Synergistic effects of D-arginine, D-methionine and D-histidine on the inhibition and eradication of Porphyromonas gingivalis biolm

Porphyromonas gingivalis (P. gingivalis) biolm is involved in peri-implantitis and periodontitis. Whether a mixture of D-AAs has synergistic effects on P. gingivalis biolm remains unknown. The aim of this study was to investigate the effects of multiple D-AAs on P. gingivalis biolm. D-arginine (R), D-methionine (M) and D-histidine (H) were used. The bacterial growth activity and minimum inhibitory concentrations (MICs) were determined. The effects of D-AAs mixture on biolm biomass, extracellular polymeric substances (EPS), biolm morphology, cytotoxicity, biolm structure and bacterial membrane integrity were determined. D-AAs mixture delayed the proliferation of P. gingivalis below MICs. Mixtures of D-AAs displayed denite effects on the inhibition and disassembly of biolm than single D-AAs. The EPS content increased with D-AAs concentrations. D-AAs mixture damaged the integrity of biolm, while RMH showed the best effects. 8 mM RH and 4 mM RMH had no cytotoxicity. 4 mM RMH decreased biolm thickness and altered bacteria membranes structure. Hence, the combined mixture of 4 mM RMH presents the best synergistic effects on the inhibition and eradication of P. gingivalis biolm. Our study provides new insights of application of D-AAs for the treatment of peri-implantitis and periodontitis.


Introduction
Peri-implantitis, characterized by bone resorption and peri-implant in ammation, is the major cause leading to implant failure. Among the etiologies for peri-implantitis, plaque bio lm is a de nite and crucial factor 1 . Similarly, periodontitis is also usually caused by the accumulation of plaque bio lm. Bio lm is formed by multiple bacterial strains, while bacteria are encapsulated in matrix in order to prevent antibacterial agents to penetrate the bio lm, leading to hundreds of times of tolerance to antibacterial agents 2 . Hence antibiotic treatment is not effective to eliminate bio lm and embedded bacteria 3 . Other methods like mechanical treatment, laser therapy and photodynamic therapy also did not acquire ideal result yet, for either damaging the implant surface or causing substance residue. Therefore, nding an optimal method to eradicate plaque bio lm would be an innovation to treat peri-implantitis as well as periodontitis.
P. gingivalis is a gram-negative, black-pigmented anaerobic bacterium, which belongs to the red complex. Once incorporating into bio lm, P. gingivalis could colonize subgingivally and contact with surrounding tissues directly, then it secretes lipopolysaccharide, peptidoglycan, gingipain and other virulence factors to stimulate the host's innate immune response to produce in ammatory factors such as IL-6, IL-8, INF-γ and TNF-α, resulting in tissue necrosis 4,5 . Furthermore, gingipain could enable P. gingivalis to escape the host defense system by degrading antimicrobial peptides 6 . In general, P. gingivalis has been proven to be an important pathogen causing peri-implantitis or periodontitis, eradicating its bio lm phenotype is crucial for the elimination of its toxicological effects.
In bio lm communities, the EPS matrix is mainly composed of polysaccharides, proteins, lipids, and extracellular DNA. In most cases, the EPS matrix presents around 90% of the total bio lm biomass 7 . Therefore, strategies focusing on disruption of the EPS matrix have attracted much attentions recent years, which may increase bacterial susceptibility to antibacterial agents. Due to the characteristics of EPS matrix, those strategies can be classi ed into matrix disruptive agents, nanocarriers and bio lm physical removal technologies (magnetic eld, photodynamic therapy, ultrasounds) 8 . But they usually have common disadvantages, such as low bioavailability at later stage 9 or less effective during eradicating mature bacterial bio lms 10 . Additionally, these mentioned strategies mainly focus on how to eradicate existing mature bacterial bio lms, few studies concerned about preventing bio lm formation, which is equally important for combat bio lm infections.
In recent decades, D-amino acids (D-AAs) have been found in microorganisms, plants, and humans 11 .
Especially in microorganisms, D-AAs are essential components of cell wall peptidoglycan, within which D-AAs participate in synthesis and assembly process, as well as the metabolism of bacterium.
Subsequently, D-AAs were found during the bio lm lifecycle, that was, D-AAs were produced and accumulated rapidly at the dispersion phase 12 . Afterwards, several D-AAs were found effective in inhibiting bio lm formation of classic pathogenic bacteria [12][13][14] . Our previous research revealed that Darginine, D-valine, and several other D-AAs had the abilities of both inhibiting P. gingivalis bio lm formation and triggering the disassembly of mature P. gingivalis bio lm 15,16 . Considering the cytotoxic effects of D-AAs at certain concentrations 17 , D-AAs should be used biocompatibly to combat bio lm and nding an ideal strategy to avoid the cytotoxicity of D-AAs is urgent. Whereas previous studies neither investigated the biocompatibility of different concentrations of D-AAs to mammalian cells nor the effects of a mixture of multiple D-AAs on P. gingivalis bio lm.
Therefore, the aim of this paper is to investigate the combined effects of different D-AAs on P. gingivalis bio lm and EPS matrix below the concentrations of inhibiting P. gingivalis viability, and the cytotoxicity of effective D-AAs mixture to mouse broblast L929. In order to provide an optimal strategy for combined application of D-AAs.

