Evaluation of antimicrobial and antibiofilm efficacy of different antimicrobial peptides on multispecies biofilm of endodontic pathogens

Abstract

Background:

Assessing and evaluating the function and activity of different types of antimicrobial peptides (AMPs) in suppressing multispecies endodontic pathogens is necessary.

Aim:

The study was conducted to assess the antimicrobial and antibiofilm efficiency of gramicidin S, D-cateslytin (D-Ctl), GH-12, and DJK-5 AMPs on multispecies biofilm formed by endodontic pathogens.

Methodology:

Multispecies biofilm comprising Enterococcus faecalis, Actinomyces naeslundii, Lactobacillus salivarius, and Streptococcus mutans were formed on 80 hydroxyapatite disk samples. Sixteen samples were allocated for each peptide in the experimental group (n = 16) and eight samples each for the control group, 2% sodium hypochlorite (positive control) and normal saline (negative control). The total number of colony forming units (CFUs) and biofilm intensity to fluorochrome in each study group were measured using the culture method and dual stain fluorescence microscopy method. The differences across groups were compared using Tukey’s multiple comparisons test and one-way analysis of variance (α =0.05).

Results:

DJK-5 (CFU = 110/ml), gramicidin (CFU = 110/ml), and D-Ctl (CFU = 60/ml) peptides showed statistically significant correlation with respect to CFUs and similar antibiofilm activity (P < 0.01), whereas GH-12 (CFU = 90/ml) peptide revealed differences that were not statistically significant (P > 0.05).

Conclusion:

DJK-5, gramicidin S, and D-Ctl peptides demonstrated remarkable efficacy against multispecies oral biofilms of S. mutans, L. salivarius, A. naeslundii, and E. faecalis. Saline showed least antimicrobial and antibiofilm activity. Sodium hypochlorite (NaOCl) showed strongest difference when compared to peptides or saline, validating it as a potent control. Hence, these peptides can be employed as potential antibiofilm agents in endodontic treatment procedures for better outcomes.

INTRODUCTION

Despite the greatest efforts of oral healthcare providers, endodontic treatment failures due to reinfection in the root canal and periradicular area are an unresolved issue across the globe. The major factor associated with the limited success rate is attributed to the persistence of mixed colonization of multispecies microorganisms, bestowed with high survival capability and self-protective biofilm ecology in the canal space and/or the periradicular area.[1] Biofilms account for about 65% of all human infections and represent a major health concern that needs immediate attention. These are extracellular polymeric substances with increased tolerance/adaptive resistance against the currently available antimicrobial agents and are extremely difficult to eradicate.[2,3] The natural mode of survival of these pathogens in the oral cavity is through “oral biofilm-microbe mediated phenomena or biofilm state,” in which complex polymicrobial communities are embedded in self-produced matrix/extracellular polymeric substances that consist of polysaccharides, protein metabolites, and bacterial DNA.[4,5] These biofilms are 1000-fold more resistant to disinfecting solutions or antimicrobial agents when compared to microbes in planktonic (free-floating) suspensions.[1,4,5] This could be due to specific characteristic traits such as altered phenotype, slow growth rate, and gene transcription phenomena present in the oral biofilm pathogenic niche. Moreover, they provide a well-conducive environment for mutation in bacterial cell walls, thereby promoting their survival and persistence. In addition, the complex structural configuration of biofilms allows a good nutrient supply and metabolic cooperation among the same or different species of bacteria to survive in their own highly organized internal protective microenvironment from a variety of environmental stresses such as alteration in pH, osmotic shock, ultraviolet radiation, and desiccation. Blessed with an extracellular polysaccharide matrix, it acts as a barrier and aids in trapping extracellular enzymes such as lactamase and inactivating β-lactam antibiotics.[6] It is quite a challenging and extremely daunting task for the clinician, to combat and overcome this dynamic biofilm complex structure during root canal treatment. Various disinfecting solutions and intracanal medicaments such as calcium hydroxide (CH), chlorhexidine (CHX), and sodium hypochlorite (NaOCl) were used in the past and reported limited success rates.[7]

