Introduction

Apical periodontitis (AP) is a common pathology, defined as an inflammatory process around the apex of a root, causing pain and resorption of periradicular structures (Persoon and Özok 2017). AP affects all populations with a varying prevalence. A recent review showed that AP concerned 7% of individuals in a Spanish population to 86% in a Croatian population, with a median of 52.5%. (Persoon and Özok 2017). Since a correlation between endodontically treated teeth (Joshipura et al. 2006; Caplan et al. 2009) or chronic AP (Caplan et al. 2006) and adverse cardiovascular effects has been proposed, it is essential to treat teeth with AP.

The fundamental role of bacteria within the pulp space in the development, expansion and maintenance of AP has been widely demonstrated (Nair 2006; Peciuliene et al. 2008; Siqueira and Rocas 2009; Ricucci and Siqueira 2010). Endodontic treatment aims to improve periapical health by removing the root canal pathogens (Trope and Bergenholtz 2002; El Karim et al. 2007). Although the chemomechanical preparation realized during endodontic treatment is effective in reducing the number of microorganisms, it cannot completely eliminate pathogens from the root canal (Byström and Sundqvist 1981; Haapasalo et al. 2005). Therefore, if the canal is left empty between appointments, the remaining bacteria can multiply and reach levels similar to the beginning of treatment (Byström and Sundqvist 1981; Sjogren et al. 1997; Mohammadi and Dummer 2011). To overcome this problem, the use of intracanal medicaments with antimicrobial properties is recommended to fill the root canal between treatment sessions (Mohammadi and Dummer 2011; Gupta et al. 2015).

Currently, calcium hydroxide (Ca(OH)2) is the most commonly used intracanal medicament. Ca(OH)2 is commercially available as pastes containing various vehicle. Its concentrations in these pastes vary from 0.85 g/mL to 1.7 g/mL (Fava and Saunders 1999).

The popularity of Ca(OH)2 is due to its antimicrobial effect against Gram-positive, Gram-negative bacteria and fungi from the root canal (Law and Messer 2004; Mohammadi and Dummer 2011; Mohammadi et al. 2012). These antimicrobial properties are related to the release of hydroxyl ions, which increase the pH within the root canal and directly affect the cell membrane and proteins structure of microorganisms (Mohammadi and Dummer 2011; Mohammadi et al. 2012). Indeed, the hight pH of Ca(OH)2 modifies the integrity of the cytoplasmic membrane through denaturation of proteins and destruction of phospholipids or unsaturated fatty acids. These chemical modifications may be due to peroxidation process (Mohammadi et al. 2012).

Among the numerous endodontic pathogens, Enterococcus faecalis is one of the microorganisms often recovered in root canal with persistent infections (Rocas et al. 2004; Stuart et al. 2006). It is a Gram-positive bacterium able to invade dentinal tubules (Love 2001) and to resist very harsh environmental conditions (starvation, acidic and alkaline pH) (Evans et al. 2002; McHugh et al. 2004), especially by maintaining pH homeostasis (Figdor et al. 2003; Stuart et al. 2006). Because E. faecalis has shown resistance to Ca(OH)2 (Evans et al. 2002), its association with other antimicrobial agents was proposed (Mohammadi and Dummer 2011).

In our laboratory, since several years, we have identified antimicrobial peptides (AMPs) derived from the processing of Chromogranin A (CgA) (Aslam et al. 2012). These AMPs are part of the innate immune response and are released into the circulation shortly after an infection (Radek et al. 2008). They are short, stable in a wide range of pH and temperature and not toxic for host cells (Aslam et al. 2012). Furthermore, they display bacteriolytic activity against a broad spectrum of pathogens, including oral microorganisms, play a crucial role in modulating the immune response (Lugardon et al. 2001; Briolat et al. 2005; Aslam et al. 2012, 2013; Zaet et al. 2017) and induce less resistance than antibiotics (Zaet et al. 2017). We recently observed that by substituting all L-amino acids from Cateslytin, one of the CgA-derived AMPs, with D-amino acids, we could generate D-Ctl, which has improved antimicrobial efficiency against several oral pathogens such as Parvimonas micra, Prevotella intermedia, Fusobacterium nucleatum and Candida albicans (Zaet et al. 2017; Dartevelle et al. 2018).