Results
Effects of D-AAs mixture on P. gingivalis bacterial growth activity and minimum inhibitory concentrations (MICs) The bacterial growth activities of P. gingivalis within mixture of D-AAs were shown in Fig. 1. Different concentrations of D-AAs had the ability of delaying P. gingivalis proliferation. Particularly, RMH expressed the most notable effects at equal concentrations as RM, RH and MH. Nevertheless, P. gingivalis could proliferate in the form of logarithmic phase. The MICs of each group were: RM 20 mM, RM 20 mM, MH 16 mM, RMH 10 mM. Taking into consideration that completely inhibition of bacterial proliferation might causing bacterial resistance to biological agents 18 , the concentrations of D-AAs used in subsequent experiments were below the MICs.
Effects of D-AAs mixture on P. gingivalis bio lm formation and bio lm disassembly The concentrations of D-AAs using for all groups in this part were below the MICs. This experiment was divided into two parts, namely bio lm formation and bio lm disassembly. As shown in Fig. 2a, b, c, d, the biomass of P. gingivalis bio lm formation displayed descending tendency with the increase of concentrations of RM/RH/MH/RMH, so as the same tendency in bio lm disassembly experiments (Fig. 2e, f, g, h). Whereas the exception was that 4 mM MH and 8 mM MH did not show statistical difference with 0 mM (P > 0.05). Hence, the minimum effective concentration of each group was used for subsequent experiments. Figure 2i, j, k showed that RM (4 mM), RH (4 mM), MH (12 mM) were more effective in inhibiting bio lm formation than the single use of corresponding D-AAs respectively (P < 0.05). Figure 2l showed the difference among different D-AAs mixture groups on inhibiting bio lm formation at equal concentration of 4 mM, which revealed that RM was more effective than RH/MH/RMH and RMH was more effective than RH/MH respectively (P < 0.05). Next, the effects of 4 mM D-AAs on disassembling mature bio lm were investigated, 4 mM of RM, RH and MH showed enhanced effects on disassembling P. gingivalis mature bio lm than the single use of corresponding D-AAs respectively ( Fig. 2m, n, o). Figure 2p showed the different effects among mixtures of D-AAs: RH MH RM/RMH (P < 0.05).
Effects of D-AAs mixture on P. gingivalis bio lm EPS matrix The concentrations of D-AAs that both below MICs and signi cantly affected bio lm were used in this part. As shown in Fig. 3a, at 4 mM and 8 mM, the mixture of RMH signi cantly inhibited EPS matrix production in bio lm formation phase than the mixture of RM or RH (P < 0.05). At 12 mM, the effects of RM seemed to attenuate than RH/MH. With the increase of concentration, RM displayed promoting effects on EPS matrix, while RH displayed reducing effects on the contrary. In bio lm disassembly experiment (Fig. 3b), the EPS matrix of RM/RH/MH/RMH raised with concentration consistently. When comparing with 0 group, MH/RMH did not show notable effects on EPS matrix, even resulted in more EPS matrix biomass. However, RH showed the most signi cant effects on reducing EPS matrix comparing with 0, RM, MH, RMH respectively at each concentration (P < 0.05).
Effects of D-AAs mixture on P. gingivalis bio lm morphology The morphological characteristics of P. gingivalis bio lm treated with D-AAs mixture was further analyzed by SEM (Fig. 4). The left two columns represented the bio lm formation phase: 4 mM RM notablely inhibited bio lm formation, the integrity of bio lm completely disappeared, while remaining few scattered cells. 4 mM RH did not present manifest difference with 0 group, but 8 mM RH evidently prevent the accumulation of cells, and furtherly, 12 mM RH completely inhibited bio lm formation. 4 mM and 8 mM MH did not wreck the junction among cells, but 12 mM MH seemed to reduce the EPS matrix, in which cells trended to separate with each other. There was also a similar tendency in group RMH, with the increase of concentrations, the adhesion became insecure and cells tended to sparse. The morphology change tendency was consistent with the experiment of bio lm biomass (Fig. 2). The right two columns represented the mature bio lm disassembly phase: without D-AAs, mature bio lm was compact and multilayer. 4 mM RM damaged the integrity, and the bio lm was thoroughly invisible under 8 mM RM.
Despite the effects of 4 mM and 8 mM RH on the bio lm formation were not obvious, their effects on bio lm disassembly were notable, especially at 8 mM, only scattered EPS matrix and few cells could be observed. With at 12 mM, the bio lm completely disappeared. 4 mM and 8 mM MH showed similar effects on disassembling mature bio lm, which the bio lm remained compact under SEM (5 k). While at 12 mM MH, bio lm separated into pieces, but cells remained adhering by EPS matrix. Low concentrations of RMH showed prominent effects on disassembling mature bio lm. Brie y, 2 mM and 4 mM RMH reduced the thickness and compactness of bio lm, additionally 6 mM and 8 mM RMH obviously eradicated bio lm with very few cells being observed. The morphology change tendency of bio lm disassembly was also consistent with the experiment of bio lm biomass (Fig. 2).

