- Understanding the physiology and genetics

of microorganisms are important to

produce therapeutic agents
- Knowledge of mechanisms whereby
microorganisms are able to resist
antibiotics, colonize medical devices are
essential in the production of new drugs
and in healthcare practices

Chapter 1: Introduction to
Pharmaceutical Microbiology
- A modern medicine must be effective,
safe, and of good quality
- These medicines consists of active
ingredients, stable, and safe during storage
- Analytical Chemists and Pharmacists

Chapter 2: Fundamental features of

microbiology
o

MICROORGANISMS
- microscopic, living, single-celled
organisms
-more versatile than mammals in breaking
down foods.

Pharmaceutical Microbiology
- Foundation
- Encompasses the subject of sterilization
and preservation against microbial spoilage
- Pharmacist must be responsible for the
safe and hygienic manufacturing of
medicines

Differ in:

Antibiotics

1.Shape
2.Size

- Major importance in pharmacy

3.Genetic Characteristics

- Naturally occurring substance that would

inhibit or kill microorganisms

4. Metabolic Characteristics
- Major groups:
o

Bacteria

Fungi

Protozoa

Viruses

CLASSIFICATION OF MICROORGANISMS:
o

Viruses
- do not have cellular structure
- Composition:

- Microbial metabolite
- Synthetic agents that are normally used
systematically to treat infection
- Antibiotic production began with the
discovery of penicillin in the 1940s
- Attack and kill bacteria without harm to
the host

Ribotyping
- Genetic technique used to identify crossinfection, reduce transmission and optimize
management of hospital-acquired infections

-No nucleic acid

Nucleic acid surrounded by

proteins

Some posses lipid envelope

associated w/ glycoprotein

Absent:

-atypical form of mammalian protein

-can interact w/ normal protein molecule
and cause it to undergo conformational
change and make it into a prion and ceases
normal function

-recognizable chromosomes

-responsible for transmissible spongiform

encephalopathies

-cytoplasm
-cell membranes

Ex. Creutzfeldt-jakob disease

-incapable of independent replication

Bovine spongiform encephalopathy

- intracellular parasites

-simplest and most recognized agents of

infectious disese

-reproduced using metabolic capabilities of

host cell

-extreme resistance to conventionall

sterilizing agents (steam,gamma
radiation,disinfectant,etc.)

-smaller than bacteria

- Variation in:

PROKARYOTIC MICROORGANISMS:
-

No true nucleus

Usually single chromosomes

Haploid

Asexual reproduction

Archea

Shape (helical, linear, or

spherical)

Size (20-400nm)

Nucleic acid composition (single

or double-stranded,linear or
circular RNA or DNA)

-Viewed using electron microscope.

Examples : Bacteria, Archea

Viroids (Virusoids)
-simplier than viruses

-no pharmaceutical importance

-infectious particles

-capable of living in extreme environment

-single stranded RNA w/o associated

proteins

-exhibit specialized modes of metabolism

Ex. Plant phatogens

Bacteria
-unicellular
-posses prokaryotic properties

Prions
-infectious agents

- contaminate or cause spoilage of

pharmaceutical products

-exhibit great diversity in form, habitat,

metabolism, pathogenicity

- obtain energy by decomposition

of animal and vegetable materials

-Bacteria of interest in medicine and

pharmacy: Eubacteria

- described as parasites or
pathogens

-many bacteria would be described as

facultative anaerobes or microaerophils
-most bacteria important in medicine and
pharmacy :

Rickettsia and Chlamydia

posses cell wall

- obligate intracellular parasites
grow well at temperatures
between ambient and human
body temperature

- incapable of growing outside host

cell

exhibit wide variation in

requirement for oxygen

EUKARYOTIC MICROORGANISMS:

Strict aerobes = require

atmospheric oxygen

- true cell nucleus

- chromosomes separated from
cytoplasm by nuclear membrane

the

- diploid

Strict anaerobes = oxygen is toxic

Examples:

- sexual reproduction

Eubacteria
- Bacteria of interest in medicine
and pharmacy

Examples: fungi,protozoa,algae,etc.

- types:

1. Bacillus- rod shaped

Fungi

2. Cocci-spherical

- structurally more complex and varied

appearance than bacteria
- non-photosynthizing
- some fungi exhibit unicellular(yeast like)
or mycelial (mould-like) upon cultivation

3. Curved or spiral cell

approximately 0.5 to 5 mm
- Divided into two groups acc. to
Christian Grams 1884 staining procedure.
1. Gram positive

- Most are saprophytes w/ few pathogenic

potential
- ability to make spores that are resistant
in drying = pharmaceutical contaminants

2. Gram negative

Saprophytes

- Fungus: covers both terms yeast and

moulds

Microorganisms are more versatile than humans

in breaking down food, Many can use alternative
methods in breaking down food depending on the
environment, and some can obtain energy from
carbohydrates, digestion of proteins and other noncarbohydrate materials.
o

Chemoheterotrophs
- obtain carbon from nitrogen
- gets energy from breaking down
organic compounds
- organisms of interest in pharmacy
in medicine

Catabolic Reactions
- energy is liberated by digestion of
food materials.

Anabolic Reactions
- use liberated energy to make
complex cellular
polymers,protein,carbohydrates,
and nucleic acids.

Oxidation
- removal or loss of electrons

Yeast
- normally unicellular
- divide by budding
- larger than bacteria
- divide by binary fission or budding

Moulds
- imprecise term to describe fungi
that doesnt form fruiting bodies to
naked eyes
- grow by extension or branching of
hyphae
- excluding toadstool and
mushrooms
- 1 to 50 um wide
- Differentiated for special
function
- consist of tangled mess or
filaments of thread

Protozoa
- predominantly unicellular
- mostly animals than plants
- free living motile organisms

- when food materials are oxidized

energy are released.
- does not invariably involve oxygen
- when oxidizing molecules accept
electron the other molecule is
reduced; reduction and oxidation is
linked and called Redox reaction.
o

Redox Potential
- indicates whether oxidation or
reduction will prevail
- Anaerobic organism = low redox
potential
Aerobes = high redox potential

- occur in water and soil

- not normally found as contaminants
-potential to cause disease
Ex. Organism responsible for malaria and
ameobic dysentery
*for further comparison of eukaryotes and
prokaryotes check Table 2.1 page 11

NAMING OF MICROORGANISMS:
- known by two names genus and species
- written in italic or underlined

MICROBIAL METABOLISM:

Some pathogenic bacteria can

ferment:
different sugar, acids, alcohols,
acetone, butane, etc.