Antimicrobial peptides (AMPs)/host-defense peptides are short cationic amphipathic molecules with broad-spectrum anti-biofilm activity and potent immunomodulatory capabilities that provide early protection against invading microbes.[2,8] Hence, AMPs are considered potential alternatives to conventional pharmaceuticals against multidrug-resistant pathogens and biofilm-related infections.[9] The principal mode of action is by dissipating the transmembrane potential and permeability of the bacterial cell wall, resulting in cell death.[10,11,12] Various authors have proposed the pore model (barrel stave pore model and toroidal pore model), the nonpore model, and the mineralization self-assembled peptide nanofibers model to contribute to its mechanism of action.[9,10,11,12,13] AMPs have proved to be better than most commonly used irrigant solutions when compared to their toxicity and other damaging effects on adjacent soft tissues.[14] Recent studies have reported great potential in the application of these AMPs in endodontics. Nisin,[13] D-amino acids,[15] human-β-defensin-3,[16] LL-37, linezolid, polylysine, KSL, modified KSL (KSL-W), L-K6, chrysophsin-1, lacticin 3147, GL13K, specifically targeted AMPs such as C16G2, M8G2, C16-33, and M8-33)[2,6,17] have received a great deal of attention as therapeutic applications against both single-species and/or multispecies dental biofilms. However, they are extremely costly and have a low success rate in clinical trials due to their intrinsic vulnerability to the proteolytic destruction process. This dictated the need to assess and evaluate the role and activity of various different categories of AMPs in inhibiting endodontic pathogens. Few in vitro studies were conducted applying a combination of DJK-5 and ethylenediaminetetraacetic acid (EDTA),[15,17] D-cateslytin (D-Ctl), and CH[18,19] on oral biofilms. However, the antimicrobial and antibiofilm efficiency of these peptides in endodontic multispecies biofilm of known composition is yet to be evaluated. Based on this uncertainty, we identified the research gap and applied FINGER criteria to formulate the research question as “Are AMPs effective as both antimicrobial and antibiofilm in inhibiting endodontic pathogens?” Therefore, our objective was to assess and compare the antibacterial and antibiofilm efficacy of four potential peptides, DJK-5, GH-12, D-Ctl, and Gramicidin S, on multispecies biofilms produced by endodontic pathogens.

METHODOLOGY

An in vitro study was conducted after due approval from the ethical committee bearing the protocol number YEC-1/2022/052. The study aimed to evaluate the antibacterial and antibiofilm properties of four promising peptides – Gramicidin S, D-Ctl, GH-12, and DJK-5 on multispecies biofilm formed by endodontic pathogens. The sample size calculation was based on study conducted by Ye et al.,[8] in which the efficacy of DJK-5 peptide in terms of percentage of dead cell volume in root canal was 55% after 3 min of irrigation. Considering the above information and fixing a 10% level of significance,

Z 1 is 1.96 with 95% CI, P is 0.5, and n is 80; α error is 0.5 with a d² value of 0.0121 with a margin of error of 0.11_. The total sample size estimated was 80.

A total of six groups, i.e., four experimental groups for each peptide and two control groups were as follows: Group 1: DJK-5 peptide, Group 2: gramicidin peptide, Group 3: D-Ctl, Group 4: GH 12 peptide, Group 5: 0.9% sterile saline (negative control), and Group 6: 2.5% sodium hypochlorite (positive control). Sixteen samples were allocated for each peptide in the experimental group (n = 16) and eight samples (n = 8) each for the control group. The null hypothesis was set, stating “there was no difference in antimicrobial and antibiofilm efficiency of gramicidin S, D-Ctl, GH-12, and Djk-5 AMPs on multispecies biofilm formed by endodontic pathogens” and was tested at 0.05 level significance.

Determination of minimum inhibitory concentration

For determining the minimum inhibitory concentration (MIC) level, 10 dilutions of each peptide in thioglycollate broth were prepared. In the initial tube, 400 µl of peptide was added as neat, subsequently followed by 200 µl of thioglycollate broth from the 2^nd to 12^th tube, finally, transferring the initial 200 µl of peptide to the 2^nd tube, making it a l0:2 dilution. The serial dilution method was followed up to the 11^th tube. The 12^th tube, which consisted of broth and only organisms was considered a growth control and 11^th tube, which consisted of broth and compound, was considered a compound control. The tubes were incubated according to the growth requirement for 48–72 h at 37°C and observed for turbidity.