In the present study, we analyzed the activity of several CgA-derived peptides including Chromofungin (CHR), Catestatin (CAT) and the L- and D-form of its active domain, the Cateslytin (D-Ctl and L-Ctl), against E. faecalis. We then combined D-Ctl with Ca(OH)2 to improve its antimicrobial efficiency and develop a new stable, non-toxic combination therapy efficient against endodontic pathogens, including E. faecalis.

Materials and Methods

Antimicrobial Agents

The following peptides were purchased from Proteogenix: CHR (bCgA47-66: RILSILRHQNLLKELQDLAL), CAT (bCgA344–364: RSMRLSFRARGYGFRGPGLQL), L-Ctl and D-Ctl (bCgA344–358: RSMRLSFRARGYGFR).

Preparation of Ca(OH)2 Solutions

Ca(OH)2 was purchased from Sigma-Aldrich. A saturated solution of Ca(OH)2 (1.7 mg/mL) was diluted to ½ (0.85 mg/mL) and ¼ (0.425 mg/mL). Ca(OH)2 was diluted in milli-Q water, Anaerobe Basal Broth, Sabouraud medium or Dulbecco’s Modified Eagle’s Medium (DMEM F12, Dutscher) depending on the experiment performed.

Microorganisms and Mammalian Cell Line

Fusobacterium nucleatum (ATCC© 49256TM), Prevotella intermedia (ATCC© 49046TM), and Parvimonas micra (ATCC© 33270TM) were purchased from ATCC. Enterococcus faecalis (CCM 2541) was obtained from the Czechoslovac Collection of Microorganisms. Bacteria were cultured in Anaerobe Basal Broth (Oxoid) at 37 °C in anaerobic conditions. Candida albicans (ATCC© 10231TM) was cultured in Sabouraud medium (Becton–Dickinson, Germany), supplemented with tetracycline (10 μg/mL; Sigma Aldrich) and cefotaxime (10 μg/mL; Sigma Aldrich).

Human gingival fibroblasts (HGF-1; ATCC® CRL-2014TM) were commercially obtained from ATCC and cultured at 37 °C and 5% CO2 in DMEM F12 (Dutscher) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 100 units/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher Scientific).

Broth Dilution Assays

An overnight culture of each pathogen was diluted (OD600nm = 0.001) and incubated at 37 °C in 96-plates in the presence of different concentrations of antimicrobial agents. After 24 h incubation, the OD600nm was evaluated with a spectrophotometer (Multikan EX, Thermo Fisher Scientific).

Determination of the Minimal Inhibitory Concentration (MIC)

The MIC, defined as the lowest concentration of peptide able to inhibit 100% of the inoculum, was calculated using a modified Gompertz model as described by Lambert and Pearson (2000).

Cytotoxicity Assays

The cytotoxicity of the antimicrobial agents was examined by MTT [3(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] assays (Sigma Aldrich) using HGF-1 cells as a model. HGF-1 cultured in DMEM F12 (Dutcher) supplemented with 10% FBS (Gibco), 100 units/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher Scientific) were incubated at 37 °C and 5% CO2 for 24 h in a 96-wells plate before being treated with various concentrations of Ca(OH)2 alone or supplemented with D-Ctl. Untreated cells were used as a control. After 24 h, 48 h and 72 h incubation, cells were carefully washed with phosphate-buffered saline (PBS) and treated with MTT at a final concentration of 0.25 mg/mL. HGF-1 cells were then incubated for 2 h at 37 °C before being lysed with isopropanol/HCl (48:2, v/v). Cell viability was assessed by reading the OD540nm with a Multiskan EX microplate spectrophotometer (Thermo Fisher Scientific).

Stability Assays of D-Ctl in the Supernatant of E. faecalis

The supernatant of E. faecalis was prepared as follows: a single colony of E. faecalis was suspended in 5 mL of Anaerobe Basal Broth and incubated at 37 °C overnight. The culture was centrifuged at 10,000×g for 1 min and filtered (Millex GV filter unit 0.22 µm, Millipore SAS). The supernatant (1 mL) was then incubated at 37 °C for 24 h with D-Ctl (300 µg). As controls, bacterial supernatant (1 mL) and D-Ctl (300 µg) diluted in milli-Q water (1 mL) were incubated separately in similar conditions.

