Effects of Tea Tree Oil on Biofilm Formation

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RIRDC Publication No. 08/140

RIRDCInnovation for rural Australia

Effects of Tea Tree Oil on Biofilm Formation

by KA Hammer, CF Carson, T-J Tan & TV Riley

September 2008

RIRDC Publication No 08/ 140 RIRDC Project No PRJ-000451

ISBN 1 74151 729 X ISSN 1440-6845

Effects of Tea Tree Oil on Biofilm Formation

Publication No. 08/140 Project No. PRJ-000451

The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances.

While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication.

The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors.

The Commonwealth of Australia does not necessarily endorse the views in this publication.

This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165.

Researcher Contact Details Katherine A. Hammer Microbiology and Immunology School of Biomedical, Biomolecular and Chemical Sciences The University of Western Australia Phone: (08) 9346 1986 Fax: (08) 9346 2912 Email: [email protected]

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.

RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6271 4100 Fax: 02 6271 4199 Email: [email protected]. Web: http://www.rirdc.gov.au

Published in September 2008 by Union Offset

Foreword

The ability of tea tree oil to inhibit and kill a wide range of microorganisms means that it has great potential not only as a topical antimicrobial agent for use in humans but also as a biocide in industrial applications. One of these potential applications is in the prevention of biofilm formation or the remediation of surfaces on which biofilms have formed. This report provides data on the ability of tea tree oil to prevent and disinfect or disrupt biofilm. It provides additional evidence for the antimicrobial activity of the oil and yet another avenue for future product development.

Biofilm formation is becoming recognised as a key step in many infections. Prevention of this process as well as destruction of pre-formed biofilm are key strategies in controlling infectious disease. Apart from the key role of biofilm formation in many infectious disease processes, biofilm formation is a major problem in industrial settings such as water reticulation, sewage treatment, food manufacture and any setting where sufficient water and nutrients are available for microorganisms to grow.

With careful formulation designed to preserve the antimicrobial activity of tea tree oil, followed by large-scale testing of these products on biofilm formation and biofilm destruction, this may represent another property of tea tree oil that has widespread application in medical and industrial settings. The purpose of this project was to provide initial data characterising the effect of tea tree oil on biofilms which is why RIRDC has invested in this report.

The importance of this report is that it provides basic data demonstrating the ability of tea tree oil to inhibit the formation of biofilms and to destroy pre-existing ones. It will be a useful basis for those contemplating investment or formulating products and will help support the marketability of tea tree oil internationally.

This project was funded from industry revenue which is matched by funds provided by the Australian Government.

This report, an addition to RIRDC’s diverse range of over 1800 research publications, forms part of our Tea Tree Oil R&D program, which aims to investigate the efficacy of tea tree oil and develop new products.

Most of our publications are available for viewing, downloading or purchasing online through our website:

• downloads at www.rirdc.gov.au/fullreports/index.html • purchases at www.rirdc.gov.au/eshop

Peter O’Brien Managing Director Rural Industries Research and Development Corporation

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Acknowledgments

This work was supported with an industry contribution and tea tree oil samples from Pat and Paul Bolster, Gelair Pty. Ltd.

We are grateful for the technical, financial and institutional support of the Discipline of Microbiology & Immunology, School of Biomedical, Biomolecular and Chemical Sciences at The University of Western Australia and the Division of Microbiology and Infectious Diseases at PathWest Laboratory Medicine WA.

Abbreviations

ATCC American Type Culture Collection GRAS Generally recognized as safe MCC Minimum cidal concentration MIC Minimum inhibitory concentration NCTC National Collection of Type Cultures nm Nanometres OD Optical density PBS Phosphate buffered saline RT Room temperature SBF Specific biofilm formation TSB Trypticase soy broth TSBG Trypticase soy broth with 0.25% glucose TSBS Trypticase soy broth supplemented with 1% sodium chloride v/v Volume for volume w/v Weight for volume XTT 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide YEPD Yeast extract peptone dextrose

Contents Foreword...... iii Acknowledgments ...... iv Abbreviations ...... iv Contents ...... v List of Tables...... vi List of Figures ...... vii Executive Summary...... viii Introduction ...... 1 Materials and Methods...... 2 Tea tree oil...... 2 Organisms ...... 2 Determination of minimum inhibitory and cidal concentrations ...... 2 Biofilm formation...... 3 Destruction of formed biofilm...... 3 Quantification of biofilm...... 4 Crystal violet staining ...... 4 XTT metabolism ...... 4 Regrowth of viable biofilm organisms...... 4 Standardisation of data and statistical analyses...... 5 Results ...... 6 C. albicans...... 9 V. harveyi...... 9 S. maltophilia ...... 9 Destruction of pre-formed biofilm ...... 25 Discussion ...... 29 Implications ...... 31 Recommendations ...... 31 References ...... 32

List of Tables

Table 1. Composition of the tea tree oil used throughout the study...... 2 Table 2. Susceptibility data for test organisms determined by broth microdilution ...... 7 Table 3. Lowest concentrations resulting in a significant decrease in biofilm formation by S. epidermidis compared to controls...... 8 Table 4. Lowest concentrations resulting in a significant decrease in biofilm formation by P. aeruginosa compared to controls ...... 8 Table 5. Lowest concentrations of tea tree oil (% v/v) resulting in a significant decrease in biofilm formation by C. albicans compared to controls...... 9

List of Figures

Figure 1. Relative biofilm formation (mean and standard deviation) by five different isolates of S. epidermidis...... 10 Figure 2. Relative biofilm formation (mean and standard deviation) by five different isolates of S. epidermidis...... 11 Figure 3. Relative biofilm formation (mean and standard deviation) by (1) P. aeruginosa ATCC 27853 and (2) P. aeruginosa NCTC 8505 under three different incubation conditions...... 12 Figure 4. Relative biofilm formation (mean and standard deviation) by (3) P. aeruginosa NCTC 10662 and (4) P. aeruginosa PAO1 under three different incubation conditions ...... 13 Figure 5. Relative biofilm formation (mean and standard deviation) by (5) P. aeruginosa 27399 (clinical isolate) and (6) P. aeruginosa 27455 (clinical isolate) under three different incubation conditions...... 14 Figure 6. Relative biofilm formation (mean and standard deviation) by (7) P. aeruginosa 4969 (clinical isolate) and (8) P. aeruginosa 5040 (clinical isolate) under three different incubation conditions...... 15 Figure 7. Relative biofilm formation (mean and standard deviation) by (9) P. aeruginosa 5052 (clinical isolate) and (10) P. aeruginosa 4938 (clinical isolate)...... 16 Figure 8. Relative biofilm formation (mean and standard deviation) by (1) C. albicans ATCC 90028 and (2) C. albicans ATCC 90092...... 17 Figure 9. Relative biofilm formation (mean and standard deviation) by (3) C. albicans ATCC 10231 and (4) C. albicans 42J (clinical isolate)...... 18 Figure 10. Relative biofilm formation (mean and standard deviation) by (5) C. albicans 68E (clinical isolate) and (6) C. albicans 57X (clinical isolate)...... 19 Figure 11. Relative biofilm formation (mean and standard deviation) by (7) C. albicans KE216 (clinical isolate) and (8) C. albicans LL031 (clinical isolate)...... 20 Figure 12. Relative biofilm formation (mean and standard deviation) by (9) C. albicans 45E (clinical isolate) and (10) C. albicans FF225 (clinical isolate) ...... 21 Figure 13. Relative biofilm formation (bars, mean and standard deviation) and planktonic growth...... 22 Figure 14. Relative biofilm formation (mean and standard deviation) by S. maltophilia ATCC 13637 after 24 h in TSB with tea tree oil...... 25 Figure 15. Effects of tea tree oil on pre-formed S. epidermidis...... 26 Figure 16. Effects of tea tree oil on pre-formed P. aeruginosa NCTC 10662 biofilm ...... 27 Figure 17. Effects of tea tree oil on pre-formed S. maltophilia ATCC 13637 biofilm ...... 28

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Executive Summary

What the report is about This report details the results of an investigation into the ability of tea tree oil to prevent the formation of or disrupt existing biofilm, known in lay terms as “slime”. The importance of biofilm in industrial and commercial settings has been appreciated for decades since its formation fouls production lines, disrupts production and its management is often problematic contributing significantly to production time and costs. In medical settings, the importance of biofilm is becoming more obvious; it is now implicated in many types of infections including those of wounds, the oral cavity, ears and on devices such as catheters and pacemakers.

