AQA (B) A2 Module 7: Microbes and Disease: Microbes Bacteria. Industrial Biotechnology

Module 7 - Microbes and Disease - page 1
AQA(B) A2 Module 7: Microbes and Disease Contents

Specification Microbes Bacteria Viruses Practical Microbiology Industrial Microbiology Industrial Enzymes Antibiotics Antibiotic Resistance Infectious Disease Examples of Disease Defence Against Infectious Disease Immunisation p1 p3 p7 p10 p18 p22 p25 p31 p36 p42 p45 p54
Disease
Module 7 Specification
Microbes Bacteria. Characteristic features of bacteria, including cell wall, cell membrane, nuclear zone, 70S ribosomes, mesosomes, flagella, plasmids and capsule; and the major functions of these structures. Bacteria may obtain energy from sunlight, inorganic substances or from organic substances. (Details of processes not required.) Binary fission as the main method of reproduction. Viruses. The structural features of a virus, exemplified by the influenza virus and HIV. The process of replication of the influenza virus and of the human immunodeficiency virus (HIV). Practical Microbiology Aseptic techniques and health and safety procedures in handling, culturing and disposing of bacteria. The techniques of haemocytometry, turbidimetry and dilution plating. The distinction between total and viable counts. Bacteria Population Growth Typical growth curve of a bacterial population and reasons for lag phase, exponential phase, stationary phase and death phase. Effect of temperature, pH, nutrient availability and oxygen availability on growth. HGS A-level Biology Analyse and interpret experimental data from microbial growth investigations. Industrial Biotechnology The advantages of using microorganisms for industrial processes. Screening procedures are carried out to identify the most suitable microorganisms to use for a particular process. The relative advantages and disadvantages of continuous and batch culturing techniques. Downstream processing is carried out to extract and purify the end-product of fermentation. Candidates should be able to use information provided to explain the purpose of various parts of industrial fermenters and the stages involved in biotechnological processes. Industrial Enzymes It is more efficient to use isolated enzymes than whole cells because enzyme concentration is higher and there are no unwanted enzymes present. Casein can be used to identify protease production by bacteria. Isolated enzymes can be immobilised so that they do not contaminate the end-products and can be used over and over again. Immobilisation can be achieved by bonding the enzyme with a cross-linking agent, entrapment inside a gel, and by binding the enzyme on to the surface of an absorbing agent. NCM/1/03
Module 7 - Microbes and Disease - page 2 stroying, and preventing the spread of, microorganisms that enter the blood and other tissues. Antigens Antigens, as substances that are foreign to the individual organism exposed to them. They usually take the form of a protein, polysaccharide or glycoprotein structure on the cell surface of a microorganism on the surface of a virus on the cell surface of a tissue or organ transplant as a free molecule, such as a toxin. Cell-mediated Immunity Production of T lymphocytes in bone marrow and activation by the thymus. Recognition of antigens by specific, membrane-bound receptor molecules on T lymphocytes. Cloning of T lymphocytes, and destruction of bacteria and cells with antigens. Antibody-mediated Immunity Production of B lymphocytes, and activation by antigens attached to macrophage membrane. Cloning of B lymphocytes, and production of specific antibodies. Antibody structure, and formation of antibody-antigen complex. The role of antibodies in: agglutination of antigens stimulation of phagocytosis precipitation of soluble toxins preventing pathogenic bacteria attaching to cell membranes. Immunological memory The production of memory T cells and B cells. The primary and secondary responses. The role of immunological memory in producing a quicker and stronger immune response to a subsequent infection by the same antigen. The effects of antigenic variability in some pathogens, such as influenza virus, on immunity. Active Immunisation The principle that vaccines contain antigens derived from pathogens, which can protect against infection by that organism. The principles involved in the production of, and the method of administering, the following types of vaccine: live non-virulent strains, exemplified by rubella killed virulent organisms, exemplified by whooping cough isolated antigens from a pathogen, exemplified by influenza genetically engineered antigens, exemplified by hepatitis B. modified toxins, exemplified by diphtheria Passive Immunisation The use of antibodies to counteract possible infection, and the reason for its temporary effectiveness. Natural passive immunity in young babies.
Antibiotics The mode of action of antibiotics in the treatment of diseases caused by bacteria and fungi. Effects of antibiotics on cell walls and membranes, and on nucleic acid and protein synthesis. Fungi can be screened for antibiotic production. The microorganisms used and the main stages in the production of penicillin. Use of a bacterial lawn as a bioassay technique to determine the effectiveness of disinfectants and antibiotics on inhibition of bacterial growth. The principle of selection of particular antibiotics for treatment of specific diseases, and the roles of broad spectrum and narrow spectrum antibiotics. Antibiotic resistance Mechanisms of resistance, such as the production of penicillinase. The process by which an antibiotic resistant strain of bacterium may develop. Transfer of resistance genes between bacteria. The impact of resistant strains on the treatment of disease and the use of antibiotics. Disease Bacterial Disease. The factors affecting pathogenicity of bacteria, to include: Transmission of food-borne and water-borne infection, exemplified by Salmonella food poisoning and E.coli. Precautions to avoid contamination of food and water. Human carriers. features of cell wall and capsule that affect attachment and entry to host cells infectivity, exemplified by numbers required to cause Salmonella food poisoning and typhoid fever invasiveness, the ability of bacteria to spread within the host. exotoxins and endotoxins produced by bacteria Treatment of diarrhoea. Viral Disease. Damage to host cells due to viral replication and the production of toxins. Reasons for the difficulty in treating viral infections. The course of infection, signs, symptoms and transmission of influenza. Candidates should be able to suggest strategies for reducing the incidence of influenza. How HIV causes AIDS. The course of infection, signs, symptoms and transmission of acquired immune deficiency syndrome (AIDS). Candidates should be able to suggest strategies for preventing the spread of HIV. Defence against Disease The roles of the skin, tears, mucus, saliva and cilia in preventing microorganisms gaining access to living cells. Phagocytosis The process of phagocytosis and the roles of phagocytic white blood cells (neutrophils and macrophages) in deHGS A-level Biology
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Bacteria
Bacteria are all prokaryotes. The main structures of a bacterial cell are: Cytoplasm. Contains all the enzymes needed for all metabolic reactions, since there are no organelles Ribosomes. The smaller (70 S) type, all free in the cytoplasm, not attached to membranes (like RER). For protein synthesis. Nuclear Zone (or Nucleoid). The region of the cytoplasm that contains DNA. It is not surrounded by a nuclear membrane. DNA. Always circular (i.e. a closed loop), and not associated with any proteins to form chromatin. Sometimes confusingly referred to as the bacterial chromosome. Plasmid. Small circles of DNA, used to exchange DNA between bacterial cells, and very useful for genetic engineering. Flagellum. A rigid rotating helical-shaped tail used for propulsion. The motor is embedded in the cell membrane and is driven by a H+ gradient across the membrane. Clockwise rotation drives the cell forwards, while anticlockwise rotation causes a chaotic spin. This is the only known example of a rotating motor in nature. Cell Membrane. made of phospholipids and proteins, like eukaryotic membranes. Mesosome. This is now thought to be an artefact of the electron microscope and not a real structure. A tightly-folded region of the cell membrane containing all the membrane-bound proteins required for respiration and photosynthesis. Can also be associated with the nucleoid. Capsule (or Slime Layer). A thick polysaccharide layer outside of the cell wall, like the glycocalyx of eukaryotes. Used for sticking cells together, as a food reserve, as protection against desiccation and chemicals, and as protection against phagocytosis. In some species the capsules of many cells in a colony fuse together forming a mass of sticky cells called a biofilm. Dental plaque is an example of a biofilm. Cell Wall. Made of murein (not cellulose), which is a glycoprotein or peptidoglycan (i.e. a protein/carbohydrate complex). There are two kinds of bacterial cell wall, which are identified by the Gram Stain technique when observed under the microscope. Gram positive bacteria stain
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capsule (slime layer) 70S ribosomes cell wall cell membrane mesosome nucleoid DNA
plasmid
flagellum
Not all prokaryotic cells have all the parts shown here
1 m
purple, while Gram negative bacteria stain pink. The technique was discovered by Christian Gram in 1884 and is still used today to identify and classify bacteria. We now know that the different staining is due to two types of cell wall: Gram positive cell wall Gram negative cell wall (stains purple) (stains pink)
cell membrane cell membrane periplasm thin cell wall outer membrane lipopolysaccharides capsule
Gram positive bacteria have a thick murein cell wall outside their cell membrane. The cell wall is very strong and allows these bacteria to withstand severe physical conditions. There may be a capsule outside the cell wall. Gram negative bacteria have a more complex structure, with a thin layer of periplasm (like cytoplasm but outside the cell), a thin murein cell wall, and then a second, outer membrane, which contains lipopolysaccharides instead of phospholipids in its outer layer. This layer resists antibiotics and lysozyme enzymes, so gram negative bacteria are more difficult to kill.
thick cell wall
Bacteria have a variety of distinctive shapes when seen under a microscope:

