BIOC0004 Section A Q1 Section B Q1 Q2 PDF

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Section A – Question 1Discuss the mechanisms by which a virus undergoes antigenic shift andantigenic drift. Using an example of each, provide an overview of theepidemiology associated with antigenic shift and antigenic driftpathogenesis and its effects in the population.Viruses can undergo both anti...

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Section A – Question 1 Discuss the mechanisms by which a virus undergoes antigenic shift and antigenic drift. Using an example of each, provide an overview of the epidemiology associated with antigenic shift and antigenic drift pathogenesis and its effects in the population. Viruses can undergo both antigenic shifts and antigenic drifts. Antigenic shift is a process whereby a new subtype of a virus is formed by the combination of two or more different strains of a virus. In influenza viruses, the single-stranded RNA genome is divided into 8 distinct segments. In order for the virus to be infective, these segments are then randomly packaged, each one with a copy of the 8 distinct segments. More than one of these packaged viruses can infect an animal simultaneously. If two or more viruses infect the same cell, both viral genomes will be replicated, thus combining the genetic information of both strains. This is called reassortment, and results in the formation of a new genetically unique virus strain. Reassortment viruses are the cause of antigenic shift because the new genome causes changes to the structure of the virus, specifically to the glycoprotein antigens haemagglutinin (HA) and neuraminidase (NA) on the surface of the influenza virus. Antigenic shift can occur when an influenza virus in an animal gains the ability to infect humans. This is exhibited in influenza pandemics such as the 2009 swine flu caused by Influenza A subtype H1N1. This was caused by the reassortment of bird and human influenza A virus in pigs forming a new highly virulent virus strain (H1N1), displayed in the diagram below. 2 of the 8 influenza genes were traced back to the pigs in Eurasia, whilst 6 genes originated from the pigs in North America. Because antigenic shifts cause the formation of unique strains with widen and complete structural changes, the human immune system has no compatible antibodies to eradicate the disease in the body. This leads to a wave of new infections, thereby making humans more vulnerable to severe symptoms. Additionally, a weakened immune system makes the host more susceptible to secondary bacterial infections, some of which can be fatal, like severe pneumonia. The swine flu lasted about 19 months with 491,382 lab-confirmed cases, of which 18,449 were deaths. However, the suspected cases were approximately 700 million to 1.4 billion with around 284,000 deaths.

Antigenic drift occurs more frequently than antigenic shift. Antigenic drift is the accumulation of multiple, random mutations. Point mutations occur in the viral genes that code for the HA and glycoprotein antigens. The mutations may change the structure of said antigens; consequently, the antibodies of the host cell cannot bind successfully to them.

Antigenic shift is, therefore, the progressive changes in the structure of the HA and NA proteins. An example of antigenic drift is prominent in Influenza A/H3N2 and Influenza B. During 1996/7, influenza B viruses were isolated and found to be antigenically closely related to B/Harbin/7/94: the prototype vaccine strains. However, the 1996/7 isolates from Germany represented a completely different group of viruses, as they differed by 9 amino acids from B/Harbin/7/94. This indicates antigenic drift because the vaccine strain prototype was no longer effective for all strains, due to mutations causing a different sequence of amino acids in the genome. Antigenic drift has a relatively more minute effect on the population compared to antigenic shift as the small changes usually produce closely related virus strains. This means that the immune system’s antibodies will likely recognize the new strains; this process is called cross-protection. However, this is not always the case as some mutations cause greater changes as demonstrated in the example provided above where antigenic drift may lead to a diminishing population. Word count: 572

SECTION B – QUESTION 1 Describe three different ways in which growth in a biofilm can benefit bacteria. For each example, explain why bacteria may be protected against some antibacterials but remain susceptible to others Biofilms are formed by the attachment of collections of bacterial cells to a surface forming an adhesive matrix consisting of polysaccharides, proteins and nucleic acids that are secreted by said bacteria. The first way in which growth in a biofilm can benefit bacteria is that it acts as a self-defense mechanism that promotes bacterial survival. Biofilms resist physical forces that would otherwise cause detrimental damage to the bacteria by removing them from the surface because without the biofilm the bacteria would be very weakly attached to it. Moreover, biofilms provide protection against penetration by the cells of the immune system such as phagocytes (therefore, protection against phagocytosis is provided). Additionally, resistance against the penetration of toxic molecules such as antibiotics is displayed. However, the bacteria may still be susceptible to invasion by antibacterials that are bactericidal with T6SS that deliver lipases that degrade the membrane. For instance, triclosan inhibits fatty acid biosynthesis that is necessary for building and repairing the membrane. Hence, eventually the biofilm may become susceptible to deterioration. The second way in which growth in a biofilm can benefit bacteria is that biofilms allow for the bacteria to grow in nutrient-rich environments making it favorable for bacterial growth. The bacteria attach to animal tissues or flowing systems that provide the bacteria with a constant source of nutrients as the surfaces are often replenished. However, there may be selective pressures that decrease the influx of nutrients or other factors contributing to a lower concentration of nutrients available. For instance, the death of the animal tissue or the flowing systems slowing down. As a result, the biofilm is weakened and makes it susceptible to possible penetration by toxic molecules such as antibacterials. Lastly, biofilms allow for bacteria to be grown closely together. This facilitates cell-to-cell communication by providing the basis for the exchange of nutrients and genetic material. Bacteria reproduce by binary fission and as the cells are in closer proximity to each other, the process is sped up. Additionally, the exchange of nutrients is faster by forming a mutualistic symbiosis relationship between the cells. The biofilms benefit the growth of bacteria in this way, thus strengthening the assemblages of them. Although this provides them with protection against antibacterials, the bacteria are still susceptible to some antibacterials. Furanones for instance, inhibit the development of biofilms and the colonization of bacteria by interfering with bacterial quorum-sensing, this is done by interfering with the AI-2 signaling system. AI-2 is an autoinducer signal molecule, produced by the protein LuxS, which is found in multiple bacterial species and facilitates interspecies communication in both gram-negative and gram-positive bacteria. Furanones also inhibit

