preserve foods,
antibiotics, microorganisms, and
other biologic materials
 Filtration
• Removes microbes and spores from
liquids and air
• Air filtration using high efficiency
particulate air (HEPA) filters. Effective
to 0.3 m
• Membrane filters for fluids.
• Pore size for bacteria: 0.2 – 0.4 m
• Pore size for viruses: 0.01 m
• Applications
- Liquids that are sensitive to heat:
Serum, vaccines, media
an example of a filtration system
 Radiation
• Ionizing
o Gamma rays (High energy)
o X-rays (Intermediate energy)
o Cathode rays (least energy)
• Non-ionizing
• Ultraviolet
• Radiation: Mode of action
- Ionizing radiation ejects orbital
electrons (e-) from an atom
o High energy
- deep penetrating power that
has sufficient energy to
cause e- toleave their orbit,
breaks DNA
- penetrates liquids and solids
effectively
- Non-ionizing radiation raises atoms
to a higher energy state
o Low energy
- Less penetration capability
- Formation of pyrimidine
(thymine) dimers which
interfere with replication
• The effects of ionizing and non- - Salmonella and
ionizing radiation on DNA. Pseudomonas are particularly
sensitive
- Sterilization of heat sensitive
materials: drugs, vitamins,
herbs, suture material
- Sterilization of some foods
(fruits, vegetables, meats)
- Commonly use Cobalt-60
radioisotope

o Non-ionizing radiation
- Most effective wavelength = ~
260 nm
- Alternative disinfectant
- Used to limit air and surface
contamination. Little
penetrating power must be
used at close range to directly
exposed microorganisms
• Ultraviolet (UV) radiation can cause - Germicidal lamp in hospitals,
the formation of pyrimidine dimers on schools, food preparation
DNA. areas (inanimate objects, air,
water)

• Nonionizing Radiation: Microwave

- Wavelength: 1 mm – 1m
- H2O quickly absorbs energy →
release as heat to environment
- Indirect killing of bacteria through
heat

 Chemical Methods of Microbial Control

• Disinfectants, antiseptics, sterilants,
degermers, and preservatives.
• Chemical disinfection refers to the use
of chemical agents to inhibit the growth
of pathogens, either temporarily or
permanently.
• Radiation: Applications
o Ionizing radiation  Factors that Affect Germicidal Activity of
- Alternative sterilization Chemicals
method • Prior cleaning of the object or surface
 • The organic load (e.g., feces, blood,
pus)
• The bioburden; types and numbers of
microbes
• Concentration of the disinfectant
• Contact time or time of exposure
• Physical nature of the object being
disinfected
• Temperature and pH
• Degree of contamination
• Nature of the material being treated
• Strength and chemical action of the
germicide
 Chemical Methods of Microbial Control
• Characteristics of an ideal chemical
antimicrobial agent:
1. Should have a broad
antimicrobial spectrum
2. Fast acting
3. Not affected by the presence of
organic matter
4. Nontoxic to human tissues and
noncorrosive
5. Should leave a residual
antimicrobial film on surface
6. Soluble in water and easy to
apply
7. Inexpensive and easy to prepare
8. Stable as both a concentrate and
a working solution
9. Odorless
 Levels of Chemical Decontamination
1. High-level germicides
- kill endospores; may be sterilants
- Devices that are not heat
sterilizable and intended to be used
in sterile environments (body
tissue)
2. Intermediate-level
- kill fungal spores (not endospores),
tubercle bacillus, and viruses
- Used to disinfect devices that will
come in contact with mucous
membranes but are not invasive
3. Low-level
- eliminate only vegetative bacteria,
vegetative fungal cells, and some
viruses
- Clean surfaces that touch skin but
not mucous membranes
 Types of Disinfectants
• Phenolics: Cresols (Lysol) – disinfectant
• Bisphenols
o Hexachlorophene (pHisoHex,
prescription), hospitals, surgeries,
nurseries
o Triclosan (toothpaste, antibacterial
soaps, etc.)
• Disrupt cell walls and membranes and
precipitate proteins
• Low to intermediate level – bactericidal,
fungicidal, virucidal, not sporicidal
 Halogens
• Chlorine
- Oxidizing agent
- Widely used as disinfectant
- Forms bleach (hypochlorous acid)
when added to water.
- Broad spectrum, not sporicidal
(pools, drinking water)
o Denaturate proteins by disrupting
disulfide bonds
o Intermediate level
o Unstable in sunlight, inactivated
by organic matter
o Water, sewage, wastewater,
inanimate objects
• Iodine
 - More reactive, more germicidal.
Alters protein synthesis
membranes.
o Tincture of iodine (solution with
alcohol) → wound antiseptic
o Iodophors combined with an
organic molecule → iodine
detergent complex (e.g.
Betadine®). Occasional
sensitivity, partially inactivated by
organic debris, poor sporicidal
activity.
- Interferes with disulfide bonds
of proteins
- Intermediate level
- Milder medical and dental
degerming agents,
disinfectants, ointments
• Alcohols
- Ethyl (60 – 80% solutions) and
isopropyl alcohol
- Act as surfactants dissolving
membrane lipids and coagulating
proteins of vegetative bacterial cells
and fungi
- No activity against spores and poorly
effective against viruses and fungi
- Easily inactivated by organic debris
- Also used in hand sanitizers and
cosmetics
- Intermediate level
• Hydrogen Peroxide
- Produce highly reactive hydroxyl-free
radicals that damage protein and
DNA while also decomposing to O2
and gas – toxic to anaerobes
- Antiseptic at low concentrations;
strong solutions are sporicidal
- Weak (3%) to strong (35%)
- Quick method for sterilizing medical
equipment
skin
 Heavy Metals
• Oligodynamic action: toxic effect due to
metal ions combining with sulfhydryl (—
SH) and other groups  proteins are
denatured.
o Mercury (HgCl2, Greeks & Romans
for skin lesions); Thimerosal
o Copper against chlorophyll
containing organisms → Algicides
o Silver (AgNO3): Antiseptic for eyes
of newborns
o Zinc (ZnCl2) in mouthwashes, ZnO
in antifungal in paint
Solutions of silver and mercury kill
vegetative cells in low
concentrations by inactivating
proteins
• Oligodynamic action
• Low level
• Merthiolate, silver nitrate, silver
Applications of Heavy Metals
 Surface Acting Ingredients / Surfactants
• Soaps and Detergents
- Major purpose of soap: Mechanical
removal and use as wetting agent
- Definition of Detergents
o Acidic-Anionic detergents
 - Anion reacts with plasma • Aniline dyes are very active against
membrane. gram-positive species of bacteria and
- Nontoxic, non-corrosive, various fungi
and fast acting. (Laundry - Sometimes used for antisepsis
soap, dairy industry.) and wound treatment
o Cationic detergents - Low level, narrow spectrum of
- Quaternary ammonium activity
compounds (Quats).
- Strongly bactericidal
against wide range, esp.
Gram+ bacteria
- Alter membrane
permeability of some  Acids and alkalis
bacteria and fungi • Low level of activity
- Organic acids prevent spore
germination and bacterial and fungal
growth
- Acetic acid inhibits bacterial growth
- Propionic acid retards mold
- Lactic acid prevents anaerobic
For detergents, the positive charge region bacterial growth
binds bacteria and the uncharged region - Benzoic and sorbic acid inhibit yeast
integrates into the cell membrane.
• Food preservative
 Aldehydes and Chemical Sterilants
• Aldehydes (alkylating agents)  Microbial Characteristics and Microbial
• Inactivate proteins by cross-linking with Control
functional groups (–NH2, –OH, –COOH,
–SH)
• Glutaraldehyde: Sterilant for
delicate surgical instruments
(Kills S. aureus in 5 min, M. tuberculosis
in 10 min)
• Formaldehyde: Virus inactivation for
vaccines
• Chemical Sterilants for heat sensitive
material
• Denature proteins

 Gases
• Ethylene oxide, propylene oxide
• Reacts with functional groups of DNA
and proteins
• Sterilizes and disinfects plastic
materials, prepackaged devices, foods

 Dyes
• Crystal violet
- Effective against Gram positive
bacteria
- Ointments

 Disk-diffusion Method
- Disk of filter paper is soaked with a
chemical and placed on an inoculated
agar plate; a zone of inhibition indicates
effectiveness.
 Antibiotic – substance produced by a
microorganism that kills or inhibits growth of
other microorganisms
 Antibiotics that have been chemically
modified to kill a wider variety of pathogens
or reduce side effects = semisynthetic
antibiotics
o Ex., semisynthetic penicillins – ampicillin
and carbenicillin

The discovery of penicillin by Alexander

Fleming. (A) Colonies of Staphylococcus
aureus are growing well in this area of the
plate. (B) Colonies are poorly developed in this
area of the plate because of an antibiotic
(penicillin) being produced by a colony of
Using anti-microbial agents to control Penicillium notatum (a mould), shown at C.
microbial growth
 The History of Chemotherapy
Terms defined:  Paul Ehrlich and Sahachiro Hata
developed Salvarsan (Arsphenamine)
 Chemotherapy = the use of any chemical
against syphilis in 1910: The concept of
(drug) to treat any disease or condition
chemotherapy to treat microbial
 Chemotherapeutic agent = any drug used
diseases was born.
to treat any condition or disease
 Sulfa drugs (sulfanilamide) discovered in
 Antimicrobial agent = any chemical (drug) 1932 → against Gram+ bacteria
used to treat an infectious disease, either  1928: Fleming discovered penicillin
by inhibiting or killing pathogens in vivo  1940: Howard Florey and Ernst Chain
o Some antimicrobial agents are performed first clinical trials of penicillin.
antibiotics.
 Therapies to combat causal agents of
 Drugs disease
o used to treat bacterial diseases –  Supportive Therapies:
antibacterial agents o Reduction in Stress
o used to treat fungal diseases – o Improvement in Diet • Exercise
antifungal agents o Good Hygiene
o used to treat protozoal diseases –
antiprotozoal agents  Preventive Therapies: (fending off,
o used to treat viral diseases – antiviral interception/ prevention of causal
agents agents)
o including antimicrobial chemotherapy
o Prevent transmission of causal
agents
- Disinfection (inanimate objects)
- Eradication of reservoirs
- Elimination of vectors
- Antisepsis
o Prevent infection
- Restriction & localization by
enforcing physical, chemical and
biological barriers
 Antimicrobial Chemotherapy  Most antimicrobial and antiviral drugs
 Goal o interfere with the function of
- administer a drug to an infected enzymes required to synthesize or
person, which destroys the infective assemble macromolecules or
agent without harming the host’s o destroy structures already formed in
cells the cell
 Chemotherapeutic agents are described
with regard to their origin, range of  Antimicrobial drugs should be selectively
effectiveness, and whether they are toxic in that they destroy or inhibit
naturally produced or chemically microbial cells without damaging host
synthesized tissues.