Coccus (spheres)
staphylococi e.g. staphylococcus aureus causes food poisoning.
diplococci e.g.diplococcus pneumoniae casues pneumonia
streptococci e.g. streptococcus pyogenes causes sore throats
Bacillus (rods)
e.g. Salmonella typhi causes typhoid fever
e.g. Azobacter is a nitrogen-fixing e.g. Treponema pallidum causes syphilis e.g. Vibrio cholerae causes cholera
Spirillum (helical)
Vibrio (crescent)
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Bacterial Nutrition
Bacteria have a large range of different metabolic reactions at their disposal, far more than in the eukaryotes, who are confined to just respiration or photosynthesis. Bacteria can be classified according to their source of energy: Phototrophs obtain their energy from sunlight. Many bacteria are photosynthetic and use the same process of photosynthesis as plants. These phototrophic bacteria were some of the earliest forms of life on the planet, and their metabolic reactions increased the oxygen content of the atmosphere from 1% to 20%. Organotrophs obtain their energy by oxidising organic compounds like glucose. Most bacteria are organotrophs, using the same respiration reactions as animals and fungi. Lithotrophs obtain their energy by oxidising inorganic compounds like ammonia, nitrite, methane or hydrogen sulphide. These bacteria use a variety of unusual metabolic reactions and many are able to synthesise carbohydrates from carbon dioxide the chemosynthetic bacteria. There are whole ecosystems on the deep ocean floor with no light, based on lithotrophic bacteria as producers. Although rare, lithotrophic species are enormously important in ecology, as they are responsible for much of the cycling of matter (e.g. the nitrifying bacteria). They could also be useful in biotechnology as they can synthesise useful organic compounds from waste inorganic ones (e.g. methylomonas can make carbohydrates from methane).
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Bacterial Reproduction
Bacteria always reproduce asexually, using binary fission ("splitting in two").
The DNA in a bacterium is always circular (or rather a closed loop), and is often attached to an invagination of the cell membrane.
Before cell division the DNA is replicated. Unlike eukaryotic cells, there are no histone proteins, and the DNA is not coiled into chromosomes.
The cell elongates from the middle, separating the two DNA molecules, which are attached to different parts of the membrane.
A new cell wall, or septum, is formed down the middle of the elongated cell.
Eventually the septum meets, dividing the cell in two.
Although this looks a bit like mitosis in eukaryotic cells, it isn't. There is no nucleus, no chromatids, no centrioles and no spindle. Binary fission is very fast and bacteria can double every 10 min under optimum conditions, though the doubling time is usually slower.
Bacterial Spores
Many bacteria can produce spores. These are specialised dormant cells formed in response to adverse conditions, and they can survive long periods of high temperatures (even boiling), desiccation, low nutrients, radiation and chemicals. When conditions return to normal the spores "germinate" and develop into normal cells. Although spores are a little like plant seed they are not used for reproduction. Because they are so difficult to kill, spores can cause problems for sterilisation.
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Viruses
Viruses are the smallest and simplest of the microbes. They are acellular (not made of cells) and, since they cannot reproduce (or do any of the seven characteristics of life) on their own, they are considered non-living. They are in fact more like complex chemicals than simple living organisms. Viruses are obligate parasites who can only reproduce inside host cells which get damaged in the process, leading to disease. A virus has been famously described as "a piece of bad news wrapped up in protein". Viruses are thought to have arisen from lengths of DNA that became separated from their cells.
Virus Structure
A complete virus particle is called a virion. A virion is the dormant form of a virus that is transmitted between cells. Virions are too small to see with a light microscope and were first seen in the 1930s using the electron microscope. Once inside a host cell virions dismantle into their separate parts, and the virus can be reproduced. All virions contain: Nucleic acid, which can be DNA or RNA, and single or double-stranded. Viruses are classified according to the type of nucleic acid they contain. The nucleic acid typically codes for 5-100 proteins (by comparison, the bacterium E. coli has about 4000 genes). A protein coat called a capsid, made of subunits called capsomeres. If the capsid proteins are closely bound to the nucleic acid, then the combination is called a nucleocapsid. Because capsids are composed of many repeating subunits, they tend to have simple geometrical shapes, such as helix or icosahedron (20 triangular faces). Some virions have very simple structures containing nothing else, but many virions have more complex structures, including: Enzymes, required to replicate the viral nucleic acid or incorporate it into a host. A lipid envelope, not made by the virus itself, but derived from a host cell membrane. Matrix proteins to attach the capsid to the envelope. Glycoproteins to allow the virus to attach to host cells. Some examples of virion structures are shown on the next page.
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Tobacco mosaic virus (TMV) has a very simple structure: just a coil of RNA surrounded by a helical capsid.
capsomeres single-stranded RNA helical capsid
icosahedral capsid Adenovirus contains singlestranded DNA surrounded by an icosahedral capsid. single-stranded DNA protein fibres icosahedral head helical sheath baseplate tails The human influenza A virus is enveloped virus. It contains 8 single-stranded RNA segments combined with capsomeres to form helical nucleocapsids surrounded by a sphere of matrix proteins attached to a lipid envelope. 8 nucleocapsids (single-stranded RNA + helical capsid) RNA polymerase enzymes matrix proteins lipid envelope membrane glycoproteins
Bacterial viruses (or bacteriophages), like this T2 virus have complex structures that combine icosahedral and helical capsids.
The human immunodeficiency virus (HIV) is an enveloped retrovirus. It comprises 2 copies of single-stranded RNA together with some enzymes, surrounded by an icosahedral capsid, which is in turn surrounded by a sphere of matrix proteins attached to a lipid envelope.
single-stranded RNA (2 copies) reverse transcriptase enzymes icosahedral capsid matrix proteins lipid envelope membrane glycoproteins
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Viral Reproduction
Viruses can only reproduce inside host cells. The general strategy is to use the host cell enzymes to replicate and translate viral DNA, making more virus particles, which then burst out of the cell. Two different mechanisms are shown here.
Influenza Virus
1
The virus attaches to the host cell membrane, which stimulates endocytosis.
nucleus
HIV
1
The virus attaches to the host cell membrane, and the viral and cell membranes fuse. This releases the nucleocapsid into the cytoplasm.
nucleus
2
nucleocapsids
The viral envelope fuses with the vesicle membrane, releasing the nucleocapsid into the cytoplasm. The RNA enters the nucleus, where it is replicated to form mRNA, using the viral RNA polymerase enzyme. The mRNA is now used to synthesise more viral RNA and capsid proteins. The RNA and proteins are assembled into new virus particles (without envelopes). The glycoproteins migrate to the cell membrane. The virus particles are released by exocytosis, collecting the lipid envelope from the cell membrane. This kills the host cell.
2
RNA DNA
The viral enzyme reverse transcriptase is used to synthesise double-stranded DNA from the singlestranded viral RNA. The DNA enters the nucleus and is incorporated into the host's genome, where it is called a provirus. The provirus remains dormant for years. It is replicated every time the host cell divides.
mRNA
DNA proteins
proteins
At some trigger signal the provirus DNA is transcribed to RNA and viral proteins are synthesised. The RNA and proteins are assembled into new virus particles (without lipid envelopes). The glycoproteins migrate to the cell membrane. The virus particles are released by exocytosis, collecting the lipid envelope from the cell membrane. This kills the host cell.
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Practical Microbiology
Microbiology experiments require special techniques and precautions, for two good reasons: Health and Safety to prevent the escape of any experimental microbes to the surrounding environment, where they may cause disease. Validity to prevent contamination of experiments by microbes from the environment. These other microbes could out-compete the experimental microbes or otherwise invalidate the results. Before and after a practical session, all equipment (glassware, cultures, surfaces, etc.) must be sterilised: Equipment and cultures are best sterilised by using an autoclave. This is a large pressure cooker, which heats up steam under pressure to 121C for at least 15 min. This kills all microbes and bacterial spores. All cultures are sealed and autoclaved after an experiment before being discarded with normal waste. Surfaces, spillages and discard pots (e.g. for slides and syringes) are best sterilised using a disinfectant such as Virkon, hypochlorite (bleach) or ethanol. This isn't as thorough as sterilisation, but it kills likely pathogens and the inhibits the growth of most microbes. Hands should be washed with a disinfectant soap at the end of a practical session. Any cuts must be protected with a plaster or disposable gloves. The area where the microbiology work is to be done should be tidy and should have a lit Bunsen burner. This is used to sterilise equipment, and also to provide a convection current, which draws air and spores up and away from the experimenter. Read and obey the safety instructions given. You are responsible for your own safety and the safety of everyone else in the lab.
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Growing Microbes
Microbes for experiments can be obtained from natural sources (e.g. soil, food, skin, water, air), or bought from suppliers in agar slopes. You don't need much, since a dot may contain millions of viable cells, each of which could grow into a whole colony. Medium The mixture that the microbes are grown (cultured) on. The medium must contain all the nutrients (sugars, minerals, proteins) needed for the microbes to grow, and by adjusting these nutrients, the medium can be made selective for one type of microbe. Culture media can be made up by mixing together known amounts of specific chemicals (a defined or synthetic medium), or they can be made from a natural source such as boiled meat or yeast extract, which generally contains the nutrients required by most microbes (an undefined or complex medium). Nutrient Medium Broth Agar A cheap general-purpose complex medium used for most school experiments. A liquid medium (i.e. without agar). Agar is mixed with a liquid medium to make a solid medium, which is very useful to observe, separate and store bacteria cultures. A solid medium in a petri dish is known as an agar plate, while a solid medium in a Universal bottle is called an agar slope. Agar is actually a polysaccharide extracted from seaweed. It melts at 41C (so can be incubated at 37C without melting), is reasonably transparent, and is not broken down by microbes, so it remains solid. Aseptic Transfer Also called aseptic technique. The transfer of a sample of bacteria from one vessel to another. This is the most common and basic technique and is used in almost all microbiological experiments. The bacteria are usually transferred using a wire or glass inoculating loop, which can carry a tiny volume of culture (10 l) or a scraping of cells from an agar plate. Larger volumes are transferred using a sterile syringe or pipette. Inoculate Incubate To add few cells to a medium, so that they may grow. To leave a culture to grow under defined conditions. Cultures are usually incubated in an incubator, which is basically an oven with a very good thermostat. Broth cultures may be incubated in a water bath, preferably with constant stirring. Culture A growth of microbes in a medium. The culture can be pure (one species of microbe) or mixed (many species). Colony A visible growth of bacteria on an agar plate containing many millions of cells. The key point is that each colony grows from a single original cell, so is a pure sample.
Streak Plate
A method of inoculating an agar plate with bacteria so that the bacteria are gradually diluted. The aim is to separate individual cells, which can grow into visible colonies. This is used to separate pure cultures of bacteria from a mixed starting culture, such as soil, food or bodily fluids.
Lawn
A layer of bacteria growing on the surface of an agar plate. This is useful to test the effectiveness of antimicrobial substances such as antiseptics or antibiotics, using a disk diffusion test (p31). There are two ways of making a bacterial lawn. In the pour plate method a universal bottle of warm, sterile, molten agar is inoculated with bacteria, stirred, and then the agar is poured into a plate, where it sets. The bacteria will grow uniformly on the surface. In the spread plate method an agar plate is inoculated with a small volume of broth culture, which is then spread even over the surface using a glass spreader or a cotton bud. The pour plate method gives a more even lawn, but requires universal bottles of agar at just the right temperature, while the spread plate is easier to do but may give an uneven lawn.
Growing Viruses
Because viruses are parasites, they cannot be cultured in the lab like other microbes. Instead they must be grown inside their specific living host cells. In the past this meant infecting whole animals or plants, but with improvements in tissue culture techniques almost any host cell can now be grown in the lab and infected with a virus. Bacteriophages are perhaps the most-studied of all viruses, simply because their hosts, bacteria, are so easy to grow in the lab.
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Measuring the Growth of Microbes

Growth of cells in a liquid culture is generally measured by simply counting the number of cells. There are various techniques for doing this. Some give total cell counts, which include both living and dead cells, while others give viable cell counts, which only include living cells. 1. Cell Counter (Haemocytometer) This counts the total cells by observing the individual cells under the microscope. This is reasonably easy for large cells like yeast, but is more difficult for bacterial cells, since they are so small. The cell counter (or haemocytometer) is a large microscope slide with a very accurate grid drawn in the centre. The grid marks out squares with 1 mm, 0.2 mm and 0.05 mm sides. There is an accurate gap of 0.1 mm between the grid and the thick coverslip, so the volume of liquid above the grid is known. The number of cells in a known small volume can thus be counted, and so scaled up. The units are cells per cm3. For example:
A 0.2 mm square has an average of 80 cells in a 1000x dilution Volume above square = 0.2 x 0.2 x 0.1 = 0.004 mm 80 cells in 0.004 mm = 20 000 cells per mm in the diluted suspension which is 20 000 x 1000 = 2 x 107 cells per mm of undiluted suspension or 2 x 1010 cells per cm
2. Turbidometry This technique also counts the total cells. It is quicker than using a haemocytometer, but less accurate. A sample of the liquid culture is placed in a cuvette in a colorimeter, and the absorbance of light is measured. The greater the concentration of the cells, the more cloudy or turbid the liquid is, so the more light it scatters, so the less light is transmitted to the detector. A wavelength of 600nm is normally used. Although the absorbance scale of the colorimeter is used, light is not actually absorbed by the cells (as it is by pigment molecules), but scattered. If the same sample is counted in a haemocytometer and its absorbance measured, than a calibration curve can be plotted. From this calibration curve the concentration of cells can be read off for any absorbance. calibration curve
absorbance concentration of cells
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0.82
lamp cuvette filter detector absorbance
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3. Dilution Plating This technique counts viable cells. A sequence of ten-fold dilutions is taken from the original culture flask, using sterile medium. This is called a serial dilution, and allows large dilutions to be made using small volumes. From each dilution a 1 cm sample is taken and spread evenly onto an agar plate. Each viable cell in the sample will multiply and grow into a colony. In most of the samples there will be too many colonies to count, but in one of the dilutions there will be a good number (20-200) of individual colonies. From this we can calculate the concentration of viable cells in the original culture.
X1 dilution X10 dilution X100 dilution X1 000 dilution X10 000 dilution X100 000 dilution
1 cm
1 cm
1 cm
1 cm
1 cm
culture flask spread 1cm on to agar plate
+ 9 cm sterile medium spread 1cm on to agar plate
far too many colonies

For example:
too many colonies
just right
too few colonies
suppose there were 83 colonies in the x10 000 dilution agar plate. This means there were 83 viable cells in the 1 cm sample of the x10 000 dilution So there were 83 x 10 000 = 8.3 x 105 cells per cm in the original culture
This method is very accurate, but tedious. It is the only way to count viable cells, because only those cells that grow into living colonies are counted.
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Bacterial Growth Curves

