Carnosic Acid as an intracanal medicament performs better than triple antibiotic paste and calcium hydroxide to eradicate Enterococcus faecalis from root canal: An in vitro confocal laser scanning microscopic study.

Background: Carnosic acid is an herbal derivative with potent antioxidant, anti-inflammatory, antimicrobial, and anticancer properties. Aim: Comparative evaluation of the antimicrobial potential of carnosic acid, calcium hydroxide, and triple antibiotic paste as intracanal medicaments against Enterococcus faecalis. Settings and Design: Department of Conservative Dentistry and Microbiology, an in vitro study. Materials and
Methods: Fifty-two extracted single-rooted human teeth were decoronated and chemomechanical preparation was performed. The specimens were secured in the center of screw-capped vials and autoclaved. A strain of E. faecalis was inoculated into the canals and grown for 72 h. The teeth were divided into: Group I-Ca(OH)2, Group II- triple antibiotic paste (TAP), Group III-Carnosic acid, and Group IV-Negative control. The medicaments were applied in the canal and left for 14 days. The specimens were sectioned transversely at three levels to create dentinal discs and observed under the confocal laser scanning microscopic (CLSM). Images were analyzed, and quantification of bacteria was done using the Image J software. Statistical Analysis: Mean percentage of live/dead bacteria was analyzed using One-way ANOVA and Post hoc Tukey test.
Results: Mean percentages of live and dead bacteria were seen under CLSM in Group I, Group II, and Group III were (4.44 ± 2.87, 4.56 ± 2.93, 1.61 ± 1.90), and (4.59 ± 3.04, 4.25 ± 2.98, 1.70 ± 1.99), respectively, with least mean percentages for live and dead bacteria in carnosic acid (Group III).
Conclusion: Carnosic acid showed better antimicrobial efficacy against E. faecalis than TAP and Ca(OH)2 by showing a low percentage of both live and dead bacteria. Copyright:

INTRODUCTION

The microorganisms that enter the root canal system are responsible for the initiation and sustenance of periapical disease.[1] The reinfected root canal is dominated by anaerobic bacteria microflora, especially facultative anaerobes like Enterococcus faecalis.[2] Apart from its resistance to antibiotics, other specific virulence factors: quorum-sensing, adhesins, secretion factors, capsular polysaccharide, and collagen-binding protein are attributed to the persistence of E. faecalis in the root canal microenvironment system.[34]

Calcium hydroxide (Ca(OH)2 is the intracanal medicament of choice for many years, owing to its antimicrobial properties. When mixed into a paste, it has a solubility of <0.2% and a pH of 12.5. Ca(OH)2 exhibits its antimicrobial activity by releasing hydroxyl ions (OH-), thereby creating a nonconducive alkaline environment for the microorganisms to survive. However, the rate of hydroxyl ion diffusion is prolonged due to the buffering capacity of the dentin.[5]

The polymicrobial nature of microorganisms is responsible for root canal infections. Therefore, triple antibiotic paste (TAP), a combination of antibiotics (Metronidazole, ciprofloxacin, and minocycline), is routinely used to eliminate the microorganisms.[6] The major concern with the usage of this drug is its potential to discolor the tooth, bacterial resistance, and altered root dentin structure.[78]

Plant extracts and their components possessing antimicrobial properties are used in medical treatments. Rosmarinus officinalis L.(Lamiaceae) is an edible evergreen shrub that possesses antioxidant, anti-inflammatory, anticancer, and antimicrobial properties for medicinal and culinary purposes. The main extracts are: Rosmarinic acid, carnosic acid, carnosol, ursolic acid, oleanolic acid, genkwanin, apigenin, and luteolin.[9] An in vitro study evaluated commercially available rosemary extract formulations against a few bacterial species. The Gram-positive bacteria were more sensitive than Gram-negative bacteria, especially to oil-soluble extracts that contained carnosic acid.[10] There is a paucity of research regarding its use as an intracanal medicament. Hence, we planned to conduct this preliminary study to evaluate the antimicrobial efficacy of Carnosic acid and compare it with that of TAP and Ca(OH)2 when used as intracanal medicaments against E. faecalis using confocal laser scanning microscopy (CSLM). The null hypothesis stated no difference in the antimicrobial efficacy of the various experimental intracanal medicaments.