 Features of Antimicrobial Drugs  Major Mechanisms of Drug Action

 Selective toxicity: Drug kills pathogens  The 5 most common mechanisms of
without damaging the host. action of antimicrobial agents are:
 Therapeutic index: ratio between toxic 1. Inhibition of synthesis and
dose and therapeutic dose – or ratio of interference with structure and
LD50 to ED50 function of nucleic acids (either DNA
 High therapeutic index – less toxic or RNA)
 Antimicrobial action – Bacteriostatic 2. Inhibition of synthesis and
vs. bactericidal interference with structure and
 Activity Spectrum – Broad-spectrum function of proteins
vs. narrow- spectrum 3. Inhibition of cell wall synthesis
 Tissue distribution, metabolism, and 4. Interference with cell membrane
excretion – BBB; Unstable in acid; half- structure or function
life duration 5. Inhibition of specific metabolic
pathways (such as folic acid
 Origins of Antimicrobial Drugs
 Antibiotics – secondary metabolic
products of aerobic bacteria and fungi
o Bacteria: Streptomyces and Bacillus
o Molds: Penicillium and
Cephalosporium

 Chemists have created new generations

of drugs by altering the structure of
naturally occurring antibiotics (semi-
synthetic drugs)

 Researchers continue to search for new synthesis) and enzyme activity

antimicrobial compounds also in
organisms other than bacteria and fungi  Antimicrobial Drugs that affect Nucleic
(bioprospecting) Acid Synthesis
 Block synthesis of nucleotides
 Interactions between Drug & Microbe  Inhibit replication (DNA synthesis)
 Goal of antimicrobial and antiviral drugs:  Inhibit (prevent or stop) transcription
o Disrupt cellular processes or (RNA synthesis)
structures of bacteria, fungi, and
protozoa  Antimicrobial Drugs that block Protein
o Inhibit virus replication Synthesis
  Inhibit translation by reacting with the
ribosome‐mRNA complex
 selective effect is possible because:
bacterial ribosomes are different from
eukaryotic ribosomes
PABA – para-aminobenzoic acid
 Major Antimicrobial Drug Groups
 Antibacterial Agents
 Antimicrobial Drugs that Affect the
Bacterial Cell Wall
 Active cells with a cell wall must
constantly synthesize new NAM‐NAG
units, transport them across the plasma
membrane to the proper place and
incorporate them into the existing
peptidoglycan layer in the cell envelope
 Penicillins and cephalosporins react with
one or more of the enzymes required to
complete this process (PBPs)
- Bactericidal antibiotics
 Antimicrobial Drugs that Disrupt Cell
Membrane Function
 Damaged cell membranes invariably
result in death from elimination of
gradients or lysis
 Specificity is possible because particular
microbial groups have differences in the
types of lipids in their cell membranes
 Antimicrobial Drugs that Inhibit Folic
Acid Synthesis
 Sulfonamides and trimethoprim‐ act via
competitive inhibition
• Supplied to cells in high concentrations
to make sure enzyme is constantly
occupied with the metabolic analog
inhibitor rather than the true substrate
 About 260 different antimicrobial drugs
 Classified in 20 drug families
 Largest number of antimicrobial drugs
are used to combat bacterial infections
 Bacteriostatic drugs inhibit growth of
bacteria, whereas bactericidal drugs kill
bacteria.
 Sulfonamide drugs inhibit production of
folic acid (a vitamin) in those bacteria
that require ρ-aminobenzoic acid to
synthesize folic acid; without folic acid
bacteria cannot produce certain
essential proteins and die.
o Sulfa drugs are competitive
inhibitors; they are bacteriostatic.
 In most Gram-positive bacteria,
penicillin interferes with the synthesis
and cross-linking of peptidoglycan, a
component of cell walls. By inhibiting
cell wall synthesis, penicillin destroys
the bacteria.
 Colistin and nalidixic acid destroy only
Gram-negative bacteria; (narrow-
spectrum antibiotics)
 Antibiotics that are destructive to both
Gram-positive and Gram-negative
bacteria are called broad-spectrum
antibiotics (examples: ampicillin,
chloramphenicol and tetracycline).
 Multidrug therapy
 - Sometimes one drug is not sufficient;  Antiprotozoal agents are usually toxic to
2 or more drugs may be used the host.
simultaneously, as in the treatment
of tuberculosis.  Antiprotozoal agents work by:
 Some Major Categories of Antibacterial
Agents o Interfering with DNA and RNA
 Penicillins (bactericidal; interfere with synthesis (e.g., chloroquine,
cell wall synthesis) pentamidine, and quinacrine)
 Cephalosporins (bactericidal; interfere o Interfering with protozoal
with cell wall synthesis) metabolism (e.g., metronidazole)
 Tetracyclines (bacteriostatic; inhibit
protein synthesis)  Antiviral Agents
 Aminoglycosides (bactericidal; inhibit  Antiviral agents are the newest weapons
protein synthesis) in antimicrobial methodology.
 Macrolides (bacteriostatic at lower  Difficult to develop agents because
doses; bactericidal at higher doses; viruses are produced within host cells.
inhibit protein synthesis)  Some drugs have been developed that
 Fluoroquinolones (bactericidal; inhibit are effective in certain viral infections,
DNA synthesis) but not others; they work by inhibiting
viral replication within cells.
 Antibacterial Agents  Antiviral agent “cocktails” (several drugs
 Synergism vs Antagonism that are administered simultaneously)
- Synergism is when 2 antimicrobial are being used to treat HIV infection.
agents are used together to produce
a degree of pathogen killing that is Drug Resistance “Superbugs”
greater than that achieved by either
drug alone. Synergism is a good  Superbugs are microbes (mainly
thing! bacteria) that have become resistant to
- Antagonism is when 2 drugs actually one or more antimicrobial agent.
work against each other. The extent Infections caused by superbugs are
of pathogen killing is less than that difficult to treat!
achieved by either drug alone.  Bacterial superbugs include:
Antagonism is a bad thing! o methicillin-resistant Staphylococcus
aureus (MRSA);
 Antifungal Agents o vancomycin-resistant Enterococcus
 Most antifungal agents work in one of 3 spp. (VRE)
ways: o multidrug-resistant Mycobacterium
1. By binding with cell membrane
tuberculosis (MDRTB);
sterols (e.g., nystatin and
o multidrug-resistant strains of
amphotericin B)
Acinetobacter, Burkholderia, E. coli,
2. By interfering with sterol
Klebsiella, Pseudomonas,
synthesis (e.g., clotrimazole and
Stenotrophomonas, Salmonella,
miconazole)
Shigella. and N. gonorrhoeae
3. By blocking mitosis or nucleic
o β–lactamase-producing strains of
acid synthesis (e.g., griseofulvin
and 5-flucytosine) Streptococcus pneumoniae and
 Antifungal agents and antiprotozoal Haemophilus influenzae
agents tend to be more toxic to the o carbapenemase-producing
patient because, like the infected Klebsiella pneumoniae
human, they are eucaryotic organisms.
 How Bacteria Become Resistant to
 Antiprotozoal Agents Drugs
  Some bacteria are naturally resistant  chromosomal mutation or by the
because they lack the specific target acquisition of new genes by
site for the drug or the drug is unable to transduction, transformation and, most
cross the organism’s cell wall or cell commonly, by conjugation
membrane and thus, cannot reach its
site of action. Resistance of this type is  Drug Resistance
known as intrinsic resistance. β-Lactamases
 If bacteria that were once susceptible to  Every penicillin and cephalosporin
a particular drug become resistant, this molecule contains a double- ringed
is called acquired resistance. structure (referred to as a “house and
 Before a drug enters a bacterial cell it garage”). The “garage” is known as the
must first bind to proteins on the β-lactam ring.
surface of the cell; these proteins are  Some bacteria produce enzymes, β-
called drug- binding sites. lactamases, that destroy this ring; when
o a chromosomal mutation that the β–lactam ring is destroyed, the drug
affects the structure of a drug- no longer works.
binding site can prevent the drug o 2 types of β-lactamases-
from binding, resulting in drug penicillinases and
resistance. cephalosporinases; some bacteria
 To enter a bacterial cell, a drug must be produce both types.
able to pass through the cell wall and  Drug companies have developed special
cell membrane drugs that combine a β–lactam antibiotic
o chromosomal mutations may alter with a β-lactamase inhibitor.
the structure of the cell membrane,
thus preventing the drug from  Sites of β-lactamase Attack on Penicillin
entering the cell; this results in drug and Cephalosporin Molecules.
resistance.

 Bacteria can develop the ability to

produce an enzyme that destroys or
inactivates a drug.
o Many bacteria have become
resistant to penicillin because they
have acquired the gene for
penicillinase production during  Some Strategies in the War Against Drug
conjugation.

 A plasmid that contains multiple genes

for drug resistance is known as a
resistance factor (R-factor).
 Bacteria can also become resistant to
drugs by developing the ability to
produce multidrug-resistance (MDR)
pumps (also known as MDR Resistance
transporters or efflux pumps).  Education of healthcare professionals
o An MDR pump enables the cell to and patients
pump out drugs before they can  Patients should stop demanding
damage or kill the cell. antibiotics every time
 Physicians should not be pressured by
Summary: Bacteria can acquire resistance to patients and should prescribe drugs only
antimicrobial agents by: when warranted
  Clinicians should prescribe a narrow-
spectrum drug if lab results indicate that
it kills the pathogen
 Patients should destroy any excess or
out-dated medications
 Antibiotics should not be used in a
prophylactic manner
 Healthcare professionals should practice
good infection control
 Patients should take drugs in manner
prescribed
 Empiric Therapy
 Empiric therapy is when drug therapy is
initiated before laboratory results are
available (i.e., before the pathogen is
identified and/or before susceptibility
test results are available).
o Empiric therapy is sometimes
necessary to save a patient’s life.
o Clinicians make an “educated
guess” based on past experience
with the type of infectious disease
and the most effective drugs.
 Clinicians must take a number of factors
into consideration before prescribing
antimicrobial agents.
 Undesirable Effects of Antimicrobial
Agents
 Reasons why antimicrobial agents
should not be used indiscriminately:
1. Organisms susceptible to the
agent will die, but resistant ones
will survive; this is known as
selecting for resistant organisms.
2. The patient may become allergic
to the agent.
3. Many agents are toxic to humans
and some are very toxic.
4. With prolonged use, a broad-
spectrum antibiotic may destroy
the normal flora, resulting in an
overgrowth of bacteria known as
a superinfection.

 Factors to be Considered
 If pathogen identity is known, use the
“pocket chart” of antimicrobial
susceptibility test data from past year.
 Is the patient allergic to any
 Selecting for drug-resistant organisms:
antimicrobial agents?
A. Indigenous microflora of patient before
 What is the age of the patient?
antibiotic therapy. (S = susceptible; R =
 Is the patient pregnant?
resistant)
 Inpatient or outpatient?
B. After antibiotic therapy has been
 In the hospital formulary?
initiated
 Site of the infection?
C. Resistant organisms multiply and
 What other medication(s) is the patient
become the predominant organisms.
taking?
 What other medical problems does the
patient have?
 Is the patient leukopenic or
immunocompromised?
 What is the cost of the drug(s)?

