To find out how four antibiotic agents- Penicillin G, Streptomycin, fresh garlic and odourless garlic- effect gram-positive and gram-negative bacteria. The two different bacteria used will be Bacillus subtilis and Escherichia coli. After transferring the antibiotics to the bacteria I will be able to look for zones of inhibition in the bacteria where the antibiotic agents were placed.
Scientific knowledge & understanding
Bacteria are prokaryotes, which means they are organisms that lack a nuclei and are a lot smaller in volume than Eukaryotes (animals, plants, fungi & protoctists), on average only 0.5-5 m in diameter. Bacteria are a lot simpler in their structure. Structures always found in bacteria include a cell wall, plasma membrane, cytoplasm, ribosomes and circular DNA. Other structures that are present in some bacteria can be shown in the diagram below.
Bacteria can be found almost anywhere as different types have different optimum temperatures e.g. Thermophilic bacteria work best at temperatures above 45 C whereas Psychrophilic bacteria grow best at temperatures below 20 C. Bacteria can also be identified by their shape. Spherical bacteria are called cocci, bacteria that have a rod like shape are known as bacilli, corkscrew shaped bacteria are called spirilla and bacteria with a thread like appearance are termed filamentous. Bacteria are important in most habitats as they help to decay and recycle organic waste. However, some bacteria can be very harmful. For example, Mycobacterium tuberculosis and M.Bovis are responsible for Tuberculosis, an infectious disease. Most bacteria are harmless and are very useful. Bacitracin is an antibiotic produced by Bacillus subtilis that prevents wall synthesis in some bacteria.
The purpose of bacteria is to grow and reproduce. This division usually occurs by binary fission. First the DNA is replicated and then attaches to mesosomes, which help to separate the DNA. The septum (cross-wall) is made to divide the cell and eventually grows right across the cell, forming two daughter cells.
Bacteria can be differentiated into gram positive and gram negative by using the “Gram stain” procedure, originally developed by a Danish physician, Hans Christian Gram. The technique involves using crystal violet and Gram’s solution (1 part iodine, 2 parts potassium iodide and 300 parts water) to stain the bacteria and then decolourise with 95% ethanol until the thinnest parts of the smear are colourless. The smear is then flooded with Safarin (a pink colour) for 10 seconds and then blotted with absorbent paper. Bacteria that retain the violet iodine colouration after washing in ethanol are termed gram positive and those that lose the purple colouration and are stained red from the safarin are termed gram negative.
The reason for the difference in staining is due to the structure of the cell walls. Both types of bacteria contain peptidoglycan in the cell wall, but in gram-negative bacteria, various other layers protect it, including a more complex outer membrane and so the stain cannot reach it to cause the colouration.
Below is a picture of Escherichia coli after it has been gram-stained. It is not stained purple; therefore we know it is a gram-negative bacterium.
The complex outer membrane of the gram-negative bacteria is a lipid bilayer similar to the inner membrane except that it contains lipopolysaccharides. These always face the outside and increase the barrier for molecules entering the cell. This outer membrane also contains porins, which are proteins that form pores in the membrane and allow small hydrophilic molecules to pass into or out of the cell. Hydrophobic and larger molecules cannot pass through the porins and this is how the Gram Stain is prevented from reaching the peptidoglycan layer to colour it. As well as this protective membrane, gram-negative bacteria also have a thinner layer of peptidoglycan and a periplasmic space between the cell wall and the membrane, as shown in the diagram below.
The cell wall of gram-positive bacteria lacks the components found in the outer membrane of the cell wall above. The cell wall of gram-positive bacteria consists of a large layer of peptidoglycan as well as polysaccharides and/or teichoic acids. This peptidoglycan is multilayered as it is varied in its structure and composition. The teichoic acids consist mainly of glycerol, ribitol and mannitol and are covalently linked to the peptidoglycan through phosphodiester bonds. These acids are found in some actinomycetes, bacilli, lactobacilli, listeria and staphylococci. A diagram of a typical gram-positive cell wall can be seen below.