Who the report is targeted at This report is targeted at manufacturers and marketers of tea tree oil products. It provides information regarding yet another mechanism by which tea tree oil can potentially control microorganisms. Further testing of appropriate formulations may lead to products for this significant market.

Background Tea tree oil has a well-characterised spectrum of antimicrobial activity and some of the mechanisms of action have been elucidated. However, no previous work has systematically investigated the activity of the oil against the formation of biofilm by a number of isolates of Gram positive and negative bacteria and the yeast Candida albicans. Since biofilm formation is an important process used by microorganisms to colonise surfaces, compounds such as tea tree oil that disrupt and prevent this process have many potential applications.

Aims/Objectives The aims of this work were to: 1. examine the effect of tea tree oil on the formation of biofilm, 2. investigate the effects of tea tree oil on existing biofilm, 3. investigate the mechanism(s) by which biofilm formation is influenced and 4. explore potential medical and industrial applications of biofilm inhibition.

Methods The methods used in this study are well-established models of biofilm formation. Briefly, test microorganisms are inoculated into growth medium in 96-well trays and incubated in conditions conducive to biofilm formation. In some experiments tea tree oil is added at the same time as the microorganisms in order to assess the effect on biofilm formation. In other experiments, biofilms are allowed to form and then treated with tea tree oil before the amount and viability of remaining biofilm is measured.

Results When tea tree oil was added at the same time as microorganisms and at concentrations known to inhibit the growth of microorganisms, it was able to significantly compromise the formation of biofilm. That is, it could reduce the amount of biofilm produced as long as the concentration was high enough to also inhibit growth of the microorganism. When tea tree oil was added to previously formed biofilms and allowed to treat the biofilm for 24 hours, significant amounts of the biofilm were removed and the viability of the microorganisms in the remaining biofilm was greatly reduced. Reductions in viability were generally more pronounced that reductions in biomass measured by crystal violet staining, suggesting that tea tree oil reduces the viability of the organisms within the biofilm but has less of an effect the structure or matrix of the biofilm.

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Notably the concentrations of tea tree oil required to inhibit the formation of, cause the destruction of and inactivate the organisms in biofilm were all similar to those that could inhibit the growth of free-living, planktonic microorganisms. This is in marked contrast to other antimicrobial agents for which the concentrations that prevent, destroy and inactivate biofilm are generally much higher than those that inhibit the growth of planktonic microorganisms.

Implications The results of this work suggest that tea tree oil may be useful as an active ingredient in anti- biofilm products. With appropriate product development, this may represent another potential market for tea tree oil. The wide spectrum of applications in industrial and medical situations renders this a massive opportunity to broaden markets for tea tree oil.

Recommendations The results of this study deserve further attention; the spectrum and degree of anti-biofilm activity demonstrated means that tea tree oil should be investigated as an active anti-biofilm ingredient in plastic products. It should also be further investigated as an active ingredient in products formulated to treat, remove and/or prevent the accumulation of biofilm. Product developers, manufacturers and marketers should consider this potential.

Introduction

Tea tree oil has well characterised antimicrobial activity against a broad spectrum of bacteria, fungi and viruses (Carson et al. 2006). Most of the in vitro data for the oil’s activity against bacteria and fungi have been determined against microorganisms growing in suspension in broth cultures; that is, planktonic or free-living organisms (Carson et al. 1995; Carson et al. 1996; Hammer et al. 1996). It is now recognised that the majority of bacteria and fungi grow in matrix-enclosed communities, or biofilms, adherent to surfaces and that these sessile organisms are significantly different from their planktonic counterparts (Donlan and Costerton 2002). Biofilms may be defined as microbially derived sessile communities characterised by cells that are irreversibly attached to substrata or interfaces or to each other, are embedded in a matrix of extracellular polymeric substances that they have produced, and exhibit an altered phenotype with respect to growth rate and gene transcription (Donlan and Costerton 2002). A lay term for biofilm is “slime”. The altered phenotype displayed by biofilms includes changes in the susceptibility of their member microorganisms to antibiotics and biocides, rendering them highly resistant. This makes biofilms difficult to prevent as well as difficult to disrupt or destroy.

Biofilms occur wherever sufficient water, nutrients and microorganisms occur and can fix themselves to any available surface. Some settings where biofilms are problematic include food manufacturing equipment (Lebert et al. 2007), external surfaces of marine vessels and fixings (Cooney and Tang 1999), sewage treatment plants, air-conditioning units and cooling towers (Walker et al. 1999), prosthetic devices used in human and animal health, medical equipment such as endoscopes and colonoscopes and dental irrigation units (Donlan and Costerton 2002).

There is a need for safe and effective biocides for biofilm prevention and destruction. There is also interest in natural compounds for this purpose. Plant essential oils and their components have been suggested as potentially useful anti-biofilm agents (Ramage et al. 2002; Ouhayoun 2003; Niu and Gilbert 2004; Alviano et al. 2005; Filoche et al. 2005; Masako et al. 2005; Jabra-Rizk et al. 2006; Jabra-Rizk et al. 2006; He et al. 2007), particularly in the food industry (Knowles et al. 2005; Lebert et al. 2007; Sandasi et al. 2008) where they are often ingredients in the foodstuffs being manufactured. Many are considered safe to ingest and, within specified concentration limits, are classified with therapeutic or chemical regulatory authorities as “generally recognized as safe” (GRAS) making them particularly attractive as control agents in food manufacturing or topical pharmaceuticals.

This study aims to investigate whether tea tree oil can inhibit the formation of microbial biofilm, and to investigate the effects of tea tree oil on existing biofilm. Further aims were to investigate the mechanism(s) by which biofilm formation is inhibited and to explore potential medical and industrial applications of biofilm inhibition.

Materials and Methods

Tea tree oil Tea tree oil (batch #1216) was provided by Gelair Pty Ltd. and its composition is listed below in Table 1.

Table 1. Composition of the tea tree oil used throughout the study

Component Composition (%) Component Composition (%) terpinen-4-ol 42.4 limonene 1.1 γ-terpinene 20.1 aromadendrene 1.1 α-terpinene 9.0 δ-cadinene 0.8 1,8-cineole 3.7 ledene 0.6 terpinolene 3.2 sabinene 0.3 ρ-cymene 3.1 globulol 0.2 α-terpineol 3.1 viridiflorol 0.1 α-pinene 2.4

Organisms In assays to determine the effect of tea tree oil on biofilm formation, ten isolates each of Pseudomonas aeruginosa, Staphylococcus epidermidis and Candida albicans and nine isolates of Vibrio harveyi were selected. These included the reference strains P. aeruginosa NCTC 10662 and ATCC 27853, S. epidermidis ATCC 12228, ATCC 27626 and NCTC 11047 and C. albicans ATCC 10231, 90028 and 90029. The organism Stenotrophomonas maltophilia ATCC 13637 was also used.

In assays to determine the ability of tea tree oil to destroy or inactivate pre-formed biofilm the clinical isolate S. epidermidis 4513735E and the reference isolates P. aeruginosa NCTC 10662 and S. maltophilia ATCC 13637 were used.

Determination of minimum inhibitory and cidal concentrations

The minimum inhibitory and minimum cidal concentrations (MIC and MCC, respectively) were determined using previously published methods (Hammer et al. 2004; Papadopoulos et al. 2006). Briefly, bacteria were tested in Mueller-Hinton broth and Candida albicans was tested in RPMI 1640 medium. Both media were supplemented with 0.001% Tween 80 to enhance oil dispersion. Serial two-fold dilutions of oil were inoculated with test organisms and incubated. MICs were determined visually by identifying the lowest concentration of tea tree oil that prevented growth as indicated by turbidity. MCCs were determined by sub-culturing 10 μl samples from each well onto fresh medium and, after 24 h incubation, determining the lowest concentration at which 99.99% of the inoculum did not grow.

MICs of V. harveyi were determined during assays on biofilm formation. After the first 24 h incubation at 28°C, the wells were visualised and the presence or absence of growth as indicated by turbidity recorded. Samples of 10 μl were removed from each well, sub-cultured onto fresh media and incubated at 28°C for 24 h after which the MCC was determined as the concentration at which 99.99% of the inoculum concentration did not grow.

Biofilm formation Methods for growing and quantifying biofilms were adapted from methods previously published (Yassien et al. 1995; Ramage et al. 2001; Pitts et al. 2003).