Pocket chart for aerobic Gram- negative

bacteria. The chart is a quick reference
whenever empiric therapy is necessary.
Epidemiology and Public Health
Terms Defined:
1. Pathology – study of disease
2. Pathogenesis – development of disease
3. Pathogens – agents of disease, have
properties that allow them to invade the
human body or produce toxins.
4. Etiology – the study of the cause of a
disease.
5. Infection – invasion and growth of
pathogens in the body.
6. Disease – abnormal state in which the
body is not functioning normally.
i.e.: infectious agent overcomes body’s
defenses.
The Normal Microbiota (Flora)
 Microbial antagonism due to competition
between microbes.
 Resident flora – normal microbiota
 acquired at passage through birth canal
 establish permanent colonies on/inside
body without producing diseases.
 protect the host by:
1. occupying niches that pathogens
might occupy (competitive exclusion)
2. producing acids
3. producing bacteriocins (toxins)
4. stimulation of immune system
Transient Microbiota (Flora)
 Certain microbes are present for various
periods (days, weeks, or months) – then
disappears.
 Advantageous: Probiotics – live microbes
applied to or ingested into the body,
intended to exert a beneficial effect.
 Dynamic nature of resident flora: changes
due to age (they grow old with us), type of
food consumed, hormonal state,
antibiotics.
Various Co-existence Relationship between
Bacteria and Host
 Symbiosis
1. Mutualism – microbe and host benefit
from co-existence, neither suffers
2. Commensalism – microbe benefits but
host is unaffected
3. Parasitism – microbe benefits, host
suffers
 Opportunistic Pathogens cause disease
under special conditions (mutualistic
relationship becomes parasitic)
 Healthy carriers of pathogenic organisms
 Cooperation among microorganisms: One
microorganism may make it possible for
another to cause disease or produce more
severe symptoms.
Koch’s Postulates: Proof of Etiology and
Infectious Diseases
1. The same pathogen must be present in
every case of the disease.
2. The pathogen must be isolated from the
diseased host and grown in pure culture.
3. The pathogen from the pure culture must
cause the same disease when it’s
inoculated into a healthy, susceptible lab
animal.
4. The pathogen must be isolated from the
inoculated animal and must be shown to
be the original microbe.
Exceptions to Koch’s Postulates
Modification of Koch’s postulates were
necessary:
1. to establish disease etiology for viruses
and bacteria, which cannot be grown on
artificial media.
2. some diseases, e.g.: pneumonia and
nephritis, may be caused by a variety of
microbes.
3. some pathogens, such as S. pyogenes,
cause several different diseases.
4. certain pathogens, such as HIV, cause
disease in humans only.
Classifying Infectious Diseases
 Symptom – a change in body function that
is felt by a patient as a result of disease
(from patient)
 Sign – a change in a body that can be
measured or observed as a result of
disease. (temperature, blood pressure,
number of antibodies (RT-PCR) etc.)
 Syndrome – a specific group of signs and
symptoms that accompany a disease.
 Communicable vs. Non-communicable vs.
Contagious
o Communicable disease – A disease
that is spread from one host to another
o Contagious disease – A disease that is
easily spread from one host to another
o Non-communicable disease – A
disease that is not transmitted from one
host to another
Symptoms of a Disease versus Signs of a
Disease
 A symptom of a disease is defined as some
evidence of a disease that is experienced
by the patient; something that is subjective.
For example, aches or pains, ringing in the
ears, blurred vision, nausea, dizziness, etc.
o In a symptomatic disease, the patient
is experiencing symptoms.
o In an asymptomatic disease, the
patient is not experiencing any
symptoms.
 A sign of a disease is defined as some type
of objective evidence of a disease. For
example, elevated blood pressure,
abnormal heart sounds, abnormal pulse
rate, abnormal laboratory results, etc.
Severity or Duration of a Disease
Scope of disease can be defined as:
 Acute: Disease develops rapidly, and is
usually followed by a relatively rapid
recovery. Examples are measles, mumps,
and influenza
 Chronic: Disease develops slowly and
lasts a long time. Examples are
tuberculosis, leprosy, and syphilis.
 Subacute: Symptoms between acute and
chronic. One that comes on more suddenly
than a chronic disease, but less suddenly
than an acute disease. An example would
be bacterial endocarditis.
Latent Infections
 Disease with a period of no symptoms
when the causative agent is inactive.
 Infectious diseases that go from being
symptomatic to asymptomatic, and then
later, go back to being symptomatic.
 examples: syphilis, herpes virus infections
such as cold sores, genital herpes, and
shingles.
Extent of Host Involvement
 Toxemia – toxins in the blood
 Viremia – viruses in the blood
 Primary Infection – acute infection that
causes the initial illness.
 Secondary Infection – opportunistic
infection after a primary (predisposing)
infection.
 Subclinical disease – no noticeable signs
or symptoms (inapparent infection)
Primary versus Secondary Infections
 One infectious disease may commonly
follow another. In such cases, the first
disease is referred to as a primary infection
and the second disease is referred to as a
secondary infection.
 Example: serious cases of bacterial
pneumonia frequently follow mild viral
respiratory infections.
 During the primary infection, the virus
causes damage to the ciliated epithelial
cells of the respiratory tract, leading to the
secondary infection.

Extent of Host Involvement: An Infection
can be:
1. Local: limited to small area of body
2. Systemic: spread throughout body via
blood
3. Focal: spread from local infection to
specific areas of the body
 Sepsis: toxic inflammatory condition
arising from spread of microbes or their
toxins, from a focus
 Bacteremia: bacteria in the blood
 Septicemia: growth of bacteria in the
blood
Localized Versus Systemic Infections
1. Localized Infections
 Once an infectious process is initiated,
the disease may remain localized or it
may spread;
 examples of localized infections are
pimples, boils and abscesses.
2. Systemic Infections
 When the infection spreads throughout
the body it is said to have become a
systemic or generalized infection; an
example is miliary tuberculosis caused
by Mycobacterium tuberculosis.
Interactions Among Pathogens, Hosts, and
the Environment
 Whether an infectious disease occurs
depends on:
1. Factors pertaining to the pathogen
(e.g., virulence of pathogen, mode of
entry, number of organisms)
2. Factors pertaining to the host (e.g.,
health status, nutritional status,
hygiene, age, travel, lifestyle, etc.)
3. Factors pertaining to the environment
(e.g., physical factors such as climate,
season, geographic location;
availability of appropriate reservoirs;
sanitary and housing conditions; and
availability of potable water)
Patterns of Disease: Predisposing Factors
 Variable susceptibility (of person) due to
 Genetics
 Gender
 Climate and weather
 Age
 Stress and fatigue
 Lifestyle
 Chemotherapy
The Chain of Infection
 There are 6 components in the infectious
disease process:
1. a pathogen
2. a source of the pathogen (a
reservoir)
3. a portal of exit
4. a mode of transmission
5. a portal of entry
6. a susceptible host
Disease Development and Stages
 To cause a disease, a pathogen must
replicate and grow inside a host.
 A well-adapted pathogen lives in balance
with its host.
o Chronic infections: Host and
pathogen survive.
 New pathogens sometimes emerge for
which the host has no resistance
o Acute infections: Pathogen can be
selective force.
Disease Development and Stages
1. Incubation period: Time interval
between initial infection and first
appearance of signs and symptoms.
2. Prodromal period: Characterized by
appearance of first mild signs and
symptoms.
3. Period of illness: Disease at its height:
all disease signs and symptoms
apparent.
 4. Period of decline: Signs and symptoms
subside.
5. Period of convalescence: Body returns
to prediseased state, health is restored.
Why Infection Does Not Always Occur
 The microbe may land at an anatomic
site where it is unable to multiply.
 Many pathogens must attach to specific
receptor sites before they are able to
multiply and cause damage.
 Antibacterial factors may be present at
the site where the pathogen lands.
 Indigenous microflora of that site may
inhibit growth of the foreign microbe
(i.e., microbial antagonism).
 The indigenous microflora may produce
antibacterial factors (i.e., bacteriocins)
that destroy the pathogen.
 The individual’s nutritional and overall
health status often influences the
outcome of the pathogen-host
encounter.
 The person may be immune to that
particular pathogen.
 Phagocytes present in the blood may
destroy the pathogen.
The Spread of Infection: Reservoir of
Infections
 continual source of infectious agents
 the sources of microorganisms that
cause infectious diseases
 are many and varied
Reservoirs of Infection
 sites in which infectious agents remain
viable and from which individuals can
become infected
 A number of infectious diseases are
caused by pathogens that propagate in
humans and animals.
 For other pathogens, nonliving matter
serves as reservoirs.
 For example, soil is a reservoir for
Clostridium tetani, the cause of tetanus.
 Living reservoirs – humans, pets, farm
animals, insects, arachnids
 Human carriers:
 Passive carriers
 Incubatory carriers
 Convalescent carriers
 Active carriers
 Human carriers:
o Carrier – an individual who
inconspicuously shelters a
pathogen and spreads it to others;
may or may not have experienced
disease due to the microbe
o Passive carrier – contaminated
healthcare provider picks up
pathogens and transfers them to
other patients
o Asymptomatic carrier – shows no
symptoms
 Incubatory carriers – spread
the infectious agent during the
incubation period
 Convalescent carriers –
recuperating without
symptoms
 Chronic carrier – individual
who shelters the infectious
agent for a long period
 Animals – Infectious diseases that
humans acquire from animal sources =
zoonotic diseases or zoonoses.
 Zoonoses may be acquired by direct
contact with an animal, inhalation or
ingestion of the pathogen, or injection of
the pathogen by an arthropod
o Rabies
o Lyme disease
o Many others
 Arthropods – many different types of
arthropods serve as reservoirs of
infection, including:
o insects (e.g., fleas, mosquitoes,
lice)
o arachnids (e.g., mites and ticks)
 Vectors = arthropods involved in the
transmission of infectious diseases
o Examples of arthropod-borne
diseases:
 Lyme disease
 Malaria
 Nonliving Reservoirs – Air, soil, dust,
contaminated water and foods, and fomites
  Fomites - inanimate objects capable of
transmitting pathogens (e.g., bedding,
towels, eating and drinking utensils,
hospital equipment, telephones,
computer keyboards, etc.)
 Inanimate Vectors of Infection
(Fomites)
Most Common Modes of Transmission of
Infectious Diseases
Infectious Disease Transmission
 Pathogens can be classified by their
mechanism of transmission, but all
mechanisms have the following stages
in common.
1. escape from host
2. travel
3. entry into new host
 Pathogen transmission can be direct or
indirect.
Modes of disease transmission
1. Direct host-to-host transmission
o Infected individual transmits a
disease directly to a susceptible
host without the assistance of an
intermediary (e.g., flu, common
cold, STDs, ringworm).
2. Indirect host-to-host transmission
o occurs when transmission is
facilitated by a living or nonliving
agent
 Living agents are called
vectors.
 Nonliving agents are called
fomites.
3. Contact Transmission
o Direct: close association between
infected and susceptible host
o Indirect: spread by fomites Droplet
transmission: via airborne droplets
from saliva or mucus (coughing or
sneezing)
4. Airborne Transmission
1. pathogens carried on water droplets
or dust for a distance greater than 1
meter
5. Vehicle Transmission
2. water, food, air
6. Vector Transmission
3. arthropods carry pathogens from one
host to another (mechanical vector
vs. biological vector)
 Direct skin-to-skin contact
 Direct mucous membrane-to-mucous
membrane contact by kissing or sexual
intercourse
 Indirect contact via airborne droplets of
respiratory secretions, usually produced
by sneezing or coughing
 Indirect contact via food and water
contaminated by fecal matter
 Indirect contact via arthropod vectors
 Indirect contact via fomites
 Indirect contact via transfusion of
contaminated blood or blood products or
by parenteral injection using nonsterile
syringes or needles
The Language of Epidemiology
Epidemiology
 the study of the occurrence, distribution,
and determinants of health and disease in a
population
Public health – the health of the population as
a whole
The Beginning of Epidemiology
1. John Snow (1848–1849) – Mapped the
occurrence of cholera in London
2. Ignaz Semmelweis (1846–1848) –
Showed that handwashing decreased
the incidence of puerperal fever
3. Florence Nightingale (1858) – Showed
that improved sanitation decreased the
incidence of epidemic typhus
Epidemiology
 Major goal:
 Identifying the nature of a disease and
its transmission
 In developed countries, infectious
diseases cause fewer deaths than
noninfectious diseases.
 In developing countries, infectious
diseases account for nearly half of all
deaths.
 Effective control of infectious disease
remains a challenge.
 Epidemiologists
 rely on surveillance: the observation,
recognition, and reporting of diseases as
they occur
 can trace the spread of disease to
identify its origin and mode of
transmission
 study the factors that determine the
frequency, distribution, and
determinants of diseases in human
populations
 also develop ways to prevent, control, or
eradicate diseases in populations
 Disease incidence and prevalence
 to describe any given disease in a
population
 The incidence of a disease is the
number of new cases of the disease in a
given period of time.
 The prevalence of a disease is the total
number of new and existing cases in a
population in a given time.
o Period prevalence is the number of
cases of a disease existing in a given
population during a specific time
period (e.g., during the year 2009).
o Point Prevalence is the number of
cases of a disease existing in a given
population at a particular moment in
time (e.g., right now).
The scope of disease
 An endemic disease is constantly present
in a population, usually at low incidences,
of a particular geographic area. (ex.
gonorrhea)
o Individuals that are infected with a
pathogen that causes endemic
disease are called reservoirs – may be
human or non-human animals.
 A disease is an epidemic when it occurs in
a large number of people in a population
at the same time, usually within a short
period of time. (ex. the Legionnaire’s
disease epidemic of 1976)
 A sporadic disease is one that occurs only
occasionally within the population of a
particular geographic area. (ex. tetanus)
 A pandemic is a disease that is occurring
in epidemic proportions in many countries
simultaneously.
Examples include:
 Influenza
o Examples: (1) the Spanish flu
pandemic of 1918 during which
more than 20 million people were
killed worldwide (500,000 in the
U.S.) (2) the H1N1 (“swine flu”)
pandemic of 2009-2010
 HIV/AIDS
 Tuberculosis
 Malaria
 A disease outbreak occurs when a
number of cases of a disease are reported
in a short period of time.