Oxidizing Agents
- molecules that can accept
electrons

Glycolysis
- breakdown of glucose to release
energy

Fundamental Principle of Antibiotic

Action
- drug must exploit a difference in
metabolism between organism to be
killed and the human host.

- metabolic pathway used by

pathogenic bacteria and mammals
- conversion of glucose to series of
reactions to pyruvic acid, oxygen is
not required.

- w/o difference it will be very

toxic to the patient.
o

Primary metabolites
- metabolic products that arise
during period when microbial
culture is growing.

- undertaken by both aerobic and

anaerobic
- release small amount of energy
stored in sugar molecule.

Ex. ethanol, organic acids

o

Secondary metabolites
- diverse

- have commercial or therapeutic

importance

- Oxygen in end of sequence of

respiratory reaction .

- produced after the cell

multiplication has stopped.
o
Ex. Antibiotic,enzymes,toxins,
carbohydrates

Aerobic respiration
- Used by mammals to release more
energy in sugarcompared to
glycolysis.

Fermentation
- an anaerobic process
- alternative to respiration

MICROBIAL CULTIVATION
CULTURE MEDIA
- Some microorganisms have different
sugar fermentation patterns
- Sugars in culture media are usually used
for identification purposes.
- NaCl may be incorporated to adjust
osmotic pressure.

- means of releasing more energy

from sugar
- a process in which in which the
final electron acceptor is inorganic
molecule
- production by yeast of ethanol and
carbon dioxide from sugar
- many organisms can be used as
apart from yeast and is not
restricted to common sugar
(sucrose).

-Glucose: readily employed

as nutrient

CLASSIFICATION BASED ON CONSISTENCY:

1. Solid
- gelled by addition of agar (1-1.5% w/v)
- Agar: from seaweeds ; firms at 37C and
liquid at 45C.
- For anaerobic organisms contain nontoxic
reducing agents
-low redox potential
2. Liquid
- contains no agar.

Selective or Diagnostic Media

- restrict the growth of certain
types of microorganisms while
permitting or enhancing the growth
of others.

- For yeast and moulds have lower pH (5.56.0)

- Lactic acid: used to impart lower pH

CLASSIFICATION BASED ON
COMPOSITION:
1. Truly Synthetic Media
- chemically defined
- for microorganisms that
can synthesize materials
needed for growth from
simple carbon and nitrogen.
2. Media w/ biochemicals
- used for organism that
cant synthesize
- some more commonly
used
- complex w/ precise
chemical composition
- vary slightly batch to
batch
Ex. Routine laboratory media,
General Purpose meda, Complex
Media
- aq. Soln of animals and plants
extracts that contain:

- use additives for identification

o

Enrichment Medium
- designed to permit a particular
type of organism to grow while
others, so the one that grows
increase in relative numbers and is
enriched.

CULTIVATION METHODS
o

Binary Fission
- division pattern
- cell enlarges or elongates then
forms cross wall called septum and
is followed by constriction until
finally broken and daughter cells
separate.

Hydrolysed protein
-inexpensive sources are meet
extracts, milk, and soya
-hydrolysed by varying degree to
give peptones or amino acids
-trypsin and other proteolytic
enzymes preffered to acids

B-group vitamins
-requirement is satisfied by
yeast extract

Carbohydrates
-in form of starch and yeast

- Several situations where number of microbial

cells in culture, sample or specimen are needed to
be measured:
Measuring levels of microbial
contamination in raw material or
manufactured medicine
Evaluating the effects of
antimicrobial chemical or
decontamination process
Using microorganisms in
manufacture of therapeutic agents
Assessing the nutrient capability of
growth medium

-In pyrogen testing and vaccine manufacture both

number of dead and living cells are required
o

Total count
is a counting procedure enumerating both
living and dead cells

Viable count
-records living cells alone

Total Viable Count (TVC)

-used in most pharmacopoeias and by many
regulatory agencies
-mean a viable count that records all
different species or types of microorganism
that might be present in sample.

Three Traditional Methods of Viable

Counting:
Basis: Living cell will give rise to colonies when
introduced w/ suitable medium and incubated.

1.Pour Plating
- surface spread method used

- In optimal conditions of laboratory

cultivation of bacteria this division
takes place every 25-30 minutes.
- growth continues until one or more
nutrient is exhausted or toxic
metabolites accumulates
o

Overnight incubation in liquid media

- culture media clear but becomes
cloudyas concentration increases
- indirect means of monitoring
culture media.

Colony
- usually arise on solid media in
petri dishes
- a collection of cells arising by
multiplication of a single original
cell or a cluster
- in microscope: hundreds to
millions
- typically 1-10 mm
- Periphery of colony; part that is
actively growing

Petri dishes incubated in a anaerobic jar

- Where anaerobic microorganisms
grows

Planktonic Cells
- Routinely used to testing procedures
designed used to assess the activity
- different susceptibilities of lethal
agents
- Reappraisal appropriate

2 Miles Misra Method

- Surface drop
- membrane filter methods used
3. MSP (Most Probable number)
- anticipated count is very low

ENUMERATION OF MICROORGANISMS

- fast ,readily automated, and eliminates

long hours of incubation and numerous petri
dishes

- more commonly used in water,

food, and dairy.
- poor accuracy
- last resort

- not capable of reproducing colonies

Operating method principles:
o Epifluorescent Techniques
o Living cells generate ATP
o Manometer Techniques
o Resistance and capacitance or
impedance of culture media

MICROBIAL GENETICS

*for more information about methods of viable

counting check page 18 Table 2.2 and page 19
Table 2.3

WAYS TO MEASURE BIOMASS:

1. Turbidity Measurements
- most common used in estimating the total
number of bacteria in sample

- Genetic material may be transferred

depending if organism is prokaryote or
eukaryote.

