Our hypothesis that we have formulated for our experiment based off of our background research and prior knowledge is:

If natural antibiotics such as garlic and honey are compared to synthetic antibiotics like ampicillin, kanamycin, and amoxicillin against Escherechia Coli (E.coli), then the natural antibiotics will show strong antibacterial properties, but the effectiveness of the commercial antibiotics will overall overpower the strength of the natural antibiotics because the commercial antibiotics have been specifically designed and perfected to be effective among a wide range of bacterial infections.

During the different stages of our research, procedure, and experiment, we will work to prove whether this hypothesis is in fact correct or incorrect.

 

 

What are antibiotics, and what role do they play in modern bacterial medicine?

Antibiotics are strong antimicrobial substances that have been utilized by us for decades now to fight infections in the human body caused by bacteria. Antibiotics do this by either killing or inhibiting the growth of harmful bacteria. Unlike other medicines, such as Tylenol or Advil, which are not classified as antibiotics, antibiotics only treat bacterial infections, but not viral infections. Viral infections such as the flu, common cold, and hepatitis b will not benefit from antibiotics. In fact, using common antibiotics on non bacterial infections and diseases may actually harm your body and contribute to the worldwide issue of antibiotic resistance. Antibiotics treat infections such as bloodstream infections, ear infections, urinary tract infections, bacterial pneumonia, and many more. They are one of the most useful and important modern bacterial medicines.

What are natural antibiotics defined as?

Natural antibiotics are types of chemical compounds found in nature that have the ability to inhibit the growth of some harmful bacteria. These can be found in different herbs, plants, fungi, and spices. A few examples of the most powerful natural medicines used are honey, garlic, thyme, and ginger, a few of which have been used in traditional medicine for centuries now for treating different species of bacterial infections. These types of antibiotics can be easily accessible to generally anyone, and are effective against bacterial infections both on the exterior and interior of the human body, depending on the species and strain of antibiotic. 

What are the antibacterial properties of honey?

Honey is a very powerful natural antibiotic that is most commonly used for healing sore throats and coughs, wounds, and burns. Overall, it is most commonly used for its soothing properties; whether that be the interior or exterior of the human body. Honey is given most of its antibacterial properties from its high sugar content and low water content. The average content of honey is 17% water, 82.5% sugars. Bacteria need water to survive, and because of honey's low water concentration, the bacteria has a difficult time surviving in the environment that honey creates. Because of its high sugar concentration, this causes osmosis, which is the action of water flowing from a place of high  concentration to low concentration. Undiluted honey has been evident to kill bacteria because of the osmotic pressure exerted. Through osmosis, even more extra water gets transported out of the bacterial cell. In the hypertonic solution that the honey has placed the cells in, which is a solution that causes the cell to shrink and deflate, the cell becomes unable to grow because of immense dehydration. Honey is given most of its external antibacterial property from a chemical compound called hydrogen peroxide. When applied to the affected area, oxygen compounds in the hydrogen peroxide get released, which causes the foaming white bubbles to appear on the surface of the applied area. The main purpose of this process is to deep clean the affected area, preventing further infection from the cut or wound. Specific types of honey contain a high concentration of a chemical known as methylglyoxal, shortened as MGO, which comes from dihydroxyacetone (DHA), which is highly concentrated in the manuka flowers. MGO fights bacteria by directly damaging and inhibiting the formation of flagella and fimbriae, which are biological nanomachines used for locomotion, the movement and ability to move from one place to another. Flagella and fimbriae work as a propeller, driving the bacteria cell through a liquid environment. By causing alterations in the flagella and fimbriae, the bacterias motility, which means the ability of the bacteria to move independently using metabolic energy, becomes limited. Another main antibacterial compound of honey is its high concentration in bee defensin. Coming from its name, bee defensin, also known as defensin-1, comes from honeybees, which is produced in honeybees to aid them in their immune system, protecting them from things that could cause them harm such as bacteria and other pathogens. It plays a key role in the honey bee hive, fighting off microbial organisms.When honey is produced by bees, the bee defensin gets transferred to the honey, therefore filling the honey with natural antibacterial compounds. Bee defensin has potent antibacterial qualities when working with the other components of honey such as hydrogen peroxide. The bacterial cell membrane is the semipermeable outer membrane on the bacterial cell that surrounds the cytoplasm. The action of being semipermeable means to let certain things like sugars and amino acids in, while keeping chemicals and toxins that are harmful to the bacteria out. This membrane is not always 100 percent accurate, letting bee defensin-1 work actively to insert itself into the cell membrane, creating pores in the cell membrane. This can lead to cell dysfunction and/or death. This occurs  when the vital ions and molecules get leaked out of the cell because of the pores created by the bee defense. Overall, the antimicrobial properties of honey come from the compound hydrogen peroxide,  methylglyoxal, and bee defensin. According to an experiment and report done by Cheung Yi-Chen on Antimicrobial Effect of Honey Phenolic Compounds against E. coli, several types of honey significantly decreased the growth of E.coli on agar plates after incubation.

What are the antibacterial properties of garlic, one of our natural antiboitcs?

Garlic is a species of bulbous flowering plant, usually known as a vegetable or herb, and a popular ingredient in cooking that can also offer a variety of health benefits. It is said that variants of Romanian red and Georgian fire garlic deliver the most health benefits.

