Assignment 4

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May 30 th , 2023 Assignment 4: (covers Units 13-15; Chapters 21-28, 18, 20, and 9 of the textbook) Due: After Unit 15 Weight: 10% of course final grade This assignment is out of 100 marks. Your mark for this exercise will be 10% of your final grade for the course. This assignment is made up of one short-essay question worth 20 marks; a set of short-answer questions, worth 40 marks; and a set of questions based on four research articles, worth 40 marks. A. Short-Essay Question (20 marks) Length: 300-500 words The essay should be 300-500 words long. Marks will be deducted if it is shorter than 300 words. The essay must include in-text citations and a full bibliography (reference list). For your referencing format, see the “ Instructions to Authors ” from the American Society for Microbiology (online). Your citations and references must follow the ASM format. You may use figures or tables in the essay component to illustrate concepts; these must also be formatted according to ASM requirements. 1. Identify one body system, and describe an infection of this system according to the following criteria: a. site of infection b. etiology c. virulence factors d. pathology e. outcome of disease f. treatment g. prevention Respiratory System – Tuberculosis Tuberculosis is a serious infection that primarily afflicts structures of the lower respiratory system. It predominantly attacks the lungs, 75% of the time, but may also take the form of extrapulmonary tuberculosis - when it occurs outside of the lungs such as in bones and joints, the kidney, the liver, cerebral spinal fluid, and in the lymph nodes (1,2). 1

May 30 th , 2023 Tuberculosis is caused by the bacterium Mycobacterium tuberculosis and is spread from infectious individuals via bacterial aerosol and sputum through speaking, coughing, sneezing, or contaminated surfaces (2,3). An individual contracts tuberculosis after inhaling droplets contaminated with M. tuberculosis and are phagocytized by macrophages present within the alveoli (2). Hosts are usually unaware when infected with tuberculosis pathogens as a healthy individual is able to defend against it 90% of the time (2). Mycobacterium tuberculosis is an obligate aerobe that sometimes forms filaments as it tends to grow in clumps, in a mold-like appearance (1). It’s cell wall contains large amounts of lipids (indicating that it is acid-fast) enabling it to be resistant to environmental stressors such as desiccation and chemical antimicrobials such as antiseptics and disinfectants (1). When macrophages ingest but fail to kill pathogens, infection occurs (4). M. tuberculosis multiplies within macrophages forming a tubercle (1,4). The mycolic acids in the cell wall stimulate an inflammatory response in the host releasing enzymes and cytokines that cause damaging inflammation to the lung (1). After a few weeks of infection, some macrophages die forming a anaerobic caseous center in the tubercle, this leads to many tubercle bacilli remaining dormant causing latent tuberculosis, a reactivation of the disease (1). In some individuals a mature tubercle is formed as the caseous center enlarges through liquefaction and produces and air-filled tuberculous cavity (4). This allows the pathogenic bacilli to multiply outside of macrophages. The tubercule may then rupture, spreading the infectious bacilli into a bronchiole then subsequently throughout the lungs, and the circulatory and lymphatic systems (4). This stage is referred to as miliary tuberculosis (1). Symptoms of tuberculosis include low fever, chills, a general sense of malice, tiredness, difficult and/or painful breathing, coughing (blood filled with progression of the disease), chest pain, and sudden and severe headaches (1). With more advance stages significant weight loss is typically observed followed by blood vessel rupture and hemorrhaging that may lead to death without intervention (4,5). Diagnoses can be preformed with a tuberculin skin test, where patients are injected with a diluted antigen (3,5). If the individual has had or has tuberculosis, 2