exo-enzyme expression. These are responsible for degrading the cells of the human immune system. This inhibition enhances the immune systems response, making biofilms more susceptible to being attacked by the immune system. Word count: 468

SECTION B – QUESTION 2 i) Briefly describe (i.e. two or three sentences for each technique, with drawings if appropriate) each of the different experiments you carried out in the lab to reach your conclusions. The bacterium is identified as gram-negative using the gram staining technique. The primary stain, Hucker’s Crystal Violet, is added to a heat-fixed smear followed by the addition of iodine solution. A mixture of alcohol and acetone is then added to decolorize the sample (from purple to colorless). The sample is then counterstained with safranin and the sample turns pink due to the presence of gram-negative bacteria that have cells walls containing only a thin layer (about 10%) of peptidoglycan and a high lipid content. Bacteria are identified as obligate aerobes using a thioglycolate broth. The bacteria being examined gather at the top of the test tube containing the broth, as the oxygen concentration is the highest, meaning that they require oxygen for survival. This shows that the bacteria cannot respire anaerobically or ferment as they are obligate aerobes.

The bacterium is described as a natural lysine auxotroph. This is examined by subjecting it to an experiment where it is plated in a medium with minimal nutrient requirements lacking lysine. In this environment, the bacterium does not grow. If the bacteria are placed in a minimal medium with arginine, it does not grow either. This indicates that the bacterium requires the presence of lysine to grow, so it is a lysine auxotroph.

The catalase test indicates that the bacterium is catalase positive. The technique for this experiment involves suspending the sample in a solution of hydrogen peroxide. When catalase is present, bubbles of oxygen are released, confirming the decomposition of hydrogen peroxide into water and oxygen. The oxidase test is used to indicate that the bacterium produces the cytochrome oxidase enzyme. Smear a sample of the bacterium onto disk with dried filter paper containing Kovács oxidase reagent; as the bacterium is positive for the enzyme, the color changes to dark purple within 5-10 seconds.

The bacterium can be tested for sensitivity to the four antibiotics using antibiotic susceptibility testing where a petri dish containing nutrient agar is streaked with a sample of the emulsified bacteria in a sterile saline solution. An antibiotic disk containing ampicillin, rifampicin, gentamicin and tetracycline is placed on the surface of the petri dish. This dish is then incubated and the zones of inhibition of each antibiotic is measured and compared with a standard table to determine its sensitivity. For gentamicin and tetracycline, the zones of inhibition are small indicating that the bacteria are less sensitive and therefore, resistant to these antibiotics. However, for ampicillin and rifampicin the zones of inhibition are larger indicating higher sensitivity towards these antibiotics. Lastly, PCR is used to amplify the 16s ribosome gene and cell morphology through visual examination to identify the yellow color, irregular colonies and crusty edges is used to classify the new species of the genus Pseudomonas.

ii) Suggest, again in a couple of sentences, three additional tests you might carry out to further characterize your isolate. Firstly, I would determine what the optimum pH for bacterial growth is in this case. I would do this by subjecting the bacteria to different pH values ranging from 1-14 and determine at which pH the population of the bacteria is the greatest i.e., how big the colony is. This can be done using serial dilution or by measuring the turbidity of the culture. Secondly, I would determine the optimum temperature for bacterial growth. I would do this by incubating the bacteria at different temperatures ranging from 10-50C and measure the bacterial growth in the same way. Thirdly, I would measure the optimum salinity for bacterial growth by adding samples of the bacteria to different concentrations of saline solutions ranging from 0.1-20% concentration. I would measure the growth at each concentration to determine the preferred concentration for optimum bacterial growth. Word count: 599...