- measured using spectrophotometer or

colorimeter
- not used in fungi

- Mutation is very important for resistance

of antibiotics
o

Bacteria (Prokaryote)
- genes for growth and metabolism:
chromosomes of double stranded
DNA

Bacterial Chromosomes
- tightly coiled
- 1mm contain 1000-3000
genes
- additional genes for
survival advantage under
certain circumstances:
Plasmids

Plasmids
- smaller and replicate
independently
- 0.1-1% size of bacterial
chromosomes
- not essential for normal
functioning
- replicate independently

2. Dry Weight Determination

-for fungi biomass
3.Direct Microscopic Counting
- for bacteria, yeast , and fungal spores
- not for moulds and indirect measure of
biomass

Limitations of traditional method of viable

counting:

Relative labour intensive

Not easy to automate

Slow due to to the need for incubation

May require relative large volumes of

culture media many petri dishes and
incubator spaces

Rapid Methods of detecting and counting

microorganisms:
- enumerate viable organisms(usually
bacteria and yeast)
- employ various means of indirect
detection of living cells

- can be passed to one cell

or another by various means

PHARMACEUTICAL IMPORTANCE OF MAJOR

CATEGORIES OF MICROORGANISMS
o

Genes received from other sources:

Viruses
- importance is based on Pathogenic
potential

Bacteriophages
- Genome of Cell
= Bacterial Chromosomes +
Plasmids + Bacteriophages

- not susceptible to antibiotics

- Hazard Category 4

o
- easy to destroy by heat, radiation,
or toxic chemicals

Eukaryote
- nucleus that contain one or more
pairs of linear chromosomes

Prions

- DNA complexed w/ protein

- withstand sterilizing conditions
- Cells may divide asexually and
undergo mitosis but many have the
potential to undergo sexual
reproduction and undergo meiosis

- ability to cause incurable and

fatal disease

Bacteria
- important as pathogens

- based on RNA instead of DNA

- ability to resist activity of

antibiotics and biocides; long
standing notoriety

- possibility of creating new gene

combination

o
- streptomycetes bacteria produce
antibiotics
- grow on diverse substrates ensure
potential as agents of spoilage

- describes genetic composition

regardless if expressed or not

- can produce bacterial spores

Fungi
- survive in drying
- produce spores
- contaminants

Phenotypic Adaptation
- non-genetic adaptation
- bacteria adopt a phenotypic
change to counter environmental
stress.

- survive well in drying, dust, and

other adverse environments
- contaminants

Genotype

Genetic Adaptation
- Acquire new genes either by
mutation or conjugation
- process of selection ensure that
mutant organisms that are better
suited for new environment
becomes numerically dominant.

organisms in order to minimize both

formation of damaging ice crystals
and osmotic stress that accelerate
cell death during freezing and
thawing.

- Less degree of resistance than

bacteria
- little threat to immunocompetent
individuals

Chapter 3: Bacteria

Protozoa
- significantly large owning to the
pathogenic potential of few species

Prokaryotes
- Smallest free living organism (bacteria
and archaea)
- Lack a true nuclear membrane
* Eukaryotic cells presence of a nuclear
membrane and internal
compartmentalization
Major feature: cytoplasm of membraneenclosed organelles

- Do not poses cell wall

- Do not survive drying well
- Do not display resistance to
sterilization to match bacterial
spores
- More troublesome in veterinary

Differences (Bacteria and Archaea):

- Cell wall composition (major difference)
- Lipid structure making up their
cytoplasmic membranes
- Metabolic patterns

Bacteria:
- Vast majority of
prokaryotes of medical
and pharmaceutical
significance

Archaea:
- most are anaerobes
- inhabit extreme
environments
- greater stability under
extreme conditions
- no disease-causing
archaea have yet been
identified

PRESERVATION OF MICROORGANISMS
- Manufacture of Medicines: microorganisms are
employed in variety of test and assays to
measure activity of antimicrobial chemicals.
- Aim of Culture Preservation: maintain viability of
the highest possible percentage of cells and to
minimize risk of selecting atypical mutants.

- Gram positive bacteria tends to survive better

than gram negative ones.
Most common procedure for long term storage:

Bacteria
- Represent a large diverse group of
organism that can exist as single cells or as
cell clusters
- Have the ability to carry out their life
processes of growth, energy generation and
reproduction independently of other cells
* Very different from the cells of animals
and plants (unable to live alone in nature,

1. Freezing at -80C in refrigerators

2. Storage in liquid nitrogen at -196C special
vessels
3. Lyophilization or freeze-drying
o

Cryoprotectant Chemicals
-compounds like glycerol or
dimethylsulphoxide
- incorporated at concentrations
10% v/v in liquid culture of

Rarer morphological forms:

a. Actinomycetes
rigid bacteria resembling fungi that may
grow as lengthy branched filaments
b. Mycoplasmas
lack a conventional peptidoglycan
(murein) cell wall
highly pleomorphic organisms of indefinite
shape
c. Some miscellaneous bacteria stalked,
sheathed, budded, and slime producing forms
often associated with aquatic and soil
environments

Cellular components
- Simple base cell structure compared with
eukaryotic cells

Reasons to have a good knowledge of the

bacterial cell structures and functions:
- Provides an excellent route for probing the
nature of bacterial processes many of which are
shared by multicellular organisms
- Normal bacterial processes can be customized to
benefit society on a mass scale
- To know how to destroy bacterial contaminants
and disease-causing organisms (pharmaceutical and
healthcare perspective)

Cell Wall

exist only as a part of a multicellular

organism)

- Capable of growing in a range of different

environments
- Cannot only cause contamination and
spoilage but also a range of different
diseases

Bacterial Diversity and Ubiquity

- Bacteria are diverse in shape and sizes
(morphology), adaptation to environment,
survival strategies, and metabolic processes
- The presence of bacteria may be
considered ubiquitous. There is no natural
environment that is free from bacteria.