Mortality, Morbidity, and DALY
 Mortality is the incidence of death in a
population.
 Morbidity of a disease refers to the
incidence of disease, including fatal and
nonfatal diseases.
 Disability-Adjusted Life Year (DALY)
quantitatively measures disease burden in
terms of lost years due to the disease,
disability due to disease, and premature
death.
o While hundreds of millions of people
have infectious diseases, most survive,
but may have significant impacts, as
measured by DALY.
The Host Community
 Coevolution of a host and a pathogen
– Coevolution of a host and its parasite
is common.
o Virulence of the parasite in host-
to-host transmission diminishes,
and resistance of the host
increases (e.g., myxoma virus
introduced to control rabbits in
Australia).
 A host-to-host pathogen that
kills its host before it can
infect another host may
become extinct.
o If a pathogen does not rely on
host-to-host transmission, it may
remain extremely virulent (e.g., E.
coli in hospitals).
 Herd immunity
– defined as the resistance of a group to
infection due to immunity of a high
proportion of the group
o If a high proportion of individuals
are immune to an infection, then
the whole population will be
protected.
o Immunized people protect non-
immunized people because the
pathogen cannot be passed on,
and the cycle of infectivity is
broken.
– Diseases such as influenza tend to
occur in cycles.
Characteristics of Disease Epidemics
 Major epidemics are usually classified as:
1. Common-source epidemic – usually
arises from contamination of water or
food
example: cholera
2. Host-to-host epidemic – the disease
shows a slow, progressive rise and a
gradual decline
examples: influenza and chicken pox
Carriers
 pathogen-infected individuals showing no
signs of clinical disease
 potential sources of infections may be
individuals in the incubation period of the
disease
 can be identified using diagnostic
techniques, including culture and
immunoassays
• Typhoid Mary is an example of a carrier.
Epidemiology and Public Health
Public Health and Infectious Disease
 Common vehicles include
1. food
2. water
3. air
 Controls directed against common
vehicles
 Food laws lowered incidence of
foodborne pathogens.
 Water purification reduced incidence of
typhoid fever.
 Airborne pathogens are difficult to
control.
 Controls directed against the reservoir
 If reservoir is animal, it can be
immunized or destroyed.
 When humans are the reservoir,
eradication can be difficult.
o Those with disease can be
quarantined, immunized, and
treated.
o used by the WHO to eradicate
smallpox

 Isolation, quarantine, and surveillance
 controls directed against transmission
of the pathogen
o immunization
- diseases have been controlled
- examples: smallpox, rubella,
and tetanus
o quarantine
- restricts the movement of an
individual with an active
infection
o surveillance
- the observation, recognition,
and reporting of diseases
 Pathogen eradication
o Goal: to remove all of a pathogen
from any reservoir (e.g., smallpox,
polio, and potentially rabies,
leprosy, and others)
o In 2014, only 359 cases of polio
were reported worldwide.
Global Health Considerations
 Infectious diseases in Americas versus
Africa
 Death rate in Africa is much higher.
 Most African deaths are due to
infectious diseases (10x as many as in
the Americas).
 Concern for people traveling to other
areas
 Travelers can be immunized.
 Drink only decontaminated water.
Emerging and Reemerging Infectious
Diseases
• Worldwide distribution of diseases changes
rapidly.
• Diseases that suddenly become prevalent are
called emergent.
• Reemerging diseases are those that have
become prevalent after having been under
control.
1. Emerging Infectious Diseases (EIDs)
• Diseases that are new, increasing in
incidence, or showing a potential to
increase in the near future
• CDC, NIH, and WHO are responsible for
surveillance and responses to emerging
diseases
Emergence factors:
 human demographics and behavior
 technology and industry
 economic development and land use
 international travel and commerce
 microbial adaptation and change
 breakdown of public health measures
 abnormal natural occurrences
Addressing emerging diseases
• Key elements
 recognition of the disease
 intervention to prevent pathogen
transmission
• Preventing the spread of emerging
infections must be a public health
response employing various
methods.
 Methods include quarantine,
immunization, and drug
treatment.
Contributing factors for EIDs
• Genetic recombination (E. coli 0157; H5N1
avian flu)
• Evolution of new strains (V. cholerae 0139)
• Inappropriate use of antibiotics and
pesticides (Antibiotic resistant strains)
• Changes in weather patterns (Hantavirus)
• Modern Transportation (West Nile virus)
• Ecological disaster, war, and expanding
human settlement (Coccidioidomycosis)
• Animal control measures (Lyme disease)
• Public Health failure (Diphtheria)
• Improved case reporting
Pandemics
• Examples of Pandemics: HIV/AIDS,
Cholera, and Influenza
1. Acquired immunodeficiency syndrome
(AIDS)
- viral disease that attacks the immune
system
 - First reported cases were in the United
States in 1981.
- At least 70 million people have been
infected worldwide with HIV.
- More than 25 million people have died
from AIDS.
- Studies in the United States suggest the
virus was transmitted through sexual
contact or blood.
2. Cholera
- causes severe, water-loss diarrhea
- typically transmitted through ingestion
of contaminated water containing Vibrio
cholerae
- largely restricted to developing
countries
- endemic in Africa, Southeast Asia, the
Indian subcontinent, and Central and
South America
- can be controlled by application of
water treatment
Epidemic cholera
- occurs where sewage treatment is
inadequate or absent
- may develop into pandemic
- Travelers carry pathogen to new
location.
3. Influenza
- Influenza pandemics occur every 10 to
40 years.
- Caused by major change in the
influenza genome
o antigenic drift – minor change in
influenza virus antigens due to gene
mutation
o antigenic shift – major change in
influenza virus antigen due to gene
reassortment
- 2009 swine flu pandemic (H1N1
influenza)
o swine in Mexico simultaneously
infected with swine influenza, bird
influenza, and human influenza
o example of a reassortment
Public Health Threats from Microbial
Weapons
Characteristics of microbial weapons
• Biological warfare is the use of biological
agents to kill a military or civilian
population.
• Biological weapons are organisms or
toxins that are
 easy to produce and deliver
 safe for use by the offensive soldiers
 able to incapacitate or kill individuals
under attack in a consistent and
reproducible manner
• Virtually all pathogenic bacteria or
viruses are potentially useful for
biological warfare.
• Bacterial toxins, such as botulinum toxin
from Clostridium botulinum, are also
potential biological weapons.
Smallpox and anthrax
 Smallpox virus has intimidating potential
as a biological warfare agent.
o can easily be spread by contact or
aerosol spray.
o debilitating, causing a disfiguring rash
and fever.
o has a mortality rate of 30 percent or
more
 Bacillus anthracis is a preferred agent for
biological warfare and biological terrorism.
o Endospores enhance ability to
disseminate B. anthracisin aerosols.
 Three Forms of the Disease
1. cutaneous anthrax
2. gastrointestinal anthrax
3. pulmonary anthrax
 preparations of B. anthracis that exhibit
properties to enhance dissemination.
 Particles are small and interspersed with a
very fine particulate agent (usually talc).
o Vaccination is given only to people at
risk.
o Anthrax is treated with ciprofloxacin
Healthcare Epidemiology
 the study of the occurrence, determinants,
and distribution of health and disease
within healthcare settings facilities
 primary focus: is on infection control and
the prevention of healthcare-associated
infections
 it includes any activities designed to study
and improve patient care outcomes
Healthcare-Associated Infections
 Infectious diseases can be divided into 2
categories:
1. Those acquired within healthcare
facilities (health care associated
infections)
2. Those acquired outside of healthcare
facilities (community acquired
infections)
Nosocomial (Hospital-Acquired) Infections
 Acquired as a result of a hospital stay.
 5-15% of hospital patients acquire
nosocomial infections.
 Aseptic techniques can prevent
nosocomial infections.
 Hospital infection control staff members
are responsible for overseeing the proper
cleaning, storage, and handling of
equipment and supplies.
Relative Frequency of Nosocomial
Infections
Common Causes of Nosocomial Infections
Patients Most Likely to Develop HAIs
 Elderly patients
 Women in labor and delivery
 Premature infants and newborns
 Surgical and burn patients
 Diabetic and cancer patients
 Patients receiving treatment with steroids,
anticancer drugs, antilymphocyte serum,
and radiation
 Immunosuppressed patients
 Patients who are paralyzed or are
undergoing renal dialysis or catheterization
Major Factors Contributing to HAIs
 The 3 major factors that combine to cause
HAIs are:
1. An ever-increasing number of drug-
resistant pathogens
2. The failure of healthcare personnel to
follow infection control guidelines
3. An increased number of
immunocompromised patients
The Three Major Contributing Factors
in Healthcare-Associated Infections
Additional Factors Contributing to HAIs
 Overcrowding of hospitals and shortages of
healthcare staff
 The indiscriminate use of antimicrobial
agents
 A false sense of security about antimicrobial
agents
 Lengthy and more complicated types of
surgery
 Increased use of less-highly trained
healthcare workers
 Increased use of anti-inflammatory and
immunosuppressant agents
 Overuse and improper use of indwelling
devices
What Can be Done to Reduce the Number of
HAIs?
 Strict compliance with infection control
guidelines
 Handwashing is the single most important
measure to reduce the risks of transmitting
pathogens from one patient to another or
from one anatomic site to another on the
same patient!
 Other means of reducing the incidence of
HAIs include disinfection and sterilization
techniques, air filtration use of ultraviolet
lights, isolation of especially infectious
patients, and wearing gloves, masks, and
gowns whenever appropriate.
Healthcare professional washing her hands –
the most important and most basic technique in
preventing and controlling infections and
preventing the transmission of pathogens is
handwashing.
Infection Control
 Infection control – the numerous measures
taken to prevent infections from occurring in
healthcare settings.
 Asepsis means “without infection”
2 types:
1. Medical asepsis
 Precautionary measures necessary to
prevent direct transfer of pathogens from
person to person and indirect transfer of
pathogens through the air or on
instruments, bedding, equipment, and
other inanimate objects (fomites)
2. Surgical asepsis or sterile technique
 Practices used to render and keep objects
and areas sterile
 Surgical aseptic techniques are practiced in
operating rooms, labor and delivery areas,
and during invasive procedures (e.g.,
drawing blood, injecting medications,
urinary and cardiac catheterization, lumbar
punctures)
Differences between medical and
surgical asepsis:
1. Medical asepsis is a clean technique
whereas surgical asepsis is a sterile
technique
2. The goal of medical asepsis is to
exclude pathogens, whereas the goal of
surgical asepsis is to exclude all
microorganisms
Standard Precautions – are to be applied to
the care of ALL patients in ALL healthcare
settings, regardless of the suspected or
confirmed presence of an infectious agent
o They provide infection control guidelines
regarding:
 hand hygiene
 wearing of gloves, masks, eye
protection, and gowns
 respiratory hygiene/cough etiquette
 safe injection practices
 lumbar puncture
 cleaning of patient-care equipment
 environmental control
 handling of soiled linens
 resuscitation devices
 patient placement
 disposal of used needles and other
sharps
Healthcare Professional Donning Personal 2. indirect contact (i.e., transfer of
Protective Equipment (PPE) - microorganisms by a contaminated
sterile gown (A), mask (B), and gloves (C) intermediate object)

