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Module 3: Global Health •

History of microbiology and the idea of spontaneous generation; Pasteur’s germ theory Historians are unsure who made the first observations of microorganisms, but the microscope was available during the mid-1600s, and an English scientist named Robert Hooke made key observations. He is reputed to have observed strands of fungi among the specimens of cells he viewed. He described the structure of blue moulds. He allowed observations of around 30X magnification. In the 1670s and the decades thereafter, Anton van Leeuwenhoek made careful observations of microscopic organisms such as bacteria and protozoa, which he called animalcules. He revealed the microscopic world to scientists of the day and is regarded as one of the first to provide accurate descriptions of protozoa, fungi, and bacteria. He did not make the connection between these processes and microorganisms but by using a microscope he did establish that there were forms of life that were not visible to the naked eye. In those years, scientists debated the theory of spontaneous generation, which stated that microorganisms arise from lifeless matter such as beef broth. John Needham advanced spontaneous generation, but Lazzaro Spallanzani disputed the theory by showing that boiled broth would not give rise to microscopic forms of life. Pasteur had to disprove spontaneous generation to sustain his theory, and he therefore devised a series of swan-necked flasks filled with broth. He left the flasks of broth open to the air, but the flasks had a curve in the neck so that microorganisms would fall into the neck, not the broth. The flasks did not become contaminated (as he predicted they would not), and Pasteur's experiments put to rest the notion of spontaneous generation. His work also encouraged the belief that microorganisms were in the air and could cause disease. Pasteur postulated the germ theory of disease, which states that microorganisms are the causes of infectious disease. Pasteur's attempts to prove the germ theory were unsuccessful. However, the German scientist Robert Koch provided the proof by cultivating anthrax bacteria apart from any other type of organism. He then injected pure cultures of the bacilli into mice and showed that the bacilli invariably caused anthrax. The procedures used by Koch came to be known as Koch's postulates. They provided a set of principles whereby other microorganisms could be related to other diseases.

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Describe Koch’s postulates They were that if an organism is to be the cause of a disease it is to be found in all cases of the disease examined, isolated from the host with the disease and grown in pure culture, capable of producing the same disease in a healthy, susceptible animal and be re-isolated from the infected animal and identified as the same organism infecting the original host. Used to prove the causative relationship between most bacterial pathogens and their respective diseases.

Module 3: Global Health

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Describe normal flora and ubiquity of microbes Ubiquity of microbes means that microbes are found everywhere. They are very diverse and include bacteria, fungi and protists. The microbes that cause disease are called pathogens. Normal flora is found regularly at any anatomical site of the human body. They are mostly bacteria but also some yeasts and they are found on surface tissues such as skin. They are acquired at birth and from the environment. They can never cause diseases but some may in sick people or wrong site.

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Describe one health as a unifying principle in infectious disease microbiology Ø Around 60% of all human diseases originate in animals Ø Urbanisation, poor water, pollution – encourage disease vectors Ø Diseases in food producing animals cause loss in production; antibiotics used in animal production cause resistance in human pathogens

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Describe transmission in infection; direct and indirect contact and the role of vehicles and vectors Diseases can spread through direct contact such as through sexual contact, contaminated blood and sharing needles are different forms that can spread disease. Person to person transmission can also occur from mother to child during pregnancy Diseases can spread through indirect contact as well such as contaminated objects, food and water. Here non-living agents that transmit disease are disease vehicles. Living agents that transmit disease are disease vectors such as insect vectors where they are spread by blood sucking insects. Diseases can also be spread through animal and environmental reservoirs such as soil and water. Also zoonoses occur when diseases pass from animal to humans.

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Provide examples of infectious diseases causing major problems today § Malaria Causes flu like symptoms such as fatigue, headache, nausea and can cause cerebral malaria which is common in children with around 20% mortality. Although now the deaths caused by malaria are declining due to better mosquito control, use of bed nets, urbanisation of human population, there are still a lot of people dying where mostly are children. §

Tuberculosis (TB) The disease is spread by person to person via infected droplets. It is a latent disease where no major symptoms are evident for many years before becoming active. It is shown that indigenous Australians are more likely to get it than non-indigenous Australians. Recently it has become a more and more drug resistant.

