Microbiology Flashcards
Describe conjugation, including the plasmid and genes required
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+.
Describe natural transformation
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.
Describe artificial transformation
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.
What is the difference between generalised and specialised transduction?
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).
How could a single substitution result in reduced growth/division of a cell?
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).
What is the difference between composite transposons and insertion sequences?
Composite transposons also carry additional genes (e.g. for antibiotic resistance).
What is the difference between PD, NPD, and TT?
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)
How do you tell if genes are linked or unlinked based on tetrad organisation?
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.
Describe the epidemiological aspects of a microbial disease
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)
Describe the coagulase test with results examples
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.
Describe the indole test with results examples
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
Briefly describe direct and indirect immunofluorescence
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.
How can ELISA be used to detect a viral specimen?
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).
What occurs during a haemagglutination assay?
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).
What are the principles of qPCR with example techniques?
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).
Describe the drug diffusion assay
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.
What is the difference between whole cell and nucleic acid vaccines?
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)
What would be the steps for testing a patient with a suspected enterobacterial infection?
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).
What would be the outcome of mating between Hfr and F-?
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-.
What is phage therapy and how could it be used?
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.
Why might a pathogen be limited in the cells it can adhere to?
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.
Describe the activity of example AB toxins.
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.
What is quorum sensing?
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.
Describe the role of quorum sensing in the bioluminescence of Vibrio fischeri.
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.