Garlic is widely recognised for its phenomenal anti-fungal, antibacterial, and antiviral properties, acting on both gram-positive and gram-negative bacteria. Its active compound is allicin. Allicin is extracted when fresh garlic is crushed or chopped, releasing an enzyme called allinase, leading it to convert alliin into allicin. While allicin is a compound with various properties, alliin is the amino acid leading to the development of allicin. The chemical compound, allicin, was proven to be effective towards a large range of both Gram-negative and Gram-positive bacteria. The main antibacterial properties in allicin comes from its chemical reactions with thiol groups found in various enzymes such as alcohol dehydrogenase, RNA polymerase, and thioredoxin reductase. Thiol groups are similar to alcohol groups, containing chemicals like a sulfur atom and a hydrogen atom attached to a carbon atom. Thiol groups are crucial for the survival of bacteria  because they are a main target for antimicrobial action, essentially being the tool that the enzyme needs to function.  Because of the high reactive rate of allicin, when it encounters enzymes with thiol groups, the allicin will chemically modify them, making the enzymes crucial for the bacteria to survive inactive, damaged or killed.  Alcohol dehydrogenase is an enzyme responsible for helping the bacterium survive in anaerobic (low-oxygen) conditions by assisting with ethanol formation. Because this process is not very energy efficient, it is only used when oxygen is scarce, and when it isn’t usually a process called aerobic respiration. RNA polymerase is the most essential component of bacterial transcription. This enzyme is crucial for the multiplying of cells, without it, bacteria would not be able to multiply. All bacteria use the same RNA polymerase to transcribe their genes and DNA. Thioredoxin reductase is an enzyme found in bacteria that reduce Trx, or thioredoxin, which is responsible for control of cell death and DNA synthesis. These two enzymes; thioredoxin reductase and thioredoxin fight against each other for their opposite functions.

 

How do the properties of synthetic antibiotics fight and influence their effectiveness against different bacteria?

Synthetic antibiotics contain many bacterial compounds that have been researched and created by scientists in labs. These antibiotics often contain elements similar to natural antibiotics because certain plants and molds contain the chemicals used to form these drugs, meaning that many types of synthetic antibiotics ingredients are derived from nature. What influences their effectiveness are different components such as the mechanism of action of the drug. As was mentioned before, most synthetic antibiotics are designed to target the bacterial cell wall, but others interfere with the cell ribosome's digestive process which inevitably prevents the cell from getting energy and ultimately killing them. As mentioned in our hypothesis, commercial or synthetic antibiotics are expected to eliminate the infection and bacteria more efficiently because they have been more articulately modelled and created to be very effective.

What are the antibacterial properties of amoxicillin.

Amoxicillin is a semi synthetic antibiotic a part of the penicillin family. It acts on both gram-negative and gram-positive bacteria and is popularly known for being a strong antibiotic that is digested more easily and leads to less diarrhea than other popular antibiotics. This antibiotic destroys bacteria by preventing the cell from building its protective outer layer. It can prevent various different bacteria from spreading such as colds or ear infections which are gram positive, and others that can cause bladder or skin infections which are gram negative. Gram positive bacteria have thicker cell walls while gram negative bacteria have thinner cell walls. Antibiotics like amoxicillin tend to have an easier time killing gram positive bacteria. Although Gram negative bacterias have thinner cell walls, they have an extra layer of protection which makes it more resistant to some medications. Amoxicillin works to fight bacteria by preventing the growth and synthesis of bacterial cell wall mucopeptide, which are structures composed of amino acids and sugar that make up the bacterial cell wall. The general structure of this process is similar to that of honey or garlic, with most antibiotics functioning by breaking down or inhibiting the repair of the bacterial cell wall. The amoxicillin exerts antibacterial action by binding one of the penicillin binding proteins. Penicillin binding proteins are enzymes found on the surface of bacterial membranes used to modify and polymerize, simply known as assembling the peptidoglycan of the cell wall. They do this by forming the peptide bonds, which are covalent bonds that form proteins using amino acids. The peptide bonds then work to cross link the peptidoglycan chains and strands.  Amoxicillin resembles the components of PBP, essentially tricking the PBP to bind to the antibiotic instead of its usual precursors, which are  essential building blocks or starting materials used for complex transformations through chemicals, as explained before. If the PBP is unable to bond to its usual precursors, and instead to the antibiotic, the protein is unable to form bonds to create and maintain the structure of peptidoglycan. The antibacterial properties of amoxicillin instantly disables the PBP. The PBP gets its name (penicillin binding proteins), because of its ability to bind to β-lactam antibiotics in the class of penicillin, such as amoxicillin and ampicillin. A β-lactam antibiotic is an antibiotic that contains a β-lactam ring in their chemical structure. The β-lactam ring works to inactivate beta-lactamases. This process can kind of contradict each other because the role of the beta-lactamases is to provide resistance to the beta-lactam antibiotic, so both enzymes are fighting against each other in a β-lactam antibiotic. Amoxicillin is classified as a β-lactam antibiotic. When bound to the antibiotic and disabled, the PBP can perform lysis, self-destruct of the cell or result in complete cell death.The key feature of amoxicillin is its beta-lactam ring. This is a 4-membered lactam ring that contains carbon, nitrogen, and oxygen. It mimics the PDP by mimicking the D-Ala-D-Ala portion of the building blocks of the cell wall peptidoglycan. The D-Ala-D-Ala is an essential target for antibacterial drugs. 