Cytotoxicity of D-AAs mixture
The cytotoxicity of D-AAs mixture was then measured. As shown in Fig Effects of 4 mM RMH on the thickness and density of P. gingivalis bio lm The bio lm formation of P. gingivalis treated with 0 or 4 mM RMH were analyzed by confocal laser scanning microscopy. Figure 6 showed the whole state of P. gingivalis bio lm. After culture for 3 days without D-AAs, the bio lm formed a thick and dense mature structure, with dominant green (live) signals ( Fig. 6a, b). While the bio lm green (live) signals decreased signi cantly (Fig. 6c, d) in the 4 mM RMH treated bio lm. The thickness of bio lms was then measured, which revealed that 4 mM RMH decreased the thickness of P. gingivalis bio lm compared with D-AAs untreated group (P < 0.05), and the density of bio lm was also decreased (Fig. 6e).
Effects of 4 mM RMH on the structure of P. gingivalis bacteria in bio lm As shown in Fig. 7a-c, P. gingivalis bacteria in 0 group presented regular shapes, as either rod-like along the long axis or round-like along the transverse axis. The membranes were intact, and the intracellular structures could be clearly recognized. Moreover, there was no intracellular content over owed and intracellular staining was few. On the contrary, however, 4 mM RMH signi cantly changed the cell structure ( Fig. 7d-f). Brie y, the shapes tended to become irregular under low magni cation, and the cell volume became larger. In addition, the membranes were damaged, and the contents of the bacteria obviously over owed. Massive intracellular staining could be observed compared with 0 group.

