Different antibiotics exhibit different reactions towards varied bacteria

Different antibiotics exhibit different reactions towards varied bacteria, thus exhibit different degree of effectiveness towards different bacteria. The effectiveness is due to their mode of action and spectrum of action. In part one of this experiment, antibiotic discs composed of 30ug kanamycin, 25ug ampicillin, 50ug chloramphenicol, 100ug tetracycline, 50ug nitrafuratonin, 30ug nalidixic acid, 100ug colistin sulphate and 25ug streptomycin were pressed onto three different agar plates made of Staphylococcus aureus, Escherichia coli and Serratia marcescens using a pair of sterilized forceps. The area of inhibition of the clear zone around the paper disc was measured and recorded to form relevant conclusions on the nature of the antibiotics. It was concluded that tetracycline, nitrafuratonin, nalidixic acid and chloramphenicol are broad spectrum antibiotics. Chloramphenicol, tetracycline and nitrafuratonin are bacteriostatic, whereas nalidixic acid is bacteriocidal. Collistin sulphate and the aminoglycosides of streptomycin and kanamycin are bactericidal and have a narrow antibacterial spectrum. Ampicillin is defined as a narrow spectrum bactericidal agent.

Introduction
Antibiotics are chemotherapeutic substances produced by living organisms, that destroy or inhibit the growth of micro organisms, primarily bacteria in living tissue (N., J., O., G., K., & P., S., 2014). Resistance to antimicrobial compounds is due to innate structural features of microorganisms such as an impermeable outer membrane which resists penetration of antibiotics (N., J., O., G., K., & P., S., 2014). Gram-negative bacteria contain a thick lipopolysaccharide layer which acts as a barrier to limit diffusion of antibiotic molecules into the cell while gram-positive bacteria are characteristically defined by lipophilic substances in their cells walls which retards penetrations of hydrophilic, cationic and antimicrobial compounds (Silhavy, T. J., Kahne, D., & Walker, S., 2010). In addition to barriers, microorganisms may possess a variety of other resistant mechanisms. A prominent example, is that the organism may lack a transport system necessary for antibiotic uptake or be lacking the biochemical target required for attachment and proper functioning of the antimicrobial compound (Silhavy, T. J., Kahne, D., & Walker, S., 2010).

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Mode of Action
Antibiotics are classified as bacteriostatic or bactericidal based on their mode of action. Bactericidal agents inhibit the growth of cells in addition to triggering pathways within the cell that lead to irreversible cell death (N., J., O., G., K., & P., S., 2014) so have more powerful antibacterial action than bacteriostatic agents. In contrast, bacteriostatic agents, inhibit the growth and multiplication of bacteria (Silhavy, T. J., Kahne, D., & Walker, S., 2010). Upon exposure to a bacteriostatic agent, cells in a susceptible population stop dividing. However, if the agent is removed, the cells once again multiply (N., J., O., G., K., & P., S., 2014). Therefore, bacteriostatic antibiotics are thought to be less effective which has led to the recommendation that severely ill patients with bacterial infections should be treated with bactericidal antibiotics (Silhavy, T. J., Kahne, D., & Walker, S., 2010).

Spectrum of Action
Antibiotics are classified as narrow spectrum or broad spectrum. Broad-spectrum antibiotics, can be used to treat a wide variety of infections, inhibiting both gram-positive and gram-negative bacteria. Narrow spectrum antibiotics are defined as being selective in their spectrum of action (Silhavy, T. J., Kahne, D., & Walker, S., 2010).

Purpose
From pressing antibiotic discs composed of composed of 30ug kanamycin, 25ug ampicillin, 50ug chloramphenicol, 100ug tetracycline, 50ug nitrafuratonin, 30ug nalidixic acid, 100ug colistin sulphate and 25ug streptomycin onto agar plates composed of S. aureus, E. coli and S. Marcescens and viewing the ingredient discs with different sized clear zones around the disc shows the antibiotics had varied effectiveness towards the different bacteria. Therefore, the area of inhibition of the clear zone around the paper disc was measured and recorded to form relevant conclusions on the nature of the antibiotics. Drug sensitivity tests similar to this one are utilised on a wide scale in the medical industry to determine which drugs are likely to be effective against particular strains isolated from patients (Silhavy, T. J., Kahne, D., & Walker, S., 2010).