What are the antibacterial properties of Kanamycin: 

Kanamycin is used to treat severe bacterial infections, typically those caused by gram negative bacteria. It belongs in the aminoglycoside class of drugs which work by inhibiting the growth of bacteria. It attaches itself onto the 30s subunit of the bacterial ribosome which disrupts the process of translating energy into proteins, which would eventually kill the cell for it has no food intake. When the protein synthesis cannot proceed, the E.coli cannot replicate nor repair itself, and reduces in quantity until it is eventually all gone. Sometimes Kanamycin is used to combat certain strains of E.coli that are resistant to most antibiotics. It has a Aminoglycoside sugar backbone that is predominantly made up of monosaccharides (sugar molecules) that are chained together, hence the name. The backbone helps to control how the antibiotic can interact with the other bacteria’s ribosomes. The aminocyclitol ring is a ring-like structure that helps to form the core of the molecule. This component of the cell is crucial, for it is the part that actually latches onto the ribosomes and stops the protein synthesis of other bacterins. Kanamycin contains two functional groups, hydroxyl groups (–OH) and amino groups (–NH₂). Hydroxyl groups are made up of a hydrogen atom bonded to an oxygen atom. This group helps to ensure that the drug gets absorbed into the body, and it is able to do this by increasing solubility, so that the water is able to dissolve into solvents in the surrounding environment more swiftly. It also can partake in the hydrogen bonding when it comes to attaching to ribosomes. As for the amino groups, they are made up of a nitrogen atom attached to two hydrogen atoms. Amino groups are what inhibit the cell from producing food after attaching to the ribosome, it forms bonds with the chemicals in the bacterial ribosome which is how it sticks on. 

What are the antibacterial properties of Ampicillin:

Ampicillin is a beta-lactam antibiotic in the aminopenicillin class of penicillin antibiotics, similar to amoxicillin. It was scientifically developed to be more potent than regular penicillin and overcome drug resistance. Its mechanism of action is generally the same as amoxicillin and all antibiotics in the class of penicillin, the mechanism being: 

• Binding to the primary receptors in the cell, known as a PBP, which forms the cell wall and are involved in an important factor in peptidoglycan synthesis, maintaining the rigidity of the cell wall
• When bound to the PBP proteins, (penicillin binding proteins), the antibacterial activity instantly inactivates the PBP.
• PBP disruption causes cell lysis (bursting or self-suicide of the cell), leading to cell death.

B-lactam vs B-lactamase

B-lactam is a type of antibiotic, unique for its b-lactam ring in their chemical structure, while B-lactamase is the enzyme produced by bacteria to counteract the beta-lactam antibiotics.

What is bacteria?

Bacteria, or bacterium, are microscopic living organisms made of only one cell, unlike most other living organisms. Found in many places such as water, plants, soil, animals, humans, and numerous other places, bacteria aren't always harmful. Beneficial bacteria can be found in your skin, digestive system and your gut. These types of bacterias, called flora, make up your microbiome, which contrasts the doing of harmful bacteria and keeps you healthy to prevent the growth of harmful bacteria.

However, bacteria like Escherichia coli, which is the bacteria we will be testing in our experiment, are harmful to the human body and cause infections leading to diarrhea, stomach cramps, nausea, red, swollen skin, sores, and blisters, which are only some of the many different symptoms of bacterial infections. For our topic, we will be mostly focusing on the harmful bacteria.

How do antibiotics combat bacterial infections, and what are their key components and active compounds? How do bacteria combat antibiotics in return?

Antibiotics combat bacterial infections by killing bacteria in order to stop them from multiplying and harming the body further. To accomplish this, antibacterial properties of the antibiotic must destroy the crucial parts of the bacteria that help it survive.

The average bacterial cell contains organelles such as the cytoplasm, ribosome, plasmid, cell wall and cell membrane. These organelles all have specific roles in keeping the bacteria alive and functioning. A bacterial strain, which is defined as a specific type of bacteria characterized by its unique features such as size, detectability, and morphability, is typically categorized as either Gram-positive or Gram-negative. One of the key differences between the two variants is the thickness of the peptidoglycan layer. While gram-positive bacteria possess a thick, multilayered , gram-negative bacterial strains have a rather thin, monolayered peptidoglycan, meaning that they have only one layer. Peptidoglycan, also known as murein, is a polymer or a large macromolecule composed of sugar and amino acids that forms a mesh like structure, scientifically known as sacculus surrounding the bacterial plasma membrane of the bacteria. The sugar component of the peptidoglycan consist of alternating residues of β-(1, 4), a glycosidic bond covalent bonding between the oxygen to the C1 of one glucose ring and the C4 of the connecting ring

Parts of the bacterial cell such as the cell wall or their DNA walls are mostly made up of conjoined peptidoglycan/murein and other components, which we defined earlier. The cell wall provides the bacterial cell with rigidity which acts as the main connection between the inside components of the bacterial cell and the external environment. Antibiotics such as penicillin or glycopeptides hinder peptidoglycan biosynthesis of the cell wall, which then proceed to make the cell vulnerable to osmotic pressure and bacterial autolysis. Osmosis is when water moves from a place that has less dissolved particles to a place that has more dissolved solute to create homeostasis. A bacterial strain, which is defined as a specific type of bacteria characterized by its unique features such as size, detectability, and morphability, is typically categorized as either Gram-positive or Gram-negative. One of the key differences between the two variants is the thickness of the peptidoglycan layer. Osmotic pressure, as explained before, is the excess of pressure applied or exerted on the cell membrane or cell wall due to the process of osmosis, caused by a difference in concentration inside and outside of the cell. When this force is applied to a solution, it prevents the solvent from moving across the semipermeable membrane. This means that the water will stop moving and therefore cannot balance out the solute. The cell wall plays a big role when it comes to protecting the cell from internal pressure that comes from osmosis pressure, but if an antibiotic were to make it so that the surrounding area had a lower concentration of solutes, then all the water would rush into the cell to maintain balance. Though if there is too much water that inevitably causes the pressure in the cell to build up until the cell can no longer handle it, causing it to burst. This is called lysis. Just like cell lysis, plasmolysis is the same but reversed. If the environment's solute concentration is too low, the water from inside the cells will exit into the space around, making the cell shrink. It also causes the cell membrane to slowly detach from the cell wall, leaving the cell with no protection. Osmotic pressure can also disrupt cellular functions for it can disturb the balance between ions and molecules in the bacteria. Once this imbalance happens, the cell can no longer do vital processes like protein synthesis or energy production. Antibiotics such as those in the penicillin group work by weakening the cell wall which makes it more susceptible to osmotic pressure. 

