A. actinomycetemcomitans ATCC 43717 (Guangdong Microorganism Culture Collection Center, Guangzhou, China) was used as the representative bacterial strain. After the growth of the microorganism on a Columbia blood agar plate, a single colony was extracted by inoculation loop and cultured in brain heart infusion broth (Hopebio Qingdao, Qingdao, China) containing 5 mg/L hemin and 1 mg/L menadione for 48 hours in an anaerobic environment at 37°C and 5% CO₂. According to the previous study, expression and purification of Est816 was successfully prepared (Fan et al., 2012). Based on our previous study findings (Zhao et al., 2024), 12 U/mL of enzyme Est816 exerted a significant anti-biofilm effect against A. actinomycetemcomitans with minimal effect on bacterial growth and was used in this study.

The minimal inhibitory concentrations (MICs) were defined as the lowest concentrations of antibiotics that resulted in no visible growth of A. actinomycetemcomitans. Antimicrobial susceptibility testing was performed using the broth microdilution method. 50 μL of the bacterial suspension (1× 10⁶ CFU/mL) (OD_600 = 0.1) was added to equal volume of metronidazole (MTZ), amoxicillin (AMX), and minocycline (MINO) on the 96-well plate (MedChemExpress, NJ, U.S.A.) (Lachica et al., 2019). The three antibiotics were diluted in brain heart infusion broth with final concentrations ranging from 0.125µg/mL to 128µg/mL. In the experimental groups, each group of antibiotics containing bacterial suspension was supplemented with enzyme Est816 at a final concentration of 12 U/mL. Negative and positive controls were administered with equal volumes of inactivated enzyme Est816 and PBS. The plates were incubated for 24 h at 37°C. The MICs were determined as previously described in the literature (Goodwine et al., 2019; Allkja et al., 2020). The inhibitory effects of the antibiotics in the presence of specific concentrations of enzyme Est816 (12 U/mL) were determined using spectrophotometry at 600 nm. Each experiment was repeated in triplicate. The concentrations that significantly reduced the growth and concentrations of A. actinomycetemcomitans without growth inhibition (sub-MIC) were selected for further experiments.

A suspension of A. actinomycetemcomitans in mid-exponential phase was diluted to OD_600 = 0.1 (1.0 × 10⁶ CFU/mL), and 500 µL were seeded onto round coverslips (14 mm diameter) on the bottom of 24-well plate, which was cultured with MTZ (sub- MIC or MIC), AMX (sub-MIC or MIC) and MINO (sub-MIC or MIC) alone or in combination with enzyme Est816 for 48 hours to form biofilms. Scanning electron microscopy (SEM) (Zhao et al., 2024) was used to investigate the biofilm morphology. A. actinomycetemcomitans was cultured with MTZ (sub-MIC or MIC), AMX (sub-MIC or MIC) and MINO (sub-MIC or MIC) alone or in combination with the enzyme Est816 for 48 hours to form a biofilm, subsequently washed with PBS and fixed in 2.5% (vol/vol) cold (4°C) glutaraldehyde overnight. The samples were then dehydrated using a graded ethanol series (30, 50, 70, 80, and 90%) for 15 minutes. After critical point drying and ion coating with gold (Ion Sputtering, Cressington 108Auto), SEM analysis was performed to assess the morphology of A. actinomycetemcomitans.

For crystal violet semi-quantitative biofilm assay, 500 μL of A. actinomycetemcomitans suspension (1 × 10⁶ CFU/mL) was added to 24-well plates, and 500 μL PBS, antibiotics (sub-MIC or MIC), and enzyme Est816 were added to each well individually to co-culture with the biofilm. After incubation for 48 hours at 37 °C under microaerophilic conditions, the biofilm was fixed with 400 μL paraformaldehyde solution for 15 minutes. The solution was then removed, and the biofilms were stained with crystal violet solution (0.1%, 300 μL) for 20 minutes. Next, the biofilms were washed thrice, and the crystal violet stain was dissolved with 400 μL of 95% ethanol. The optical density values of the semi-quantitative analysis of the biofilm were detected at 570 nm using a microplate reader. To determine biofilm reduction (Shakya et al., 2022), the percentages of biofilm was calculated as follows: B i o f i l m   R e d u c t i o n % = Abs Control  −  Abs Sample Abs Control    ×   100 % (Abs Control represented the group without the addition of drugs). For testing biofilm eradication rates, A. actinomycetemcomitans suspension in the 96-well plate was incubated at 37^◦C for 48 hours to form a mature biofilm. Then, the antibiotics at concentrations of sub-MIC, MIC with or without enzyme Est816 were added to disassemble mature biofilm for another 24 h. The procedure for the detection and calculation of residual biofilm was the same as described above.