Proper Procedure for Glove Removal

Examples of Diseases Requiring Contact

Precautions
 acute viral (hemorrhagic) conjunctivitis
 acute respiratory infectious diseases or
aseptic meningitis in infants and young
children
 chickenpox
 cutaneous diphtheria
 disseminated shingles
 extrapulmonary tuberculosis with draining
lesion
Transmission-Based Precautions  gastroenteritis in diapered or incontinent
 Transmission-Based Precautions are used persons
for patients who are known or suspected to  impetigo
be infected or colonized with highly  infection or colonization with multidrug-
transmissible or epidemiologically important resistant organisms
pathogens for which additional safety  major draining abscesses or wound
precautions beyond Standard Precautions infections
are required to interrupt transmission within  monkeypox
healthcare settings  poliomyelitis
 severe mucocutaneous herpes simplex
 The three types of Transmission- infections
Based Precautions are:  smallpox
1. Contact Precautions  staphylococcal scalded skin syndrome
2. Droplet Precautions  major staphylococcal or streptococcal
3. Airborne Precautions disease of skin, wounds, or burns
 viral hemorrhagic fevers due to Lassa,
 Contact transmission is divided into Ebola, Marburg, or Crimean-Congo fever
2 subgroups: viruses
1. direct-contact (i.e., transfer of
microorganisms by body surface-to-
body surface)
  confirmed or suspected pulmonary or
laryngeal tuberculosis
 extrapulmonary tuberculosis with draining
lesions
 disseminated shingles in any patient
 localized shingles in immunocompromised
patients
 measles
 Monkeypox
 severe acute respiratory syndrome (SARS)
 smallpox
Examples of Diseases Requiring Droplet
Precautions Note that some of these diseases also require
 adenovirus infection in infants and young Droplet Precautions and/or Contact
children Precautions
 adenovirus pneumonia
 epiglottitis or meningitis caused by A type N95 respirator is used when Airborne
Haemophilus influenzae type B Precautions are indicated
 major skin, wound, or burn infections due to
group A streptococcus Airborne Infection Isolation Room
 scarlet fever in infants and young children  The preferred placement for patients who
 influenza are infected with pathogens that are spread
 meningitis or pneumonia caused by via airborne droplet nuclei (5 m or less in
Neisseria meningitidis diameter), and therefore require Airborne
 mumps Precautions, is an airborne infection
 Mycoplasma pneumonia isolation room (AIIR)
 parvovirus B19 skin infection  An AIIR is under negative pressure to
 whooping cough prevent room air from entering the corridor
 pharyngeal diphtheria  The air evacuated from an AIIR passes
 pneumonic plague through a HEPA (high-efficiency particulate
 German measles air) filter
 severe acute respiratory syndrome (SARS)
 strep throat in infants and young children Protective Environments
 rhinovirus infection  Patients who are especially vulnerable to
 viral hemorrhagic fevers due to Lassa, infection are placed in a Protective
Ebola, Marburg, or Crimean-Congo fever Environment – patients with severe burns or
viruses leukemia, transplant or immuno-suppressed
patients, patients receiving radiation
treatment, leukopenic patients, premature
infants
 The room is under positive pressure and air
entering the room passes through HEPA
(high-efficiency particulate air) filters

Handling Food and Eating Utensils

 Some of the regulations for safe handling of
food and eating utensils include:
 Using high-quality, fresh food
Examples of Diseases Requiring Airborne  Properly refrigerating and storing food
Precautions  Properly washing, preparing, and
 chickenpox cooking food
 Properly disposing of uneaten food
  Covering hair and wearing clean clothes
and aprons
 Thoroughly washing hands and nails
before handling foods
 Keeping all cutting boards and other
surfaces scrupulously clean
 Washing cooking and eating utensils in
a dishwasher with a water temperature
> 80oC
Handling Fomites
 Fomites are nonliving, inanimate objects,
other than food, that may harbor and
transmit microbes.
Examples: patients’ gowns, bedding,
towels, hospital equipment, telephone,
computer keyboard, etc.
 Transmission of pathogens by fomites can
be prevented by observing certain rules:
 Use disposable equipment and supplies
whenever possible
 Disinfect or sterilize equipment soon
after use
 Use individual equipment for each
patient
 Use disposable thermometers or
thermometer covers
Medical Waste Disposal
 General Regulations
o Follow standards for disposal of
medical wastes
 Disposal of Sharps
o Sharps should be handled and disposed
of properly
o Dispose of sharps in specifically
designed puncture-resistant containers
(“sharps containers”)
Infection Control Committees and Infection
Control Professionals
 All healthcare facilities should have
some type of formal infection control
program in place.
 The Infection Control Committee (ICC)
is composed of representatives from
most of the hospital’s departments,
including medical and surgical services,
pathology, nursing, hospital
administration, risk management,
pharmacy, housekeeping, food services,
and central supply.
o The chairperson is usually an infection
control professional such as an
epidemiologist or infectious disease
specialist, an infection control nurse, or
a microbiologist.
Role of the Clinical Microbiology Laboratory
(CML) in Hospital Epidemiology and
Infection Control
 CML personnel participate in infection
control by:
o Monitoring the types and numbers of
pathogens isolated from hospitalized
patients
o Notifying the appropriate infection control
person should an unusual pathogen or
an unusually high number of isolates of a
common pathogen be detected
o Processing environmental samples,
including samples from hospital
employees, that have been collected
from within the affected ward(s)
In conclusion:
 HAIs can add several weeks to a patient’s
hospital stay and may lead to serious
complications and even death.
 HAIs can be avoided through proper
education and disciplined compliance with
infection control practices!
 All healthcare workers must fully
comprehend the problem of HAIs, must be
completely knowledgeable about infection
control practices, and must personally do
everything in their power to prevent HAIs
from occurring!
Diagnosing Infectious Diseases
 The proper diagnosis of an infectious
disease requires:
1. Taking a complete patient history
2. Conducting a thorough physical
examination of the patient
3. Carefully evaluating the patient’s signs
and symptoms
4. Implementing the proper selection,
collection, transport, and processing of
appropriate clinical specimens
Clinical Specimens
 Specimens collected from patients such as
blood, urine, feces, and cerebrospinal fluid
(CSF)
 Specimens commonly submitted to the
hospital’s Clinical Microbiology Laboratory
(CML) include: blood, bone marrow,
bronchial washings, sputum, CSF, cervical
and vaginal swabs, feces, hair and nail
clippings, pus, skin scrapings, synovial fluid,
throat swabs, tissue specimens, urethral
discharge material, urine, and urogenital
secretions.
 All specimens should be of the highest
possible quality
The Role of Healthcare Professionals in the
Submission of Clinical Specimens:
 There should be a close working
relationship among the members of the
healthcare team to ensure a proper
diagnosis of infectious diseases.
 Healthcare professionals who collect and
transport specimens should exercise
extreme caution during the collection and
transport of specimens.
 In the laboratory, all specimens are handled
carefully, exercising Standard Precautions.
Although laboratory professionals do not
themselves make diagnoses, they make
laboratory observations and generate test
results that assist clinicians to correctly
diagnose infectious diseases and initiate
appropriate therapy.
Importance of High-Quality Clinical
Specimens
 High-quality clinical specimens are
required to achieve accurate, clinically
relevant laboratory results.
 The 3 components of specimen quality
are:
1. Proper specimen selection
2. Proper specimen collection
3. Proper transport of the specimen to the
laboratory
 The laboratory must provide written
guidelines (“Laboratory Policies and
Procedures Manual”).
 The person who collects the specimen is
ultimately responsible for its quality.
Proper Selection, Collection, and Transport
of Clinical Specimens
 Specimens must be properly selected.
 Specimens must be collected properly.
 Material (i.e., specimens) should be
collected from a site where the suspected
pathogen is most likely to be found.
 Specimens should be obtained before
antimicrobial therapy, if possible.
 The acute stage of the disease is the
most appropriate time to collect a
specimen.
 Specimen collection should be performed
with care and tact to avoid harming the
patient.
 A sufficient quantity of the specimen must
be obtained to provide enough material for
all required diagnostic tests.
 All specimens should be placed or
collected into a sterile container to prevent
contamination.
 Specimens should be protected from heat
and cold and promptly delivered to the
laboratory.
 Hazardous specimens must be handled
with even greater care to avoid
contamination of couriers, patients, and
healthcare professionals.
 Whenever possible, a sterile, disposable
specimen container should be used.
 The specimen container must be properly
labeled and accompanied by an
 appropriate request slip with adequate
instructions.
 Specimens should be collected and
delivered to the lab as early in the day as
possible to allow sufficient processing
time.
loop) to determine the number of
colony-forming units (CFUs).
• # Colonies x dilution factor = #
CFUs/mL
- Obtaining a Urine Colony Count

Types of Clinical Specimens Usually

Required to Diagnose Infectious Diseases
1. Blood
- Usually sterile
- The presence of bacteria in the
bloodstream is known as bacteremia.
- Septicemia is a serious disease
involving chills, fever, prostration, and
3. Cerebrospinal Fluid (CSF)
the presence of bacteria or their toxins
- Meningitisis inflammation or infection
in the bloodstream.
- To prevent contamination of a blood of the membranes (meninges) that
surround the brain and spinal column.
specimen with indigenous skin flora,
- Encephalitisis is the inflammation or
extreme care must be taken to use
aseptic technique. infection of the brain.
- Meningoencephalitis is inflammation
2. Urine or infection of both the brain and
- Normally sterile in the bladder; meninges.
becomes contaminated by indigenous - CSF is collected by lumbar puncture
microflora of the distal urethra during into a sterile tube; this is a surgically
voiding. aseptic procedure performed by a
- Contamination is reduced by collecting physician.
a clean-catch, midstream urine. - CSF is considered a STAT
- Urine culture involves 3 parts: (emergency) specimen in the lab
• A colony count (using a calibrated
loop) - Technique of Lumbar Puncture
• Isolation and identification of the
pathogen
• Antimicrobial susceptibility testing

- Urine Colony Count

• The colony count is a way of
estimating the number of viable
bacteria that are present in a urine
specimen.
• A calibrated loop, either 0.01 mL or
0.001 mL, is used to inoculate the
entire surface of a blood agar
plate.
• After incubation at 37oC overnight,
the colonies are counted and the
number is multiplied by the dilution
factor (either 100 for the 0.01 mL
loop, or 1000 for the 0.001 mL
4. Sputum
- Sputum is pus that accumulates deep
within the lungs of a patient with
pneumonia, tuberculosis, or other
lower respiratory infection.
- Often, specimens labeled “sputum” are
actually just saliva; saliva specimens
don’t provide clinically relevant
information.
 - If TB is suspected, extreme care - Bacteria in fecal flora are obligate-,
should be taken aerotolerant-, and facultative
- Better specimens can be obtained by anaerobes.
bronchial aspiration or transtracheal - A combination of direct microscopic
aspiration. examination, culture, biochemical
tests, and immunologic tests may be
5. Throat Swabs performed to identify Gram-negative
- Routine throat swabs are used to and Gram-positive bacteria, fungi,
determine whether a patient has strep intestinal protozoa, and intestinal
throat helminths isolated from fecal
- Specific cultures may be necessary specimens.
when Neisseria gonorrhoeae or
Corynebacterium diphtheriae are The Pathology Department
suspected  Clinical specimens are submitted to the
Clinical Microbiology
6. Wound Specimens  Laboratory (CML), which is a part of the
- Whenever possible, a wound specimen Pathology Department.
should be an aspirate (i.e., pus  The Pathology Department is under the
collected by needle and syringe), direction of a pathologist (a physician who
rather than a swab has specialized training in pathology).
- Specimens collected by swab are  The pathology department is divided into 2
frequently contaminated with major divisions:
indigenous microflora