May 30 th , 2023 sensitized T cells active at the injection site producing a hardening and reddening of the area (3,5). Treatment varies based on the sensitivity of the organism and other factors but typically consists of prolonged antibiotic usage (3,6). A multiple-drug therapy approach is predominantly used for 6 months to minimize the emergence of resistant stains – this includes drugs such as isoniazid, rifampin, ethambutol, and pyrazinamide, termed first-line drugs (6). Second line drugs are commonly used for multi-drug resistant (MDR) strains – these include several aminoglycosides, fluoroquinolones, streptomycin, and para-aminosalicylic acid (PAS) (6). Prevention of tuberculosis can be achieved through limiting contact with known contagious individuals along with proper personal protective equipment when interacting with sick individuals (1). More specifically, the Bacille Calmette-Guerin (BCG) vaccine is commonly used to prevent tuberculosis, so much so it is the most widely used vaccine in the world (6). The BCG vaccine uses a live, attenuated strain of Mycobacterium bovis to provide immunity (6). Though researchers have noted issues with the effectiveness of the BCG vaccine and it wide usage in populations (6). New vaccines are now currently in development and testing phases (2,3,6). References 1. Tortora GJ, Funke BR, Case CL. 2019. Microbiology: An Introduction, 13th ed. Pearson, Boston, United States of America. 2. Guinn KM, Rubin EJ. 2017. Tuberculosis: just the FAQs. MBio, 8(6):e01910-17. 3. Suárez I, Fünger SM, Kröger S, Rademacher J, Fätkenheuer G, Rybniker J. 2019. The diagnosis and treatment of tuberculosis. Dtsch Arztebl Int, 116:43. 4. Churchyard G, Kim P, Shah NS, Rustomjee R, Gandhi N, Mathema B, Dowdy D, Kasmar A, Cardenas V. 2017. What We Know About Tuberculosis Transmission: An Overview. J Infect Dis. 5. Koegelenberg CFN, Schoch OD, Lange C. 2021.Tuberculosis: The Past, the Present and the Future. Respiration. 100(7):553-556. 6. Liebenberg D, Gordhan BG, Kana BD. 2022. Drug resistant tuberculosis: Implications for transmission, diagnosis, and disease management. Front Cell Infect Microbiol. 12:943545 3

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May 30 th , 2023 B. Short-Answer Questions (40 marks) Length: (Relate the length to the number of marks alloted to the question.) 1. Define meningitis . What three bacteria cause meningitis? ( 4 marks ) Meningitis is the inflammation of the meninges, the three membranes covering the brain and spinal cord, and can be caused by numerous pathogens such as viruses, bacteria, fungi, and protozoa. Only three bacterial species have been historically reported to cause meningitis. Each which posses a capsule, enabling them to evade phagocytosis and replicate rapidly in bloodstreams, entering the cerebral spinal fluid. Haemophilus influenzae type B, once prevalent, has now been nearly irradicated in the United States due to a highly effective vaccine. Now Neisseria meningitidis and Streptococcus pneumonia are primarily the cause of 80% of the meningitis cases in adults over 16. A vaccine against S. pneumoniae is now coming into more common use to help provide immunity. 2. Compare the etiologies of bacterial pneumonia, viral pneumonia, and influenza. ( 6 marks ) 4 1. Influenza 2. ◦ Caused by influenzavirus ; several serotypes 3. ◦ HA and NA spikes – allows recognition and attachment to cells 4. ◦ high rates of genetic alterations increasing virulence 5. Viral pneumonia 6. ◦ usually resultant of influenzavirus , but can be from measles or chicken pox 7. ◦ complication of viral infections - when it progresses and goes untreated, invading the lungs causing them to swell 8. 9. Bacterial pneumonia 10. ◦ Typical pneumonia 11. ◦ From: Haemophilus influenzae type B, - gram-negative, coccobacillus Neisseria meningitidis, or - Streptococcus pneumonia - gram-positive