A. actinomycetemcomitans cells were stained using a LIVE/DEAD Baclight Bacterial Viability Kit L7012 (Thermo Fisher Scientific, Waltham, MA, USA) by immersion in equal volumes of SYTO9 dye and PI dye for 10 min (The final working concentrations were 11 μM for SYTO 9 and 66 μM for PI). The biofilms were rinsed with PBS and examined under a confocal laser scanning microscope (LSM880, Zesis, Germany) (Qi et al., 2021). The lense used in the microscope is Plan-Apochromat 20×/0.8 M27 and the filters of SYTO and PI is 488 and 543nm, respectively. The COMSTAT computer program was used to analyze the structural organization of the microbial communities by quantifying three-dimensional biofilm image stacks.

A. actinomycetemcomitans was cultured with or without enzyme Est816, antibiotics or both for 48 hours. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized from 200 ng of RNA using the Prime Script RT Reagent Kit (Takara, Kusatsu, Japan). The sequences of the PCR primers used in this study are listed in Supplementary Table 1 . The PCR reaction parameters were as follows: initial denaturation step of 95°C for 30 seconds, and another 40 cycles of melting step at 95°C for 3 seconds, followed by 60°C for 30 seconds. The 2 ^−ΔΔCt method was used to analyze the relative expression levels of target genes.

The biocompatibility of Est816 on Human gingival fibroblasts (HGF) was tested in advance of the in vivo rat experiments. HGF were procured from Anhui Hanjin Science and Technology Co. and utilized as experimental cells to assess cell morphology via immunofluorescence staining. The cells were treated with MINO (MIC), Est816 (12 U/mL), and a combination of MINO (MIC) and Est816 (12 U/mL) for three days. Untreated cells were used as a control group. Fibrillar actin (F-actin) structures were detected using standard TRITC-phalloidin staining (Sigma, St. Louis, MO, USA) (Zhu et al., 2023). Subsequently, the cells were examined under a fluorescence microscope.

The 36 Sprague-Dawley rats aged 6-8 weeks (weighing 180 – 200 g) used in this study were obtained from the Animal Experimental Center of Anhui Medical University (Anhui, China). The protocol for all experiments described herein adhered strictly to the ARRIVE and the Use of Laboratory Animals of the National Institutes of Health guidelines. This study was approved by the Animal Ethics Committee of the Anhui Medical University, China (Protocol No. LLSC20230821). Pluronic F127 (Sigma-Aldrich) serves as a topical drug delivery system that improves in situ drug adhesion without compromising drug efficacy or achieving stable and prolonged drug delivery (Almoshari et al., 2020). The rats were anesthetized, and silk ligatures were placed around the cervical part of the first upper molar. The rats were divided into six groups, each consisting of six animals: an untreated control group, a ligated control group, an experimental periodontitis group (0.15 ml of 1.0 × 10⁶ CFU/mL A. actinomycetemcomitans suspension every 4 days), an experimental periodontitis group treated with enzyme Est816 (a mixture of A. actinomycetemcomitans and 12 U/mL of enzyme Est816 with Pluronic F127 every 4 days), an experimental periodontitis group treated with MINO at the MIC, and an experimental periodontitis group treated with the combination of antibiotics and enzyme Est816.

After 8 weeks, the rats were sacrificed, and their maxillae were removed for further evaluation. Micro-CT scanning of the maxillae was performed using (Micro-CT; SkyScan, Kontich, Belgium), and the data were analyzed using the CT Analyzer software (version 1.15.4.0+; Skyscan). The area from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) of the molar was defined as the vertical alveolar bone resorption.

