Antibacterial Properties Of Aloe Vera Tea Tree Oil Biology Essay

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Abstract

This study investigates the in vitro activity of Aloe barbadensis Miller (Aloe vera) and Malaleuca alternifolia ( Tea Tree Oil) against a number of clinical bacterial isolates. The isolates included Methicillin-Resistant Staphylococcus aureus (MRSA), S. epidermidis, E. coli, S. aureus and S.saprophyticus. The two methods utilised were the disc diffusion and broth microdilution assays. No antibacterial activity was demonstrated with the use of Aloe vera juice, aqueous and ethanolic extracts on all five isolates tested using an agar diffusion method. Using the same method, TTO was found to be effective and had inhibitory effects on the growth of all isolates. The size of inhibition zones of TTO all isolates tested were 13.5- 34.5mm. A broth microdilution method was used to determine Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of S. aureus, E. coli, S. saprophyticus. S. aureus and S. saprophyticus were the 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.

Introduction

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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 (ref). 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 (ref). 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 remain largely anecdotal and scientific evidence is often sparse and inconsistent (ref). 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 publicized 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 the juice to be more effective against Gram positive bacteria with the exception of S. aureus. An inhibitory effect was observed against Gram positive bacteria, Klebsiella pneumonia (K. pneumonia) 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. Pneumonia.

Tea tree Oil (TTO) is derived from the leaves of the Melaleuca alternifolia plant and has been used for centuries as a botanical medicine (ref). This oil has also attracted considerable interest for potential use in wound therapy. The main chemical component to have antimicrobial activity in tea tree oil is attributed to terpinen-4-ol. 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 (year). Of the 64 MRSA isolates examined (33 of which were mupirocin-resistant), all were establish as being susceptible to TTO. The work of .... also supports the evidence gathered by Carson ( ) in the susceptibility of MRSA 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 broad range of bacteria have now 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 using agar or broth dilution methods. However, activity has also been demonstrated using time-kill assays (refs), suspension tests and "ex vivo"-excised human skin (ref).

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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 therefore is 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 and a commercial health drink preparation of Aloe vera (containing undiluted and unfiltered whole leaf extract and inner gel) obtained from the company Forever Living Products distributed by Holland & Barrett.

In the study, the powder was weighed and dissolved by two methods, using ethanol and sterile water. The Aloe vera solutions (aqueous and ethanol) was 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 5 ml LB broth. A spectrophotometer was used to measure turbidity at 625nm. 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 clinical isolates except E. coli which was diluted 1:100.

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 (ref). This antibacterial susceptibility test is routinely used in hospitals to test for antibiotic-resistant bacteria. The test microorganisms were spread onto Iso-sensitest medium 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 placed on test organism plates by pressing gently. Antibiotics Neomycin and Fusidic acid were used as positive control and as negative control. 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 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% was carried out on TTO within the 96-well microtitre plate. The positive control comprised of LB broth and test organism, and the negative control comprised of LB broth only. The microtitre plate covered and incubated at 37oC for 24 h. 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 (MBC). The MBC is defined as the lowest concentration of the essential oil at which inoculated microorganisms were completely killed. 10 μL taken from specific wells and spread onto agar, incubation at 37oC and observed whether any growth took place.

Statistical Analysis

Results

Antibacterial effectiveness- Agar disc diffusion assay

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The full results of the agar disc diffusion assay to determine the antibacterial effectiveness of Aloe vera and TTO are presented in Table 2 and Table 3.Values from Table 1 (below) was used to evaluate the bacterial response to each compound (Johnson and Case, 1995).

Table 1.

Diameter of zone of inhibition (mm)

Resistant

10 or less

Intermediate

11-15

Susceptible

16 or more

Table 2 Antibacterial activity of Aloe vera by the disc diffusion method.

Type of Bacteria

Bacteria

Zone of Inhibition (mm)

Ethanolic extract

Aqueous extract

Juice

Neomycin

-g/ml

Fusidic acid

Gram positive

Staphylococcus aureus

- a

-

-

20

38

MRSA

-

-

-

-

40

Staphylococcus epidermidis

Staphylococcus saprophyticus

-

-

-

-

-

-

21

26

12

33

Gram negative

Escherichia coli

-

-

-

17

-

a-, no inhibition detected

As shown in Table 2, Aloe vera in various forms demonstrated no antibacterial activity against the 5 clinical isolates tested.

Table 3 Antibacterial activity of TTO (%v/v) at various concentrations by the disc diffusion method

Type of Bacteria

Bacteria

Zone of Inhibition (mm)

Concentration of TTO (%v/v)

100

80

60

40

20

10

Gram positive

Staphylococcus aureus

34.5

30.5

20

7

-

-

MRSA

13.5

-

-

-

-

-

Staphylococcus epidermidis

Staphylococcus saprophyticus

16

22

-

21

-

16

-

12

-

8.5

-

-

Gram negative

Escherichia coli

22.5

20

17

10.5

8

-

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

Of the 5 clinical isolates tested, 4 were deemed to be susceptible and 1 intermediate to TTO (%v/v) with zone sizes of 13.5-34.5 mm. At 100% v/v TTO, S. aureus, E. coli and S. saprophyticus were found to be the most susceptible to TTO having zone sizes of 34.5, 22.5 and 22mm respectively. These three isolates underwent further susceptibility testing at lower TTO concentrations. All were relatively susceptible (zone sizes >16mm) up until a TTO concentration of 40% where there were at an intermediate level. At 20% TTO, only S. saprophyticus and E. coli were resistant to TTO.

