Antibacterial Properties Of Aloe Vera And Tea Tree Biology Essay

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This study investigates the antibacterial activity of Aloe barbadensis Miller (Aloe vera) and Malaleuca alternifolia (Tea Tree Oil) against a number of clinical bacterial isolates in vitro. The isolates included Methicillin-Resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis (S. epidermidis), Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Staphylococcus saprophyticus (S. saprophyticus). The two methods utilised were the agar disc diffusion and broth microdilution assay. No antibacterial activity was demonstrated with the use of Aloe vera juice, aqueous and ethanolic leaf extracts on all five isolates tested via the agar disc diffusion method. Using the same method, TTO was found to be effective and had inhibitory effects on the growth of all isolates. The sizes of the inhibition zones of all isolates tested using TTO were 13.7- 34.57mm±SD. A broth microdilution method was used to determine the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of S. aureus, E. coli and S. saprophyticus. S. aureus and S. saprophyticus were equally susceptible to TTO, with MICs and MBCs of 1.56% and 6.3%, respectively. E. coli was the most susceptible with an MIC of 0.39% and MBC of 3.13%. The retained results support the use of TTO as a therapeutic agent but also highlight the need for a standardised method to test antibacterial properties.

CONTENTS

Page No.

General Introduction

1-3

Materials and Methods

2.1 Aloe vera

4

2.2 Tea tree oil

4

2.3 Growth and Maintenance of test microorganisms

4

2.4 Preparation of Bacterial Concentrations

4

2.5 Antibacterial activities- Agar disc diffusion assay

4-5

2.6 Antibacterial activities- Broth microdilution method

5

2.7 Minimum bactericidal concentration

6

2.8 Statistical analysis

6

Results

3.1 Antibacterial effectiveness- Agar disc diffusion assay

7-9

3.2 Antibacterial effectiveness- Broth microdilution method

9-10

Discussion

11-15

References

16-17

APPENDICES

COSHH forms

I

Ethics form

II

ABBREVIATIONS

v/v Volume per volume

% Per cent (age)

µg Microgram

oC Degree centigrade or Celsius

ml Millilitre

mm Millimetre

µL Microlitre

SD Standard deviation

ACKNOWLEDGEMENTS

I would like to thank my supervisor Dr. Patrick Kimmitt at the University of Westminster for his inspiring valuable guidance and his manifold assistance, timely suggestions and above all for his encouragement throughout the present investigation.

INTRODUCTION

Globally, antibiotic and multi-drug resistance is becoming an increasing public health concern in hospitals and the community (Bax et al., 2000). This evolution in resistance is attributed to widespread injudicious use of antibiotics as well as selective pressures on bacterial strains. This resistance has lead to increasing health care costs, morbidity and mortality due to treatment failures hence there is an urgent global call for solutions to manage this problem. In recent years, various strategies have been implemented by national, international and professional bodies to limit the unprecedented growth in bacterial resistance. Execution of these measures has enabled the recognition that, drug resistant infections cannot be properly managed as there is a deficit of innovative drugs, vaccines and diagnostic aids. This predicament has encouraged the pharmaceutical industry and fledging biotechnology companies to put renewed efforts into the development of new therapeutic agents from natural resources (Bax et al., 2000).