By starting a new culture with a small number of cells, and using one of the counting methods to measure the number of cells over a period if time, a growth curve can be obtained.
total cells no. of cells
Log (no. of cells)
4
living cells
3 2 1
2 1
time
time
This growth curve is typical of unicellular microbes such as bacteria and yeast that increase their numbers by binary fision. The curve is called a sigmoid growth curve, and can be plotted on a linear or logarithmic scale. It has four phases: 1. Lag phase, while the microbes adjust to the new conditions (e.g. develop from spores, synthesise new RNA and enzymes). Cell division and cell death rates are both low, so the population does not change much. 2. Exponential phase, while the cells divide without any limiting factors (i.e. there is plenty of space and food). If the division rate is constant then the population increase is proportional to the population, and the population has a constant doubling time (or generation time), e.g. if it takes one hour for the population to double from 100 to 200 cells, then it will also take one hour to double from 1000 to 2000 cells. One of the fastest growth rates recorded is the thermophilic bacterium Bacillus stearothermophilus, which grows at 60C with a doubling time of 8 minutes. Most bacteria have a doubling time of less than an hour under optimal conditions. The population after n generations = 2n. In practice exponential growth only lasts a short time until some factor limits growth rate. If the growth curve is plotted on a log scale the exponential phase is linear, so it is sometimes called a log phase. 3. Stationary phase, when cell division slows down and cell death increases until the two rates are equal, so the population of viable cells is constant. The growth rate may be limited by lack of nutrients, lack of oxygen, lack of space, or accumulation of toxic metabolic waste products. 4. Death phase, when cell death rate is greater than cell division rate, so number of viable cells decreases. Note that the total number of living and dead cells may increase during the stationary and death phases.
Factors Affecting Cell Growth Rate

Temperature In general growth rate increases with temperature until the microbes' enzymes denature. Bacteria are found in an astonishing variety of habitats, sometimes with extreme temperatures, where no other organisms can survive. Different bacteria can grow in the range -7C to 118C. These cells are adapted so that their enzymes have unusual optimum temperatures. Bacteria can be classified according to their optimum temperature: optimum temperature 20-45C >45C name of group Mesophiles Thermophiles examples Mammalian symbiotic bacteria. These are the commonest bacteria. Rotting vegetation, hot springs, volcanic vents. These are useful in biotechnology, since there enzymes do not denature easily (e.g. in PCR or biological detergents). <20C Psychrophiles Soil bacteria or polar ocean dwellers. These bacteria can cause food spoilage in refrigerators. In school labs bacteria must not be grown above 30C by law. This prevents the growth of human pathogenic bacteria (which are mostly mesophiles). pH Most microbes grow best in neutral pH and die in extreme acid or alkali conditions as their enzymes slow down. The optimum pH for different microbes varies from 5 to 7.5. A few acid-tolerant species can grow at pH 2, but almost all microbes are killed below pH 4, which is why human stomach acid is so effective. The pH of a growth medium can change over time due to production of waste acids or alkalis, so it is important to use a pH buffer when growing microbes. Nutrients All microbes need to be provided with the major elements CHONSP in an appropriate form. Organotrophs require simple carbohydrates such as glucose or sucrose for respiration (and some may be able to digest starch), together with minerals, especially a source of nitrogen such as ammonia or amino acids to make proteins. Lithotrophs require mush simpler inorganic nutrients, such as methane, carbon dioxide, water and minerals.
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Oxygen
Many microbes need molecular oxygen for respiration, just as animals do, and are termed obligate aerobes. In a solid agar medium these microbes will only grow on the surface, and a liquid medium must be well aerated. Some microbes are facultative anaerobes, which means they will use aerobic respiration if oxygen is available, but can switch to anaerobic respiration if oxygen is absent. These include yeasts and lactic acid bacteria, and their metabolic products can be controlled by varying the amount of oxygen. For example in brewing, the vessel is left open for the first day to allow aerobic respiration and rapid growth of yeast cells (but no alcohol is produced), but then closed for the next 5 days to encourage anaerobic respiration and the production of alcohol. A few microbes are obligate anaerobes, which means they die in the presence of oxygen. Anaerobes are useful industrially as they will grow throughout a medium, whereas aerobes will only grow on surfaces, or in well-stirred liquid media.
Any of these factors can be controlled to select particular microbes, or particular metabolic products, or to control the growth rate. You don't always want the fastest growth of cells, but the fastest production of a metabolite, which may occur under different conditions.
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Industrial Microbiology
Microbes produce many useful products, and humans have made use of this for thousands of years. Today there is a wide range of products made by microbial biotechnology, most of which are too complex to be synthesised by purely chemical techniques. These include: food (bread, cheese, yoghurt, single cell protein (SCP)); drink (beer, wine, vinegar); fuels (ethanol, methane); enzymes; hormones; antibiotics; chemicals (citric acid, amino acids, steroids); plastics; etc. Microbes are particularly useful for industrial purposes because: They have fast growth rates. They have simple nutritional requirements and can often be fed on cheap or even waste substrates such as molasses, whey, wood pulp, etc. They can be grown indoors and their growth does not depend on seasons, climate, latitude, etc. They are often tolerant of a wide range of temperatures and pH. There are fewer ethical problems, when compared to animals. Prokaryotes can be more easily genetically modified than eukaryotic cells, since they don't have a nucleus. Genes from other species can easily be inserted into the bacterial DNA to produce a range of gene products by fermentation, or the microbe can be altered to produce far more of the product than normal.
Screening
Screening means selecting a suitable species and strain of bacteria for a particular job. Basically it means testing every bacterium you can find to see if it makes the product you want in the right quantities and at the right speed. This can take years and can account for most of the cost of development.
Primary and Secondary Metabolites

Most of the useful products produced by microbes are produced as part of their normal growth. These subsugars, amino acids, ethanol, citric acid, and protein. A few of the useful products are only produced by microbes under unusual or extreme conditions, such as lack of food. These substances are not required for normal growth and are called secondary metabolites.
time food no. of cells 1 metabolite production 2 metabolite production cells
stances are called primary metabolites, and include
The most important of these are certain enzymes and antibiotics, which some microbes produce in
overcrowded conditions to kill competing microbes. The distinction between primary and secondary metabolites is important because they must be harvested at different stages of cell growth.
To maximise primary metabolite production: To maximise secondary metabolite production: keep in exponential phase keep in stationary phase use contimuous fermenter use batch fermenter
Industrial Fermenters
Traditionally, fermentation meant anaerobic respiration in microbes, such as that used for centuries to make wine and beer. Since modern biotechnology is ultimately based on these traditional techniques, the term fermentation now refers to any process where microbes are grown on a large scale, whether it is anaerobic or aerobic. There are two kinds of industrial fermenter: batch and continuous fermenters.
Batch Fermenters
A batch fermenter is a closed tank. Industrial batch fermenters are large tanks holding up to 105 dm (100 m). It is made of stainless steel alloy to resist the corrosive effect of acidic fermentation products and to withstand pressure. A typical batch fermentation process is like this: 1. The fermenter is sterilised using high pressure steam to kill any other microbes that may infect the batch. Mistakes are very expensive. All materials added to the fermenter must also be sterile, so additives are heated and then cooled before being fed in. 2. The culture medium is added and sterilised by heating in the fermenter. 3. For aerobic fermentation the medium is aerated with sterile air bubbling up from the bottom (sparging). Air is sterilised by filtration, radiation or steam. An antifoaming agent is often added to prevent frothing. If anaerobic conditions are required this step is missed out. 4. The medium is warmed to the correct initial temperature for maximum growth by an electrical heater or using hot air, prior to inoculation with microbes. 5. The microbe culture (inoculum) is added. 6. The medium is stirred using impellers driven by a powerful motor to prevent clumping of cells, especially with thick fungal cultures. Alternatively, media can be stirred by high-pressure air. 7. Once growth is underway, the fermenter needs to be cooled using a flow-through water jacket. Microbial respiration can easily raise the temperature by a few C per hour, and without cooling the enzymes would soon denature. 8. Gases are produced by microbial metabolism, and these must be vented to prevent pressure building up. These vented gasses must pass through a fermentation lock to prevent non-sterile air from entering, and are often sterilised for safety.
gas vent Stirring Motor nutrients/acid/alkali steam for sterilising sterile air computer control system
fermentation lock
cooling water jacket
environmental probes (eg pH, O2, CO2, temperature, pressure, etc) Heater
fermentation mixture
impeller (for stirring) Sparging ring (for aeration)
drain / sample port
9. The environmental conditions (e.g. temperature, pH, O2) are constantly monitored and adjusted using automatic computer control. Many metabolic processes produce acids such as lactic acid, citric acid, and acetic acid (indeed these are often the desired products), while deamination of amino acids can produce alkaline ammonia. The medium should be buffered, but acid or alkali can also be added if required. 10. Samples of the culture may also be taken for lab testing of product concentration, cell count, etc. 11. Once the product has reached its maximum concentration, the fermentation is stopped and the medium is run off. 12. The fermenter is sterilised and a new batch is started. In a batch fermentation process the cells show the sigmoid growth curve discussed earlier. If left long enough the nutrients will be used up and the cells will die. A variation is the fed-batch process, where nutrients are added at intervals to prolong the stationary phase.
Continuous Fermenters.
This takes place in an open or flow-through fermenter. The culture is continuously run off to make product, while nutrients are continuously added at the same rate. The rate is set to match the growth rate so the number of cells in the fermenter stays constant. This is very efficient, since the microbes are kept at their exponential growth phase.
start flow no. of cells flow too slow
flow just right flow too fast stop flow time
Batch Fermenters Simple to set up Versatile - fermenter suitable for many purposes Slow production labour intensive due to stop-start cycle Large vessels needed to be cost-effective
Continuous Fermenters Difficult to get right Fermenter only suitable for one process Faster production Automated, so cheaper to run Small vessels give high productivity
Downstream Processing
Products in a fermenter are impure and dilute, so need to be purified by downstream processing. This usually involves filtration to separate the microbial cells from the liquid medium, followed by chemical purification and concentration of the product (detailed examples later). Downstream processing can account for 50% of the cost of a process. We'll look at all these aspects of two particular products: Enzymes and Antibiotics.
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Industrial Enzymes
Some of the most important of microbial products are enzymes, which have numerous uses: commercial uses- e.g. biological washing powders medical uses- e.g. biosensors, therapeutic enzymes, drugs industrial uses - e.g. bioconversion, fruit juice extraction, sweeteners Enzymes are being used more and more for industrial bioconversion i.e. making a chemical product using purified enzymes rather by pure chemical methods (e.g. citric acid production) or using whole cells (e.g. yeast in brewing). Advantages of using isolated enzymes for industrial bioconversion only one reaction, no by-products products easier to purify since no cells higher concentration of enzyme possible no substrate wasted to make microbial biomass Dont confuse the fermentation process that produces the enzyme with the bioconversion process that produces the industrial product. Both take place in vats and can use batch or continuous processes. Fermentation: microbes
enzyme
Disadvantages of using isolated enzymes for industrial bioconversion more expensive to purify enzyme can only do 1 (or a few) reaction steps
enzyme
Bioconversion:
substrate
product
Selecting and Screening Microbes for Enzyme Production

There are two considerations to take into account when selecting a strain of bacteria for the production of an enzyme: does the bacterium grow under the right conditions? does the bacterium produce the correct enzyme? So firstly, bacteria are selected for their ability to grow at high temperatures, alkali pH, high salt, etc. These will grow quickly in a fermenter, and their enzymes will function under industrial conditions. Secondly the selected bacteria are screened for the production of extracellular enzymes using an agar plate technique we used in module 1. For example to test for protease production a milk agar plate is used. The milk protein casein makes the plates cloudy , and if bacteria growing on the agar
plates secrete a protease enzyme they will digest the casein and turn the agar clear. The larger the clear zone the better the protease production.
milk agar plate innoculate with bacteria to be screened incubate at 40C for 24 hours
clear zones indicate protease production
Enzyme Production Methods

