Antibacterial efficacy of Azadirachta indica, Mimusops elengi and 2% CHX on multispecies dentinal biofilm.

AIMS: To check the antimicrobial activity of Azadirachta indica (Neem), Mimusops elengi (Bakul), and Chlorhexidine gluconate (CHX) on multispecies biofilm of common endodontic pathogens such as Streptococcus mutans, Enterococcus faecalis, Staphylococcus aureus and Candida albicans. SETTINGS AND
DESIGN: In vitro dentin disinfection model used to check the antimicrobial efficacy of herbal extracts.
MATERIALS AND METHODS: The in vitro dentin disinfection model was used to check the antimicrobial activity of the methanolic extracts of the medicinal plants along with Chlorhexidine gluconate. The polymicrobial biofilm was allowed to grow on extracted teeth sections for a period of 21 days. Remaining microbial load in the form of CFU/ml after the antimicrobial treatment was tabulated, and data were statistically analyzed using ANOVA and Bonferroni post-hoc tests. STATISTICAL ANALYSIS USED: SPSS version 17, one-way ANOVA, Bonferroni post-hoc test.
RESULTS: Both the plant extracts showed considerable antimicrobial efficacy as compared to negative control. 2% CHX was the most effective antimicrobial agent having statistically significant difference against plant extracts and negative control (saline).
CONCLUSION: The methanolic extract of A. Indica, M. elengi, and Chlorhexidine Gluconate has considerable antimicrobial activity against polymicrobial dentinal biofilm of S. mutans, E. faecalis, S. aureus and C. albicans.

INTRODUCTION

Microorganisms are the primary etiological factor in the development of pulpal and periapical diseases.[12] Endodontic treatment is aimed at complete elimination of microbes form the pulp space. This goal is achieved by thorough chemo-mechanical preparation followed by three dimensional obturation of the root canal system. While mechanical instrumentation can remove a significant number of bacteria from the root canal system,[3] the bacteria remaining in the intricacies of the pulp space can cause or sustain periradicular tissue inflammation.[4] Therefore, mechanical instrumentation of the pulp space is accompanied by the use of different types of irrigation solutions. According to current literature, sodium hypochlorite and 2% chlorhexidine remains to be the most preferred irrigating solutions.[5] Chlorhexidine gluconate has a wide antimicrobial spectrum. However, at 2% concentration, which is most commonly used in endodontics, may have toxic effects on host tissues if expressed beyond the confines of the root canal and may impair healing.[6] It also lacks tissue dissolving capacity.[7]

Natural products are known to play an important role in human life. Various parts of the plants like root, bark, seed and leaves have been an important source of medicine for thousands of years. In recent years, a predominant interest has been observed in evaluating different plant extracts for their antimicrobial properties against bacteria causing dental caries and periradicular pathology. Murray et al. evaluated the possible use of Morinda citrifolia juice as an alternative to sodium hypochlorite as an irrigant.[8] Prabhakar et al. evaluated herbal alternatives such as Triphala and green tea polyphenols for their antimicrobial efficacy against E. faecalis biofilm.[9] Berberine, a plant alkaloid isolated from many medicinal plants when combined with CHX was found to be comparable to sodium hypochlorite in its bactericidal efficacy.[10] India has a rich flora of medicinal plant species that are widely distributed throughout the country.

Mimusops elengi, locally known as Bakul is a small to large tree found all over India. The plant finds an important place in the indigenous system of medicine, and its various parts are used in the treatment of various systemic diseases including dental problems. It has shown significant anti-inflammatory, analgesic, and antipyretic activity.[11] The bark of M. elengi is acrid, astringent and is used as a gargle for odontopathy, inflammation and bleeding gums.[12] The tender stems are used as toothbrushes.[13]

Azadirachta indica (Neem) is perhaps the most useful traditional medicinal plant. Every part of the tree has been used as traditional medicine for a household remedy against various human ailments. The tree is still regarded as “village dispensary” in India. Most of the plant parts such as fruits, seeds, leaves, bark, and roots contain compounds with proven antiseptic, antiviral, antipyretic, anti-inflammatory, antiulcer, and antifungal properties.