Historically, medicinal plants have been the source of numerous therapeutic agents however the potentiality of many plants as a source of new drugs is an area lacking thorough investigation. Among the medicinal plants, Aloe barbadensis Miller (Aloe vera) is of great interest as it is has been used therapeutically for many centuries and has a lengthy historic reputation as a curative agent (Pandey & Mishra, 2009). The majority of these claims however are mostly anecdotal and scientific evidence is often sparse and inconsistent (Pandey & Mishra, 2009). As one of few documented studies, Ferro et al. (2003) has shown that the inner-leaf gel from Aloe vera inhibits the growth of Streptococcus pyogenes and Shigella flexneri species in vitro and an alternate study by Shilpakala et al. (2009) further demonstrate the susceptibilities of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) to this component. Habeeb et al. (2007) has also publicised that Aloe vera has an inhibitory effect on the growth of MRSA 9 and MRSA 6. Nonetheless research on the inner gel of Aloe vera is still limited therefore more studies are required to give credence to the popular use of Aloe vera gel (Pandey & Mishra, 2009). Studies have also been carried out on the juice of Aloe vera; Alemdar & Agaoglu (2009) has suggested that the juice is more effective against Gram-positive bacteria with the exception of S. aureus. An inhibitory effect was observed against Gram-positive bacteria, Klebsiella pneumoniae (K. pneumoniae) and Candida albicans (C. albicans) and Enterococcus faecalis (E. faecalis). Furthermore Aloe vera juice exhibited no inhibitory activity on the proliferation of Gram-negative bacteria (E. coli) with the exception of K. Pneumoniae.

Tea tree Oil (TTO) is derived from the leaves of the Melaleuca alternifolia plant and has been used for centuries as a botanical medicine. This oil has also attracted considerable interest for potential use in adjunctive wound therapy. The main chemical components to have antimicrobial activity in TTO are terpinen-4-ol and α-terpineol. Several studies have been conducted to evaluate the activity of TTO against Methicillin-Resistant Staphylococcus aureus (MRSA) an example being the work of Carson et al. (1995). Of the 64 MRSA isolates examined (33 of which were mupirocin-resistant), all were establish as being susceptible to TTO. TTO has been shown to inhibit cellular respiration in E. coli by disrupting the permeability barrier of microbial membranes the oil causes the cells to die (Cox et al., 1998). De Prijck et al. (2008) indicated death of E. coli, and S. aureus after exposure to a mixture of TTO and jojoba oil. Furthermore, a large variety of bacteria have currently been tested for their susceptibilities to TTO and numerous studies give support the powerful antibacterial activity of this oil against E. coli, S. aureus (Raman et al., 1995) (Carson et al., 1995) and Staphylococcus epidermidis (S. epidermidis). There is little known of the effect of TTO on Staphylococcus saprophyticus (S. saprophyticus). Antibacterial activity has been determined through use of the agar or broth dilution method however, it had been reported that activity has also been demonstrated using time-kill assays, suspension tests and "ex vivo"-excised human skin (Carson et al., 2006).

This research will investigate the antibacterial effectiveness of Aloe vera juice, aqueous and ethanolic extracts as well as TTO using the agar disc diffusion and broth microdilution methods. The aim of this study is therefore to provide evidence for the growth inhibitory property of both compounds on E. coli, S. aureus, S. epidermidis, S. saprophyticus and MRSA in order to supplement the findings other studies. This research is important as if proven effective, this would validate to use of Aloe vera and TTO in new drug/therapeutics development as a way to retard or cease the problem of antibiotic resistance world-wide. The research hypothesis is that TTO will exhibit greater antibacterial effects than Aloe vera. The second hypothesis is that MRSA, E. coli and S. aureus will be more susceptible to TTO as shown by previous studies.

2. MATERIALS AND METHODS

2.1 Aloe vera

The experiments described in this paper have used Aloe vera powder from capsules (containing leaf extract) obtained from the company Forever Living Products and a commercial health drink preparation of Aloe vera: Maximum Strength Aloe Vera Juice Drink (containing undiluted and unfiltered whole leaf extract and inner gel) acquired from Holland & Barrett.

In the study, Aloe vera powder was weighed and dissolved by two methods, using ethanol and sterile water. The Aloe vera solutions (aqueous and ethanolic) were heated in a 45°C water bath to encourage dissolving. The juice was used without dilution in the trial of antibacterial activity.

2.2 Tea Tree Oil

The TTO was purchased from Boots and was deemed to be 100% pure. The TTO was prepared and contained 0.25% (v/v) Tween 80. Use of this emulsifier enhanced TTO solubility.

