The phyletic relationship of four bacterial pathogens causing dental cavities has been studied by utilizing ITS1 sequences. The standard strains were aligned using the ClustalW computing machine program. The essential oil obtained from the leaves of Micromeria biflora Benth. was obtained by hydrodistillation.
The chemical compositions of the essential oil from Micromeria biflora Benth were analyzed by gas chromatography-mass spectroscopy (GC-MS). The GC/MS analysis showed eight major active components in the leaf essential oil of Micromeria biflora Benth.
The antibacterial activity of the oil was evaluated against four bacterial pathogens causing dental cavities, namely Streptococcus mutans (MTCC 890), Lactobacillus acidophilus (MTCC 447), Streptococcus mitis (MTCC 2695), and Streptococcus salivarius (MTCC 1938), using the broth microdilution method recommended by the Clinical Laboratory Standards Institute (CLSI), formerly known as NCCLS.
It showed excellent activity against Streptococcus mutans with its minimal inhibitory concentration (MIC) of 0.15 mg/ml and its IC50 of 0.10 mg/ml, and was less effective against Lactobacillus acidophilus. The essential oil of Micromeria biflora Benth from leaves played a significant role in inhibiting bacterial pathogens causing dental cavities.
The relationships of the bacterial pathogens causing dental cavities to the toxicity of the oil vis-à-vis phylogeny using molecular data of pathogens have also been discussed.
Key words: Antibacterial activity; Dental cavities; Essential oil; Micromeria biflora Benth; Phylogenetic Analysis; Streptococcus mutans.
Introduction
Dental cavities are a multifactorial infectious disease, commonly associated with increased numbers of Streptococcus mutans at the site of the disease. In addition, other microflora like Lactobacillus acidophilus, Streptococcus salivarius, Streptococcus mitis, and Streptococcus sanguis are also involved in the process of causing dental cavities.
Assessment of the salivary levels of these bacteria may be useful for evaluating cavities risk in patients and for monitoring their response to preventive measures. Streptococcus mutans is an important component of the biofilms on teeth (dental plaque) associated with many forms of dental cavities.
Streptococcus mutans adheres firmly to the smooth tooth surfaces and produces sticky water-insoluble dextran from dietary sucrose, forming plaque, which facilitates the accumulation of microorganisms. Streptococcus mutans and other organisms in the plaque produce organic acids such as lactic acid that gradually destroy the enamel and form a cavity.
The Micromeria biflora Benth, known as Indian wild thyme, belongs to the household Lamiaceae found in the tropical and temperate Himalayas and Western Ghats. It is a sturdy plant that grows up to district 0, flowers from June to August, and its seeds ripen from August to September.
The flowers are hermaphroditic (holding both male and female organs) and are insect-pollinated. A paste of the root was applied to treat odontalgia. The plant was rubbed and the aroma inhaled to treat nose bleeds. A paste of the plant was used as a poultice to treat lesions. The juice of the plant is taken internally and also inhaled in the treatment of sinusitis.
The aim of this study was to investigate the toxicity of the Micromeria biflora Benth. leaf essential oil vis-à-vis phylogeny using molecular data of pathogens. In particular, 16S rDNA sequences have been widely used to construct bacterial phylogenetic relationships.
Materials and Methods Collection of Plant Materials and Extraction of Essential Oil
The essential oil was extracted from the fresh leaves of Micromeria biflora Benth. collected from Himachal Pradesh, India by hydrodistillation using Clevenger’s setup (6). A clear dark reddish-yellow colored oily layer was separated and dried with anhydrous sodium sulfate.
Physicochemical Properties
The essential oils obtained from Micromeria biflora Benth were studied on various parameters of physicochemical properties such as plant height, oil output, color, specific gravity, optical rotation, refractive index, and solubility in 90% alcohol. The results are given in Table 2.
GC-MS Analysis
Gas chromatography analysis of the oil was performed on a Perkin-Elmer GC 8500, utilizing a amalgamated capillary column (25m x 0.55 mm ID, film thickness 0.25um), coated with dimethyl siloxane (BP-1).
The oven temperature was programmed at 60°C to 220°C at 50°C/min, then held isothermal at 220°C for 15 min. Injector temperature was 250°C, sensor temperature was 300°C, and the carrier gas was N at a linear speed of 10 pounds per square inch: split, 1:80.
GC-MS Data
Data were obtained on a Shimazdu QP-2000 mass spectrometer at 70 electron volts and 250°C. The GC column was Ulbon HR-1 equivalent to OV-1, fused silica capillary column 0.25 mm x 50m, film thickness 0.25 um. The initial temperature was 100°C for 7 min and heated at 5°C/min to 250°C. The carrier gas was He at a flow rate of 2 ml/min. The percentage composition of Micromeria biflora Benth leaves oil is given in Table 1.
Dental Cavities Causing Bacterial Pathogens
Four dental cavities causing bacterial pathogens were selected for this survey: Streptococcus mutans (MTCC 890), Lactobacillus acidophilus (MTCC 447), Streptococcus mitis (MTCC 2695), and Streptococcus salivarius (MTCC 1938). Cultures were obtained from Microbial Type Culture Collection (MTCC), Chandigarh, India.
The cultures of bacteria were maintained on Nutrient agar angles at 4°C throughout the survey and used as stock cultures.
Determination of Minimum Inhibitory Concentration (MIC) and IC50 by Broth Microdilution Method
The antimicrobial activity of compounds was determined by the broth microdilution method recommended by the Clinical Laboratory Standards Institute (CLSI) once NCCLS (7) using Mueller Hinton Broth. All the standard waterborne bacterial cultures were maintained on Nutrient agar at 37°C. The 96-well tissue culture plates were used for double consecutive dilution.