7. GC Cultures (for Neisseria

gonorrhoeae)
- N. gonorrhoeae is a fastidious,
microaerophilic, and capnophilic
bacterium.
- Only Dacron, calcium alginate, or
nontoxic cotton swabs should be used
to collect GC specimens.
- Specimens (e.g., vaginal, cervical,
urethral, throat, and rectal swabs) are
cultured on special medium (e.g.,
Thayer-Martin medium) and incubated
in a CO2 incubator.
- Special transport media are available
1. Anatomical Pathology
and GC swabs should never be
• Diseased organs, stained tissue
refrigerated.
sections, and cytology specimens are
examined here.
8. Fecal Specimens
• Cytogenetic technologists,
- Ideally, fecal (stool) specimens should
cytotechnologists, histologic
be collected at the laboratory and technicians, histotechnologists, and
processed immediately to prevent a pathologist’s assistants are employed
decrease in temperature, which would in this division.
allow the pH to drop and cause the • In addition, autopsies are performed in
death of many Shigella and Salmonella the morgue and some Pathology
species. Departments have an Electron
Microscopy Laboratory.
 identify the bacterial pathogens, and
2. Clinical Pathology antimicrobial susceptibility testing is
• Consists of several laboratories in addition performed whenever appropriate to do
to the Clinical Microbiology Laboratory: so.
Clinical Chemistry, Urinalysis, - CML professionals are very much like
Hematology/Coagulation, Blood Bank, and detectives and crime scene
Immunology. investigators, in that they gather clues
• Personnel include pathologists, chemists about a pathogen until they are able to
and microbiologists, clinical laboratory identify it.
scientists (also known as medical - Numerous phenotypic characteristics
technologists MTs), and clinical laboratory are used to identify the bacteria (e.g.,
technicians (also known as medical Gram reaction, cell shape, motility,
laboratory technicians - MLTs). presence and
location of spores, presence or absence of
various enzymes,
etc.)

 CML professionals are very much like

detectives and crime scene investigators
-- gathering clues about a pathogen until
they have enough information to identify
it.

• The CML may be under the direction of a Minisystems Used to Identify Bacteria
pathologist, a microbiologist, or a senior 1. API-20E for identification of
clinical laboratory scientist. Enterobacteriaceae
2. Enterotube II for identification of
• Responsibilities Enterobacteriaceae
- Primary mission of the CML is to
assist clinicians in the diagnosis Diagram Ilustrating the 3 types of
and treatment of infectious Hemolysis that Can be Observed on a
diseases. Blood Agar Plate
- The 4 major day-to-day
responsibilities are to:
1. Process various clinical
specimens that are
submitted to the CML
2. Isolate pathogens from those
specimens
3. Identify (speciate) the
pathogens
4. Perform antimicrobial
susceptibility testing, when
appropriate to do so  Mycology Section
- Responsibility is to assist clinicians in
Isolation and Identification (Speciation) of the diagnosis of fungal infections
Pathogens (mycoses)
- The specimens processed here are
• Bacteriology Section the same as those that are
- Bacterial pathogens are isolated from processed in the Bacteriology
specimens, tests are performed to Section, with the addition of hair and
nail clippings and skin scrapings.
 - A variety of procedures are used to
identify fungal pathogens, including
special media, KOH preps, tease
mounts, biochemical tests (for
yeasts), and a combination of
microscopic and macroscopic
observations (for moulds).
Mould Aspergillus fumigatus - a Common
Cause of Pulmonary
 Parasitology Section
- Assists clinicians in the diagnosis of
parasitic diseases
- Parasites are identified by observing
and recognizing various parasite life
cycle stages (e.g., trophozoites,
cysts, microfilariae, eggs, larvae,
adult worms) in specimens –
identified primarily by their physical
appearance (e.g., size, shape,
internal details)
• Virology Section
- Assists clinicians in the diagnosis of
viral diseases
- Techniques used in the identification
of viruses include immuno-
diagnostic tests, cytologic or
histologic examination, electron
microscopy, molecular techniques,
virus isolation by cell cultures, and
cytopathic effect (CPE)
• Mycobacteriology Section (also called
the TB Lab)
- Assists clinicians in the diagnosis of
tuberculosis (TB)
- Various types of specimens are
submitted, but sputum is the most
common type
- Mycobacterium spp. are identified by
the acid-fast staining procedure and
by using a combination of growth
characteristics (e.g., growth rate,
colony pigmentation, photo reactivity,
and morphology) and a variety of
biochemical tests
Pathogenesis
Terms defined
• Infection
- situation in which a microorganism is
established and growing in a host,
whether or not the host is harmed
• Pathogens
- microbial parasites that cause disease,
or tissue damage in a host
• Pathogenicity
- the ability of a parasite to inflict
damage on the host
Human-Microbial Interactions
Microbial Adherence
• Adherence is the enhanced ability of
microbes to attach to host tissues. It is
necessary, but not sufficient, to start
disease.
The infection process:
1. Exposure to pathogens
2. Adherence to skin or mucus
3. Invasion through epithelium
4. Multiplication – growth and
production o virulence factors and
toxins.
The disease process:
1. Toxicity – local and systemic toxin
effects
2. Invasiveness – further growth at
original and distant sites
3. Tissue or Systemic Damage
  Adherence Structures: Capsules
• bacterial capsule forms a thick coating
outside the plasma membrane and cell
wall and serves two important functions
in bacterial pathogenicity
o The capsule is both sticky and
contains specific receptors to
facilitate attachment on host tissues
o Capsules, such as those found in
Streptococcus pneumoniae, protect
the bacteria from ingestion by white
blood cells
 Adherence Structures: Adhesins
e many different receptors coating both the pathogen
es where the bacteria or virus binds
are glycoproteins or lipoproteins found on the
’s surface that enable it to bind to host cells
 Adherence Structures: Fimbriae, Pili,
and Flagella
• Fimbriae, Flagella, and pili are bacterial
cell surface protein structures that
function in attachment
Colonization and Invasion
• Colonization is the growth of
microorganisms after they’ve gained
access to host tissues.
o The process begins at birth.
• Typically starts with mucous
membranes, or tightly packed epithelial
cells coated in mucus, a thick liquid
secretion of glycoproteins
• Growth of the Microbial Community: An
Example from Human Dental Caries
o Dental caries, or cavities, are an
oral microbial disease
o After initial contact, Streptococcus
sobrinius and Streptococcus
mutans attach and reproduce and
form a biofilm called plaque
• Invasion and Systemic Infection
o Invasiveness
- ability of a pathogen to grow in
host tissue at densities that inhibit
host function
- Bacteremia: the presence of
bacteria in the bloodstream
- Septicemia: blood borne
systemic infection; may lead to
massive inflammation, septic
shock, and death
- Infection: any situation in which
a microorganism (not a member
of the local flora) is established
and growing in a host
Becoming Established: Portals of Entry
• Microbe enters the tissues of the body by a
portal of entry
- Usually a cutaneous or membranous
boundary
- Normally the same anatomical regions
that support normal biota
• Source of infectious agent
1. Exogenous
2. Endogenous
(Preferred) Portals of Entry
• Mucous membranes
1. Conjunctiva
2. Respiratory tract: Droplet
inhalation of moisture and dust
particles. Most common portal of
entry.
3. GI tract: food, water, contaminated
fingers
4. Genitourinary tract
5. Skin: Impenetrable for most
microorganisms; can enter through
hair follicles and sweat ducts.
6. Parenteral Route
o Trauma (S. aureus, C. tetani)
o Arthropods (Y. pestis)
o Injections
Numbers of Invading Microbes
ID50: Infectious dose for 50% of the test
population
LD50: Lethal dose (of a toxin) for 50% of the
test population
  Mumps
Bacillus anthracis  Rubella
Portal of Entry ID50  Chickenpox
Skin 10–50 endospores  Common cold
Inhalation 10,000–20,000  Bacteria and fungi causing pneumonia
endospores
Ingestion 250,000–1,000,000 Urogenital portals of entry
endospores • Sexually transmitted diseases (STDs)
• Enter skin or mucosa of penis, external
Infectious agents that enter the Skin genitalia, vagina, cervix, and urethra
• Nicks, abrasions, and punctures • Some can penetrate an unbroken surface
• Intact skin is very tough " few microbes can • Examples
penetrate  Syphilis
• Some create their own passageways using  Gonorrhea
digestive enzymes or bites  Genital warts
 Chlamydia
• Examples  Herpes
 Staphylococcus aureus Pathogens that infect during Pregnancy and
 Streptococcus pyogenes Birth
 Haemophilus aegyptius • Some microbes can cross the placenta (ex.
 Chlamydia trachomatis the syphilis spirochete)
 Neisseria gonorrhoeae • Other infections occur perinatally when the
child is contaminated by the birth canal
The Gastrointestinal tract as Portal  TORCH (toxoplasmosis, other
• Pathogens contained in food, drink, and diseases, rubella, cytomegalovirus, and
other ingested substances herpes simplex)
• Adapted to survive digestive enzymes and
pH changes Portals of Exit
• Examples • Respiratory tract: Coughing and sneezing
 Salmonella • Gastrointestinal tract: Feces and saliva
 Shigella • Genitourinary tract: Urine and vaginal
 Vibrio secretions
 Certain strains of Escherichia coli • Skin
 Poliovirus • Blood: Biting arthropods and needles or
 Hepatitis A virus syringes
 Echovirus
 Rotavirus Steps in the Pathogenesis of Infectious
 Entamoeba histolytica Diseases
 Giardia lamblia • A common sequence of steps in the
pathogenesis of infectious diseases is:
1. Entry of the pathogen into the body.
The Respiratory portal of entry 2. Attachment of the pathogen to some
• The portal of entry for the greatest number tissue(s) within the
of pathogens 3. body
• Examples 4. Multiplication of the pathogen.
 Streptococcal sore throat 5. Invasion or spread of the pathogen.
 Meningitis 6. Evasion of host defenses.
 Diphtheria 7. Damage to host tissue(s).
 Whooping cough
 Influenza Virulence
 Measles • The term “virulent” is sometimes used as a
synonym for pathogenic.
• The degree of pathogenicity
• Determined by its ability to
 Establish itself in the host
 Cause damage
 There may be virulent (pathogenic) strains
and avirulent (nonpathogenic) strains of a
particular species.
 Virulent strains are capable of causing
disease; avirulent strains are not.
 Example: toxigenic (toxin-producing)
strains of Corynebacterium diphtheriae
can cause diphtheria, but nontoxigenic
strains of C. diphtheriae cannot.
 Thus, the toxigenic strains are virulent,
but the nontoxigenic strains are not.
 Sometimes, the term virulence is used to
express the measure or degree of
pathogenicity.
Examples:
 It only takes 10 Shigella cells to cause
shigellosis, but it takes between 100 and
1,000 Salmonella cells to cause
salmonellosis. Thus, Shigella is more
virulent than Salmonella.
 Some strains of Streptococcus
pyogenes (e.g., the “flesheating” strains)
are more virulent than other strains of S.
pyogenes.
 Some strains of S. aureus produce toxic
shock syndrome, but other strains of S.
aureus do not. Those that do are
considered more virulent.
Virulence Factors
 any characteristic or structure of the
microbe that contributes to its virulence are
attributes that enable pathogens to attach,
escape destruction, and cause disease are
phenotypic characteristics that are dictated
by the organism’s genotype.
 Different healthy individuals have widely
varying responses to the same
microorganism: hosts evolve
 Examples:
 Adhesins (ligands) - special molecules
on the surface of pathogens – are
considered to be virulence factors
because they enable pathogens to
recognize and bind to particular host cell
receptors.
 Pili (bacterial fimbriae) are considered
to be virulence factors because they
enable bacteria to attach to surfaces,
such as tissues within the human body.
Adhesins and Receptors
Examples of Virulence Factors
Capsules and Flagella
 Capsules and flagella are considered to
be virulence factors.
 Examples of encapsulated bacteria:
Streptococcus pneumoniae, Klebsiella
pneumoniae, Haemophilus influenzae and
Neisseria meningitidis.
 Flagella are virulence factors because
they enable flagellated bacteria to invade
aqueous areas of the body; may also help
the bacterium to escape phagocytosis.
Exoenzymes
• The major mechanisms by which pathogens
cause disease are the exoenzymes or
toxins that they produce.
• Exoenzymes released by bacteria include:
1. Necrotizing enzymes Coagulase
2. Kinases Hyaluronidase
3. Collagenase Hemolysins
4. Lecithinase
Exoenzymes
• Coagulase: Blood clot formation. Protection
from phagocytosis (virulent S. aureus)
• Kinase: Dissolve blood clot (e.g.:
streptokinase)
• Hyaluronidase: (Spreading factor)
Digestion of “intercellular cement”  tissue
penetration
• Collagenase: Collagen hydrolysis
• IgA protease: IgA destruction
How Pathogens Damage Host Cells
1. Use host’s nutrients; e.g.: Iron
2. Cause direct damage
3. Produce toxins
4. Induce hypersensitivity reaction
Toxins
• Toxins are poisonous substances released
by various pathogens.
• 2 general types:
  Plasmodium spp. (which cause
malaria) and Babesia spp. (which
cause babesiosis) are examples of
intraerythrocytic pathogens; they
live within red blood cells.