May 30 th , 2023 3. Travellers to other countries may aquire an infectious disease of the gastrointestinal tract. Some illnesses are self-limiting, while others are more severe and require medical intervention. Contrast two diseases of the digestive tract, Traveller’s diarrhea and cholera, in terms of causative agent, method of transmission, symptoms, and treatment. ( 8 marks ) Traveller’s Diarrhea Cholera Causative Agent Escherichia coli, Salmonella spp. , Shigella sonnei, Campylobacter jejuni, etc. Vibrio cholerae O:1 and O:139 Method of Transmission Ingesting contaminated food and beverages Ingesting contaminated substances; usually from stale water after natural disasters Symptoms Mild to severe abdominal pain, diarrhea, fever, vomiting Diarrhea with large water loss Treatment Rehydration; Azithromycin, Fluoroquinolones, Rifamycins Rehydration; Doxycycline 4. Name one protozoal disease of the reproductive system, and indicate the causative agent. ( 2 marks ) Trichomoniasis is a reproductive system disease caused by the disruption of the normal acidity within the vagina resulting in the overgrowth of the protozoan Trichomonas vaginalis . 5. Define bioremediation and bioaugmentation, and give an example of the use of bacteria to remove pollutants. ( 5 marks ) Bioremediation is the use of microbes to detoxify or degrade pollutants. This can occur naturally as some bacteria break down petroleum in aerobic environments. This bioremediation can be utilized in instances of oil spills from wrecked tankers and drilling accidents, one of the most dramatic examples of chemical pollution. 5

May 30 th , 2023 Additional nutrients, such as nitrogen and phosphorus (bioenhancers), are added to a contaminated water source to supplement and enhance oil degradation. Bioaugmentation, similar to bioremediation, is the addition of microbes that have been specifically selected for growth on a certain pollutant or of genetically modified bacteria that are specially adapted to metabolize petroleum products. For example, bioaugmentation is typically used in municipal water treatment to restart activated sludge bioreactors. 6. Describe one method of food preservation. What food is preserved by this method? Name at least one contaminating organism that this method is used to prevent. ( 5 marks ) Industrial food canning involves heating the product enough to kill dangerous microbes and spoilage organisms without degrading the palatability or appearance of the food. Fruit, vegetables, soup, and meat are commonly preserved by canning to prevent the growth of harmful microbes such as the endospore-forming Clostridium botulinum . Commercial sterilization begins with washing and sort followed by blanching through hot water or steam to denature enzymes that might affect colour, flavor, or texture. From there cans are filled leaving minimal room, steam is used to remove dissolved air, and then sealed. Sealed cans are then sterilized and cooled then labeled and sent to distributors. 7. What is a toxoid? How are toxoids used as vaccines? Give an example of a toxoid vaccine. ( 4 marks ) Toxoids are inactivated endotoxins that have been treated with heat, formaldehyde, iodine, or other chemicals so that that can no longer cause disease in individuals. Toxoids are still able to stimulate the body to produce antitoxins thus making them an effective vaccine. Diphtheria and tetanus can be prevented by a toxoid vaccine. 6

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May 30 th , 2023 8. What is the minimal inhibitory concentration of an antibiotic, and how does this relate to treatment of disease? ( 3 marks ) The minimal inhibitory concentration (MIC) of an antibiotic is the lowest concentration of a chemotherapeutic agent that will prevent the growth of a test bacteria. This can be conducted through E tests or broth dilution tests. Antibiotic concentrations higher than the MIC should be free of the bacteria when cultured. Determining the MIC allows doctors to avoid the underuse of antibiotics, not effectively treating the pathogenic agent but also the overuse and toxic reactions associated with larger-than-necessary dosages of antibiotics. 9. What are restriction enzymes, and why are they used in recombinant DNA technology? ( 3 marks ) Restriction enzymes are DNA-cutting enzymes found in many bacteria that protect bacterial cells by hydrolyzing bacteriophage DNA. In today’s laboratories restriction enzymes are specified so that the enzyme only recognizes and cuts a particular sequence of nucleotide bases (4-8 long) in DNA, cutting DNA the same every time. Hundreds of restriction enzymes are known, each able to cut and produce DNA fragment with characteristic ends. These restriction enzymes allow scientist to cut DNA in desired locations to modified organismal DNA to obtain enhanced or desired characteristics by adding or remove sections of DNA. This is known as recombinant DNA technology. 7

