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THE EFFECT OF DIFFERENT ANTIBIOTICS ON BACTERIA

Antibiotics are medicines that kill bacteria. Bacteria can cause infections such as strep throat, ear infections, urinary tract infections, and sinus infections (sinusitis). There are many types of antibiotics. Each works a little differently and acts on different types of bacteria.

Don't antibiotics cure everything?

Antibiotics are powerful medicines, but they cannot cure everything. Antibiotics do not work against illnesses that are caused by a virus. They do not help illnesses such as:

Common colds.

Influenza (flu).

Most cases of acute bronchitis.

Most sore throats not caused by strep.

Runny noses.

These illnesses usually go away by themselves. If you take antibiotics when you do not need them, they may not work when you do need them. Each time you take antibiotics, you are more likely to have some bacteria that the medicine does not kill. Over time these bacteria change (mutate) and become harder to kill. The antibiotics that used to kill them no longer work. These bacteria are called antibiotic-resistant bacteria.

These tougher bacteria can cause longer and more serious infections. In order to treat them you may need different, stronger antibiotics that cost more. A stronger antibiotic may have more side effects than the first medicine.

Antibiotic-resistant bacteria also can spread to family members, children, and friends. Your community then will have a risk of getting an infection that is harder to cure and costs more to treat. Some antibiotics that doctors once prescribed to treat common infections no longer work. Taking antibiotics you do not need will not help you feel better, cure your illness, or keep others from being infected. On the other hand, take them when unnecessary may cause harmful side effects. Those side effects include:

Nausea.

Diarrhea.

Stomach pain.

When antibiotics kill the normal bacteria in your intestine and allow the C.difficilebacteria to grow, this causes diarrhea, fever, and belly cramps. In some rare cases, it can even cause death. This condition is known as Clostridium difficilecolitis or C. difficile colitis which is the swelling and irritation of the large intestine, or colon. Women may develop vaginal yeast infections from taking antibiotics. Rarely, antibiotics can cause a dangerous allergic reaction that requires emergency care.

The right way to take antibiotic:

Take it exactly as directed. Always take the exact amount that the label says to take. If the label says to take the medicine at a certain time, follow these directions.

Take it for as long as prescribed. After the first few days of taking the medicine, you might feel better. However, it is important to keep taking the antibiotic as directed and usually until it is finish. Full prescription is always needed to get rid of those bacteria that are a bit stronger and able to survive the first few days of treatment. Bacteria that an antibiotic cannot kill (antibiotic-resistant bacteria) can develop if only part of an antibiotic prescription is being taken. Most importantly, leftover medicine must not be saved for the purpose of future use.

Antibiotics are usually safe despites of all the side effects. Common side effects include nausea, diarrhea, and stomach pain. In women, antibiotics can lead to vaginal yeast infections. Some minor side effects are inevitable. In rare cases, antibiotics can cause a dangerous allergic reaction that requires emergency care.

Antibiotics are among the most frequently prescribed medications in modern medicine. Antibiotics cure disease by killing or injuring bacteria. After the first antibiotic, penicillin which was accidentally been discovered from a mold culture, there are now over 100 different antibiotics available to cure minor discomforts as well as life-threatening infections.Although antibiotics are useful in a wide variety of infections, it is important to realize that antibiotics only treat bacterial infections. Antibiotics are useless against viral infections such as the common cold and fungal infections ringworm.

Types of Antibiotics

Although there are well over 100 antibiotics, the majority come from only a few types of drugs. These are the main classes of antibiotics. 

-Penicillins such as penicillin andamoxicillin 

-Cephalosporins such as cephalexin(Keflex) 

-Macrolides such as erythromycin (E-Mycin), clarithromycin (Biaxin), andazithromycin (Zithromax) 

-Fluoroquinolones such as ciprofloxacin(Cipro), levofloxacin (Levaquin), andofloxacin (Floxin) 

-Sulfonamides such as co-trimoxazole (Bactrim) and trimethoprim (Proloprim) 

-Tetracyclines such as tetracycline (Sumycin, Panmycin)

-Aminoglycosides such as gentamicin (Garamycin) and tobramycin (Tobrex)

The use of right antibiotic is crucial as each antibiotic cures only certain types of infections but not all. Also, a person may have allergies that eliminate a class of antibiotic from consideration, such as a penicillin allergy which should not prescribe amoxicillin. Other factors may be considered when choosing an antibiotic. Medication cost, dosing schedule, and common side effects are often taken into account. Patterns of infection in your community may be considered too.