The maximum enzyme production is usually in stationary phase of microbe growth, so a batch or fed-batch process are usually used. The medium must be chosen to stimulate the microbe into synthesising the correct enzyme. For example to stimulate a microbe to synthesise amylase enzymes, a medium with starch but no sugars is used. Similarly, to stimulate a microbe to synthesise protease enzymes, a medium with proteins but no amino acids is used. In all cases, the concentration of the substrate should be kept low, so that the microbe synthesises a lot of enzyme to try to digest the substrate quickly. Production of purified enzyme requires a great deal of downstream processing. The nature of the downstream processing depends on two considerations: Whether enzyme is intracellular (stays in cell), or extracellular (secreted from cell). Most industrial enzymes are extracellular because they are easier to purify, they tend to be more robust and they are produced in concentrate by evaporation at low How pure the final product needs to be. In- temperature and pressure dustrial enzymes can be quite crude, but or by osmosis/dialysis larger quantities. medicinal enzymes must be extremely pure. The purer the enzyme, the more complex the downstream processing, and the more expensive it is. A typical downstream processing sequence is shown on the right.
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extracellular enzyme
intracellular enzyme break open cells by grinding or by ultra-sonics
filter
cell biomass (useful wasteproduct)
enzyme in solution
crude enzyme solution e.g. protease in chemical industry powdered crude enzyme e.g. pectinase pure enzyme for medicine e.g. glucose oxidase
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precipitate
chromatography
Immobilised Enzymes
In normal enzyme-catalysed industrial bioconversion reactions the enzyme is in solution, mixed in with the substrate and product, and so the product needs to be separated from the enzyme by downstream processing. This can be difficult and expensive. In recent years industrial technology has turned towards using immobilised enzymes, i.e. enzyme molecules that are attached to a support matrix rather than free in solution. They still function properly but can be kept separate from the reactants and the products. Immobilised enzymes are usually used in continuous flow-through reactors, which have a low volume. E E E E E E E E E E E E E E E E E E
substrate in
product out
The advantages of immobilised enzymes are that the purification of product is easier; the enzyme can be re-used without costly re-purification; smaller amounts of enzyme are needed; and the immobilised enzyme may be more stable and resistant to extremes to pH and temperature. The disadvantages of immobilised enzymes are that the immobilisation process can cause the enzyme to loose activity, and development of successful immobilisation methods is expensive. Methods of Immobilisation Entrapment The enzyme is entrapped within an inert matrix, such as alginate, silica or collagen, and cannot be washed out. The substrate and product molecules can diffuse in and out of the matrix, but this diffusion may limit the rate of the reaction. This is the most gentle method of entrapment, and does little damage to the enzymes. Adsorption The enzyme molecules are attached by weak physiparticles. This does not chemically modify the enzyme molecules, but the adsorption process may cause the enzymes to loose their shape and therefore their activity. The molecules may also become detached during the bioconversion reaction. Cross-linking Enzyme molecules are chemically cross-linked by covalent bonds using glutaraldehyde: E=CHCH2CH2CH2 CH=E. This only works for some enzymes, but when it does work, it works well.
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E E E EE E EE E EE
alginate beads
lipid vesicles
E E E E E E
E E E
E E
E E E
E E E
silica gel
collagen fibres
E E
cal forces to a support matrix, such as glass beads or carbon
adsorbing matrix
E E
E E E
E E E E E
E
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Antibiotics
Antibiotics are antimicrobial agents produced naturally by other microbes (usually fungi or bacteria). The first antibiotic was discovered in 1896 by Ernest Duchesne and "rediscovered" by Alexander Flemming in 1928 from the filamentous fungus Penicilium notatum. Neither investigator appreciated the importance of what he had found, and the antibiotic substance, named penicillin, was not purified until the 1940s (by Florey and Chain), just in time to be used at the end of the second world war. Today there are hundreds of different antibiotics, though many are modified forms of naturally-produced antibiotics (semi-synthetic antibiotics). There are also other completely synthetic antimicrobial drugs (i.e. not derived from microbes) in use, notably the sulphonamides. The word "antibiotic" is sometimes used to mean any antimicrobial agent (natural or synthetic), but this use is wrong in A-level biology.
Action of Antibiotics
Many chemicals kill microbes. But a therapeutically useful antimicrobial agent must be selectively toxic i.e. it must kill pathogenic microbes already growing in human tissue, without also killing the host human cells. The tables on the next two pages give some examples of how selective toxicity is achieved. Some antibiotics are cidal (bacteriocidal, fungicidal, etc.), which means they kill the microbes, while others are static (bacteriostatic, fungistatic, etc.), which means they stop further growth, but don't kill existing cells. Both are useful medically, because if the growth of an infective pathogen is stopped, the body's immune system will be able to kill it. These graphs show how the two kinds of drug affect bacterial growth curves. bacteriostatic agent
Log (no. of viable cells)
agent added agent removed
bacteriocidal agent
Log (no. of viable cells)
agent added agent removed
Time
Time
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Antibacterial Drugs
Bacteria, as prokaryotes, have many unique features not present in eukaryotes, so antibiotics can be selectively toxic by targeting such features as the bacterial cell wall, 70S ribosomes, and enzymes that are specific to bacteria. In this way the human eukaryotic cells are unaffected. This table shows some of the ways in which common antibacterial drugs work. Name of drug Mode of action Effect Notes penicillin, ampicillin, Inhibits enzymes involved in syn- Cidal Narrow spectrum- little amoxycillin, methicillin thesis of peptidoglycan for effect on Gram negative (-lactam group) bacterial cell wall, causing cell cells. lysis. cephalosporin vancomycin Cell membrane polymyxin gramicidin sulphonamides DNA replication Inhibits enzymes involved in syn- Cidal made by fungus thesis of peptidoglycan. Cephalosporium acremonium Inhibits peptide bond formation in Cidal Effective against Grampeptidoglycan chains. positive bacteria Proteins forming channels in cell Cidal Toxic to humans, not membrane causing lysis. used much now Proteins forming channels in cell Cidal Toxic to humans, not membrane causing lysis. used much now Stops DNA replication by inhibit- Static Synthetic (not an antibiing folic acid synthesis, a otic). Humans don't have precursor of nucleotides. this enzyme so need folic acid in diet.
Cell wall
norfloxacin, ciproflox- Inhibit bacterial DNA gyrase, pre- Cidal Eukaryotes have a difacin (quinolone group) venting replication. ferent DNA gyrase. rifampicin Inhibits bacterial RNA polymer- Cidal Eukaryotes have a difase, preventing transcription ferent RNA polymerase (except in mitochondria).
Transcription
streptomycin, Stop 70S ribosome formation, pre- Static Broad spectrum. Toxic gentamicin, neomycin venting translation. to humans in large doses. (aminoglycoside group) Made by the bacterium
Streptomyces griseus
tetracycline Translation
Prevents binding of tRNA to 70S Static Resistance common. ribosomes. Made by the bacterium
Streptomyces aureofaciens
Fusidic Acid
Prevents release of tRNA from Static Steroid antibiotic used 70S ribosomes for Gram-positive infections. Inhibits 70S ribosome enzyme that Static Broad spectrum natural forms peptide bonds antibiotic now made synthetically. Inhibits 70S ribosome enzyme that Static Common alternative to forms peptide bonds penicillin
chloramphenicol
erythromycin
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Antifungal Drugs
Fungi, as eukaryotes, have similar cell structures and processes to human cells, so it is more difficult to make a selectively-toxic agent. Fortunately, there are not many serious fungal pathogens of humans, and most fungal diseases are minor skin infections. A far bigger problem (in economic terms) is the effect of fungal infection on crops, and many antifungal agents are used in agriculture. One obvious candidate for selective toxicity is the chitin cell wall, which is unique to fungi. However, despite much research, few effective inhibitors of chitin synthesis has yet been found. Current therapeutic antifungal drugs target unique lipids in the fungal cell membrane or unique fungal enzymes. There are both naturally-occurring antifungal drugs (fungal antibiotics) and synthetic ones. Name of drug Cell wall polyoxin D, nikkomycin Z Mode of action Effect Notes
Inhibits chitin synthase enzyme, Static Natural antibiotic from bacteso cell wall doesn't form. rium Streptomyces cacaoi. Used in agriculture but not in medicine.
Cell Membrane
nystatin, amphoteri- Bind to fungal lipids in mem- Static Natural antibiotic from bactecin B (polyene brane, causing it to leak ria Streptomyces spp. group) clotrimazole, mico- Inhibit synthesis of ergosterol in Cidal Synthetic. Used for skin innazole, fluconazole fungal membrane, causing it to fections. Can be effective (azole group) leak. against bacteria and toxic to humans terbinafine Inhibit synthesis of ergosterol in Cidal Synthetic. Uses different fungal membrane, causing it to mechanism from azole drugs, leak. and less toxic to humans. Disrupts microtubule formation, Cidal Natural antibiotic from fungus preventing chromosome sepaPenicillium griseofulvum. Narration at mitosis. row spectrum. Competitive inhibitor of an en- Cidal Synthetic nucleotide. Humans zyme involved in nucleotide have different enzyme. Resissynthesis. tance common.
griseolfulvin DNA
flucytosine
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Screening Microbes for Antibiotic Production

Antibiotics are the most-prescribed drugs and are big business, so there is a constant search for new antibiotics. There are over 10 000 different antibiotics known, but only about 200 in commercial use, since most new antibiotics are no better than existing ones. Finding a new antibiotic and getting it on to the market is a very long process and can take 15 years. 1. 2. Start with some natural sample containing microbes, e.g. soil, sea water, other organisms, faeces, etc. Isolate pure cultures by streak plate method. Test microbes for antibiotic activity using a cross-streak plate:
this bacterium is resistant
1 year
streaks of common bacteria
incubate
6 months
streak of new microbe inhibition zone around microbe
If microbes shows antibiotic activity, perform further cross-streak investigations to find if it is broad-spectrum (kills many species of microbes) or narrow-spectrum (kills only a few species of microbes). Both are useful. 3. Perform animal toxicity tests. The antibiotic must kill bacteria, but not the host animals. Find: The therapeutic dose - the concentration required to kill bacteria The toxic dose - the concentration required to kill the animal host Then calculate the Therapeutic Index = toxic dose therapeutic dose
5 years
The higher the index the better the antibiotic is. Most antibiotics fail at this stage. 4. 5. 6. Small-scale human trials using healthy volunteers to test for side-effects. Large-scale clinical trials to test effectiveness against disease. Apply for a government licence to sell in each country.
3 years 5 years 2 years
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Antibiotic Production Methods

Antibiotics are produced on an industrial scale using a variety of fungi and bacteria. We'll look at penicillin proPenicillin is produced by the fungus Penicillium chrysogenum which requires lactose, other sugars, and a source of nitrogen in the medium to grow well. Like all antibiotics, penicillin is a secondary metabolite, so is only produced in the stationary phase. It therefore requires a increase production.
Medium corn steep liquor (sugars) lactose yeast extract (nitrogen) pH buffers minerals
0 1 2 3 4 5 6 7 lactose cells
amounts
duction as an example of how antibiotics are made.
penicillin
Time (days)
batch fermenter, and a fed batch process is normally used to prolong the stationary period and so Downstream processing is relatively
Starter Culture Penicillium
easy since penicillin is secreted into the medium (to kill other cells), so there is no need to break open the fungal cells. However, the product needs to be very pure, since it being used as a therapeutic medical drug, so it is dissolved and then precipitated as a potassium salt to separate it from other substances in the medium.
10 times in 6 days remove 30% culture add 30% fresh medium
rotating filter filtrate fungal cells
The resulting penicillin (called penicillin

dissolve in butylacetate poassium ions added to precipitate salt of penicillin animal feed G) can be chemically and enzymatically
modified to make a variety of penicillins with slightly different properties. These semi-synthetic penicillins include penicillin V, penicillin O, ampicillin and amoxycillin. When penicillin was first made at the end of the second world war using the fungus Penicilium notatum, the process
wash, filter and dry
99.5% pure penicillin chemical and enzymatic modification to make new antibiotics
made 1 mg dm-3. Today, using a different species (P. chrysogenum) and a better extraction procedures the yield is 50 g dm-3. There is a constant search to improve the yield.
Selecting Antibiotics to Treat Infections