Cell Size and Shape

- Majority of bacteria are 1-5 um long and
1-2 um in diameter
* >5 um (ex: Thiomargarita namibiensis)
extremely rare
* bacterial size increased, efficient, and
rapid transport and growth rates
- Classification of bacteria is made through
morphological grounds
- Mostly unicellular and possess simple
shapes: round (cocci), cylindrical (rod,
bacillus), or ovoid

- LPS determines the antigenicity of the

bacteria
*extremely toxic to animals
- LPS is made up of lipid A, core
polysaccharide and O-specific
polysaccharide
- The cells lose the crystal-violet iodine
complex and are rendered colorless (gram
stain)
- Appear red under the light microscope

Cytoplasmic membrane
- Fragile phospholipid bilayer with protein
distributed randomly throughout
- Involved in various transport and enzyme
functions associated with the membrane
- Transports of nutrients, energy generation
and electron transports
- Selective barrier between the cytoplasm
and the cell environment

- Essential for the maintenance of the shape

and integrity of the bacterial cell
- An obvious target for antibiotics (Cell
Lysis)
- Provide a strong, rigid structural
component that can withstand the osmotic
pressure caused by high chemical
concentrations of inorganic ions in the cell
- Most bacterial cell walls have
peptidoglycan layer (murein/ glycopeptide)
exceptions include the Mycoplasmas,
extreme halophiles, and the archaea
- Peptidoglycan is composed of Nacetyl
muramic acid (NAM) and Nacetyl
glucosamine (NAG)

Bacteria can be divided into two large

groups (on the basis of a differential staining
technique called the Gram stain): Gram-positive,
Gram-negative

Gram-positive Cell Wall

Cytoplasm
- Consists of 80% water and contains
enzymes that generate ATP
- Compose of the ribosomes, nucleoid and
inclusion granules

Nucleoid
- Singular, covalently closed circular
molecule of double stranded DNA

Plasmids
- Relatively small circular pieces of double
stranded extrachromosomal DNA

- Consist primarily of a single type of

molecule
- Contains teichoic acids and lipoteichoic
acids (negatively charged)
- During an infection, lipoteichoic acids
molecules trigger an inflammatory response
- Retain the dye (gram stain)
- Appear purple under the light microscope

Gram-negative Cell Wall

- Multilayered structure, quite complex

- For autonomous replication

- Encode many auxiliary functions that are
not usually necessary for bacterial growth
(antibiotic resistance)

- Compose of proteins, lipoproteins,

phospholipids and lipopolysaccharide that
are unique to gram negative bacteria

- Biofilm formation begins with attaching to

surface and form cement cells to protect
the bacteria from hazardous materials

- transfers, increasing the spread of

resistance

Ribosomes
BACTERIAL SPORULATION

- The site of protein synthesis

- Process in which the vegetative cell undergoes a

profound biochemical change to give rise to a
specialized structure called an endospore or spore
- Not part of a reproductive cycle

- Two subunits: 30S and 50S

Spore

Inclusion granules
- Serves as the storage material for carbon,
nitrogen, and sulphur or phosphorus

Highly resistant

Cell surface components

Enables producing organism to survive in

adverse environmental conditions (lack of
moisture or essential nutrients, exposure
to toxic chemicals, radiation or high
temperatures)
All sterilization process for pharmaceutical
products have been designed to destroy
the bacterial spore

Flagella
- Bacterial motility
a. Monotrichous a single polar flagellum
b. Lophotrichous two or more flagella at one pole
of the cell
c. Amphitrichous single/tuft of flagella at each
end of the cell
d. Peritrichous flagella distributed over the
entire cell

Endospore structure
Endospores
Differentiated cells that possess a
grossly different structure to that
of the parent vegetative cell in
which they are formed
Exosporium
Outermost layer

Cortex

Composed of protein; within are

the spore coats (proteinaceous but
with a high cysteine content)

Consists of loosely cross-linked

peptidoglycan

Central core
Contains the genome

Partially dehydrated (dehydration

shown to increase resistant to both
heat and chemicals)
Containing only 10-30% of the
water content of the vegetative
cells

Pili and Fimbriae

a. Pili (pilus) join bacterial cell in preparation of
DNA and to environmental surfaces
involved in the genetic exchange
process of conjugation
b. Fimbriae for adherence of cells to one another
and to environmental surfaces
responsible for hemaglutination
and cell clumping in bacteria
Glycocalyx (Slime Layer and Capsule)
- General substances that surround cells
- Gelatinous polymer of polysaccharide,
polypeptide, or both
a. Slime Layer unorganized and loosely attached
to the cell wall
b. Capsule substance is organized and firmly
attached to cell wall

Biofilms
- Any surface for microbial habitat
- Usually contains more than one species of
bacteria which exist and cooperate
together

Classified as either endotoxin

(ex. Cell wall-related,
exotoxin, products released
extracellularly as the organism
grows)

Lipid A component of LPS

Possesses multiple biological

properties including ability to
induce fever, initiate the
complement and blood
cascades
Activate B lymphocytes and
stimulate production of tumor
necrosis factor
Released from lysed or
damaged cels
Depyrogenation; process where
care must be taken to
eliminate or exclude such heatresistant material from
parenteral products and their
delivery systems.