In some cases, laboratories may help to decide which antibiotic to be used. Special techniques such as Gram stains may help narrow down which species of bacteria is causing infection. This is because certain bacterial species will take a stain while the others will not. Cultures may also be obtained. In this technique, a bacterial sample from infection is allowed to grow in a laboratory. The way bacteria grow or what they look like when they grow can help to identify the bacterial species. Cultures may also be tested to determine antibiotic sensitivities. A sensitivity list is the roster of antibiotics that kill a particular bacterial type. This list can be used to double check that you are taking the right antibiotic.

Escherichia coli

E. coli is a common type of bacteria that can get into food, like beef and vegetables. E. coli is short for the medical term Escherichia coli. The strange thing about these bacteria and lots of other bacteria — is that they're not always harmful to you.

Theodor Escherich first described E. coli in 1885, as Bacterium coli commune, which he isolated from the feces of newborns. It was later renamed Escherichia coli, and for many years the bacterium was simply considered to be a commensal organism of the large intestine. It was not until 1935 that a strain of E. coli was shown to be the cause of an outbreak of diarrhea among infants. The GI tract of most warm-blooded animals is colonized by E. coli within hours or a few days after birth. The bacterium is ingested in foods or water or obtained directly from other individuals handling the infant. The human bowel is usually colonized within 40 hours of birth. E. coli can adhere to the mucus overlying the large intestine. Once established, an E. coli strain may persist for months or years. Resident strains shift over a long period (weeks to months), and more rapidly after enteric infection or antimicrobial chemotherapy that perturbs the normal flora. The entire DNA base sequence of the E. coli genome has been known since 1997.

E. coli normally lives inside your intestines, where it helps the body to break down and digest the food. Unfortunately, certain types of E. coli can get from the intestines into the blood. This is a rare illness, but it can cause a very serious infection.

Someone who has E. coli infection may have these symptoms:

bad stomach cramps and belly pain

vomiting

diarrhea, sometimes with blood in it

One very bad strain of E. coli was found in fresh spinach in 2006 and some fast-food hamburgers in 1993. Beef can contain E. coli because the bacteria often infect cattle. It can be in meat that comes from cattle and it's also in their poop, called manure.

E. coli is a consistent inhabitant of the human intestinal tract, and it is the predominant facultative organism in the human GI tract; however, it makes up a very small proportion of the total bacterial content. The anaerobic Bacteroides species in the bowel outnumber E. coli by at least 20:1. However, the regular presence of E. coli in the human intestine and feces has led to tracking the bacterium in nature as an indicator of fecal pollution and water contamination. As such, it is taken to mean that, wherever E. coli is found, there may be fecal contamination by intestinal parasites of humans. Physiologically, E. coli is versatile and well-adapted to its characteristic habitats. It can grow in media with glucose as the only organic constituent. Wild-type E. coli has no growth factor requirements, and metabolically it can transform glucose into all of the macromolecular components that make up the cell. The bacterium can grow in the presence or absence of O2. Under anaerobic conditions it will grow by means of fermentation, producing characteristic "mixed acids and gas" as end products. However, it can also grow by means of anaerobic respiration, since it is able to utilize NO3, NO2 as final electron acceptors for respiratory electron transport processes. In part, this adapts E. coli to its intestinal (anaerobic) and its extra intestinal (aerobic or anaerobic) habitats.

