1.

Introduction to Medical Microbiology – History and Subject

What is microbiology?

Microbiology is the study of organisms too small to be seen by the naked eye (<1mm diameter). Includes
bacteria (bac), fungi, viruses, protozoa and algae. Microbes can be unicellular, cell clusters or multicellular.

- Medical microbiology = concerned w/microbes which are pathogenic to humans

History

Robert Hooke created a microscope in the 17th century by changing the lens of a telescope. In 1665 =
published Microphagia describing observations he made and he coined the term cell for describing biological
organisms

Anton Van Leeuwenhoek = 1st person to observe microorganisms (MOs) using a microscope. 1684 = placed a
toothpick in his mouth and prepared a slide and observed what he saw using his microscope. He saw bac
which he called animalcules and also observed motility. He didn’t understand what he saw.

Lazzaro Spallanzani = in 18th century identified that boiling fluids would sterilise and kill MOs, and that these
MOs had a relationship w/air

Louis Pasteur = proved that MOs couldn’t spontaneously grow, instead they have to travel via spores,
removing the theory of spontaneous generation and supporting germ theory instead.

Ferdinand Cohn = founder of the field of bacteriology. He discovered bacillus, described their lifecycle and
endospore formation. Learnt that bacillus produces an endospore that allows survival in harsh
environments. In the mid 19th century he classified bac into 4 groups based on shape – spherical (cocci),
rods, threads, and spirals

Robert Koch = showed that MOs caused disease by studying anthrax Bacillus anthracis.

2. Bacterial Taxonomy: Classification, Nomenclature and Identification

Classification

The term “taxonomy” refers to the branch of science that is concerned with classification of organisms.
Bacteria are grouped and named depending on their morphological, biochemical and metabolic differences
as well as their immunological and genetic characteristics.

Bacterial taxonomy  the rank-based classification of bacteria established by Carl Linnaeus – Domain,
Kingdom, Phylum, Class, Order, Family, Genus, and Species.

- MNEMONIC = Keep Ponds Clean Or Fish Get Sick

- 3 domains = eukaryotes (animals, plants, and fungi), prokaryotes (bacteria), and archaea

Phylogeny  studying the evolutionary relationships between organisms. Phylogenetic relationships can be
understood by comparing the genetic info that exists in their nucleic acids/proteins

The characteristics used in bacterial taxonomy: morphological (structural differences), physiological and
metabolic, ecological/environment, pathogenicity, and genetic analysis
Endosymbiotic theory  suggests mitochondria and plastids were once independent prokaryotic cells.
Originally 3 prokaryotic cells: one capable of aerobic respiration, one capable of photosynthesis and one
incapable of doing either. This last cell engulfed the 1st 2 cells. It was now able to make energy and convert
energy from the sun into stored chemical energy. This cell now has a source of energy and a way to convert
that energy whilst the other 2 cells have a safe place to live and grow = protection and nutrients.

Identifying bacteria

List the criteria for classification of bacteria

- Growth on media – non-selective (blood/chocolate agar – support growth of many different

bacteria), selective (growth of only certain bacteria), and differential media (used to distinguish
between closely related organisms)
- Bacterial microscopy
- Biochemical tests
- Immunological tests

Some common microbial biochemical tests used to differentiate among bacteria

- Gram staining = used to distinguish between G+ and G- bac

- Carb breakdown = tests for fermentation of sugars – for anaerobic bac
o Uses Hiss media
o Lactose, glucose, sucrose, mannitol, and maltose = common 5 sugars tested
o E.coli is the most catalytic bac – ferments all 5 sugars
- Catalase test  breakdown of H2O2 to H2O and O2; a positive result produces O2 bubbles
o E.g. of an immediate test. Can see O2 bubbles
o Used to differentiate staphylococci (+ve) from streptococci (-ve)
- Citrate utilisation test = uses citric acid media. If bac can utilise citric acid, indicator turns blue as the
pH rises
- Urease test  urea-containing broth. If urea is cleaved, NH4 is released and colour change observed
(orange > fuchsin). Detection of H. pylori
- Decarboxylases and deaminases = decarboxylation and deamination of AA Lys, Orn, and Arg is
detected by the effects of the amino products on the pH of the rxn mixture – G- rods
- Oxidase test  shows whether bac is oxidative or not

Nomenclature

Bacteria are named according to Genus (capital letter) and species (lowercase) ;Written in italics or
underlined. Correct name has to be written in Latin. For example; Haemophilus influenzae. The Genus is
haemophilus, and the species is influenzae.

Methods of identification

- Cellular morphology, Staining characteristics, Motility, Growth characteristics

- Serological tests, Genetic analysis
3. Bacterial Morphology
Overview

Bacteria are prokaryotes with no true nucleus or membrane bound organelles. Most are 0.5-5um in size; e.g.
staphylococcus aureus and Escherichia coli.

- Smaller ones are 0.1-0.5um in size; e.g. H. influenzae (not the influenza virus)
- Larger ones are 5-100um and are called giants; refers to spore-forming bacteria such as Clostridia
bacillus

Shape

Bacteria can be classified according to their

general shape:

- Cocci  spherical bacteria with all

dimensions equal in diameter
o Monococcus  one sphere
o Diplococcus  2 spheres
o Tetracoccus  4 spheres
o Streptococcus  spheres
arranged in a line
o Staphylococcus  spheres
arranged in a bunch
o Sarcins  spheres arranged in
a cube shape
- Bacillus  rod-shaped; includes both
spore-forming and non-spore forming
bacteria. They may or may not have flagella that allows them to be motile.
o E.g. e.coli, proteus mirabilis
o Some rods resemble cocci due to the small size and have very similar diameters; these are
called coccobacillus.
- Spirillum  comma-shaped; e.g. the vibrio species
- Pleomorphic  no distinct shape

Cell envelope

The cell envelope consists of a cytoplasmic membrane and a cell wall. The cytoplasmic membrane is a
phospholipid bilayer similar to that of eukaryotic membrane, although it contains no cholesterol (except for
mycoplasmas).

Gram staining separates bacteria into 2 categories; gram positive or negative. Bacteria stain on the basis of
the differences in cell wall structure, specifically peptidoglycan.

- Peptidoglycan surrounds the cytoplasmic membrane of most prokaryotes and is essential for the
structure, replication and survival of the bacterial cell. It is made up of glycan strands that are cross-
linked by short oligopeptides.

The gram stain depends on the ability of bacteria to retain gentian violet dye and iodine after washing with
alcohol; hence why gram positive bacteria are stained blue and gram negative are stained red (fuchsin).
 - In gram positive bacteria, the peptidoglycan layers make up 50% of the cell wall; the many layers of
peptidoglycan can retain the gentian violet stain.
- Gram positive cell walls also contain teichoic and lipoteichoic acids;
o Teichoic acids are important for virulence and are essential for cell viability
o Lipoteichoic acids are common surface antigens that distinguish bacterial serotypes and
promote attachment to other bacteria and specific receptors on human cells

Gram negative bacteria have a thinner peptidoglycan layer (5-10% of the cell wall) but the overall structure
of the cell wall is more complex. Between the outer membrane and inner cytoplasmic membrane is the
periplasmic space, which contains;

- Thin peptidoglycan layer

- Hydrolytic enzymes for metabolism; proteases, lipases, nucleases

Gram negative cell walls have a bilayer outer membrane with the inner layer resembling a cell membrane,
and the outer layer containing a distinctive lipopolysaccharide component; this makes the bilayer
asymmetrical.

- Gram negative walls consist of a complex glycolipid called lipid A, which is embedded in the outer
layer of the membrane. It is known as the endotoxin of gram-negative bacteria because it is only
released when the cells die.
o When LPS splits into lipid A and polysaccharide, all toxicity is associated with lipid A.

Mycoplasmas have no cell wall (peptidoglycan) and cannot be stained by the gram staining method.
Mycobacteria have cells walls that contain large amounts of waxes called mycolic acids; they also cannot be
gram stained because of their high lipid content. Acid fast staining is used to stain mycobacteria.

4. Bacterial Ultrastructure – Eukaryotes and Prokaryotes, Cytoplasmic structures

Eukaryotes vs Prokaryotes

Both eukaryotic and prokaryotic cells consist of a cytoplasmic membrane, cytoplasm, ribosomes and genetic
information.

