Microbiology

Question 1

Bacterial Cell

Answer 1

Around 0.5 um.
Typical bacteria contains nucleoid, ribosomes, fimbriae, plasma membrane and a peptidoglycan cell wall.
May also have flagella

Question 2

Cell Wall

Answer 2

Peptidoglycan wall function:

1. Rigid macromolecular layer that provides strength to the cell.
2. Protects the cell from osmotic lysis and confers cell shape.

Structure:
Alternating NAM and NAG carbohydrates forms a chain. Peptide forms a cross bridge to form a multilayer meshwork.
Transpeptidase is the enzyme which cross links the peptidoglycan chains to form rigid cell walls.

Question 3

Gram-positive Bacteria

Answer 3

Bacteria have a thick peptidoglycan layer (20-80nm) which will trap and retain crystal violet. It will therefore appear purple from a gram stain.

Question 4

Gram-negative Bacteria

Answer 4

Bacteria have a cell wall with 2 layers; a thin peptidoglycan layer (5-10nm) as well as an outer membrane. Crystal violet is easily washed away. Therefore this bacteria will appear pink (colour of the red safranin counter stain).

Question 5

Bacterial Flagella

Answer 5

Motile bacteria produce flagella (around 5-10 per cell).
These are long appendage resembling ‘tails’.
They’re proteinaceous (made of protein) and will act like a propeller as the cell rotates them.

Structure: 3 major sections
1. Long filament (F)
Extends into surrounding medium, composed of flagellin subunits.
2. Hook (H)
Curve section connecting the filament to cell surface.
3. Basal Body (or motor)
Anchors the flagellum into the cell membrane if the bacterium by special disc-shaped structures called plates or rings. This motor forces the propeller to spin / turn.
Fuel for the motor = protons.

Propeller can turn in either direction:
Anticlockwise = bacteria zooms off in the direction it’s facing. This is called run.
If one filament stops turning, it breaks up the bundle of flagella and the cell begins to tumble.

Question 6

Tactic Response

Answer 6

Chemotaxis - bacteria move along a concentration gradient towards a chemical attractant (positive) or away from a chemical repellent (negative).
Bacteria sense a change in chemical concentration outside of the cell overtime “temporal gradients” so respond as it moves.
This means they will sense the environment in one place, and then sense whats in a different place a period of time later and compare before responding.

On the outside of the bacteria = sensors
On the inside = signal transduction pathway which tells the flagella to spin.
As a result there will be a turnover of signals inside the cell, if its going in the right direction = reinforcement, if its going in the wrong direction = reduction of the reinforcement. Thus the movement can be a little random but will accumulatively go in the right direction.

Question 7

Bacterial Adherence Factors

Answer 7

Fimbrae = structures with adhesive properties that cause bacteria to stick / adhere to surfaces.
Fimbrae are hair (1um in length) like with 100 - 1000s per cell.

Glycocalyx = capsules and slime layers.
A gelatinous polysaccharide and / or polypeptide outer covering which forms a sticky meshwork of fibres allowing it to stick to surfaces.
Gycocalyx capsule functions:
- Virulence factors = protecting the bacteria from phagocytosis and engulfment by immune cells.
- Adherence to cell surfaces and structures eg medical implants.
- Prevent cell from desiccation

Question 8

Bacterial Endospores

Answer 8

Formed during unfavourable growth conditions and germinate under favourable growth conditions.
Only in some gram-positive bacteria.
Protect cells from stress (ie nutrients starvation, high cell density).
Endospore is formed in Mother cell and will become free when the mother cell breaks down.
They sit in the environment and eventually germinate if conditions are right before going back into binary fission with a vegetative cells.
They’re resistant to heat, harsh chemicals, antibodies, disinfectants and radiation = very hard to kill.
This is the dormant stage of bacterial lifecycle.

Question 9

Binary Fission

Answer 9

Asexual Reproduction.
Binary fission results in the formation of 2 cells which are genetically identical.
Process:
1. Chromosome replication begins
2. One copy of the origin moves to each end of the cell
3. Replication finishes
4. Cell divides into 2 daughter cells

Occasionally things will go wrong during this process resulting in mutations which allow for selection.

Question 10

Closed batch culture system

Answer 10

Refers to a form of cell culturing.