The samples (1 mL) were analyzed by HPLC (Dionex, Ultimate 3000) using a Nucleosil reverse-phase 300-5C18-column (4.6 × 250 mm; particle size: 5 µm; porosity, 300 Å) (Macherey–Nagel). The two solvents used were: 0.1% (v/v) Trifluoroacetic acid (TFA) in water (solvent A) and 0.09% (v/v) TFA in 70% (v/v) acetonitrile–water (solvent B). Absorbance was measured at 214 nm (A214nm). The flow rate for elution was 0.7 mL/min with a gradient of solvent B as indicated on the chromatograms. For each sample, material (1 mL) was directly injected on the HPLC column.

Stability Assays of D-Ctl in Ca(OH)2

D-Ctl (156 µg or 78 µg) corresponding to MIC or ½ MIC was incubated in 1 ml of a Ca(OH)2 solution (1.7 mg/mL or 0.85 mg/mL) at 37 °C for 24 h. The pH of the solution was buffered with HCl to be the same as for the broth dilution assays and MTT tests (pH 9). As a control, D-Ctl (156 µg or 78 µg) was also incubated in milli-Q water (1 mL) at 37 °C for 24 h. The samples (1 mL) were analyzed by HPLC as previously reported.

Statistical Analysis and Graphs

Each assay was done at least in triplicate. For broth dilution and cytotoxicity assays, data are presented as mean ± standard deviation. Statistical analysis was performed using SigmaPlot (release 12.5, Systat Software, Inc). Statistical significance between two groups was determined using the Mann–Whitney Rank Sum Test or the t-test. Statistical significance between three groups or more was determined using the Kruskal–Wallis one-way analysis of variance on ranks. Two-way analysis of variance was used when the experimental setups involved two independent variables. For each experiment, the statistical test used is indicated in the figure legend. p-values < 0.05 were considered statistically significant and noted by * on the graphs. Artworks were created using SigmaPlot (release 12.5, Systat Software, Inc).

Results

Antibacterial Assays of Solution of Ca(OH)2

In a preliminary experiment, the efficiency of a saturated solution of Ca(OH)2 (1.7 mg/mL, pH 9) was assessed by broth dilution assays on E. faecalis and F. nucleatum. Our results confirmed previous studies showing that a saturated solution of Ca(OH)2 could not completely inhibit the growth of E. faecalis (Ferreira et al. 2007). In our hands, the inhibition rate was only 58% (± 5%). In addition, as previously described (Ferreira et al. 2007), we confirmed that a saturated solution of Ca(OH)2 was able to inhibit 100% (± 1%) of F. nucleatum growth (Fig. 1).

Fig. 1
figure 1

Inhibition of E. faecalis (n = 9) and F. nucleatum (n = 3) growth by a saturated solution of Ca(OH)2

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Antibacterial Activity of Several CgA-Derived Peptides Against E. faecalis

We then tested the antibacterial activity of several CgA-derived peptides such as Chromofungin (CHR), Catestatin (CAT) and Cateslytin (L-Ctl or D-Ctl) against E. faecalis using broth dilution assays (Fig. 2). All peptides were tested at 200 µg/mL. Our results demonstrated that no natural peptides derived from CgA (CHR, CAT and L-Ctl) display antimicrobial activity against E. faecalis. In contrast, D-Ctl was able to achieve bacterial growth inhibition (Fig. 2a). Its MIC on E. faecalis, determined by using the Gompertz model, was evaluated to 156 µg/mL (Fig. 2b). D-Ctl was therefore chosen as the best candidate to combine with Ca(OH)2.

Fig. 2
figure 2

Activity of antimicrobial CgA-derived peptides against E. faecalis.a Antimicrobial activity of CgA-derived peptides at 200 µg/mL against E. faecalis. For CHR, CAT and L-Ctl n = 3 and for D-Ctl n = 9. Statistical significance was determined by a Kruskal–Wallis one-way analysis of variance on ranks (p = 0.007 indicated by “*”). b Determination of the MIC (156 µg/mL) of D-Ctl against E. faecalis using a modified Gompertz model

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D-Ctl is Stable in a Saturated Solution of Ca(OH)2

The stability of D-Ctl at the MIC in a saturated solution of Ca(OH)2 (1.7 mg/mL, pH 9) was assessed by HPLC (Fig. 3a). Under our experimental conditions, D-Ctl was eluted after 38 min (Fig. 3a, chromatogram 1). The same peak was identified in a saturated solution of Ca(OH)2, suggesting that D-Ctl at the MIC remains stable under these conditions. The shoulder in the peak corresponds to an oxidized form (oxidation of methionine residue) of D-Ctl. Indeed, the first major peak is the oxidized D-Ctl and the second one to the non-oxidized form (Fig. 3a, chromatogram 2). The areas under the curves were calculated in three independent experiments for each condition. The ratio of areas under the curve of chromatogram 2 to chromatogram 1 was 1.01 ± 0.12.