This study is aimed to check the antimicrobial efficacy of methanolic extracts of selected medicinal plants such as A. Indica (Neem) and M. elengi (Bakul), along with 2% chlorhexidine gluconate against polymicrobial biofilm formed on root canal walls of extracted human teeth.

MATERIALS AND METHODS

Procurement of material

The leaves of Neem and bark of Bakul were collected from the courtyard. All the plant materials were identified by the senior professor of Department of Pharmacognosy at L. M. College of Pharmacy, Ahmedabad. Plant materials were washed with distilled water and dried under shade for 10-12 days. All the material was ground in an electric grinder to produce a powder.

Preparation of extracts

The powdered material was again dried in an oven at 40°C for 4 h. and used for extraction. Accurately weighed 50 g of powdered leaf sample was extracted with 500 ml methanol. This process was repeated until the residual marc got exhaustively extracted, and finally extracts were pooled and evaporated in rota-evaporator. The extracts were concentrated under partial vacuum at 80°C to dryness, leaving behind thick semi-solid residue. This extract was dissolved in dimethyl sulfoxide (DMSO) to get six different concentrations to be tested.

Procurement of microorganisms

The microbial strains investigated in the study were obtained from IMTECH-Chandigarh. The strains used are E. faecalis (MTCC 439), Streptococcus mutans (MTCC 497), Staphylococcus aureus (MTCC 737), and Candida albicans (MTCC 227).

As a preliminary screening test, the antimicrobial efficacy of the plant extracts and CHX was checked on all the microorganisms in their planktonic form using agar diffusion test before evaluating them against polymicrobial biofilm. The test microorganisms were subcultured on specific media procured by Hi-Media Laboratory Pvt. Ltd., Mumbai, India and incubated aerobically at 37°C for 24 h. A total of six wells were made into a nutrient agar plate using sterile cork borer (6 mm in diameter) and inoculums containing 1 × 105 CFU/ml of bacteria were spread on the solid plate with the bacterial suspension. Hundred microliters of the working solution of different medicinal plant extract carrying different concentration of the medicine was filled in the wells with the help of micropipette. The plates were then incubated at 37°C for 24 h in an aerobic environment. After overnight incubation, the plates were observed for the zone of inhibition and the diameter of the inhibition zone in millimeters were measured using a scale. Each extract was tested 3 times, and mean values were recorded. Minimum inhibitory concentration and minimum bactericidal concentration were determined using micro broth dilution method.

Biofilm formation on the root canal

A total of 31 single rooted, intact, noncarious human anterior teeth with fully formed apices were selected for the study. The presence of single canal was confirmed by preoperative radio-visiography images. The teeth were cleaned to remove superficial debris, calculus and tissue tags and were stored in normal saline to prevent dehydration before use. The pulp chamber was opened with the help of a round bur in an airotor. The working length was determined as the negotiating file was just visible at the apical foramen. The root canals were instrumented using the crown-down technique with rotary instruments and canals were prepared to an apical size 30 by using ProTaper F3 (ProTaper, Dentsply Maillefer, Ballaigues, Switzerland). During the preparation 2 ml of 3% NaOCl (Vishal Dento Care, Ahmedabad, Gujarat, India) was used as an irrigant between each instrument. The teeth were then sectioned at the cemento-enamel junction as well as vertically along the mid-sagittal plane into two halves. The concave tooth surfaces were then minimally grounded to achieve a flat surface, simultaneously exposing the root canal surface to allow formation of a biofilm.

The sterilized tooth sections were stored in 2 ml ependrof tubes. The vials containing tooth sections were inoculated with 2 ml of a bacterial suspension of all four microorganisms and incubated at 37°C for a period of 21 days to allow biofilm formation. The culture medium was replaced every alternate day to avoid nutrition depletion and accumulation of toxic end products. The viability and purity of the culture were checked during every change of the culture medium. The samples were taken from each well with a sterile paper point, inoculated onto Mueller-Hinton agar plates, and incubated at 37°C for 24 h to check for cell viability and purity of culture.