Discussion
Herein, we investigated the combined effects of D-arginine, D-methionine, and D-histidine on P. gingivalis bio lm for the rst time. As is known, peri-implantitis is one of the most important causes for implant failure, and plaque bio lm accumulation is a major initial factor for peri-implant mucositis and subsequent peri-implantitis [19][20][21][22] . Likewise, periodontitis is also tightly related to subgingival plaque bio lm, which is mainly composed of anaerobes. Hence, the inhibition as well as the eradication of bio lm should be an ideal strategy for treating these bio lm-associated diseases. Antibiotics have always been the rst choice for combating against infection by their bactericidal e cacy for centuries. However, massive evidences have indicated that extensive use of antibiotics leads to bacterial resistance 18,23,24 . Meanwhile, bacterial bio lms increase tolerance to antibiotics by preventing antibiotics penetrating bio lm matrix 25 . Therefore, we conceived to damage bio lm either through inhibition or eradication by agents, and meanwhile not causing bacterial resistance, in order to increase bacterial susceptibility to additional acknowledged antibiotics.
We previously reported that D-arginine and D-valine were effective in inhibiting P. gingivalis bio lm formation and promoting the disassembly of mature P. gingivalis bio lm respectively 15,16 . Considering that different D-AAs might be involved in different metabolic forms of bacteria, we speculated that combined application of D-AAs would present synergistic effects on bio lm. After con rming the effects of R/M/H on bio lm respectively, we evaluated the effects of D-AAs mixture on bacterial growth activity and MICs. Gradient concentrations of D-AAs mixture that below MICs merely delayed cell proliferation, but it nally grew as typical logarithmic growth form (Fig. 1). The growth curve of 20 mM D-Met on V.
cholerae presented the similar performance 26 , which inferred that D-Met regulated cell wall synthesis in V. cholerae. Hence, we supposed that the mixture of R/M/H might affect distinct aspects of P. gingivalis synthesis, which needs further research.
CV staining was used to determine the bio lm biomass combining with crystal violet. While SEM was used to observe P. gingivalis bio lm morphology more intuitively. The results in Fig. 2  Confocal laser scanning microscopy (CLSM) results similarly showed that 4 mM RMH inhibit bio lm formation both by decreasing bio lm thickness and density (Fig. 6), consistent with the study of Shiwen 27 , which revealed that the thickness of the DANA-treated P. gingivalis bio lm was signi cantly lower than that of the untreated group. Kinds of micromolecules may possess competitiveness for the target protein or binding sites with others 28,29 . Therefore, we assumed that M and H were likely to act on the same site during P. gingivalis anabolism. Cells embedded in bio lm without D-AAs displayed oval or rod-like shape, whereas within the notable effects of D-AAs on bio lm formation, cells became elongated from 1 µm to 2 µm (Fig. 5). Likewise, the wild-type Vibrio cholerae could produce M, and 1 mM M did not alter morphology of cells. But 2 mM D-Ala stimulated the conversion of rod-shaped cells to spheres 30 . Hence,we speculate that R/M/H may act on P. gingivalis cell membranes, resulting in the weaken of the scaffold function of cell membranes. Without the encapsulation of EPS matrix, cells become slender.
In order to verify the above presume, we further observed the bacterial structure by transmission electron microscopy. The results showed that 4 mM RMH obviously changed the normal shapes of P. gingivalis bacteria. Speci cally, the cell volume became enlarged and membranes were disrupted resulting in the leakage of intercellular contents. Since the integrity of bacterial membranes were weakened, additional staining easily permeated into intracellular environment. S. gordonii membranes were damaged when treated with TBP-1-GGG-hBD3-3 for 12 h, which indicated that TBP-1-GGG-hBD3-3 presented the antibacterial and anti-bio lm activities by acting on the S. gordonii membrane 31 . AmyI-1-18 had a bactericidal effect on F. nucleatum owing to its membrane-disrupting properties, resulting in membranes destruction and inner structures leaking out 32 . These previous studies prove our mentioned presume that D-R/M/H might misincorporate in P. gingivalis cell membrane synthesis or directly act on membrane, subsequently weakens the integrity or elasticity of membrane. Whereas, the mechanisms need further research.
EPS assay seemed not corresponding to the results of bio lm biomass (Fig. 2) or bio lm morphology (Fig. 4). In the bio lm formation experiment, RM promoted EPS matrix production, whereas RH reduced EPS matrix production on the contrary. Considering the mentioned speculation that M and H might act on the same site during P. gingivalis anabolism, we further deduce that M and H display the opposite effect on the production of EPS matrix (Fig. 3). 1 mM D-Alanine or 1 mM D-Serine strikingly reduced EPS production, but different concentrations of D-Glutamic acid did not have similar inhibitory effect on bacteria EPS production 33 , which revealed that D-AAs could reduce bio lm formation independent of affecting EPS production. In the bio lm disassembly experiment, the EPS matrix of all combinations of D-AAs mixture increased with concentration. Interestingly, the EPS matrix of RH was signi cantly lower than other groups at each concentration (P < 0.05). Retrospecting that 4 mM RH notablely disassembled bio lm under SEM, we put forward a point of view that after totally disassembling bio lm, bacteria lose the environment of forming bio lm. Contrarily, other D-AAs triggered bio lm disassembly moderately, the dispersed planktonic cells rejoined the edges of intrinsic bio lm by secreting EPS matrix. Owing to the high molecular weight and viscosity of polysaccharides, the scattered polysaccharides still adhered to remaining bio lm or the bottom of plate. Therefore, the EPS content of RM/MH/RMH might be more than 0 group, consistent with our previous research 16 . In summary, there are multiple factors in uencing the content of EPS matrix, hence, it is not an appropriate index to measure the effects of D-AAs on P. gingivalis bio lm.