Endotoxin

Exotoxin

A-B toxins; consists of a B

subunit that binds to a host
cell receptor and is also
covalently bound to the A
subunit that mediates the
enzymic activity responsible
for toxicity (ex. Diphtheria
toxin, cholera toxin)
Cytolytic toxins; do not have
seperable A and B portions but
work by enzymatically
attacking cell constituents,
causing lysis (ex. haemolysins
and phospholipases)
Superantigen toxins; lack an AB type structure and act by
stimulating large numbers of
immune response cells to
release cytokines, resulting in
a massive inflammatory
reaction (ex. Staphylococcus
aureus)

pH; 1 unit lower than the

cytoplasm of the vegetative cell
and contains high levels of corespecific proteins that bind tightly
to the DNA and protect it from
potential damage
Core-specific proteins; function as
an energy source for the outgrowth
or germination of a new vegetative
cell from the endospore

Spore

Presence of dipicolinic acid and

high levels of calcium ions which
complex together

Endospore formation
Vegetative cell undergoes a complex series of
biochemical events in cellular differentiation
Sporulation; accomplished by activation of a
variety of spore-specific genes such as spo and
ssp
Leads to the production of a dry, metabolically
inert but extremely resistant endospore
Endospore germination
Reversion of endospore back to a vegetative cell
Removal of the stress inducer that initiated
sporulation
Germination loss of resistance properties; occurs
along with a loss of calcium dipicolinate and
cortex components, and degradation of the corespecific proteins

BACTERIAL TOXINS
- Organisms, if presented with the correct set of
conditions, can cause disease
- (ex. Opportunist pathogens; Staphylococcus
epidermis,non-pathogenic environmental
organism; Ps. Aeruginosa)

Toxins

Products of bacteria that

produce immediate host cell
damage

Growth and solid surfaces

Solidified growth media are deployed to
separate different types of bacteria and
also as an aid to enumerating viable cell
numbers in the laboratory
Agar media are used in the laboratory
either poured as a thin layer into a
covered dish or contained within a small,
capped bottle
The colour, size, shape and texture of
colonies of different species of bacteria
very considerably and form a useful
diagnostic aid to identification

BACTERIAL REPRODUCTION AND GROWTH

KINETICS
- Multiplication and division cycle
Binary fission; process where the majority
of cells multiply in number
Each daughter cell will automatically
contain those materials that are dispersed
through the mother cell (mRNA, rRNA,
ribosomes, enzymes, cytochromes, etc.)
Bacterial chromosome; circular and
attached to the cytoplasmic membrane
where it is able to uncoil during DNA
replication

Growth in liquids
Growth ceases when the rate of
consumption of nutrients exceeds the rate
of supply
Bacteria (being of colloidal dimension and
sometimes highly motile, are dispersed
evenly through the fluid (nutrients are
equally available to all cells)

Liquid batch culture (closed)

Logarithmic growth phase (B); during
active growth a logarithmic plot of cell
number against time gives a straight line
Lag period (A); the inoculum adapts its
physiology to that required for growth on
the available nutrients
Late logarithmic phase; rate of growth

Stationary phase (C); eventual halt

Decline phase (D); starvation, death of
some of the cells and adaptation to a
dormant state

DNA replication if based on the number of

base pairs within it and the growth
temperature (ex. Escherichia coli;
replication of chromosomes will take
approximately 45 minutes)
Gram-negative cells; do not have rigid cell
wall, must develop a cross-wall that
divides the cell into two equal halves
Rod-shaped organisms; maintain their
diameter during the cell cycle and
increase their mass and volume by a
process of elongation
Coccal forms; increase in size by radial
expansion
Population growth
Cell numbers will increase exponentially
as a function of time
Generation time; the time interval
between one cell division and the next
A mean generation time is usually
calculated when considering a growing
culture containing thousands of cells

 F-factor (fertility factor); simplest form of

plasmid
F-factor will simply transfer a copy to a
recipient cell

ENVIRONMENTAL FACTORS THAT INFLUENCE

GROWTH AND SURVIVAL
Gram-negative bacteria tend to be aquatic
Gram-positive bacteria then to prefer
more arid conditions such as the skin
Physiochemical factors that affect
growth and survival of bacteria
Temperature
Permissive temperature; range of
temperatures under which bacteria can
actively grow and multiply
Chemical and enzymic reactions within
the cell proceed more rapidly, and growth
becomes faster until an optimal rate is
achieved as temperature rises
Beyond such temperature, certain
proteins may become irreversibly
damaged through the thermal lysis,
resulting in a rapid loss of cell viability
pH
PH effects on growth are bell-shaped
Extremes of pH can be lethal
Growth optima of 7.4 and 7.6;
microorganisms that have medical or
pharmaceutical significance
May dictate the range of microorganisms
that could potentially cause its spoilage
Water activity/ solutes
Gram-negative cell envelope cannot
withstand the high internal osmotic
pressures associated with rapid
rehydration after desiccation
Water activity (Aw); vapor pressure of
water in the space above the material
relative to the vapor pressure above pure
water, can markedly affect its
vulnerability to spoilage contaminants

Availability of oxygen
Oxygen acts as the terminal electron
acceptor in respiration and is essential for
growth

Growth in open culture

Bacteria make up >90% of the dry mass of
faeces
In many situations the bacteria become
immobilized, as a biofilm, upon a surface
and extract nutrients from the bulk fluid
phase

Growth and genetic change

Transformation
Ability of certain types of bacteria to
absorb small pieces of naked DNA from the
environment that may recombine into
recipient chromosome
Means of transferring genes between
different types of bacteria
Transduction
Bacterial DNA having moved between cells
Temperate phage; rather than enter a
replication cycle, the viral DNA becomes
incorporated by recombination into the
chromosome of the bacterium
Conjugation
Thought to have evolved through
transduction
Plasmids; DNA strands

concentration, do not
discriminate between living
and dead cells
Microcalorimeters; time taken
to detect such heat can be
directly related to the numbers
of viable cells present