The full results of the broth micro dilution assay for MIC and MBC determination are presented in table 4. It is clear that E .coli isolates were the most susceptible to TTO with MICs of 0.39% 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.

Table 4 MIC and MBC of TTO (%v/v) against different Gram positive and Gram-negative bacteria.

Type of Bacteria

Bacteria

MIC

MBC

Gram positive

Staphylococcus aureus

1.56

6.3

Staphylococcus saprophyticus

1.56

6.3

Gram negative

Escherichia coli

0.39

3.13

Graph 1. MIC values gained through the broth microdilution assay of TTO alone and TTO supplemented with Tween-80.

The general trend shown by graph 1 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. The MICs retained with and without tween-80 for E. coli was 4 times less that of the other two isolates.

Discussion

The finding of the study suggests that Aloe vera juice, aqueous and ethanolic extracts has no antibacterial activity 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. Regardless of this, the investigation has its weaknesses as well as strengths. One of the strengths is the use of tween-80 which helped to solve the problem of TTO insolubility. This enabled the oil to be better absorbed in the disc diffusion method and allowed better incorporation in solution for the broth microdilution method. The weakness however is that, although Tween makes TTO more soluble, in a study by..... it was shown that varying concentrations of Tween compromises the effects of TTO thus greatly affects the size of the zones of inhibition. However it is also mentioned that these effects vary between organisms. Contrary to this, a single in vitro study reported that the antibacterial activity of tea tree oil is decreased by the presence of organic matter or the surfactants Tween 20 and Tween 80. It has therefore been recommended by so and so that the concentration be kept to a minimum within the range..... however for this study a concentration of 0.25% v/v Tween 80 was used. Furthermore the range of bacterial isolates tested was limited an it was also difficult to visibly judge the MIC for the broth microdilution method as TTO in aqueous solution was quite cloudy.

In the case of Aloe vera, difficulties were encountered in dissolving of the capsule using distilled water and ethanol. Additionally, in this study Aloe vera juice and capsule dissolved in sterile water and ethanol was used rather than the pure plant extract thus lacks the constituents which possess antibacterial activity. It is quite difficult to directly compare the results retained in this study to the findings gathered by other researchers. This is due to the fact that a range of methodologies have been used by various researchers in the evaluation of antibacterial activity such as use of pure Aloe vera plant or various parts, whether leaf or gel.

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 various among studies, some researchers use Tween whilst other use dimethylsulphoxide (DMSO) and ethanol. Due to the reasons stated above, there is a need for a standard and reproducible method for assessing the antibacterial activity of the TTO. Nevertheless, the finding of the ineffectiveness of Aloe vera juice on S. aureus and E. coli has been replicated by Alemdar & Agaoglu (2009). In addition, several published reports have addressed minimum inhibitory and bactericidal concentrations of TTO against clinical isolates of S aureus. Mean MICs and MBCs of 0.5% (range: 0.12-0.5%) and 2% (range: 0.25-2%), respectively have been reported for clinical isolates of S. aureus via the broth microdilution assay. A study of 105 clinical isolates of S aureus using a broth microdilution method found the MIC90 of TTO to be 0.5%. 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, MIC being 1.56. The difference however lies in that....

Staphylococcus aureus

0.5-1.25

1-2

13, 32, 126

Escherichia coli

0.08-2

0.25-4

13, 32, 67, 104

These findings are important as it disproves the use of Aloe vera as a potential antibacterial for the bacteria types 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.

Comparing the results between previous studies has been difficult due to use of different methods to measure the antibacterial activities of TTO. However in this study, two standardised methods were used, the disc diffusion and broth microdilution method. This allows a direct comparison to be made between the results retained in this study to others that have utilised the same techniques

Clear statement of conclusions:

In this study, E.coli was particularly sensitive to the antimicrobial action of TTO. These antimicrobial properties now must be evaluated thoroughly in vivo, and comprehensive safety data obtained, before TTO can be accepted as a therapeutic topical antimicrobial agent.

This research suggests that Aloe vera juice has no antibacterial properties whereas TTO has proven to be a very effective antibacterial agent. This reinforces its use in various therapies, wound therapy?... However this study does highlight the need for a standard and reproducible method for assessing the antibacterial activity of the TTO

Without a standardized reliable method it is virtually impossible to elucidate a true picture of the bioactivity, therapeutic potential and clinical utility of essential oils.

Proposed future work: Errors/improvement

Test the actual gel of aloe vera

Use different testing methods

Increase the number of bacterial strains to test

Test other natural products,

Note: can say in discussion, the value retained for E coli was within the range of documented studies.