Bacterial autolysis is an important process in the killing of bacterial cells. It is the process in which a cell breaks its own components down in reaction to the activation of the enzymes in itself. Typically this happens once a cell dies, and the enzymes in digestive organelles like lysosomes begin to digest the cell it once belonged to. This can also occur if a food breaks down naturally it is often because of those same enzymes. When bacteria is exposed to antibiotics or other harmful conditions. When breaking down bacteria with autolysis, the cell’s wall gets broken down by the enzyme known as autolysins. This particular enzyme hydrolyzes peptidoglycan. This means that it degrades the cell wall with a chemical reaction that incorporates hydrogen. It is believed that bacterial autolysis has connections to something called PCD which is when a cell is genetically regulated to undergo death. It controls when and why it meets its demise. The similarities between these two concepts has led scientists to believe that autolysis is a form of PCD. Now, although PCD is most commonly associated with controlled death of a cell, it can also occur under stressful situations, for example, being exposed to antibiotics. When this happens, the homeostasis between cell wall synthesis and degradation is disrupted. All of this means that interacting with antibiotics could cause bacteria to experience the process of autolysis.

Peptidoglycan biosynthesis is the process of strengthening and building the bacterial cell's cell wall, which, as mentioned earlier, is the main wall protecting the bacterial cell from the antibiotic components.

This process of peptidoglycan biosynthesis can be separated into four sets of reactions of the key precursors: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylmuramic acid (UDP-MurNAc), and UDP-N-acetylmuramyl-pentapeptide. all of which are produced through several biosynthetic steps. 

1. The first main step of the process takes place in the cytoplasm, the gel-like structure in the bacterial cell that holds the functions for cell growth and replication. It is forming the UDP-N-acetylglucosamine, more simply known as  UDP-GlcNAc. UDP-GlcNAc is an amino sugar nucleotide donor that makes up a large part of peptidoglycan of the bacterial cell wall. This formation of  UDP-GlcNAc is derived from fructose 6-phosphate, a sugar intermediate in carbohydrate metabolism, which is the first starting molecule of peptidoglycan biosynthesis. 
2. The next step is the formation of UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine​​. UDP-N-acetylmuramic acid plays a pivotal role in the carrying of peptidoglycan building blocks. This transformation occurs by adding a lactyl group in order to create the muramic cell structure. 
3. Next, a short chain of pentapeptide is added to UDP-N-acetylmuramic acid (UDP-MurNAc), which are amino acids that help to make the UDP-MurNAc stronger and better prepared for the next step of the process. It provides the foundation for cross-linking the peptidoglycan in the final steps. 
4. The new molecule created by the series of steps beforehand is attached to a lipid, which are fatty compounds that carry things called bactoprenol. The bactoprenol facilitates its traveling to across the bacterial cell membrane 
5. The final step of the process consists of the final molecule being flipped across the membrane by a special enzyme. Once outside the membrane and in the cell wall, enzymes link the molecule together with other pieces, using a cross linking process known as transpeptidation which cross links the peptide chains and strengthens the peptidoglycan wall.

Antibiotics work to hinder and stop this process of strengthening this cell wall, making the bacterial cell weaker and not able to protect itself from antibacterial activity such as osmotic pressure and lysis. 

What are the similarities and differences between natural and synthetic antibiotics?

The biggest difference between natural and synthetic antibiotics is that natural antibiotics are not manufactured in a lab, while the antibiotics that were are typically prescribed by a doctor. Overtime we have accumulated what classifies something as an antibiotic. The criteria being:  source, chemical structure, mechanism of action, and type of action and spectrum of activity. Depending on the source, these antibiotics can be categorized into Natural compounds that come from microorganisms, semi- synthetic members which can be defined as natural products that scientists and chemists have modified, and lastly, synthetic antibiotics which are completely man-made in labs. Both synthetic and natural antibiotics do well at eliminating bacteria. A study done by Lego, Rimmo Loyi proved that the synthetic antibiotics completely created in a lab were most effective at fighting a wider range of bacterial strains, but just by a hair. The natural antibiotics were proven to be almost just as strong and effective. It is expected that the synthetic antibiotics did better because we have spent a lot of time and money on and developing them and carefully crafting them for their special traits. Although lab-crafted antibiotics have been proven to be stronger and more consistent, side effects are more likely to occur through them like allergic reactions such as coughing, difficulty breathing, rashes, and sometimes severe, life threatening reactions known as anaphylaxis; and a common issue in the world: antibiotic resistance. 