When treating an infectious disease, doctors have to select the right antibiotic. The wrong choice could be ineffective against the disease, and could even make the symptoms worse. There are two stages: 1. Identify the infective organism and start an "empirical treatment" immediately. This usually means prescribing a broad-spectrum antibacterial, antifungal or antiviral agent chosen in the light of the patient's symptoms and site of infection. This should not be continued too long, as a broadspectrum antibiotic is more likely to have side-effects by disturbing the commensal microbial flora. It also encourages resistance. Bacteria, fungi and viruses can only be treated by their specific antimicrobial agent and antibiotics should never be prescribed for viral infections (such as flu). 2. Test for antibiotic sensitivity using a disk diffusion test. A sample of the patient's blood or tissue fluid is taken and spread onto a nutrient agar plate to make a lawn (using aseptic technique). Samples of different antibiotics are then applied to the agar on sterile filter paper disks. The plate is incubated for 24 hours and then examined. The antibiotic diffuses out from the filter paper through the agar forming an invisible ring. If the antibiotic is effective against the bacteria causing this disease there will be a clear inhibition zone round the filter paper with no bacterial growth. If the bacteria are resistant to this antibiotic there will be growth right up to the paper. Special disks, called multidisks or mast rings, can be used to test 8 different antibiotics at once.
bacteria lawn on agar plate filter paper disks soaked in antibiotic incubate at 35C for 24 hours antibiotic effective at a low concentration antibiotic only effective at a high concentration mast ring. (8 different antibiotics)
In general, the larger the inhibition zone around the disk, the more effective the antibiotic is against these particular bacteria, because the concentration of antibiotic is lower the further away from the disk, so a large inhibition zone means the antibiotic kills bacteria at a low concentration. However, care needs to be taken when comparing different antibiotics, as the size of the inhibition zone can depend on a number of factors: the concentration of antibiotic on the filter paper the solubility of the antibiotic in the agar the rate of diffusion of the antibiotic through the agar the sensitivity or resistance of the bacterium to the antibiotic. The final choice of antibiotic may also depend on allergies, side-effects and cost.
Antibiotic Resistance
When antibiotics were first introduced after the second world war, they were seen as "miracle drugs" because they killed all bacteria and cured all bacterial diseases. However, within a few years, some antibiotics lost their effectiveness as bacteria became resistant to them.
Mechanisms of Antibiotic Resistance

There are several ways in which bacteria can resist the effects of antibiotics: Bacteria can be resistant because they have enzymes that destroy antibiotics. For example some bacteria have the enzyme penicillinase that breaks the -lactam ring in the -lactam antibiotics such as penicillin, rendering the antibiotic useless. Antibiotics are often similar to normal bacterial metabolites, and a small mutation in a existing enzyme can modify its active site to fit an antibiotic. Bacteria can be resistant because the antibiotics can't enter the cell. The outer membrane of the complex cell wall of Gram-negative bacteria stops antibiotics such as penicillin. Modifications to the penicillin structure can overcome this problem, and some semi-synthetic penicillins such as ampicillin have a much broader spectrum, killing Gram-negative as well as Gram-positive bacteria. Bacteria can be resistant because they don't have peptidoglycan in their cell walls. Rickettsia and Chlamiydia don't have peptidoglycan, so are resistant to penicillin and all the -lactam antibiotics. Bacteria can be resistant because they have active transport pumps that pump antibiotics out of the cell. Bacteria can be resistant because the enzyme affected by the antibiotic has mutated. The enzyme still does its job, but is unaffected by the antibiotic. For example bacteria with a mutated DNA gyrase are resistant to quinilone antibiotics, and bacteria with mutated ribosome proteins are resistant to streptomycin. Bacteria can be resistant because they have alternative metabolic pathways. For example folic acid can be synthesised by different enzymes, or absorbed from the environment, making these bacteria resistant to sulphonamides.
Development of Antibiotic Resistance

How do bacteria become resistant to antibiotics? Some species may be resistant because they don't posses the antibiotic target, such Rickettsia with no peptidoglycan. But in general, resistance first develops due to a mutation. Bacteria reproduce asexually, so all the offspring should be the same, but sometimes, at random, mutations occur when DNA is replicated. These mutations may have any effect (and most will be fatal), but just occasionally a mutation occurs that makes that bacterium
resistant to an antibiotic. For example a mutation could slightly alter a ribosome protein so that streptomycin can no longer bind, or a mutation could slightly alter an enzyme, changing its substrate specificity so that its active site will now bind penicillin. Such mutations are very rare, but bacteria reproduce so rapidly, and there are so many bacterial cells, that new resistance mutations do crop up at a significant rate (a few times per year somewhere on the planet). Remember that development of antibiotic resistance is a random event, and is not caused by the presence of the antibiotic. It is certainly not an adaptation that bacteria acquire. As an example, imagine a community of different bacterial species living in your gut, and one particular cell has just mutated to become resistant to penicillin. What happens next? It will reproduce by binary fission and pass on its resistance gene to all its offspring, forming a new strain of bacteria in your gut. If there is no antibiotic present in your gut (most likely) this mutated strain may well die out due to competition with all the other bacteria, and the mutation will be lost again. However, if you are taking penicillin, then penicillin will be present
These cells win competition if no antibiotic These cells win competition if antibiotic present mutation
in the bacteria's environment, and these mutated cells are now at a selective advantage: the antibiotic kills all the normal bacterial cells, leaving only the mutant cells alive. These cells can then reproduce rapidly without competition and will colonise the whole environment. This a good example of natural selection at work.
Spread of Antibiotic Resistance

How do these resistant bacteria spread to other people? In the example above the bacteria will contaminate faeces and may then infect other individuals through poor hygiene. In fact the resistant bacteria can spread by any of the normal methods of spreading an infection: through water, food, sneezing, infected instruments, etc. Some bacteria can form spores to aid their dispersal, and so a mutated strain can survive long journeys and long periods of time. In most new environments the mutated strain will die out through competition, but whenever it encounters penicillin it will thrive, out-competing all other bacteria. This is how resistance of one bacterial strain to one antibiotic can spread, but unfortunately resistance can also spread between species. Bacteria have a trick that no other organisms can do: they can transfer genes between each other; even between different species. In this way a resistance gene
can spread from the bacterium in which it arose to other, perhaps more dangerous, species. There are three methods of gene transfer in bacteria: Conjugation. This is the transfer of DNA between bacterial cells via a cytoplasmic bridge or pilus. From time to time two bacterial cells can join together (conjugate), and DNA passes from one (the donor) to the other (the recipient). The transferred DNA can be one or more plasmids, or can be all or part of the whole bacterial chromosome (in which case the donor cell dies). Conjugation is sometimes referred to as bacterial sex or mating, but it is quite distinct from sexual reproduction, because the gene exchange is not equal, it can take place between different species, and bacteria do not use conjugation for reproduction. It is better thought of as an alternative to sex, where these asexual organisms gain some of the advantages of genetic exchange. Donor DNA Pilus
Donor Cell
Recipient Cell
Donor dies
Recipient contains new genes
Transduction. This is the transfer of DNA between cells via a virus. When viruses (or bacteriophages) infect bacteria they often incorporate their DNA into the bacterial chromosome, to form a provirus. When the virus reproduces and new virus particles are formed, they can contain some of the bacteria's DNA as well as the viral DNA. This bacterial DNA is then incorporated into the chromosomes of the next bacterial cell the virus infects.
1 Virus infects bacterium species A

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2 Viral DNA incorporated into bacterial DNA
3 New virus assembled with some bacterial DNA
4 Virus infects bacterium species B
5 Bacterium species B contains genes from species A

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Transformation. This is the uptake of DNA from the environment. When a bacterial cell dies, its DNA is often released into its surroundings, and certain other bacterial species can simply take up this naked DNA and incorporate it into their own chromosome. Although a natural process, transformation is now most important as an artificial process used extensively in genetic engineering. DNA, usually in the form of a plasmid, is removed from one cell and injected into another one. Under the right conditions the injected DNA is taken up by the new host cell and replicated and expressed.
plasmid
plasmid DNA incorporated into bacterial DNA
In the past genetic engineers used R-plasmids (R for Resistance) with antibiotic-resistance genes in them as vectors to carry useful genes, because these antibiotic-resistance genes could be used as markers (see module 2). At the time this seemed sensible, but with hindsight it can be seen as an unfortunate way to spread antibiotic resistance around the world. Most scientists have stopped using antibiotic resistance genes as markers, but a few continue to do so. Multiple Resistance. These methods of gene transfer are the main cause of the most worrying aspect of antibiotic resistance: multiple resistance. It is highly unlikely that a single strain will mutate twice to develop resistance to antibiotics, but it is perfectly likely that it could receive genes for resistance to different antibiotics by gene transfer. This has led to strains of bacteria that are resistant to many (or even all) antibiotics.
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Impact of Resistance on the use of Antibiotics