Defined supply of nutrients is provided, once used cells cannot proliferate (ie evolve rather than reproduce rapidly).

Question 11

Microbial growth in closed batch culture system

Answer 11

1. Lag phase = time is required to get biosynthetic reactions running and organisms will need to adapt to new conditions.
2. Exponential phase = cells are actively dividing, nothing is limiting for growth. Population will be doubling in a constant time interval.
3. Stationary phase = Cells stop growing cryptic growth is observed. This is when organisms survive by consuming lysed cell constituents of other dead cells within the culture.
   The population will be dynamic. There will be an equilibrium between dying cells and growing cells. (rate of growth = rate of death).
4. Death phase = Equilibrium between growing cells and dying cells is skewed towards death (rate of death > rate of growth)

Question 12

Penicllin

Answer 12

Antibiotic which kills bacteria by blocking cell wall synthesis. Therefore only growing / replicating bacteria will be affected.

Question 13

Persistent Bacterial Infections

Answer 13

Persisters are insensitive to penicillin because they are in a dormant, no dividing phase, similar to that of bacteria in the lag phase.
How persisters lead to persistent bacterial infections:
Large amount of population is normal and will be killed by antibiotics as its growing. This will leave you with a small population of bacteria which weren’t reproducing. Once you remove the antibiotic because you think you’ve killed the infection, the second population will re grow causing a reinfection.

Question 14

What do prokaryotes need to multiply?

Answer 14

Microorganisms need 3 things to grow

1. Carbon source = building blocks
2. Energy source = drive anabolic and catabolic reactions
3. Reducing power = carriers of electrons / energy

How do they harvest energy?
Chemical energy is stored in bonds. Broken chemical bonds can release energy which can be captured in new bonds (ADP + P = ATP).
ATP = most common energy currency. Its bonds can be broken again later to release that energy. Thus reduction and oxidation of coupled compounds can be applied to many compounds and forms, the basis of redox.

We can define microbes by where they get these compounds and what strategies they use for survival

Question 15

Matabolism

Answer 15

The sum of all chemical reactions in an organism.

Anabolism + Catabolism = Metabolism.

Question 16

4 Key Trophic groups of microorganisms

Answer 16

Energy source:
1. Light (photo-)
Use solar energy

2. Chemical compounds (chemo-)
   Use chemical energy from either carbon compounds eg glucose or non carbon compounds (inorganic) eg H2S

Carbon source:
1. Carbon dioxide (auto-)
Harvest CO2 from the environment and build it up to more complex forms of carbon.
Primary producers, self sufficient

2. Organic compounds (hetero-)
   Harvest carbon from organic compounds.
   Dependent on primary producers

Question 17

Photoautotrophs

Answer 17

Light energy, Fix carbon (ie use CO2 to produce organic C molecules)

Question 18

Chemoautotrophs

Answer 18

Chemical energy, Fix carbon

Question 19

Photoheterotrophs

Answer 19

Light energy, Need carbon provided to them (organic compounds)

Question 20

Chemoheterotrophs

Answer 20

Chemical energy, Need carbon provided to them.
Basically break down things which other organisms have built.
Humans - Most animals are chemoheterotrophs

Question 21

Microbial Ecology

Answer 21

The study of the interrelationships among microorganisms and their environment.

Question 22

Microbiome

Answer 22

All microorganisms and their genes within a particular environment.

Question 23

Enrichment culture

Answer 23

Providing the temperature and chemical conditions in the laboratory that encourage the growth of specific groups of microbes.

Question 24

Microbial Metabolism

Answer 24

One process -> 2 potential goals
Breaking a bond can be used to do 2 particular things:
- harvest energy
- harvest building blocks (or both)
This process can run in reverse (if you have the energy and building blocks available)

Question 25

Redox in Bacteria

Answer 25

The basis for energy transfer in cells.
For every action eg oxidation, there is an equal and opposite reaction eg reduction.
Energy from oxidation is shuttled through an intermediate (NADH / NADPH). (We don’t use the energy directly from the reaction, we trap it in something).