MATERIALS AND METHODS

The present study was conducted in the Department of Conservative Dentistry and Endodontics, Department of Microbiology and School of life sciences after obtaining the Institutional Ethics Committee clearance (protocol ref no: 16091).

Selection and preparation of samples

Fifty-two extracted human single-rooted teeth with Type I root canal anatomy were chosen for the study. Teeth with restorations, stains, cracks, noncarious lesions, attrition, white spot lesions, and hypoplasia were excluded from the study. The collected teeth were stored in 0.01% sodium hypochlorite (NaOCl) solution (Vishal Dentocare, Gujarat, India) until use. Debris, soft tissue, and calculus were mechanically removed from the root surface, following which teeth were decoronated using a highspeed diamond bur (TF-21, Mani Inc, Japan) under water cooling. The root length of all the teeth was maintained at 16 mm. After confirming apical patency with #15 K-file (Dentsply Maillefer, Ballaigues, Switzerland), root canals were enlarged to #20 K-file (Dentsply Maillefer, Ballaigues, Switzerland). Chemomechanical preparation of the samples was performed using 2.5% NaOCl to flush out debris and to shape the canals. Protaper rotary nickel-titanium system (Dentsply Maillefer, Ballaigues, Switzerland) up to the size F3 finishing file was used. On completion of instrumentation, the canals were irrigated with 17% ethylene diamine tetraacetic acid to ensure smear layer removal, followed by 2.5% NaOCl and a final rinse with sterile saline for a minute. The apical foramina were sealed with cyanoacrylate, and specimens were secured in the center of screw-capped vials and autoclaved twice at 121°C for 20 min.

Bacterial introduction and biofilm generation

E. faecalis strain ATCC 29212 was the microorganism used in this study. It was cultivated in the microbiology laboratory. A single colony of E. faecalis was collected from the agar plate and suspended in the sterile brain heart infusion broth supplemented with 1.5%(wt/vol) agar and incubated anaerobically at 37°C for 24 h. The plastic vials containing the autoclaved teeth were opened in a Biohazard Cabinet (ESCO Airstream) to maintain the aseptic environment. The experimental root canals were inoculated with 10 μl of E. faecalis suspension using sterile 1 ml tuberculin syringes. Specimens were then placed in stainless steel boxes and incubated at 37°C for 72 h within Orbital Incubator (Sanyo). The specimens were then divided into four experimental groups according to the intracanal medicaments to be used.

Preparation of intracanal medicaments

Group I-Commercially available CA(OH)2 paste (Calcicur, VOCO America. Inc) was used as an intracanal medicament.

Preparation of triple antibiotic paste

Group II-The ingredients of TAP: Ciprofloxacin (Ciplox 500 mg tablet, Cipla India), Metronidazole (Metrogyl 400 mg tablet, JB Chemicals and Pharmaceuticals) and minocycline (Minoz, 100 mg tablet, Ranbaxy India) Each of these powders were prepared separately by removing the enteric coatings of the tablet and crushing it to a fine powder using sterile mortar and pestle. The three antibiotic powders were weighed individually using a digital weighing machine to obtain a desired 1:1:1 proportion. Then the powders were mixed to obtain Triple antibiotic powder.[6] One mg powder was mixed with one ml of sterile water to obtain a paste consistency.

Preparation of carnosic acid paste

Group III-≥91% Carnosic acid (Sigma Aldrich) is available in a powder form, 10 mg of powder was dispensed on the paper pad and mixed with two drops (0.1 ml) of propylene glycol to a paste consistency.