Preparation of inocula Inocula were prepared by culturing organisms overnight in the appropriate growth medium with shaking except for V. harveyi which was not shaken. P. aeruginosa, S. maltophilia and S. epidermidis were grown in trypticase soy broth at 37°C and C. albicans was grown in yeast extract peptone dextrose broth (YEPD) at 35°C. V. harveyi were grown in trypticase soy broth supplemented with 1% sodium chloride (TSBS) and incubated at 28°C.

Cells were collected, washed once in saline and then resuspended in 0.85% saline. Cells were adjusted with 0.85% saline using a nephelometer so that final inocula concentrations were approximately 5 × 106 cfu/ml for P. aeruginosa, S. maltophilia, S. epidermidis and C. albicans. Final inocula concentrations for V. harveyi were approximately 1 × 106 cfu/ml with the final concentration in each well being approximately 5 × 105 cfu/ml.

Preparation of tray for biofilm formation Preliminary investigations for this part of the study indicated that the most biofilm was formed when the polystyrene 96-well flat-bottomed trays manufactured by Nunc™ (Roskilde, Denmark) were used. A series of doubling dilutions of tea tree oil were made in TSB, RPMI 1640 or YEPD broth with a final concentration of 0.001% Tween 80. TSB was supplemented with 0.25% glucose (TSBG) for tests with S. epidermidis and TSBS was used for tests with V. harveyi. Tests with C. albicans were performed in RPMI 1640 and YEPD broths. Tea tree oil concentrations ranged from 8 – 0.12% for P. aeruginosa, from 4 – 0.125% for S. maltophilia, from 2 – 0.031% for S. epidermidis, from 1 – 0.016% for C. albicans and from 1 – 0.0095% for V. harveyi. Three incubation conditions were used for P. aeruginosa: 1) 24 h at 37°C, 2) 6 h at 37°C and 3) 24 h at room temperature. For S. maltophilia, C. albicans and S. epidermidis tests were incubated at 37°C for 24 h.

Destruction of formed biofilm Inocula were prepared by inoculating 1-2 colonies of S. epidermidis or P. aeruginosa into TSBG and 1-2 colonies of Stenotrophomonas maltophilia into TSB and incubating overnight with shaking. Overnight cultures were adjusted to approximately 108 cfu/ml in TSBG. Volumes of 200 μl of prepared inocula were aliquoted into the wells of a 96 well microtitre tray. TSBG without microorganisms was also aliquoted into several rows of the tray to serve a negative control. Trays were placed inside a plastic bag to reduce evaporative loss during incubation. S. epidermidis and S. maltophilia were incubated at 37°C for 24 h while P. aeruginosa was incubated at room temperature for 24 h.

After incubation, the wells of the trays were emptied by inversion and wells were washed once with 200 µL PBS. Volumes of 200 µl of tea tree oil in PBS, at concentrations ranging from 4 – 0.125% (v/v) was added to the tray wells, resulting in 9 replicate test wells and three replicate control wells per tea tree oil concentration. Trays were then re-incubated for 24 h at 37°C. After treatment with tea tree oil well contents were removed by inversion and 200 μl of neutraliser solution (Messager et al., 2005) was added to each well. After 5 min contact with neutraliser, wells were rinsed with 200 μl of PBS. The remaining biofilm was then quantified by crystal violet staining (after air drying and fixing with methanol) or by the regrowth of viable organisms.

Quantification of biofilm Crystal violet staining Trays were stained by aliquoting 200 μl of crystal violet solution into each well of the microtitre tray. Crystal violet stain was prepared as Hucker’s Crystal violet (2% w/v) and was then diluted with distilled water to a final crystal violet concentration of 1.0% (w/v) for C. albicans, 0.3% (w/v) for P. aeruginosa, S. maltophilia and S. epidermidis and 0.1% (w/v) for V. harveyi.

Trays were stained for 5 minutes and then excess stain was rinsed off under running tap water. Trays were air dried and the 200 μl of a 33% (v/v) solution of glacial acetic acid was added to each well to resolubilise the dye. The absorbance of the trays was then read at 540 nm. Trays were shaken prior to reading.

Averages of the duplicate rows were calculated and the relevant blank value (tea tree oil without organism) was subtracted from each optical density reading.

Replication of the method by Niu & Gilbert In assays examining the effect of tea tree oil on V. harveyi biofilm formation, a modified version of the method of Niu & Gilbert (2004) was replicated. This method attempts to control for effects on bacterial growth when determining whether or not biofilm formation is affected by tea tree oil. Trays are prepared, fixed and stained in the same manner except that an additional control of wells containing tea tree oil but no bacteria is included. This control is referred to as the abiotic control and controls for the turbidity of the tea tree oil. In addition, after incubation but prior to emptying the contents of the tray, the optical density of each well is read spectrophotometrically at 600 nm in order to quantify bacterial growth. The tray is also read visually with the aid of a magnifying mirror to determine the MIC (see above) and a 10 μl sample is removed from each well and plated onto fresh agar to determine the MCC. Thereafter, the tray is treated as above.

XTT metabolism A solution of 1 mg/ml XTT was made in PBS and filter sterilised using a 0.22 μM filter. The solution was aliquoted and stored at –80°C until required. A solution of 0.4mM menadione was prepared in ethanol and was stored at 4°C protected from light. Prior to the staining of the trays, a fresh solution of XTT/menadione was prepared in PBS. Volumes of 200 μL of XTT/menadione were added to each tray well and trays were incubated at 37°C for 2 h in the dark. After incubation, volumes of 100 μL were transferred to new trays and the absorbance was read at 490 nm.

Regrowth of viable biofilm organisms The ability of tea tree oil to inactivate pre-formed biofilm was investigated using one isolate each of S. epidermidis, S. maltophilia and P. aeruginosa. The method was based on that of Chambers et al. (2006). Briefly, biofilm was established as in assays for the destruction of pre-formed biofilm, the wells emptied and then treated with 200 μl of tea tree oil in PBS over the range 4 – 0.25%. After 24 h incubation at 37°C or room temperature, respectively, the well contents were discarded and 200 μl of neutralising broth (Messager et al. 2005) added to the wells for 5 min. Once the neutralising broth was discarded, the wells were rinsed with 200 μl of PBS and 200 μl of TSBG was added to all wells. The tray was then incubated for 6 h at 37°C to allow re-growth of viable organisms.

Another tray with one column of sterile TSBG was prepared to act as a control for the growth medium. Both trays were read at 600 nm. Using the sterile TSBG tray as a ‘control blank’, the average and standard deviation was found. If the blanks in the inoculated tray exceeded the control blank average by more than 3 standard deviations, they were considered contaminated.

Standardisation of data and statistical analyses In all experiments, to correct for inter-test variation, optical density values in test wells were made relative to those in control wells. This was done by dividing test values by control values.

A student’s t-test (two-tailed, paired) was used to compare values at each tea tree oil concentration to the inoculated well without tea tree oil (control). Significance was set at p < 0.05.

Results

Susceptibility data The susceptibility of all test isolates was determined for comparative purposes. Results are shown in Table 2.

A comparison of MICs determined for S. maltophilia in both Mueller Hinton broth and trypticase soy broth did not show any differences (ie 1.0% for both MIC and MCC in both media).

Biofilm formation S. epidermidis

Figures 1 and 2 show that biofilm formation was reduced in the presence of tea tree oil. However, as can be seen in Table 3, the lowest concentration resulting in a significant reduction in biofilm formation did not differ dramatically from the MIC, suggesting that the inhibition of biofilm may be a function of reduced growth, rather than a specific inhibition of biofilm. It is not unexpected that biofilm formation was inhibited in all isolates at concentrations of 0.25% and above, since the MIC was either 0.25 or 0.5% for all of the test isolates. It is to be expected that biofilm formation would be inhibited at concentrations that severely restrict microbial growth.

For 7 of the 10 test isolates biofilm formation appears to be increased at low concentrations. These differences were not significant using a two-tailed Student’s t-test. Using a one-tailed test, increases in biofilm formation were significant for isolates 11047 and O1 at the concentration of 0.03% tea tree oil.

P. aeruginosa

Results of biofilm formation for 10 isolates of P. aeruginosa are shown in Figs. 3-7. Each isolate was tested under three different conditions; 24 h at 37°C, 6 h at 37°C and 24 h at room temperature (approx. 22°C). Preliminary experiments demonstrated that experiments incubated for 24 h at 37°C showed a high degree of variability. This was due to the formation of a pellicle at the air-liquid interface of each microtitre tray well. This slimy material would stick to the sides of each well when the well contents were removed by inverting the tray, leading to experimental inconsistencies. Alternative incubation conditions such as only 6 h at 24°C and 24 h at room temperature did not result in the same degree of pellicle formation and as such, more consistent results were achieved.