Question 26

Photosynthesis

Answer 26

Two types :
Non-cyclic photophosphorylation:
Electron flow from H2O -> Photosystem II -> Photosystem I -> NADP+, Generates O2, ATP and NADPH. (oxygenic)

Cyclic photophosphorylation:
Photosystem I can work in absence of photosystem II. This generates ATP but O2. (anoxygenic)
eg H2S -> S produces ATP used to fix Carbon

Both processes use light for energy. Both processes fix carbon. However only one produces oxygen which has repercussions for the planets atmosphere.

Question 27

How can so many microorganisms share the same metabolism?

Answer 27

It’s to do with diversification and specialisation.

Example - microbial mat in a marsh. There are layers of different coloured microbes because each layer is able to absorb different types of light by using different pigments so they’re not competing for exactly the same type of light. ie they tune their antenna to different wavelengths.

Oxygen dependent organisms can exploit anoxic environments such as cable bacteria by creating cables which shuttle electrons from anoxic into oxic zones. This allows them to breathe oxygen whilst living in anoxic conditions (where there may be plenty of nutrients).

Exploitation of different redox potential derives organisation of global microbial communities. Different redox gradients select for different microbes.

Question 28

Human Microbiome Project (HMP) Goals

Answer 28

1. Develop a reference set of microbial genomes sequences and to perform preliminarily characterisation of the human microbiome.
2. Explore the relationship between disease and changes in the human microbiome.
3. Develop new technology and tools for computational analysis.
4. Establish a resource repository
5. Study the ethical, legal and social implications of human microbiome research.

Question 29

Human Bacteria

Answer 29

Different sites = different bacteria in healthy humans.
Human microbial communities are dominated by 4:
1. Firmicutes
2. Bacteroidetes
3. Actinobacteria
4. Proteobacteria

Question 30

Human gut

Answer 30

Contains the highest density and diversity of microbes in the human body.
Differences in microbes reflect the different sites and conditions throughout the gut, eg small number in stomach due to low pH.
50% of faecal biomass = bacteria.

Question 31

Functional Foods

Answer 31

Food claimed to have a health-promoting or disease preventing property beyond the basic function of supplying nutrients.

Question 32

Probiotics

Answer 32

Live microorganisms (fermented foods - yogurt)
Eg Lactic acid bacteria (LAB), Bifidobacteria.
They can survive the transit through stomach and duodenum.
Potential benefits: chronic intestinal inflammatory diseases, prevention and treatment of pathogen-induced diarrhoea, urogenital diseases.

Question 33

Prebiotic

Answer 33

An ingredient (form of fibre) that beneficially nourishes the good bacteria already in the large bowel or colon.
Prebiotics stimulate the growth of probiotics.
Mostly obtained from a carbohydrate fibre called an oligosaccharide.
Examples include bananas, wholegrain, honey.

Question 34

Fecal transplants for CDI

Answer 34

Fecal matter transplants (FMT) is a highly successful treatment for multiple recurrences of clostridium difficile infection (CDI). It is done using a stool from a healthy donor to replace a patients gut microbial flora.

Question 35

Viruses

Answer 35

Acecellular microorganisms that cannot survive without a host, as they have no metabolic abilities of their own. They rely completely on biosynthetic machinery of infected cell to multiply.
They infect all cell types

Question 36

Basic Structural characteristics of Viruses

Answer 36

Consists of 2 parts:
1.Genetic material (DNA or RNA)
2.Capsid
+ In some cases a third part: Envelope of lipids.

Question 37

Viral Capsid

Answer 37

Capsids are made of multiple units of the same protein building block known as capsomers arranged in a precise and highly repetitive pattern around the nucleic acid.
3 types:
- Helical (each capsomer is slightly offset forming a spiral)
- Icosahedral (20 face polyhedron)
- Complex (Composed of a head and a tail)

Question 38

Viral Genome

Answer 38

Made up nucleic acid - DNA or RNA
May be either - Linear, circular or segmented

They can all 4 forms - Single stranded RNA, double stranded RNA, single stranded DNA, double stranded DNA.

Question 39

Bacteriophage

Answer 39

Bacteriophage are viruses that infect and replicate in Bacteria.
Types of bacteriophage infections:
1. Lytic Infection = host cell will die
2. Lysogenic Infection = host cell will survive

Question 40

Lytic Cycle

Answer 40

1. Virus must recognise and bind to the receptor on the host cell surface.
2. Phage DNA will enter the cell causing degradation of the host cell’s DNA
3. Synthesis of viral genomes and proteins. Virus will use host’s nucleotides and machinery to replicate its genome and ribosomes to make its proteins.
4. Self assembly - Virus particle will assemble driven by the concentration of proteins.
5. Release - Virus produces an enzyme called lysosome which breaks down peptidoglycan in the cell wall, causing the cell to burst, releasing the virus.