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HIV/AIDS There were rare infections associated with very compromised immune systems and a rare skin cancer. There are two subtypes and HIV-1 causes most AIDS globally and HIV evolves rapidly. It is spread via sexual contact, contaminated blood and from mother to child during pregnancy. HIV infects and kills immune system cells and is treated by ART which is drugs that block different viral stages and reduced the virus load and does not cure infection.

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Appreciate how microbes impact every step of the food production For good: Ø Fungi are mutual with plants to promote growth Ø Microbes recycle debris and promote soil health Ø Good gut microbial promotes health Ø Some microbes like mushrooms and seaweed are important food sources For bad: Ø Pathogens infect animals and plants Ø Irrigation water can be contaminated with pathogens Ø Factories can be contaminated by microbes Ø Poor gut microbiota causes poor health Ø Poor hygiene in restaurants, shops and in home can cause spoilage and food poisoning Soil: Microbes maintain health by: Ø Transforming and releasing important elements such as nitrogen and iron Ø Breaking down soil matter into forms that can be used by plants Ø Suppressing pathogenic microbes that can cause plant and animal disease Ø Breaking down toxic molecules like pollutants Animals: Ø Cellulose – sugars – fermented to acids, esters, CO2 and methane Plants: Ø Have fungi that increase water, mineral and nutrient intake and receive sugars from the plant

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Describe the fermentation process used to create beer and other foods Beer: Fermentation is the process by which yeast(Saccharomyces) converts glucose in the wort(liquid extracted from the mashing process during the brewing of beer) to ethyl alcohol and carbon dioxide gas giving the beer broth its alcohol content and its carbonation. Once in the bottle the yeast continues to make carbon dioxide which makes the beer fizzy. Chocolate: Wild yeasts convert the pulp’s sugar into alcohol and then bacteria takes over converting the alcohol into acid. Acid and heat produced by fermentation kills the bean and release enzymes and the enzymes convert bitter compounds into milder tasting compounds. Soy sauce: Steamed soybeans are fermented with Aspergillus where the spores germinate, grow and produce enzymes. The mouldy beans are converted in brine where it kills the mould, lactic acid bacteria and yeast grows. The sauce is then pasteurised, clarified and bottled.

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Explain how food spoils as a result of microbial action and provide examples of pathogens Crop pathogens cause global food loss. Animal pathogens: foot and mouth disease – does not infect people but causes huge economic losses Bird flu: transmission to people is rare but can be fatal Ø Ø Ø Ø Ø

Refrigeration may be needed to stop microbial growth Irrigation water can be contaminated with pathogens Post harvest contamination can run food crops Factories can be contaminated with microbes Poor hygiene in restaurants, shops and homes can cause food poisoning and spoilage.

Due to growth of microbes and or enzymes that release moulds, yeasts and bacteria. Around 20% of harvested food is lost to spoilage Some food spoilage results in food poisoning from foods such as cheese, milk, fish. E coli. – found in gut, often contaminates water Salmonella – carried by humans and animals Listeria – found in soil, water, cows, chickens, and can contaminate food plants Campyblobacter – found in the gut of chickens, cows and other animals Staphylococcus – found on the skin Food poisoning usually involves diarrhoea/vomiting, cramps and fever but can be life threatening. Some food spoilage can cause mycotoxins. Fungi can grow on drier foods. Mycotoxins are complex molecules a bit like antibiotics. Can be highly toxic and carcinogenic. Can be heat stable and no inactivated by cooking.

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Describe the gut microbiome and the role of microbes in nutrition and obesity Gut microbiome is made up of trillions of microorganisms and their genetic materials. These bacteria live in the digestive system and play a key role in digesting food and help absorbing nutrients. The development of microbiota starts at birth. Importance: Ø Helps the body to digest foods that the stomach and small intestine are not able to

Module 3: Global Health Ø Helps with the production of some vitamins Ø Helps combat aggressions from other microoganisms, maintain the wholeness of the intestinal mucosa Ø Important role in the immune system, performing a barrier effect Depends on what we eat: Children from Africa eating high fibre diet: increase bacteriodetes, decrease firmicutes Children from Europe eating western high protein and high fat diet: decrease bacteriodetes, increase firmicutes

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Describe the role of microbes in the carbon cycle The amount of carbon dioxide in the atmosphere is controlled by the carbon cycle. Processes that remove carbon dioxide from the air: Ø Photosynthesis by plants Ø Dissolving in the oceans Processes that return carbon dioxide from the air: Ø Respiration by plants, animals and microbes Ø Combustion Cellulose: All cells contain carbon, because they all contain proteins, fats and carbohydrates. For example, plant cell walls are made of cellulose, a carbohydrate. Decomposers: Decomposers such as microbes and fungi play an important role in the carbon cycle. They break down the remains of dead plants and animals and in doing so release carbon dioxide through respiration.