For tests conducted for 24 h at 37°C, for three isolates of the 10 tested, there were no significant differences in biofilm formation in the presence of tea tree oil. For an additional three isolates only one or two concentrations differed significantly from the controls. Very few isolates showed a dose- dependent decrease in biofilm formation with increasing tea tree oil concentration. This was largely due to the experimental variability which resulted in large standard deviations when data were analysed.

For tests incubated for only 6 h at 37°C, some experimental variability was still present but for many of the isolates a step-wise decrease in formed biofilm was evident with increasing tea tree oil. For all isolates, the three highest concentrations of tea tree oil (ie 2, 4, and 6%) resulted in significantly less biofilm being formed. However, this would be expected as these concentrations are similar to, if not the same as the MICs and MCCs (Table 2).

Table 2. Susceptibility data for test organisms determined by broth microdilution

Organism Strain/isolate Tea tree oil (% v/v) designation MIC MCC Stenotrophomonas maltophilia ATCC 13637 1 1 Pseudomonas aeruginosa ATCC 27853 2 2 NCTC 8505 1 1 NCTC 10662 1 1 PAO1 2 2 27399 2 2 27455 2 2 4969 2 2 5040 1 1 5052 2 2 4938 1 2 Staphylococcus epidermidis NCTC 11047 0.5 1 ATCC 12228 0.5 1 ATCC 27626 0.5 0.5 735E 0.25 1 I2 0.5 1 26025 0.5 2 27314 0.5 1 S2 0.25 1 O1 0.5 0.5 835B 0.25 1 Candida albicans ATCC 90028 0.25 0.5 ATCC 90029 0.25 0.5 ATCC 10231 0.5 0.5 137442J 0.5 1 147168E 0.5 0.5 142457X 0.5 0.5 KE216 0.5 0.5 LL031 0.5 0.5 137645E 0.5 1 FF225 0.5 0.5 Vibrio harveyi 1 0.125 0.25 2 0.125 0.125 3 0.125 0.25 4 0.125 0.25 5 0.125 0.125 6 0.06 0.125 7 0.125 0.125 8 0.125 0.125 9 0.125 0.25

Table 3. Lowest concentrations resulting in a significant decrease in biofilm formation by S. epidermidis compared to controls. MICs and MCCs for planktonic cells are shown for comparison.

Isolate Biofilm inhibitory MIC MCC concentration NCTC 11047 0.25 0.5 1 ATCC 12228 0.03 0.5 1 ATCC 27626 0.03 0.5 0.5 735E 0.03 0.25 1 835B 0.12 0.25 1 S2 0.06 0.25 1 26025 0.12 0.5 2 27314 0.25 0.5 1 O1 0.25 0.5 0.5 I2 0.25 0.5 1

Tests incubated for 24 h at room temperature were also more reproducible than those incubated for 24 h at 37°C. Results generally showed a dose-dependent decrease in formed biofilm with increasing tea tree oil, similar to results obtained after 6 h at 37°C. All isolates showed significantly decreased biofilm at the three highest tea tree oil concentrations although for seven of the isolates biofilm was inhibited at no less than one other tea tree oil concentration.

For tests incubated for 24 h at 37°C, many isolates showed what appears to be increased biofilm formation at 4 and/or 8% tea tree oil. However, these ‘increases’ were not statistically significant, with the exception of isolate PAO1 at 4% tea tree oil. It is difficult to know whether these were true increases in biofilm formation or an experimental artefact as these increases are not seen under the other incubation conditions and occurred at concentrations that are generally severely growth inhibitory, if not bactericidal.

Table 4. Lowest concentrations resulting in a significant decrease in biofilm formation by P. aeruginosa compared to controls (determined by three methods). MICs and MCCs for planktonic cells are shown for comparison.

Biofilm inhibitory concentrations Isolate 24 h, 37°C 6 h, 37°C 24 h, RT MIC MCC 27853 0.25* 0.12 0.12 2 2 8505 0.25* 0.5 2 1 1 10662 0.12* 1 0.25 1 1 PAO1 - 2 2 2 2 27399 1* 1 2 2 2 27455 0.25* 2 0.12* 2 2 4969 - 1 0.25 2 2 5040 0.12* 1 0.5 1 1 5052 - 2 0.25* 2 2 4938 - 1 0.5* 1 2 * One or more concentrations above this were NOT significant (see graphs)

Similar to S. epidermidis, it is also difficult to know whether the decreased biofilm was due to a non- specific decrease in microbial growth or a specific anti-biofilm effect. However, for most isolates, significant biofilm inhibition tended to occur nearest the MIC and/or MCC, suggesting that growth inhibition was the main cause of biofilm inhibition.

C. albicans Figures 8-12 show that there was a dose-dependent decrease in biofilm formation, whereby increasing levels of tea tree oil resulted in less biofilm being formed by C. albicans. This same trend was evident regardless of which growth medium or staining method was used. The ten strains tested were also relatively uniform in their susceptibility to tea tree oil. For some of the test isolates (eg see Figs 9 and 12, graphs 4a and 10a, respectively) biofilm was significantly inhibited at the lowest tea tree oil concentration tested. It is therefore likely that biofilm formation would be inhibited at concentrations even lower than those tested. There were also very few instances where biofilm formation appeared greater in the presence of tea tree oil (see Figs 9 and 10, graphs 4b and 5b, respectively) but these increases did not reach statistical significance.

Table 3 shows a comparison of the lowest concentrations resulting in a significant reduction in levels of biofilm when compared to the relevant control, and also shows MICs determined by the broth microdilution method. It is apparent that the concentrations inhibiting biofilm are lower than the MICs.

Comparison of biofilms grown in RPMI 1640 stained with crystal violet and XTT showed very close correlation, indicating that for C. albicans, biomass (determined by crystal violet) appears to correlate with viability (determined by XTT).

Table 5. Lowest concentrations of tea tree oil (% v/v) resulting in a significant decrease in biofilm formation by C. albicans compared to controls. MICs and MCCs for planktonic cells are shown for comparison.

Growth medium and quantification method Isolate RPMI YEPD RPMI MIC MCC crystal violet crystal violet XTT ATCC 90028 0.03* 0.016 0.03 0.25 0.5 ATCC 90029 0.016* 0.016 0.016 0.25 0.5 ATCC 10231 0.016 0.12 0.016 0.5 0.5 42J 0.016 0.12 0.016 0.5 1 68E 0.016 0.06 0.016 0.5 0.5 57X 0.03 0.12 0.06 0.5 0.5 KE216 0.016 0.03 0.016* 0.5 0.5 LL031 0.03 0.03 0.06 0.5 0.5 45E 0.03* 0.06 0.12 0.5 1 FF225 0.016 0.12 0.016 0.5 0.5 * One or more concentrations above this were NOT significant (see graphs)

V. harveyi Nine isolates of the Gram negative organism V. harveyi were examined using similar methodology as for S. epidermidis. These results are shown in Figure 13. On occasion, biofilm formation was inhibited by almost all of the tested concentrations of tea tree oil (see Fig 13, graph 4) but this did not occur frequently.

In addition to the basic method, elements of a recently described method that attempts to factor in the amount of bacterial growth when estimating biofilm formation (Niu and Gilbert 2004) were used. The results are also shown in Figure 13. These results show that the amount of biofilm produced generally mirrors the amount of growth; where the trend for growth increases or decreases, the trend for biofilm production follows suit.

S. maltophilia One isolate of S. maltophilia was investigated as a Gram negative organism that unlike P. aeruginosa is susceptible to lower concentrations of tea tree oil. As would be expected, concentrations at and above the MIC for this organism significantly inhibited the formation of biofilm (see Fig. 14).

Figure 1. Relative biofilm formation (mean and standard deviation) by five different isolates of S. epidermidis. Bars marked with an asterisk differ significantly from the control. Relative biofilm formation was calculated by dividing all absorbance readings (570 nm) by the absorbance reading for the control.

Figure 2. Relative biofilm formation (mean and standard deviation) by five different isolates of S. epidermidis. Bars marked with an asterisk differ significantly from the control. Relative biofilm formation was calculated by dividing all absorbance readings (570nm) by the absorbance reading for the control.