The most common sources for antibiotic-resistant bacteria (and especially multiple-resistant bacteria) are hospitals. This is partly because they have a high concentration of people with bacterial infections, but also because the environment is awash with antibiotics. This provides a strong selection pressure for any antibiotic-resistant strains, which multiply in the absence of much competition. A good example is Staphylococcus aureus, a bacterium responsible for a variety of diseases from staphylococcal food poisoning to toxic shock syndrome. This species has been resistant to penicillin for years, due to the possession of the penicillinase enzyme. Semi-synthetic penicillins such as methicillin are unaffected by penicillinase and so were effective against S. aureus. However, within a year of the introduction of methicillin, methicillin-resistant strains of S. aureus (MRSA) were found in hospitals where methicillin was in regular use. Infections by MRSA were very difficult to treat, responding only to the antibiotic vancomycin. In 1997 vanocmycin-resitant, methicillinresistant Staphylococcus aureus appeared in Japan. These bacteria are effectively untreatable at present. So what can be done? The simple solution is to use a wider range of antibiotics, and this is why there is a constant industrial search for new anti-microbial agents. However, as the example of MRSA has shown, sooner or later the bacteria will develop resistance, so this is only a short-term solution. A better long-term strategy is to stop using antibiotics (or at least minimise their use). This will end the selection pressure in favour of resistant strains. Most resistant strains are inferior to the "wild type" strains in other respects (perhaps they reproduce more slowly), so in an antibiotic-free environment the mutants are generally out-competed and will die out. The antibiotic resistance genes will therefore disappear from the gene pool of that population. Unfortunately we are now in an "antibiotic culture" where many doctors prescribe antibiotics routinely for common ailments such as the flu (even though they have no effect), simply to keep the patient happy. And farmers, especially in the USA, routinely feed their livestock small concentrations of antibiotics, just in case they come into contact with an infection. If this overuse of antibiotics continues, then most antibiotics will become useless, and we will revert to the pre-antibiotic age, where common bacterial infections will once again become untreatable.
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Infectious Disease
Disease is a general term meaning a disorder of the body. To most people disease means an infectious disease, but in fact there are several different kinds of disease: 1. Infectious Diseases are caused by other organisms (usually microbes) living in or on the body and so causing harm (e.g. cold, TB, AIDS). These are the diseases you can "catch". 2. Genetic Diseases are caused by genes inherited from parents. These are really characteristics that are unusual in the population and life-threatening (e.g. muscular dystrophy, cystic fibrosis, haemophilia). 3. Dietary Deficiency Diseases are caused by a lack of specific nutrients in the diet, e.g. kwashiorkor (protein), scurvy (vitamin C), rickets (vitamin D). 4. Environmental Diseases are caused by non-living factors in the environment. They include skin cancer, lung cancer, asthma, asbestosis, caused by radiation or pollution, prion diseases such as CJD, and can also include the "self-inflicted" disorders of modern society such as alcoholism, coronary heart disease, anorexia and even accidents. 5. Ageing Diseases are caused by degeneration of body tissues and include arthritis, arteriosclerosis and cataracts. For the rest of this module, we shall only be concerned with infectious diseases. Infectious diseases are caused by a variety of microbes, and a few of the common ones are shown in this table: Viral Diseases Disease common cold influenza smallpox measles hepatitis B AIDS tuberculosis typhoid food poisoning cholera tetanus dysentery whooping cough pneumonia thrush athletes foot ringworm malaria amoebic dysentery sleeping sickness Microbe Rhinovirus Myovirus Variola virus Paramyxovirus DNA virus HIV Mycobacterium tuberculosis Salmonella typhi Salmonella spp. Vibrio cholerae Clostridium tetani Shigella sonnei Bordetella pertussis Streptococcus pneumoniae Candida albicans Tinea pedis Tinea capititis Plasmodium vivax Entamoeba histolytica Trypanosoma spp.
Bacterial Diseases
Fungal Diseases Protoctist Diseases
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Pathogenicity of Microbes
Some pathogens are more harmful than others; in other words they are have a greater pathogenicity or virulence. For a pathogen to cause a disease five steps must take place: 1. Transmission 2. Invasion 3. Attachment 4. Entry 5. Harm The pathogen must be transmitted to the human host The pathogen must invade the human body The pathogen must attach to a specific host cell The pathogen often enters the host cell and grows there The pathogen must damage the host
We'll look at each of these in turn.
1. Transmission
The pathogens that cause infectious disease can be transmitted to humans in several different ways: Transmission by Water. Many pathogens can survive (though not reproduce) in water and so infect humans drinking that water. Water-borne infections include cholera, typhoid, dysentery, gastroenterisis and food poisoning (salmonellosis). The link between drinking water and disease was demonstrated in 1849, when a London doctor, John Snow, stopped a cholera epidemic by simply removing the handle of the water pump in Broad Street. The Public Health Act of 1875 resulted in proper sewage treatment and clean water supplies to cities and towns in the UK, and there are now strict laws governing the quality of public water supplies (though not bottled water). In developing countries the simplest way to improve public health is often a simple water pump to deliver clean water. Water is readily contaminated by human waste in towns and villages where there is no sewage system, so infectious diseases like cholera are quickly transmitted to everyone using that water supply. Sewage treatments range from simple strategies such as discharging sewage into rivers downstream of drinking water abstraction, to advanced sewage treatment works in large cities, where sewage is filtered to remove particles and bacterial cells and sterilised with chorine to kill most remaining microbes. Transmission by Food. Food is an even better way of transmitting pathogens, since microbes can grow and reproduce in food, which they can't do in water. A small quantity of food can therefore contain millions of bacterial cells, some of which can reach the lower intestine. Food can become contaminated by contact with humans or insects. Food-borne infections include TB, botulism, cholera, amoebic dysentery, typhoid and gastroenteritis.
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Microbes grow best in the temperature range 30-40C, so this is the "danger zone", which must be avoided as much as possible. Food can be stored for short periods in fridges (0-10C), which slows microbial growth and enzyme activity, or for longer periods in freezers (below -5C), which stops growth and kills many microbes, but can affect taste. Neither method kills microbes, so cooking is still required. Food contamination can be prevented by following a few simple rules: Keep food preparation area This reduces the risk of contaminating food in the kitchen. clean Wash hands before han- This prevents transmission of microbes from faeces to food. dling food Use food by its use-by date Wash fruit and vegetables Store food in the fridge Store raw and foods separately This ensures that microbes do not have time to multiply too much. This removes soil microbes as well as agricultural sprays. The temperature of domestic fridges (1-10C) slows down microbial growth, but does not kill them,
cooked Contact with raw meat could pass microbes on to others foods that will be eaten without further cooking.
Don't refreeze thawed fro- Freezing does not kill all microbes, and each thaw cycle gives microbes a chance to grow. zen food Thaw frozen food in the The centre of a frozen item can be much cooler than the outside, so microbes may not be killed. fridge thoroughly Cook food thoroughly The high temperature denatures proteins both in the food, making it more palatable, and in any resident microbes, killing them. Large items need a longer cooking time to allow the centre to reach the same temperature as the outside.
Transmission by Aerosol Droplet. When an infected person coughs, sneezes, talks or breathes, they are transmitting their pathogens inside droplets of mucus, saliva or water. These droplets can be suspended in the air for long periods, or they can dry out, releasing the microbes to be carried by air currents. The pathogen can then be inhaled or enter the body by other opening in the skin, such as cuts. Air-borne infections include many of the viral infections such as cold, flu, rubella, and some bacterial infections such as TB, diphtheria and strep throat. Droplet infection is most effective where there is a high density of people and in enclosed spaces such as aircraft and office buildings. Good ventilation systems can help, but they can also be a reservoir of pathogens, leading to "sick building syndrome". Recent outbreaks of Legionnaires disease have been traced to infected air conditioning units. In hospital operating theatres droplet infection is reduced by the wearing masks and keeping the air pressure slightly above atmospheric pressure to stop air-borne droplets from getting in.
Transmission by Direct Contact. The skin is a good barrier to microbial entry, but some infections are still possible by contact with the skin. These are called contagious infections. Fungal infections such as athletes foot and ringworm are infections of the skin and are transmitted by direct contact with infected clothes or surfaces. Sexual intercourse is a special case of direct contact, and sexually-transmitted diseases (STDs) include the viral infections Hepatitis B and AIDS and the bacterial infections gonorrhoea and syphilis. The purpose of sexual intercourse is to transmit small, delicate cells from one person to another without exposing them to the harsh outside environment, so it is a very effective way of transmitting pathogens that cannot survive in the air. The transmission of STDs is increased by sexual promiscuity (having many partners) and reduced by using barrier methods such as condoms. Transmission by Vectors. In the context of disease, a vector is an animal that transmits a pathogenic microbe between other animals. Examples include rabies (transmitted by dogs), malaria (transmitted by mosquitoes), sleeping sickness (transmitted by tsetse flies) and yellow fever (transmitted by mosquitoes).
2. Invasion
Once the pathogen has been transmitted to the human host, it must enter the host's body and spread within the host till it reaches its target tissue. This is called invasiveness. Some pathogens can enter the blood or lymph vessels and so get carried to other parts of the body. This can cause tissue damage far away from the original site of infection. Bacterial toxins are also readily carried all over the body in the bloodstream. Some pathogens, such as salmonella enteritidis, (which causes food poisoning) need a large number of cells to infect the host before they can grow and cause disease. This is called low infectivity and means that the disease is comparatively difficult to catch. Other microbes, such as salmonella typhi (which causes typhoid), only need a small number of cells to infect the host before the disease occurs. This is called high infectivity and means that the disease is comparatively easy to catch.
3. Attachment
As soon as microbes infect a new host they need to attach themselves firmly to the host's cells in order to avoid being removed by cilia, sneezing, vomiting, diarrhoea, etc. To do this microbes use molecules called adhesins, which are protein or carbohydrate molecules on the cell wall of bacteria or the capsid or envelope of viruses. These adhesin molecules bind to specific protein receptor molecules on the cell membranes of host cells forming adhesin-receptor complexes, so firmly attaching (or adhering) the microbe to the host cell. Because this involves specific binding, this attachment is called specific adherence.
bacterium adhesins
virus
receptors cell membrane Specific adherence explains why microbes tend to be specific for particular species and tissue. For example the malaria parasite only grows in red blood cells; the diphtheria bacterium only colonises the membranes of cells in the trachea; and the polio virus only invades nerve cells. It also explains one reason why different people have susceptibility to certain infections. Individuals without the "correct" receptor cannot attach the pathogen and so cannot catch that particular disease.
4. Entry
Some bacterial and fungal pathogens remain attached to the outer surface of human cells or colonise extracellular spaces in humans, such as the lumen of the gut, alveoli, or tissue fluid. These are termed extracellular pathogens. But in many cases (all cases with viruses), the pathogen needs to enter the host cell. These are called intracellular pathogens. Remember that bacterial cells, as prokaryotes, are much smaller than eukaryotic cells and are often similar is size to organelles like mitochondria. Entry is achieved in different ways: Some bacteria secrete invasin enzymes, which digest the cell membrane of the host cell, allowing the bacterial cell to enter it. invasin enzymes Sometimes the specific adherence described above stimulates endocytosis by the host cell, which brings the microbe cell inside the host cell. The host cell may then try to digest the microbe cell, but if the bacterium has a capsule it may be protected. Viruses with lipid envelopes fuse with the host cell membrane, allowing the rest of the virus into the cytoplasm of the host cell. bacterium
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Viruses without lipid envelopes may attach to the cell surface and inject their DNA into the cell. This DNA is then used to replicate whole new viruses.
virus
DNA
5. Harm
Pathogens harm their hosts in two ways. 1. By reproducing inside host cells. Viruses, bacteria and protoctists can all colonise living cells and reproduce inside them, using up cellular resources and preventing the cell from carrying out its normal reactions. The microbes then usually burst out of the host cell, rupturing the cell membrane and killing the cell in the process. 2. By producing toxins chemicals that interfere with the body's reactions. These may be enzyme inhibitors, mutagens... Many viral proteins used for replicating the virus act as toxins. Some bacteria secrete protein toxins while they are growing. These are called exotoxins and each different exotoxin has a specific effect. For example the tetanus bacterium colonises nerve cells and secretes a toxin that inhibits motor neurones, causing muscle paralysis; while the cholera bacterium colonises the intestine and secretes a toxin that causes violent inflammation of the intestine and diarrhoea. Exotoxins are also produced by bacteria growing in food, and these toxins can cause food poisoning even if the bacteria themselves are killed in cooking. This kind of food poisoning is marked by a very short incubation period, 30 min-6 h after eating, whereas normal food poisoning (such as salmonellosis) has an incubation time of 1-3 days while the bacteria replicate in the gut. Some bacterial exotoxins (e.g. cholera) are actually proteins made by viruses that are infecting the bacteria that are infecting the human! Gram negative bacteria also release the lipopolysaccharides from their cell wall when they die. These are called endotoxins and they all cause the same general effects, which include fever, weakness and aching.
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Examples of Diseases
We shall now examine four diseases in detail as examples of infectious diseases. Disease
Typhoid Fever (Enteric Fever)
Pathogen Salmonella typhi bacterium Transmission Through faecal contamination of water due to untreated sewage contaminating drinking water supplies, or to poor hygiene. Other sources are milk and food contaminated by infected person or carrier, and flies. High infectivity (105-106 cells required for infection). Infection Bacteria colonise small intestine, are absorbed into the lymphatic system, multiply mechanism in lymph glands for 10 days, then enter blood stream colonising white blood cells and causing blood poisoning due to production of a powerful endotoxin. Signs and After a two-week incubation period symptoms include fever, headache, muscle Symptoms pains, followed later by diarrhoea, "rose-spot" rash on the skin and mental confusion. If untreated recovery takes about a month but death is possible in rare cases. Treatment Treatment with antibiotics is effective. Prevention Vaccination is available for UK travellers to risky areas. Prevention is by ensuring clean water, by separating sewage from drinking water supplies and by personal hygiene. A major killer in the UK until the public health act of 1875. Now very rare in UK, and all case come from abroad.
Disease
Salmonellosis (Salmonella Food Poisoning)
Pathogen Salmonella enteritidis bacterium Transmission Through poultry and their eggs, especially when uncooked (e.g. mayonnaise and ice cream), or lightly-cooked (e.g. meringue and soft-boiled eggs); or through unpasteurised milk. Other sources are contamination of foods and water by human or household pet faeces. Low infectivity (106-107 cells required for infection). Infection The bacteria colonise small intestine and remain there producing exotoxins. mechanism Signs and Sudden signs 15-48 h after eating infected food. This is much faster than typhoid Symptoms because the bacteria remain in the gut. Symptoms include moderate diarrhoea,
vomiting, abdominal pain, mild fever and headache. Recovery usually in 2-3 days. Treatment Treatment usually by fluid replacement. Antibiotics unnecessary and may increase infectivity of faeces. Prevention Prevention is mainly by hygiene in food preparation; thorough cooking of poultry and eggs; and pasteurisation of milk. Less crowded poultry farming methods also reduce infection rates in hens. Eggs in UK are now always pasteurised. Disease
Influenza
Pathogen Human Influenza virus (type A, B or C). Transmission Through air-borne droplet infection from the coughs and sneezes of infected individuals. Infected people are infectious from a day before symptoms show themselves until a week afterwards. Infection The virus invades the epithelial cells lining the upper respiratory tract (nose, mechanism mouth, throat, trachea and bronchi) and reproduces inside them, killing many cells in the process. These dead cells increase the amount and thickness of mucus produced during an infection, which irritates the throat, causing coughing. Signs and The onset is sudden after an incubation period of 1-4 days, and the symptoms inSymptoms clude fever, shivering, headache, muscular pain, coughing of excess mucus and a loss of appetite. Recovery normally takes about 4 days, unless there are secondary infections, which can be fatal if untreated. The influenza pandemic of 1918 killed 22 million people world-wide, making it the greatest killer disease ever. Treatment Treatment is by bed rest with plenty of fluids and analgesic drugs like asprin or paracetamol. Since this is a viral infection, antibiotics are useless. Prevention Vaccination is difficult because of genetic changes in the influenza virus, but vaccinations based on a variety of antigens are now used to protect at-risk groups (babies and elderly). Prevention would require the isolation of flu victims, which is not practical.
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Disease
AIDS
stranded RNA and the enzyme reverse transcriptase).
Pathogen Human immunodeficiency Virus (HIV), a retrovirus (i.e. one that contains single-
Transmission Through infected semen or vaginal secretions during sexual activity, or through infected blood in transfusions or contaminated needles. Infected mothers can also pass the virus on to their children through the placenta or milk. Before 1985 many hospital patients, especially haemophiliacs, became infected through blood transfusions, but since 1985 all blood donations in the UK are tested for HIV. Many drug addicts have been infected through sharing needles. By far the most important method of transmission of HIV world-wide is unprotected sexual intercourse. HIV cannot survive in air and therefore cannot be transmitted by skin contact or kissing. Infection HIV in the blood attaches to cells that carry the "CD4" antigen, including the T mechanism lymphocyte and macrophage white blood cells. After entering these cells it becomes a provirus in the nuclear DNA, remaining dormant but being replicated for a long latency period of 8-10 years. Eventually the virus particles are re-assembled and emerge into the blood, rupturing and killing the T cells in the process. The lack of T cells leaves the immune system severely compromised. Signs and Like other retroviruses, HIV has a long latency of 8-10 years, during which time Symptoms there are no symptoms, but the individual is infectious. After this period the person starts to shows mild symptoms of the AID-related complex (ARC), such as tiredness, fever, weight loss and diarrhoea. This is followed by the more serious symptoms of full-blown AIDS. Since the immune system no longer functions the patient has no defence against a variety of opportunistic infections. The most common of these are Kaposi's sarcoma (a skin cancer), TB and pneumonia, which is usually fatal. Treatment There is as yet no cure or vaccination for AIDS, though drugs like AZT can delay its onset for many years. Prevention Vaccinations are difficult because the HIV genome is highly variable (probably because reverse transcriptase make many base copying errors). Prevention of AIDS has concentrated on "safe sex" education (using condoms and reducing promiscuity), not sharing needles, and screening blood transfusions.
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Defence Against Infectious Disease