The rat jaws were fixed in a 10% formaldehyde solution at 4°C for 1 week. After rinsing with distilled water, the samples were decalcified in 10% thylenediaminetetraaceticacid for 8 weeks. Subsequently, they were rinsed in PBS buffer for 12 hours, dehydrated, embedded, and cut into 3 – 4 μm sections. These sections were subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry to detect matrix metalloproteinase-9 (MMP-9) expression in the periodontal tissues. The results were analyzed to assess the integrity and inflammatory response of the alveolar bone and cementum by histological observation using an optical microscope (OLYMPUS AX80, Olympus Co., Tokyo, Japan).

The data were presented as the mean ± the standard error of the mean. One-way or two-way analysis of variance (ANOVA) followed by Bonferroni’s test were used to test for significance among groups. P values< 0.05 indicated statistical significance.

In order to determine whether enzyme Est816 enhanced the sensitivity of A. actinomycetemcomitans to antibiotics, the MICs of MINO, MTZ, and AMX in the presence and absence of enzyme Est816 were assessed ( Figures 1A–C ). In this study, it was observed that inactivated enzyme Est816 did not have a significant impact on the MICs ( Figure 1 ). The MIC of MINO when combined with enzyme Est816 was 0.25 μg/mL, while the MIC for the control and inactivated enzyme groups was 0.5 μg/mL ( Figure 1A ). Both MTZ and AMX exhibited a MIC of 4 μg/mL in the control and inactivated enzyme groups, whereas the MIC of the combination group was 2 μg/mL ( Figures 1B, C ). These findings indicate that the addition of enzyme Est816 synergizes with antibiotics to inhibit the growth of the growth of A. actinomycetemcomitans.

In the untreated group, the bacterial monolayer adhered to the slide surface and formed an overlapping structure of multiple layers ( Figure 2A ). However, in group Est816, the biofilm structures of A. actinomycetemcomitans collapsed, confirming that enzyme Est816 had a dispersing effect on biofilm formation. The combination of antibiotics with enzyme Est816 was significantly more synergistic than antibiotics alone in inhibiting biofilm formation. The biofilm grown in the presence of antibiotics and enzyme Est816 was evenly distributed in a single layer. Notably, a reduction in the biofilm biomass was observed even at sub-MIC concentrations of the synergistic antibiotics when combined with enzyme Et816. These results highlight the synergistic enhancement of the antibacterial effects of the antibiotics in the presence of enzyme Est816, effectively hindering A. actinomycetemcomitans biofilm formation.

Subsequently, crystal violet staining and quantitative analysis were used to determine the biofilm inhibition and eradication rates. The combined treatment exhibited superior inhibition of A. actinomycetemcomitans biofilm formation compared to the antibiotics alone (p< 0.01) ( Figure 2B ). When combined with enzyme Est816, each antibiotic effectively prevented biofilm formation at sub-MIC concentrations, demonstrating significant biofilm inhibition compared with its use alone (p< 0.01). AMX alone showed limited efficacy in removing biofilms; however, in combination with enzyme Est816, it significantly reduced biofilm formation. Disruption of mature biofilms by the combination of antibiotics with enzyme Est816 was also measured ( Figure 2C ). Notably, the three antibiotics showed weak activity at the sub-MIC and MIC values against mature A. actinomycetemcomitans biofilms. The synergistic effect of the enzyme Est816 significantly increased the eradication rate of antibiotic treatment compared to antibiotics alone (p< 0.01).