Bacterial cells and blue-green algae are prokaryotes, whereas cells form animals, plants and fungi are
eukaryotes and have a “true nucleus.”

- Prokaryotic cells are simpler than eukaryotic cells at every level, except for their cell envelope, which
is more complex.
Prokaryotes

In addition to lacking a nucleus and other organelles, prokaryotes also have a smaller ribosome (70S, vs 80S
in eukaryotes) and have a mesh-like peptidoglycan call wall surrounding the cell membrane for protection
against the environment.

Essential structures

Prokaryotic structures can be separated into essential and non-essential for survival.

The essential structures are the chromosome, ribosomes, cytoplasmic membrane and the cell wall. Nucleoid
contains the genetic material of a bacterial cell and is contained in a single double-stranded DNA molecule
that is circular. It can be seen with a light microscope and has no surrounding membrane.

- Exception  Borrelia; these bacteria have a linear chromosome.

- The DNA is not associated with a protein like in eukaryotic nuclei.
- The lack of nuclear membrane simplifies the mechanisms for protein synthesis and allows
transcription and translation to occur simultaneously.
o Ribosomes can bind mRNA and protein can be made as mRNA is being synthesised.
- Rapidly growing bacteria have more nucleoids per cell than slowly growing ones
- Prokaryotes also have no eukaryotic-like mitotic apparatus
- Prokaryotes are haploid

The nucleoid along with the other DNA structures make up the bacterial chromosome. In terms of genetic
material, only the nucleoid is essential for the survival of the bacteria. Other extrachromosomal DNAs such
as plasmids and other may also be present but are not essential.

- Plasmids are most commonly found in gram-negative bacteria. They may provide a selective
advantage such as resistance to antibiotics, enterotoxin production, enhanced pathogenicity,
resistance to toxic metal ions etc.
 - Plasmids can replicate independently of the chromosome or may be integrated with it; in either
case, they are normally passed onto the progeny

Bacterial ribosomes are smaller than eukaryotic ones; consists of 30S + 50S subunits, forming a 70S ribosome
(eukaryotic are 40S + 60S; 80S ribosome). They have some differences compared to eukaryotic, so they can
be used as targets for antibacterial agents.

- E.g., streptomycin interferes with the bacterial ribosome without disturbing human ribosomal
function

Other prokaryotic structures

The mitochondria, Golgi complex and endoplasmic reticulum are absent in prokaryotes.

Bacterial inclusion  serve as reserve nutrient substances, and reduce osmotic pressure by tying up
molecules in particulate form. They consist of volutin (acid polyphosphates), lipid, glycogen, starch or
sulphur.

- Volutin granules (metachromatic) are found in Corynebacterium diphtheriae, which causes

diphtheria and also in Lactobacillus.

Flagella  long, hollow, helical filaments which are responsible for locomotion. They are composed of
helically coiled protein subunits called flagellin, which are highly antigenic (H-antigens; some immune
responses to infection are directed against flagellins).

Each flagellum consists of 3 parts: filament, hook and basal body.

- Filament  the longest part that extends from

the cell surface to the tip.
- Hook  a short, curved segment that joins the
filament to the basal body and acts as a joint
between these structures.
- Basal body  it is embedded into the cell
membrane; ATP-dependent rotation generated in
the basal body allows the bacteria to move
towards food and away from poisons (positive
and negative chemotaxis).
o in gram positive it has one pair of rings
(total of 2), in gram negative it has 2 pairs
(total of 4).
o The rings are connected to a central rod
or associated with LPS and PG layers.
- Flagella can be arranged in 4 ways:
o Monotrichous  one polar flagellum (e.g., Vibrio cholerae)
o Lophotrichous  multiple polar flagella (e.g.,
Bartonella baciliformis)
o Amphitrichous  flagella at both ends (e.g.,
Spirillum serpens)
o Peritrichous  multiple flagella all over the
cell (e.g., Escherichia coli)
Pili (also called fimbriae)  surface appendages that are shorter and finer than flagella but are not
responsible for movement of a bacterium. They are composed of protein subunits called pilins. Several
hundred fimbriae are usually arranged peritrichously over the entire surface of a bacterial cell. Minor
proteins located at the tips of pili are responsible for attachment properties.

- Two classes of pili:

o Ordinary  play a role in adherence of symbiotic and pathogenic bacteria to host cells
o Sex pili  allows for bacteria to exchange genetic info via conjugation; F pili promote
transfer of large segments of bacterial chromosomes between bacteria.
- Adherence of pili is an important virulence factor for N. gonorrhoeae and other bacteria.
- Pili of different bacteria are antigenically distinct; some bacteria are able to make pili of different
antigenic types and can still adhere to cells in the presence of antibodies to its original type of pili.
o Antibodies against the pili of one type of bacterium will not prevent the attachment of
another bacterial species

Spores  go to point 7.

5. Bacterial Ultrastructure – Cell Wall, Structure and Biosynthesis of Bacterial Cell

Wall, Bacterial exceptions in the Cell Wall Structure
Cell wall structure

Bacteria are classified according to their cell wall; as gram positive or negative. There are other species that
do not have cell walls, and some have chemically unique cell walls.

The cell wall is an essential structure that protects the bacteria from hostile environments including inside
and outside of the human host. It enables bacteria to survive in fluid environments that differ from their
intracellular environment.

- Bacteria are often in environments where the outside of dilute and inside is concentrated
- Target for antibiotics
- Source of identification by other bacteria, organisms and the immune system

Peptidoglycan is what the cell wall is composed of; it is a structure that Is unique to bacterial cells and is a
meshwork of carbohydrates and proteins arranged in a lattice-like formation; these carbohydrates are cross-
linked by oligopeptides in a material called murein. The functions of murein are:

- Protects the plasma membrane from damage and

provide structural integrity.
o The true plasma membrane of the cell is
enclosed by the peptidoglycan
- Maintains rigidity and shape of the bacterial cell
- Stimulates immunity reactions (both innate and
acquired) and inflammation
- Hence it is essential for the structure, replication
and survival of the bacterial cell
- Peptidoglycan can be degraded by lysozymes
(exists in human tears, mucus and is produced by
some bacteria), penicillin and other beta-lactam antibiotics
If the peptidoglycan is the most internal layer, this causes gram staining.

Gram positive  peptidoglycan layer is thick and multi-layered; it is the most external layer. Underneath it is
the cell plasma membrane; gram staining causes purple dye to be retained by the wall.

- Gram positive cell walls may also include other components such as teichoic acid and lipoteichoic
acids. Antigenic specificity in gram positive bacteria is facilitated by teichoic acids.
o Lipoteichoic acids are shed into the host and can initiate responses similar to endotoxins.
These acids promote attachment to other bacteria and to specific receptors on mammalian
cell surfaces
- Gram positive streptococci polysaccharides in the cell wall, such as C polysaccharides, allow for
medically significant grouping.

Gram negative  the thinner peptidoglycan layer is enveloped within the inner plasma cell membrane and
an outer membrane, which is unique in all cells. The area between the cytoplasmic and outer membranes is
called the periplasmic space; this is where the thin peptidoglycan layer is located. It contains:

- Hydrolytic enzymes for metabolism; proteases, lipases, carbohydrate-degrading enzymes

- Lytic enzymes (virulence factors); collagenases, hyaluronidases
- Components for sugar transport.

The outer membrane is an asymmetric bilayer made of phospholipids, lipopolysaccharides, porins and outer
membrane proteins.

- LPS is an endotoxin composed of a polysaccharide and a lipid composed o-antigen; it is a powerful

stimulator of innate and immune responses
- Porins allow diffusion of metabolites and small hydrophilic antibiotics, but is a barrier for large or
hydrophobic antibiotics.
- Multiple layers that surround gram negative cells allows them to withstand more harsh
environmental conditions vs gram positive.
o Hence, they are more capable of developing resistance to antibiotics; the additional outer
membrane allows them to develop more complex mechanisms of defence.
o Also provides an additional physical barrier for more defence.
- Outer membrane contains lipid A, which is an endotoxin that is released when gram negative
bacteria die

Gram stain is a way to distinguish gram positive and negative bacteria. Gram positive stain purple and
negative stain red. Gram positive microbes are killed by penicillin and cephalosporin. Gram negative are
generally more resistant.