Group IV-No intracanal medicament was placed (Control).

Treatment of biofilms

Thirty-nine samples were irrigated with sterile saline for 2 min and dried with sterile paper points, and randomly divided into three groups (n = 13). Control specimens (n = 13) were placed in the incubator for the entire duration of the experiment [Table 1]. The root canals were filled with the allocated experimental intracanal medicament using a Lentulospiral (Dentsply Maillefer, Ballaigues, Switzerland). Zinc oxide eugenol was used to seal the coronal access cavity.

Table 1

Comparison of coronal, middle, and apical live and dead bacterial counts (expressed as a percentage) using the one-way ANOVA test (P=0.05)

Intra-canal level	Medicament group (mean±SD)				P
	Calcium hydroxide	Triple antibiotic paste	Carnosic acid	Saline
Live bacterial percentage
Coronal	5.04±3.01∞	5.09±3.22ⴃ	3.07±2.53ⴆ	12.43±1.43ⴆ,ⴃ,∞	<0.001
Middle	5.02±2.6∞,ⴊ	4.62±2.94ⴃ,ⴂ	1.28±0.99ⴆ,ⴂ,ⴊ	10.08±2.48ⴆ,ⴃ,∞	<0.001
Apical	3.28±2.84∞,ⴊ	3.98±2.74ⴃ,ⴂ	0.49±0.47ⴆ,ⴂ,ⴊ	11.62±1.2ⴆ,ⴃ,∞	<0.001
Dead bacterial percentage
Coronal	5.44±3.41∞	5.4±3.23ⴃ	3.18±2.7	1.68±0.55∞,ⴃ	<0.001
Middle	4.92±2.68∞,ⴂ	5.03±2.94ⴃ,ⴊ	1.45±1ⴂ,ⴊ	1.23±0.6∞,ⴃ	<0.001
Apical	3.4±2.84ⴂ	2.33±1.7ⴊ	0.46±0.41ⴂ,ⴊ	0.7±0.44	<0.001

ⴆ,ⴃ,∞,ⴂ,ⴊ: Similar symbols represent significant difference (P<0.05) between the subgroups using Posthoc Tukey test. SD: Standard deviation

Preparation of specimens for microbiological analysis

The root canals were washed with 20 ml sterile saline and dried with absorbent paper points on the 14th day.

Confocal laser scanning microscopic examination

The specimens (n = 13 in each group) were sectioned transversely to obtain dentin discs of approximately 1 mm thickness at three levels (coronal, middle, and apical), using a rotating diamond disk under water cooling. After the specimens were washed using phosphate-buffered saline, they were stained with fluorescent LIVE/DEAD BacLight Bacterial Viability stain [Molecular Probes, Eugene, OR, USA]. The SYTO™9 (ThermoFisher Scientific, USA) (green-fluorescent stain) that labels live bacteria and Propidium Iodide (PI) (Himedia, India) (red-fluorescent stain) that labels dead bacteria were used. These fluorescent stains were diluted in saline to give final concentrations of 10 and 60 μmol L−1, respectively. The specimens were placed in the tubes containing the stain and stored in the dark for 30 min. The specimens were rinsed thoroughly using PBS solution and blot dried before confocal laser scanning microscopic (CLSM) analysis.

A confocal laser scanning microscope [Leica DMi] was used to view the specimen cross-sections. The coronal, middle, and apical thirds of the root specimens were scanned at ×40 magnification. The excitation/emission wavelengths were 480/500 nm for SYTO9 and 490/635 nm for PI. CLSM images were acquired and analyzed using Leica Application Suite (Leica Microsystems, Germany) at a resolution of 1024 × 1024 pixels. Images were processed for background noise reduction (Leica Application Suite software). Quantification of the CLSM images was done using the Image J software. The area percentage of live cells (green fluorescence) and dead cells (red fluorescence) was calculated at coronal, middle, and apical third levels for all samples.