2.3 Growth and maintenance of test microorganisms

Clinical isolates of S. aureus, MRSA, E. coli, S. epidermidis and S. saprophyticus were obtained from University College Hospital London and laboratory stocks at the University of Westminster. The bacterial isolates were maintained by subculturing weekly onto LB agar and incubation at 37°C for 24 hours.

2.4 Preparation of bacterial concentrations

For the assays described in this paper, a single colony was taken from an agar plate and diluted in 10 ml LB broth within a McCartney bottle. A spectrophotometer was used to measure optical density at A625 nm. Further dilution or addition of colonies was used to achieve a 0.5 McFarland standard. After which a 1:20 dilution was carried out on all suspensions using LB broth except E. coli which was diluted 1:100 in order to ascertain semi-confluent growth.

2.5 Antibacterial activities- Disc diffusion assay

Aloe vera juice, ethanolic and aqueous extracts as well as TTO were tested by the disc diffusion method. This antibacterial susceptibility test is routinely used in hospitals to test for antibiotic-resistant bacteria. The test microorganisms were spread onto Iso-Sensitest agar (Oxoid, Basingstoke, UK) using separate sterile cotton buds. Sterile filter paper discs (Whatman: 5mm) were individually impregnated with the undiluted Aloe vera juice, extracts and oil (30 μL disc-1) and firmly placed on the test organism plates. Antibiotics, neomycin and fusidic acid (Oxoid) were used as positive controls. The antibacterial assay plates were incubated at 37°C for 24h.The presence of zone of inhibition was regarded as the presence of antibacterial action and activity and was expressed in terms of average diameter measured in millimetre. Each test was carried out in triplicate.

2.6 Antibacterial activities- Broth microdilution method

A broth microdilution method was used to determine the minimum inhibitory concentration (MIC) according to the National Committee for Clinical Laboratory Standards (NCCLS, 1999). The MIC is defined as the lowest concentration of the essential oil at which the microorganism does not demonstrate visible growth. This method was utilised on S. aureus, E. coli and S. saprophyticus. Using LB broth as the diluent, a 2-fold serial dilution ranging from 100%-0.2% (v/v) was carried out on TTO within the 96-well microtitre plate. To the series of dilutions 5µl of the bacterium was added. The positive control comprised of LB broth and the test organism, and the negative control comprised of LB broth only. The microtitre plate was covered and incubated at 37oC for 24 hours. Broth microdilution assays were performed in triplicates.

2.7 Minimum bactericidal concentration (MBC) assay

The broth microdilution method was also used to determine the Minimum Bactericidal Concentration (MBC). The MBC is defined as the lowest concentration of the essential oil at which inoculated microorganisms were completely killed. 10 μL of the test inoculum was taken from specific wells of the microtitre plate and spread onto agar. This inoculated plate was incubated at 37oC for 24 hours and then observed whether any growth had taken place.

Statistical analysis

Mean values were determined for all clinical isolates tested. Data was expressed as mean ± SD.

RESULTS

3.1 Antibacterial effectiveness- Agar disc diffusion assay

The full results of the agar disc diffusion assay to determine the antibacterial effectiveness of Aloe vera and TTO are presented in Table 1 and Table 2.

Table 1 Antibacterial activity of Aloe vera juice, ethanolic and aqueous extract obtained by the disc diffusion method.

Type of Bacteria

Bacteria

Zone of Inhibition (mm)

Ethanolic extract

Aqueous extract

Juice

Neomycinb (10µg)

Gram-positive

S. aureus

- a

-

-

20

MRSA

-

-

-

-

S. epidermidis

S. saprophyticus

-

-

-

-

-

-

21

26

Gram- negative

E. coli

-

-

-

17

a- = no inhibition detected

b = controls

As shown in Table 1, undiluted Aloe vera juice, ethanolic and aqueous extract demonstrated no inhibitory activity against the clinical isolates of Gram-positive bacteria, S. aureus, MRSA, S. epidermidis, and S. saprophyticus as well as Gram-negative bacteria, E. coli.

