Wizardhawk's memorial thread (a cautionary tale of dental care and garlic)

Keywords:
tea tree oil;Melaleuca alternifolia;minimum inhibitory concentration;time kill

Abstract
The in vitro activity of Melaleuca alternifolia (tea tree) oil against 161 isolates of oral bacteria from 15 genera was determined. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) ranged from 0.003 to 2.0% (v/v). MIC90 values were 1.0% (v/v) for Actinomyces spp., Lactobacillus spp., Streptococcus mitis and Streptococcus sanguis, and 0.1% (v/v) for Prevotella spp. Isolates of Porphyromonas, Prevotella and Veillonella had the lowest MICs and MBCs, and isolates of Streptococcus, Fusobacterium and Lactobacillus had the highest. Time kill studies with Streptococcus mutans and Lactobacillus rhamnosus showed that treatment with ?0.5% tea tree oil caused decreases in viability of >3 log colony forming units/ml after only 30 s, and viable organisms were not detected after 5 min. These studies indicate that a range of oral bacteria are susceptible to tea tree oil, suggesting that tea tree oil may be of use in oral healthcare products and in the maintenance of oral hygiene.

The essential oil of Melaleuca alternifolia, also known as tea tree oil, has been used medicinally in Australia for more than 80 years (5). The tree itself has been used therapeutically for even longer, being one of the plants used in traditional medicine by the Bundjalung aborigines of northern New South Wales (5). The essential oil is obtained by steam distillation and contains approximately 100 components, which are mostly monoterpenes (4). Commercial oils must comply with the percentage composition ranges for components stipulated in the International Standard 4730 for 'Oil of Melaleuca, terpinen-4-ol type' (12). Tea tree oil, and several of the components, has broad-spectrum antimicrobial (5) and anti-inflammatory (3, 14) activity in vitro. These properties have formed the basis of its use in the treatment of a range of superficial complaints such as cuts, insect bites, boils, acne and tinea (2, 5). Furthermore, data from recent clinical studies indicate that superficial infections or conditions caused by bacteria (2), fungi (13, 22) and viruses (6) respond clinically to treatment with tea tree oil. Anecdotal and scientific evidence also suggest that tea tree oil may be useful in the maintenance of oral hygiene and the prevention of dental disease (9, 19, 23). However, the in vitro activity of tea tree oil against the organisms found in the oral cavity, or the potential uses of tea tree oil in the oral cavity have not been studied extensively. Therefore, the purpose of this study was to investigate the in vitro susceptibility of a wide range of oral bacteria to tea tree oil.


Material and methods
Tea tree oil
Tea tree oil (batch 97/1) was kindly donated by Australian Plantations Pty Ltd., Wyrallah, NSW. The oil composition was determined by gas-chromatography mass spectrometry performed by the Wollongbar Agricultural Institute, Wollongbar, NSW, as described previously (11). Levels of terpinen-4-ol were 41.5% and levels of 1,8-cineole were 2.1%, in compliance with the International Standard 4730 (12).

Bacterial isolates
A collection of 161 bacterial isolates was obtained from the culture collections of the Discipline of Microbiology at The University of Western Australia (n= 9), the Division of Microbiology and Infectious Diseases at the Western Australian Centre for Pathology and Medical Research (n= 123) and from the oral cavities of volunteers (n= 29) (Tables 1 and 2). Reference isolates were Escherichia coli NCTC 10418, Veillonella parvula NCTC 11810, Actinobacillus actinomycetemcomitans ATCC 43718 and Lactobacillus rhamnosus NCTC 10302. Oral bacteria were isolated by rubbing a sterile cotton-tipped swab over the teeth, gums and tongue and then placing the swab into a glass Bijou bottle containing 1 ml of phosphate buffered saline. The bottle was mixed thoroughly using a vortex mixer and the resulting bacterial suspension was inoculated onto a range of selective and non-selective culture media. Media were incubated anaerobically at 35?C for 3-10 days and pure cultures obtained. Isolates were identified using biochemical profiles determined with API 32A strips, antibiotic susceptibilities determined using Microring AN discs (Medical Wire and Equipment Co. (Bath) Ltd., Wiltshire, England) and other standard microbiological methods (20).

