Microbiology

Question 1

Describe conjugation, including the plasmid and genes required

Answer 1

F plasmid containing the tra operon is required for conjugation to take place.
This allows an F pilus to be formed from the donor cell, which binds to specific receptors on the surface of the recipient cell.
Cells are pulled together by disassembly of pilin subunits.
A single strand of plasmid DNA is transferred after the plasmid is cut by the TraI enzyme allowing it to unwind and transfer to the recipient, making the recipient F+.

Question 2

Describe natural transformation

Answer 2

Bacterial cell produced competence factors which are released into the environment. These interact with receptors on the bacterial cell surface to increase membrane permeability.
Double stranded DNA begins to cross the cell membrane, where it is cleaved by a nuclease enzyme, leaving a single strand to enter the cell.
Once in the cell the ssDNA is protected from other enzymes by DNA binding proteins, allowing it to recombine into the cells genome.

Question 3

Describe artificial transformation

Answer 3

Chemical transformation: Cells incubated with CaCl2 to make them competent. Competent cells are mixed with selected DNA and subjected to heat shock treatment to allow DNA uptake by the cell.

Electroporation: Electroporation is used to increase membrane permeability, making cells competent. Cells are mixed with selected DNA and subjected to high voltage electrical impulses to allow uptake of DNA by the cell.

Question 4

What is the difference between generalised and specialised transduction?

Answer 4

Generalised: Random fragments (1) of bacterial chromosome (i.e. any gene can be transferred) are package within phage particles during assembly and then transferred to another bacterial cell during infection cycle (1). Only occurs during the lytic cycle (1).

Specialised: Requires lysogenic cycle (1). Incorporation of bacterial genes within the genome of a lysogenic/temperate phage when it excises incorrectly from within bacterial genome (1). Specific genes are transferred by the phage (1) as they are next to the integration site for the phage in the bacterial chromosome (1).

Question 5

How could a single substitution result in reduced growth/division of a cell?

Answer 5

If an essential base in a protein required for growth/division is changed this could result in that outcome. A missense mutation could change an essential aa or a nonsense mutation could cause a premature stop codon (1). This could result in gene being transcribed/translated producing a partial/inactive protein which could be non-functional (1).

Question 6

What is the difference between composite transposons and insertion sequences?

Answer 6

Composite transposons also carry additional genes (e.g. for antibiotic resistance).

Question 7

What is the difference between PD, NPD, and TT?

Answer 7

Parental ditype - tetrad contains parental meiotic products (ab, ab, AB, AB)

Non-parental ditype - tetrad contains 2 parental and 2 recombinant meiotic products (aB, aB, Ab, Ab)

Tetratype - tetrad contains 2 types of meiotic products different from parents (ab, aB, Ab, AB)

Question 8

How do you tell if genes are linked or unlinked based on tetrad organisation?

Answer 8

Unlinked
If two genes are unlinked, the possible tetrad types produced are PD and NPD in equal frequency and some TT.
Frequency of PD tetrads = frequency of NPD tetrads.
Genes far apart, can easily change during crossing over.

Linked
If two genes are linked, the possible tetrad types produced are mostly PD and TT. NPD are the lest frequent and quite rare.
Frequency of PD tetrads > frequency of NPD tetrads.
Genes close together, less recombinants.

Question 9

Describe the epidemiological aspects of a microbial disease

Answer 9

Cholera caused by Vibrio cholerae. It is transmitted via faecal-oral route as a result of contaminated food/water. It can also be transmitted via naturally contaminated shellfish if eaten raw/undercooked. The disease is rare in industrialised countries, with most cases in Africa (e.g. DR Congo, Ethiopia, Nigeria), Asia (e.g. India, Bangladesh, Nepal) and Haiti. Symptoms include dysentery/watery diarrhoea, vomiting, muscle cramps, circulatory collapse. Results in death in ~20% cases if left untreated. Treated by oral rehydration solution (sometimes IV fluid replacement required). Vaccination: oral vaccines can provide short term/4-6 months protection with an effectiveness of 90%. (1)
Prevention/Education: WHO and CDC promote the Global Water, Sanitation and Hygiene (WASH) programme to encourage use of house water treatment systems. (1)

Question 10

Describe the coagulase test with results examples

Answer 10

Coagulase is an enzyme which converts fibrinogen to fibrin, resulting in blood plasma clotting. The test distinguishes the presence of this enzyme.

