Comparative evaluation of antimicrobial efficacy of calcium hydroxide, triple antibiotic paste and bromelain against Enterococcus faecalis: An In Vitro study.

Context: Considering the effectiveness and safety concerns of synthetic intracanal medicaments (ICM), alternative usage of herbal ingredients in the root canal treatment may prove to be beneficial. Hence, the naturally available potent proteolytic, anti-inflammatory, and antimicrobial pineapple extract Bromelain as an ICMs needs to be studied. Aim: The aim of this study is to evaluate and compare the antimicrobial efficacy of calcium hydroxide (CaOH) and triple antibiotic paste (TAP) with Bromelain against Enterococcus faecalis bacteria using the turbidity method. Materials and
Methods: Forty-four freshly extracted single-rooted teeth were collected and 6 mm sections were prepared. These samples were infected with E. faecalis bacteria (ATCC 29212), randomly divided into four groups. Negative control, CaOH paste, TAP, and Bromelain paste. After the removal of medicament, the optical density (OD) of the dentinal shavings was measured using a spectrophotometer. Statistical Analysis: The Chi-square and Kruskal - Wallis ANOVA test were used. The confidence interval was set to 95% at 5% significance level.
Results: TAP showed less OD than CaOH2 paste which is statistically significant. (P = 0.0022). The OD reading of Bromelain paste was less than CaOH2 with P = 0.007. The OD of Bromelain and TAP were statistically insignificant (P = 0.095), indicating their comparable antibacterial efficacy. Conclusions: The antimicrobial efficiency of Bromelain was found to be more effective than Ca (OH) 2 paste and comparable to TAP against E. faecalis. Copyright:

INTRODUCTION

Microorganisms have a major role in the occurrence of pulp and periapical diseases and eventually in failures of endodontic treatment. The success of endodontic treatment depends primarily on the elimination of the microbial flora using various irrigants, activation techniques, and judicious use of intracanal medicaments (ICM). However, certain bacteria such as Enterococcus faecalis may stay even after thorough disinfection and hence may cause endodontic failures.[12] Reinfected or failed endodontically treated teeth are found to be more likely to embed E. faecalis than the primary infection.[34] E. feacalis is a Gram-positive facultative anaerobic bacteria which has the ability to exist without oxygen in the changing environment and also have the ability to deeply penetrate into the dentinal tubules.[5] As it is also able to tolerate various antibacterial agents, hence, it is provocating to eradicate it from the root canal (RC) system.[6]

ICMs such as formocresol, glutaraldehyde, calcium hydroxide (CaOH)[7] chlorhexidine,[8] and propolis[9] have been studied to limit the residuum of microorganisms. Desirable properties of ICMs include continuous and extended antimicrobial effect, stability in the solution, biocompatibility, anti-bacterial, anti-fungal, and nonstaining to the tooth. However, till date, there is no such ideal ICM available.[10] CaOH being the most frequently used ICM over the period of time has been proved to compromise the strength of the RC system.[210] On the other hand, triple antibiotic paste (TAP) which was also more likely found to be effective than CaOH and chlorhexidine gel caused bacterial resistance and tooth discoloration.[11] Considering the quantum of efficacy, the harmful effects and safety concerns of synthetic agents; the replacement with herbal agents might prove to be advantageous.

Various herbal agents such as Propolis, Triphala, Camelliasinensis (Chai – Tea), Curcuma longa Linn. (Haridra -Turmeric), orange oil, guava, and neem with potent antimicrobial properties have been used to disinfect the canals.[12] Although the literature has very few reports regarding the adverse effects of these natural products such as digestive tract irritation, allergic conjunctivitis, and allergic contact dermatitis,[13] they are considered safe. Similarly, an unexplored agent Bromelain is well known for its proteolytic, antibacterial, antifungal, anti-inflammatory, antithrombotic, and fibrinolytic properties.[14]

Bromelain is a proteolytic enzyme derived from the stem part and fruit of pineapples (Ananas comosus). It is derived from Bromeliaceae family.[15] It is used for curing various conditions such as allergies, inflammation, burns, blood coagulation, enhancement of antibiotic action, blocked sinuses, cardiovascular diseases, osteoarthritis, diarrhea, and cancer.[16] It is also known to improve the absorption of antibiotics which leads to effective diffusion of the drug in tissues and thus lowers the probable side effects associated with its toxicity.[17] It is rich in flavonoids and protease that provide the antibacterial property. It has been studied in various areas of dentistry such as against periodontal pathogens[18] and bleaching.[19] Considering its variable role with eminent properties, its usage in the field of endodontics cannot be neglected.

Till date, no studies have been conducted using the naturally available potent proteolytic, anti-inflammatory, and antimicrobial pineapple extract Bromelain in endodontics as an ICM. Thus, this in vitro study was conducted to comparatively evaluate the antibacterial efficacy of CaOH paste and TAP with bromelain against E. faecalis bacteria as an ICM.

MATERIALS AND METHODS

The research protocol was approved by the Ethical Committee of the institution. (CDCRI/DEAN/ETHICS/COMMITTEE/CONS-04/2018). To avoid bias, all the procedures were carried out by a same operator who was blind with the study materials.