Table 1. In vitro susceptibility of viridans streptococci (n= 78) to tea tree oil
Organism (n) MIC (% v/v) MBC (% v/v)
Range 901 Range 901
1
Percentage tea tree oil inhibitory or bactericidal to 90% of isolates.
S. bovis (1) 0.5 1
S. constellatus (8) 0.25-1 0.25-1
S. gordonii (2) 0.5 0.5-1
S. intermedius (6) 0.12-2 0.25-2
S. mitis (11) 0.25-1 1 0.25-1 1
S. mutans (2) 0.25-2 0.25-2
S. oralis (5) 0.25-1 0.25-1
S. parasanguis (3) 0.25-0.5 0.25-0.5
S. salivarius (2) 0.25 0.25
S. sanguis (19) 0.25-1 1 0.25-2 2
S. sobrinus (1) 1 2
Streptococcus spp. (18) 0.25-1 1 0.25-2 2
Table 2. In vitro susceptibility of oral bacteria (n= 83) to tea tree oil
Organism (n) MIC (% v/v) MBC (% v/v)
Range 901 Range 901
1
Percentage tea tree oil inhibitory or bactericidal to 90% of isolates.
Actinobacillus actinomycetemcomitans (1) 0.06 0.06
Actinomyces spp. (13) 0.1-1 1 0.1-2 1
Branhamella sp. (1) 0.06 0.06
Capnocytophaga sp. (3) 0.03-0.06 0.03-0.06
Clostridium glycolicum (1) 0.05 0.1
Eikenella corrodens (5) 0.03-0.06 0.03-0.06
Fusobacterium spp. (5) 0.25-2 0.25-2
Lactobacillus spp. (18) 0.03-2 1 0.06-2 2
Neisseria sp. (1) 0.25 0.25
Peptostreptococcus asaccharolyticus (3) 0.25-0.5 0.5-1
Porphyromonas endodontalis (8) 0.025-0.1 0.025-0.1
Prevotella intermedia (15) 0.003-0.1 0.1 0.003-0.1 0.1
Prevotella spp. (3) 0.016-0.06 0.016-0.06
Stomatococcus sp. (1) 0.5 0.5
Veillonella spp. (5) 0.016-1 0.03-1
In vitro susceptibility assays
Inocula for susceptibility tests were prepared by growing organisms for 24-48 h on Rogosa agar (Oxoid Ltd., Basingstoke, England) for Lactobacillus spp. or blood agar for the remaining organisms and suspending colonies in sterile distilled water. All isolates were grown anaerobically, except for Streptococcus spp. and A. actinomycetemcomitans, which were grown in air plus 5% CO2. Suspensions were adjusted to the turbidity of a 0.5 McFarland standard using a nephelometer and diluted as necessary to result in final inocula concentrations of approximately 5 ? 105 colony forming units (cfu)/ml, as confirmed by viable counts.

For the microdilution assay, doubling dilutions of tea tree oil ranging from 4 to 0.004% (v/v) were prepared in 100 ?l volumes in a 96-well microtiter tray in the relevant growth medium. Todd Hewitt Broth (Oxoid) was used for Streptococcus spp., de Mann, Rogosa and Sharpe (MRS) broth (Oxoid) was used for Lactobacillus spp. and Brain Heart Infusion Broth (BHIB) (Oxoid) was used for all other organisms. A final concentration of 0.001% (v/v) Tween 80 was included to enhance tea tree oil solubility. After inoculation, tests were incubated for 24 h under anaerobic conditions except for tests with Streptococcus spp. and A. actinomycetemcomitans, which were incubated for 24 h in air plus 5% CO2. After incubation, microtitre trays were subcultured by mixing the contents of each well, removing 10 ?l aliquots and spot inoculating onto pre-dried Rogosa agar or blood agar. MICs were determined from subcultures as the lowest concentration resulting in the maintenance or reduction of the inoculum, and the MBC was determined as the concentration resulting in the death of 99.9% of the inoculum. The reference isolate E. coli was included in all assays as a control. Assays were repeated at least twice for each isolate and modal MIC and MBC values were selected.

Isolates of Porphyromonas endodontalis, Prevotella intermedia and one isolate each of Actinomyces viscosus and Clostridium glycolicum did not produce sufficient growth in the microdilution format and were tested by broth macrodilution. Dilutions of tea tree oil were prepared in 1 ml volumes of BHIB and, after inoculation, final concentrations of tea tree oil ranged from 0.5-0.001% (v/v). Controls were prepared with no tea tree oil. Macrodilutions were pre-reduced for approximately 60 min, inoculated with 1 ml volumes of inocula prepared as described above, then incubated for 48 h. MICs and MBCs were determined by subculturing as described above.