E. coli can respond to environmental signals such as chemicals, pH, temperature, osmolarity. Therefore, in a number of very remarkable ways considering it is a unicellular organism. For example, it can sense the presence or absence of chemicals and gases in its environment and swim towards or away from them. It can stop swimming and grow fimbriae that will specifically attach it to a cell or surface receptor. In response to change in temperature and osmolarity, it can vary the pore diameter of its outer membrane to accommodate larger molecules (nutrients) or to exclude inhibitory substances. With its complex mechanisms for regulation of metabolism the bacterium can survey the chemical contents in its environment in advance of synthesizing any enzymes that metabolize these compounds. It does not wastefully produce enzymes for degradation of carbon sources unless they are available, and it does not produce enzymes for synthesis of metabolites if they are available as nutrients in the environment.

Figures 1.1 & 1.2: Escherichia coli

Staphylococcus aureus

Figure 1.3: Electron micrograph of Staphylococcus aureus

The Staphylococci

Staphylococci (staph) are Gram-positive spherical bacteria that occur in microscopic clusters resembling grapes. Bacteriological culture of the nose and skin of normal humans invariably yields staphylococci. In 1884, Rosenbach described the two pigmented colony types of staphylococci and proposed the appropriate nomenclature: Staphylococcus aureus (yellow) and Staphylococcus albus (white). The latter species is now named Staphylococcus epidermidis. Although more than 20 species of Staphylococcus are described in Bergey's Manual (2001), only Staphylococcus aureus and Staphylococcus epidermidis are significant in their interactions with humans. S. aureus colonizes mainly the nasal passages, but it may be found regularly in most other anatomical locales, including the skin, oral cavity and gastrointestinal tract. S epidermidis is an inhabitant of the skin. 

Taxonomically, the genus Staphylococcus is in the Bacterial family Staphylococcaceae, which includes three lesser known genera, Gamella, Macrococcus and Salinicoccus. The best-known of its nearby phylogenetic relatives are the members of the genus Bacillus in the family Bacillaceae, which is on the same level as the family Staphylococcaceae. The Listeriaceae are also a nearby family. 

Staphylococcus aureus forms a fairly large yellow colony on rich medium; S. epidermidis has a relatively small white colony. S. aureus is often hemolytic on blood agar; S. epidermidis is non hemolytic. Staphylococci are facultative anaerobes that grow by aerobic respiration or by fermentation that yields principally lactic acid. The bacteria are catalase-positive and oxidase-negative. S. aureus can grow at a temperature range of 15 to 45 degrees and at NaCl concentrations as high as 15 percent. Nearly all strains of S. aureus produce the enzyme coagulase: nearly all strains of S. epidermidis lack this enzyme. S. aureus should always be considered a potential pathogen; most strains of S. epidermidis are non-pathogenic and may even play a protective role in humans as normal flora. Staphylococcus epidermidis may be a pathogen in the hospital environment. 

Staphylococci are perfectly spherical cells about 1 micrometer in diameter. The staphylococci grow in clusters because the cells divide successively in three perpendicular planes with the sister cells remaining attached to one another following each successive division. Since the exact point of attachment of sister cells may not be within the divisional plane and the cells may change position slightly while remaining attached, the result is formation of an irregular cluster of cells. 

The shape and configuration of the Gram-positive cocci helps to distinguish staphylococci from streptococci. Streptococci are slightly oblong cells that usually grow in chains because they divide in one plane only, similar to a bacillus. Without a microscope, the catalase test is important in distinguishing streptococci (catalase-negative) from staphylococci, which are vigorous catalase-producers. The test is performed by adding 3% hydrogen peroxide to a colony on an agar plate or slant. Catalase-positive cultures produce O2 and bubble at once. The test should not be done on blood agar because blood itself contains catalase. 

Figure 1.4: Gram stain of Staphylococcus aureus in pustular exudate

Figure 1.5: Staphylococcus aureus

Problem statement: Which antibiotic is most effective on bacteria?

Hypothesis: Different antibiotics have different effect on bacteria. Ampicillin is the most effective antibiotic against Escherichia Coli and Staphylococcus aureus compared to other antibiotics.

Variables:

Manipulated Variable: Types of antibiotics and types of bacteria.

Responding Variable: The diameter of clear zone around the paper discs.

