Effect of a Natural and Synthetic Antibiotic on the Growth of Staphylococcus Epidermidis Bacteria: Analysis of Penicillin

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1.0 Introduction:

Bacteria are microscopic, single-celled organisms that thrive in diverse environments. They constitute a large domain of prokaryotic microorganisms and can be divided into two groups: gram-positive or gram-negative. Staphylococcus epidermidis is a gram-positive bacterium mostly found on human skin. Although most strands of S. Epidermidis are harmless, S. Epidermidis can become pathogenic and cause serious health implications if not treated correctly. The most common infection that S. Epidermis causes is golden staph. Golden Staph infections usually commence with the introduction of bacteria from the skin of the patient or that of health care personnel during device insertion (peripheral or central intravenous catheters) and have increased in number most likely owing to the increased use of such devices. Those with weakened or compromised immune systems are also at a greater risk of contracting a S.Epidermis infection.

The cell walls of gram-positive bacteria are composed predominantly of peptidoglycan; a polymer consisting of sugar and amino acids that forms a mesh-like layer outside the plasma membrane of S. Epidermis refer to figure 1. Peptidoglycan can represent up to 90% of the cell wall, with numerous layers forming around the cell membrane. NAM tetrapeptides are crosslinked with peptide interbridge where complete cross-linking is achieved. These components combine together to create a strong cell wall. An additional component within gram-positive cell walls is teichoic acid. Opposite to peptidoglycan, teichoic acid is glycopolymer and is embedded within the peptidoglycan layers around the cell membrane. Teichoic acid generates the net negative charge of the cell contributing to the overall rigidity of the cell wall which is important in playing a role in resistance to adverse conditions such as high temperature and salt conditions and also B-lactam antibiotics. Teichoic acids can be covalently linked to peptidoglycan or via a lipid anchor refer to figure 1.

Figure 1: Schematic of the gram-positive cell wall showing that wall teichoic acids are covalently anchored to peptidoglycan and lipoteichoic acids are tethered to the membrane. WTA’s are important as they play a role in antibiotic resistance.

Synthetic antibiotics are microbes that can kill or inhibit the growth of microorganisms, specifically bacteria. They are a type of antimicrobial that is either categorized as bactericidal or bacteriostatic. Bacteriostatic and bactericidal antibiotics have two different actions. Bacteriostatic antibiotics limit the growth of bacteria by interfering with bacterial protein production, DNA replication, and bacterial cellular metabolism. Bactericidal antibiotics inhibit cell wall synthesis meaning they kill the bacteria. Antibiotic effectiveness can be measured by the zone of inhibition. The zone of inhibition is the clear region created around the antibacterial agent created on an agar plate refer to figure 2.

Figure 2: Zone of inhibition created around the antibiotic mask ring.

Antibiotics from different classes affect the death of a bacteria cell differently. In the method of cell lysis – the process which lysis puncture the membrane of the cell; antibiotics have differing effects on this process that either allow or block antibiotics from being able to diffuse across the cell membrane.

Penicillin is a B- Lactam bactericidal antibiotic. This particular class of antibiotics act to restrict specific steps in homeostatic cell wall biosynthesis, resulting in cell lysis, therefore, killing the bacteria cell completely when it is treated against a bacterium. B- Lactam antibiotics consist of all antibiotic agents that contain a beta-lactam ring in their molecular structures. The beta-lactam ring is the part of the core structure of several antibiotic families including penicillin. S. Epidermis is a natural pathogen which produces penicillinases. Penicillinases is an enzyme which inactivates penicillin through breaking the penicillin B-lactam ring which can cause S.Epidermis to have a resistance to penicillin refer to figure 3.

Figure 3: Mechanism of action of beta-lactam antibiotics. Top: In the absence of drug, transpeptidase enzymes (also known as Penicillin Binding Proteins; PBP) in the cell wall catalyze cross-links between adjacent glycan chains, which involves the removal of a terminal D-alanine residue from one of the peptidoglycan precursors (highlighted by the broken oval). Glycosyltransferases (GT), which exist as either separate subunits, or tightly associated with transpeptidases (e.g. as is the case for PBP-2) create covalent bonds between adjacent sugar molecules NAM & NAG. The net result of covalent bonds between both the peptide and sugar chains creates a rigid cell wall that protects the bacterial cell from osmotic forces that would otherwise result in cell rupture. Bottom: Beta-lactam antibiotics, which include penicillins (Pen), cephalosporins (Ceph), monobactams (Mono) and carbapenems (Carba) bear a structural resemblance to the natural D-Ala-D-Ala substrate for the transpeptidase, and exert their inhibitory effects on cell wall synthesis by tightly binding to the active site of the transpeptidase (PBP).