Fig. 3
figure 3

Stability of D-Ctl in a saturated solution of Ca(OH)2 and in the supernatant of E. faecalis.a The chromatograms 1 and 2 correspond to D-Ctl diluted in milli-Q water and D-Ctl diluted in a saturated solution of Ca(OH)2 (pH 9), respectively. A black arrow indicates presence of D-Ctl. b The chromatograms 1, 2 and 3 correspond to D-Ctl diluted in milli-Q water, D-Ctl diluted in the supernatant of E. faecalis and the supernatant of E. faecalis alone, respectively. A black arrow indicates presence of D-Ctl

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D-Ctl Remains Stable in the Supernatant of E. faecalis

The stability of D-Ctl in the supernatant of E. faecalis was assessed by HPLC (Fig. 3b). In our experimental conditions, D-Ctl in milli-Q water was eluted at 38 min (Fig. 3b, chromatogram 1). The same peak was still observed when D-Ctl was incubated with the bacterial supernatant (Fig. 3b, chromatogram 2). The areas under the curves were calculated in three independent experiments for both conditions. The ratio of areas under the curve of chromatogram 2 to chromatogram 1 was 0.97 ± 0.04. The chromatogram 3 represents the bacterial supernatant alone (Fig. 3b, chromatogram 3). Notably, the other peaks on the chromatograms 2 and 3 correspond to proteins included in the media and the proteases secreted by the bacteria. In conclusion, D-Ctl is resistant to the degradation by the virulence factors of E. faecalis, allowing a prolonged action of the peptide against this pathogen.

Ca(OH)2 is Cytotoxic for HGF-1 at High Concentration

Cytotoxicity of solutions of Ca(OH)2 at various concentrations (0.425 mg/mL, 0.85 mg/mL, 1.7 mg/mL) was assessed by MTT assays on HGF-1 grown for 72 h in culture. Cell viability was assessed and expressed as a percentage of control (without Ca(OH)2). Ca(OH)2 at 0.85 mg/mL and 0.425 mg/mL showed no statistically significant toxicity (4% ± 5%), even after 3 days of incubation. Meanwhile, only 50% (± 2%) of the cells were alive after 72 h of incubation with a saturated solution of Ca(OH)2 (1.7 mg/mL) (Fig. 4). Based on these results, diluted solutions of Ca(OH)2 (0.425 mg/mL, 0.85 mg/mL) constituted better choices than a saturated solution for subsequent assays.

Fig. 4
figure 4

Cytotoxicity of Ca(OH)2 towards human gingival fibroblasts (n = 5). The bars represent cytoxicity for a solution of Ca(OH)2 at 0.425 mg/mL (light grey bars), 0.85 mg/mL (dark grey bars) and 1.7 mg/ml (black bars). Statistical significance was determined by the Two-Way Analysis of Variance (p < 0.05 is indicated by “*”)

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Combination of D-Ctl and Ca(OH)2 Inhibits Key Endodontic Pathogens

Since D-Ctl displayed antimicrobial activity against E. faecalis and was stable in Ca(OH)2, experiments were conducted to determine the most efficient and the least toxic combination of these antimicrobial agents to eradicate root canal pathogens.

To identify the most efficient combination of Ca(OH)2 and D-Ctl to inhibit the growth of E. faecalis, broth dilution assays were performed with different concentrations of Ca(OH)2 and D-Ctl. Specifically, a solution of Ca(OH)2 at non-toxic concentrations (0.85 mg/mL, 0.425 mg/mL) was combined with the MIC, ½ MIC and ¼ MIC of D-Ctl (Fig. 5). As depicted, the combination using the lowest concentration of Ca(OH)2 and D-Ctl able to inhibit E. faecalis growth was a solution of Ca(OH)2 at 0.85 mg/mL with ½ MIC of D-Ctl (78 µg/mL) (Fig. 5).