Cell wall biosynthesis

- Peptidoglycan monomers are synthesised in the cell and transported to the cell wall
- The monomers are joined together by transglycosidase enzymes to form peptidoglycan chains
o Glycopeptides such as vancomycin inhibit this step
- The peptidoglycan chains are linked by transpeptidase enzymes
o Beta-lactams such as penicillins inhibit this step.

Exceptions in the cell wall structure

The exceptions are Archaeobacteria (contains pseudoglycans and pseudomureins related to peptidoglycans)
and mycoplasmas, which have no cell wall; mycoplasmas incorporates steroids from the host into their cell
membranes.

Mycobacteria have cells walls that contain large amounts of waxes called mycolic acids; they have a
peptidoglycan layer surrounded by the wax but they cannot be gram stained because of their high lipid
content. Acid fast staining is used to stain mycobacteria.

6. Bacterial Ultrastructure – External Structures

Capsules

Some bacteria are closely surrounded by loose polysaccharide or protein layers called capsules. A capsule is
a type of glycocalyx (sticky sugar coat) that increases the virulence of bacteria by evading phagocytosis. They
are located outside the cell envelope.

Capsules are found most commonly in gram-negative bacteria, but some gram-positive bacteria may also
have a capsule.

There are 3 general types:

- thick capsule  3-5um in size; visible after staining with optical microscope by the exclusion of India
ink particles
- microcapsule  thin polysaccharide or protein layer formed from fimbriae; exists in E coli. These
capsules are too small to be seen with an ordinary microscope.
- Slime layer  loosely adherent polysaccharide liberating from bacteria; it is important for their
adhesion to mucosae, prosthetic devices, plastic tubes and other instruments in ICUs.

The function of the capsule is mostly protective to maintain survival within the host; it provides protection
against phagocytosis, complement, antibiotics and detergents.

- in this role, the capsules are a major virulence factor. Bacteria become avirulent without their
capsules
o e.g., bacillus anthracis; if it has a capsule it can cause anthrax
o s. pneumoniae can cause pneumonia, sepsis, meningitis but only if it has a capsule; if there is no
capsule it cannot cause these diseases

The capsule also functions as a K-antigen or “capsular antigen;” anti-capsule IgG antibodies opsonise bacteria
and aid in their phagocytosis and killing. K-antigens are also important diagnostic antigens.

The capsule also promotes adherence to other bacteria or host tissue surfaces. E.g., for Streptococcus
mutans, the dextran and levan capsules are the means by which the bacteria attach to the tooth enamel.

Biofilms

Bacterial biofilms are clusters of bacteria that are attached to a surface and/or to each other and embedded
in a self-produced matrix. The biofilm matrix consists of substances like proteins (e.g., fibrin), polysaccharide
(e.g., alginate), as well as eDNA. In addition to the protection offered by the matrix, bacteria in biofilms can
employ several survival strategies to evade the host defence systems.

- By staying dormant and hidden from the immune system, they may cause local tissue damage and
later cause an acute infection.
 - Within the biofilm, the bacteria adapt to environmental anoxia and nutrient limitation by exhibiting
an altered metabolism, gene expression, and protein production, which can lead to a lower
metabolic rate and a reduced rate of cell division.
- In addition, these adaptations make the bacteria more resistant to antimicrobial therapy by
inactivating the antimicrobial targets or reducing the requirements for the cellular function that the
antimicrobials interfere with.

Cells in the biofilm formations demonstrate a higher level of resistance in comparison to their planktonic
forms; it has been estimated that they are generally 1000x less susceptible to common antimicrobial agents
and are highly resistant to phagocytosis by immune system phagocytes.

This makes bacterial biofilm formation a cause for many chronic infections. An example of a biofilm-
mediated infection is bacterial vaginosis.

- Gardnerella vaginalis has a strong ability to adhere to the vaginal epithelium by its glycocalyx; it
forms a significantly thicker biofilm than other vaginal pathogens, hence why it has a large role in BV.

Biofilm formation begins with adhesion of plantonic microbes to a surface via their capsule (or slime layer),
followed by colonisation, co-adhesion, growth and maturation.

- tooth plaque is a type of biofilm that forms be facultative anaerobic bacteria such as Streptococcus
mutans; can spread in dentinal tubules and colonise them because the morphology of the tubules
provides a good environment for microbial growth

7. Bacterial Ultrastructure – Bacterial Spores

https://www.youtube.com/watch?v=0mHHQ85vhIY

Function

Only gram-positive bacteria can form spores; they are resistant dormant structures that develop in
unfavourable environmental conditions, such as the depletion of nutrients. Bacteria convert from a
vegetative state to a dormant state known as a spore, and allows bacteria to continue to exist in
unfavourable conditions.

The medically significant bacteria that can form spores are Bacillus and Clostridium (i.e., soil bacteria)

- The location of spores in a cell is a characteristic of the bacteria that can be used to assist in its
identification.
o Spore can be located centrally, sub-terminally or terminally.
o Appearance can be spherical, ovoid or elongated
o Bacillus  the diameter of the spore is the same or less than the width of the bacterium
o Clostridium  the diameter is wider than the bacillary body; produces and bulge in the cell.
- Spores contain a complete copy of the chromosome, as well as minimal concentrations of essential
proteins and ribosomes and a high concentration of calcium bound to dipicolinic acid
- In this form, bacteria can exist for centuries.

Structure
Spores look refractile (bright) under a microscope. The structure of the spore, particularly the protein coat,
protects the bacterial genome from insults by intense heat, radiation, enzymes and chemical agents. A spore
consists of:

- Core  contains the normal cell structures but is metabolically inactive

- Wall  consists of an inner membrane and two layers of peptidoglycan known as a core.,
- Spore coat  an outer layer of a keratin-like protein coat that surrounds the cortex.
- Exosporium  spores of some species have an additional loose covering known as the exosporium.

Sporulation

Spore formation commences when growth of bacteria stops due to lack of nutrients from a growth medium.
The process takes 6-8 hours and can be divided into several stages:

- Spore septum  the bacterial chromosome is

duplicated and the newly-formed chromosome is
isolated along with a small portion of cytoplasm by
an ingrowth of plasma membrane called the spore
septum
- Forespore  the septum becomes a double-layered
membrane that encloses a smaller compartment
within the cell to form a forespore.
- Spore coat  a peptidoglycan layer forms between
the two membranes that now surround the
forespore. This layer is the cortex. It is then
surrounded by the keratin-like protein coat
- Endospore  the exosporium disintegrates and the
endospore is freed.

Germination

This the process of conversion of a spore back into the vegetative state under suitable conditions. It occurs in
3 phases: activation, initiation and outgrowth.

- Activation  transformation of spores into the vegetative state is stimulated by disruption of the
protein coat by mechanical stress, pH, heat or another stressor.
o This disruption of the coat allows water and nutrients to come in and trigger germination.
- Initiation  once activated, the spore will initiate germination if conditions are favourable (i.e.,
warm temperature and rich in nutrients).
- Outgrowth  the spore swells and the spore wall is shed. The cell elongates to form the vegetative
bacterium again.

Clostridium difficile endospores can result in repeated infections; individuals are treated with antibiotics in
an intermittent manner to allow the endospores to germinate. E.g., the antibiotic is taken for a week, then
stopped for another week to allow the endospores to germinate, then the antibiotics are taken for another
week to kill the newly germinated bacteria and so on. This is an uncommon method of treatment.

8. Bacterial Metabolism
Metabolic characteristics
Bacteria can be classified into 2 groups depending on their metabolic properties;

- How the organism deals with oxygen

- What the organism uses as a carbon and as an energy source
o Another property that characterises them are the different metabolic end products that
bacteria produce, such as acids and gases.

How bacteria deal with oxygen is a major factor for classification; when molecular oxygen is reduced it can
form toxic by-products such as H2O2 and superoxide radicals and hydroxyl radicals. These are all toxic unless
they are broken down by:

- Catalase  breaks H2O2 into H2O + O2

- Peroxidase  also breaks down H2O2
- Superoxide dismutase  breaks down the superoxide radical into H2O2 + O2.