Antibiotic resistance

Antibiotic resistance is the term used to describe the action of germs and bacteria developing the ability to defeat and become resistant or immune to the antibiotic designed to kill them. Although there is a liability for this to develop against natural antibiotics, chemicals contained in synthetic derived from a lab that are specifically modified to fight the bacteria, make it much easier and simpler for bacteria to develop resistance, making synthetic antibiotics more effective as well as containing more risks. Some examples of synthetic 

 

Escherichia Coli (E.coli):

What is E.coli?

E.coli is a gram-negative, rod-shaped bacteria usually found in the intestines of warm-blooded organisms, like animals and humans. Usually, this bacteria lives harm free in your intestines, but few strains can be dangerous and cause disease and infections. Some symptoms caused by harmful strains of E.coli are: diarrhea, which can be bloody in severe cases, vomiting, fever, and stomach cramps. E.coli can be exposed to you from: dirty or contaminated water, contaminated food, undercooked meat and raw vegetables. More severe or fatal cases are most commonly found in youth and the elderly, while most middle aged adults recover pretty quickly. An estimated amount of 48 million people are affected by this bacteria each year.

How is e.coli contracted?

As stated earlier E.coli comes from contaminated food or water, especially and most commonly in ground beef, fresh produce, and unpasteurized dairy products. E.coli is commonly present in the intestines of cows, which when slaughtered for consumption, can be transferred to the beef. Although all beef can be contaminated by this, ground beef is more likely to be contaminated because it is essentially a combination of meat from many different sources and animals, increasing the risk. When fully cooked well-done, the E.coli and harmful bacteria present is killed, but if the ground beed if undercooked some e.coli bacteria may still be in the beef.

How does E.coli work and affect you?

Most e.coli causes mild symptoms and infections, but some strains of E.coli can lead to stronger effects and symptoms, such as damaged kidneys. Some strains of dangerous E.coli bacteria make a toxic or poisonous material that harms and damages the lining of the small intestine. The damage of the small intestine is the cause of diarrhea following E.coli. These toxins are known as shiga toxins. (Stx). This specific toxin comes from strains of E.coli known as “Shiga toxin-producing E. coli (STEC).” Shiba toxins are ribotoxic, which results in the inhibition of some protein synthesis.

Backup antiboitc (Neosporin)

Neosporin is made up of active ingredients such as neomycin, polymyxin B, and bacitracin. 

Neomycin: An aminoglycoside antibiotic which is an antibiotic that belongs to a class of drugs known for its ability to eliminate bacteria by interfering with protein synthesis. This antibiotic begins by binding itself to the 30s subunit which is one of the two components of a bacterial ribosome. The Ribosomes main purpose in a cell, is to translate genetic information into certain types of amino acids which then form into proteins. So, by latching onto a part of the ribosome, it disrupts the protein synthesis. This inconvenience results in incomplete or defective proteins, cells need proteins and energy to live, so this of course would lead to the cell’s demise. Neomycin actually kills bacteria rather than just inhibiting its growth; plus it works on a wide range of bacteria, including gram positive and gram negative species. 

Polymyxin B: This antibiotic mostly focuses on the outer membrane of gram negative bacteria. The outer membrane is made up of a lipid bilayer, which is a helpful barrier which stops harmful substances from entering the cell. Polymyxin B binds itself onto lipopolysaccharides which are essentially just large molecules made up of Lipid A attached to a chain of carbohydrates. When it latches on, it destabilizes the structural integrity of the membrane. The lack of stability within the membrane creates holes, and all the vital parts of the bacteria leak out, such as the proteins and ions. But, it also gives an opportunity for harmful toxins to enter the cell. Ultimately killing the bacteria. Much like the neomycin, polymyxin kills the bacteria itself rather than just stopping its growth.

When these components collaborate, they can effectively kill a variation of bacteria, especially ones that cause skin infections. 

Bacitracin: What bacitracin does to stop bacteria from growing is it prevents an enzyme called bactoprenol from transporting peptidoglycan building blocks to the actual cell wall. The cell wall is mainly made up of peptidoglycan, so of course it cannot work effectively when protecting the cell from outside particles and such. After a while without any more “building blocks”, the cell becomes too weak to withstand the pressure that comes from all the components of the cell which leads to cell lysis and the cell reaches the end of its life. In simple terms, cell lysis is when the cell bursts due to the pressure. (NOT EFFECTIVE AGAINST E.COLI).

Neosporin vs Polysporin, what is the difference? 

These two antibiotic ointments are very similar, and can often be mistaken for each other. The main difference between these two antibiotics is their ingredients and the way you use them. Polysporin is a double antibiotic ointment, while Neosporin is a triple antibiotic, making neosporin more effective on a wider range of infections and wounds. Double antibiotics amalgamate bacitracin zinc and polymyxin B sulfate antibiotics, or various other combinations. Polysporin is recommended for external use only. This ointment can help to speed up the healing process of minor cuts, scrapes or burns. Triple antibiotics contain neomycin, bacitracin, and polymyxin. They are typically used to prevent or treat exclusively bacterial skin infections, and would not work for fungal infections or viruses. Both are for external use only.

 

There are three types of variables included in our project, the controlled, dependent/manipulared variables, and independant/responding variables.

 

Controlled variables are referring to the variables that will not be changed or adjusted throughout the whole experiment. These aspects of the experiment make sure that each manipulated variable has the equal chance: size of agar plates, type of agar, amount of agar added to each plate,  incubator type and temperature, 

 

Dependent/manipulated variables are variables that are changed throughout the project. This is the main component of the experiment that will provide us our results: Type of antibiotic, natural or commercial; different types of each category.