Method
Part one: Preparation of the agar plates
Initially, 3 sterile nutrient agar petri dishes were labelled with the respective name of the bacteria, initials, date and project number. The bottle containing bacterial broth was opened using the last finger, prior to flaming the bottle to kill any pathogens present. Utilising the aseptic technique, 0.2mL of the bacterial broth was pipetted to 7mL of the tube of molten agar, where the bacterial broth and molten agar were both flamed with the bunsen burner to kill any pathogens present. The knob of the pipette was gently pressed until the first pressure exerted was felt and released, preventing any air bubbles forming. The solution was mixed gently and thoroughly to further avoid air bubbles forming then dispensed into a petri dish, where the lid of the petri dish was opened enough only to allow the tip of the pipette and closed quickly. This procedure was repeated for S. aureus, E. coli and S. marcescens broth. The agar solution was handled using the aseptic technique, where around 18-20mL was poured into each the petri dishes. The bacterial broth and agar was then gently mixed together by swirling the petri dish in all directions. The plates were allowed to dry for approximately 5 minutes. Whilst the plates were drying, the forceps were sterilized by dipping them into ethanol, then flaming until the alcohol is burnt off. The sterile forceps were used to remove a multodisk from the box and place in the middle of each of the agar plates. The plates were inverted and incubated for around 24 hours at 37

Part two: Examining Growth
A metric ruler was used to measure the diameter of the zone of inhibition for each anti microbial on each plate, measuring from the edge of the disk to the edge of bacterial growth. Three readings were taken, then the average was taken.

Results
Table one showing the zones of inhibition from the edge of the disk to the edge of bacterial growth for S. aureus.

Radius (mm)
Type
Trial 1
Trial 2
Trial 3
Average
Kanamycin (30ug)
3
4
3
3.67
Amphicillin (25 ug)
12
13
14
13
Chloramphenicol (50 ug)
10
10
10
10
Tetracyclin (100 ug)
12
13
12
12.33
Nitrafurantonin (50 ug)
10
10
10
10
Nalidixic Acid (30 ug)




Colistin Sulphate (100 ug)
1
1
1
1
Streptomycin (25 ug)
2
2
3
2.33

Table two showing the zones of inhibition from the edge of the disk to the edge of bacterial growth for E. coli.

Radius (mm)
Type
Trial 1
Trial 2
Trial 3
Average
Kanamycin (30ug)
14
13
13
13.67
Amphicillin (25 ug)
7
6
7
6.67
Chloramphenicol (50 ug)
5
4
5
4
Tetracyclin (100 ug)
3
3
4
3.33
Nitrafurantonin (50 ug)
5
4
3
4
Nalidixic Acid (30 ug)
13
13
13
13.67
Colistin Sulphate (100 ug)
10
9
12
10.33
Streptomycin (25 ug)
4
4
3.5
3.83

Table three showing the zones of inhibition from the edge of the disk to the edge of bacterial growth for S. Marcescens.

Radius (mm)
Type
Trial 1
Trial 2
Trial 3
Average
Kanamycin (30ug)
9
8
8
8.33
Amphicillin (25 ug)
2
3
2
2.33
Chloramphenicol (50 ug)
3
2
1
2
Tetracyclin (100 ug)
2
2
1.5
1.83
Nitrafurantonin (50 ug)
2
1
1
1.33
Nalidixic Acid (30 ug)
10
10
10
10
Colistin Sulphate (100 ug)
7
8
7
7.33
Streptomycin (25 ug)
9
8
9
8.33