Table 2 shows the zones of inhibition formed by TTO when used neat in the disc diffusion method. All clinical isolates were inhibited by 100% v/v TTO with zone sizes of 13.7-34.7 mm.

Table 2 Antibacterial activity of TTO at 100 (%v/v) at various concentrations obtained by the disc diffusion method.

Type of Bacteria

Bacteria

Zone of Inhibition (mm)

100% v/v Neomycin b Fusidic acid b

(10µg) (5µg)

Gram- positive

S. aureus

34.7± 0.58 19 38

MRSA

13.7± 0.58

Gram-negative

S. epidermidis

S. saprophyticus

E. coli

15.7± 0.58

21.7± 0.58

23.7 ± 0.58

Values are mean inhibition zone (mm) ± S.D of three replicates

a = no inhibition detected

b= controls

S. aureus had the largest zone of inhibition of 34.7mm± 0.58 which was also larger than the control antibiotic neomycin. 100 % v/v TTO had the least inhibitory effect on MRSA which had the smallest zone of inhibition 13.7mm± 0.58. E. coli and S. saprophyticus were relatively sensitive to TTO having zone sizes of, 23.7mm± 0.58 and 21.7 mm± 0.58 respectively.

S. aureus, E. coli and S. saprophyticus were the inhibited the most than the other two clinical isolated test so they underwent further susceptibility testing at lower TTO concentrations (see figure 1). At TTO concentration of 20% v/v, the growth of S. saprophyticus and E. coli only were subdued by TTO with inhibition zone sizes of 8.5mm± 0.7 and 8mm±0 respectively. At 10%v/v TTO no zones of inhibition was formed for the three clinical isolates.

Error bars indicate ±SD

S. aureus formed the largest zones of inhibition, 31 mm± 0.71 and 20mm±1.41 at concentrations of TTO 80% v/v and 60% v/v in comparison to the other bacteria. At 80% v/v TTO E. coli had the smallest zone of inhibition thus was the inhibited the least by the activity of TTO. It was noted however that margin of error indicated by the SD value was fairly large.

Antibacterial effectiveness- Broth microdilution assay

The full results of the broth micro dilution assay for MIC and MBC determination are presented in Table 3. It is clear that E .coli isolates were the most susceptible to TTO with MIC and MBC of 0.39% (v/v) and 3.13 %; S. aureus and S. saprophyticus isolates were comparably less susceptible. Both obtained the same MIC and MBC values of 1.56% (v/v) and 6.3% respectively. The MBC were usually four times higher than the MIC for S. aureus and S. saprophyticus whereas the MBC for E. coli was eight times more that the MIC.

Table 3 MIC and MBC of TTO (%v/v) against different Gram-positive and Gram-negative bacteria obtained by the broth microdilution method.

Type of Bacteria

Bacteria

MIC

MBC

Gram-positive

S. aureus

1.56

S. saprophyticus

1.56

Gram- negative

E. coli

0.39

In comparing of the agar disc diffusion method and the broth microdilution assay, it is clear that the latter method is more effective as it shows that a small concentration of TTO has antibacterial activity.

The general trend shown by Figure 2 is that addition of Tween 80 to TTO reduces MIC values by 4-folds. The MIC values of TTO with and without Tween 80 is the same for S. aureus and S. saprophyticus (6.3%, 1.56% respectively). The MIC retained with and without Tween 80 for E. coli was four times less than that of the other two isolates.

DISCUSSION

The finding of the study suggests that Aloe vera juice, aqueous and ethanolic extracts have no antibacterial properties whereas TTO has high levels of antibacterial activity on the species of bacteria tested. This research therefore supports the first hypothesis that TTO would exhibit greater antibacterial activities than Aloe vera. The second hypothesis that MRSA, E. coli and S. aureus would be more susceptible to TTO than the other isolates tested is partly supported as E. coli and S. aureus were two of the three most susceptible clinical isolates tested.