The bacteria being used in this investigation are Bacillus subtilis and Echerichia coli. B.sub and E.coli have many common features but they have important differences in their metabolism and gene regulatory mechanisms.
Bacillus subtilis is a gram-positive bacterium. It is rod-shaped and is an endospore-forming aerobic bacterium. It is found in soil and rotting plant material and is non-pathogenic. B.sub is one of the most studied gram-positive bacteria mainly because of its ability to differentiate and form endospores. Many strains of B.sub are used in the commercial production of extra cellular enzymes. Other strains produce insect toxins, peptide antibiotics and antifungals. Some of these have been used in agricultural crop production.
E.coli is a gram-negative bacterium, as the diagram relevant to gram staining on the previous page proved. E.coli are cylindrically shaped bacteria, about 0.3-1.0 m in diameter and
1.0-6.0 m long. This shape bacterium is usually termed rods or bacilli as opposed to spherical bacteria, which are known as cocci. This is because they do not form spores or retain dyes after being treated with organic solvents. Bacteriologists describe E.coli as gram-negative, non-acid-fast non-sporing rods or bacilli.
E.coli can develop resistance to antibiotics due to plasmids that may be carried. These plasmids are extra chromosomal pieces of DNA, which code for the resistance to a number of antibiotics. Resistance can also be developed within the bacterium’s chromosome.
The antibiotics being used in the investigation are Penicillin G, Streptomycin, fresh garlic and
Odourless garlic. These will be used to observe how effective different antibiotics are at inhibiting the gram-positive and gram-negative bacteria that have been described.
Antibiotics are found throughout nature and interfere with specific activities in certain types of organisms. A narrow spectrum antibiotics will only affect either gram-positive or
gram-negative bacteria whereas if an antibiotics has a widespread effect on both types of bacteria, it is said to be a broad spectrum antibiotic. Different antibiotics work in different ways. Different functions include:
> Cell wall synthesis inhibitors
These target the formation of peptidoglycan cell walls by interfering with the cell wall component D-alanyl-D-alanine, which is a dipeptide that is required for normal synthesis of peptidoglycan cell walls.
> Cell membrane inhibitors
These are less common and attack the integrity of the bacterial membranes e.g. polymoxin.
> Protein synthesis inhibitors
These target activities occurring at the ribosome. There are no known antibiotics that interfere with amino acid activation or attachment to a tRNA.
> Nucleic Acid Effectors
These attack the DNA or RNA of a cell, preventing DNA synthesis and blocking the natural growth of the cell.
> Competitive inhibitors
These do not alter the enzyme itself, but occupy the enzymes so that there is less chance of it binding with normal substrates e.g. Sulfonamides
Examples of antibiotics with different functions can be seen in the diagram below:
Penicillin was discovered in 1929 by Sir Alexander Fleming who observed the inhibition of staphylococci on an agar plate contaminated with a Penicillin mould as shown below.
It was then discovered that Penicillin was effective against all sorts of infections caused by gram-positive bacteria although it was to be found deadly when given to those who were allergic to the medicine.
Penicillin is produced by the organism, Penicillium chrysogenum and it works by preventing the cross-linking of small peptide chains in peptidoglycan. Cells already existing are unaffected by the drug but all newly produced cells grow to be abnormal and so are unable to maintain their wall rigidity.
The two natural penicillin’s, Penicillin G and Penicillin V, were extremely useful in treating the wounded during the Second World War.
It was also discovered that removing the acyl group and then adding new acyl groups to give new properties to the antibiotic could modify penicillins. These new semi-synthetic penicillin’s had new specific properties such as resistance to stomach acids and an extent of activity against some gram negative bacteria.
Penicillin is still used clinically although not as often as it once was due to the development of resistance by microorganisms and people’s allergic reactions. However it is still prescribed to treat syphilis, gonorrhoea, meningitis and anthrax. The success of Penicillin led to a search for other antibiotic-producing microorganisms. It was this search that led to the discovery of Streptomycin.