Statistical analysis

The mean percentage of live and dead bacteria was statistically analyzed using One-way ANOVA and Post hoc Tukey test. Paired t-test was applied for intragroup comparison of the colonization variation between the coronal, middle, and apical thirds. Analysis of data was performed using SPSS version 20.0 software (IBM Corp, Somers, NY) and the P value was set at P ≤ 0.05.

RESULTS

From the data collected using open J software analyses of CLSM images, it revealed that among the intracanal medicament groups, Carnosic acid group showed very low live bacterial percentage (1.61 ± 1.90) followed by Ca(OH)2 group (4.44 ± 2.87) and TAP group (4.56 ± 2.93). The Control group had the highest live bacterial percentage (11.38 ± 2.00). The highest mean dead bacterial percentage values were for the Ca(OH)2 group (4.59 ± 3.04) followed by the TAP paste group (4.25 ± 2.98), Carnosic acid group (1.70 ± 1.99), and the least in the control group (1.20 ± 0.66) [Figure 1]. Interestingly, the Carnosic acid group exhibited a low percentage of both live and dead bacteria in the present study.

Figure 1

Live and dead bacterial percentage values

The intragroup comparison showed that the live mean bacterial percentage was highest in the coronal third of the root canal and least in the apical third among all the experimental groups. Similarly, the mean dead bacterial percentage was highest in the coronal third section and least in the apical one-third [Table 1].

The intergroup comparison showed that the live and dead bacteria percentage was comparable in all three groups in the coronal regions [Figure 2]. The live bacterial percentage was significantly more in the middle and apical regions in Ca(OH)2 and TAP groups than in the Carnosic acid group, with no significant difference in Ca(OH)2 and TAP groups. A similar observation was noted with the dead bacteria percentage (P = 0.001) [Figure 2 and Table 1]. Carnosic acid showed the lowest live and dead bacteria in all three sections of root.

Figure 2

CLSM images showing live (L)/dead (D)/Consolidated(C) bacteria for different groups at coronal third level:1A- Ca(OH)2 (L),1B- Ca(OH)2 (D), 1C- Ca(OH)2 (C),2A- TAP (L),2B- TAP(D), 2C- TAP(C),3A-Carnosic acid(L),3B- Carnosic acid(D),3C- Carnosic acid(C). CLSM images showing live (L)/dead (D)/Consolidated(C) bacteria for different groups at middle third level.: 1D- Ca(OH)2 (L),1E- Ca(OH)2 (D), 1F- Ca(OH)2 (C),2D- TAP (L),2E- TAP(D), 2F- TAP(C),3D-Carnosic acid(L),3E- Carnosic acid(D),3F- Carnosic acid(C). CLSM images showing live (L)/dead (D)/Consolidated(C) bacteria for different groups at apical third level. 1G- Ca(OH)2 (L),1H- Ca(OH)2 (D), 1I- Ca(OH)2 (C),2G- TAP (L),2H- TAP(D), 2I- TAP(C),3G-Carnosic acid(L),3H- Carnosic acid(D),3I- Carnosic acid(C)

DISCUSSION

Chemomechanical preparation of the canal system will eradicate most organisms and their biofilms, but some chronic organisms in the root canal are challenging to eradicate, especially E. faecalis.[11] The study results imply that the antibacterial activity of Ca(OH)2 and TAP was similar in all three regions of the roots [Table 1]. The antibacterial effect produced by calcium hydroxide is primarily dependent on the diffusion of the hydroxyl ions into the dentinal tubules and their high pH.[5] Therefore, it is evident that to achieve any antibacterial effect within the tubules, the ionic diffusion capacity of Ca(OH)2 must exceed the dentin's inherent buffering ability.[12] According to Siqueira et al., the bacterial cells at the periphery of the colonies can have a protective effect on the colonies present deep within the tubules.[13] This could explain why a more significant number of dead cells were observed at the periphery/at the circumpulpal dentin than the deeper parts of the dentin, as seen in the CLSM images.