We are surrounded by microbes in the air, on the ground and all other surfaces, and in our food and water. The only reason we are still alive is because humans (and all other animals) have a very powerful defence mechanism The Immune System.
The Immune System

The parts of the immune system are spread all over the body. They include: The lymph and blood vessels. These transport pathogens and leukocytes all over the body. The lymph nodes. These contain millions of phagocyte and lymphocyte cells, which identify and lymph. The spleen. This also contains millions of phagocyte and lymphocyte cells, which identify and remove pathogens from blood. The thymus. This is where blood stem cells are differentiated into T-lymphocytes. The cells of the immune system are the white blood cells (or leukocytes). Leukocytes are derived from stem cells, which are produced in huge numbers in the bone marrow (the soft centre of large bones). These stem cells differentiate to form dozens of different kinds of leukocytes, which fall into four categories: White Blood cells (Leukocytes)
lymph nodes in groin lymph nodes in neck
remove pathogens
from
lymph nodes in armpits
thymus
spleen
Phagocytes for phagocytosis Macrophages Neutrophils Monocytes
Granulocytes for inflamation Mast cells Eosinophils Basophils
T Lymphocytes for cell-mediated immunity Helper T-cells Killer T-cells Memory T-cells
B Lymphocytes for antibody-mediated immunity B-cells Plasma B-cells Memory B-cells
Non-Specific Immune System

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Specific Immune System

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The phagocytes and the granulocytes form the non-specific immune system, which kills pathogens quickly and indiscriminately. Although effective, the non-specific immune system does not "learn from experience", so it does not lead to immunity to a disease. The lymphocytes form the specific immune system, which is a more complex and sophisticated collection of reactions that not only kill invading pathogens, but also remember the pathogen's features so that it can be killed quickly on subsequent infections. While all animals have a non-specific immune system, only vertebrates have a specific immune system, so it must be a later evolutionary advance. The key difference of the specific immune system is that it is capable of recognising foreign cells as distinct from its own cells, an ability called self/nonself recognition. It does this by making use of antigens. An antigen is a large molecule (protein, glycoprotein, lipoprotein or polysaccharide) on the outer surface of a cell. All living cells have these antigens as part of their cell membrane or cell wall. The capsid proteins of viruses and even individual protein molecules can also be classed as antigens. Their purpose is for cell communication, and cells from different individuals have different antigens, while all the cells of the same individual have the same antigens. Antigens are genetically controlled, so close relative have more similar antigens than unrelated individuals. Blood groups are an example of antigens on red blood cells, but all cells have them. There are two kinds of lymphocyte B-lymphocytes (or just B-cells) and T-lymphocytes (or just Tcells). B-cells are called that because they mature from stem cells in the bone marrow. T-cells are called that because they mature from stem cells in the thymus. B-cells make antibodies. An antibody (also called an immunoglobulin) is a protein molecule that can bind specifically to an antigen. Antibodies all have a similar structure composed of 4 polypeptide chains (2 heavy chains and 2 light chains) joined together by strong disulphide bonds to form a Y-shaped structure. The stem of the Y is called the constant region because in all immunoglobulins it has the same amino acid sequence, and therefore same structure. The ends of the arms of the Y are called the variable regions of the molecule because different immunoglobulin molecules have different amino acid structure and therefore different structures. These variable regions are where the antigens bind to form a highly specific antigen-antibody complex, much like an enzymesubstrate complex.
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variable region
disulphide bridges
constant region
antigen-binding site
stylised diagram
Rasmol molecular model
Each B-cell has around 105 membrane-bound antibody molecules on its surface and can also secrete soluble antibodies into its surroundings. Every human has around 108 different types of B cell, each making antibodies with slightly different variable regions. Between them, these antibodies can therefore bind specifically to 108 different antigens, so there will be an antibody to match almost every conceivable antigen that might enter the body. T-cells have receptor molecules on their surfaces which are very similar, but not identical, to antibodies. These receptors also bind specifically to antigens to form antigen-receptor complexes. Each T-cell has around 105 receptor proteins, and again there are about 108 different types of T-cell, each with slightly different receptor molecules, so they can also specifically bind to any conceivable antigen. T-cells do not secrete soluble proteins. B-cell antigens membrane-bound antibodies soluble antibodies The B and T cells are exposed to so many "self" antigens on every normal cell they come across, that they quickly "learn" to recognise them very early in life. From then on self antigens are ignored, but any non-self antigens are recognised and stimulate an immune response as described below. membrane receptors T-cell
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The Three Lines of Defence

Humans have three lines of defence against invading pathogens: 1. Barriers the skin and associated chemicals stop microbes entering the body 2. The non-specific immune system phagocytes quickly destroy microbes that pass the first line of defence 3. The specific immune system lymphocytes kill any microbes that pass the second line of defence, and remain on guard for future attacks.
The First Line of Defence Barriers

The body has many mechanism to try to stop microbes entering the body, particularly the bloodstream. The skin is a tough, impenetrable barrier (which is why we use it to make leather shoes). The outer layer, the epidermis, is 20-30 cells thick (about as thick as a sheet of paper) and its cells are toughened by the protein keratin. The next layer, the dermis, is 20-40 times thicker and provides the main structure for the skin as well as all the receptor cells, blood vessels and hairs. Cells are constantly being lost from the surface of the skin (to form dust) and are replaced by new cells from further down. Sweat and tears, secreted by glands in the skin, contain lysozyme enzymes, which destroy (lyse) bacteria growing on the surface of the skin by digesting their peptidoglycan cell walls. The digestive tract is a potential entry route for pathogens, but it is protected by concentrated acid in the stomach, which denatures microbial enzymes and cell surface proteins, as well as protease enzymes. Saliva also contains lysozymes. The respiratory tract is another potential entry route, but it is protected by sticky mucus secreted by glands in the bronchi and bronchioles, which traps microbes and other particles in inhaled air before they can reach the delicate alveoli. Mucus contains lysozymes, and cilia constantly sweep the mucus upwards to the throat, where it is swallowed so that the microbes are killed by the stomach acid. The human body is home to billions of bacterial cells called variously the natural microbiota, the normal flora, the commensal flora (because they have a non-harmful or commensal relationship with their host) or even the "friendly bacteria". There are more bacteria cells in a human than there are human cells. These commensal bacteria colonise the skin, mouth, lower digestive tract, respiratory tract and vagina, and they help prevent infection by out-competing pathogenic microbes for food and space.
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The Second Line of Defence The Non-Specific Immune System

The second line of defence is the non-specific immune system, a host of quick, non-specific methods of killing microbes that have passed the first line of defence and entered the body. Some of the main methods are: Phagocytosis. Phagocytes are large, irregularly-shaped leukocyte cells that remove bacteria, viruses, cellular debris and dust particles. The phagocytes are constantly changing shape, and they flow over microbes, surrounding and ingesting them through the process of phagocytosis to form a phagosome. The phagosome then fuses with lysosomes - small vesicle containing lysozymes, which are released into the phagosome, killing and digesting the microbe. Different phagocyte cells work in different locations: neutrophils circulate in the blood, while macrophages are found in lymph, tissue fluid, lungs and other spaces, where they kill microbes before they enter the blood. Complement System. This comprises more than 20 different proteins, which kill microbes by making pores in their cell membranes and can also inhibit viral reproduction inside cells. They are also involved in activating other parts of the immune system. Inflammation. This is a localised response to an injury or infection. The granulocyte cells and the affected cells release chemicals, including histamines and prostaglandins, which stimulate: vasodilation to increase the flow of blood to the area (so the area turns red); capillary leakage so that phagocytes and granulocytes can enter the local tissue fluid (so the area swells); sensory neurone impulses (so the area is tender or painful); blood clotting to seal a wound (so a scab is formed). The dead pathogens and phagocytes, together with excess tissue fluid, are release as pus. The chemicals also help to stimulate the specific immune response (see below). Fever. This is caused by pyrogen chemicals, which include some of the inflammation chemicals as well as bacterial endotoxins. These stimulate the hypothalamus of the brain to increase the body's temperature from 37C up to 39C. This helps the immune system and inhibits growth of some pathogenic bacteria. lysozome phagosome bacterium nucleus
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The Third Line of Defence The Specific Immune System