This investigation has its strength in that two methods were utilised in the testing, both of which are commonly used in susceptibility tests. Moreover each testing was performed in triplicates in order to verify the results retained. Furthermore the addition of the surfactant, Tween 80 to TTO increased its solubility thus enabled the oil to be better absorbed in the disc diffusion method and allowed better amalgamation within the broth microdilution assay. Consequently the antibacterial effect of TTO was enhanced as shown by Figure 2. Regardless of this, as with any investigation, this study has a few weaknesses. Firstly, only a limited number of bacteria were tested so this study only provides verification of antibacterial activities on a narrow spectrum of bacteria. Secondly, of the five clinical isolates tested in the agar disc diffusion method further testing with different concentrations of TTO was carried out on the three most susceptible isolates. Third, difficulties were encountered in dissolving the Aloe vera capsule using both distilled water and ethanol. The powder from the capsules did not dissolve completely therefore the liquid fraction was used when the leaf extract had settled. The possible implication is that the antibacterial components was not fully released so would explain the lack of activity. Another setback was during the broth microdilution method, the dispersion of the TTO resulted in a turbid suspension which made determination of the MIC difficult.

It is quite complicated and in some case impossible to compare the finding of studies relevant to this investigation as a range of methodologies has been used by various researchers in the evaluation of antibacterial activity. However, the usefulness of this study is that it utilised two of the most popular methods which enable comparisons to be made with a few other studies. The finding of the ineffectiveness of Aloe vera juice on S. aureus and E. coli has been replicated by Alemdar & Agaoglu (2009). The aqueous and ethanolic extracts of Aloe vera capsules used in this study are evidently deficit of constituents which possess antibacterial activity unlike other studies which utilised the pure Aloe vera plant (leaf or gel) extract. Considering the work of Pandey & Mishra (2010), it was shown that leaf extract of Aloe vera (both aqueous and ethanolic) inhibited Gram-positive bacteria, S. aureus. It was generally found that the inhibitory activity was extremely low in aqueous extract (3-4±SE mm) in comparison to ethanolic extract (20-30±SE mm). Additionally, the same pattern was also observed with the Gram-negative bacterium, E. coli with ethanol extract but no inhibitory effect has been noted for aqueous extract. This could possibly be due to active component of Aloe vera being extracted in ethanol rather than water. The success of this investigation lies in the fact that the actual plant material (the leaf extract) was used with the added benefit of an effective extraction strategy that preserved the antibacterial constituents of the Aloe.

In the case of TTO, several published reports have addressed minimum inhibitory and bactericidal concentrations of TTO against clinical isolates of S. aureus. Using the broth microdilution assay in this study, the MIC and MBC for clinical isolates of S. aureus was found to be 1.56% and 6.3%. However, this cannot be regarded as fully conclusive as research by Banes-Marshall et al. (2001) presented MIC and MBC to be 0.5% and 1 %, respectively for S. aureus isolates. Although it should be noted that the testing was carried out in duplicate. A study of 105 clinical isolates of S. aureus using a broth microdilution method found the MIC90 of TTO to be 0.5%. This is further supported by various researchers that used the same method and reported mean MICs and MBCs of 0.5% (range: 0.12-0.5%) and 2% (range: 0.25-2%), respectively. Another study examining several species of normal skin flora reported MIC90 for 4 isolates of S. saprophyticus, 0.25% to 1%. The result retained is relatively similar to that gained within this study although the MIC is slightly higher (1.56%). In this study, E. coli was particularly sensitive to the antimicrobial action of TTO. The possible reason for this has been explored in a report by Gustafson et al. (1998) which states that TTO is able to stimulate autolysis particularly in the exponential but also in the stationary phase of E. coli. Carson et al. (2006) demonstrated that quite a few studies have documented MICs and MBCs of E. coli to be within the range 0.08-2% and 0.25-4% respectively. The value retained for E. coli in this investigation was within the range of documented studies, 0.39% and 3.31% MIC and MBC in that order. The problem however lies in that there are slight variations in the methodologies used by each researcher. Some researchers also used a coloured dye as an indicator of the MIC in the broth microdilution however the success rate of this method has been mixed.