According to how they deal with oxygen, bacteria are classified as:

- Obligate aerobes  use aerobic respiration; TCA cycle, oxidative phosphorylation. They need 21%
O2 at least. Aerobes in general counteract the effect of O2 radicals by utilising the enzymes
mentioned above.
o E.g. mycobacterium tuberculosis
- Facultative anaerobes  although “anaerobes” they still use O2 as an electron acceptor and still use
catalase and superoxide mutase, but they can grow without O2 via fermentation.
o Highly adaptable; most clinically important bacteria like E. coli, staphylococcus and
streptococcus.
- Micro-aerophilics  use fermentation but have no electron transport system; they can tolerate low
amounts of oxygen because they have superoxide dismutase but no catalase.
o E.g. helicobacter pylori (causes gastritis, gastric ulcers and gastric cancers)
- Obligate anaerobes  cannot grow in presence of oxygen because they have no enzymes to defend
against lethal O2 radicals.
o Causative agents of severe infections such as gas gangrene and abscesses.
- Capnophiles  bacteria that thrive in presence of high CO2 concentrations; an example of
campylobacter, which are capnophiles that are more easily identified because they are also micro-
aerophiles. Campylobacter species can cause intestinal disorders.

According to how bacteria deal with their carbon and energy source:

- Phototrophs  light as an energy source

- Chemotrophs  chemical compounds as energy source; these are further divided into:
o Autotrophs  use inorganic chemical energy sources e.g., sulphide
o Heterotrophs  use organic carbon sources of energy.

Energy generation

All biochemical reactions in cells are either catabolic or anabolic. Pyruvate is a key intermediate that can be
used for cell synthesis and as a target for different enzymes; it allows metabolism to be flexible.

- Catabolism  molecules are broken down from complex to simpler ones and energy is released in
the form of energy carriers (e.g. ATP) and electron carriers (e.g. NAD, FAD).

Energy is also generated by:

 - Substrate level phosphorylation  synthesis of ATP from ADP directly joined to the breakdown of
high energy organic substrates; ATP generated by ADP phosphorylation
- Oxidative phosphorylation  electrons are transported down a chain of proteins that synthesise
ATP by ATPase; the final electron acceptor is H+ and H2O is made.
o Combined with glycolysis and TCA cycle in aerobes and facultative anaerobes.

Anaerobic  in fermentation, an organic molecule is the terminal acceptor; glucose is broken down to
pyruvic acid, which yields ATP. Pyruvate is then broken down and the end product is formed; e.g., acetone,
lactic acid, ethanol.

- In fermentation, organic molecules are both electron donors and acceptors. Energy yield is low and
results in an excess of NADH.
- An example of an organism that ferments is Lactobacillus bulgaricus; the product is yogurt.

9. Bacterial Growth and Cell Division

Requirements for growth

Environmental factors influence rate of bacterial growth such as acidity (pH), temperature, water activity,
macro and micro nutrients, oxygen levels, and toxins. Conditions tend to be relatively consistent between
bacteria with the exception of extremophiles.

- Effect of pH  optimal growth differs with acidophiles (need acid pH), neutrophils (neutral pH) and
alkaliphiles (alkaline pH).
- Water availability  halophiles (need NaCl for growth), halotolerant (grow at moderate salt
concentrations, but grow best without it; e.g., S. aureus).
o This is why some foods are best preserved in salt or when dehydrated
- Temperature  psychrophiles (can grow at -20C), mesophiles (grow best at 37C; accounts for most
pathogenic bacteria), thermophiles (grow best between 40-120C)

Binary fission is the process through which asexual reproduction happens in bacteria. During binary fission, a
single organism becomes two independent organisms. Binary fission also describes the duplication of
organelles in eukaryotes.

- Bacterial DNA is uncoiled, duplicated, and pulled to opposite poles of the cell
- The growth of a new cell wall begins to separate the bacterium into 2
- The new cell wall fully develops, resulting the complete split of the bacterium
o The new daughter cells have tightly coiled DNA rods, ribosomes, and plasmids.

Growth phases

Lag phase  the phase in which the bacteria do not multiply; they only become aware of the condition they
are in so they figure out if they have all of the nutrients needed for development, so their concentration
does not change.

- After they have a good idea of their environment, they begin to metabolise and divide in such a way
that the amount of new bacteria that are being produced is higher than the amount of bacteria that
are dying; this is the exponential phase

Exponential phase  due to the division of bacteria into 2 whenever they reproduce. Around 18-24 hours is
needed for the colony to be seen by the naked eye.
The generation time is the time needed for one cell to divide into 2 cells. For most bacteria, the generation
time is 15-30mins. After another 30, they divide again, then again after another 30 and so on. Hence in 24
hours a full colony will be visible.

- There are some exceptions; e.g. V. cholerae is faster; has a generation time of around 5-10 minutes,
so a colony is seen after 6-8 hours.
o Ureaplasma and mycoplasma are slower; generation time is more than 20 minutes so 3-5
days are needed to see a colony.
o Mycobacteria tuberculosis is also slower; generation time is 30-40 hours so a colony is seen
after one month or even longer.

Stationary  concentration of bacteria that are newly formed is equal to the concentration of bacteria that
are dying. Death occurs due to the close conditions that the microorganisms live in; they are given only a
specific amount of nutrients that they will start competing for, and because during metabolism,
microorganisms are producing metabolites that suppress the development of other organisms. This phase
can last several weeks

Death phase  the new bacteria produced is less than the amount of bacteria that are dying. This occurs
mostly due to depletion of nutrients, or due to an environmental stressor (e.g., change in pH)

Bacteria that are only in exponential phase or stationary phase can receive new genetic information either
spontaneously or artificially, mostly in the logarithmic phase.

10. Bacterial genetics: DNA; the genetic material; structure, replication and
function (control)
Structure

The bacterial genome consists of the bacterial chromosome and extrachromosomal non-essential genetic
structures known as plasmids and bacteriophages.

The bacterial chromosome is a single haploid circular molecule of double-stranded and supercoiled DNA. A
collection of enzymes (DNA gyrase, topoisomerase, etc.) is responsible for winding the DNA

- A fully supercoiled chromosome is around 1um in diameter and is small enough to fit inside a
bacterium. The gyrase is a target for antibiotics; novobiocin and fluoroquinolones.
o DNA gyrase twists the DNA about itself, causing it to fold over at every 200 bps.
o Supercoiling creates the tertiary structure of the bacterial chromosome.

In prokaryotes, only the chromosome is important for the survival of bacteria. Extrachromosomal structures
are not essential for survival, but they give these bacteria an advantage in more environments, such as
resistance against antibiotics, or ability to produce exotoxins.

- Plasmids  take on the same shape is the bacterial chromosome; i.e. if it is circular and ds, the
plasmids are also circular and ds. However, plasmids replicate independently of chromosomal DNA.
- Plasmids can be classified into small or big;
o small  encode and carry genetic information only for their own replication.
o big  carry info for their own replication plus some extra genetic structures, such as info for
resistance, attachment, conjugation, biochemical activity, toxin production etc.
 o Small plasmids are still important; if they receive info from insertion sequences or
transposons, they will become big plasmids.

Bacteriophage  a virus that infects and replicates within a bacterial cell;

- Lytic phage  infects sensitive bacteria to produce many copies of itself, before lysing and killing the
host bacterial cell
- Lysogenic  the virus infects the cell and integrates its DNA into the bacterial genome without
killing the bacteria
- Temperate phage  DNA incorporated into the host DNA and replicates independently of the host.

Transposons  a DNA sequence that can change its position within a genome, sometimes creating or
reversing mutations and altering the cell's genetic identity and genome size. Transposition often results in
duplication of the same genetic material.

Replication

DNA continuously replicates; the process is semiconservative, which results in two DNA molecules, each
having one parental strand of DNA and one newly synthesized strand.

- Replication is initiated at a specific sequence on the chromosome called Ori C

- Helicase unzips the DNA double helix to produce a replication fork
- Primase synthesizes a short RNA primer, providing a free 3′-OH group to which DNA polymerase III
can add DNA nucleotides.
- During elongation, the leading strand of DNA is synthesized continuously from a single primer. The
lagging strand is synthesized discontinuously in Okazaki fragments, each requiring its own primer.
o The RNA primers are removed and replaced with DNA nucleotides by bacterial DNA
polymerase I, and DNA ligase seals the gaps between these fragments.
- Termination of replication in bacteria involves the resolution of circular DNA concatemers by
topoisomerase IV to release the two copies of the circular chromosome.