The actions of the specific immune system are summarised in this diagram:
virus viral-infected cell toxin bacterium phagocyte transplant organ tumour cells
Antigen Presentation Clonal Selection
Cellmediated immunity T-Cells cloning
chemical signaling B-Cells
Antibodymediated immunity
cloning
CytoxicT-cells
Memory T-cells
Helper T-cells
Memory B-cells
1. Antigen Presentation Infection is started when cells with non-self antigens enter the blood of tissue fluid. The antigens can be from a variety of sources: a virus capsid protein or envelope protein on the surface of a bacterial cell a toxin released from a bacterium on a macrophage that has ingested a pathogen on the surface of cells of a transplanted organ. on a cancerous cell on a cell infected with a virus so that it has viral proteins on its surface
Macrophages are the most important antigen-presenting cells because they are the most numerous. They constantly inspect the surface of every cell they come into contact with in the blood, spleen, lymph nodes, tissue fluid and alveolar spaces. If the antigens are not recognised as self antigens, then the macrophage ingests the antigen and its cell by phagocytosis. Some of the antigens pass to the surface of the macrophage, which thus becomes an antigen-presenting cell. This amplifies the
number of antigens. The macrophage also secretes cytokine chemicals (also called lymphokines or interleukins) to stimulate the lymphocytes. 2. Clonal Selection At birth we have less than 100 copies of each type of B or T lymphocyte. Whenever a particular antigen enters the body it comes into contact with all the various cells in the blood and lymph, including the lymphocytes. Sooner or later the antigen will encounter a lymphocyte with a matching receptor molecule, to which it can bind tightly. It's a bit like Prince Charming trying the glass slipper on all the girls in the kingdom until eventually he finds Cinderella, who is an exact fit. As soon as a match is found, the binding of the antigen to the receptor stimulates the lymphocyte to divide repeatedly by mitosis, making an army of about 106 identical cloned B and T lymphocyte cells. This is called clonal selection, because only the selected cell is cloned. This army of clones can now destroy the infecting microbe, as described below. 3. T-Cells and Cell-Mediated Immunity The T-lymphocytes differentiate into cells with different functions. Cytoxic T-cells (or killer T-cells) bind to antigens on infecting cells and kill the cells by releasing perforin proteins. These insert into the cell membrane of the other cell, where they make a pore, which allows water to diffuse in so that the cell bursts. Helper T-cells bind to antigens on infecting cells and secrete chemicals called cytokines. These stimulate all the other white blood cells (phagocytes, granulocytes and B lymphocytes) and speed up the immune response. The AIDS virus HIV destroys these helper T-cells, and the immune system doesn't work nearly as well without them. Memory T-cells remain the blood for many decades after the infection. This means that the same antigen will be identified much more quickly in a subsequent infection, when the memory T-cells will quickly divide to form cytoxic T-cells and helper T-cells. 4. B-Cells and Antibody-Mediated Immunity (or humoral immunity) The B-lymphocytes also differentiate into cells with different functions. Plasma cells secrete free soluble antibodies. These antibodies are carried around the blood, lymph and tissue fluid binding to any antigens they come into contact with and forming antibody-antigen complexes. A single B-cell can divide to form 106 plasma cells, each of which can release 103 antibodies each second for 4 days. So during the immune response to an infection there is an enormous number of antibodies in the body and it is highly likely that every antigen will be targeted by one.
The antibodies help to kill cells in various ways. By binding to antigens on viruses and bacteria they prevent the viruses or bacteria attaching to cells and so infecting them. Similarly, when antibodies bind to free toxin proteins, they change the shape of the active region so that these proteins can no longer take part in the reactions that caused disease. Because each antibody molecule has two antigen-binding sites (one on each arm of the Y), antibodies can stick antigens together into large clumps. This process, called agglutination, immobilises viruses and cells, and precipitates soluble toxins so that they can easily be destroyed by phagocytes or killer T-cells. Large antigenantibody complexes also stimulate the various activities of the non-specific immune response, such as phagocytosis, complement production and inflammation. Memory B cells continue to secrete antibodies in small quantities for many decades, and can multiply rapidly to produce an instant supply of plasma cells if the same pathogen invades again. Primary and Secondary Immune Responses The first time a new antigen is encountered there are only a few lymphocyte cells of each kind (<100) for the antigen to encounter, so it can take several days for clonal selection to take place and the clone army to be assembled. Furthermore the clone army tends to be fairly small. This slow and weak response to a first infection is called the primary immune response. It is during this period that the symptoms of the disease are shown, partly due to toxins and cell death due to the pathogen, and partly due to the immune response itself (e.g. fever, inflammation). After a primary response memory cells (both T and B lymphocytes) remain in the blood. This means that after a subsequent infection by the same antigen the clonal selection stage can be bypassed and the specific immune response is much faster and much greater (i.e. more clone B and T lymphocytes and antibodies are produced). This is called the secondary immune response, and is so fast that the pathogen is destroyed before it reproduces enough to cause disease. In other words the individual is immune to that disease. Note that the non-specific immune response is the same in all infections.
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Concentration of antibody in blood
secondary response primary response

antibody concentration required for immunity Time after 40 infection (days)
0
First infection
10
20
30
40
Delay of years
10
20
30
Second infection
This immunity works well for many diseases, such as chicken pox, measles or mumps. We think of these as childhood diseases because it is common to catch them once as children and never catch them again. However it appears that you can keep on catching some diseases, such as the common cold and the flu. Why does the secondary immune response not work with these diseases? Because these microbes have constantly-changing antigens. This is referred to as antigenic variability, and it is caused by mistakes in DNA or RNA replication (mutations) due to poor polymerase enzymes. The result is that each infection causes a new primary response, with all the trappings of the accompanying disease. With some diseases the pathogen is so active and the toxins so effective that the first infection causes a disease that is fatal (e.g. cholera, smallpox, diphtheria, AIDS).
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Immunisation
We have been able to make use of the immune system's memory to artificially make people immune to certain diseases even without ever having caught them. The trick is to inject with an antigen that will promote the primary immune response, but has been modified so that it is novirulent (or non-pathogenic), i.e. will not cause the disease. The immune system is thus fooled into making memory cells so that if the person is ever infected to the real virulent pathogen, the more powerful secondary immune response is triggered and the pathogen is killed before it can cause the disease. This technique is called vaccination and is commonly used to provide artificial immunity to a number of potentially-fatal diseases. In the UK children are commonly vaccinated against diphtheria, tetanus, whooping cough, polio, measles, mumps, rubella and TB. There are several different ways of making vaccines. In each case the aim is to make an antigen that is good enough to bind to antibodies and so stimulate an immune response, but defective in some way so that it will not cause a disease. Five different kinds of vaccine are used: 1. Live non-virulent strains. The microbe is sub-cultured for many generations in the laboratory, each time selecting the least virulent and slowest growing cells to start the next generation. This is an example of selective breeding, and it results in microbial cells that still contain the same antigens as their pathogenic relatives, but are deficient in some aspect of their pathogenicity. They may be slow-growing, unable to make toxins, unable to attach, etc. One vaccine made this way is for rubella. 2. Killed virulent organisms. The pathogens are first grown in culture to create a large number of cells, then killed using chemicals (such as formaldehyde) or radiation so as not to denature the antigen. This is the oldest technique, but there is a danger that any cells surviving the treatment are still pathogenic. Vaccines made this way include whooping cough and influenza. 3. Isolated antigens from a pathogen. The pathogens are first grown in culture to create a large number of cells, then the cells are disrupted and the antigen proteins are chemically isolated from the cell membranes. One vaccine made this way is for influenza. 4. Genetically engineered antigens. If the gene for the antigen has been identified it can be inserted into another microbe (bacterium or yeast) using genetic engineering techniques. The new transgenic microbe is then grown in culture, where it will make and secrete the antigen into its surroundings, from where it can easily be isolated. This is the newest method and also the safest, but it only works for antigens that are pure proteins, not glycoproteins. One vaccine made this way is for hepatitis B.
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5. Modified toxins (Toxoids). Bacterial toxins can be purified and chemically modified so that they are no longer toxic, but still function as antigens in promoting an immune responses. One vaccine made this way is for diphtheria. The choice of vaccine depends on several factors including safety, cost, ease of delivery (injection, oral) and side effects. Passive Immunity Injecting antigens to promote an immune response is called active immunity, but it is also possible to inject antibodies against certain pathogens into the blood. This is called passive immunity and is used when someone has already been infected (or is likely to become infected) with a pathogen. The antibodies in it assist the body's normal immune response and help it deal with serious diseases. Antibodies are either prepared from the blood serum of an infected human (or rarely animal), called an antiserum, or are made by genetic engineering. Passive immunisation is not very common, but can be used for rabies, tetanus, measles and hepatitis B, and is being tried to combat AIDS. Passive immunity also occurs naturally when a mother passes antibodies to her child. Antibodies can pass across the placenta to the foetus and are also found in colostrum, the milk produced in the first few days after birth. Since the baby's digestive system does not function at this stage, the immunoglobulin proteins can be absorbed intact. This passive immunity helps the new-born baby survive in a world full of pathogens, and is one reason why breast feeding is so important. The different kinds of active and passive immunity are summarised in the table. Active Immunity (antigens received) Natural Passive Immunity (antibodies received)
Achieved through the passing of antiAchieved through the primary immune bodies from mother to child through the response following an infection placenta and milk. Achieved through injection of modified Achieved through injection of antibodies antigens (vaccination). (antiserum).
Artificial
HGS A-level Biology
NCM/1/03

AQA (B) A2 Module 7: Microbes and Disease: Microbes Bacteria. Industrial Biotechnology

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AQA (B) A2 Module 7: Microbes and Disease: Microbes Bacteria. Industrial Biotechnology

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Module 7 - Microbes and Disease - page 1

AQA(B) A2 Module 7: Microbes and Disease Contents

Module 7 - Microbes and Disease - page 3

Module 7 - Microbes and Disease - page 4

thick cell wall

Bacteria have a variety of distinctive shapes when seen under a microscope:

staphylococi e.g. staphylococcus aureus causes food poisoning.

diplococci e.g.diplococcus pneumoniae casues pneumonia

streptococci e.g. streptococcus pyogenes causes sore throats

e.g. Salmonella typhi causes typhoid fever

HGS A-level Biology

Module 7 - Microbes and Disease - page 5

HGS A-level Biology

Module 7 - Microbes and Disease - page 6

Eventually the septum meets, dividing the cell in two.

HGS A-level Biology

Module 7 - Microbes and Disease - page 7

HGS A-level Biology

Module 7 - Microbes and Disease - page 8

capsomeres single-stranded RNA helical capsid

HGS A-level Biology

Module 7 - Microbes and Disease - page 9

HGS A-level Biology

Module 7 - Microbes and Disease - page 10

HGS A-level Biology

Module 7 - Microbes and Disease - page 11

Module 7 - Microbes and Disease - page 12

HGS A-level Biology

Module 7 - Microbes and Disease - page 13

Measuring the Growth of Microbes

Module 7 - Microbes and Disease - page 14

culture flask spread 1cm on to agar plate

+ 9 cm sterile medium spread 1cm on to agar plate

+ 9 cm sterile medium spread 1cm on to agar plate

+ 9 cm sterile medium spread 1cm on to agar plate

+ 9 cm sterile medium spread 1cm on to agar plate

+ 9 cm sterile medium spread 1cm on to agar plate

far too many colonies

far too many colonies

far too many colonies

too many colonies

too few colonies

HGS A-level Biology

Module 7 - Microbes and Disease - page 15

Bacterial Growth Curves

Log (no. of cells)

Module 7 - Microbes and Disease - page 16

Factors Affecting Cell Growth Rate

HGS A-level Biology

Module 7 - Microbes and Disease - page 17

HGS A-level Biology

Module 7 - Microbes and Disease - page 18

Primary and Secondary Metabolites

stances are called primary metabolites, and include

Module 7 - Microbes and Disease - page 19

Module 7 - Microbes and Disease - page 20

cooling water jacket

impeller (for stirring) Sparging ring (for aeration)

drain / sample port

Module 7 - Microbes and Disease - page 21

flow just right flow too fast stop flow time

HGS A-level Biology