Conclusions
Within the limitations of this study, this work has shown that the combined mixture of 4 mM RMH presents the best synergistic effects on the inhibition and eradication of P. gingivalis bio lm without inhibiting bacterial proliferation, which suggests that the combination application of D-AAs could be an optimal strategy for the treatment of peri-implantitis and periodontitis.

Materials And Methods
Bacterial culture and reagents P. gingivalis ATCC 33277 was used in this study, which was subcultured on sterilized BHI (HopeBio) supplemented with hemin (5 µg/mL), menadione (0.5 µg/mL), and yeast extract (LP0021, Oxoid) and incubated at 37 °C under anaerobic conditions (80% N 2 , 10% H 2 , and 10% CO 2 ). D-AAs (Sigma-Aldrich) Bacterial growth activity and MICs Single P. gingivalis colonies were inoculated into BHI bacterium liquid medium, then the inoculum was adjusted to 10 7 colony-forming unit (CFU) counts (CFU/mL) 15 . Afterwards, the suspension was inoculated to 96-well plates (Nest) and mixed with a series of concentrations of D-AAs, consequently incubated for 72 hours at 37 °C under anaerobic conditions. Simultaneously, the OD 600 value was measured every 4 hours with a spectrophotometer (Bole Life) 34 . The minimum concentration of sample showing no turbidity at 72 hours was recorded as the minimal inhibitory concentrations (MICs).
Bio lm biomass assay This experiment was divided into two parts, namely bio lm formation and bio lm disassembly. The concentrations of D-AAs for all groups were below the MICs referred to above results.
Bio lm formation inhibition experiment: Different concentrations of D-AAs and their mixture were added in 96-well plates which inoculated with P. gingivalis at a concentration of 10 8 CFU/mL for 72 hours.
Mature bio lm disassembly experiment: P. gingivalis were inoculated in 96-well plates at a concentration of 10 8 CFU/mL for 72 hours to form mature bio lm, then different concentrations of D-AAs and their mixture were added in each well, subsequently cultured for 72 hours.
At the end of above experiments, planktonic bacterial supernatant was removed and the culture plate was rinsed with phosphate-buffered saline (PBS) (Boster), carefully air-dried for 20 min, and then xed with 2.5% (v/v) glutaraldehyde for 20 minutes. 0.1% Crystal violet (CV) added subsequently. Then the excess CV was rinsed with PBS. The CV bounded with bio lms were decolorized by 95% ethanol, then transferred to another 96-well plate. Finally, the bio lm biomass was evaluated at OD 600 nm using a microplate reader (Synergy HT, BioTek) 35 .

EPS matrix detection
The concentrations of D-AAs mixture that both below MICs and signi cantly affected bio lm biomass were used in this part. The extraction of EPS from bio lm based on the method of Albuquerque 36 . Brie y, bio lms were sequentially rinsed with PBS, distilled water, 6% phenol solution and 97% sulfuric acid.
Afterwards, samples were air-dried for 20 min, followed by incubation for 20 min at room temperature. Then, the amount of polysaccharides in the bio lm was determined by recording the absorbance at OD 490 nm with glucose as standard 37 .