Positive: Staphylococcus aureus.
Negative: Staphylococcus epidermidis.

Question 11

Describe the indole test with results examples

Answer 11

Determines the ability of a bacterial species to convert the amino acid tryptophan into indole. Results in red colonies in media.

Positive: E. coli., Proteus vulgaris, Haemophilus influenzae, Vibrio sp.
Negative: Enterobacter, Klebsiella

Question 12

Briefly describe direct and indirect immunofluorescence

Answer 12

Direct/Primary: A primary antibody that is fluorescently tagged is incubated with the sample. This antibody specifically binds to an antigen present in the sample.

Indirect/Secondary: An unlabelled primary antibody specific against the antigen is incubated with the sample. A secondary fluorescently tagged antibody specific against the primary antibody is then applied.

Question 13

How can ELISA be used to detect a viral specimen?

Answer 13

ELISA (Enzyme-linked immunosorbent assays) can be used to test patient sera (1) for the presence of specific viral antigens (1). Present of viral antigens will result in binding to monoclonal antibodies against the specific antigen (1). This is detected via a labelled monoclonal antibody (e.g. fluorescence, enzyme causing colour change) (1).
This can be used to detect viruses such as: any appropriate example e.g. HIV, Hepatitis B, rotaviruses and SARS-CoV-2 (1).

Question 14

What occurs during a haemagglutination assay?

Answer 14

Haemagglutination is when a microorganism directly causes agglutination of red blood cells (1). The presence of antibodies against the microorganism prevents haemagglutination (1). Inhibition assays screen serum for the presence of specific antibodies against the microorganism, with a positive result being the inhibition of haemagglutination (1).

Question 15

What are the principles of qPCR with example techniques?

Answer 15

Quantitative PCR technique (1).
There are two approaches to this:
Use of dyes which bind to dsDNA. As reaction progresses and more dsDNA is formed, a stronger fluorescent signal is produced (1).
Use of probes which have a 5’ flurophore and 3’ quencher. These bind a target sequence and as amplification proceeds, DNA polymerase cleaves the probe releasing the fluorophore, resulting in an increase in fluorescent signal (1).
Signal detected by a computer which can measure the amount of nucleic acid target and plot it against a standard curve of known quantity of target gene, allowing quantification (1).
This can be used to detect pathogens in patient samples by isolating DNA/RNA (1) and using specific pathogen primers to amplify and quantify pathogen in sample (1).

Question 16

Describe the drug diffusion assay

Answer 16

Lawn of microorganism to be tested grown on an agar plate.
Filter paper discs of known size covered in antimicrobial agent (e.g. antibiotic) placed on plate.
Plates incubated for a standard timeframe (normally 24 hours).
Diameter of inhibition zone is measured.
Measured against standards (known) to indicate resistance or susceptibility.

Question 17

What is the difference between whole cell and nucleic acid vaccines?

Answer 17

Whole cell - A dead or weakened/attenuated version of the microorganism can be used to stimulate an immune response against the antigens on this harmless version of the pathogen.

Nucleic acid - DNA/RNA encoding a pathogen antigen (1) is injected into muscle cells (1). The pathogen antigen protein is produced and expressed on the surface of host cells (1). This stimulates an immune response against the antigen, providing immunity (1). (Covid-19)

Question 18

What would be the steps for testing a patient with a suspected enterobacterial infection?