 

Independent/responding variables are the results, or the aspect that responds to the manipulated variable. The independant/manipulated variable will help and provide us with countable/recordable results, giving us our final data: Number of bacterial colonies counted and their average.

 

Materials: 

• Bacterial culture: E.coli 
• Commercial antibiotics (amoxicillin, kanamycin and, ampicillin)

• Natural antibiotics (garlic, honey)
• Incubator
• Agar plates
• Heat block
• Pipettes 
• Spreaders
• 100% ethanol 
• Shaker 
• Syngene G Box gel documentation system 
• Ice

The process of our experiment consisted of a 3-day process at the science lab.

• Before the experiment at the lab, garlic and amoxicillin solutions had to be prepared beforehand at home in a sterile environment. Ampicillin and kanamycin was provided by the lab
•  1mLof juice was extracted from garlic, as well as 1 mL for honey as well.

Day 1:

1. All antibiotics brought in the lab
2. Make sure to wear gloves when touching materials (especially agar plates). Also tie back long hair for the sterilization of the spreaders, as well as to avoid contamination
3. Honey pasteurized for 5 minutes at -82 degrees celcius to expel any contamination, using a heat block 
4. With the slurry of the 250 mg capsule of amoxicillin combined with 1ml of water, it created a 250mg/ml solution. This slurry was highly over-saturated compared to the actual concentration we needed to spread on plates. To resolve this, we diluted this solution by 1000 times, at a ratio of 1:1000. We did this by taking one μl of the solution and diluting it in one mL of water. This method would be effective because one mL is equivalent to 1000 μl (microlitre). According to our calculations, this would make it a 250 μl/ml solution. 
5. Antibiotics placed into ice to slow the bacteria from growing (Though you do this continuously once you are finished plating the antibiotic) 
6. Agar plates are labeled with a type of antibiotic (two for amoxicillin, two for ampicillin, two for kanamycin, two for honey, two for garlic and two controls. Twelve total)
7. Use pipette with sterile tips to drop 50 μl of antibiotic onto each agar plate. Making sure to switch out the tip after every individual antibiotic is placed 
8. Drop 50 microlitres of garlic juice onto 2 plates
9. (repeat for all plates. Keep in mind you may need to continuously heat up the honey even after a single drop.)
10. Use spreaders to spread antibiotics evenly on all plates (Spreaders were soaked in 100% ethanol and set on fire to sterilize between each spreading of the different antibiotics 

    Things to consider:

    • The honey was viscous, and garlic had small chunks in it because of difficulty extracting juice out of a small amount of garlic. The honey was heated up to reduce the viscosity, and the tips were cut so garlic juice with small chunks would be easier to get out.
    • Why didn't we dilute the honey? (osmosis full potential. Luna will explain)
    • Had to sterilize a petri dish
    • Garlic was not evenly spread 

    
    Day two:

    1. Before plating we had to decide how much to dilute our e.coli solution so that the colonies grown were countable. An experiment was done before-hand seeing the result of diluting the E.coli luria broth by 10 times, 100 times, and 1000 times. (show picture) The experiment showed that the dilution of 10 times had uncountable colonies, with the result of the colony growth being in streaks with colonies bunched up instead having separate colonies. The dilution of 100 times showed results filled with colonies. Although theoretically countable, counting 12 plates with that amount of colonies would not have been efficient. The power of 1000 only had a few bacterial colonies, which we came to the conclusion that 1000 would be too inaccurate. Decided to go in between to the power of 3 and 4. This means diluted by 5000 times and a 0.0005 ratio. 
    2. Put the E.coli luria broth solution into the shaker 10 minutes prior to put air into the solution
    3. Robyn plated the E.coli using the same process as last time. We could not participate in this step due to the fact that we are underage. 
    4. After plating the E.coli we put the petri dishes into the incubator at about 37 c to replicate the environment as if it were growing in vivo. This is because 37c is the normal body temperature, where bacterial growth is most optimal, as if it was in a human body. 

    
    Day 3:

    1. Take all plates out of incubator
    2. Took pictures using the Syngene Gbox gel documentation system 
    3. Counted all the colonies using the photos 

 

Kanamycin 

• The plate was almost completely clear of any bacterial growth
• 5 bacterial colonies grew on the first plate and 4 on the other
• Created an average of 4.5 colonies
• Colonies generally grew around the sides of the plates 

Amoxicillin

• Contradicting our predictions and hypothesis, a large amount of colonies on the both amoxicillin plates, 409 grew on the first plate and  240 grew on the second
• One plate had significantly more bacterial growth than the other
• The average for both plates was 324.5 colonies of bacteria 

Garlic

• 320 bacterial colonies grew on the first plate and 395 colonies on the second.
• The average for both plates is 357.5.
• Colonies looked significantly smaller than other types of antibiotics, although size was uneven for all the colonies.
• When seen in a clearer picture, we saw that most of the larger colonies came with a small colony right beside it. 

Control

• The control was as we expected, with 426 colonies for one and 440 for the other, and an average of 433 

Honey

• 500 bacterial colonies grew on the first plate and 368 colonies on the second
• The average for both plates is 434
• We observed that the honey was very viscous at first, leading us to heat it up twice 
• When we were plating the second plate, it was too thick and we kept pulling up air bubbles
• Several large circular shaped colonies were seen spread around the plate, although unsure whether these were big bunched upcoloniesora reflection/air bubbles in the light. (Included in sources of error)
• The plate that had less growth has more large dots, which were more spread out 
• The plate with more colony growth had more clustering in the middle. 