Question 41

Lysogenic Cycle

Answer 41

1. Phage DNA integrates into bacterial chromosome forming a prophage.
   A phage genome will remain ‘silent’, transcription of the prophage gene is inhibited.
2. Prophage will be copied with the bacterial chromosome during replication.
3. Many cell divisions (binary fission) create many infected bacteria.
4. Occasionally a prophage will exit the bacterial chromosome, initiating a lytic cycle.

Question 42

HIV Virus

Answer 42

1. Glycoprotein of the virus will bind to CD4 T Cells (host) membrane. This is cause the fusion the 2 membranes, releasing the virus particle into the cytoplasm. IN doing so the virus’s capsid will be broken down.
2. The virus will convert its RNA into a DNA-RNA hybrid (synthesis of a DNA strand complementary to the viral RNA). This process is called reverse transcriptase. Another cycle of replication forms a double stranded DNA copy of the virus.
3. This DNA will be translocated into the nucleus and gets integrated into the host chromosome. Its now called a provirus. This will start producing 2 forms of RNA, 1 form is the full length genome and the other is RNA to make the viral proteins. 1 type of protein produced - glycoproteins will secrete out and become embedded in the surface of our cell membrane. Other proteins include the capsid a d reverse transcriptase.
4. All of these proteins will assemble with the new viral RNA in a virus particle.
5. Viral particle recognises the glycoproteins in the membrane surface and will bud out through the host cell membrane, acquiring the membrane as an envelope, forming a new virus.

Question 43

Influenza virus

Answer 43

Like all other viruses, influenza are obligate intracellular parasites which get inside of a host cell to use its machinery and replicate itself before leaving and killing the cell. The virus is able to swap its genetic material with other viruses which have invaded the same host cell to produce new types of virus.
Influenza = H1N1 (reflects the spike proteins) is single stranded RNA enveloped virus. It is easily spread but rarely fatal.

Question 44

Other Medically important pathogens

Answer 44

• Fungi = Trichophyton spp (ie ringworm)
• Prions = Kuru
  (only microbe which is just a protein)
• Protozoa = Plasmodium spp (ie Malaria)
• Helminths = Anclyostoma duodenale (ie Hook worm)
• Bacteria = Bacillus anthracis

Question 45

Koch’s Postulates

Answer 45

Guidelines to demonstrate that a specific pathogen causes specific disease symptoms.

1. Pathogen must be present in every individual with the disease
2. A sample of the microorganism taken from the diseased host can be grown in pure culture
3. A sample of the pure culture causes the same disease when injected into a healthy host.
4. The microorganisms can be recovered from the experimentally diseased host.

Exceptions:

• Microbes that can’t be cultured
• Pathogens that can’t be found in healthy individuals

Question 46

Key Stages of Microbial Pathogenesis

Answer 46

1. Adherence to host cell
2. Invasion of host tissues
3. Replication within host tissues
4. Disease causing damage to host tissues (pathology)

(Thus infected and disease are two different things).

Question 47

Bacterial Virulence Factors

How they relate to the key stages of microbial pathogenesis

Answer 47

1. Adherence to host cell:
   Adhesions such as fimbrae (bind to host cell)
2. Invasion:
   Motility such as flagella - Move through the mucus
   Internalin-related proteins (InIB) (bacteria produce a range of enzymes which dissolve and let them attach better to the cells they want to invade)
3. Replication within host tissue
   Siderophores (bind iron) - small molecules produced and secreted by bacteria which mediate the uptake of essential iron into the cell.
   Capsules - Resist phagocytosis
4. Disease causing damage to host cell tissues
   Endotoxins
   Exotoxins
   ( = toxic virulence factors)

Question 48

Endotoxins

Answer 48

Lipopolysaccharides (LPS) found in the outer membrane of gram-negative bacteria, and elicit strong immune responses.
When the bacteria get destroyed, it releases this LPS.
Potential effects of endotoxin Lipid A = Fever, inflammation, blood clotting and shock.