Time kill assays
A clinical isolate of Streptococcus mutans and L. rhamnosus NCTC 10302 were used in time kill studies. Inocula were prepared by growing each isolate on the appropriate solid media for 48-72 h, suspending growth in 0.85% saline and adjusting to 0.5 McFarland to result in final inocula concentrations of approximately 107 cfu/ml. Treatments containing tea tree oil ranging from 4-0.12% (v/v) were prepared in 1 ml volumes of double-strength BHIB (S. mutans) or MRS (L. rhamnosus) with 0.002% Tween 80. Treatments were pre-reduced for at least 30 min before 1 ml volumes of prepared inocula were added. Samples were removed at 30 s, 5 and 10 min for viable counts. Samples were diluted 10-fold in 0.85% saline and 10 ?l aliquots were spot inoculated in duplicate onto pre-dried blood agar or Rogosa agar. Inoculation, sampling and viable counting was performed in the anaerobic chamber. Agar plates were incubated anaerobically for 48-72 h and viable counts were calculated from each replicate 10 ?l spot having one or more colony. The limit of detection was 1 ? 103 cfu/ml. Assays were repeated at least twice for each tea tree oil concentration and the mean, standard deviation and standard error of the viable count data were calculated. Data were compared using a Student's t-test, two-tailed, two-sample assuming unequal variance. P-values of <0.05 were considered significant.


Results
Results of in vitro susceptibility assays are shown in Tables 1 and 2. MICs and MBCs were similar for all Streptococcus species, and MICs ranged from 0.12 to 2% and MBCs ranged from 0.5 to 2%. The MIC90 for all streptococci was 1% and the MBC90 was 2%. Some of the non-streptococcal bacteria were considerably more susceptible to tea tree oil, with MICs and MBCs as low as 0.016% for isolates of Prevotella and Veillonella. Both the MIC90 and MBC90 for all non-streptococcal bacteria was 1%. For reference isolates, MIC/MBC values were 0.06/0.06% for A. actinomycetemcomitans, 0.25/0.5% for L. rhamnosus, 0.016/0.03% for V. parvula and 0.25/0.25% for E. coli.

Time kill data are shown in Fig. 1. Significant decreases in the viability of S. mutans occurred after 30 s treatment with 4, 2, 1 and 0.5% tea tree oil. Numbers of viable organisms recovered from the 2 and 4% treatments after 30 s were 3.2 ? 103 and 3.3 ? 103 cfu/ml, respectively (data not shown in Fig. 1). Viable counts of S. mutans after treatment with 0.25% tea tree oil differed significantly from controls at 30 s, 5 and 10 min, whereas viable counts of cells treated with 0.12% differed significantly from controls at 5 and 10 min only. For L. rhamnosus, numbers of viable cells were below detectable limits after 30 s treatment with 1% tea tree oil. Treatment with 0.5% tea tree oil reduced the numbers of viable organisms by >3 log within 30 s and, at 5 min, viable organisms were no longer detectable. Treatment with 0.25% tea tree oil resulted in a modest reduction in viability, which was significant at 5 and 10 min, whereas treatment with 0.12% had no significant effects.

image
Figure 1. Time kill curves for S. mutans (A) and L. rhamnosus (B). Cells were treated with 0.5% (?), 0.25% (?), 0.12% (?) and 0% (?) tea tree oil and viable counts were determined after 30 s, 5 min and 10 min. Mean ? standard error. For S. mutans, the MIC and MBC was 2% and for L. rhamnosus the MIC was 0.25% and the MBC was 0.5%.


Discussion
The tea tree oil susceptibility data obtained in the present study are similar to those from previous studies of oral bacteria, which found MIC ranges of 0.03-1.25% (15) and 0.11?>0.6% (19) for nine and six isolates, respectively. Data were also similar to the MIC range of 0.02-0.08% found for 12 isolates of oral bacteria by Walsh & Longstaff (23), although these MICs were of Melasol, a solution containing 40% tea tree oil and 13% isopropyl alcohol (23). In addition, a range of anaerobic and microaerophilic vaginal bacteria, similar to those found in the oral cavity, have been shown to be susceptible to tea tree oil, with MICs ranging from 0.03-2% (10). Although data from the current and previous studies were generally similar, notably different was the MIC of 1.25% for P. intermedia obtained by Kulik et al. (15) which was considerably higher than the MICs determined for this organism in the present study and, conversely, the values obtained in the current study for Fusobacterium spp. were considerably higher than those found by previous authors (19, 23). Factors that may have contributed to these divergent results include differences between the types and numbers of bacterial isolates tested in each of the studies, and the methods used, including the criteria for determining MICs. In particular, where possible, multiple isolates from each species were tested in the current study, whereas previous studies often tested only single isolates.

