Assessment of Antimicrobial Activity of LL-37 Peptide Against Periapical Pathogens: A Cross-Sectional Study

Abstract

Background:

Reinfection in the root canal and periradicular area due to the emergence of antibiotic resistance and/or multidrug-resistant strains of mixed colonization of multispecies periapical endodontic is the main reason for endodontic treatment failures. This has imposed a serious public health concern, contributing to decreased clinical efficiency and poor treatment outcomes. To combat these issues, “antimicrobial peptides are considered as potential alternatives and a promising agent” in endodontics.

Objective:

To evaluate and compare the antimicrobial efficiency of LL-37 peptide against periapical endodontic pathogens (Streptococcus mutans, Lactobacillus salivarius, and Actinomyces naeslundii).

Methodology:

A multispecies biofilm of S. mutans, L. salivarius, and Actinomyces naeslundii was created on 100 hydroxyapatite disks samples (n = 100). Sixty samples (n = 60) were allocated for the peptide group and 20 samples each (n = 20) for the control group treated with disinfecting solution (sodium hypochlorite and normal saline). The minimum inhibitory concentration (MIC) and colony-forming units (CFUs) were assessed.

Results:

LL-37 peptides showed statistically significant correlation w.r.t CFUs and antimicrobial activity (P < 0.01).

Conclusion:

LL-37 peptides were highly effective against periapical endodontic pathogens and hence could be used as a potential therapeutic agent in the future for endodontics.

INTRODUCTION

Despite advancements in endodontic procedures, treatment failures due to persistent reinfection of the root canal and periradicular tissues remain a significant concern in clinical practice. This is primarily attributed to the presence of polymicrobial communities forming biofilms that exhibit high survival capacity and self-protective mechanisms in the canal system and periapical area.[1] The extracellular matrix of these biofilms, composed of polysaccharides, proteins, and bacterial DNA, facilitates mutation, altered gene expression, and enhanced microbial persistence.[1,2,3] Furthermore, their adaptive resistance makes eradication extremely difficult.[4,5]

This has led to the exploration of antimicrobial peptides (AMPs), or host defense peptides, which possess broad-spectrum anti-biofilm and immunomodulatory properties.[4,6,7] AMPs disrupt bacterial membranes via several mechanisms, including pore and non-pore models.[8,9] Agents, such as Nisin, D-AAs, Linezolid, KSL variants, GL13K, and STAMPs, have shown efficacy against multispecies endodontic biofilms.[4,10,11,12]

METHODOLOGY

A cross-sectional study was conducted to evaluate the antimicrobial efficiency of LL-37 peptide on a multispecies biofilm of Streptococcus mutans, Lactobacillus salivarius, and Actinomyces naeslundii. The sample size calculation was based on the formula derived as follows: n = Z_1-α/2² × P × (1 − P)/d² and total sample size estimated was 100 samples (n = 100). The null hypothesis was tested at a 0.05 level significance, stating “there was no antimicrobial efficiency of LL-37 antimicrobial peptides on multispecies biofilm formed by endodontic pathogens.”

A total of 100 hydroxyapatite disks (HA) were sterilized by autoclaving. Each disc was 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 [Figure 1].

Figure 1.

Depicts a three-week-old multispecies biofilm on hydroxyapatite disks

The preparation and quantification of microbial load was done using the blood agar method [Figure 2]. The results were expressed in OD values as detected by a spectrophotometer at 590 nm. One-way ANOVA and Tukey’s multiple comparisons test were used to compare the differences amongst groups (a = 0.05).

Figure 2.

Depicts multispecies colonies in a blood agar plate

RESULTS

LL-37 peptide exhibited a highly significant reduction in the total number of colonies in blood agar when compared to sodium hypochlorite and normal saline disinfecting solution. The MIC at different concentrations of (500, 250, 125, 62.5, 32, 16,8, 4, 2) μg/mL for multispecies biofilm of S. mutans, L. salivarius, and A. naeslundii [Figure 3]. The intergroup comparisons were done using the Tukey’s multiple comparisons test and inferred a statistically significant P value. Heatmap depicts the sensitivity/resistance of the microorganisms to LL-37 peptide [Figure 4].

Figure 3.

Depicts a heat map demonstrating the sensitivity/resistance of multispecies microorganisms to LL-37 peptide at different concentrations

Figure 4.

Depicts line graph demonstrating the sensitivity/resistance of multispecies microorganisms to LL-37 peptide at different concentrations

DISCUSSION

LL-37, a 37-amino acid peptide and the only member of the human cathelicidin family, has emerged as a promising AMP due to its dual role in antimicrobial defense and immunomodulation.[13,14] Its anti-biofilm activity is particularly valuable in endodontics, as it interferes with quorum-sensing genes and enhances bacterial surface motility, thereby limiting adherence and biofilm thickness.[15]

Our findings confirmed a statistically significant reduction in CFUs for all three pathogens when treated with LL-37, particularly at a concentration of 500 μg/mL (P < 0.01). These results are consistent with previous studies by Pereira et al. and Yoshida et al., who also reported potent antimicrobial and antibiofilm effects of LL-37.[16,17]

CONCLUSION

LL-37 peptides were highly effective against periapical endodontic pathogens and hence could be used as a potential therapeutic agent in the future for endodontics.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.