Facultative Intracellular Pathogens

1. Endotoxins • Facultative intracellular pathogens are
- Part of the cell wall structure of capable of both an intracellular and
Gram-negative bacteria extracellular existence.
- Can cause serious, adverse • Intracellular Survival Mechanisms
 Possess a cell wall composition that
physiologic effects such as fever and
resists digestion (e.g., Mycobacterium
shock
tuberculosis)
 Fusion of lysosomes with phagosomes
2. Exotoxins
is prevented
- Poisonous proteins secreted by a
 Production of phospholipases that
variety of pathogens
destroy the phagosome membrane,
- Examples: neurotoxins, enterotoxins,
thereby preventing lysosome-
exfoliative toxin, erythrogenic toxin, phagosome fusion
and leukocidins  Other unknown mechanisms

Pathogenic Properties of Viruses

Evasion of IS by
• Growing inside cells
• Rabies virus spikes mimic Ach
• HIV hides attachment site → CD4 long
and slender

Visible effects of viral infection = Cytopathic

Effects
1. cytocidal (cell death)
2. noncytocidal effects (damage but not death)

Pathogenic Properties of Fungi

 Fungal waste products may cause
symptoms
 Chronic infections provoke allergic
responses
Pathogenic Properties of Microorganisms
 Proteases
Obligate Intracellular Pathogens
o Candida, Trichophyton
• Pathogens that must live within host cells
 Capsule prevents phagocytosis
in order to survive and multiply (examples:
o Cryptococcus
Rickettsia and Chlamydia spp.)
 Intraleukocytic pathogens (e.g.,
Ehrlichia spp. and Anaplasma Fungal Toxins
phagocytophilum) live within white • Ergot toxin
blood cells, causing diseases known o Claviceps purpurea
as ehrlichiosis and anaplasmosis • Aflatoxin
o Aspergillus flavus
Pathogenic Properties of Protozoa &
Helminths
• Presence of protozoa
• Protozoan waste products may cause
symptoms
• Avoid host defenses by
o Growing in phagocytes
o Antigenic variation
• Presence of helminths interferes with host
function
• Helminths metabolic waste can cause
symptoms

Microbial Mechanisms of Pathogenicity –

Overview

Mechanisms by Which Pathogens Escape

Immune Responses
• Antigenic Variation
o Some pathogens evade the immune
system by changing their surface
antigens – antigenic variation;
examples, Neisseria gonorrhoeae
and Borrelia recurrentis

• Camouflage and Molecular Mimicry

o Some organisms conceal their
foreign nature by coating themselves
with host proteins – like camouflage
(e.g., adult schistosomes)

• Destruction of Antibodies
o Some pathogens produce IgA
protease, an enzyme that destroys
some of the host’s antibodies;
example, Haemophilus influenzae
Host Defense Mechanisms and Immunology chemical factors, microbial
antagonism, fever, the inflammatory
Host Defense Mechanisms response, and phagocytic white
 ways in which the body protects itself from blood cells
pathogens – referred to as 3 lines of
defense First Line of Defense
o first 2 lines of defense are  Skin and Mucous Membranes as
nonspecific Physical Barriers
o the 3rd line of defense, the immune
response, is very specific  Cellular and Chemical Factors
- in the 3rd line of defense, special  In addition to the skin as a physical
proteins called antibodies are barrier, there are other factors (e.g., pH
produced in response to foreign and temperature of skin, temperature,
substances called antigens perspiration, cilia, and various enzymes
in secretions such as lysozyme) that
Lines of Defense are components of the first line of
defense.

 Microbial Antagonism
 When indigenous microflora prevent
colonization of “new arrivals” as a result
of competition for sites and nutrients and
production of lethal substances.

Second Line of Defense

 Transferrin
 Levels of this glycoprotein increase in
response to systemic bacterial
infections; binds to iron, depriving
Categories of Host Defense Mechanisms pathogens of this vital nutrient

 Fever
 Stimulated by pyrogenic (fever-
producing) substances (e.g., pathogens
and Interleukin 1 [IL-1])
 Augments host’s defenses by
stimulating leukocytes, reducing
available free plasma iron, and inducing
the production of IL-1

 Interferons
Nonspecific Host Defense Mechanisms
 nonspecific host defense mechanisms
are general and serve to protect the
body against many harmful substances
o example: innate or inborn resistance
(exact factors that produce innate
resistance are not well understood.)
o other nonspecific host defense
mechanisms: mechanical and
physical barriers to invasion,
 Small antiviral proteins produced by
virus-infected cells; they prevent viruses
from multiplying
 There are 3 types (alpha, beta and
gamma), produced by 3 different types
of cells
 The 3 types are induced by different
stimuli (e.g., viruses, tumors, bacteria,
and foreign cells)
  Interferons are not virus-specific, but
they are species-specific Interferons can
cause nonspecific flu-like symptoms
 The Complement System
 A group of about 30 different proteins
found in normal blood plasma
–“complementary” to the immune
system
 Complement components interact with
each other in a stepwise manner known
as the complement cascade
 The complement system assists in the
destruction of many different pathogens
 Opsonization is a process by which
phagocytosis is facilitated by the
deposition of opsonins (e.g., antibodies
or certain complement fragments) onto
objects (e.g., pathogens)
Opsonization
Phagocyte,
having no
surface
receptors
which can
attach to the
bacterial
capsule
 Acute-Phase Proteins
 Plasma proteins that increase rapidly in
response to infection, inflammation, or
tissue injury; one example is C-reactive
protein
 Cytokines
 Chemical mediators released from many
different types of cells in the body;
enable cells to communicate with each
other – within the immune system and
between the immune system and other
systems of the body
 Some cytokines are chemoattractants;
they recruit phagocytes to sites where
they are needed
 Inflammation
 the body responds to any local injury,
irritation, microbial invasion, or bacterial
toxin by a complex series of events
referred to as inflammation
 the 3 major events in acute inflammation
are:
o An increase in the diameter of
capillaries (vasodilation) which
increases blood flow to the site
o Increased permeability of the
capillaries, allowing the escape of
plasma and plasma proteins
o Exit of leukocytes from the capillaries
and their accumulation at the site of
injury
 The primary purposes of the
inflammatory response are to:
 Localize an infection
 Prevent the spread of microbial
invaders
 Neutralize any toxins being produced
at the site
 Aid in the repair of damaged tissue
 The 4 major signs and symptoms of
inflammation are: redness, heat,
swelling (edema), and pain
 Plasma that escapes from the capillaries
into the site causes the area to become
edematous (swollen)
Sequence of Events in Inflammation
1. Tissue Injury
2. Vasodilation
3. Increased Permeability
4. Emigration of Leukocytes
5. Chemotaxis
6. Phagocytosis
 The accumulation of fluid, cells, and cellular
debris at the inflammation site is known as
an inflammatory exudate.
 If the exudate is thick and greenish-yellow,
containing many live and dead leukocytes,
it is known as a purulent exudate or pus.
 In many inflammatory responses (e.g.,
arthritis or pancreatitis), there is no exudate
and no invading microorganisms.
 Pyogenic microorganisms (pus-producing
microorganisms) like staphylococci and
streptococci result in additional pus
formation.

Phagocytosis Digestion:
 Phagocytic white blood cells are called
phagocytes, and the process by which
they surround and engulf (ingest) foreign
material is called phagocytosis.
 The 3 major categories of leukocytes
(white cells) found in blood are
monocytes, lymphocytes, and
granulocytes.
o The 3 types of granulocytes are:
eosinophils, basophils, and Mechanisms by Which Pathogens Escape
neutrophils. Destruction by Phagocytes
 The most important groups of  Capsules; initially serve to protect the
phagocytes in the human body are organism from phagocytosis (they serve
macrophages and neutrophils. an antiphagocytic function)
 Some bacteria produce an exoenzyme
Cellular Elements of Human Blood called leukocidin, which kills phagocytes.
 Some bacteria (e.g., Mycobacterium
tuberculosis) are not destroyed within the
phagolysosome.
 The mechanism by which each pathogen
evades digestion by lysosomal enzymes
differs from pathogen to pathogen, and is
not yet fully understood.

Disorders and Conditions that Adversely

Affect Phagocytic and Inflammatory
Processes
 Leukopenia - an abnormally low number of
circulating leukocytes
 Disorders and conditions affecting
Four Steps in Phagocytosis leukocyte motility and chemotaxis
o Inabililty of leukocytes to migrate in
response to chemotactic agents may
be related to a defect in the
production of actin, a structural
protein associated with motility
 Disorders and conditions affecting
intracellular killing by phagocytes (e.g.,
chronic granulomatous disease, CGD)

Additional Factors that Can Impair Host

Defense Mechanisms
Ingestion:  Nutritional status
 Increased iron levels
 Stress
 Cancer and cancer chemotherapy
  Various genetic defects • Acquired immunity = immunity that results
 Age from the active production or receipt of
 AIDS antibodies during one’s lifetime
 Drugs (e.g., steroids)
 Active acquired immunity:
Immunology  Antibodies are produced within the
• Immunology is the scientific study of the person
immune system and immune responses.  Usually provides long lasting
• The primary functions of the immune protection
system are to: Two types of active acquired immunity:
 Differentiate between “self’ and “non- 1. Natural active acquired immunity
self” - occurs naturally
 Destroy that which is “non-self” 2. Artificial active acquired immunity
- artificially induced
• Cells involved in immune responses - Artificial active acquired immunity
originate in bone marrow; results when a person receives a
• 3 lines of lymphocytes are derived from vaccine.
lymphoid stem cells of bone marrow: o A vaccine is defined as material
 B lymphocytes (or B cells), T that can artificially induce
lymphocytes (or T cells) and natural immunity to an infectious disease,
killer cells (NK cells) usually following injection or
 There are 2 categories of T cells: Helper ingestion of the vaccine.
T cells and Cytotoxic T cells o Most vaccines are made from
living or dead pathogens or the
• There are 2 major arms of the immune toxins that they produce.
system:
1. Humoral immunity; where special How Vaccines Work
glycoproteins called antibodies are • Vaccines stimulate the recipient’s immune
produced by B cells to destroy specific system to produce protective antibodies
microbes (i.e., antibodies that will protect the person
2. Cell-mediated immunity; involves a from disease).
variety of cell types, with antibodies only
playing a minor role, if any Types of available vaccines
• Attenuated vaccines
The Two Major Arms of the Immune System • Inactivated vaccines
• Subunit vaccines
• Conjugate vaccines
• Toxoid vaccines

 Passive acquired immunity:

 Antibodies are received that were
produced by another person or
persons or by an animal
 Usually provides only temporary
protection
 Antibodies produced in one person
are transferred to another person to
protect the latter from infection –
Immunity provides temporary protection.