According to Mohammadi and Abbott, tetracyclines present in the TAP (minocycline) can create a strong and reversible bond with the hard tissues by forming complexes with trivalent and bivalent cations, resulting in slower release of the drug over a period of time.[6] Sato et al. demonstrated that Metronidazole could penetrate the deeper layers of the dentin and indicated in the treatment of obligately anaerobic bacteria.[14] Due to the inherent discoloration potential and its effect on dentin structure, TAP must be used cautiously.[15]

In the present study, the carnosic acid group has displayed good antimicrobial efficacy by showing a low percentage of the live and dead bacterial count, suggesting that carnosic acid did not allow the bacteria to multiply [Table 1]. The exact antimicrobial mechanism of carnosic acid is not entirely known but could be attributed to the lipophilic nature of the compounds that allows it to insert into the bacterial membrane.[16]

Carnosic acid interacts with the ethidium bromide efflux system and acts as a modulator for membrane permeability.[17] According to Nakagawa et al., carnosic acid is a potential quorum sensing inhibitor against Staphylococcus aureus; it inhibits the activation of genes involved in virulence and biofilm formation. Quorum sensing inhibition has been suggested as an alternative approach to combat infection and the same mechanism of action of carnosic acid may also have been effective against E. faecalis.[18] The pathogenesis of Enterococcal infection at the molecular level is oxidative stress due to the production of free radicals. E faecalis produces superoxide and hydrogen peroxide, which is essential for its survival. The antioxidant activity of carnosic acid could have acted as scavengers of free radicals and curbed its proliferative ability.[19]

E. faecalis uses dentin serum to adhere to the dentin and enter the dentinal tubules and multiply, so it can be postulated that carnosic acid could have prevented the growth of E faecalis by modifying the dentinal serum.[20] Hence, we speculated that carnosic acid reduces adhesion and does not provide an environment to allow microorganism growth. Moreno et al., concluded that the antimicrobial efficacy of R. officinalis L. was associated with their specific phenolic composition. According to their findings, 25–200 μg of carnosic acid inhibits bacterial growth.[21]

An intragroup comparison revealed that all the experimental intracanal medicaments showed the highest dead bacteria percentage in the coronal third, followed by the middle and apical third region [Table 1]. Intracanal medicaments are conveniently and adequately applied in the coronal third, enabling better action at this region.

Recently, CSLM has emerged as an excellent method to study the biofilm structure, primarily because it does not disturb the bacterial ecosystem and permits noninvasive investigation of these ecosystems.[2223] However, we performed our experiments on an in vitro biofilm model that may not accurately represent or mimic in vivo biofilm behavior. The limitation of in vitro models is that they lack the external factors that challenge in vivo biofilm, primarily due to the host immune response.

The standardization of fluorescence between the optical slices and obtaining a consistent balance between the red and green fluorescent signals is a limitation with CSLM. The intensity of fluorescence depends on various factors: The depth of the optical slice, the thickness of the sections, and the degree of dentin mineralization.[24] Hence, these parameters need to be adjusted separately for each image, resulting in slight variations. These shortcomings have been highlighted previously by Hope and Wilson.[25]

To our knowledge, very few studies have been conducted to evaluate the effectiveness of carnosic acid as an intracanal medicament. Further clinical studies are required to evaluate the potential effect of carnosic acid on clinical outcomes. Hence within the study's limitations, it can be concluded that all three experimental intracanal medicaments are capable of producing an antimicrobial effect and are effective against E. faecalis. Since Carnosic acid is a new biomaterial used in endodontics, further studies evaluating its physical, chemical, and biological properties and their effects on substrate would be beneficial.

CONCLUSION

Based on the results obtained, all three intracanal medicaments tested in the study showed significantly reduced E faecalis count in 14 days. Carnosic acid is a promising intracanal medicament with a good antibacterial effect, where E. faecalis is a significant pathogen.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.