A natural antibiotic contains antibacterial, antimicrobial, and antioxidant properties that inhibit the growth of bacteria. Garlic is an antibiotic (Bergner, 1995). In a study conducted by School of Molecular Biosciences in 2011, garlic concentrate was found to be effective against bacteria. Dr. Tariq Abdullah, a prominent garlic researcher stated in the August 1987 issue of Prevention, “Garlic has the broadest spectrum of any antimicrobial substance that we know of — it is antibacterial, antifungal, antiparasitic, antiprotozoan and antiviral.” In the study the bactericidal effect increased along with an increase in the concentration of the garlic concentrate shown in figure 4. Garlic’s antibiotic comes from its natural antimicrobial Allicin. Allicin is a compound produced when garlic is crushed or chopped. The main antimicrobial effect of allicin is due to its chemical reaction with thiol groups of various enzymes, e.g. alcohol dehydrogenase, thioredoxin reductase, and RNA polymerase, which can affect essential metabolism of cysteine proteinase activity involved in the virulence of E. histolytica. Thioredoxin is an enzyme protein found in S. Epidermis therefore the allicin released from garlic granules causes the reaction between the allicin and Thioredoxin which causes the metabolism of the Thioredoxin to break down allowing the allicin to diffuse into the bacteria (S. Epidermis) membrane and inhabit the growth, therefore, making it an effective natural antibiotic.

Figure 4: Effect of garlic concentrates on the growth and survival of Campylobacter jejuni in sterilized aline water at different temperatures and times.

With taking in consideration from the resistance that S. Epidermis builds towards penicillin, it is uncertain whether a natural or synthetic antibiotic would be most effective in limiting bacteria growth.

2.0 Aim

To investigate the effect of a natural antibiotic (garlic) and synthetic antibiotic (penicillin) on the growth of S. Epidermis bacteria to determine which is the most effective in limiting bacteria growth. The effectiveness of the natural antibiotic (garlic) and synthetic antibiotic (penicillin) will be measured by the diameter of the zone of inhibition (mm).

3.0 Hypothesis

In the treatment of S. Epidermis bacteria, the synthetic antibiotic; penicillin and natural antibiotic; garlic will both be effective in inhibiting bacterial growth. However, the synthetic antibiotic (penicillin) will produce a larger zone of inhibition therefore making it more effective against S. Epidermis bacteria than a natural antibiotic (garlic) that will produce a smaller zone of inhibition.

4.0 Materials

1mL Staphylococcus Epidermis
Antibiotic mast rings containing penicillin
Garlic granules
Distilled water
9 agar plates
Forceps
Bunsen burner
Heat mat
Box of matches
Sterile spreader
200mL pipet
Pipet tips
Vernier callipers
Bleach 10%
Ethanol 70%
Tweezers
Incubator
Lab coat
Safety glasses
Filter paper disks

All these materials were sourced from the Sheldon College Science Department. A pestle and mortar was brought from home

5.0 Method – reference to the original method

5.1 Setup of Apparatus

This experiment was based upon the investigation conducted by Paul Bergner at Medical Herbalism 1995 who tested the effectiveness of garlic against Staphylococcus Aurerus. In this investigation, the bacteria was changed to S. Epidermis as to Sheldon College’s availability. Penicillin was also tested against S.Epidermis to compare the effect of a natural antibiotic (garlic) and synthetic antibiotic (garlic). The method was altered slightly to introduce a sterile workspace. The method used is as follows.

All materials were collected. A sterile work zone was created through spraying 70% ethanol onto the workbench and let soak for one minute. This was then wiped clean. The heat mat was placed underneath the Bunsen burner and the Bunsen burner was then lit using the matches. A lab coat, safety glasses, and gloves were worn throughout the process.

A pestle and mortar was used to crush the garlic granules into a thin powder. Distilled water was then added to create a garlic paste. This was let sit.

100L of S.Epidermis was ejected using the 200mL pipet onto 9 agar plates. 100L in total was ejected onto each of the plates. The bacteria was then spread using the sterile spreader to cover the entire agar plate.