The proper growth control, drug control, and the space were adjusted onto the plate. Essential oil of Micromeria biflora Benth was dissolved in 5-10% DMSO at a concentration of 50 mg/ml stock solution in the case of natural disinfectants. 20 µl of the drug was added into the 4th well of 96-well tissue culture plate horizontally having 180 µl Mueller Hinton Broth.
Thus, the maximum concentration of the trial indispensable oil was 2.5 mg/ml. From here, the solution was serially diluted up to the 4th well to the 11th well, resulting in half of the concentration of trial indispensable oil.
The bacterial inoculum was prepared at 0.5 McFarland standards; the optical density was equal to the inoculum suspension containing 1×107 cells per milliliter for bacterial isolates. Then, the standard bacterial inoculum was added and kept for incubation at 37°C in a damp chamber.
The Minimum Inhibitory Concentration (MIC) and Inhibitory Concentration at 50% (IC50) were recorded spectrophotometrically at 492 nanometers using SpectraMaxplus 384 after 24 hrs incubation.
Determination of Minimal Disinfectant Concentration (MBC)
A 100 µl aliquot of inoculant was taken aseptically from incubated 96-well plates that did not show turbidity and poured onto Nutrient agar plates. The plates were then incubated for 24 hours at 37°C. MBC was defined as the lowest concentration of the essential oil at which 99.99% or more of the initial inoculant was killed.
If there was no growth, it means the concentration was cidal. The number of surviving organisms was determined by viability counts. All tests were performed in triplicate.
Phylogenetic Survey
To find out why the essential oil is more effective against bacteria causing dental cavities, phylogenetic relationships were studied, including Streptococcus mutans, Lactobacillus acidophilus, Streptococcus mitis, and Streptococcus salivarius, using the Clustal W computer program and GENETYX-MAC 10.1 software (Software Development Co., Ltd., Tokyo, Japan).
Phylogenetic trees were then constructed by the DNA maximum-likelihood (ML) method in the PHYLIP program (Phylogeny Inference Package), version 3.5c and the neighbor-joining (NJ) method in the NJPLOT program. Bootstrap analysis with the Clustal W program was performed.
Nucleotide Sequence Accession Numbers
Data for the phylogenetic analysis were obtained from sequences contained in the GenBank nucleotide sequence database. The ITS1 sequences of the standard strains used in this survey, Streptococcus mutans (accession no. AF204255), Lactobacillus acidophilus (accession no. HM162411), Streptococcus mitis (accession no. NC013853), and Streptococcus salivarius (accession no. S41233), were aligned.
Consequences and Discussion
Plant indispensable oils and infusions have been used for many thousands of years (15), particularly in food preservation, pharmaceuticals, alternative medicine, and natural therapies.
In the present study, the composition and comparative percentages of the indispensable oil of Micromeria biflora were determined. Four major components were identified, with high content of thymol (54%), iso-thymol (9.9%), gurjurene (3.3%), and β-caryophyllene (6.6%), with retention times (RT) of 8.06, 8.83, 10.20, and 12.86 respectively.
However, earlier studies suggested that the Micromeria biflora sp. Arabica K. Walth, indispensable oil was analyzed by GC-MS (17), and 30 constituents were identified, representing 98.2% of the entire oil. The major components were trans-caryophyllene (43.7%), caryophyllene oxide (18.0%), spathulenol (8.5%), β-humulene (4.6%), β-myrcene (3.1%), and germacrene-D (3.1%).
The present investigation of GC-MS analysis of Micromeria biflora indispensable oil revealed that the chemical components were quite different due to different agroclimatic alterations. The major constituents and their retention times are summarized in the table.
Filoche et al. reported that the essential oil of cinnamon showed antimicrobial activity (1.25-2.5 mg/mL) against Streptococcus mutans and Lactobacillus plantarum.
However, in the present study, the antibacterial activity of Micromeria biflora Benth leaf indispensable oil was assayed in vitro by a broth micro-dilution method against four bacteria causing dental cavities, such as Streptococcus mutans (MTCC 890), Lactobacillus acidophilus (MTCC 447), Streptococcus mitis (MTCC 2695), and Streptococcus salivarius (MTCC 1938).
According to the results, Micromeria biflora Benth leaf indispensable oil was found to be active against all bacteria causing dental cavities. The strongest antibacterial activity was seen against Streptococcus mutans with a minimum inhibitory concentration (MIC) value of 0.15 mg/mL and an IC50 value of 0.10 mg/mL.
While the MIC value of Lactobacillus acidophilus, Streptococcus mitis, and Streptococcus salivarius was 0.35 mg/mL, 0.20 mg/mL, and 0.19 mg/mL respectively. These results are shown in Table 3. Nascimento et al. also reported that the Hyptis pectinata indispensable oil exhibited a considerable inhibitory effect against either all the clinical isolates obtained from patients’ saliva or the ATCC strains tested, with minimal inhibitory and disinfectant concentrations of 200 μg/mL.
Conclusions
The present study clearly demonstrates that the leaf indispensable oil of M. biflora Benth exhibited powerful disinfectant action and can be used as a therapeutic remedy against bacteria causing dental cavities. The effectiveness of the oil was equal to those bacteria causing cavities, which are close in the phylogenetic tree.
As such, in the future, the oil can be used as a potential source of effective and inexpensive herbal preparation after undergoing successful multicentral topical testing.