Enrichment culture
Intended to increase the dominance of a
numerically minor component of a mixed
culture such that it can be readily
detected on an agar plate
Enrichment media; always liquid, intended
to provide conditions that are favourable
for the growth of other likely isolates
MacConkey broth; contains bile salts that
will inhibit the growth of non-enteric
bacteria and may be used to enrich for
Enterobacteriaceae

Selective media
Solidified enrichment broths,
intended to suppress the growth of
particular groups of bacteria and to
allow the growth of others
Counts of colonies obtained on
selective solid media are often
documented as presumptive counts
Identification media
(diagnostic)
Contain nutrients and reagents that
indicate, usually through some form
of colour formation, the presence
of particular organisms

Microscopy
Simple stains (such as the Gram stain)
Size, shapes, arrangement into clusters,
chain and tetrads, specific stains for the
presence of endospores, capsules, flagella
and inclusion bodies

Fermentation; carbon substrate is in

excess

Nutrition and growth

Chemolithotrops; simple inorganic forms
of elements, can utilize atmospheric
carbon dioxide and nitrogen as sources of
carbon and nitrogen
Diauxic growth; second lag phase during
the logarithmic growth period while such
adaption takes pace

DETECTION, IDENTIFICATION AND

CHARACTERIATION OF ORGANISMS OF
PHARMACEUTICAL AND MEDICAL
SIGNIFICANCE
Culture technique
- Diluting the sample to varying degrees and
inoculate the surface of a predried nutrient
agar with known volumes of those dilutions
- Enumeration media
Will only ever culture a subset
of cells towards which the
medium and incubation
conditions are directed
Simple salts media with
relatively simple sugars as
carbon sources and trace levels
of amino acids; often used to
enumerate bacteria associated
with water
Psychrophilic Gram-negative
bacteria; can be a major
source of bacterial pyrogen
Highly nutritious media are
also used as enumeration
media (ex. blood agar)
Rapid enumeration techniques

Bioluminescence

Epifluorescence

Impedance techniques

Coulter counters; used to

determine bacterial

Bread

Enzymes

Antibiotics

Recombinant proteins

Biochemical testing and rapid

identification
Differing ability of bacteria to ferment
sugars, glycosides and polyhydric alcohols
(widely used to differentiate the
Enterobacteriaceae and in diagnostic
bacteriology generally)
Results of oxidase and catalase tests
performed directly on isolated colonies
Molecular approaches to identification
Have not yet become routinely adopted in
the analytical or diagnostic laboratory
Denaturing gradient gel electrophoresis
(DGGE); isolates and amplifies 16S
ribosomal DNA and, following sequencing
of the bases, compares this with known
sequences held in a reference library
Gene probes carrying fluorescent dyes;
can be used in hybridization procedures
with the collected clinical material

FUNGI
O
O
M
Y
C
E
T
E
S

A
S
C
O
M
Y
C
E
T
E
S

B
A
S
I
D
I
O
M
Y
C
E
T
E
S

T
E
L
I
O
M
Y
C
E
T
E
S

U
S
T
O
M
Y
C
E
T
E
S

D
E
U
T
E
R
O
M
Y
C
E
T
E
S

Pharmaceutically and medically

relevant microorganisms
Broadly classified into those organisms
that are harmful or problematic, and
those that can be used to our advantage

The Kingdom Fungi can be subdivided into six

classes:

Oomycetes

Refer to Table 3.3 of book; examples

of some pharmaceutically useful bacteria

-contains the mildews and water moulds

Ascomycetes
-contains the mildews, some moulds and
most yeast species (including
Saccharomyces cerevisiae)

Chapter 4: Fungi
Fungi

Basidiomycetes

Eukaryotic organisms

-contains the mushrooms and bracket fungi

Widely distributed in nature

Teliomycetes

Extremely important group of microbes in

the medical field

Responsible for a number of potentially

fatal diseases in humans

Great benefit in humans in terms of:

-contains the rust fungi (plant pathogens)

Ustomycetes
-contains the smuts (plant pathogens)

Deuteromycetes

Production of alcoholic beverages

Average thickness varies from 100 to 300

nm

COMPOSITION OF FUNGAL CELL

WALL

Cell Wall

Glucan, the main structural component of

fungal cell wall, is a branched polymer of
glucose.
The innermost layer is rich in glucan and chitin
which provides rigidity to the wall and its
important in regulating cell division.

5060%

G
L
U
C
A
N

S
T
R
U
C
T
U
R
A
L

M
A
N
N
A
N

POLYSACCHARIDES

25
%

15-23%

C
H
I
T
I
N

P
R
O
T
E
I
N
S
AND

1-9%

L
I
P
I
D
S

-contains species such as Aspergillus,

Fusarium and Penicillium
There are four distinct phyla within the fungal
kingdom; these are the Chytridiomycota,
Zygomycota, Ascomycota and Basidiomycota.

Periplasmic space

a thin region that lies directly below the

cell wall

contains secreted proteins

Grow as single cells

location for a number of enzymes required

for processing nutrients prior to entry into
the cell

Reproduce asexually by budding, although

a minority of species reproduce by fission

Many yeast species are capable of sexual

production and formation of spores

Cell membrane / Plasmalemma

Yeast

Moulds

located directly below the periplasmic

space

Grow as masses of overlapping and

interlinking hyphal filaments

a phospholipid bilayer which contains

phospholipids, lipids, protein and sterols.

Reproduce by producing masses of spores

Approximately 10 nm thick

Nucleus

A discrete organelle

Structure of the fungal cell

Oval in shape

Surrounded by a rigid cell which contains

structural polysaccharides

suppressed as a result of therapy or

disease.