New gloves were put on to risk cross-contamination.

2 antibiotic mask rings (penicillin) were placed on 4 agar plates making a total of 2 antibiotic mask rings to 1 agar plate. They were spread evenly from each other within the plate.

Filter paper disks were coated with the garlic pasted created earlier. They were placed on 4 agar plates with 2 garlic filer papers to 1 agar plate. They were spread evenly from each other within the plate. Antibiotics were not placed on one agar plate for control. The 9 agar plates were then placed in the incubator at 30. After three consecutive days after the experiment was conducted, the zone of inhibition was measured.

5.2 Variables of Experiment

Table 1: The experimental variables (constant, independent, dependent) of this experiment.

Constant
Independent
Dependent
Temperature of incubator (30)
S. Epidermis Bacteria
Zone of inhibition (mm) -measured using electric vernier callipers
100L of bacteria (S. Epidermis) on each agar plate
Antibiotics- Penicillin and Garlic Granules formulated into a garlic paste (coles homebrand)
Length of time that each agar plate was incubated (3 days – 72 hours)

6.0 Results

Photo:

Key: Big number = Plate no.
A or B = the disk name
Disk Number & penicillin a/b
Diameter of zone of inhibition (mm)

Disk 1 A 22.6

Disk 1 B 22.4

Disk 2 A 20.3

Disk 2 B 21.1

Disk 3 A 21.9

Disk 3 B 21.2

Disk 4 A 26.7

Disk 4 B 23.3

Table 2: Zone of inhibition diameter (mm) results recorded at 72 hours of Synthetic antibiotic (penicillin) against S.Epidermis bacteria.

Table 3: Line graph showing the zone of inhibition diameter results recorded at 72 hours of Synthetic antibiotic (penicillin) against S.Epidermis bacteria across 4 disks and 8 penicillin antibiotic mask rings. The largest zone of inhibition recorded was disk 4 penicillin mask no. a at 26.7 mm.

Photo:

Key: Big number – agar plate no.
A or B= disk name
Disk Number & garlic a/b

Diameter of zone of inhibition (mm)

1a 9.7

1b 15.6

2a 14.6

2b 12.7

3a 12.8

4a 15.7

4b 11.3

Table 4: Zone of inhibition diameter (mm) results recorded at 72 hours of natural antibiotic (garlic) against S.Epidermis bacteria.

Table 5: Line graph showing the zone of inhibition diameter results recorded at 72 hours of natural antibiotic (garlic) against S.Epidermis bacteria across 4 disks and 8 garlic mask rings. The largest zone of inhibition recorded was disk 4 garlic mask no. a at 15.7mm. The smallest zone of inhibition recorded was disk 3 garlic mask no. b at 0mm.

Table 6: S. Epidermis average results for garlic antibiotic and penicillin antibiotic when the zone of inhibition was recorded at 72 hours.

Antibiotic (synthetic or natural)

Average Zone of Inhibition radius (mm)

Penicillin 22.44

Garlic 11.56

Table 7: Average zone of inhibition of S. Epidermis bacteria caused by the natural and synthetic antibiotics 72 hours after the commencement of the investigation. Penicillin had the largest effect on the bacteria with an average zone of inhibition of 22.44mm. Garlic had an average zone of inhibition of 11.56mm.

Standard Deviation * Standard Error *

7.0 Discussion

The results were measured 72 hours after the commencement of the investigation.

8.0 Conclusion

9.0 Appendix

Zone of inhibition from synthetic antibiotic (penicillin) on S. Epidermis Bacteria

1a 1b 2a 2b 3a 3b 4a 4b 22.6 22.4 20.3 21.1 21.9 21.2 26.7 23.3 Agar plate and corresponding disk number

Diameter of the zone of inhibitions (mm)

Zone of Inhibition from natural antibiotic (garlic) on S. Epidermis Bacteria

1a 1b 2a 2b 3a 3b 4a 4b 9.6999999999999993 15.6 14.7 12.7 12.8 0 15.7 11.3 Agar plate and corresponding disk number

Diameter of the zone of inhibition (mm)

Average Zone of Inhibition from measuring antibiotic effectiveness against S. Epidermis

Zone of Inhibition Penicillin Garlic 22.44 11.56 Antibiotics

Zone of inhibition radius (mm)

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