The ability of the synergia of the antibiotics and enzyme Est816 to disperse pre-formed biofilms was further explored using CLSM. There was a significant difference in the overall structure and thickness of biofilms between the combination and control groups ( Figure 3A ). The biofilms in the control group were dense and thick. After treatment with the antibiotics and enzyme Est816, the overall structure of the biofilm was disrupted. Compared with the antibiotic-only group, the enzyme Est816-antibiotic combination group showed a greater decrease in cell density, indicating that enzyme Est816 enhanced the inhibition of biofilm formation. For a comprehensive assessment of the impact of combination agents on bacterial biofilms, CLSM imaging with COMSTAT analysis was employed to analyze the architecture of the biofilms. This allowed for the visualization and characterization of the in-vitro biofilms. After 48 hours of treatment with or without enzyme Est816, the biomass and average thickness of the biofilm decreased at varying concentrations of antibiotics. The changes in the trend observed in each group were consistent with the results of biofilm reduction in crystal violet biofilm quantification ( Figure 3B ). When antibiotics (sub-MIC) were combined with enzyme Est816, the average thickness, biofilm biomass, and diffusion distance of A. actinomycetemcomitans biofilm were significantly lower and statistically different from those treated with antibiotics alone, except for MINO (p< 0.01). Furthermore, regardless of the sub-MIC or MIC concentration of the antibiotics, there was no statistically significant difference in the three quantitative measurements of biofilms in the combined group with enzyme Est816 (p > 0.05).

To understand the mechanisms underlying antibacterial and anti-biofilm activities, we conducted qRT-PCR on two genes responsible for pathogenicity and biofilm formation.

Compared to control group, the combination of antibiotics with enzyme Est816 showed a strong inhibitory effect on the expression of virulence factors ltxA and rcpA (p< 0.01) ( Figure 4 ). The increase in rcpA caused by AMX indicates a different mechanism of action for antibiotics on bacteria. The decrease in rcpA expression observed during combination therapy is a result of the Est816 effect.

Immunofluorescence staining of HGF cells was performed to determine compatibility with enzyme Est816. As shown in Figure 5 , after 3 days of incubation, there was no significant difference in cell proliferation between the control group and group Est816. In addition, no changes in DAPI fluorescence intensity were detected in any of the study groups compared with the control group. The results showed that cells cultured with MINO at the MIC and enzyme Est816 (12 U/mL) did not exhibit significant morphological or nuclear damage compared with the control group. Additionally, structural reorganization of the actin cytoskeleton was not detected at the end of cultivation with enzyme Est816, indicating acceptable biocompatibility.

Micro-CT analysis was performed to assess the therapeutic effects of the combination of enzyme Est816 and MINO on periodontitis in vivo. Bone resorption was minimal in the ligation group, whereas bone loss was most pronounced in the periodontitis group, as was evident in the root furcation area and alveolar septum ( Figure 6A ). To accurately assess alveolar bone resorption, we measured the distance from the CEJ to the ABC and the loss of bone in the bifurcation area. Compared to the control group, the experimental periodontitis group showed a significant increase in periodontal bone resorption (p< 0.01), while the MINO and enzyme Est816 groups showed a decrease in bone resorption compared to the experimental periodontitis group ( Figure 6B ). Compared with periodontitis, the combination of Est816 and MINO resulted in a significant reduction in bone loss (p< 0.01). H&E staining showed that the gingival epithelium and alveolar bone between the first and second molars of rats in the control group were both present and healthy ( Figure 7A ). In the ligated control group, the gingival epithelium was detached from the tooth at the ligation site (arrow), and the alveolar bone was only slightly lower than that in the control group. In the periodontitis group, sequestrum formation and deep periodontal pockets were observed under the microscope. In the antibiotic group, loss of gingival tissue and an increase in the number of inflammatory cells were observed, while in the enzyme group, the gingival tissue was partially destroyed, and alveolar bone was resorbed. The combined Est816 intervention showed no evidence of periodontal pockets. Thus, alveolar bone levels were essentially unaffected, and only epithelial damage caused by mechanical stimulation was observed. Immunohistochemical staining for MMP-9 showed no obvious inflammatory reactions in the control, ligation, or combined groups ( Figure 7B ). In addition, the group that received antibiotics and enzyme Est816 showed reduced expression of MMP-9 compared to the periodontitis group; however, the individual effects of enzyme Est816 and MMP-9 were far less pronounced than their combined effect. When taken together, these results indicate that the components of the combination treatment had a marked synergistic effect in treating periodontitis, even in the absence of debridement or irrigation.