Sample preparation

Forty-four freshly extracted single-rooted human mandibular premolar teeth were collected and kept in 3% sodium hypochlorite (NaOCl) solution for 24 h to disinfect and clear the organic debris. The crowns and apical third of each sample were cut using a diamond disc under running water to obtain a section of 6 mm thickness from the mid root. The internal diameter of each RC was enlarged to a uniform size using gates glidden (GG) drill #3 (Dentsply, United States.). This procedure was carried out under water to keep the samples hydrated. These samples were again kept in 3% NaOCl solution for 24 h to remove debris from the RC.

The samples were autoclaved for 30 min at 121°C to make them sterile and then incubated in a glass flask of narrow mouth containing brain − heart infusion (BHI) broth for 24 h at 37°C. These samples were confirmed of their sterility through the absence of turbidity in the BHI broth. The samples were then removed from the broth and RCs were blotted dry with sterile paper points. Each sectioned sample was coated from outside with varnish and allowed to dry and then mounted in a sterile petri dish with the help of the sticky wax.

Inoculation

An inoculum of E. faecalis bacteria (ATCC 29212) was injected into RC space and each sample was incubated at 37°C for 21 days. Fresh inoculum was added to each RC every day to keep the canals full. The viability of inoculums was tested weekly through culture. After inoculation for 21 days, the debris from the RC of any two random samples was incubated for 24 h at 37°C in separate test tubes containing fresh BHI broth to check for turbidity that confirmed the positive samples. At the end of inoculation period of 21 days, 10 ml of sterile saline was used to irrigate each RC and sterile paper points were used to blot dry the canals. The samples were then randomly divided into four experimental groups of 11 samples [Figure 1] each as follows:

Figure 1

Samples divided into four groups according to the intracanal medicament to be used. (A) Negative control, (B) Calcium hydroxide, (C) Triple antibiotic paste, (D) Bromelin

Group A: No placement of ICM in the RCs (n = 11) (negative control)

Group B: Placement of CaOH paste as an ICM in the RC (n = 11) (ApexCal, Ivoclar Vivadent, Liechtenstein) (positive control)

Group C: TAP (n = 11) (freshly prepared powder of Tab Metronidazole, Ciprofloxacin, and Minocycline, mixed with saline in the ratio of 1:1:1)

Group D: Bromelain paste (n = 11) (Bromelain powder with enzymatic activity of 2400 Gelatin digestion unit per Gram was mixed with saline in 1:1 proportion 1 g powder was mixed with 1 ml distilled water).

Under all aseptic conditions, the prepared RCs of the samples were filled with ICM except for the control group which was kept empty. The RC of all the samples was sealed with wax on both the sides of orifices and incubated at 37°C for 7 days. After the completion of medication period for 7 days, all RC were irrigated with 10 ml of saline to remove all ICM from the canals. The canals were dried with sterile paper points. The dentin chips from each canal of the samples were obtained by enlarging the canals with GG drill #4 (Dentsply, United States). The dentin chips samples were then separately collected in test tubes containing 3 ml of sterile fresh BHI broth. These test tubes were incubated at 37°C for 24 h to allow any bacteria that harbored in dentin to grow. After 24 h, the turbidity of samples was checked and optical density (OD) values were recorded using the spectrophotometer (Labman 600 nm wavelength) [Figure 2]. Before the above procedure, the same spectrophotometer (Labman) was used to measure the OD of fresh BHI broth which was used as the baseline values for the samples.

Figure 2

The turbidity of samples was checked and optical density values were recorded using the spectrophotometer (Labman 600 nm wavelength)

Statistical analysis

The collected data from all groups were transferred to the Statistical Package for the Social Sciences (SPSS) software version 16.0 (SPSS Inc., Chicago, IL, USA). The mean and standard deviation were calculated to identify the characteristics of the sample. The Chi-square test was applied to compare the variables among the groups. The nonparametric Kruskal − Wallis ANOVA test was used for the pairwise comparisons. The confidence interval was set at 95% and 5% was considered for statistical significance.

RESULTS

The turbidity of the samples was measured through OD analysis. OD values for all groups (mean and standard deviation) are shown in Table 1. The turbidity is directly proportional to the OD value and indirectly proportional to its antibacterial efficacy.

Table 1

Optical-density values

OD values	Group A (control)	Group B (CaOH)	Group C (TAP)	Group D (bromelain)
Mean	0.559	0.557	0.441	0.495
SD	0.085	0.041	0.085	0.054

CaOH: Calcium hydroxide, TAP: Triple antibiotic paste, OD: Optical density, SD: Standard deviation

Intergroup comparison and level of significance between four groups is shown in Table 2. TAP showed less OD values than CaOH paste which was statistically significant (P = 0.0022). TAP was more effective than CaOH2 against E. faecalis. The OD reading shown by Bromelain paste was less than CaOH indicating more effectiveness of bromelain against E. faecalis than CaOH. This difference was statistically significant (P = 0.007). However, the OD of Bromelain and TAP were comparable but statistically insignificant (P = 0.095), indicating their comparable antibacterial efficacy.