At the end of 21 days, two tooth sections were selected randomly and sent for scanning electron microscopic evaluation for confirmation of polymicrobial biofilm growth. Remaining 60 sections were divided into four groups according to the medicaments to be tested. Normal saline was served as negative control. The four groups (15 each) were divided as follows:

Group 1: 2% chlorhexidine gluconate (CHX).

Group 2: A. Indica extract (75 mg/ml in 10% DMSO).

Group 3: M. elengi extract (75 mg/ml in 10% DMSO).

Group 4: Normal saline.

All the samples were immersed in the 2 ml of test solutions according to their groups for 10 min. Then, the biofilm on the root canal portion was scraped off with the help of a scalpel to collect well-attached, deeply seated as well as loosely attached superficial bacteria from the formed biofilm. The scrapings were inoculated on Muller-Hinton agar plates and incubated for 24 h at 37°C for qualitative analysis where n = 2 for each group. The quantitative analysis was performed by vortexing the scraped biofilm material of the treated tooth samples with sterile saline for a few minutes, followed by serial dilution method and CFU counts, where n = 13 for each group. The mean and standard deviations were calculated.

The collected data was tabulated and analyzed using SPSS software version 17 (SPSS Inc. Chicago, USA) for ANOVA. Multiple comparisons were carried out using Bonferroni post-hoc test to check the difference between the groups. To maintain the normality of data the log transformation was done. Parametric tests assume a normal distribution. If this assumed distribution is correct, then a small number of samples are sufficient to characterize the mean and variance.

RESULTS

Both the plant extracts showed considerable antimicrobial activity against S. mutans, E. faecalis and S. aureus in agar diffusion test. However, they were ineffective against C. albicans at the highest concentration (3 mg) used in our study. 2% CHX was the most consistent of all the tested agents, showing antimicrobial effects against all the microorganisms. However, the difference between the CHX and plant extracts was not statistically significant [Tables 1 and 2].

Table 1

The zone of inhibition, MIC and MBC of test solutions against individual microorganisms in their planktonic forms

Table 2

Quantitative analysis of 3-week polymicrobial biofilm formed on tooth substrate for different groups

In the quantitative analysis with 3-week old polymicrobial biofilm on the canal portion, chlorhexidine proved to be the best antimicrobial agent with a statistically significant difference with plant extracts and Control (saline). Among the plant extracts, M. elengi was more effective than A. Indica and saline and the difference between them was statistically significant.

DISCUSSION

Biofilms are matrix enclosed microbial communities in which cells adhere to each other and/or to surfaces.[14] The intracanal microbiota in primarily as well as secondarily infected teeth exists as biofilm structures, consisting of cocci, rods, and filamentous bacteria.[15] The selection of microorganisms in our study was based on their significant presence in primary endodontic infections as well as their contribution in the failure of the endodontic therapy. Most of the studies performed to evaluate the antimicrobial efficacy of various medicaments have been done either on planktonic cultures or monoculture biofilm of E. faecalis. However, It has been well established that endodontic infections are polymicrobial in nature.[16] and E. faecalis has been reported to coexist with other several taxa in root canal treated teeth.[1718] Although most commonly implicated in recurrent infections, E. faecalis has also been a member of root canal flora of preliminary endodontic infections. Using various molecular methods, the prevalence of E. faecalis in primary endodontic infections were 7.5% by checkerboard hybridization,[19] 18%,[20] 7.5-15% by standard polymerase chain reaction (PCR),[2122] 11% by genotyping,[22] 7% and 13% by cultivation and qPCR, respectively has been reported.[23]

The recovery rates of S. aureus from acute dental abscess range from 0.7% to 15% using conventional culture-based methods.[19232425262728] Interestingly, S. aureus has been reported to occur more frequently in severe dental abscesses from children.[26] Along with S. aureus, the presence of S. mutans is also found to be more strongly associated with preoperative symptoms and with the presence of swelling.[2124] Moreover, it has been shown that S. mutans may have a major influence on both the initial pulpal lesion and subsequent pulpal pathology.[29] Fungi are considered normal inhabitants of the oral cavity, but may produce disease when there are local or systemic factors predisposing the individual to infection. C. albicans has been the fungi most commonly isolated from the oral cavity of both the healthy and medically compromised patients. The role of C. albicans in the etiology of primary as well as the secondary endodontic infection is well established.[3031] Therefore, in the current study, we have used a polymicrobial biofilm model consisting of these microorganisms to investigate the antimicrobial activity of methanolic extracts of A. Indica and M. elengi along with 2% CHX.