Fig. 5
figure 5

Inhibition of E. faecalis growth by various combinations of Ca(OH)2 and D-Ctl. The combinations tested are indicated on the graph. The number of replicates (n) is indicated on the graph for each combination. Statistical significance was determined by the two-way analysis of variance (p < 0.05 is indicated by “*”)

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The stability of D-Ctl after 24 h in such a solution was confirmed by HPLC (Fig. 6a). Indeed, D-Ctl eluted at 38 min (Fig. 6a, chromatogram 1) was stable in a solution of Ca(OH)2 at 0.85 mg/mL (pH 9) (Fig. 6a, chromatogram 2). Shoulder on the peaks corresponds to an oxidized form of D-Ctl. The areas under the curves were calculated in three independent experiments for both conditions. The ratio of areas under the curve of chromatogram 2 to chromatogram 1 was 1.075 ± 0.06. We also assessed the cytotoxicity of this combination towards HGF-1 by MTT assays during 72 h. No statistically significant decrease in cell viability was observed (Fig. 6b).

Fig. 6
figure 6

Stability, cytotoxicity and antimicrobial activity of the combination between D-Ctl and Ca(OH)2. a The chromatograms 1 and 2 correspond to D-Ctl diluted in milli-Q water and the combination of Ca(OH)2 at 0.85 mg/mL and ½ MIC of D-Ctl (pH 9) respectively. b The cytotoxicity of the combination between Ca(OH)2 at 0.85 mg/mL and ½ MIC of D-Ctl on HGF-1 cells was assessed with MTT assays (n = 4). The Kruskal–Wallis One-Way Analysis of Variance on Ranks showed no statistical significance (p = 0.239). c The efficacy of the combination between Ca(OH)2 at 0.85 mg/mL and ½ MIC of D-Ctl (striped bars) compared with Ca(OH)2 at 0.85 mg/mL (grey bars) was assessed on four other main endodontic pathogens (n = 3). Statistical significance was determined by t-test (p < 0.05 is indicated by “*”)

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Finally, we verified that this combination was also efficient against the most common endodontic pathogens. To this aim, in addition to E. faecalis, we performed broth dilution assays against P. micra, P. intermedia, F. nucleatum and C. albicans (Fig. 6c). Our results showed that Ca(OH)2 at 0.85 mg/mL was able to inhibit 87% (± 2%) of C. albicans growth, 15% (± 6%) of P. micra growth, 77% (± 6%) of P. intermedia growth and 30% (± 10%) of F. nucleatum growth, whereas the combination inhibited respectively 97% (± 1%), 98% (± 2%), 98% (± 3) and 97% (± 5%) of the pathogen’s growth. Therefore, the combination of D-Ctl and Ca(OH)2 is efficient against other key endodontic pathogens like C. albicans, P. micra, P. intermedia and F. nucleatum.

Discussion

The use of antimicrobial agents able to eliminate resistant species from root canals and to modulate the periapical inflammatory-immune response may improve the success rate of endodontic treatments (Turner et al. 2004; Lima et al. 2015). In the field of endodontics, AMPs have been studied as irrigants, used during chemomechanical preparation and as intracanal medicament, applied between treatment sessions.

Concerning endodontic irrigants, DJK-5, a cationic peptide, was shown to be effective in improving the antimicrobial efficacy of a rinse of 6% sodium hypochlorite followed by rinse with ethylenediaminetetraacetic acid (EDTA) (Wang et al. 2018). Furthermore, nisin, an AMP derived from Lactococcus lactis (Severina et al. 1998), was demonstrated to improve the antimicrobial efficacy of two conventional endodontic irrigants (Mixture of A Tetracycline Isomer, An Acid and A Detergent (MTAD) and sodium hypochlorite), against some Gram-positive bacteria associated with persistent intracanal infection, including E. faecalis (Tong et al. 2012, 2014; Kajwadkar et al. 2017). Therefore, nisin has potential as an alternative to conventional irrigation with sodium hypochlorite.

Another aspect of endodontic disinfection is the use of an intracanal medicament, such as Ca(OH)2, to prevent the multiplication of residual microorganisms in the root canal between treatment sessions. Two peptides have been studied as intracanal medicaments: Human β-defensin-3 (HBD3), an epithelial derived cationic AMP (Harder et al. 2001), widely expressed in the oral cavity and nisin. In recent studies, HBD3 demonstrated better antimicrobial activities against E. faecalis biofilms and multispecies biofilms than Ca(OH)2 and chlorhexidine (Lee et al. 2013a, b; Ahn et al. 2017). Nisin has also been shown to be effective in inhibiting E. faecalis with a minimum bactericidal concentration (MBC) of 70 mg/mL and Streptococcus gordonii with a MBC of 20 mg/mL (Turner et al. 2004). However, nisin alone may not be an appropriate intracanal medicament, because it is less effective against Gram-negative bacteria, which are also present in infected root canals (Turner et al. 2004).