Answer 18

A faecal sample is taken and sent to the microbiology diagnostic lab (1).
A suspension is made and spread aseptically on a range of selective media for enterobacteria, such as MacConkey’s agar (1).
Isolates likely to be facultative anaerobes (1), grow aerobically/anaerobically/ facultatively at 37oC for 24–48 hours (1).
Colony morphology and microscopic analysis to inform empirical therapy (1).
DNA extraction and analysis to confirm (1).

Question 19

What would be the outcome of mating between Hfr and F-?

Answer 19

Hfr strains have the plasmid incorporated into the bacterial chromosome. As a result, conjugation between a Hfr and F- strain would result in some of the bacterial chromosome being transferred and the full F plasmid would not be transferred. The recipient cell would therefore remain F-.

Question 20

What is phage therapy and how could it be used?

Answer 20

Bacteriophage are viruses which infect specific bacterial strains, replicate inside the bacteria resulting in lysis of the cell. Phage therapy involves taking advantage of this, which could be beneficial as antibiotic resistance is becoming more of an issue in bacterial populations. There is the potential to genetically modify phage to target specific bacterial pathogens. While bacteria may evolve resistance to phage, phage would also have the potential to evolve and overcome any resistance.

Question 21

Why might a pathogen be limited in the cells it can adhere to?

Answer 21

Specific pathogens capable of interacting with specific receptors in host cells. Distribution of receptors determines the range of host tissues the pathogen can adhere to and infect.

Question 22

Describe the activity of example AB toxins.

Answer 22

Botulinum toxin produced by Clostridium botulinum. The A domain is the active enzyme (a protease) while the B domain is involved in binding to host cell membranes and allows A to be delivered. The A subunit is responsible for cleaving receptors and preventing release of acetylcholine. This prevents muscle contraction causing flaccid paralysis, which can lead to suffocation and death.

Tetanospasmin is an AB toxin produced by Clostridium tetani. The A domain is the active toxin while the B domain is involved in binding to host cell membranes and allows A to be delivered. The A subunit is responsible for binding to inhibitory interneurons in the spinal cord causing continuous release of acetylcholine. This prevents muscle relaxation causing spastic paralysis.

Question 23

What is quorum sensing?

Answer 23

Method of intercellular communication used by bacterial cell to determine population density via secretion of autoinducer molecules into the environment. Once a critical level of autoinducer is present, cells respond in a coordinated way through changes in gene expression. This causes changes in bacterial behavior.

Question 24

Describe the role of quorum sensing in the bioluminescence of Vibrio fischeri.

Answer 24

Bioluminescence is controlled by the lux operon. Bacterial cells produce AHL autoinducer molecule. Once cells reach a high enough density, AHL reaches a high extracellular concentration. This results in AHL diffusing back into the cell where it binds to and activates LuxR. LuxR is responsible for activating genes to produce more AHL and the genes required for bioluminescence. This is not able to happen when cells are at a low density, as only low concentrations of AHL are present in the environment.

Question 25

Describe the process of biofilm formation.

Answer 25

A small number of cells attach to a surface. Intercellular signalling/quorum sensing between cells results in formation of a colony and production of a polysaccharide matrix. The biofilm develops, growing over time through the recruitment of new cells and expansion of the matrix. Cells are able to detach from the biofilm.

Question 26

Describe antigenic drift and shift in relation to influenza virus.

Answer 26

Drift - accumulation of point mutations in existing antigens, common and gradual over time/humans can retain partial but not complete immunity.

Shift - during co-infection viruses can exchange similar sections of their genome - gene reassortment. Results in rapid change and a new strain with a combination of structural proteins from both strains.

Question 27

Describe antigenic variation in relation to Trypanosoma brucei and how it allows evasion of the host immune response.