Fixed Variable: Surrounding temperature, humidity, light intensity, size of paper discs.

Apparatus: Agar plate, Bunsen burner, marker pen, autoclaved forceps.

Materials: Bacteria E. coli, bacteria S. aureus, bench spray of disinfectant, 1% Virkon, soap and dettol, paper towels, antibiotic- impregnated paper disc, adhesive tape.

Procedure:

Hands are washed with dettol handwash. Disinfectant spray is sprayed thoroughly to the working area. Paper towels are then used to wipe the working area.

Two sterile Petri dishes are labeled correctly. One is filled with the bacteria S. aureus and another one with E. coli. The label is pasted at the side of the Petri dishes.

The apparatus needed: bottle containing sterile nutrient agar, micropipette with sterile tips, Bunsen burner, bottle containing bacteria cultures and sterile Petri dishes labeled correctly.

200ml of E. coli bacteria culture is pipetted into a sterile Petri dish beside a burning Bunsen burner.

Molten agar is poured into the Petri dish until the bottom of the Petri dish is covered by the agar.

The Petri dish is then covered and gently pushed back and forth and in all four directions to mix the bacteria well with the agar.

The agar is then allowed to set.

Steps 4 to7 are repeated for S. aureus.

The 2 Petri dishes containing the agar lawn are allowed to set.

One paper disc is placed in a solution of antibiotics named Ampicillin using sterile forceps.

The paper disc is then soaked into Petri dish containing the agar.

Steps 10- 11 are repeated for antibiotics Tetracyclin, Carbenicillin, and sterilized distilled water.

The Petri dish is closed and the bottom of the Petri dish is labeled to identify the position of each paper disc.

The agar plates are then left in 30.0 0C incubator for 24 hours.

Hands are washed thoroughly again after working with the bacteria culture.

After 24 hours, the agar plates are observed with the Petri dishes closed. The diameter of the clear region around the paper discs are measured and recorded. The results are recorded in a table

Precautions:

The bacteria must be pipetted into the agar before the agar is set so that the bacteria can mix well with it.

During observation, the lid of the Petri dishes must not be lifted up as the bacteria are harmful to our healths.

After working with the bacteria culture, hands must be washed with disinfectant in order to avoid any infections.

Result:

Antibiotics

Diameter of the clear zone, cm

Escherichia coli

Staphylococcus aureus

Ampicillin

3.2

3.0

Tetracycline

2.3

2.7

Carbenicillin

1.3

2.8

Distilled water

0.5

0.5

Discussions:

Analysis of data

From the result, it is found that the largest inhibition zone or clear region is formed around the paper disc soaked in Ampicillin for E. coli bacteria lawn, followed closely by Tetracyclin and Carbenicillin. Ampicillin’s paper disc caused the largest area of inhibition zone in E. coli. This showed that Ampicillin is most effective in inhibiting the growth of the E. coli. Meanwhile, Carbenicillin’s paper disc which caused the smallest area of inhibition zone in E. coli showed that it is the weakest antibiotic against E. coli.

Similarly, Ampicillin is also the most effective antibiotic against bacteria Staphylococcus aureus as the area of inhibition zone around the paper disc soaked with Ampicillin solution is the greatest, which has a diameter of 3.0 cm. Followed by that is the antibiotic Carbenicillin and the least effective antibiotic is Tetracyclin which has a slightly smaller diameter of clear zone than Cabenicillin, which is 2.7cm.

Therefore, it can be concluded that Ampicillin is the most effective antibiotic against both type of bacteria and being the broad spectrum antibiotic while the effectiveness of Tetracyclin and Carbenicillin towards both bacteria varied. This showed that different antibiotic has different effect on different bacteria as well.

The inhibition zones are all circular. If it is not circular, it is sensible that the diameter should be measured by using two points which are furthest from each other within the clear region. The diameter of the inhibition zones is affected by the strength of antimicrobial properties of the antibiotics towards different bacteria. It is important not to always choose the antibiotic with the largest inhibition zone to treat the patients as some other factors should be considered as well such as the side effect caused by the antibiotics, the conditions of the patients and the risk of that particular antibiotics.