Regulation

Bacteria do not have introns and exons; genes of bacteria are organised in operons, which are groups of
genes that are all controlled by transcription from a single promoter. The regulatory region of an operon
includes the promoter itself and the region surrounding the promoter to which transcription factors can bind
to influence transcription.

Although some operons are constitutively expressed, most are subject to regulation through the use of
transcription factors (repressors and activators).

- A repressor binds to an operator, which is a sequence within the regulatory region between the RNA
polymerase binding site in the promoter and first structural gene, thereby physically blocking
transcription of these operons.
- An activator binds within the regulatory region of an operon, helping RNA polymerase bind to the
promoter, thereby enhancing the transcription of this operon.
- An inducer influences transcription through interacting with a repressor or activator.

The trp operon is an example of a repressible operon. When tryptophan accumulates, tryptophan binds to a
repressor, which then binds to the operator, preventing further transcription.
11. Bacterial genetics: Mutation, Recombination, and DNA exchange
Mutation

A mutation is defined as any change in the genetic material, including changes in gene expression and
changes that do not lead to any phenotypic differences (i.e., silent mutations). Gene mutations occur due to
errors arising during DNA replication or errors in DNA repair mechanisms.

Gene mutations specifically alter the nucleotide sequence of a DNA molecule by:

- Substitution  a nucleotide in a sequence is replaced by different one; also called point mutation.
o Transition  substitution of purine by purine (A-G) or pyrimidine to pyrimidine (C-T).
o Transversions  purines are substituted by pyrimidines or vice versa.
- Insertion  a new base is inserted into the sequence
- Deletion  a nucleotide is removed.

The consequences of these types of mutations can be classified according to the functional change (if any):

- Missense  a single nucleotide changes results in a codon that codes for a different amino acid
- Nonsense  the single nucleotide change creates a stop codon
- Silent  no change in amino acid
- Frameshift  this occurs if the number of inserted/deleted nucleotides is not divisible by 3. If
divisible by 3, there is a loss/gain of amino acids in a sequence.
o Due to the triplet nature of gene expression by codons, the insertion or deletion can change
the reading frame, resulting in a completely different translation from the original.
o The earlier in the sequence the deletion or insertion occurs, the more altered the protein.

Recombination

Bacterial recombination is the incorporation of foreign (extrachromosomal) DNA into the

chromosome/plasmid. There are 2 types:

- Homologous (legitimate)  occurs between closely related DNA sequences and generally
substitutes one sequence for another. Exchange occurs by crossing-over.
o Process requires a set of enzymes produced by the recA genes.
- Non-homologous (illegitimate)  occurs between dissimilar (non-homologous) DNA sequences and
generally produces insertions, deletions, or both. Usually requires specialised recombination
enzymes produced by many transposons and lysogenic bacteriophages

Exchange

Horizontal gene transfer is the transfer of genes between organisms in a manner other than reproduction –
transfer is not between bacteria of the same lineage (vertical). It is the primary reason for AB resistance and
in evolution, maintenance and transmission of virulence.

Transformation  when a donor cell dies, its DNA is released into the environment and under specific
conditions, the DNA is taken up by a recipient cell; under the action of specific enzymes the DNA enters the
cell through the cell wall

- the enzymes will cut the donor DNA in small particles which then enter the recipient cell.
- The transformation process only occurs under specific environmental conditions:
o The donor DNA has to be natural, not artificially-made.
o DNA has to be small/short
 o DNA must be similar to DNA that already exists in the recipient cell.
- Predominantly, gram negative bacteria undergo this transformation process
- This process occurs naturally

Conjugation  the process by which genetic information is transferred between bacterial cells.
predominantly occurs in gram negative bacteria. It also occurs naturally but it can be stimulated artificially.

- Bacteria B releases pheromones to attract bacteria A; specific conjugative plasmids start to appear
over the surface of B until it forms a bridge with A
o Replication of A’s plasmid occurs and the newly formed plasmids passes through the bridge
from A to B, hence B receive genetic information identical to that of A
o Afterwards the bridge is destroyed and the bacteria separate again.

Transduction  occurs naturally but can be stimulated artificially. In order for transduction to occur, viruses
are needed, specifically bacteriophages (viruses that attack bacteria), When a virus attacks a bacterium, it
injects its genetic information into it, and this insertion has 2 possible outcomes;

- The viral DNA replicates within the cell and is translated to produce proteins, until the host cell is
destroyed and then the newly synthesised viruses are released into the environment and they can
attack more host cells.
- The viral DNA integrates itself into the genome of the host cell; when this cell begins to divide, it will
also cause the viral DNA to multiply.
o During replication of the chromosome, it is possible that only some parts are replicated
correctly; hence the new bacterial cell that is made is not genetically identical to the parent.
o The newly formed virus may then leave the bacterial cell along with some genetic
information from the bacterial chromosome, so it is genetically different from the initial
virus that attacked the cell. It then goes on to attack more host cells, which then receives
info from more bacterial cells and the cycle continues.

12. Bacterial genetics: Genetic Engineering

Recombinant DNA technology

Genetic engineering involves recombinant DNA technology, the process by which a DNA sequence is
manipulated in vitro, creating recombinant DNA molecules that have new combinations of genetic material.

- The recombinant DNA is then introduced into a host organism.

- If the DNA that is introduced comes from a different species, the host organism is now considered to
be transgenic.

The components in genetic engineering are:

- Cloning and expression vectors: plasmids, phages

- DNA sequence to be cloned, amplified and expressed
- Restriction enzymes used to cleave DNA and DNA ligase to link the fragments to the cloning vectors
- Recipient cells

Process

- Obtain foreign DNA

- Remove plasmids (vector) from bacterial cells
 - Restriction enzymes break the DNA at specific restriction sites producing restriction fragments (sticky
ends). The same restriction enzyme cuts the plasmid at specific restriction sites, producing
complementary sticky ends
- DNA + plasmid → recombinant plasmid
- DNA ligase seals the nicks in the DNA – DNA and plasmid attach permanently

Recombinant DNA is then introduced into a bacterial host, usually E. coli, and undergoes transformation
(process by which foreign DNA is introduced into a cell)

- The plasmid containing bacteria is selected and placed on an agar medium containing ampicillin.
Hundreds of recombinant strains grow on the agar plate.
o Cells that did not take up the recombinant DNA are killed by ampicillin
- Colonies are screened to identify the bacteria containing the appropriate DNA (DNA hybridisation,
ELISA)

Hybridisation

Hybridization refers to the joining together of two complementary single strands of DNA. Blunt ends can also
attach together, but less efficiently than sticky ends due to the lack of complementary overhangs facilitating
the process.

DNA-DNA hybridisation measures the degree of genetic similarity between pools of DNA sequences

FISH  method of using fluorescently labelled probes to detect and localise the presence/absence of
specific DNA sequences on chromosomes. Probes are often made from modified fragments of DNA

- Method = Cells fixed on a slide, fluorescently labelled probe is added, cell and probe treated w/high
temp. to become a single strand
o probe finds a complementary sequence and is hybridised, non-hybridised probe is washed
off, nucleus counterstained w/DNA specific fluorescent dye
o nuclei observed w/fluorescent microscopy.

Southern blot

Technique that combines the use of restriction enzymes and electrophoresis to generate, separate, and
detect pieces of DNA

- DNA extracted from cells and cleaved using restriction enzymes/endonucleases

- Fragments are separated by electrophoresis
- DNA fragments in the gel are denatured and transferred to a nitrocellulose membrane
- DNA fragments of interest are identified by a probe

PCR

PCR is a DNA amplification-based technique used to amplify a DNA sequence of interest.

Process  different samples are inserted into different wells. DNA is negatively charged so if it is put into an
electrolyte solution and an electrical field is applied, it will separate according to size so bands will form in
the agarose gel. The smaller DNA molecules will travel further up the gel vs the larger ones.

- A marker/standard is used containing DNA molecules with known dimensions so they can be used to
compare sample DNA
- Steps of PCR are repeated (30-40 cycles) to amplify DNA  denaturing, annealing and extension
o Denaturating  heating to separate strands
 o Annealing  cooling down so primers and taq polymerase can bind
o Extension  at 72*C
- Only the DNA of interest is amplified by using specific primers that correspond to this DNA sequence.
If this DNA molecule is not in the sample, the primers will not attach to DNA cannot replicate
(negative result).