Answer 27

T. brucei has >1000 varient surface glycoprotein (VSG) genes, with only one VSG being expressed at a time. Antigenic variation involves changing which VSG is expressed on the surface of the pathogen. VSG genes are actively transcribed at telomeric expression sites; other VSG genes are at internal chromosomal loci and are silent. During infection, the host produces antibodies against the VSG antigens which would allow T. brucei to be recognised and killed. Changing which VSG is expressed means that the pathogen is no longer recognised by the immune response/antibodies. This results in continuous waves of parasitaemia as new antibodies are produced and therefore the VSG coat changes again.

Question 28

Briefly explain ways bacterial pathogens can overcome phagocytosis.

Answer 28

Avoid - e.g., downregulate gene expression involved in the recruitment of phagocytes.

Neutralise - e.g., prevent phagocytes from crossing cell layers.

Survive - e.g., prevent phagolysosome formation.

Kill - e.g., prouce toxins/cytolysins/streptolysins/leukociding to destroy phagocytosis.

Question 29

Explain why a pathogen may be limited in the cells it can adhere to.

Answer 29

Specific pathogens capable of interacting with specific receptors in host cells. Distribution of receptors determines the range of host tissues the pathogen can adhere to and infect.

Question 30

Identify two benefits of biofilms.

Answer 30

Protection from antibiotics/predators, increased resistance.
Enhanced adhesion to surfaces.
Source of food by remaining in a favourable environment/utilising waste from other bacteria.

Question 31

Describe the role of antigenic variation in developing drug resistance in Trypanosoma brucei.

Answer 31

Single nucleotide polymorphisms result in incorporation of alternative nucleotides. This is common in microsatellite regions, which are made of repetitive sequences with high rates of mutation. These mutations can result in changes in the structure of drug targets, leading to resistance.

Question 32

Describe the mechanism of action of penicillin.

Answer 32

Interferes with the linking of interpeptides of peptidoglycan. Inhibits transpeptidases enzyme which is responsible for linking peptidoglycan strands. Reduces crosslinks which weakens the cell wall structure making it prone to rupture during cell division. Weakened cell wall creates osmotic imbalance due to water being able to flow into the cell resulting in lysis and death.

Question 33

Describe the mechanism of action of the antibiotic tetracycline.

Answer 33

Inhibits elongation of growing polypeptide chains by binding to 30S ribosomal subunit. Prevents the binding of tRNA to mRNA-ribosome complex. Prevents addition of new amino acids, thus inhibiting protein synthesis.

Question 34

Describe the mechanism of action of the antiviral acyclovir.

Answer 34

Nucleoside analogue, which is phosphorylated in host cell. Competitively inhibits viral DNA polymerase (high affinity) which decreases replication of viral DNA. Can also be incorporated into growing viral DNA which results in chain termination.

Question 35

List some consequences of mutations.

Answer 35

Beneficial
- Gain-of-function mutations to enhance gene product.
- Increased fitness (e.g., antibiotic resistance)
- Change of surface proteins to evade recognition

Detrimental
- Loss-of-function mutations resulting in reduced or less of a gene product (e.g., change in an essential base could result in non-functional/inactive protein.
- Lethal mutations.

Question 36

Difference between direct, indirect, and sandwich ELISA?

Answer 36

Direct - Primary antibody labelled with reporter enzyme or tag. Reacts directly with the antigen immobilised on the plate.

Indirect - Labelled secondary antibody with specificity for the primary antibody. The primary antibody binds to immobilised antigen, secondary antibody binds to primary.

Sandwich - The antigen binds to an immobilised capture antibody, and the primary antibody binds to the antigen. Labelled secondary antibody with specificity to the primary binds.

Question 37

Describe a specific fimbriae.

Answer 37

Fimbriae are types of short pilus involved in attachment. They have thin, rod-like structures composed of protein.

Type 1 fimbriae are commonly found in Gram negative enterobacteria (like e coli, salmonella, klebsiella). They are composed primarily of FimA protein which is a major structural protein. Minor protein component includes FimH which is found at the tip and interspersed along the length of the fimbriae. FimH is an adhesin - promotes specific adhesion to D-mannose residues (e.g., in host cell surface glycoproteins).