Time kill studies demonstrated rapid killing of both S. mutans and L. rhamnosus after 30 s treatment with as little as 0.5% tea tree oil, suggesting that tea tree oil may be an effective active ingredient in antiseptic mouthrinses. Time kill studies of 30 s have been recommended for evaluating mouthrinses in vitro (24) and many mouthrinses are recommended by manufacturers to be used for 30 s twice daily, with volumes of approximately 20 ml (8, 17). However, the significant bactericidal effects seen with ?0.5% tea tree oil in vitro may not necessarily reflect what may be occurring in the oral cavity when rinsing with a tea tree oil mouthwash solution, for several reasons. Firstly, in vitro and in vivo findings may not directly correlate, as found previously for another mouthrinse product, which gave promising in vitro results but did not perform well in vivo (17). Secondly, the presence of exogenous protein in the oral cavity may adversely affect the activity of tea tree oil (11) and it is therefore important to repeat the time-kill studies in the presence of protein to examine this effect (24). Ultimately, clinical trials are required to determine in vivo efficacy and investigate optimal tea tree oil concentrations.

Traditional or alternative oral hygiene measures, such as mastic chewing gum and traditional chewing sticks, have been the subject of recent studies (7, 21). The use of mastic chewing gum was shown to significantly reduce bacterial numbers in saliva, plaque index and gingival index in 20 volunteers, compared to placebo gum (21). Similarly, use of a eucalyptus-extract gum significantly reduced the plaque index in 15 volunteers when compared to control gum (18). It has been suggested that the effectiveness of these traditional oral hygiene tools may be due, in part, to the antimicrobial plant compounds found within these traditional medicaments (7, 18). Furthermore, it has been suggested for some time that tea tree oil may be an effective agent for both the treatment and prevention of oral infections or conditions, although scientific reports are few. In 1937, Penfold & Morrison reported that oral conditions such as thrush, aphthous stomatitis, mouth ulcers, gingivitis and pyorrhoea all responded favorably to treatment with tea tree oil (16). More recently, the effect of mouthwashes containing tea tree oil (approximately 0.34%) or 0.1% chlorhexidine on plaque formation was compared to placebo in eight volunteers (1). However, the plaque index and plaque vitality after use of the tea tree oil mouthwash did not differ from placebo mouthwash on any day, whereas the chlorhexidine mouthwash group differed significantly from placebo on all days (1). Another study compared tea tree oil (0.2%), garlic (2.5% solution) and chlorhexidine (0.12%) by determining levels of S. mutans and other oral microorganisms in saliva samples after the use of each mouthwash (9). All products produced significant immediate reductions in viable counts and reductions were maintained for 2 weeks following the cessation of mouthwash use, but only for the tea tree oil and garlic mouthwashes, not chlorhexidine (9). Tea tree oil mouthwash has also been evaluated for the treatment of oral candidiasis in two different studies with overall clinical response rates of 67% (13) and 60% (22). These clinical data illustrate that tea tree oil can be used effectively in the oral cavity for yeast infections, however, whether tea tree oil can reduce or prevent the formation of microbial plaque has not yet been established. A further rationale for the oral use of tea tree oil is that it has anti-inflammatory effects (3, 14) and may therefore be helpful in the treatment or prevention of gingivitis. Many oral hygiene products may reduce gingival inflammation indirectly by reducing plaque, whereas tea tree oil has the potential to reduce both gingivitis and plaque mass simultaneously.

Safety issues associated with the use of tea tree oil in the oral cavity need to be addressed. Adverse effects such as a burning sensation (9, 13, 22), stinging (22) and unpleasant taste (9) have all been noted previously by patients using tea tree oil mouthwashes. However, in two of these studies the tea tree oil solution used was alcohol-based, and it is possible that the alcohol may have caused the burning, rather than the tea tree oil. Also, the mild to moderate burning sensation was noted largely during the first week of therapy and gradually decreased thereafter (13, 22), which may be due to accommodation. Patients using Listerine? (which contains 26.9% alcohol and 0.26% essential oils) have reported very similar reactions of an initial burning sensation and bitter taste, both of which decreased over subsequent days (24). The possibility of reactions to tea tree oil within the oral cavity or systemic toxicity from ingestion of products requires further research.

In conclusion, in vitro susceptibility data from this study show that the bacteria found in the mouth are susceptible to, and rapidly killed by, tea tree oil. As such, tea tree oil, incorporated into appropriate oral hygiene products such as mouthrinses, dentifrices and dental gels or irrigators, may be suitable for use in the oral cavity.


Acknowledgments
This work was supported by grants UWA-58A and UWA-55A from the Rural Industries Research and Development Corporation, and Australian Bodycare Pty. Ltd., Vissenbjerg, Denmark.


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