Figure 3. Relative biofilm formation (mean and standard deviation) by (1) P. aeruginosa ATCC 27853 and (2) P. aeruginosa NCTC 8505 under three different incubation conditions; (a) 24 h at 37°C, (b) 6 h at 37°C and (c) 24 h at room temperature (approx. 22°C). Bars marked with an asterisk differ significantly from the control. Relative biofilm formation was calculated by dividing all absorbance readings (570nm) by the absorbance reading for the control.

Figure 4. Relative biofilm formation (mean and standard deviation) by (3) P. aeruginosa NCTC 10662 and (4) P. aeruginosa PAO1 under three different incubation conditions; (a) 24 h at 37°C, (b) 6 h at 37°C and (c) 24 h at room temperature (approx. 22°C). Bars marked with an asterisk differ significantly from the control. Relative biofilm formation was calculated by dividing all absorbance readings (570nm) by the absorbance reading for the control.

Figure 5. Relative biofilm formation (mean and standard deviation) by (5) P. aeruginosa 27399 (clinical isolate) and (6) P. aeruginosa 27455 (clinical isolate) under three different incubation conditions; (a) 24 h at 37°C, (b) 6 h at 37°C and (c) 24 h at room temperature (approx. 22°C). Bars marked with an asterisk differ significantly from the control. Relative biofilm formation was calculated by dividing all absorbance readings (570nm) by the absorbance reading for the control.

Figure 6. Relative biofilm formation (mean and standard deviation) by (7) P. aeruginosa 4969 (clinical isolate) and (8) P. aeruginosa 5040 (clinical isolate) under three different incubation conditions; (a) 24 h at 37°C, (b) 6 h at 37°C and (c) 24 h at room temperature (approx. 22°C). Bars marked with an asterisk differ significantly from the control. Relative biofilm formation was calculated by dividing all absorbance readings (570nm) by the absorbance reading fo the control.

Figure 7. Relative biofilm formation (mean and standard deviation) by (9) P. aeruginosa 5052 (clinical isolate) and (10) P. aeruginosa 4938 (clinical isolate) under three different incubation conditions; (a) 24 h at 37°C, (b) 6 h at 37°C and (c) 24 h at room temperature (approx. 22°C). Bars marked with an asterisk differ significantly from the control. Relative biofilm formation was calculated by dividing all absorbance readings (570nm) by the absorbance reading for the control.

Figure 8. Relative biofilm formation (mean and standard deviation) by (1) C. albicans ATCC 90028 and (2) C. albicans ATCC 90092 determined by three different methods all incubated for 24 h at 37°C; (a) culture medium RPMI 1640, stained with 1% crystal violet, (b) culture medium YEPD, stained with 1% crystal violet and (c) culture medium RPMI 1640, biofilm quantified by XTT reduction . Bars marked with an asterisk differ significantly from the control. Relative biofilm formation was calculated by dividing all absorbance readings by the absorbance reading for the control.

Figure 9. Relative biofilm formation (mean and standard deviation) by (3) C. albicans ATCC 10231 and (4) C. albicans 42J (clinical isolate) determined by three different methods all incubated for 24 h at 37°C; (a) culture medium RPMI 1640, stained with 1% crystal violet, (b) culture medium YEPD, stained with 1% crystal violet and (c) culture medium RPMI 1640, biofilm quantified by XTT reduction. Bars marked with an asterisk differ significantly from the control. Relative biofilm formation was calculated by dividing all absorbance readings by the absorbance reading for the control.

Figure 10. Relative biofilm formation (mean and standard deviation) by (5) C. albicans 68E (clinical isolate) and (6) C. albicans 57X (clinical isolate) determined by three different methods all incubated for 24 h at 37°C; (a) culture medium RPMI 1640, stained with 1% crystal violet, (b) culture medium YEPD, stained with 1% crystal violet and (c) culture medium RPMI 1640, biofilm quantified by XTT reduction. Bars marked with an asterisk differ significantly from the control. Relative biofilm formation was calculated by dividing all absorbance readings by the absorbance reading for the control.

Figure 11. Relative biofilm formation (mean and standard deviation) by (7) C. albicans KE216 (clinical isolate) and (8) C. albicans LL031 (clinical isolate) determined by three different methods all incubated for 24 h at 37°C; (a) culture medium RPMI 1640, stained with 1% crystal violet, (b) culture medium YEPD, stained with 1% crystal violet and (c) culture medium RPMI 1640, biofilm quantified by XTT reduction. Bars marked with an asterisk differ significantly from the control. Relative biofilm formation was calculated by dividing all absorbance readings by the absorbance reading for the control.

Figure 12. Relative biofilm formation (mean and standard deviation) by (9) C. albicans 45E (clinical isolate) and (10) C. albicans FF225 (clinical isolate) determined by three different methods all incubated for 24 h at 37°C; (a) culture medium RPMI 1640, stained with 1% crystal violet, (b) culture medium YEPD, stained with 1% crystal violet and (c) culture medium RPMI 1640, biofilm quantified by XTT reduction. Bars marked with an asterisk differ significantly from the control. Relative biofilm formation was calculated by dividing all absorbance readings by the absorbance reading for the control.

) 2.0 2.0 2.5 2.5 ) 2.5 2.5 ) 540 540 540 ) ) )

2.0 2.0 600 2.0 2.0 600 1.5 1.5 600

1.5 1.5 1.5 1.5 1.0 1.0 1.0 1.0 1.0 1.0 22

0.5 0.5 Relative growth (OD growth Relative Relative growth (OD growth Relative 0.5 0.5 0.5 0.5 (OD growth Relative Relative biofilm formation (OD 0.0 0.0 0.0 0.0 Relative biofilm formation (OD

Relative biofilm formation (OD 0.0 0.0 1 1 1 0.5 0.5 0.5 0.03 0.06 0.25 0.03 0.06 0.25 0.03 0.06 0.25 0.015 0.125 0.015 0.125 0.015 0.125 0.0019 0.0037 0.0075 0.0019 0.0037 0.0075 Control Control 0.0019 0.0037 0.0075 Control 0.00095 0.00095 0.00095

1 Tea tree oil (% v/v) 2 Tea tree oil (% v/v) 3 Tea tree oil (% v/v)

Figure 13. Relative biofilm formation (bars, mean and standard deviation) and planktonic growth (line, mean and standard deviation) by (1) V. harveyi 1, (2) V. harveyi 2 and (3) V. harveyi 3 in TSBS. Biofilm was quantified by staining with crystal violet and reading the optical density at 540 nm. Growth was quantified by measuring the optical density at 600 nm. Open bars and line markers differ significantly from the control. Relative biofilm formation and relative growth was calculated by dividing all absorbance readings by the absorbance reading for the control.

2.4 2.4 1.6 1.6 ) )

) 1.0 1.0 540 540 1.4 1.4 540 ) ) 2.0 2.0 ) 600 600 0.8 0.8 600 1.2 1.2 1.6 1.6 1.0 1.0 0.6 0.6 1.2 1.2 0.8 0.8 0.4 0.4 0.6 0.6 23 0.8 0.8 0.4 0.4

0.2 0.2 (OD growth Relative Relative growth (OD growth Relative 0.4 0.4 (OD growth Relative 0.2 0.2 Relative biofilm formation (OD 0.0 0.0 Relative biofilm formation (OD

Relativebiofilm formation (OD 0.0 0.0 0.0 0.0 1 1 1 0.5 0.5 0.5 0.03 0.06 0.25 0.03 0.06 0.25 0.03 0.06 0.25 0.015 0.125 0.015 0.125 0.015 0.125 0.0019 0.0037 0.0075 Control 0.0019 0.0037 0.0075 0.0019 0.0037 0.0075 Control 0.00095 Control 0.00095 0.00095

4 Tea tree oil (% v/v) 5 Tea tree oil (% v/v) 6 Tea tree oil (% v/v)

Figure 13 (cont.) Relative biofilm formation (bars, mean and standard deviation) and planktonic growth (line, mean and standard deviation) by (4) V. harveyi 4, (5) V. harveyi 5 and (6) V. harveyi 6 in TSBS. Biofilm was quantified by staining with crystal violet and reading the optical density at 540 nm. Growth was quantified by measuring the optical density at 600 nm. Open bars and line markers differ significantly from the control. Relative biofilm formation and relative growth was calculated by dividing all absorbance readings by the absorbance reading for the control.