Question 38

Autoinducer molecules.

Answer 38

Also known as quormone. They are signalling molecules secreted by bacteria. The bacteria must have a receptor (GS receptor) which detects and binds the autoinducer. It results in activation of certain genes (transcription) including those for signalling/autoinducer synthesis leading to an increased concentration of autoinducer in the environment.

Question 39

Pathogenicity and virulence

Answer 39

Pathogenicity is the ability of a microorganism to cause disease. i.e., it is successfully evaded the host defences.

Virulence is the degree of pathogenicity determined at the species level.

Quorum sensing has been shown to be important for virulence ina number of pathogenic microorganisms. e.g., pseudomonas aeruginosa, staph aureus, vibrio cholera.

It might be beneficial for a pathogen to have toxin production controlled by quorum sensing as it allows bacteria to overcome the immune response and establish infection first. Once there are sufficient numbers present, toxins will be produced and have their maximum effect. Saves wasting energy on low levels of toxin production.

Question 40

Pathogenicity and virulence in pseudomonas aeruginosa

Answer 40

Opportunistic pathogen that is a problem in cystic fibrosis patients. Uses AHL and 2-alkyl-4-quinolones as quorum sensing molecules. Disruption of quorum sensing reduces virulence.

Question 41

Pathogenicity and virulence in staphylococcus aureus.

Answer 41

Low population density; produces surface protein required for attachment and colonisation.

High population density; production of above factors repressed, toxins and proteases are secreted, switch regulated by the Arg quorum sensing system.

Question 42

Antigenic variation in influenza virus.

Answer 42

Type A influenza viruses responsible for human pandemics. Variation in surface proteins haemagglutinin (HA) and neuraminidase (NA) make influenza viruses difficult to recognise by the immune response.

18 HA subtypes and 11 NA subtypes for different viruses like H1N1 and H5N1.

Question 43

Avoiding phagocytes - Porphyromonas gingivalis

Answer 43

P gingivalis avoids phagocytosis by downregulating expression of the host intercellular adhesion molecule-1 (ICAM-1). This is normally used as a homing beacon to direct phagocytes to infected areas. Preventing ICAM-1 expression prevents recruitment of leucocytes across the epithelial layer to destroy bacteria.

It can also downregulate host production of interleukin-8 (IL-8) which normally attracts neutrophils which results in inflammation. Preventing this allows the bacteria to colonise the infected site.

Question 44

Examples of how various bacteria neutralise phagocytes.

Answer 44

CNTF1 toxin E. coli activates the Rho protein which closes tight junctions between cells, preventing phagocyte trafficking across cell layers.

Streptococcal streptolysin (exotoxin) inhibits neutrophil chemotaxis.

Staphylococcus aureus protein A binds to the Fc region of antibodies, preventing recognition by macrophages and phagocytosis.

Helicobacter pylori can change its surface antigens by changing the reading frame in which its proteins are translated from mRNA.

Question 45

Examples of how some bacteria survive phagocytosis.

Answer 45

Chlamydia species are in phagosomes they are able to prevent lysosomes fusing with the phagosome to modify the phagosome membrane.

Mycobacterium tuberculosis blocks phagolysosome formation and inhibits acidification of the phagosome by preventing of the proton ATPase.

Lysteria monocytogenes escapes the phagosome by destroying the membrane using the enzymes listeriolysin O and phospholipase C.

Question 46

Different ways of killing phagocytes.

Answer 46

Before ingestion - production of cytolysins form pores in the phagocyte cell membrane.
Streptolysins and leukocidins cause neutrophil granules to rupture, releasing toxic substances to kill the cell.
Some exotoxins function to reduce the supply of ATP to phagocytes, preventing phagocytosis.

After ingestion -
Rlease of toxins which travel through phagosome/phagolysosome membrane.
Lysteria monocytogenes release proteins which kill macrophages but allow bacteria growth in the short term.