Antibiofilm assay on synthetic surface by crystal violet assay

To create a blend of Streptococcus mutans, Lactobacillus salivarius, Actinomyces naeslundii, and Enterococcus faecalis, equal amounts of each organism solution were combined. For each test, a series of 10 wells was used from one row, and the test was performed in triplicate. Each well received 100 µl of this suspension, which was then incubated anaerobically for 24 h. As a result, biofilms formed. Any unattached cells were removed by gentle rinsing of the plate in sterile distilled water. A stock solution of 100 µg/ml for each compound was prepared in sterile vials with appropriate diluents, and the final mixture was made by adding equal quantities of individual suspensions. One hundred microliters of each drug concentration were transferred to individual wells with biofilms that was incubated at 37°C in 5% CO₂. After removing extra peptides with a gentle water rinse, the wells were treated for 15 min with 125 µl of 0.1% crystal violet solution. The optical density (OD) values and bacterial growth index were expressed in OD values as detected by spectrophotometer at 590 nm.

Antibiofilm assay on biotic surface using hydroxyapatite disks

On 80 hydroxyapatite (HA) disks, a multispecies biofilm comprising S. mutans, L. salivarius, A. naeslundii, and E. faecalis was formed. All HA disks were prepared by sectioning of the middle third of the crown portion of single/multi-rooted noncarious, teeth extracted for orthodontic or periodontal reasons of 1 mm thickness each. Each disc was sterilized by autoclaving and later placed in individual wells of culture plates and covered with 1% bovine serum albumin for 24 h, followed by thioglycollate broth (supplemented) and culture suspensions. The plate was gently agitated for 2 h for uniform spreading of the bacteria and then incubated with 5% carbon dioxide at 37°C for 3 weeks.

Two times a week throughout the incubation phase, the culture media were replaced. Following the conclusion of the incubation time, each well’s thioglycollate broth was extracted, and the specimens were gently rinsed in sterile distilled water to get rid of any detached cells. In triplicate, a diluted peptide mixture was added to each well at three distinct doses for microscopy and culture. For 24 h, the plates were incubated at 37°C with 5% CO₂. Excess peptide was removed from all wells; the discs were again rinsed in sterile distilled water and then processed by two different methods, the culture method and the dual stain fluorescent microscopy method, so as to measure the total amount of colony-forming units (CFUs) and biofilm intensity to fluorochrome in all the study groups [Figure 1a-f].

Figure 1.

Depicts multispecies colonies in a blood agar plate. (a) After treatment with DJK-5; (b) After treatment with Gramicidin S; (c) After treatment with D-Cateslytin; (d) After treatment with GH-12; (e) After treatment with normal saline; (f) After treatment with sodium hypochlorite

To remove the biofilm, the HA disks in each group were vortexed for 2 min in separate sterile microcentrifuge tubes containing 1 ml of sterile phosphate-buffered saline. After that, this suspension was plated onto various selective media and cultured at 37°C for 72 h without oxygen. The number of each type of colony was noted and represented as CFU/ml. To see the HA disk biofilm under a fluorescence microscope, it was double-stained with acridine orange and ethidium bromide.

Statistical analysis

To compare the differences across groups, Tukey’s multiple comparisons test and one-way analysis of variance were employed (a = 0.05). The “GraphPad Prism 9 (GraphPad Software, LLC, Boston, Massachusetts, United States) version” software was used to analyze the data.

RESULTS

The MIC levels for DJK 5, gramicidin S, D-Ctl, and GH-12 peptides in each of the six groups at various concentrations (500 μg/mL, 250 μg/mL, 125 μg/mL, 62 μg/mL, 5 μg/mL, 32 μg/mL, 16 μg/mL, 8 μg/mL, 4 μg/mL, 2 μg/mL, and 1 μg/mL) showed that E. faecalis, A. naeslundii, L. salivarius, and S. mutans showed the highest sensitivity to DJK 5 and D-Ctl followed by GH 12, and least sensitivity to gramicidin S. (MIC = 2 μg/mL for DJK 5 and D-Ctl; MIC = 8 μg/mL for GH 12; MIC = 16 μg/mL for gramicidin S).

The antibiofilm activity on synthetic surfaces was assessed by measuring OD values for DJK-5, gramicidin, D-Ctl, and GH 12 peptide. The results inferred that GH 12 showed OD = 0.12, followed by gramicidin (OD = 0.07), DJK 15 (OD = 0.06), and D-Ctl (OD = 0.05) when compared to controls, i.e., normal saline (OD = 0.15), followed by hypochlorite (OD = 0.07). The OD values revealed that DJK-5, gramicidin, and D-Ctl peptides demonstrated a notable decrease in the growth of bacteria in comparison to normal saline. Comparing the GH-12 peptide to normal saline, no discernible decrease in the bacterial growth was seen. Normal saline was the least effective among all the groups [Figure 2a].