 

Ampicillin 

• 1 bacterial colonies grew on the first plate and 5 colonies on the second
• The average for both plates is 3
• Very similar to kanamycin
• Only had about 6 colonies in total 
• 

Analyzing our results: 

The results of our experiment show that our hypothesis was correct, as the experiment showed that commercial antibiotics showed much stronger antibacterial colonies, and limited most E.coli colony growth, and although eliminating some colony growth and having less colonies than the control plate, natural antibiotics showed antibacterial properties, but did not overpower the synthetic antibiotics in terms antibacterial potenty

Over the course of 3 days, our results showed that ampicillin showed the strongest antibacterial properties out of the synthetic antibiotics and well as all the antibiotics in total, averaging only 3 colonies of bacteria being able to grow. Amoxicillin was least effective out of the three synthetic, with an average of 321.5 colonies of bacteria being grown. Although this antibiotic worked, it only worked to some extent. With the control growing an average of 433, the amoxicillin theoretically stopped 111.5 colonies from growing. Kanamycin was a close second to ampicillin  in terms of effectiveness, with an average of 4.5 colonies. In terms of the natural antibiotics, garlic turned out to be more effective than honey.

Why did natural antibiotics and amoxicillin show less antibacterial activity than expected? 

Our natural antibiotics, along with the amoxicillin did not work as well as we had hoped. Although we do not know the specific reason why this was, we have many speculations. For the amoxicillin it is very plausible that we did not dilute it properly, perhaps too much. Another possibility is that it was not stored properly or had been denatured by the heat at some point. A third, yet highly unlikely reason to why this happened is that the amoxicillin had somehow expired or ran out of its shelf life. The reason why this aspect would be unlikely to happen was because the amoxicillin we used was sourced approximately one year before, and has the average shelf life of two years. Another reason for this happening, is that amoxicillin is mostly effective against gram-positive bacteria, making the antiboitic in itself not as strong and effective as others could be. Since the bacteria we tested on was classified as a gram-negative bacteria, the amoxicillin did not work as well compared to if we had tested amoxicillin on a gram positive strain. Gram-positive bacteria is known to be less harmful and a less powerful bacteria than gram-negative, making it easier for antibiotics to inactivate, and therefore needing and using antiboitcs with specific needs and potency.  Overall, we found that the main reason that the amoxicillin did not work as well as we expected was either because the amoxicillin was just not active and usable anymore (expired, denatured,), or we had gotten the dilution wrong. If the dilution we used was too big for the concentration of amoxicillin, the concentration against the plate would not be poteent enough to create any antibacterial effect. The last possibility is that amoxicillin is just not as effective against E.coli as we expected, as e.coli is a gram-negative, while amoxicillin was created tobe effective mostly against gram-positive bacteria.    As for the honey and garlic, it is our assumption that in order for them to fully work, they need to be spread onto the agar plates immediately, along with the e.coli. The reasoning behind the honey is that overtime honey can lose its potency because the antimicrobial compounds degrade. Honey kills bacteria using osmotic pressure, because of the amount of sugar it contains, and raw honey contains more sugar to combat bacteria. We decided to pasteurize the honey rather than sterilize it because sterilizing it would take away too many of the natural enzymes, but it was a necessity to pasteurize it because of where it came from. Too many germs from other people were on it.  Or mix the E.coli in with the agar and then spread the antibiotic on top. We predict the reason for this is that the natural antibiotics had to be simulated so they were “in vivo” as best as we could. In vivo meaning in the human body, while in vitro means the opposite- in a lab. We did the experiment by spreading the antiboitic solution onto the agar plates beforehand, and that could have affected the efficiancy of the honey, because it did not have a water solution/envrioment to fully function in. How we completed the experiment would be the same in vivo as if you took the honey in some form before gaining the bacterial infection, which would have little to no effect.  A method most optimal that we could have done in the future to grow the bacteria on the agar plates first, and then spreading the honey solution on top; essentially reversing the method that we did. In terms of the garlic, unlike the garlic, it did show some antibacterial properties. We predict that the reason for this happening is because the liquid extraction of the garlic was done my hand, using a sterile plastic bag. After trying several methods, we found that this method was the most effective to extract juice out of a relatively small amount of garlic. Because of the manual crushing and extraction, due to human error, there were some small chunks in the final solution. Because the antibacterial properties of garlic come from allicin, which can only be released when garlic is smashed and the enzymatic juices are released. We predict that because there were chunks in the solution, and the amount we extracted was the exact amount we needed, one microlitre, it is likely that the solution of allicin that was actually released into the solution was less than 1 microlitre, caused by the fact that the chunks by themselves had no antibacterial properties. If the concentration is not potent enough, or the amount of antiboitc is not competent compared to the amount of e.coli or mass or agar, the garlic would not have worked as well.

Why did kanamycin and ampicillin work the best when fighting E.coli?  

There are several contributing factors, a main one being the fact that they both came from a lab environment. They were both sterile and no one had interfered with them without using the proper procedures and equipment. Both of these antibiotics have broad spectrum activity, and work against a large range of gram-negative and gram positive bacteria, and target fundamental processes in gram-negative bacteria such as E.coli. While kanamycin stops growth  and ampicillin kills the bacterial cells outright, these two antibiotics both work rapidly to fight bacteria. The kanamycin is an aminoglycoside drug that has been used to treat e.coli for a very long time, same goes to ampicillin for it is widely used to treat infections caused by those such as e.coli. Meanwhile the garlic and amoxicillin came from home and the honey was provided by an adult from school. 