Most of the cells genome is concentrated

in the nucleus

The most common fungal pathogens:

Repository of the DNA

Contains proteins in the form of histones

Yeasts

Moulds

Dermatophytes

The powerhouse of the cell

Medically important fungal pathogens of

humans

Possesses its own DNA

Capable of producing its own proteins on

its own ribosomes, mitoribosomes

Candida albicans
o

Most frequently encountered

human fungal pathogen

Enzymes of tricarboxylic acid cycle are

located in the matrix of the mitochondrion

Responsible for a wide range of

superficial and systemic infections

Opportunistic fungal pathogen

which can be present as a normal
part of the bodys microflora

Electron transport and oxidative

phosphorylation occur in the mitochondrial
inner membrane

The outer membrane contains enzymes

involved in lipid biosynthesis

Mitochondrion

Exist in two morphologically

distinct forms: budding
blastospores or hyphae
Plays an important role in the
degradation of the
immunoglobulins IgG and IgA

Ribosomes

The site of protein biosynthesis

Mediates the export of proteins from the

cell

Important in allowing yeast access

iron released from ruptured red
blood cells

Vacuole

A storage space

Capable of giving rise to a variety

of interconvertible phenotypes
which can be considered as
providing an extra dimension to the
existing virulence associated with
this yeast.

Nutrients, hydrolytic enzymes or metabolic

intermediates are retained until required

Aspergillus fumigatus
o

a saprophytic fungus

the dominant fungal pulmonary

pathogen of humans

Medical significance of fungi

A significant group of pathogens capable of

causing a range of diseases

Although majority of fungi appear to be

harmless to humans but a normally nonpathogenic fungus can cause a clinically
relevant problem if the immune system is

be consumed directly as a dietary

supplement
o

Obstructs capillaries particularly in

the brain with concomitant damage
to surrounding tissues

generally presents a problem in

those with pre-existing lung
disease or damage

frequently encountered growing on

decaying vegetation and damp
surfaces

Histoplasma capsulatum
o

a dimorphic fungus

cause of histoplasmosis, the most

prevalent fungal pulmonary
infection

Penicillium marneffei
o

Very rare and inconsequential

cause of disease in humans

The most frequent cause of fungal

disease in AIDS patients who reside
in, or visited, South East Asia

No longer regarded as a GRAS

(generally regarded as safe) but is
now classified as a Biosafety Level
1 pathogen

Cryptococcus neoformans
o

an encapsulated yeast

An asexual, dimorphic fungus

growing as a mycelium at 37C in
tissue and as single cells at 28C

most frequently associated with

infection in immunocompromised
patients

meningitis is the most common

clinical manifestation

Reproduce by fission

a facultative intracellular pathogen

that is capable of surviving and
replicating within macrophages and
withstanding the lytic activity
within these cells

Non-albicans Candida species

o

Candida dubliniensis

-dominant cause of oral candidosis

of HIV-positive and negative
populations from many parts of the
world
o

group of keratinophilic fungi which

can metabolize keratin

the principal protein in skin, nails

and hair

Candida krusei
-significant cause of disease in HIVpositive patients, diabetics, and
cancer patients
-dominant fungal pathogen in
certain classes of diseases

Dermatophytes

Emerging fungal pathogens

Saccharomyces cerevisiae
o

Better known as brewers yeast

or bakers yeast

Used for the production of bread

and alcoholic beverages, and can

Candida glabrata
-a serious cause of disease in
neutropenic cancer patients and

 Viral nucleic acid:

-

The viral nucleic acid is composed of

either DNA or RNA

-Fourth most commonly isolated

Candida species

A virus can only have a single genome

Viral capsid (protein core)

-

It protects the viral nucleic acid from

detrimental, chemical and physical
conditions

It is composed of a number of subunits

named capsomeres genetically
encoded by the viral genome

Capsomeres give the shape of the

capsid, and provide the virus with
resistance to physical and chemical
agents

Viral envelope

Antibiotic production of fungi

Majority of antibiotics obtained from fungi

are produced by fermentation and most
are secondary metabolites.

Isolation of Penicillium notatum by Sir

Alexander Flemming Most important
discovery regarding the beneficial use of
fungi for humans

Antibiotic production can be maximized by

optimizing production as a result of
random mutagenesis and selection.

Chapter 5: Viruses

Most outer covering of a virus

The envelope is added during the

replication process

Viruses were first discovered at the end of

19th century

It can come from the host cell nuclear

membrane, or the cytoplasmic
membrane

They were classified as filterable agents

because they can be retained by filtration

Enveloped viruses are considered to be

the most susceptible to chemical and
physical conditions

They do not survive well on their own

outside the host cell, although they
can persist longer in organic soil

III. Virus-host cell interactions

has been responsible for mortality

rates of 5-38%

Viruses can interact with the host cell in

five different ways:
1. Multiplication of the virus and
destruction of the host cell upon
release of the viral progeny

I. Introduction

II. General structure of viruses

Viruses are extremely diverse in size and

shape

Viruses are much smaller than bacteria

Intracellular parasites

Smallest virus: poliovirus -> 28nm in size

Largest virus: mimivirus -> 750nm in size

Viruses with envelope: enveloped

nucleocapsid

Viruses with no envelope: naked

nucleocapsid Components of a virus:

Envelope fuses from

the membrane

It is a process wherein
virions can fully enter
the membrane

Endocytosis: When an
envelope nucleocapsid fully
enters the host cell, it
undergoes endocytosis.

2. Multiplication of the virus and release

of the virions without the immediate
destruction of the host cell
3. Survival of the virus in a latent stage
without noticeable changes to the
infected cell
4. Survival of the infected cell in a
dramatically altered or transformed
state
5. Incorporation of the viral nucleic acid
in the host cell genome without
noticeable changes to the infected cell

Envelope fuses with the endosome

Uncoating - releases capsin to
free nucleic acids (DNA or RNA)

IV. Multiplication of human viruses

Objective of replication cycle: to

ensure the multiplication of the virus
with the formation of identical viral
progeny

Assembly or maturation maturation of virus cells; cells

become virions again

The multiplication cycle of human

viruses is generally slow, from 4 to
more than 40 hours

Release - virions are release

outside the host cell, to infect
other cells

Bacterial viruses are generally faster

and can take a little as 20 minutes to
replicate within the bacterial host

There are six distinct phases in a

replication cycle:

Eclipse stage: replicates and

expresses genes

V. Cultivation of human viruses

1. Cell culture:

May be divided into three types

according to their history:
Primary cell lines: The cell
lines are derived directly from
an intact tissue, ex: human
embryo, kidney or monkey
kidney
Secondary cell cultures:
derived from primary cultures,
usually those arising from
embryonic tissue
-

The cells are more

homogenous, better

Adsorption - attachment to the

host cell receptor
Penetration - penetrates the
virus through the cell
membrane. It has three types:
o

Direct injection: type of

virus involved -> naked
nucleocapsid
-

the virus did not fully

enter the host cell

Fusion: type of virus

involved -> envelope
nucleocapsid

The drug used for HIV

infections is called
antiretrovirals

Antiretrovirals have
considerably prolonged the life
expectancy of patients,
although not without some side
effects

These drugs aim to reduce HIV

plasma level as much and as
long as possible

characterized, but might not

be as susceptible to viral
infection as primary cell lines
Continuous cell lines: usually
derived from malignant tissue,
and have the capacity to
multiply indefinitely in vitro

Cytophatic effect: a characteristic

morphological change in the
infected cells wherein the cells
shrink, or undergo ballooning.

They usually spread to adjacent

cells and will result in the
formation of a plaque that can
easily be identified following
staining

Plaques: used for the enumeration

of viruses

Herpesvirus infections
-

Herpesviridae: a family of
viruses which include the
herpes simplex virus,
chickenpox, shingles and
cytomegalovirus
Mild herpes simplex virus ->
treated with a topical antiviral
drug

Primary herpetic
gingivostomatitis -> a change
of diet & analgesics

Severe infections -> systemic

antiviral is used

Antiviral treatments for

chickenpox: recommended in
patients at risk and in neonates
to reduce risks of severe
diseases

Antiviral treatments for

herpes: is associated with a
number of side effects which
may vary depending on the
drug

Antiviral treatments for

cytomegalovirus: usually given
to immunocompromised
patients and they tend to be
more toxic with noticeable
nephrotoxicity and a number of
side effect

2. The chick embryo

Fertile chicken eggs, (9-11 days old)
are used to grow a number of
human pathogenic viruses.

3. Animal inoculation
Animals are used to culture certain
viruses in order to study antiviral
vaccine effectiveness, and also as a
source of cell lines for cell cultures

VI. Control of viruses

1. Antiviral chemotherapy - leads to the
development of viral resistance, but is still
associated with a number of problems
a. HIV
-

The role of antivirals in HIV is

to slow or halt disease
progression

influenza vaccines rely on

chemically inactivated, virus
particles or components

Inactivated viruses
Use of viral components

Viral hepatitis
-

Treatment for acute hepatits B

-> interferons

Treatment for chronic hepatitis

B -> antivirals

Hepatitis B: viral DNA encoding for a

virus surface antigen expressed in
yeasts
Immunoglobulin: plays a role in the
protection of patients with a
compromised immunity against viral
infections

Influenza
-

Two major limitations in the

usefulness of the drug

First: the drug needs to be

taken within a few hours of the
onset of symptoms from mild
to severe symptoms reported

Second: the side effects have

been very severe

IM immunoglobulin used to protect

against hepa A virus

Viricidal effects of chemical and physical

agents on viruses

Viruses are generally transmitted

via surface and are often
associated with organic materials

In general, viruses are not

particularly resistant to chemical
or physical agents, although some
exceptions exist.

Respiratory syncytial virus

-

RSV is responsible for severe

bronchiolitis notably in infants

Treatment can be a
monoclonal antibody or an
antiviral drug

Control of viruses in pharmaceutical

products

Presence of certain viruses needs

to be controlled
Risk of a pharmaceutical product
being contaminated by viruses
depends on:

2. Vaccination
the most successful measure against
microbial and viral infections
-

The origin of the product

component
The history of the donor
The amount of material used
The manufacturing process
Its capacity to remove or
destroy contaminants

Vaccines are preparations

containing antigents that elicit
a specific and active immunity
against an infecting agent

Vaccines can induce the innate and the

adaptive parts of the immune system

Viral vaccines prepare using 3 methods:

Live attenuated viruses: will
cause a strong immune
response without causing the
disease Hepatitis A and

Use of bacteriophages to treat bacterial

infection
Introduction of antibiotics in the
early 1940s resulted in the end of
phage therapy in the West
o

Natural phages

Non-replicating phages

Genetically modified phages

Use of phages for surface

disinfection and antisepsis: further
work is still needed to develop
appropriate phage-based products,
especially the effect of the
different routes of administration
on phage viability and effectiveness

VII. Viruses as antimicrobials

1. Bacteriophages

Bacteriophages are viruses that

infect only bacteria

They were first described at the

end of the 19th century

Size: 20-200nm, and are highly

diverse in their structure and host
range and it is likely that all
bacterial species can be infected
by a phage

Phages are extremely specific in

their host range and some will only
infect a specific bacterial strain

Most studied phages are the

complex ones, tadpole-shaped
which consists of a head that
contains the viral genome, and a
tail which function is to recognize
the host receptor, attach and
subsequently serve as a nucleic
acid injection device

Two phage replication cycles:

Epidemiological uses and diagnosis

Phage typing: a method that
differentiates distinct strains of the
same bacterial species on the basis
of their susceptibility to phages

VIII. Prions

Prions devoid of nucleic acid and are

extremely resistant to heating and
ultraviolet irradiation

They fail to produce an immune response

in the host

Lytic cycle: lysis of bacterial host

Lysogenic cycle: result of a viral
nucleic acid being integrated into
the host genome
Infection with lytic phage ->
virulent phage, results in the
replication of phage within the
susceptible bacteria and the release
of infectious phage progeny from
the host cell following cell lysis
Lysogenic cycle: viral nucleic acid
which has integrated the host
genome is called prophage
Host cell that contains the viral
genome: lysiogenic