May 30 th , 2023 C. Research Questions (10 marks each article = 40 marks) These research questions are based on articles in the Scientific American special supplements. Referencing is not required in answering these questions, but you must paraphrase the information in your own words. Only a few sentences (maximum) are required for each answer. 1. Article: Taubenberger, J. K., Reid, A. H., & Fanning, T. G. (2005). Capturing a killer flu virus . Scientific American 292 (1), 62-71. a. What unusual symptoms did patients infected with the 1918 virus display that made it appear that this influenza was a new illness? How many people died of this particular strain of the flu? Doctors originally diagnosed the ailment as meningitis, but with the rapid spread of the illness along with the rapid progression of the disease, leading to death less than 48 hours after first symptoms, accompanied by blueish premortem skin and blood and/or fluid filled postmortem lungs, doctors presumed it to be a new infection or plague. Within the year following 1918, approximately 40 million people across the world between died of this strain of influenza. b. Describe the genetic structure of influenza A. Eight separate single-stranded viral RNA segments. c. How are influenza A strains classified? What is the classification of the 1918 strain? Influenza A stains are identified via their hemagglutinin (HA) and neuraminidase (NA) signature proteins on the virus’s surface. Taubenberger, Reid, and Fanning (2005) indicate that HA has 15 subtypes and NA has 9 subtypes. For example, A(H7N4) virus indicated influenza A virus subtype with an HA 7 protein and an NA 4 protein. The 1918 strain was classified to be H1N1. d. How were scientists able to characterize the 1918 virus even though no samples of the living virus were available? Following exposure to the 1918 influenza A virus, hosts develop antibodies to the specific protein subtypes preventing reinfection. Scientists were thus able to retrospectively identify the 1918 virus subtype by identifying the antibodies present in the bloodstream of the pandemic’s survivors. 8

May 30 th , 2023 e. This article was published in 2005. Based on the classification of the 1918 strain and the predictions these authors make, what connections can be made to the 2009 H1N1 pandemic? Taubenberger, Reid, and Fanning conclude from their findings that the 1918 strain was a precursor to both the human and swine H1N1 lineages. As the 1957 and 1968 pandemic stains had avian-like HA proteins in contrast to human or swine, the authors suggest these new adaptions to be a result of direct avian reassortment. The authors further dictate the importance of extended studies to examine the acquired novel genes that made the 1918 strain highly pathogenic and highly contagious, warning of public health implications. One with limited knowledge on the influenza virus can only speculate this to be the exact cause of the swine-origin H1N1 reassortment that occurred in 2009. 2. Article: Costerton, J. W., & Stewart, P. S. (2001; updated 2006). Battling biofilms . Scientific American 285 (1), 74-81. a. What is a biofilm? Biofilm is a complex and dense aggregation of organized bacterial colonies and bacterial secretions, extracellular matrix (proteins not found in free-floating cells separated by open water channels), that enable bacteria to hold together and onto wet surfaces. Biofilms allow bacteria to exchange nutrients, remove waste products, and communicate elaborately, thus producing high resistance to environmental stressors and antimicrobial agents. Biofilms can have a variety of chemical environments, sometimes even a variety of several symbiotic bacterial species and can produce toxins and other disease-causing substances. b. State the industrial and medical implications of biofilms. Biofilms increase bacterial community resistance and proliferation allowing bacteria to grow in many environments such as on your teeth, on implants, even in production facilities, contaminating products. Due to this medical and industrial implication, industries must be particularly vigilant to prevent biofilm accumulation. Additionally, within the medical field, biofilms contribute to higher antimicrobial agent resistance and thus must be taken into consideration when treating infections and cleaning equipment. 9

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May 30 th , 2023 c. Why are biofilms so resistant to antibiotics? Biofilms are particularly resistant to antibiotics in comparison to free-floating microbes. Antibiotics may fail to penetrate biofilms, especially those containing beta-lactamases. They possess both active and inactive microbes in close proximity thus, even if antibiotics kill a significant number of microbes, those that survive may utilize dead microbes as nutrients. Additionally, if a biofilm contains regions lacking certain nutrients, those microbes may be alive but inactive, surviving some antibiotic intervention. d. Studies have shown that bacteria must turn on certain genes to begin to form a biofilm. How is this achieved? Quorum sensing is the activation of dozens of genes when enough microbes assemble on a surface signaling to each other. This activation of a myriad of genes permits the production of biofilms. e. What strategies could be used to prevent biofilm formation? To prevent biofilms scientists could utilize competitive inhibition via a molecule that binds to the appendages on the pathogenic microbe’s cell surface and deters other microbes from being attracted. Additionally, scientists could interfere with the synthesis of the extracellular matrix or switch off genes the aid in matrix production. Scientists could also target pathogenic microbe communication, stopping biofilm formation and toxin production. 3. Article: Levy, S. B. (1998; updated 2007). The challenge of antibiotic resistance . Scientific American 278 (3), 46-53. a. List three bacterial species that are capable of causing life-threatening infections that have strains resistant to all available antibiotics. Enterococcus faecalis, Mycobacterium tuberculosis and Pseudomonas aeruginosa. b. What are the routes by which bacteria can become antibiotic resistant? Bacteria can become antibiotic resistant through the prevalence of resistance genes and the extent of antibiotic use. With increased use of antibiotics, the microbial community gain resistance to antibiotics as those unsusceptible are selected for and those that die off leave a vacant space for antibiotic resistant 10