Figure 2.

The antibiofilm efficacy of four peptides, namely DJK-5, Gramicidin, D-Cateslytin, and GH 12, against Entercoccus faecalis, Streptococcus mutans, Lactobacillus salivarius, and Actinomycetes sp., was studied using. (a) Crystal violet assay (b) Culture method. (b) The results are expressed in optical density and colony-forming unit values

The antibiofilm activity on the biotic surface was assessed by measuring CFUs values for DJK-5, gramicidin, D-Ctl, and GH 12 peptide. Among the six groups, sodium hypochlorite showed the least number of colonies in blood agar. The reduction of no colonies was substantially greater with sodium hypochlorite than with D cateslytin (*P < 0.05), GH12 (***P < 0.001), DJK5, and gramicidin (****P < 0.0001) [Figure 2b].

Saline showed the highest number of colonies in blood agar and was the least effective. When compared to saline, DJK5 considerably decreased colony numbers (*P < 0.05); however, Gramicidin demonstrated a highly significant decrease (**P < 0.01). In comparison to saline, GH12 also showed an extremely substantial drop in colonies (***P < 0.001), and D-Ctl exhibited a highly significant reduction compared to saline (****P < 0.0001) [Figure 2]. Despite having the fewest colonies, D-Ctl did not significantly vary from the other peptides in terms of efficacy. The proportional strength of biofilm intensity of each peptide against microorganisms tested was assessed with fluorescent microscopy. The results inferred the following findings: normal saline showed the highest biofilm intensity (368796), followed by gramicidin (96546), DJK 5 (90552), GH 12 (21308), D-Ctl (7166), and least for hypochlorite (4619) [Figure 2a, b and Tables 1, 2].

Table 1.

Depicts the Tukey's multiple comparisons test showing OD values of four peptides, namely DJK-5, Gramicidin, D-Cateslytin, and GH 12, against Entercoccus faecalis, Streptococcus mutans, Lactobacill us salivarius, and Actinomycetes sp.

Tukey's multiple comparisons test	Mean different	Below threshold?	Summary	Adjusted P value
DJK-5 peptide versus gramicidin peptide	–0.008250	No	NS	0.9993
DJK-5 peptide versus D-Cateslytin peptide	0.005500	No	NS	>0.9999
DJK-5 peptide versus GH-12 peptide	–0.06675	No	NS	0.0718
DJK-5 peptide versus hypochlorite	–0.003917	No	NS	>0.9999
DJK-5 peptide versus normal saline	–0.1019	Yes	***	0.0009
Gramicidin peptide versus D-Cateslytin peptide	0.01375	No	NS	0.9924
Gramicidin peptide versus GH-12 peptide	–0.05850	No	NS	0.1563
Gramicidin peptide versus hypochlorite	0.004333	No	NS	>0.9999
Gramicidin peptide versus normal saline	–0.09367	Yes	**	0.0029
D-Cateslytin peptide versus GH-12 peptide	–0.07225	Yes	*	0.0402
D-Cateslytin peptide versus hypochlorite	–0.009417	No	NS	0.9987
D-Cateslytin peptide versus normal saline	–0.1074	Yes	***	0.0004
GH-12 peptide versus hypochlorite	0.06283	No	NS	0.1054
GH-12 peptide versus normal saline	–0.03517	No	NS	0.6845
Hypochlorite versus normal saline	–0.09800	Yes	**	0.0016

NS: Not significant, *P<0.05 was considered significant compared to control; **P<0.01 was considered very significant compared to control; ***P<0.001 was considered highly significant compared to control

Table 2.