Discussion
S. marcescens 
Resistance of S. marcescens to ampicillin, as shown by limited inhibition was consistent with susceptibility patterns (Gupta, S., Govil, D., Kakar, P. N., Prakash, O., Arora, D., Das, S., Malhotra, A.), as S. marcescens is characterised as a gram negative bacterium, so is not within the ampicillin spectrum. Large sensitivity of S. marcescens was anticipated for collistin sulphate, kanamycin, streptomycin and nalidixic acid as these are bactericidal, narrow spectrum agents which are expected to exhibit high antibacterial action against gram negative bacteria, so lined up well with results as the clear areas of growth were largest for these agents (Gupta, S., Govil, D., Kakar, P. N., Prakash, O., Arora, D., Das, S., Malhotra, A.). A possible reason for limited inhibition of the chloramphenicol, tetracylin and nitrafurantonin antibiotics could be due to beta lactam enzyme mediated resistance, as reports indicate that reduced permeability may be combined with beta-lactamase in S. marcescens for bacteriostatic agents (Sleigh, J. D.,1983, December).
E.coli
As expected, nalidixic acid, kanamycin and collistin sulphate suppressed the rapid bactericidal activity of E. coli as represented through the large circular zone of no growth due to being categorised as bactericidal, narrow spectrum antibiotics acting only on gram negative bacteria (Kibret, M., & Abera, B., 2011). There was a moderate amount of resistance of E. coli to ampicillin shown by the small circular zone of no growth, which was anticipated due to similar results obtained in previous experiments as ampicillin is able to act on select gram negative bacteria (Kibret, M., & Abera, B., 2011). Streptomycin exhibited low-level resistance to E. coli, possibly due to streptomycin having the ability to mutate and transfer genetic material which codes for resistance (Abduzaimovic, A., Aljicevic, M., Rebic, V., Vranic, S. M., Abduzaimovic, K., & Sestic, S., December). Tetracycline, chloramphenicol and nitrafuratonin exhibited small amounts of inhibition due to being broad spectrum antibiotics, but are proven are not as effective against inhibiting growth of E. coli (Abduzaimovic, A., Aljicevic, M., Rebic, V., Vranic, S. M., Abduzaimovic, K., & Sestic, S. 2016,) comparing well with similar experiments due to these antibiotics being of a bacteriostatic nature.

S. aureus
S. aureus is the most susceptible to ampicillin, as ampicillin is a narrow spectrum, bactericidal agent so exhibits powerful antibacterial action specifically on gram positive bacteria (Gupta, S., Govil, D., Kakar, P. N., Prakash, O., Arora, D., Das, S., Malhotra, A.). Tetracycline, chloramphenicol and nitrafuratonin also exhibited large areas of inhibited growth, which these findings are consistent with similar results, as chloramphenicol, tetracycline and nitrafuratonin are categorised as broad spectrum antibiotics due to being highly polar, cationic molecules which create fissures in the outer cell membrane, resulting in leakage of intracellular contents and enhanced antibiotic uptake of gram positive and negative bacteria (Chopra, Ian, and Marilyn Roberts. Advances in Pediatrics., U.S. National Library of Medicine, 2001). In particular, tetracycline exhibited the largest amount of growth as, bacteriostatic agents are often bactericidal against some susceptible organisms at high concentrations. Nalidixic acid, colistin sulphate, kanamycin and streptomycin did not show effectiveness in inhibiting growth of S. aureus due to these antibiotics being categorised as narrow spectrum antibiotics, acting specifically on gram negative bacteria, where S. aureus is classified as a gram positive bacterium (Mingeot-Leclercq, M., Glupczynski, Y., & Tulkens, P. M.,1999).

Conclusion
It was determined that tetracycline, nitrafuratoni and chloramphenicol are broad spectrum antibiotics acting on both gram positive and gram negative bacteria. Chloramphenicol, tetracycline and nitrafuratonin are moderate inhibitors of a growth, due to being characterized as bacteriostatic, whereas nalidixic acid is a killer of the infectious agent, so is defined as bacteriocidal. Nalidixic acid, collistin sulphate and the aminoglycosides of streptomycin and kanamycin have a narrow antibacterial spectrum, mostly against common gram-negative clinical isolates and are effective in inhibiting growth on a large scale due to being bactericidal. Ampicillin is able to effectively penetrate gram-positive and some gram-negative bacteria, on a moderate scale, thus defined as a narrow spectrum bactericidal agent.