1.6 1.6 1.4 1.4 2.6 2.6 ) )

) 2.4 2.4 540 1.4 1.4 1.2 1.2 540 540

) 2.2 2.2 ) )

600 2.0 2.0 1.2 1.2 600 600 1.0 1.0 1.8 1.8 1.0 1.0 1.6 1.6 0.8 0.8 1.4 1.4 0.8 0.8 1.2 1.2 0.6 0.6 0.6 0.6 1.0 1.0

24 0.4 0.4 0.8 0.8 0.4 0.4 0.6 0.6 Relative growth (OD growth Relative Relative growth (OD growth Relative Relative growth (OD growth Relative 0.2 0.2 0.2 0.2 0.4 0.4 0.2 0.2 Relative biofilm formation (OD 0.0 0.0 0.0 0.0 Relative biofilm formation (OD

7 Tea tree oil (% v/v) 8 Tea tree oil (% v/v) 9 Tea tree oil (% v/v)

Figure 13 (cont.) Relative biofilm formation (bars, mean and standard deviation) and planktonic growth (line, mean and standard deviation) by (7) V. harveyi 7, (8) V. harveyi 8 and (9) V. harveyi 9 in TSBS. Biofilm was quantified by staining with crystal violet and reading the optical density at 540 nm. Growth was quantified by measuring the optical density at 600 nm. Open bars and line markers differ significantly from the control. Relative biofilm formation and relative growth was calculated by dividing all absorbance readings by the absorbance reading for the control.

1.8

1.6

1.4

1.2

0.8

0.6

0.4 formation biofilm Relative 0.2

Control 0.125 0.25 0.5 1 2 4

Tea tree oil (% v/v)

Figure 14. Relative biofilm formation (mean and standard deviation) by S. maltophilia ATCC 13637 after 24 h in TSB with tea tree oil. Biofilm was quantified by crystal violet staining. Bars marked with an asterisk differ significantly from the control. Relative biofilm formation was calculated by dividing all absorbance readings by the absorbance reading of the control.

Destruction of pre-formed biofilm Experiments to examine the effects of tea tree oil on pre-formed biofilm were done in two ways. Firstly, biofilms were grown in the absence of tea tree oil and then tea tree oil was added to them for 24 h after which the amount of biofilm remaining was quantified using the crystal violet staining method. Secondly, biofilms were established in the absence of tea tree oil, treated with tea tree oil for 24 h and then the viability of the cells remaining estimated by adding fresh growth medium and measuring the re-growth.

The treatment of pre-formed S. epidermidis biofilms with tea tree oil resulted in a significant reduction in biofilm at the two highest concentrations (8% and 4%) and also at 0.25%, measured by crystal violet staining (Fig. 15a). In contrast, biofilm was significantly reduced at all concentrations (0.25 - 4%) when measured by the regrowth method (Fig. 15b). Biofilms were reduced by approximately 90% at 0.5 - 4% tea tree oil when measured by regrowth, whereas when measured by crystal violet staining biofilms were reduced by approximately 20-30%.

For P. aeruginosa, biofilm was significantly reduced at only* one concentration * (0.* 25%) when measured by crystal violet staining (Fig. 16a) and no obvious trend was seen. By the regrowth method, biofilm was significantly reduced at 2 and 4% tea tree oil and a general trend of decreasing biofilm viability with increasing tea tree oil was seen (Fig. 16b).

The examination of a second Gram-negative organism, S. maltophilia, showed that biofilm was significantly reduced at all concentrations of tea tree oil (0.125 - 4%), by both methods (Fig. 17). Furthermore, whilst biofilms measured by crystal violet staining were reduced by approximately 50% at several tea tree oil concentrations, when quantified by the regrowth method these same biofilms were reduced by up to 85%.

1.6

A 1.4 1.2

1 * * * 0.8

0.6

0.4

Relative biofilm rem aining 0.2

0 Control0.250.51248

Tea tree oil (% v/v)

1.2 B 1

) 0.8 600

0.6 *

0.4 Viability (OD

0.2 ****

0 Control 0.25 0.5 1 2 4 Tea tree oil (% v/v)

Figure 15. Effects of tea tree oil on pre-formed S. epidermidis 4513735E biofilm quantified by (A) crystal violet staining and (B) regrowth after the addition of fresh growth medium. Bars marked with an asterisk differed significantly from the control.

1.6 A 1.4

1.2

1 * 0.8

0.6

0.4 remaining biofilm Relative

0.2

0 C ontrol 0.25 0.5 1 2 4 8 Tea tree oil (% v/v)

1.4 B 1.2

1 )

600 0.8

0.6 *

Viability (O D 0.4

* 0.2

0 Control 0.25 0.5 1 2 4 Tea tree oil (% v/v)

Figure 16. Effects of tea tree oil on pre-formed P. aeruginosa NCTC 10662 biofilm as quantified by (A) crystal violet staining and (B) regrowth after the addition of fresh growth medium. Bars marked with an asterisk differed significantly from the control.

27 1.2

A 1 * 0.8 *

* * * * 0.6

0.4

Relative biofilm remaining 0.2

0 Control 0.125 0.25 0.5 1 2 4

Tea tree oil (% v/v)

1.2 B

0.8

0.6

0.4

Relative biofilm remaining

0.2 * * * * * *

0 Control 0.125 0.25 0.5 1 2 4 Tea tree oil (% v/v)

Figure 17. Effects of tea tree oil on pre-formed S. maltophilia ATCC 13637 biofilm as quantified by (A) crystal violet staining and (B) regrowth after the addition of fresh growth medium. Bars marked with an asterisk differed significantly from the control.

Discussion

The first aim of this work was to investigate whether tea tree oil can inhibit the formation of biofilm. In tests with the Gram positive bacterium S. epidermidis, the Gram negative bacteria P. aeruginosa, S. maltophilia and V. harveyi and the yeast C. albicans, tea tree oil was able to significantly inhibit the de novo formation of biofilm. Clearly, tea tree oil is capable of reducing biofilm formation if present during the formative process. However, further studies conducted with V. harveyi using a modification of the specific biofilm formation (SBF) method of Niu and Gilbert (2004) indicated that the reduction in biofilm formation was likely to be a function of reduced growth, since slight changes in growth were usually mirrored by corresponding changes in the quantity of biofilm (Fig 13).

Very few previous studies have investigated the effects of tea tree oil on the de novo formation of biofilm. Even fewer have attempted to discriminate between reductions in biofilm formation due to biofilm-specific factors, or non-specific factors such as growth inhibition. In this situation it is necessary to consult literature published for other essential oils or any other similar compounds to determine if the results from the present study are typical. A previous study evaluating a commercial preparation that contained several antimicrobial components such as essential oils (including tea tree oil), butylated hydroxytoluene, triclosan (0.3%) and ethanol on biofilm formation by coagulase- negative staphylococci found that although the product inhibited biofilm, it was a non-specific effect whereby the concentrations required to inhibit biofilm formation were generally the same as those required to inhibit growth (Al-Shuneigat et al. 2005). Likewise, reductions in biofilm formation by S. aureus and S. epidermidis in the presence of oregano oil, thymol and carvacrol appeared to generally correlate with the MICs for each of these substances (Nostro et al. 2007).

Further examination of the data showed that contrary to the major trend of decreased biofilm in the presence of tea tree oil, in some tests with S. epidermidis tea tree oil appeared to actually increase biofilm formation. These increases were significant for two of the ten isolates and occurred at the sub- inhibitory concentration of 0.03% tea tree oil. Similar observations have been reported previously in the literature, whereby biofilms of S. epidermidis formed in the presence of the antimicrobial agents chlorhexidine and benzalkonium chloride were increased when compared to controls (Houari and Di Martino 2007). In another study, the essential oil components α-pinene, geranyl acetate, limonene, linalool and 1,8-cineole (1mg/ml) were also shown to enhance biofilm formation when added to 6 h biofilms of Listeria monocytogenes, as assessed by crystal violet staining (Sandasi et al. 2008). However, this study also showed that whilst amounts of biofilm were increased, the metabolic activity of the biofilms was decreased, assessed by the XTT assay (Sandasi et al. 2008).

There are a number of reasons that biofilm formation may be potentially enhanced or increased in the presence of tea tree oil. When met with hostile environmental conditions such as nutrient limitation, bacteria are able to respond in ways that enhance their survival. In E. coli, some of these responses are regulated by the rpoS regulon which coordinates the synthesis of sigma factors that mitigate the effects of stress (Donlan and Costerton 2002). The study of rpoS+ biofilms has revealed that they have higher densities with a higher number of viable organisms than their rpoS- counterparts. Thus efficient biofilm formation may be viewed as a stress response and a survival mechanism. It is likely that exposure to tea tree oil induced the formation of biofilm. However, it should be noted that this study did not examine the density, depth or strength of these biofilms. It is possible that although increased amounts of biofilm were formed, it may be atypical, with altered characteristics such as density, resistance, strength and durability. It is also possible that the components of tea tree oil increased the permeability of the microorganisms enhancing their uptake of nutrients and improving growth. The extracellular components of the biofilm contribute to its low permeability (Donlan and Costerton 2002) and the monoterpenes and sesquiterpenes contained in tea tree oil may have altered the ultrastructure of the biofilm in a manner that increases permeability.