Illustrates Tukey’s multiple comparisons test showing comparative efficacy of different peptides

Tukey’s multiple comparisons test	Mean different	95.00% CI of difference	Below threshold?	Summary	Adjusted P value
DJK-5 peptide versus Gramicidin peptide	1.853	−50.33 to 54.04	No	NS	>0.9999
DJK-5 peptide versus D-Cateslytin peptide	41.58	−7.092 to 90.26	No	NS	0.1367
DJK-5 peptide versus GH 12 peptide	20.77	−27.90 to 69.44	No	NS	0.8089
DJK-5 peptide versus Hypochlorite	107.5	48.94 to 166.1	Yes	****	<0.0001
DJK-5 peptide versus Normal saline	−78.4	−142.7 to−14.06	Yes	**	0.0083
Gramicidin peptide versus D-Cateslytin peptide	39.73	−10.05 to 89.51	No	NS	0.192
Gramicidin peptide versus GH 12 peptide	18.92	−30.86 to 68.70	No	NS	0.8731
Gramicidin peptide versus hypochlorite	105.7	46.17 to 165.2	Yes	****	<0.0001
Gramicidin peptide versus normal saline	−80.25	−145.4 to−15.07	Yes	**	0.0074
D-Cateslytin peptide versus GH 12 peptide	−20.81	−66.90 to 25.27	No	NS	0.7696
D-Cateslytin peptide versus hypochlorite	65.94	9.492 to 122.4	Yes	*	0.0129
D-Cateslytin peptide versus normal saline	−120	−182.4 to−57.58	Yes	****	<0.0001
GH 12 peptide versus hypochlorite	86.75	30.30 to 143.2	Yes	***	0.0004
GH 12 peptide versus normal saline	−99.17	−161.6 to−36.76	Yes	***	0.0002
Hypochlorite versus normal saline	−185.9	−256.3 to−115.5	Yes	****	<0.0001

*P<0.05 was considered significant compared to control; **P<0.01 was considered very significant compared to control; ***P<0.001 was considered highly significant compared to control; ****P<0.0001 was considered highly significant compared to control

DISCUSSION

Root canal reinfection and treatment failures are major concerns in the present days. The persistence of mixed colonization of multispecies microorganisms and resistance of the oral biofilms to routinely used disinfecting solutions, with complete eradication of microbes from the inaccessible areas such as fins, anastomoses, apical delta, accessory canals, isthmuses, and periradicular areas, is indicative that new categories of AMPs need to be tested and evaluated in endodontic practice.[1]

In the present study, we screened four AMPs, namely Gramicidin S, D-Ctl, GH-12 and DJK-5 for activity against multispecies biofilm of S. mutans, L. salivarius, A. naeslundii, and E. faecalis, which was formed on HA disks. Based on the observation from previous studies, we tested our hypothesis stating that these four AMPs namely Gramicidin S, D-Ctl, GH-12 and DJK-5, due to their peptidic composition, could serve as both antimicrobial and antibiofilm agents against endodontic pathogens as one of the best possible alternatives. The study results showed that DJK-5, gramicidin and D-Ctl peptides showed statistically significant correlation w.r.t CFUs and similar antibiofilm activity (P < 0.01) [Figure 2b]. However, GH-12 peptide demonstrated nonsignificant (P > 0.05) differences when compared to the control group. DJK-5, Gramicidin and D-Ctl peptides showed significant reduction in bacterial growth compared to the normal saline. Comparing GH-12 peptide to regular saline, no discernible decrease in bacterial growth was seen.

The present study results were in accordance with study conducted by Zhang et al. in which the efficiency of the DJK-5 peptide on oral multispecies biofilms from three plaque donors and two donors on E. faecalis VP3-181 biofilm was evaluated, inferring effective killing of microbes in plaque biofilm (10 μg/mL) almost exceeding 85%, thereby concluding that DJK-5 showed remarkable efficacy and could be a promising AMP for future oral antibiofilm treatments.[15] A study conducted by Kumar et al. to evaluate the cytotoxicity of DJK 5, CHX, and EDTA on L929 mouse subcutaneous fibroblast cells showed that DJK 5 had the least cytotoxic effects and additionally showed the cytotoxicity of CHX and EDTA.[17] However, our study results were in discordance with Li et al.[20] inferring GH 12 as effective in reducing the antimicrobial and antibiofilm activity. GH12 eliminated every bacterial strain in time-kill tests. S. mutans biofilm formation was reduced by GH12. Confocal laser scanning microscopy demonstrated that GH12 successfully decreased the biomass of the S. mutans biofilm that was 1 day old. Cytotoxicity assays indicated that GH12 showed little toxic effect on the viability of human gingival fibroblasts.[20,21,22,23] Li et al.[20] investigated the effects of GH12 on E. faecalis biofilm and virulence. At sub-MIC levels, GH12 markedly downregulated a number of genes for stress (dnaK, groEL, ctsR, and clpPBCEX) and virulence (efaA, esp, and gelE) in E. faecalis. Furthermore, GH12 dramatically decreased the biomass of E. faecalis biofilm that was 1 day old as well as the production of E. faecalis biofilm.