In the review of literatures, other studies have used different types of TTO; different brands have variability within batches which means, components of the oil deemed to have antibacterial properties have different concentration. The most popular methods utilised were the disc/well diffusion or the agar/broth dilution method. Although many laboratories use the disc diffusion method, for example, quite often the incubation times vary, different agar recipes are used as well as different volumes of test substance thus results by different researchers are often incomparable. Similarly published papers reporting antibacterial activities through use of the broth microdilution method demonstrate large variation in the technique and method used. Moreover the surfactant and solvents vary among studies, some researchers use Tween whilst other use dimethylsulphoxide (DMSO) and ethanol. A single in vitro study reported that the antibacterial activity of TTO is compromised by the presence of organic matter or the surfactants Tween 20 and Tween 80. It is also mentioned that these effects vary between organisms. This was not the case in this study but this highlights the possibility that the results retained by other studies may have been affected by the use as well as the choice of surfactant. It has therefore been recommended that the concentration of Tween 80 be kept to a minimum although in this study a concentration of 0.25% v/v Tween 80 was used. Perhaps Tween 80 alone should have undergone testing via the agar disc diffusion method to see whether it has any antibacterial properties. Further investigation is required to see whether this surfactant has inhibitory or stimulatory effects on TTO as there is a clear disagreement between various studies.

These findings are important as it negates the use of Aloe vera as a potential antibacterial agent for the types of bacteria used in this study. In the case of TTO, this provides further supplementary evidence for its highly effective antibacterial activity thus encourages its potential use in drug development and therapeutic management. Although extensive research in still required in order to acquire Food and Drugs Administration (FDA) approval. This research and many others have implications on policy makers as well as clinicians in that direct evidence is provided on the antibacterial effectiveness of TTO. It conveys further awareness to the potentiality of TTO in drug development to remedy various bacterial infections. Although shown to be effective, various literatures have highlighted the need for a standard and reproducible method for assessing the antibacterial activity of TTO. Without a standardized reliable method it is virtually impossible to elucidate a true picture of the bioactivity, therapeutic potential and clinical utility of this essential oil and others. Therefore future research should focus on thorough evaluation the antibacterial properties in vivo, and comprehensive safety data obtained, before TTO can be accepted as a therapeutic topical antimicrobial agent. Some studies have shown that TTO can very toxic in nature. Case reports of TTO have shown it to cause dermal sensitivity, contact dermatitis, and oral toxicity (Halcón & Milkus, 2003). For these reasons further investigation is required on the interactions of various components within TTO and also to possibly extract the most antibacterial component of TTO (terpinen-4-ol) to avoid this predicament.

Investigation of various literatures has brought awareness that specific guidelines are also required for the use of Tween as a surfactant for TTO. There needs to be stipulations on the whether it should be used and the necessary concentration for specific types of bacteria if used at all. This is highly important as a study by Ann et al. (2007) on E. coli, S. aureus and MRSA has shown that habituated cultures (exposed to sub-lethal (0.25%) concentrations of TTO) displayed reduced susceptibility to a range of clinically relevant antibiotics compared with non-habituated (control) cultures. The implication this has is that its application at excessively low concentrations may contribute to the development of even greater antibiotic resistance in human pathogens (Ann et al., 2007).

Aloe vera juice, aqueous and ethanolic extract demonstrated no antibacterial properties however this is could be attributed to the choice of material and the methodology used in the testing. Therefore more investigation is required on the actual gel of Aloe vera, by means of different testing methods such as the agar dilution method. Further investigation would also be required on a greater number of bacteria. As therapeutic management is relying greatly on discovery of innovative drugs from natural resources, this investigation could be further expanded by testing other natural compounds noted to have antibacterial properties, for example garlic and honey.

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