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In vitro effects of Melaleuca alternifolia essential oil on growth and production of volatile sulphur compounds by oral bacteria

ABSTRACT Objective Halitosis can be caused by microorganisms that produce volatile sulphur compounds (VSCs), which colonize the surface of the tongue and subgingival sites. Studies have reported that the use of natural products can reduce the bacterial load and, consequently, the development of halitosis. The aim of this study was to evaluate the antimicrobial activity of the essential oil of Melaleuca alternifolia on the growth and volatile sulphur compound (VSC) production of oral bacteria compared with chlorhexidine. Material and Methods The effects of these substances were evaluated by the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) in planktonic cultures of Porphyromonas gingivalis and Porphyromonas endodontalis. In addition, gas chromatography analyses were performed to measure the concentration of VSCs from bacterial cultures and to characterize M. alternifolia oil components. Results The MIC and MBC values were as follows: M. alternifolia - P. gingivalis (MIC and MBC=0.007%), P. endodontalis (MIC and MBC=0.007%=0.5%); chlorhexidine - P. gingivalis and P. endodontalis (MIC and MBC=1.5 mg/mL). M. alternifolia significantly reduced the growth and production of hydrogen sulfide (H2S) by P. gingivalis (p<0.05, ANOVA-Dunnet) and the H2S and methyl mercaptan (CH3SH) levels of P. endodontalis (p<0.05, ANOVA-Dunnet). Chlorhexidine reduced the growth of both microorganisms without altering the production of VSC in P. endodontalis. For P. gingivalis, the production of H2S and CH3SH decreased (p<0.05, ANOVA-Dunnet). Conclusion M. alternifolia can reduce bacterial growth and VSCs production and could be used as an alternative to chlorhexidine.


INTRODUCTION
Halitosis, also known as bad breath or malodour, is a condition caused by fetid odours present in air emanating from the mouth 1 , leading to personal and social discomfort 21 . The origin of pathological halitosis can be systemic or local 14 and should be diagnosed and treated 1,14,21 . This condition is multifactorial and may comprise both oral and nonoral causes 21,25 . Periodontal disease, peri-implantitis, deep carious lesions, tongue coating, impacted food or debris, unclean dentures, and other oral problems may contribute to the onset of halitosis 9 .
Oral bad breath can result from the degradation of proteins containing sulphur amino acids (methionine and cysteine), resulting in the production of volatile sulphur compounds (VSCs), represented by hydrogen sulfide (H 2 S), methyl mercaptan (CH 3 SH), and dimethyl sulphide [(CH 3 ) 2 S]: gases that emanate malodour 1 . Some anaerobic gramnegative bacteria present in the oral cavity, such as Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Tannerella forsythia, and Porphyromonas endodontalis, are the main species responsible for the production of VSCs 18 . In addition to the role of VSCs in generating halitosis, there is evidence suggesting that these gases are also involved in the pathogenesis of periodontal diseases 1,29 . Various oral approaches have been employed to treat halitosis, including the mechanical removal chlorhexidine, cetylpyridinium, or essential oil mouthrinses, and the application of masking products such as chewing gums and mouthrinses containing chlorine dioxide and zinc salts 5,10,23,26 . It has also been reported that natural products, such as green tea, produce effects that control halitosis and VSC production 12,28,30 . Most products used to reduce malodour have antimicrobial properties, and the decrease in VSCs is usually related to the suppression of bacterial growth.
Melaleuca alternifolia, also known as tea tree oil, has been studied because of its antimicrobial activity against oral pathogens, showing inhibitory and bactericidal effects 3,7,8 . A solution containing tea tree oil was shown to reduce the levels of malodour and production of VSCs in patients nursed in an intensive care unit 10 . Despite the antimicrobial potential of M. alternifolia, there are few studies evaluating its activity against oral pathogens that cause bad breath 4,8 . Thus, the aim of this study was to evaluate the effects of Melaleuca alternifolia oil and chlorhexidine on the viability and VSC production of P. gingivalis and P. endodontalis.

Substances tested
This study used the essential oil of Melaleuca alternifolia (Arista Industries; Wilton, Connecticut, USA) as the tested substance and chlorhexidine gluconate (Sigma-Aldrich; St. Louis, Missouri, USA) as the standard antimicrobial.