 

 

Why did ampicillin work slightly better than kanamycin

We can predict that these two antibiotics work with the same effectiveness, even though the experiment showed that ampicillin did better by just a hair, the experiment was only done with one replica, meaning there was much room for variety, and it was difficult to come to a concrete final answer about whether ampicillin is really more effective than kanamycin.

 

After analyzing our results in terms of efficiancy and colonies counted, we found that our hypothesis and predictions was correct. Although the natural antibiotics such as honey and garlic showed antibacterial properties, the synthetic antibiotics ultimately overpowered the antibacterial strength. There are two reasons for why this happened, and why there was a severe difference in colonies between the two types of antibiotics. The first being that synthetic antibiotics were created in a lab by medical professionals, meaning that they would contain more chemicals that are more potent and accurate. Some of which are just not able to be found in nature, such as in plants and herbs. These synthetic antibiotics have been crafted and perfected to target multiple aspects of the bacteria to kill or inactivate it. We found that ampicillin was the most potent and effective antibiotic against E.coli when incubated on the agar plates, with an average of only 3 colonies that were able to grow. In vivo, which means The honey had an average of 434 colonies per plate, while the garlic had an average of 357.5 colonies per plate. The Kanamycin average was 4.5 colonies and the ampicillin was 3 colonies per plate. As for the amoxicillin, it had an average of 324.5 colonies. Then lastly, the control plate had an average of 434 colonies. Although the natural antibiotics did not do nearly as well, it does not mean that they are ineffective. The way we tested the honey was as if you were to take it before you actually contracted the disease. This is why we needed to have either mixed the e coli in with the agar, or plated them at the same time. Though garlic contains an active ingredient called allicin, and it has antibacterial properties, it is our guess that the concentration was too low to actually be effective. We had a hard time squeezing out the liquid from the garlic and did not have as much for the second plate as we did for the first, which would have been the reason for the big diffference in colonies in both plates. 

 

We wanted to do this experiment to see if we would be able to find any other inexpensive options to treat e.coli. E.coli is one of the most common food related illnesses, with 48 million cases a year worldwide and over 950,000 deaths according to a study done in 2019. A lot of those deaths were in Sub-Saharan Africa, with about 230 deaths per 100,000 population. Sub-Saharan Africa is a highly impoverished area in Africa, and a majority of the people in that part of Africa do not have access to health care which explains why they cannot treat it, plus they could be suffering from many other diseases, considering the situation and the environment. This is why having cheaper and more accessible options is very important, such as honey and garlic. Perhaps you are not able to get your hands on a prescription for kanamycin, or amoxicillin because you are not a citizen yet, or your insurance doesn’t cover it, or perhaps you simply cannot afford it. It is also possible that we will be able to create more inexpensive cures using parts of these natural antibiotics and make more antibiotics that can be available to more people. 

 

The garlic had a couple chunks in it, which made it difficult to suck up, especially for the second plate. In order to deal with this, we used a sterilized razor blade to make the hole for the tips bigger. Though, the allicin comes from the garlic when it is crushed, so those very small chunks may not have had an effective amount of allicin in them. For the first plate, we had already sucked up most of the garlic juice, so there was not as much for the second plate as there was for the first. This uneven distribution of the garlic could also have impacted the growth of e.coli. We had very little garlic, meanwhile we had an abundance of honey, kanamycin, ampicillin and amoxicillin. We had to be cautious of the amount we used on each plate, but for the others we could plate them without worries. The concentrations and dilutions could be uneven. There was a hair on one of the petri dishes and so we wanted to remove it, despite wearing gloves, it was still not sterile. In order to fix this we put a small amount of 100% ethanol on the lid of the petri dish and set it on fire to sterilize it, the same procedure as the spreaders. During the plating process, we had to close up the antibiotic we were using and put it back onto the stand. Even during those few seconds of being untouched, the solution could have been settling down to the bottom, meaning that it would be very concentrated at the bottom and not as much at the top. To avoid this we shook the solution between every plate before we spread it. Spreading the antibiotics and e coli onto the petri dishes is not as accurate as mixing them in with the agar solution because they can lose their max potency even if it is just for a night. In order to sort of help us avoid this, we used a spinner for the petri dishes in order for us to maximize the amount of area we covered. We counted each colony by hand, so our numbers could be inaccurate since it was hard to tell what was a colony and what wasn’t, along with being able to differentiate what was the lighting of an actual colony. There were also numerous clumps, and we could not tell how many actual colonies were in the blobs. With the honey there were massive dots that were small colonies all clustered together, making it impossible to count how many colonies there were in those big dots. In order to maintain consistency, we counted every single plate by hand and used the same method for counting for all of them. We marked which colony we counted and then put a tally mark for it after. 

 

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We would like to extend our sincere gratitude to Dr. Robyn Flynn from the Center for geo-engineering at the University of calgary. Without your aid and support this experiment would not have been possible. She was able to get us all the supplies we needed, plus she answered all of our questions and gave us an abundance of useful information. We would also like to give big thanks to Dr. Derrick Rancourt, the lab investigator for giving us permission to do this experiment and be present in the lab while the actual experiment was being conducted, despite us being underage. We also would like to acknowledge the university of calgary, specifically the department of geo-engineering. We thank them for letting us share the space and resources. We also want to take the time to thank our parents for helping us commute and go to one another's houses to work. Along with taking us to the lab on the days we needed to go. They also helped provide us with materials such as our trifold, construction paper, a printer, markers and glue. Then, we would like to thank Ms Reinstein for organizing this event and letting us experience this opportunity, and Mr. Lahoda for also helping organize events and meetings.