13. Viruses: Taxonomy, Structure, Replication, and Cultivation

Taxonomy

A virus is a sub-microscopic infectious agent that replicates only inside the living cells of an organism. Viruses
can infect every type of host cell: plants, animals, fungi, protists, bacteria, and archaea. Most viruses will
only be able to infect the cells of one or a few species of organism. This is called the host range.

- Having a wide host range is not common and viruses will typically only infect specific hosts and only
specific cell types within those hosts.
- The viruses that infect bacteria are called bacteriophages, or just phages.

Classification of viruses depends on structural properties:

- DNA or RNA, and single- or double-stranded nucleic acid genome.

- Shape of capsid  icosahedral, spherical, helical
- Enveloped or non-enveloped (naked)
o Viruses formed from only nucleic acid and capsid are naked/nonenveloped.
o Viruses formed with a nucleic-acid packed capsid surrounded by a lipid layer are enveloped.
- Size  can range from 20 nm for small viruses, up to 900 nm for typical, large viruses

Structure

Viruses consist of a nucleic acid (either RNA or DNA, but never both) surrounded by a protein coat called a
capsid. The interior of the capsid is not filled with cytosol, as in a cell, but instead it contains the bare
necessities in terms of genome and enzymes needed to direct the synthesis of new virions.

- RNA viruses can be positive- or negative-sense; a positive-sense RNA virus can be immediately
translated into a protein by the host’s ribosomes; hence it functions like mRNA.
o Negative-RNA needs to be transcribed into a positive strand before it can be translated. The
transcription is performed by viral RNA-dependent RNA-polymerase.
- DNA viruses need to be transcribed to mRNA and translated into structural proteins and enzymes.
o Most DNA viruses have a positive and negative strand; only the negative strand is read.

Each capsid is composed of protein subunits called capsomeres made of one or more different types of
capsomere proteins that interlock to form the closely packed capsid. Virus capsids predominantly come in
two shapes: helical and icosahedral.

- Helix  a spiral shape that curves cylindrically around an axis; the viral nucleic acid coils into a
helical shape and the capsid proteins form a long tube or rod-like structure.
- Icosahedron  a geometric shape with 20 sides; these capsids somewhat resemble a soccer ball.

The viral envelope is a small portion of phospholipid membrane obtained as the virion buds from a host cell.
The viral envelope may either be intracellular or cytoplasmic in origin.

Extending outward from the capsid on some naked viruses and enveloped viruses are proteins spikes. At the
tips of these spikes are structures that allow the virus to attach and enter a cell, like the influenza virus
hemagglutinin spikes (H) or enzymes like the neuraminidase (N) spikes that allow the virus to detach from
the cell surface during release of new virions. Influenza viruses are often identified by their H and N spikes.

Replication

- Attachment  The specificity of this interaction determines the host (and the cells within the host)
that can be infected by a particular virus
- Entry  penetration and uncoating; nucleic acid of bacteriophages enters the host cell naked,
leaving the capsid outside the cell. Some enveloped viruses enter the cell when the viral envelope
fuses directly with the cell membrane.
o Once inside the cell, the viral capsid is degraded, and the viral nucleic acid is released, which
then becomes available for replication and transcription.
- Replication  mechanism depends on the viral genome. DNA viruses usually use host cell proteins
and enzymes to make additional DNA that is transcribed to mRNA, RNA viruses usually use the RNA
core as a template for synthesis of viral genomic RNA and mRNA.
- Assembly  viral proteins are built and genetic material is placed inside the new capsid.
- Release  some viruses are released when the host cell dies, and other viruses can leave infected
cells by budding through the membrane without directly killing the cell.

Cultivation

Viruses can’t be grown in culture media or agar plates alone; they must be in living cells to support their
replication. Viruses can be cultivated within suitable hosts (living cells) e.g., to study bacteriophages.

- The bacteria are grown in a suitable growth medium, then bacteriophages are added which can
multiply within the bacteria.

Purpose of virus cultivation is to isolate and identify viruses, and prepare viruses for vaccines

Tissue cultures system:

- Virus is cultivated in cell culture

- Cells (from animal e.g. fertilized eggs, living animals, or plant source) are kept alive in a suspension of
growth factors within a petri dish.
 - Thin layer of cells (monolayer) is inoculated with viruses and replication takes place

14. Fungi: Classification, Structure, Replication, and Cultivation

Classification

Fungi are eukaryotic organisms that are heterotrophs – they acquire their food by absorbing dissolved
molecules, typically secreting digestive enzymes. into their environment. They do not photosynthesize
because they lack chlorophyll.

- Part of the kingdom Fungi with chitin-containing rigid cell walls.

In addition to the well-known macroscopic fungi (such as mushrooms and moulds), many unicellular yeasts
and spores of macroscopic fungi are microscopic.

There are 4 medically important classes of fungi:

Class Description
Ascomycetes Some genera use sexually produced ascospores, or asexual spores called conidia.
Some produce an ascus (sac) containing ascospores within an ascocarp.
E.g. Aspergillus
Basidiomycetes fungi that have basidia (club-shaped structures) that produce basidiospores (spores
produced through budding) within fruiting bodies called basidiocarps.
Zygomycetes They use sporangiospores for asexual reproduction
Group name comes from the zygospores that they use for sexual reproduction, which
have hard walls formed from the fusion of reproductive cells from two individuals.
Deuteromycete No sexual reproduction; Includes most pathogenic fungi  Candida, cryptococcus
s

Fungi can also be classified morphologically:

- Moulds  multicellular fungal bodies, made up of filaments called hyphae. Hyphae can form a
tangled network called a mycelium and form the thallus (body) of fleshy fungi.
o Hyphae that have walls between the cells are called septate hyphae
o hyphae that lack walls and cell membranes between the cells are called non-septate
- Yeasts  unicellular fungi; budding yeasts reproduce asexually by budding off a smaller daughter
cell; the resulting cells may sometimes stick together as a short chain or pseudo-hypha
- Dimorphic  able to appear as yeasts or moulds, which can be important for infectivity. They are
capable of changing their appearance in response to environmental changes such as temperature:
o E.g., growing as a mould at 25 °C, and as yeast at 37 °C. This helps dimorphic fungi survive in
diverse environments.

Structure

Fungal cell wall  contains chitin, as opposed to the cellulose found in the cell walls of plants and many
protists. It surrounds the fungal cell membrane and is an antigen to the human immune system.
Cell membrane  layer surrounding the fungal cytoplasm. While animals have cholesterol in their cell
membranes, fungal cell membranes have different sterols called ergosterols. Ergosterols are often used as
targets for antifungal drugs because they do not occur in animals or bacteria.

Capsule  a coating surrounding the cell wall that is made up of polysaccharides; it acts as a virulence factor
for many fungi, such as Cryptococcus neoformans.

Replication

Fungi produce spores sexually, asexually, or both.

Fungi reproduce sexually either through cross- or self-fertilization. Haploid fungi form hyphae that have
gametes at the tips. Two different mating types (represented as “+ type” and “– type”) are involved.

- The cytoplasm of the + and – type gametes fuse (called plasmogamy), producing a cell with two
distinct nuclei (dikaryotic cell).
- Later, the nuclei fuse (called karyogamy) to create a diploid zygote.
- The zygote undergoes meiosis to form spores that germinate to start the haploid stage, which
eventually creates more haploid mycelia.
o Depending on the taxonomic group, these sexually produced spores are called zygospores
(Zygomycota), ascospores (Ascomycota), or basidiospores (Basidiomycota).

Fungi may also exhibit asexual reproduction by:

- Fission  simple cell division (mitosis)

- mitosis with budding - new organism grows from the body of the parent and detaches
- fragmentation of hyphae  mycelia are broken apart and each component grows into a new
separate mycelium.
- asexual spores  these spores are specialized cells that, depending on the organism, may have
unique characteristics for survival, reproduction, and dispersal.
o Fungi exhibit several types of asexual spores and these can be important in classification.