The second aim of this research was to examine the effects of tea tree oil on pre-formed biofilm. This work yielded particularly interesting results since pre-formed biofilms S. maltophilia and S.

epidermidis were discovered to have reduced viability after treatment with tea tree oil. Furthermore, reductions in viability were seen at approximately the MIC for each organism, whereas for many antimicrobial agents concentrations much higher than the MIC are usually required to eradicate or damage existing biofilms. Previous work has also shown tea tree to adversely affect preformed biofilm (Brady et al. 2006). Biofilms of S. aureus, S. epidermidis and several other coagulase-negative staphylococci were grown for 24 h then treated for 1 h with a solution of 5% tea tree oil. Cells were freed from biofilms by sonication and viable counts were performed. For tests with S. aureus no viable organisms were recovered whereas approximately half of the S. epidermidis and other coagulase- negative staphylococci remained viable, although there was a reduction in the numbers of viable cells. This supports the finding from the current study that tea tree oil adversely affects biofilm viability. Similarly, oregano oil and its components carvacrol and thymol have been shown to both inhibit and kill at 2-4 times the MIC and MCC, respectively, when used against S. aureus and S. epidermidis biofilms (Nostro et al., 2007).

Another interesting observation from this study was that when biofilm was measured by crystal violet staining, most reductions in biofilm were generally not large, in contrast to reductions measured by the regrowth method, which indicated that organisms were either unable, or extremely slow to regrow. These data suggest that tea tree oil adversely affects the viability of the organisms within the biofilm but may not affect the structure or matrix of the biofilm.

Existing biofilms usually exhibit marked resistance to disinfectants with the concentrations required to destroy pre-formed biofilm many-fold the MICs required for planktonic cells. However, such patterns of resistance were not observed here. Perhaps tea tree oil, by merit of its solvent properties or putative multiple mechanisms of action, may circumvent the inherent resistance of biofilms. In addition, this study has not examined the effects of tea tree oil on the ultrastructure of biofilm. Biofilm is not a homogeneous monolayer of cells fixed to a surface. Rather it is a complex collection of microorganisms and exudates that is heterogeneous in both space and time (Costerton et al. 1994). Microscopy studies to assess the physical structure of the biofilms would provide further insight into the effects of tea tree oil.

The third aim of this project was to investigate potential mechanisms by which tea tree oil may inhibit biofilm formation. The most logical aspect to examine first was to investigate whether reductions in biofilm formation occurred as a by-product of reduced growth or as a separate, specific effect. The experiments conducted using V. harveyi suggested very strongly that the reductions in biofilm were a function of reduced growth, in turn suggesting that it was unlikely that biofilm was specifically being inhibited. However, this does not rule out the possibility that tea tree oil may have specific anti-biofilm effects. In fact, there were several instances where inhibition of biofilm formation occurred several concentrations below the MIC for the test organism (eg. C. albicans 42J and KE216, S. epidermidis 735E). For these organisms where biofilm was inhibited at levels considerably lower than the MIC, it is quite possible that tea tree oil is interfering with one or more processes responsible for biofilm formation.

There are several stages of, or processes associated with biofilm formation that tea tree oil may interfere with, including the initial adhesion and attachment process, overall motility and quorum sensing. The possibility that tea tree oil may inhibit adhesion is the subject of a current RIRDC project and initial results suggest that adhesion is inhibited in the presence of tea tree oil. The possibility that tea tree oil may interfere with quorum sensing is not something that has been investigated as yet, but natural compounds such as the furanones, farnesol and cinnamaldehyde (Niu et al., 2006) have been previously demonstrated to interfere with quorum sensing.

The final aim of this work was to explore potential medical and industrial applications of biofilm inhibition. Since tea tree oil was found to have detrimental effects against both forming and formed biofilm, it seems likely that applications may be found for these useful properties.

The mindset for how microorganisms exist within the environment, including the human body has shifted in recent years. Instead of thinking of the bacteria or fungi exist largely as free-floating

planktonic organisms, evidence now suggests that microorganisms spend the majority of their time within biofilms. With regard to human disease and infection, it is now worth considering whether the particular disease or infection in question may involve biofilm. Of particular relevance to tea tree oil is the recent suggestion that biofilms play a role within many bacterial infections, particularly chronic infections of the ear and throat (Post et al. 2007) and respiratory (Moreau-Marquis et al. 2008) tract. Also, the chronicity of particular wounds such as venous leg ulcers may be in part due to the presence of biofilm within the wound (Davis et al. 2008). The ability of relatively low concentrations of tea tree oil to kill organisms within biofilm is another justification for the use of tea tree oil products to treat wounds. Also in the medical setting, biofilm is a particular problem on indwelling devices, such as catheters. The potential exists for such devices to be impregnated with tea tree oil, to prevent biofilm forming. Catheters impregnated with various antimicrobial substances including antibiotics and antiseptics have already been shown to reduce the incidence of catheter-associated infections (Eggimann 2007).

In the industrial setting, biofilms are a very common problem. Their presence in the cooling towers associated with air-conditioning systems is linked to the spread of Legionnaires disease, caused by the bacterium Legionella. Biofilm can form on the interior of pipes, leading to clogging. Biofilm is also present in food manufacturing facilities, which can result in the contamination of food products, wastage and has the potential to cause illness. Given the diverse range of situations and environments in which biofilm occurs, and the need to either eradicate or at least control biofilms in the settings, the potential exists for tea tree oil to be used. The way in which it is used for any given situation would of course need to be fine-tuned to ensure that the method of deliver is optimal, both in terms of biofilm treatment and cost-effectiveness.

This work clearly establishes that tea tree oil can inhibit the formation of biofilm and compromise pre- formed biofilm but the mechanisms by which effects occur require clarification. Further work should involve characterisation of the effects of tea tree oil and its components on the ultra-structural characteristics and properties of pre-formed biofilms that have been treated with tea tree oil or those of biofilms formed in the presence of the oil. This would include the thickness, density, topography and permeability of biofilms and the morphology, distribution and metabolic status of the organisms embedded in it (Costerton et al. 1995).

Implications

Tea tree oil is able to inhibit the formation of biofilm by bacteria and the yeast C. albicans. More importantly, data from this study suggests that pre-formed biofilms are particularly susceptible to tea tree oil at concentrations similar to the MICs. This result is intriguing since biofilms are generally much more resistant to antimicrobial agents requiring concentrations greatly exceeding those used to inhibit or kill planktonic microorganisms. If these trends are borne out in larger scale models, tea tree oil has potential as a biocide for the prevention or removal of biofilms in industrial settings. Furthermore, these results provide another mechanism to explain the clinical utility of tea tree oil as a topical antimicrobial agent for the treatment of cutaneous infections.