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Define autotroph and heterotroph and give examples Autotrophs are organisms that can produce their own food from the substances available in their surrounding using light or chemical energy. They obtain carbon from inorganic sources such as carbon dioxide. They are usually plants. Heterotrophs cannot synthesis their own food and rely on other organisms –both plants and animals for nutrition. They get their reduced carbon from other organisms. They survive by feeding on organic matter produced by or available in other organisms. There are two types: photo heterotroph – use light for energy, chemohetertroph – get energy by oxidation of preformed organic compunds eg: fungi, bacteria

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Explain the impact of microbes on global climate change

Module 3: Global Health

Module 3: Global Health

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Provide examples on the importance of microbes in - Marine ecology (coral algal symbiosis)

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Why microbes are useful for biotechnology

Module 3: Global Health

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Explain what is plasmids and define the roles A genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium. Plasmids are much used in the laboratory manipulation of genes. Key features: Selectable marker – enables us to force cells to take up plasmid Cloning site – add foreign genes here Replication functions – ensures persistence in host Role of plasmid in biotechnology: They acts as delivery vehicles to introduce foreign DNA into bacteria. Ø Scientists can force bacteria to keep them: virtually all plasmids that are used to deliver DNA contain genes for antibiotic resistance. Once bacteria have been treated with plasmid, scientists grow them in the presence of antibiotic. Only that contain plasmid will grow and reproduce. Other will be killed. Ø They are copied independently

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Define the terms DNA cloning, recombinant DNA, GMOs. Explain how they are made and the enzymes DNA cloning: Technique that makes many identical copies of a piece of DNA, such as a gene. It is the process of making multiple, identical copies of a particular piece of DNA. The gene or other DNA fragment of interest is first inserted into a circular piece of DNA called a plasmid. The insertion is done using enzymes that cut and paste DNA and it produces a molecule of recombinant DNA or DNA assembled out of fragments from multiple sources. Tools needed for DNA cloning – enzymes. Copying DNA – thermostable polymerase, cutting DNA – restriction enzyme, joining DNA – ligase

DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid. The insertion is done using enzymes that “cut and paste” DNA, and it produces a molecule of recombinant DNA, or DNA assembled out of fragments from multiple sources. What is the point of making many copies of a DNA sequence in a plasmid? In some cases, we need lots of DNA copies to conduct experiments or build new plasmids. In other cases, the piece of DNA encodes a useful protein, and the bacteria are used as “factories” to make the protein. For instance, the human insulin gene is expressed in E. coli bacteria to make insulin used by diabetics. Process: 1. Cut open the plasmid and "paste" in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA). 2. Transform the plasmid into bacteria. Use antibiotic selection to identify the bacteria that took up the plasmid.

Module 3: Global Health 3. Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the protein. Harvest the protein from the bacteria and purify it.

Recombinant DNA: DNA molecules formed by laboratory method of genetic recombination to bring together genetic material from multiple sources, creating sequences that would be found in genome. Restriction enzymes and DNA ligase: A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target sequence and cuts DNA into two pieces at or near that site. Many restriction enzymes produce cut ends with short, single-stranded overhangs. If two molecules have matching overhangs, they can base-pair and stick together. However, they won't combine to form an unbroken DNA molecule until they are joined by DNA ligase, which seals gaps in the DNA backbone. GMO: A genetically modified organism is an organism whose genetic material has been altered using techniques in genetics generally known as recombinant DNA technology.

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Why vaccines are important Vaccines are a primary defence against infectious disease. They protect against diseases such as smallpox, rabies, etc. Vaccines work by training the immune system to recognise anitgens associated with an invader. They may consist of live attenuated microbes, killed microbes and antigen produced in a GMO host.

1. Isolation of Whole genome of hepatitis-B virus: 2. Cloning of the genome with plasmid and its multiplication 3. Release of sequence coding for HBs antigen. 4. Ligate with yeast expression vector 5. Transform in sachromycis and allow for vaccine formation....