Cultivation

A fungal culture is a procedure used to determine if fungi are present in an area of the body. Fungal culture
can be referred to as a ‘fungal smear’: blood, skin, mucosal surfaces (wounds or genital region), nails

Use a cotton swab over the are infected, nail clippings (nail infection), or blood sample taken

Culture:

- Sabouraud agar
- Mycosel agar  selective for pathogenic fungi: chloramphenicol and cyclohemidie are added,
inhibiting most saprobic fungi, but C.neoformans does not grow
- CHROMagar- different candida species exhibit colonies of different colours (C.albicans has blue
colonies)

Incubation  Two cultures incubated separately- @ 25 and at 37 degrees Celsius (37- to reveal
dimorphism). Cultures are considered negative after 4 weeks incubation

15. Sterilisation, Disinfection, and Antisepsis.

Sterilisation
Sterilisation is a process that completely destroys all forms of microbial life including bacteria, viruses, fungi,
parasites, and spores.

- Disinfection  killing or removing of harmful vegetative microorganisms

- Antisepsis  destruction or inhibition of the growth of microorganisms in or on living tissue.
- Cleaning  removal of visible dirt and debris; control of microbial growth must first start with
cleaning, then sanitising, then disinfection, then sterilisation.

Sterilization protocols are generally reserved for laboratory, medical,

manufacturing, and food industry settings, where it is necessary for
items to be completely free of potentially infectious agents.

Sterilization can be accomplished through either physical or chemical

means. Chemicals that can be used to achieve sterilization are called
sterilants. Sterilants effectively kill all microbes and viruses, and, with
appropriate exposure time, can also kill endospores.

Physical methods can be categorised into 5 groups:

UV light common method; does not kill spores

- Breaks dsDNA strands; sterilises work surfaces and tools in labs and hospitals
- Most effective in the dark
- 254nm wavelength
Filtration 0.2-0.4 micrometre pore diameter stops organisms of larger diameter from passing through
- Mycoplasma can pass though
- Does not completely sterilise
- Used for surgical masks and sterilisation of solutions that would be denatured by heat
Radiation two subgroups; ionising or non-ionising radiation
- Ionising  UV, infrared (cannot be applied to humans; used to sterilise rooms or
surfaces. E.g. UV alters DNA structure)
- Non-ionising  x-rays, gamma-rays
Dry heat e.g., when sterilising the loop using the Bunsen burner
Hot air oven  stimulates oxidative destruction of the cells; 160*C for 2hrs, or 180*C for 1hr.
Used for glass, porcelain, metal items
Moist e.g., autoclave
heat

The autoclave contains a metallic box that contains everything to be sterilised. The box is closed and the
autoclave is filled with water and sealed closed

- The temperature is gradually increased from 0 to 121*C, 1-1.5atm, for 15-20min. This is enough to
kill spores of C. botulinum with an adequate margin of safety.
- The water will begin to boil at 100*C, causing evaporation but the evaporate does not escape the
machine because it has been sealed.
o The pressure from the vapour increases the pressure within the autoclave; this pressure will
kill the microorganisms inside the metallic box

It is necessary to re-sterilise the instruments through hot air oven even after using an autoclave because
spores of some soil organisms are still able to withstand this temperature and transform into vegetative
state to survive, so they are heated again using a hot air oven to kill them using dry heat.

- Prions are very resistant microbes, so they need extra care to be sterilised.
 - During the process of drying the instruments at room temperature after using the autoclave, they
may be contaminated again by open air so they need to be sterilised again.
- Another reason is that microbes may be on the material that covers instruments after they are
sterilised in the autoclave.

Chemical sterilisation:

- Ethylene oxide gas (3%) treatment is a common method used to sterilise, pasteurise, or disinfect
items because of its wide range of material compatibility.
o It is used for 70% of total sterilizations, and for > 50% of disposable medical devices.
- Hydrogen peroxide  used to sterilise heat- or temp-sensitive articles, such as rigid endoscopes.
Conc. = 35-90%
- Peracetic acid (0.2%) = sterilising medical devices such as endoscopes

Disinfection

The process of disinfection inactivates most microbes by using antimicrobial chemicals or heat. Typical
disinfection does not lead to sterilization because endospores tend to survive even when all vegetative cells
have been killed. Disinfectants should be fast acting, stable, inexpensive, and easy to use.

- An example of a natural disinfectant is vinegar; its acidity kills most microbes.

- Chemical disinfectants, such as chlorine bleach or products containing chlorine, are used to clean
non-living surfaces such as laboratory benches, clinical surfaces, and bathroom sinks.
- All disinfectants need contact time for at least 30 sec

Disinfectants have differing ranges of activity:

- High-level  kills mycobacteria, non-enveloped viruses, and fungi; Used for invasive procedures that
can’t withstand sterilisation
o Moist heat, glutaraldehyde 2%, H2O2 3-25%, peracetic acid, chlorine
- Intermediate  kills vegetative bacteria and enveloped viruses; Used for semi-critical instruments
and devices where contamination is not likely
o E.g. alcohols 70%, iodophors 30-50 ppm, free iodine
- Low  destroy bacteria but not viruses and spores (doesn’t kill mycobacterium and fungi); Used for
non-critical instruments e.g. stethoscope
o E.g. quaternary ammonium compounds 0.1-1.6%

Activity can be influenced by temperature, concentration (an optimal concentration is needed), time, and
range of action (i.e., spectrum of microbes). They may be inactivated by dirt, organic matter, blood, mucus,
pus, faeces, proteins, and some plastics.

- Hence why surfaces and instruments need to be cleaned of visible dirt before they are disinfected.

Antisepsis
Unlike disinfectants, antiseptics are antimicrobial chemicals safe for use on living skin or tissues for the
purpose of reducing the risk of infection, sepsis, or putrefaction. Examples of antiseptics include hydrogen
peroxide and isopropyl alcohol. The process of applying an antiseptic is called antisepsis.

In addition to the characteristics of a good disinfectant, antiseptics must also be selectively effective against
microorganisms and able to penetrate tissue deeply without causing tissue damage.

- Some are bactericidal, while others are bacteriostatic

- Routine hand wash removes flora; antiseptic hand wash = removes flora and ↓ resident flora, use
for at least 15s

Ethanol and isopropanol 50-70% Used on skin

Formaldehyde 8% Used on skin
Tincture of iodine 2% Disinfectant, kills endospores
Chlorine gas Disinfects drinking water, general disinfectant
Hydrogen peroxide 6% Used on skin, non-toxic, non-allergenic
Chlorhexidine Used on skin and to treat gingivitis
Phenolic compounds (Carbolic acid) Antiseptics at low conc., disinfectants at high conc.

16. Antimicrobial Chemotherapy: Antibacterial, Antiviral, and Antifungal agents;

Groups according to their spectre, activity, and molecular mechanisms.
Antibacterial agents

Antibiotics are antibacterial agents; in contrast, antimicrobials are antibacterial, antifungal, and antiviral
agents. Antibiotics (beta-lactams and non-beta lactams) and synthetic antimicrobials (sulphonamides and
fluoroquinolones) are antibacterial agents. Antibiotics can be classified according to their function:

- Cell wall inhibitors  beta-lactams, glycopeptides

- Protein synthesis inhibitors  aminoglycosides, tetracyclines, macrolides, ketolides, lincosamides,
amphenicols
- DNA synthesis inhibitors  sulphonamides, quinolones

Beta-lactams  penicillins, cephalosporins, carbapenems, monobactams; these drugs consist of a beta-

lactam ring, which irreversibly binds to the active site of penicillin-binding protein, which is required in the
process of forming cross links in the peptidoglycan wall.

- This inhibits peptidoglycan synthesis, hence killing the cell (bactericidal).

- The spectrum of action varies depending on the type of drug; e.g., natural penicillins have a broad
spectrum, but semi-synthetic penicillins can be narrow (e.g., flucloxacillin) or broad (e.g., amoxicillin)
o Resistance to beta-lactams occurs via bacterial production of beta-lactamases.

Glycopeptides  these agents do not have a beta-lactam ring but still inhibit cell wall synthesis by binding D-
ala-D-ala and N-acetylmuramic acid, which blocks cross-link formation and polymerisation of peptidoglycan
in the bacterial cell wall.