Following this model, we have done qualitative, and quantitative analysis of the teeth samples treated with 75 mg/ml. of the plant extracts and 2% CHX for a period of 10 min.

CHX is proven to be the best with statistically significant difference in terms of reduction of microorganisms against all the groups. The antibacterial effectiveness of CHX in the reduction of bacteria in infected root canals in vivo has been investigated in several other studies. In a controlled and randomized clinical trial, the efficacy of 2% CHX liquid was tested against saline using a culture technique. All of the teeth were initially instrumented and irrigated using 1% sodium hypochlorite. Then, either 2% chlorhexidine liquid or saline was applied as a final rinse. The authors reported a further reduction in the proportion of positive cultures in the CHX group. Their results showed a better disinfection of the root canals using chlorhexidine compared to saline as a final rinse.[32] CHX has been used in endodontics and proposed as both an irrigant and an intracanal medicament due to its antimicrobial efficacy against wide range of microorganisms, such as Gram- and Gram-negative bacteria, yeasts, and fungi. Its efficacy is based on the interaction between the positive charge of the molecule and the negatively charged phosphate groups on the bacterial cell wall, which allows the CHX molecule to penetrate into the bacteria with toxic effects.[33]

Plant extracts showed promising antibacterial efficacy along with 2% CHX compared with control. M. elengi is most promising among both the extracts as it has a significant difference in antimicrobial efficacy as compared to A. Indica and Control. This may be because of the solvent used for the preparation of the extract. Methanolic extracts may show greater activity because more phytoconstituents are leached in it as compared to other extracts. Different types of glycosides alkaloids, phenols, tannins, and saponins have been screened in the methanolic extract of this plant.[34]

A. Indica also showed considerable efficacy against biofilm bacteria as compared to control; however, the difference between them was not statistically significant. A. Indica has been found to be highly efficacious in the treatment of periodontal diseases, thus exhibiting its biocompatibility with human periodontal ligament fibroblasts. The use of Neem as an endodontic irrigant might be recommended because it is a biocompatible antioxidant and thus not likely to cause severe injuries to patients.[3536] A bitter taste associated with this plant can be altered by the addition of sweeteners and flavors to increase the patient compliance and acceptability.[35]

The effects of plant extracts are significantly less as compared to 2% CHX. This may be due to the fact that microorganisms develop certain genetic and phenotypic variation in biofilm growth as compared to their planktonic counterpart. These changes result in increased resistance of the microorganisms to topical antimicrobials. Biofilm grown cells have been estimated to become 1000-1500 times more resistant to antimicrobials than planktonically grown cells.[37] In our study, all the plant extracts have shown a considerable antimicrobial effect against individual microorganisms in agar diffusion test, whereas in polymicrobial biofilm the effectiveness of plant extracts has significantly reduced. The age of biofilm also plays a vital role in the resistance of microorganisms to antimicrobials.[38] In this study, we have used 3 weeks old biofilm model, which more closely simulates the in vivo condition. This factor also may have contributed to the less pronounced effect of plant extracts. A better antimicrobial efficacy can be achieved by increasing the concentration of the antimicrobial agent.

CONCLUSION

Within the limitations of this study, 2% CHX showed maximum antimicrobial activity against 3-week old polymicrobial biofilm formed on tooth substrate. Methanolic extracts of M. elengi and A. indica showed considerable efficacy as an antimicrobial agent. However, further research is required to recommend natural plant extracts as a root canal irrigant.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.