Question 49

Exotoxins

Answer 49

Produced within living bacteria, and then released into the surrounding medium. 3 different types:
1. Cytotoxins - Target cells, can cause lysis
Example - Streptolysin produced by bacteria causes the complete lysis of red blood cells (beta haemolysis)

2. Neurotoxins - Targets nerve tissue
   Example - Botulinum produced by clostridium botulinum causes paralysis (stops ACh from being released at NMJ)
3. Enterotoxins - Targets digestive system
   Example - Shiga toxin produced by shigella dysenteriae causes severe dysentery (intestinal inflammation leading to severe diarrhoea with mucus or blood in the faeces.

Question 50

Selective Toxicity

Answer 50

To kill the microbial cells but but not the host cell.

Question 51

Bacterial cell components targeted by different classes of antibiotics

Answer 51

1. Inhibition of cell wall synthesis
2. Inhibition of protein synthesis
3. Disruption of cytoplasmic membrane
4. Inhibition of general metabolic pathways
5. Inhibition of DNA or RNA synthesis (replication)

Question 52

Penicllin

Answer 52

How does penicillin work?
Penicillin interferes with the normal function of the bacteria cell wall, but inhibiting the formation of peptidoglycan cross links. It will block the enzyme transpeptidase which joins the layers together causing the bacterial wall to fall apart.
Thus growing cells will be effected by penicillin.

Question 53

Mechanisms of development of antibiotic resistance

Answer 53

Mutations are the most important cause of genetic diversity in microbial populations.

• A proportion of the population develops resistance to an antibiotic by random mutations.
• Some bacteria with the resistance survive
• Bacteria with resistance multiply, passing on the resistant phenotype.
• Resistant bacterial populations survive subsequent encounters with the antibiotic.
  (This is called vertical gene transfer)

Penicillin resistance:
Beta Lactamase - an enzyme produced by some bacteria which can be used to inactivate penicillin. It does by breaking a bond to prevent it binding to tranpeptidase.

Question 54

How to reduce the development of antibiotic resistance

Answer 54

• Decrease antibiotic utilisation = Reduce infections and outbreaks through improved hygiene and infrastructure. Also produce antibiotics that are not so general and instead target specific microbes.
• Improve diagnosis = New methods to identify resistant bacteria can make treatment more effective and reduce outbreaks.
• Identify new targets = Some cellular pathways are harder for bacteria to bypass or modify, making them ideal targets for new antibiotics.
• Combination therapies = Traditional antibiotics can be combined with molecules to block resistant mechanisms.

Question 55

Properties of the bacteria genome

Answer 55

1. Typically a single circular chromosome
2. No nuclear membrane (chromosome is restricted to a defined space called the nucleoid)
3. Contains other small circular self replicating DNA molecules which can be found in the cytosol called plasmids.

Question 56

Vertical Gene Transfer

Answer 56

From parent cell to offspring.

This passes gene down through generations.

Question 57

Horizontal Gene Transfer

Answer 57

Directly from one organism to the next within the same generation.
Resistance genes as well as genes for virulence factors can be spread by horizontal gene transfer. (These genes often found on plasmids.)

Question 58

4 strategies which generate genetic diversity in bacteria

Answer 58

1. Mutations = permanent changes in the base sequence of DNA.

(Next 3 are horizontal strategies)
2. Transformation = Involves uptake of short fragments of naked DNA (from cell lysis) by naturally transformable bacteria. This will cause a change in genotype and phenotype.

3. Conjugation = Involves transfer of DNA material either through direct contact or exchanged via the sexual pilus. Thus this requires cell to cell contact.
4. Transduction = Involves transfer of DNA from one bacterium into another via a bacteriophage.
   (During replication of the virus within the bacteria, the bacterial plasma may accidentally get packaged into a phage so when it attaches to different bacteria, it will transfer this DNA. Note - bacteriophage can go through both lytic and lysogenic cycles once it has inserted its DNA which may cause a certain type of bacteria to be sometimes pathogenic but sometimes not.)