Control

In this experiment, the control used is the sterilized distilled water. Paper discs soaked in sterilized distilled water are also put in two of the Petri dishes. This is to show that the sterilized distilled water has no effect on the bacteria. This enables us to compare the results for paper discs with the antibiotics and those with the distilled water to show that the formation of the inhibition zone or the clear region is due to the antibiotics but not because of the presence of water. In this case, clear region cannot be seen around the paper disc soaked in sterilized distilled water in both Petri dishes. Therefore, the presence of clear region around other paper discs must be due to the antimicrobial property of the antibiotics.

Variables

Three different antibiotics are used in this experiment to manipulate the types of antibiotics and to compare the effectiveness of each antibiotic to inhibit the growth of the bacteria. The antibiotics used are Tetracycline, Carbenicillin and Ampicillin. Two different types of bacteria, E. coli and S. aureus are manipulated by putting them in different Petri dishes with the agar medium. This enables us to identify the varying antimicrobial properties of the same antibiotics on different types of bacteria.

The responding variable in this experiment is the diameter of the clear region around the paper discs after 24 hours. The greater the diameter of the clear zone around the paper discs, that means the more effective the antibiotic inhibiting the growth of the bacteria. The diameter of the clear zone can be measured from one point of the circular clear region to another point through the centre point by using a ruler.

The amount of different bacteria cultures used must be the same by pipetting equal amount of the two bacteria, which is 200 ml into the agar medium. The temperature, humidity and light intensity must also be kept constant throughout the experiment. All these factors may affect the rate of growth of the bacteria. This can be done by placing the two agar plates into an incubator at 30 0 C. The size of the paper discs should also be kept constant. Paper discs which are larger will absorb more antibiotics and may lead to a greater diameter of clear zone compared to the smaller paper discs in the same bacteria culture. This is done by preparing the paper discs using the same puncher to ensure all the paper discs are of the same size.

Justification of apparatus and materials

In this experiment, the antibiotics used are Tetracycline, Streptomycin and Carbenicillin solutions. These antibiotics are more common antibiotics which are more easily available. The antibiotics have already been prepared in solutions form. This enables the paper discs to be soaked in the solutions directly and easier. The bacteria used are S. aureus and E. coli, they are practically easier to be grown and culture in agar plates. However, these two bacteria may be harmful to our health, therefore the lids of the agar plates are not allowed to be opened during observation. This is to keep us from getting any infections from these bacteria.

Bunsen burner is used in this experiment to minimize the contamination of the experimental sets while preparing them. For example, the forceps are being flamed before being used to pick up the paper discs soaked in the antibiotics solutions. Micropipette is used to transfer the 200 ml bacteria into the agar medium. The used of micropipette with sterile tips further improved the accuracy of the result.

Validity and reliability of the results

According to the result from another group, Ampicillin is the most effective antibiotic towards only E. coli bacteria. Cabernicillin is most effective towards S. aerues and this is different from the result of my group. This may properly cause by contamination which affect the accuracy of the result. However, for the bacteria E. coli, the diameter of clear zone caused by the paper disc soaked in Cabernicillin is 1.3 cm for my group while the result of another group showed no clear zone around it. Therefore, it can be concluded that Carbenicillin is the least effective antibiotics towards E. coli for two groups. Besides, in order to increase the reliability of the experiment, the variables are controlled carefully. The constant variables are kept constant while only manipulating the variables that are being studied.

Sources of errors

One possible source of errors may be the contamination of the agar medium. This occurred when saliva is accidentally being transferred to the agar during preparation. Another possible source of error could be the purity of the antibiotics used. Some of the antibiotics solutions may be contaminated by some impurities which could decrease their antibacterial properties. Also, human error can take place especially when measuring the diameter of the clear zones.

Conclusion:

The hypothesis is accepted. Different antibiotics have different effect on bacteria. Ampicillin is the most effective antibiotic against Escherichia Coli and Staphylococcus aureus compared to other antibiotics.

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