Two types of passive acquired immunity

1. Natural passive acquired immunity
 - Small antibodies, IgG, present in
mother’s blood cross the placenta to Processing of T-Independent Antigens
reach the fetus

2. Artificial passive acquired immunity

- Antibodies from an immune person
are transferred to a susceptible
person; example, hepatitis B immune
globulin
Humoral Immunity
• Antigens
- Foreign organic substances that are
large enough to stimulate the production
of antibodies
- Substances capable of stimulating
antibodies are said to be antigenic
- A bacterial cell has many molecules
(antigenic determinants) on its surface
that are capable of stimulating the
production of antibodies
• Antibodies
- Proteins produced by lymphocytes in
response to the presence of an antigen
- Are in a class of proteins called
immunoglobulins – globular
glycoproteins in the blood that
participate in immune reactions
- The processing of either T-dependent or
T-independent antigens results in B cells
developing into plasma cells, which are
capable of secreting antibodies.
- The initial immune response to an
antigen is called the primary response;
it takes 10-14 days for antibodies to be
produced.
- The increased production of antibodies
following the second exposure to a
particular antigen is called the
secondary response.
Where Do Immune Responses Occur?
• Immune responses to antigens in the blood
are usually initiated in the spleen;
responses to microbes and other antigens
in tissues are generated in lymph nodes
located near the infected area.

Antibody Structure and Function

• Antibodies are a class of glycoprotein called
immunoglobulins;
 5 types = IgA, IgD, IgE, IgG, IgM
• All antibodies are immunoglobulins, but
not all immunoglobulins are antibodies.

Basic Structure of a Monomeric

Immunoglobulin Molecule
- The amount and type of antibodies
produced by a given antigenic
stimulation depend on the nature of the
antigen, the site of antigenic stimulus,
the amount of antigen, and the number
of times the person is exposed to the
antigen.
- The majority of antigens are referred to
as T-dependent antigens because T
cells are required in their processing; the
processing of T-independent antigens
requires only B cells.

Monoclonal Antibodies
• Long-lived, antibody-producing cells called
hybridomas are produced by combining a
single plasma cell and a rapidly dividing
tumor cell.
• Hybridomas are capable of producing large
amounts of specific antibodies called
monoclonal antibodies.
• Monoclonal antibodies are used in
immunodiagnostic procedures (i.e.,
immunologic procedures used in
laboratories to diagnose diseases).
• Monoclonal antibodies are being evaluated
for possible use in fighting diseases, killing
tumor cells, boosting the immune system,
and preventing organ rejection.
Antigen-Antibody Complexes
• When an antibody combines with an
antigen an antigen-antibody complex (or
immune complex) is formed.
• Antigen-antibody complexes are capable of
activating the complement cascade; results
in some of the following effects:
 Activation of leukocytes
 Lysis of bacterial cells
 Increased phagocytosis as a result of
opsonization
Cell-Mediated Immunity
• Antibodies are unable to enter cells.
• Cell-mediated immunity (CMI)
 A complex system of interactions among
many types of cells and cellular
secretions (cytokines)
 An arm of the immune system capable
of controlling chronic infections by
intracellular pathogens (e.g., certain
bacteria, protozoa, fungi, and viruses)
 Examples of cells that participate in
CMI: macrophages, TH cells, TC cells,
NK cells, and granulocytes
• NK (Natural Killer) Cells
 NK cells are in a subpopulation of
lymphocytes called large granular
lymphocytes.
 They resemble lymphocytes, but lack
typical T or B cell surface markers.
 Do not proliferate in response to antigen
and appear not to be involved in
antigen-specific recognition.
 NK cells kill target cells, including
foreign cells, host cells infected with
viruses or bacteria, and tumor cells.
Hypersensitivity and Hypersensitivity
Reactions
• Hypersensitivity refers to an overly sensitive
immune system.
• Different types of hypersensitivity
reactions:
1. Immediate-type: occurs from within a
few minutes to 24 hours after contact
with a particular antigen; 3 types: type I,
II, and III hypersensitivity reactions
2. Delayed-type: usually takes more than
24 hours to manifest themselves
- Also known as Type IV
hypersensitivity reactions.
 Type I Hypersensitivity Reactions
• Type I hypersensitivity reactions are
also known as anaphylactic
reactions; they include:
 classic allergic responses such
as hay fever symptoms, asthma,
hives, and gastrointestinal
symptoms that result from food
allergies
 allergic responses to insect stings
and drugs
 anaphylactic shock
• The Allergic Response
 Type I immediate hypersensitivity
is probably the most common
type of hypersensitivity.
 People prone to allergies (atopic
persons) produce IgE antibodies
when exposed to allergens
(antigens that cause allergic
reactions).
 The allergic reaction results from
the presence of IgE antibodies
bound to basophils in the blood or
to mast cells in connective
tissues – IgE antibodies that were
produced in response to the
person’s first exposure to the
allergen.

• Factors in the Development of
Type I Hypersensitivity
• Events That Occur in Type I
Hypersensitivity Reactions
• Type I hypersensitivity reactions may
be localized or systemic.
1. Localized reactions involve mast
cell degranulation; they result in
allergic reactions, such as hay
fever symptoms, asthma, and
food allergies.
2. Systemic reactions involve
basophil degranulation; they
occur throughout the body, can
lead to anaphylactic shock, and
can be life-threatening.
a. Systemic Anaphylaxis
 Results from the release of chemical
mediators from basophils in the
bloodstream
 Occurs throughout the body – much
more serious than localized
anaphylaxis
 Common allergens involved are
drugs or insect venom
b. Latex Allergy
 Latex can trigger any of 3 types of
reactions: irritant contact dermatitis,
allergic contact dermatitis, and
immediate type hypersensitivity
• Allergy Skin Testing and Allergy
Shots
- Anaphylactic reactions can be
prevented by avoiding known
allergens, which is often difficult to
do.
- Skin tests (scratch tests) are used to
identify offending allergens in
patients.
o A positive test is indicated if
cutaneous anaphylaxis occurs at
the site of the scratch.
- Immunotherapy (i.e., allergy shots - IM
doses of the allergen) may be used to
treat the patient.
- IgG blocking antibodies are produced in
response to allergy shots.
 Type II Hypersensitivity Reactions
• Type II hypersensitivity reactions are
cytotoxic reactions, meaning that
body cells are destroyed during these
reactions.
• Sequence of events in a Type II
hypersensitivity reaction:
1. A particular drug binds to the
surface of a cell.
2. Anti-drug antibodies then bind to
the drug.
3. Complement activation on the cell
surface is initiated.
4. The complement cascade leads to
lysis of the cell.
 Type III Hypersensitivity Reactions
• Type III hypersensitivity reactions are
immune complex reactions – such as
those that occur in serum sickness
and certain autoimmune diseases
(e.g., systemic lupus erythematosus
and rheumatoid arthritis).
• Involve IgG or IgM antiboides,
complement and neutrophils
• Some complications of untreated or
inadequately treated strep throat and
other Streptococcus pyogenes
infections are the result of type III
hypersensitivity reactions (e.g.,
rheumatic fever and
glomerulonephritis).
 Type IV Hypersensitivity Reactions
 • Type IV hypersensitivity reactions are agammaglobulinemia; persons not
delayed-type hypersensitivity (DTH) producing a sufficient amount of antibodies
or cell-mediated immune reactions, are said to have
and are part of cell-mediated hypogammaglobulinemia.
immunity.
- Reactions are usually Immunodiagnostic Procedures
observed 24-48 hours or • Immunodiagnostic procedures (IDPs) help
longer after exposure or diagnose infectious diseases by detecting
contact either antigens or antibodies in clinical
• DTH is the prime mode of defense specimens; test results are usually available
against intracellular bacteria and on the same day!
fungi. • 3 possible reasons for the presence of
• DTH involves a variety of cell types, antibodies to a particular pathogen: present
including macrophages, cytotoxic T infection, past infection, vaccination.
cells, and NK cells -antibodies do not • A variety of different laboratory tests have
play a major role. been designed to observe the presence of
• A classic example of DTH is a an antibody-antigen reaction.
positive TB skin test. Examples of these tests include
agglutination, precipitin tests,
Autoimmune Diseases immunofluorescence, and enzyme-linked
• Autoimmune diseases result when a immunosorbent assays (ELISAs).
person’s immune system no longer
recognizes certain body tissues as “self”
and attempts to destroy those tissues as if
they were “non-self” or foreign.
• May occur with certain tissues that are not
exposed to the immune system during fetal
development and, thus, are not recognized
as “self.”
• There are more than 80 recognized
autoimmune diseases.
• Can be classified as organ-specific or non-
organ specific.
Examples: Hashimoto’s thyroiditis, Graves
disease, etc.
Skin Testing
Immunosuppression • Performed in vivo
• Persons whose immune systems are not • Antigens are injected within or beneath the
functioning properly are said to be skin
immunosuppressed. • Example: the tuberculosis skin test
• Acquired immunodeficiencies may be
caused by drugs (e.g., cancer therapeutic Procedures Used in the Diagnosis of
agents), irradiation, or certain infectious Immunodeficiency Disorders
diseases (e.g., HIV infection). • For assessment of patient’s immune
• Inherited immunodeficiency diseases can status and evaluation of immunodeficiency
be the result of deficiencies in antibody disorders
production, complement activity, phagocytic • Include B-cell deficiency states, cell-
function, or NK cell function; mediated immunodeficiencies,
Examples: DiGeorge syndrome and complement deficiencies, etc.
Wiskott-Aldrich syndrome.
• People born lacking the ability to produce Vaccination
antibodies (i.e., gamma globulins) have • The nature of vaccines
 - Immunization with live cells or a virus is - The host then mounts an immune
usually more effective than with dead or response to the protein.
inactivated material
- Most agents used for immunization are
either attenuated or inactivated
pathogens or inactivated forms of
microbial products, such as toxins
- The importance of immunizations in
controlling infectious diseases is well
established, and the Center for Disease
Control has specific recommendations
for children in the United States
- Immunizations usually involve a series
of secondary immunizations or “booster”
immunizations to produce a secondary
response and a higher antibody titer

• Synthetic and genetically engineered

vaccines
- Synthetic vaccines are the product of
genetically engineering antigenic
components to stimulate the immune
response.
- Conjugate vaccines attach a smaller
antigenic protein to a larger carrier
protein to improve immune response to
the protective antigen.
- Attaching a polysaccharide to a toxoid
improves the immune response of
polysaccharides, which are not as
immunogenic as proteins.
o pneumococcal vaccines are
made this way.

Vaccination
• DNA vaccines
- Target proteins are cloned into plasmid
vectors and injected intramuscularly.
- The DNA is taken up by host cells, and
proteins are expressed.