Determination of the chemical profile of M. alternifolia essential oil by gas chromatography-mass spectrometry (GC-MS)
The essential oil was subjected to gas chromatography analyses to obtain its chemical profile. Analyses were performed on a gas chromatograph, model: HP-6890 (HP; Palo Alto, California, USA), interfaced with a mass selective detector HP-5975. A fused silica capillary column HP-5 (length of 30 m, internal diameter of 0.25 helium as the carrier gas (1 mL min -1 ).
The oil was diluted with ethyl acetate, and 0.1 mL was injected into the device. The temperatures used were 220°C for the injector, 250°C for the detector, and 60°C -240°C for the column (3°C min -1 ). To identify the analytes, a mixture of n-alkanes was used to calculate the retention index (RI). Comparisons were performed using the National Institute of Standards and Technology (NIST) electronic library and literature data based on RI. The determination of essential oil components was based on the calculation of the area under the peaks.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
MIC and MBC were carried out according to Clinical and Laboratory Standards Institute (CLSI) 5 of MIC and MBC, serial two-fold dilutions were made for all substances tested. Concentrations ranged from 0.5% (v/v) to 0.002% (v/v) for the essential oil of M. alternifolia and from 100 μg/mL (0.01% -p/v) to 0.38 μg/mL (0.0038% -p/v) for the chlorhexidine solution. An inoculum of 40% transmittance, equivalent to approximately 8x10 8 CFU/mL, was prepared from bacterial cultures on TSA with three days of growth, and 500 μL samples of this bacterial suspension were transferred to volume of 6 mL. Moreover, tubes without tested compounds or bacterial suspension were used as controls. Cultures were maintained under anaerobic conditions for 48 hours. The lowest concentration of each substance with no bacterial growth was considered the MIC. For MBC determination, 10 μL samples from TSB cultures were transferred anaerobic conditions. The lowest concentration with no bacterial growth was considered the MBC.
The inoculum was prepared as described in item 2.3. To collect gas from inside the tubes, needles (Becton Dickinson Company; Franklin Lakes, New Jersey, USA) were coupled with the covers of the tubes. After 16 hours of growth in anaerobic conditions, 1 mL syringes were attached to the needles, and 0.1 mL of air was aspirated from inside each tube. The volume of air collected was injected into the OralChroma™ (CHM-1, Abilit Corporation; Chuo-ku, Osaka, Japan). After measurement, the device provided the concentrations of hydrogen sulfite (H 2 S), methylmercaptan (CH 3 SH), and 3 ) 2 S] in parts per billion. The tubes were also subjected to absorbance readings (Unico Inc; Dayton, New Jersey, USA).

Statistical analysis
All experiments were performed in eight replicates and reproduced at least two times. The Lilliefors test (an adaptation of the Kolmogorov-Smirnoff test) was used to check the normality of data distribution. For VSC data, analysis of variance (ANOVA) was performed, and the difference among

Chemical pro le of the essential oil of M. alternifolia
The characterization of essential oil compounds was performed by comparing retention times and MS/MS mass spectra of each peak with information the percentage range for tea tree oil recommended by ISO 4730:2004 are described in Table 1.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
The MIC and MBC values for M. alternifolia oil and chlorhexidine solution are shown in Table 2. The tea tree oil and chlorhexidine digluconate solution were able to inhibit the growth of both strains at low concentrations.

In vitro production of Volatile Sulphur Compounds
The effects of sub-MIC concentrations of tea tree oil and chlorhexidine on the growth and production of volatile sulphur gases (H 2 S and CH 3 SH) are shown P. gingivalis W83, and in Figures 3 and 4 for P. endodontalis. Concentrations tested were lower than the minimum inhibitory concentration (sub-MIC concentrations) and were The tea tree oil reduced the growth of P. gingivalis CH 3 SH at all concentrations tested (16x<MIC, 8x<MIC, and 4x<MIC). Curiously, the 16x<MIC concentration promoted less CH 3 SH production than higher concentrations (p<0.05). The production of H 2 S was not altered by the essential oil at any of the concentrations. In the presence of chlorhexidine, P. gingivalis W83 showed a reduction in growth at concentrations of 8x<MIC and 4x<MIC, and they were different from each other (p<0.05). Furthermore, it exhibited a reduction in CH 3 SH levels for all concentrations tested and in H 2 S for  8x<MIC and 4x<MIC (p<0.05). The microorganism P. endodontalis showed a reduction in growth in the presence of tea tree oil for concentrations 8x<MIC and 4x<MIC (p<0.05). Decreased gas production was observed for both H 2 S (16x<MIC, 8x<MIC, and 4x<MIC) and CH 3 SH (8x<MIC and 4x<MIC) (p<0.05). However, at the concentrations tested, chlorhexidine showed effects on P. endodontalis growth (p<0.05) but not on VSC production.