May 30 th , 2023 strains. Additionally, through the horizontal gene transfer of plasmids, bacterial virus transfer of genetic material, and with the death of bacteria releasing their genetic information, these microbes are able to pass their resistance on to pathogenic microbes that may infect the host later on. Bacteria may also naturally undergo genetic mutations spontaneously producing antibiotic resistance. c. How does antibiotic use promote the evolution of resistant bacteria? The heavy use of antibiotics promote antibiotic resistant bacteria within communities, such as hospitals, and can spread to broader communities. By attacking cells that are susceptible to antibiotics we eliminate those from environments where resistant strains take over. In other words, with improper use we evolutionarily select for antibiotic resistant bacteria to grow and proliferate. d. How does non-compliance of a patient promote the development of resistance? When patients fail to finish the full course of their treatment, and/or use leftover antibiotics to self prescribe, improper dosing fails to eliminate the infectious agent and supports the growth of more resistant strains. e. How can antibiotic resistance be reduced? To help mitigate the spread of antibiotic resistant bacteria we can first and foremost stop excessively, unnecessarily, and improperly using antibiotics. This includes minimizing use in livestock and produce, seeking other nonantibiotic therapies for minor conditions, and if resources are avaiable, identify causative agents to target specific microbe rather than use of broad-spectrum. Additionally, in hospital settings use proper and effective PPE to minimize cross contamination between patients to reduce spread of resistant stains. 4. Article: Mallin, M. A. (2006). Wading in waste . Scientific American 294 (6), 52 59. a. What has contributed to the pollution of coastal regions? The disruption of natural drainage systems in costal areas, like forests and wetlands, have been destroyed by the replacement of concrete for the construction of home, roads, shopping centers, parking lots, and more. 11

May 30 th , 2023 b. What types of organisms are of concern in these polluted regions? Fecal microbes - bacteria, viruses, and protozoa derived from feces that are disease-causing are of main concern. More specifically, species like Cryptosporidium protozoa, Escherichia coli , and Campylobacter . c. What are the implications of this contamination? Several fecal microbes have polluted drinking water causing severe disease outbreak in U.S. and Canada, leading to numerous illnesses and deaths. The ecosystem has also undergone extreme damage from the contamination effecting many wild species as sediments can physically and chemically bind with pollutants like ammonium, phosphate, metals, and fecal bacteria and viruses plaguing marinas, tidal creeks and shellfish beds. Contamination by fecal bacteria also leads to the closure of one third of U.S.’s monitored beaches. d. Discuss the relationship between impervious surfaces and microbial pollution in a particular area. Impervious surfaces are able to accumulate pollutants during dry periods and disperse high concentrations after rainfall, contaminating downstream lakes, streams, and beaches. For example, in Futch Creek, 7% of the surrounding land is covered in impervious surfaces, the average coliform count was 12 CFU per 100 milliliters. In contrast in Bradley Creek, where impervious surfaces cover 22%, the count was seven times higher. There has been noted a strong correlation between prevalence of E. coli and percentage of impervious surfaces. e. How can coastal communities fight microbial pollution? Coastal communities can help fight microbial pollution by advocating the preservation of green spaces, install filters in storm drains, and prevent the placement of septic tanks in areas with porous soils. 12

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