Even though AMPs can seem suitable to improve the root canal distinction, one barrier to their therapeutic applications is that they are expensive to manufacture (Hancock and Sahl 2006). HBD3 for example, is a cationic peptide of 45 residues with a complex tertiary structure that makes its synthesis difficult (Dhople et al. 2006). In this perspective, D-Ctl seemed to be a good candidate for this study as it is short and linear (15 amino acids).

Besides their costs, AMPs also have the drawback to be susceptible to proteolytic degradation. E. faecalis can overcome the innate immune system response and trigger persistent infections. One of its mechanisms of resistance is the degradation of AMPs (Schmidtchen et al. 2002; Nešuta et al. 2017). For example, proteases secreted by E. faecalis can degrade LL-37 and HYL-20, two AMPs known for their antimicrobial properties (Schmidtchen et al. 2002; Nešuta et al. 2017). In order to counteract an endodontic infection, D-Ctl should not be degraded by the proteases secreted by E. faecalis. Our study showed that D-Ctl maintains its integrity in presence of E. faecalis (Fig. 3b). Indeed, as D-Ctl strictly consists of D-amino acids, it is not sensitive to bacterial proteases (Zaet et al. 2017). Interestingly, D-Ctl is also stable in Ca(OH)2 and their combination (Ca(OH)2 (0.85 mg/mL) and D-Ctl (½ MIC)) is able to completely inhibit E. faecalis growth (Fig. 6). Recent studies, using Escherichia coli as a model, have shown that the bacteriolytic activity of D-Ctl is due to the rapid permeabilization of the bacterial cell membrane leading to bacterial death (Zaet et al. 2017). This mechanism is due to the positive charge and amphipathic structure of the AMPs, allowing them to interact with negatively charged phospholipid bilayers, causing the disruption of bacterial cell membranes (Nešuta et al. 2017).

The absence of cytotoxicity is also an important characteristic for an efficient intracanal medicament. From this point of view, Ca(OH)2 and chlorhexidine are the most acceptable intracanal medicaments while others, like phenol and formocresol are highly cytotoxic (Kawashima et al. 2009). In our study, Ca(OH)2 at high concentrations (1.7 mg/mL) was found to be toxic to HGF-1 after 72 h (Fig. 4). Therefore, our work focused on finding a combination including lower concentration of Ca(OH)2 (0.425 mg/mL and 0.85 mg/mL). Interestingly, Ca(OH)2 at 0.85 mg/mL alone or in combination with D-Ctl was not toxic to HGF-1.

Even though E. faecalis is strongly correlate with endodontic failure, several other pathogens are involved in endodontic infection. Here, we showed that the combination between D-Ctl and Ca(OH)2 is also efficient against four other endodontic pathogens: P. micra, P. intermedia, F. nucleatum and C. albicans. Interestingly, F. nucleatum and P. intermedia are Gram-negative bacteria, showing that the efficacy of D-Ctl does not only concern Gram-positive species. These encouraging results suggest that this combination therapy could eradicate endodontic biofilms.

Despite promising preliminary results about the antimicrobial efficacy of the combination of D-Ctl and Ca(OH)2, this drug combination was tested in vitro against planktonic bacteria and might not necessarily be effective against multi-species microbial biofilms in vivo. Therefore, further investigations are needed to test the efficiency of this combination. Interestingly, we present a comparison of sequences of Ctl with two others potent anti-biofilm AMPs (Fig. 7) (Pletzer and Hancock 2016). This comparison suggests that Ctl belongs to the same group of amphipathic peptides and could therefore be efficient against biofilms.

Fig. 7
figure 7

Comparison sequence of D-Ctl with other amphipatic anti-biofilm peptides. Basic residues are underlined and hydrophobic residues framed. Positive charges and the number of hydrophobic residues are indicated for each AMP. The final line presents a consensus sequence where basic residues (B) and hydrophobic residues (H) are pointed out

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