Our study results were in accordance with Zaet et al.[18] and Ehlinger et al.[19] inferring potent antimicrobial and antibiofilm activity of D-Ctl peptide. D-Ctl, a novel epipeptide produced from L-Ctl, was biologically characterized by Zaet et al.[18] D-Ctl has been shown to be an effective antimicrobial agent against a number of bacterial strains, including Staphylococcus aureus, Parvimonas micra, Fusobacterium nucleatum, Prevotella intermedia, Methicillin Sensitive, Methicillin Resistant, and Escherichia coli wild type and multidrug resistant. Both facultative and stringent anaerobes are represented on this panel. The MIC of D-Ctl varied from 8 to 24 μg/mL, depending on the type of bacteria. Furthermore, it was said that D-Ctl was more resistant than L-Ctl to being broken down by bacterial proteases and was not cytotoxic. D-Ctl and CH were mixed by Ehlinger et al.[19] in order to test for improved antibacterial qualities against E. faecalis. E. faecalis growth was inhibited 58% (±5%) by a saturated solution of CH (1.7 mg/mL), but it was completely inhibited by a combination of 0.85 mg/mL of CH and ½ MIC of D-Ctl.

In the present study, the mechanism of action of each AMP against each tested bacteria was explored. To kill these selected pathogens (L. salivarius and E. faecalis, A. naeslundii, and S. mutans), AMPs (DJK-5, Gramicidin S, D-Ctl and GH 12) typically interact with certain intracellular targets, impairing the cell’s membrane integrity by interacting with the negatively charged cell membrane, or block the production of proteins, DNA, and RNA. In addition, they cause the membrane to permeabilize, which can lead to either extensive damage or minor flaws that reduce the transmembrane potential and ultimately cause cell death.

The mysterious function of AMPs against the components of bacterial cells is a dynamic process that involves a number of hypotheses and models. First, the Pore model theory includes both toroidal and barrel stave pore models. In this former, AMPs create a hydrophilic channel by interacting with the bacterial cell membrane. The formation of the stalve occurs initially parallel to the cell membrane. AMPs are then placed perpendicular to the membrane bilayer plane after barrels have been created. Whereas in the latter model, the AMPs have an impact on the membrane’s curvature. With their hydrophilic regions facing the pore and their hydrophobic regions connected to the middle portion of the lipid bilayer, AMPs align perpendicularly within the bilayer structure.

Second, the nonpore model/carpet model/detergent model results in instability of the LPS layer by depositing AMPs in parallel to the cell membrane. Membrane holes are left behind when AMP molecules with hydrophobic sides pointing inward cover small portions of the membrane. The peptide micelle coats a tiny portion of the membrane after making initial contact with it. The lipid bilayer is then broken down by AMP molecules, allowing holes to develop.

The variations in the degree of sensitivity against each pathogen can be due to the fact that each pathogen varies in the virulence of oral pathogenic multispecies micro-organisms, with specific pathogenic synergy involving taxonomically distinct microbiota that have a capacity to degrade peptides.

This study examined the antibacterial effectiveness of various AMPs in an ex vivo preliminary manner. The current study’s findings have certain limitations, and they might not be applicable to a clinical setting. First, to accurately visualize the study results, the intraoral environment of an infected root canal was not recreated. Second, CFUs were used in this investigation to assess the number of bacteria. Future research might use cryopulverization of root specimens or confocal laser scanning microscopy and live-dead staining for biofilm analysis. To assess the biocompatibility and measure the bacterial resistance of AMPs, more research should be done. It is necessary to conduct additional research on the biocompatibility and measurement of bacterial resistance.

CONCLUSION

DJK-5, gramicidin S, and D-Ctl peptides demonstrated remarkable efficacy against multispecies oral biofilms of S. mutans, L. salivarius, A. naeslundii, and E. faecalis. Hypochlorite (NaOCl) showed the strongest difference when compared to peptides or saline, validating it as a potent control. Saline showed the least antimicrobial and antibiofilm activity. Hence, they can be employed as potential antibiofilm agents in endodontic treatment procedures for better outcomes.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.