Streptomycin was found in 1943 from the microorganism Streptomyces griseus, in the soil environment. It was discovered by Selman Waksman who later received the Nobel Prize in 1952. It acts by limiting bacterial protein synthesis. All normal protein synthesis inhibitors affect events that occur at the ribosome and never with amino acid activation or attachment to a particular tRNA. Streptomycin acts by binding to a specific S12 protein in the 30S ribosomal subunit and causes the ribosome to misread the mRNA so that the wrong amino acids are incorporated into the polypeptide. This results in a slow bacterial growth rate and at high concentrations can lead to cell death. The stage in translation that Streptomycin inhibits is shown below:
Streptomycin is an odourless off-white powder and is bitter to taste. It is effective against gram-negative bacilli as well as many cocci. The antibiotic is also used against tubercle bacilli and is included in a combination of other drugs to treat tuberculosis.
Streptomycin is also used in humans to treat urinary tract infections, usually through intramuscular injections for seven to ten days, as it cannot be taken orally. Studies on rats indicate that the drug does not have the potential to cause cancer but side affects can include nausea, vomiting and even injury to the kidneys and nerve damage that can result in dizziness and deafness.
The human antibiotic drug is also used as a pesticide, since it was first registered as a pesticide in 1955, to control bacteria, fungi and algae. The use of Streptomycin to control fireblight on apples and pears accounts for 58% of its total use. Other significant uses are in landscape maintenance and on tobacco. The antibiotic cannot be used for aquatic uses, as it is slightly toxic to cold water and warm water species of fish.
The other two antibiotics being used in this investigation are fresh garlic and odourless garlic.
Garlic is a herb and is most commonly known for its distinctive smell and taste and its use in various dishes. Most people are unaware of the medicinal properties that the herb possesses. These properties were first recognised thousands of years ago by Egyptians, Asians, Greeks and Indians who used it in natural remedies. Louis Pasteur first proved the antibiotic properties of garlic in 1858 when he showed how it could kill bacteria in culture dishes.
The active ingredient, which is responsible for garlic’s anti-viral and anti-fungal properties, is allicin. This is also the ingredient that is responsible for the smell. A peeled garlic clove has little smell but as soon as it’s crushed, the aroma is overwhelming. This is because alliin, a precursor molecule, found within the mesophyll cells and the enzyme alliinase, which is found in the cells around the vascular bundle are physically separated within the garlic clove. When the clove is crushed, the two come into contact and immediately produce allicin and other thiosulphates.
The allicin in garlic kills bacteria by what is known as the macroeffect. This involves interfering with the cell membrane biosynthesis; preventing the production of DNA polymerises and inhibiting RNA synthesis. By doing this, the process responsible for cell replication is disrupted.
Allicin also destroys the SH groups in proteins. These groups are found in thiol enzymes in bacteria, virus and protozoa. Antibiotics tend to target a single metabolic pathway so that the bacteria are unable to use the pathway anymore. In time, however, bacteria can find an alternative route and become resistant to the antibiotic. Resistance to the allicin in garlic cannot be accomplished, simply because it affects groups found in too many enzymes for the bacteria to find different ways around it.
Since no resistance can be built up, garlic has many uses. These include the healing of many types of common illnesses such as ear, throat and mouth infections, influenza, colds, asthma and catarrh to name a few. Lower levels of heart disease found in Mediterranean and Asian countries, where larger quantities of garlic are consumed, have led scientists to believe that the antibiotic also helps to reduce cholesterol levels and reduce the tendency of blood to clot. The body’s immune system can also be improved by taking regular garlic supplements.
Despite its many benefits, people tend to avoid taking regular supplements of fresh garlic because of its lingering odour. This is why odourless garlic capsules appear to be more favourable. However, the manufacturing of odourless garlic capsules involves removing the pungent odour which means the removal of allicin as it is this that causes the smell. Therefore the active ingredient in the garlic is not present so it is no longer effective against bacteria.
Modern technology has developed a process by which the allicin content is still present and the aroma is removed. This is achieved by blending the garlic with citrus juices and oils to deodorise the valuable ingredient. Another method is to postpone the conversion of Alliin to Allicin until after the product is digested. These new processes mean that people can benefit from the natural antibiotic without being deterred by the offensive odour.