SEM analysis of bio lms
The concentrations of D-AAs mixture used here were based on MICs, bio lm biomass and EPS matrix. The morphological characteristics of P. gingivalis bio lms treated with D-AAs mixture were observed by Scanning electron microscopy (SEM) (Hitachi S-4800). In general, 24-well plates were rinsed three times using PBS, then xed overnight in 2.5% (v/v) glutaraldehyde at 4 °C, and dehydrated by gradient ethanol solutions (30%-100%). After completely oven drying, the bio lm was observed under the SEM after the process of sputter coating with gold.

Cytotoxicity assay
The concentrations of D-AAs mixture used here were based on above experiments. Mouse broblast L929 was resuscitated and then cultured in DMEM solution, placed in an incubator of 5% CO2 at 37℃. Cells were counted when the bottom of culture bottle was covered up to 80%-85% by cells. Logarithmic growth phase cells were selected to prepare single cell suspension, and inoculated in 96-well plates for 24 hours. Afterwards, D-AAs mixture was added. After 1, 3 and 5 days of culture at 37℃, 10 µL CCK-8 (Beyotime) was added to each well, and went on culture for 2 hours at 37℃. The absorbance value was measured by recording the absorbance at OD 450 nm.

CLSM
The bio lm formation of P. gingivalis treated with 0 or 4 mM RMH were analyzed by confocal laser scanning microscopy. The method of culture of P. gingivalis bio lm was identical with bio lm biomass. After 3 days culture, the samples were gently washed twice with distilled water to eradicate planktonic bacteria on the surface of 24-well plate. Then using the Live/Dead backlight bacterial viability kit (Invitrogen, Eugene, OR) to uorescence-stained the bio lm for 30 minutes. The whole experiment was operated in dark area. Finally, the samples were observed under a confocal laser scanning microscope (FV3000, Olympus, Japan). Dual-channel scanning observations were performed with a red channel (490/635 nm for propidium iodide) and a green channel (480/500 nm for SYTO 9 stain). and. Images were drawn using Imaris software (Zeiss, Germany), the thicknesses of bio lm were calculated using ImageJ software (NIH, USA).

Transmission electron microscopic imaging (TEM)
The bacterial morphology in 4 mM RMH showed prominent change under SEM,hence we further studied the structural details under transmission electron microscope. The groups were the same as CLSM, the samples were gently washed three times with distilled water to eradicate planktonic bacteria on the surface of 24-well plate. The collection of bio lm was referred to Dejana' methods 38 . Brie y, the plates were vortexed with 0.9% NaCl for 3 min to remove the bio lm from the bottom, and collected into 5 mL centrifuge tube respectively. The specimens were centrifuged at 4000 rpm for 10 minutes (4℃) and then xed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH, 7.4) at room temperature for 1 h, subsequently post-xed with 1% osmium tetroxide for 1 h. Then, the specimens were rinsed with 0.1 M phosphate buffer (pH, 7.4) for 4 times (5 minutes per time). Graded ethanol series (50%-70%-90%-100%) and acetone were used for dehydration. Then the specimens were embedded for resin penetration and ultrathin section (90 nm). The sections were then double stained by 3% uranium acetate and lead citrate. Finally the sections were observed under a transmission electron microscope (JEM 1400 PLUS, JEOL, US).
Statistical analysis Data are expressed as mean ± SD. Dunnett's t-test was applied to compare each test group with the control group, and one-way ANOVA with appropriate post-tests was applied for multiple groups.    Effects of D-AAs mixture on P. gingivalis bio lm morphology under SEM (× 1k, × 5k). The left two columns represented the bio lm formation phase. The right two columns represented the mature bio lm disassembly phase. Bar, 50 μm in × 1k, 10 μm in × 5k   . Effects of 4 mM RMH on the structure of P. gingivalis bacteria in bio lm. P. gingivalis bacteria in 0 group presented regular shapes, as either rod-like or round-like. The membranes were intact and intracellular staining was few. (d-f)4 mM RMH signi cantly changed the cell structure. Cell shapes tended to become irregular and the cell volume became larger. Cell membranes were damaged (black arrow) and the contents of the bacteria released (black triangle).