References

Al-Shuneigat, J., S. D. Cox and J. L. Markham (2005). Effects of a topical essential oil-containing formulation on biofilm-forming coagulase-negative staphylococci. Letters in Applied Microbiology 41(1): 52-55. Alviano, W. S., R. R. Mendonca-Filho, D. S. Alviano, H. R. Bizzo, T. Souto-Padrón, M. L. Rodrigues, A. M. Bolognese, C. S. Alviano and M. M. G. Souza (2005). Antimicrobial activity of Croton cajucara Benth linalool-rich essential oil on artificial biofilms and planktonic microorganisms. Oral Microbiology and Immunology 20(2): 101-105. Brady, A., R. Loughlin, D. Gilpin, P. Kearney and M. Tunney (2006). In vitro activity of tea-tree oil against clinical skin isolates of meticillin-resistant and -sensitive Staphylococcus aureus and coagulase-negative staphylococci growing planktonically and as biofilms. Journal of Medical Microbiology 55(10): 1375-1380. Carson, C. F., K. A. Hammer and T. V. Riley (1995). Broth micro-dilution method for determining the susceptibility of Escherichia coli and Staphylococcus aureus to the essential oil of Melaleuca alternifolia (tea tree oil). Microbios 82(332): 181-185. Carson, C. F., K. A. Hammer and T. V. Riley (1996). In-vitro activity of the essential oil of Melaleuca alternifolia against Streptococcus spp. Journal of Antimicrobial Chemotherapy 37(6): 1177- 1178. Carson, C. F., K. A. Hammer and T. V. Riley (2006). Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other properties. Clinical Microbiology Reviews 19(1): 50-62. Cooney, J. J. and R.-J. Tang (1999). Quantifying effects of antifouling paints on microbial biofilm formation. Methods in Enzymology 310: 637-644. Costerton, J. W., Z. Lewandowski, D. E. Caldwell and D. R. Korber (1995). Microbial biofilms. Annual Review of Microbiology 49: 711-745. Costerton, J. W., Z. Lewandowski, D. DeBeer, D. Caldwell, D. Korber and G. James (1994). Biofilms, the customized microniche. Journal of Bacteriology 176: 2137-2142. Davis, S. C., C. Ricotti, A. Cazzaniga, E. Welsh, W. H. Eaglstein and P. M. Mertz (2008). Microscopic and physiologic evidence for biofilm-associated wound colonization in vivo. Wound Repair and Regeneration 16(1): 23-29. Donlan, R. M. and J. W. Costerton (2002). Biofilms: survival mechanisms of clinically relevant microorganisms. Clinical Microbiology Reviews 15(2): 167-193. Eggimann, P. (2007). Prevention of intravascular catheter infection. Current Opinion in Infectious Diseases 20(4): 360-369. Filoche, S. K., K. Soma and C. H. Sisson (2005). Antimicrobial effects of essential oils in combination with chlorhexidine gluconate. Oral Microbiology and Immunology 20(4): 221-225. Hammer, K. A., C. F. Carson and T. V. Riley (1996). Susceptibility of transient and commensal skin flora to the essential oil of Melaleuca alternifolia (tea tree oil). American Journal of Infection Control 24(3): 186-189. Hammer, K. A., C. F. Carson and T. V. Riley (2004). Antifungal effects of Melaleuca alternifolia (tea tree) oil and its components on Candida albicans, Candida glabrata and Saccharomyces cerevisiae. Journal of Antimicrobial Chemotherapy 53(6): 1081-1085. He, M., M. Q. Du, M. W. Fan and Z. A. Bian (2007). In vitro activity of eugenol against Candida albicans biofilms. Mycopathologia 163(3): 137-143. Houari, A. and P. Di Martino (2007). Effect of chlorhexidine and benzalkonium chloride on bacterial biofilm formation Letters in Applied Microbiology 45(6): 652-656. Jabra-Rizk, M. A., T. F. Meiller, C. E. James and M. E. Shirtliff (2006). Effect of farnesol on Staphylococcus aureus biofilm formation and antimicrobial susceptibility. Antimicrobial Agents and Chemotherapy 50(4): 1463-1469. Jabra-Rizk, M. A., M. Shirtliff, M. James and T. Meiller (2006). Effect of farnesol on Candida dubliniensis biofilm formation and fluconazole resistance. FEMS Yeast Research 6(7): 1063- 1073. Knowles, J. R., S. Roller, D. B. Murray and A. S. Naidu (2005). Antimicrobial action of carvacrol at different stages of dual-species biofilm development by Staphylococcus aureus and

Salmonella enterica serovar typhimurium. Applied and Environmental Microbiology 71(2): 797-803. Lebert, I., S. Leroy and R. Talon (2007). Effect of industrial and natural biocides on spoilage, pathogenic and technological strains grown in biofilm. Food Microbiology 24(3): 281-287. Masako, K., I. Hideyuki, O. Shigeyuki and I. Zenro (2005). A novel method to control the balance of skin microflora. Part 1. Attack on biofilm of Staphylococcus aureus without antibiotics. Journal of Dermatological Science 38(3): 197-205. Messager, S., K. A. Hammer, C. F. Carson and T. V. Riley (2005). Assessment of the antibacterial activity of tea tree oil using the European EN 1276 and EN 12054 standard suspension tests. Journal of Hospital Infection 59(2): 113-125. Moreau-Marquis, S., B. A. Stanton and G. A. O'Toole (2008). Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway. Pulmonary Pharmacology and Therapeutics in press. Niu, C. and E. S. Gilbert (2004). Colorimetric method for identifying plant essential oil components that affect biofilm formation and structure. Applied and Environmental Microbiology 70(12): 6951-6956. Nostro, A., A. S. Roccaro, G. Bisignano, A. C. Marino, M. A., F. C. Pizzimenti, P. L. Cioni, F. Procopio and A. R. Blanco (2007). Effects of oregano, carvacrol and thymol on Staphylococcus aureus and Staphylococcus epidermidis biofilms. Journal of Medical Microbiology 56(4): 519-523. Ouhayoun, J. (2003). Penetrating the plaque biofilm: impact of essential oil mouthwash. Journal of Clinical Periodontology 30(suppl 5): 10-12. Papadopoulos, C. J., C. F. Carson, K. A. Hammer and T. V. Riley (2006). Susceptibility of pseudomonads to Melaleuca alternifolia (tea tree) oil and components. Journal of Antimicrobial Chemotherapy 58: 449-451. Pitts, B., M. A. Hamilton, N. Zelver and P. S. Stewart (2003). A microtiter-plate screening method for biofilm disinfection and removal. Journal of Microbiological Methods 54(2): 269-276. Post, J. C., N. L. Hiller, L. Nistico, P. Stoodley and G. D. Ehrlich (2007). The role of biofilms in otolaryngologic infections: update 2007. Current Opinion in Otolaryngology and Head and Neck Surgery 15(5): 347-351. Ramage, G., S. P. Saville, B. L. Wickes and J. L. López-Ribot (2002). Inhibition of Candida albicans biofilm formation by farnesol, a quorum-sensing molecule. Applied and Environmental Microbiology. 68(11): 5459-5463. Ramage, G., K. Vande Walle, B. L. Wickes and J. L. López-Ribot (2001). Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrobial Agents and Chemotherapy 45(9): 2475-2479. Sandasi, M., C. M. Leonard and A. M. Viljoen (2008). The effect of five common essential oil components on Listeria monocytogenes biofilms. Food Control doi:10.1016/j.foodcont.2007.11.006. Walker, J. T., A. D. Roberts, V. J. Lucas, M. A. Roper and R. G. Brown (1999). Quantitative assessment of biocide control of biofilms including Legionella pneumophila using total viable counts, fluorescence microscopy, and image analysis. Methods in Enzymology 310: 629-637. Yassien, M., N. Khardori, A. Ahmedy and M. Toama (1995). Modulation of biofilms of Pseudomonas aeruginosa by quinolones. Antimicrobial Agents and Chemotherapy 39(10): 2262-2268.

33 Effects of Tea Tree Oil RIRDC Publication No. INSERT PUB NO. HERE RIRDC Publication No. INSERT PUBLICAITON NO. HERE RIRDC Publication No. on BiofilmRIRDC PublicationINSERT No.PUBLICAITON INSERT PUBLICAITON NO.Formation HERE NO. HERE RIRDC Publication No. INSERT PUB NO. HERE RIRDC Publication No. 08/140

This report details the results of an investigation into the ability It is targeted at manufacturers and marketers of tea tree oil of tea tree oil to prevent the formation of or disrupt existing products and provides information on how tea tree oil can biofilm, known in lay terms as “slime”. The importance of potentially control microorganisms. Further testing of appropriate biofilm in industrial and commercial settings has been appreciated formulations may lead to products for this significant market. for decades since its formation fouls production lines, disrupts The Rural Industries Research and Development Corporation production and its management is often problematic contributing (RIRDC) manages and funds priority research and translates significantly to production time and costs. results into practical outcomes for industry.

In medical settings, the importance of biofilm is becoming more Our business is about new products and services and better ways obvious. It is now implicated in many types of infections including of producing them. Most of the information we produce can be those of wounds, the oral cavity, ears and on devices such as downloaded for free from our website: www.rirdc.gov.au. catheters and pacemakers. RIRDC books can be purchased by phoning 02 6271 4160 or online at: www.rirdc.gov.au/eshop.

Contact RIRDC: Level 2 This publication can be viewed at our website— 15 National Circuit www.rirdc.gov.au. All RIRDC books can be Barton ACT 2600 purchased from:. PO Box 4776 www.rirdc.gov.au/eshop Kingston ACT 2604 Ph: 02 6271 4100 Fax: 02 6271 4199 Email: [email protected] web: www.rirdc.gov.au RIRDCInnovation for rural Australia