- Glycopeptides are generally used when infection with MRSA is suspected. Vancomycin is often
indicated in cases of penicillin allergy; there is no beta-lactam ring so cross-allergy is not seen.
Protein synthesis inhibitors are bacteriostatic, except for aminoglycosides and macrolides at high doses,
which are bactericidal. Eukaryotic and prokaryotic ribosomes differ in structure, which provides a basis for
the selective antibacterial action of these drugs:

- Eukaryotic ribosome  80S (60S + 40S).

- Prokaryotic ribosome  70S (50S + 30S).
o S refers to the sedimentation rate during centrifugation.
- These drugs work by binding and inhibiting the 50S or 30S subunits of bacteria
o 30S  aminoglycosides, tetracyclines; broad spectrum
o 50S inhibitors  macrolides, ketolides, lincosamides, amphenicols
- Both aminoglycosides and tetracyclines exhibit a post-antibiotic effect, which is when the drugs
maintain their bacteriostatic/bactericidal effect even after their serum concentration has dropped
below the minimum inhibitory concentration (MIC).

DNA synthesis inhibitors are bacteriostatic;

- Sulphonamides  structural analogues of para-amino benzoic acid (PABA), which is a substrate of

the enzyme dihydrofolate synthase; inhibition of this enzyme impairs folate synthesis, which is
required for DNA synthesis by the bacteria.
o The bacteria may resist by producing more PABA to overcome the drug, or utilising another
pathway that does not require folic acid.
- Quinolones  these inhibit DNA gyrase and topoisomerase 4, preventing DNA synthesis. DNA gyrase
has an A and B subunit; fluoroquinolones bind to the A subunit.
o These are broad-spectrum and especially effective gram-negative enterobacteria.

Antiviral agents

Inhibitors of DNA replication:

- Nucleoside analogues  converted into nucleoside triphosphate to exert anti-viral activity; e.g.,
acyclovir, valacyclovir.
- NRTIs  analogues nucleosides that lack 3’OH; chain termination of viral RNA
- NNRTIs  induces conformational change of reverse transcriptase enzyme in retroviruses.

Adamantine derivatives  blocks viral protein M2 (H+ channel), which blocks viral uncoating; e.g.,
amantadine, rimantadine.

Integrase inhibitors  prevents incorporation of HIV genome into host genome

A common feature of antivirals is that they are virostatic and work only against viruses that are replicating;
hence they do not affect latent viruses. Most antivirals are given orally, although these may be given via
parenteral routes of administration:

Antifungal agents

Antimycotic drugs that impair cell membrane permeability:

- Azoles  synthetic antifungals that inhibitor of lanosterol 14alpha-demethylase, which disrupts

ergosterol synthesis in the cell membrane. Reduced ergosterol results in leaky cell membranes that
are permeable to intracellular components.
- Allylamines  squalene epoxidase inhibitors; suppresses the early stages of ergosterol biosynthesis,
hence affecting fungal cell membrane synthesis and function.
o E.g., Terbinafine, naftifine.
 - Polyene antibiotics  natural-origin lipophilic drugs that interact with ergosterol in fungal
membranes to form artificial pores that disrupt membrane permeability, leading to leakage of
intracellular ions and other water-soluble components, which causes cell death.
o Amphotericin B, nyastatin; amphotericin B has a lesser affinity for cholesterol (on
mammalian cell membranes), but there is still some interaction, which leads to ADRs.

Antimycotic drugs that inhibit nucleic acid synthesis  flucytosine; an analogue of cytosine. It is taken up by
fungal cells via permeases and converted into 5-fluorouracil by the fungal enzyme cytosine deaminase.
Metabolites of 5-fluorouracil interfere with fungal DNA synthesis.

Antimycotic drugs that suppress synthesis of beta-glucan  echinocandins; non-competitive inhibitors of β-

1-3-glucan synthase, an enzyme critical to the synthesis of β-1-3-glucan, which is not present in human cells.

- Beta-1,3-D-glucan is a major component of the cell wall of fungi; in its absence, fungal cells lose
integrity and are lysed (osmotic lysis).

Antimycotic drugs that inhibit cell division  griseofulvin; oral fungistatic agent (natural-origin antibiotic). It
interferes with mitosis by binding to the microtubules responsible for mitotic spindle formation, leading to
defective cell wall development.

17. Antimicrobial Chemotherapy: Genetic and Biochemical Mechanisms of Bacterial

Resistance. Side effects of antibiotics; toxicity, allergy, dysbacteriosis.
Mechanisms of bacterial resistance

A common reason for resistance is over-prescription of antibiotics (e.g., in viral infections, especially
rhinosinusitis and bronchitis; bronchitis is almost exclusively viral).

- Another reason is lack of compliance; antibiotic courses should be followed through to ensure
cohorts of bacteria do not survive to mutate and develop resistance
- Since over-prescription of antimicrobial agents can cause resistance and superinfection, prophylactic
use is restricted to clinical situations in which the benefits outweigh the potential risks.

Of great concern are multidrug-resistant microbes (MDRs) and cross resistance. MDRs are also known as
“superbugs” and carry one or more resistance mechanisms, making them resistant to multiple
antimicrobials. In cross-resistance, one resistance mechanism confers resistance to multiple drugs.
Genetic mechanisms of resistance: bacteria can develop resistance by mutating existing genes, or by
acquiring mutations that confer resistance via conjugation with other bacteria. This may be the case in
situations where a course of antibiotics is not finished and the surviving bacteria are able to adapt.

Biochemical mechanisms:

- Penicillins  Resistance is due to beta-lactamase production in most cases, or modification

penicillin-binding sites. Microorganisms that lack cell walls (e.g., Mycoplasma) or are metabolically
inactive are also resistant because they are not actively synthesising peptidoglycans.
o Gram-negative bacteria  resistance can occur due to an impairment in the permeability of
the outer membrane, which limits the penetration of hydrophilic antibiotics.
o Eukaryotic cells are resistant against penicillins because they do not have a cell wall.
- Non-beta lactams  common mechanisms:
o Plasmid-mediated formation of enzymes that inactivate the drug (transferases, esterases)
o Altered permeability; reduced influx and increased efflux of the drug from the bacteria
o Changes in ribosomal binding site
- Sulphonamides  these normally compete with PABA, so bacteria can develop resistance by
increasing PABA to overcome the drug, or by using another pathway that does not require folate.
- If the target is an enzyme  reduced affinity of the enzyme for the drug (e.g., quinolones).

Methicillin (semisynthetic penicillin) was designed to resist inactivation by β-lactamases. Soon after the
introduction of methicillin, methicillin-resistant strains of S. aureus appeared and spread. The mechanism of
resistance is acquisition of a new low-affinity PBP; provides S. aureus with resistance to all β-lactams.

Side effects of antibiotics

Allergy  less likely to occur when penicillins are given orally and most likely in local application.

- There is cross-allergy between all forms of penicillin (due to the common structure) and partial
cross-allergy between penicillins and cephalosporins.
- cross reactivity  people allergic to penicillins may also be allergic to cephalosporins and vice versa
 - Tests for allergy can be performed before administration  skin prick test, intradermal test.

Some common side effects are:

- Stomach upset  N&V, cramps, diarrhoea

o Macrolides, cephalosporins, penicillins, fluoroquinolones may cause more stomach upset
than other antibiotics.
- Photosensitivity  Tetracyclines can make skin more prone to sunburn
- Tooth discolouration  Tetracycline and doxycycline can cause permanent tooth staining in children
whose teeth are still developing (younger than 8)
- Blood  Beta-lactam antibiotics and sulfamethoxazole can cause leukopenia
- Tendonitis  Ciprofloxacin can cause inflammation or rupture of a tendon
- Aminoglycosides are potentially toxic for renal and ear functions

Dysbacteriosis  a disruption to the gut microbiota homeostasis caused by an imbalance in the microflora,
changes in their functional composition and metabolic activities, or a shift in their local distribution.

- Antibiotic usage in during childhood development can lead to adverse dysbiosis in adulthood, such
as IBD, ulcerative colitis, obesity.
- The most common threat of gut microbiota alteration is increase susceptibility of intestinal infection,
such as recurrent C. difficile infection in hospitalised patients, especially if they are undergoing
antibiotic treatment.
- As well as antibiotics, alcohol misuse, poor diet, cytostatic drugs, or immunosuppressive therapies
can also cause dysbiosis.