Question 59

5 Stages of an infectious Disease

Answer 59

1. Incubation Period - Microbe has attached, incubated and just started to replicate.
   = No signs or symptoms
2. Prodromal Period - Bacteria starts to replicate and begins to produce toxins. Levels are low enough for the body to handle.
   = Vague / general symptoms
3. Illness period - Microbe reached such levels that the immune system is going crazy and has to work hard.
   = Most serve sign and symptoms
   = MOST INFECTIOUS stage (period you will most likely spread the disease.
4. Decline period - Immune system finally gets going / may have also been treated causing microbe numbers to drop.
   = Declining signs and symptoms
5. Convalescence - Pathogen usually completely eliminated
   = No signs or symptoms

Question 60

Chain / spread of infection

Answer 60

1. Causative agent - Pathogenic organism eg bacteria, virus or parasite
2. Reservoir / source - Where does the microbe usually hang out eg water, human, animal, food
3. Means of exit - Way out of the body eg excretions, droplets, secretions
4. Mode of transmission - Method of spread eg contact, airborne, vector
5. Portal of entry - Way into the body eg repiratory, mucous, skin, GI, GU
6. Person at Risk - eg elderly, young, immunocompromised.

All components in this chain must be present for the infection to spread from one person to another.

Question 61

Breaking the chain of infection

Answer 61

Ways to potentially destroy or block each stage of the chain of infection.

1. Rapid detection and treatment = Critical
2. Good hygiene, sterilisation, disinfection
3. Waste removal, disinfection
4. Airflow, bed spacing
5. Aseptic technique catheter, wound care
6. Identifying risk people and targeting them for special care, vaccination and education.

Question 62

Vocabulary of Epidemiology

Answer 62

Mortality - The incidence (number) of death in a population
Morbidity - The incidence of disease, including fatal and nonfatal cases.

Incidence - The incidence of a disease is the number of new cases of the disease in a given period of time.
Prevalence - The total number of new and existing cases in a population in a given time.

Question 63

Disease Classification

Answer 63

Endemic - Amount of a particular disease that is usually present in a community is referred to as the baseline or endemic level. (eg common cold)

Sporadic - A disease which occurs infrequently and irregularly. (eg appears often but not constantly)

Epidemic - An increase, often sudden, in the number of cases of a disease above what is normally expected in the population in that area.

Pandemic - An epidemic that has spread over several countries or continents, usually affecting a large number of people.

Question 64

HIV Virus

Answer 64

HIV is a virus that infects your CD4 T cells
Infection with this virus can cause the syndrome AIDS as a result of failure of the immune system. Untreated AIDS rapidly leads to death
Infection occurs through body fluids, esp. contaminated blood products!
Anti-retroviral therapy (ART): delay or stop progression however not a cure.
Because HIV is a virus, it has a genome (that can be sequenced)

Question 65

Sequencing Virus - By comparing DNA sequences we can understand their relationship.

Answer 65

Using PCR we can isolate viral genomes or pieces of viral genomes from infected patients.
Phylogenetic trees allow us to understand relationships between viruses and the rate of mutation. We are able to trace strains back to where they diverged.
We can also use the trees to compare the viral sequences between species to see which are more similar.

Because HIV mutates so rapidly, using the phylogenetic tree we can see multiple different sequences of the HIV genome not only between patients, but also within the same patient. It can help us understand mutation patterns and where different strains have come from.

Question 66

How do patterns of relationships between sequences provide evidence for evolution.

Answer 66

We find that sequences are more closely related within a patient than sequences between patients. This suggests there is a single point entry of a virus followed by diversification, ie infection and then mutation.

Question 67

HIV Sequence changes

Answer 67

Reverse Transcriptase (converting RNA to DNA) is more prone to errors than DNA replication, resulting in many variants formed. However we do not find any variants that are not active, working viruses.
Therefore the sequence changes must be due to evolution acting on the variation caused by the errors of reverse transcriptase.

Selective pressures which the HIV virus is under:

• The immune system
• Drug regimen
• Changes in the receptor
• Tropism in the tissue (what supports growth of a particular virus).

Due to the evolution, resistance to therapies arise rapidly, making effective vaccines is very difficult.
Patients do not just have a virus, they have a vast armada of viral variants.

Question 68

The importance of evolutionary thought on our understanding of disease

Answer 68

Evolution is a key way that pathogens respond to hosts and therapy. Therefore evolutionary thinking can help us understand and better respond with treatment regimens to pathogens.