DISCUSSION
to halitosis development, many studies involving therapies for the treatment of halitosis have focused on the inhibition and reduction of VSC production. Antimicrobial therapy aims to enhance mechanical treatments and support host defences, reducing the development of microorganisms. Some studies have M. alternifolia against oral bacteria 7,8,11,24 ; however, little is known about its effects on VSC production. Here, we showed that M. alternifolia essential oil can reduce growth and VSC levels of P. gingivalis and P. endodontalis, even at sub-MIC concentrations.
GC-MS analysis of the tea tree oil used in this study showed that the composition of this oil was consistent with the International Standard ISO of M. alternifolia oil such as quality requirements. The main components described by the ISO for M. alternifolia 20 .
All of these compounds were found in the oil used in this study, except for limonene and sabinene. However, these two compounds are generally found in small quantities in tea tree oil (0.5% -4% and trace -3.5%, respectively), and their low levels CG-MS analysis. In contrast, terpinen-4-ol and activity, were found in satisfactory percentages 4,13 . M. alternifolia have a mixture of components and their mechanisms of action are not completed elucidated. It is known that the combination of these different substances in the tea tree oil are capable of inducing loss of intracellular material, inhibition of respiration, and alterations in the homeostasis, leading to loss of bacterial membrane integrity and function 4 .
The tea tree oil showed antimicrobial activity: its MIC value was 0.007% for both bacteria, and MBC values were 0.5% for P. endodontalis and 0.007% for P. gingivalis. In a previous study, the MIC value found for P. gingivalis was 0.13% -0.25%, and the MBC value was 0.13% -0.5% 22 . Although MIC values differed between studies, MBC values were similar. Differences in MIC values may be due to different strains tested: in this study we used the W83 strain, while Takarada, et al. 22  To evaluate the activity of M. alternifolia and chlorhexidine on the production of volatile sulphur compounds, sub-inhibitory concentrations of these substances were used. The sub-MIC concentrations of the tea tree oil affected the growth of both microorganisms, reducing the growth at higher concentrations (8x<, 4x<MIC, and 16x<MIC). Furthermore, at all concentrations tested, tea tree oil reduced CH 3 SH production in P. gingivalis and both H 2 S and CH 3 SH production in P. endodontalis.
As not expected, the 16x<MIC concentration promoted a higher reduction of CH 3 SH than the 8x< and 4x<MIC concentrations. We believe that higher concentrations of tea tree oil (8x and 4x<MIC) could stress the bacteria and stimulate a little the VSC production comparing to the lower concentrations (16x<MIC). However, this hypothesis has to be The CH 3 SH and H 2 S gases are the main VSCs in intra-oral halitosis 1 , and their toxicity is associated with the development of periodontal disease 15,16,29 . Tea tree oil was able to inhibit the growth of P. gingivalis W83 and P. endodontalis, and the production of VSCs. Thus, this essential oil is a promising substance for treating halitosis. Previously, M. alternifolia oil was found to be effective as one of the components of an antiseptic mouth rinse 10 in our study, has demonstrated activity against a number of oral pathogens involved in periodontal disease and caries 17 .
Chlorhexidine is widely used in mouthrinses, causing membrane disruption and inhibition of proteolytic and glycosidic enzimes, leading to growth inhibition and cell death 27 . Despite being a potent antimicrobial, it has certain side effects such as altered taste, mucosal desquamation, tooth and a burning sensation in the oral mucosa 19 . Compared with chlorhexidine, tea tree oil showed similar antimicrobial activity, promoting bactericidal and bacteriostatic effects at low concentrations. Thus, tea tree oil could be a good alternative to chlorhexidine in oral hygiene products, mainly mouthrinses. Therefore, the development of new mouthrinses containing tea tree oil and clinical studies testing these products are necessary.
In conclusion, M. alternifolia oil showed antimicrobial activity against P. gingivalis W83 and P. endodontalis, reducing the growth and the production of VSCs at sub-MIC concentrations, comparably to chlorhexidine. Future studies can be conducted focusing on the development of pharmaceutical products containing M. alternifolia oil for halitosis treatment.