Table 2

Intergroup comparison and level of significance

Groups	Kruskal-Wallis “H” statistics	P
Control versus CaOH	0.027	0.87*
Control versus TAP	8.54	0.0034**
Control versus bromelain	3.26	0.07*
CaOH versus TAP	9.32	0.0022**
CaOH versus bromelain	7.25	0.007**
TAP versus bromelain	2.805	0.095*

*Nonsignificant, **Significant. CaOH: Calcium hydroxide, TAP: Triple antibiotic paste

DISCUSSION

The endodontic treatment aims at complete eradication of microorganisms from the RC system. It is achieved by vigorous chemicomechanical preparation followed by complete three-dimensional obturation of the RC system. ICMs are used to as an adjunct to eliminate or minimize the microbial burden in the RC system before obturation. It acts on the microorganisms which are left out during chemo-mechanical procedure, thereby reducing inflammation and preventing the re-infections. E. faecalis is the main organism seen in the refractory endodontic infection.[1] Being the most resistant intracanal pathogen in failed RC, it serves as a gold standard to represent other possible micro-organisms. As the development of efficacious therapeutic agent against E. faecalis is highly demanded, the testing of this particular microbe against its potential capabilities is highly desirable. Another advantage is that it is comparatively easy to culture.[2] Hence, in the present study, E. faecalis was used as a benchmark bacterium against which the experimental ICMs were tested.

Various methods have been experimented to assess the antimicrobial efficacy of ICMs. However in this study Spectrophotometry which was feasible, easily available, rapid screening and hassle free method to determine the turbidity of samples in terms of OD was used.[20] The microbial growth was monitored by absorbance measurements at the wavelength of 600 nm using spectrophotometer. OD values were recorded for each sample after an incubation period of 24 h. Higher turbidity and hence higher OD values indicated higher bacterial cell concentration. Tested ICM would be considered as efficient antimicrobial if it revealed lower values of OD.

CaOH one of the most commonly used ICM works in sync with the chemo-mechanical preparation. In the study by Lana et al.,[21] it was proven that CaOH pastes played an important role as an adjuvant in the eradication of enterococci during chemo mechanical preparation of RC systems. However, its efficacy was raised against E. faecalis when it was kept in the canal for 14 days as compared to 7 days’ duration. In the present study, the antimicrobial efficacy of CaOH was lesser than TAP and Bromelain both. This could be attributed to the 7 days duration for ICM. Had it been more duration probably the results would have been somewhat different. It can be considered as the limitation for this study. It has been reported in the literature that microorganisms colonize the dentinal tubules in depth and elude the direct effect of CaOH[22] that may reinfect the canal space.[23]

The result of the present study showed that TAP was superior than CaOH against E. faecalis bacteria as the antimicrobial potential of CaOH may be alleviated by dentin. This result was similar to the results by Mozayeni et al.[24] In previous studies, higher concentrations of TAP were recommended to be used as an intra-canal medicament to effectively eliminate the E. faecalis bacteria as it was discovered to have good stability and could be stored for a period of 21 days.[24]

In the present study, the results indicated that bromelain was more effective than CaOH (P = 0.007). As per the literature, Bromelain was found to bear higher efficacy against Gram positive than Gram-negative bacteria.[15] E. faecalis being a Gram-positive bacterium could be difficult to treat because of its property of being drug resistance. Bromelain being more selective on Gram-positive bacteria would have been an additional advantage for its efficiency against E. faecalis apart from its other advantages. In a previous clinical trial, oral bromelain was found to reduce the postoperative erythema, pain and inflammation after third molar extraction.[23] Bromelain shows antibacterial properties because of its chemical compounds such as saponins, tannins, flavonoids, and various enzymes. Flavonoid bears the inherited capability to create the complex bond with the extracellular protein via hydrogen bonding, thereby modifying the permeability of cell membranes.[25] A study conducted by Arsyada et al.[25] concluded that mixture of CaOH with bromelain was more effective than CaOH against E. faecalis. These results were in accordance with the present study. However, Bromelain showed statistically insignificant (P = 0.095) anti-microbial effectiveness against E. faecalis when compared with TAP. This may be attributed due to its mode of delivery into the RCs or may be it required more duration to be more efficient that TAP. No studies in the past have compared the antimicrobial efficacy of TAP with bromelain. Further clinical researches related to use of various concentrations of Bromelain are needed to establish the confirmatory results about its effectiveness against the notorious E. faecalis.

Limitations

In vitro study may give different results; hence, in vivo studies are needed to demonstrate the antimicrobial efficacy of Bromelain as ICM. Moreover, different modes of delivery of Bromelain along with different concentrations and for more duration into the RCs need to be evaluated. Only E. faecalis in not the organism responsible for RC infection; hence, studies on other microorganisms are further needed.

CONCLUSIONS

Bromelain was found to be more effective than CaOH paste against E. faecalis. The efficiency of bromelain was comparable to TAP as an ICM.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.