Potential Effect of Giant Freshwater Prawn Shell Nano Chitosan in Inhibiting the Development of Streptococcus mutans and Streptococcus sanguinis Biofilm In Vitro

An oral biofilm comprises a variety of bacteria including Streptococcus mutans and Streptococcus sanguinis that cause human infections, such as caries and periodontitis. Thus, biofilm management plays an important part in the prevention and treatment of oral diseases. Nano chitosan is a bioactive material that has antimicrobial activities. This in vitro study aimed to evaluate the effect of nano chitosan synthesized from giant freshwater prawn shells (PSNC) on S. mutans and S. sanguinis biofilm development. PSNC was prepared from the extracted chitosan of giant freshwater prawn (Macrobrachium rosenbergii) shells using the ionic gelation method. The effect of PSNC on S. mutans ATCC 25175 and S. sanguinis ATCC10556 biofilm formation was evaluated using the crystal violet assay. Both bacteria were inoculated in the presence of various concentrations (5, 2.5, and 1.25 mg/ml) of PSNC for 24 h and 48 h. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy were performed to visualize and study the biofilm architectural features. The biofilms were stained with the BacLight Bacterial Viability Kit prior to CLSM observation to monitor the viability of the biofilm. The results showed that PSNC exposure for 24 h and 48 h inhibited the formation of S. mutans and S. sanguinis biofilms. The biofilm formation inhibition percentage increased with an increase in the PSNC concentration (p < 0.05). The highest inhibitory activity was shown at 5 mg/ml PSNC (p < 0.05). Those findings were confirmed by the subsequent findings using the CLSM and SEM analyses. The biofilm architecture was strongly disrupted upon treatment with PSNC. After exposure to 5 mg/ml PSNC, the number of bacteria significantly decreased. The remaining bacteria were seen as individual cells, showing damaged cells. In conclusion, PSNC inhibits the development of S. mutans and S. sanguinis biofilm in vitro, indicating the potential of PSNC in clinical application for oral bacterial infection, prevention, and treatment.


Introduction
Oral bioflm, termed dental plaque, has been described as a structured microbial community that is attached to tooth surfaces and embedded in an extracellular polymeric matrix (EPS). Te matrix of the bioflm protects the bacterial communities and makes them withstand harsh conditions and resist antimicrobial treatments [1]. Te oral bioflm is composed of various microbial communities with approximately 700 distinct microbial species that interact with each other. Tis interaction can either positively or negatively afect the growth of the bioflm [2].
Te accumulation of bacteria on the surfaces of the teeth and the gingival sulcus is considered the primary cause of dental caries, gingivitis, and periodontitis [3]. Dental caries is a bioflm-associated infectious disease fostered by bioflm dysbiosis that causes permanent destruction of the hard tissue of the tooth [4]. Dental caries is strongly associated with frequent exposure to dietary fermentable carbohydrates, leading to the accumulation of acid-producing and acid-resistant microorganisms on the surface of the teeth, thus creating an acidic pH environment. Te overgrowth of acidproducing bacteria within bioflms leads to the formation of cariogenic bioflms that trigger caries [5]. Te destruction of mineralized tooth tissues is the end result of the acidifcation of dental bioflm that progresses over time [6].
Te sulcular and junctional epithelial cells are the frst targets of the bacteria accumulating in the gingival sulcus. Te cells react to the bacteria by altering cellular signaling, resulting in changes in cellular behavior, such as protease and cytokine production, cell proliferation, and cell migration [7]. A study by Currò et al. [8] showed that transglutaminase (TG) gene expressions in gingival tissues were altered in response to chronic injury. Transglutaminases are enzymes that contribute to determining cell shape by cross-linking proteins involved in cell adhesion and extracellular matrix stabilization. Te study demonstrated a signifcantly downregulated level of TG1 and TG3 in gingival tissues of chronic periodontitis patients. Te disintegration of the gingival epithelium makes it vulnerable to further bacterial invasion.
Furthermore, periodontitis is closely associated with various systemic diseases, including diabetes and cardiovascular diseases (CVDs) [9]. Periodontitis, through its chronic pathogenic bioflm burden, exerts a continuous negative stimulus on local host responses to secret highsensitivity C-reactive protein (hs-CRP), nitric oxide, and various infammatory mediators through mechanisms that are locally regulated by microRNAs (miRNAs).
Recent research has revealed that miRNAs have a role in a number of epigenetic processes connected to cardiovascular disease (CVD), increased oxidative stress, and also periodontitis [10]. A previous study by Isola et al. [11] showed that certain GCF miRNAs were considerably higher in individuals with periodontitis (miRNA-7 and -21), CVD (miRNA-7, -21, and -200), and individuals with periodontitis and CVD (miRNA-21).
S. mutans and S. sanguinis are important members of dental plaque and infuence each other during oral bioflm formation [2]. Streptococcus sanguinis is a novel colonizer that aids the subsequent attachment of organisms and plays a signifcant role in oral bioflm development. Studies showed that S. sanguinis is one of the most abundant species in early dental bioflms. Although this bacterium is not known to be directly involved in oral disease, it is frequently implicated in infective endocarditis [12].
Normally, S. mutans lives as a regular member of the mature dental bioflm community; however, under certain circumstances, this bacterium becomes dominant and causes dental caries. Streptococcus mutans plays a signifcant role in tooth demineralization due to its ability to adhere to the tooth surfaces, generate acid, and also resist acid [13]. In addition, the bacteria have the capability to synthesize extracellular polysaccharides such as glucans or fructans via extracellular enzymes, namely, glucosyltransferase and fructosyltransferase [14].
Prior studies showed the antagonistic interaction between S. mutans and S. sanguinis at the ecological level [15][16][17]. However, recent fndings prove otherwise. A study by AlEraky et al. [18] showed that S. sanguinis has been identifed in patients with high levels of caries. Another study by Meriç et al. [19] reported similar frequencies of S. mutans and S. sanguinis in groups of caries-and cariesfree subjects. In addition, a clinical study has shown that the interaction of S. mutans with S. sanguinis is an essential factor in caries status in children, suggesting that the relative levels of these two bacteria in the oral cavity play a signifcant role in the development of caries [20].
Oral bioflms are a major cause of oral infectious diseases; thus, bioflm management plays an essential role in the prevention and treatment of the diseases [4]. Considering that a number of studies have shown that immune response modulators such as tacrolimus are efective in the treatment of oral chronic infammatory diseases (e.g., oral lichen planus and periodontitis), their use has attracted a great deal of interest [21][22][23]. In vivo study by Guimarães et al. [23] revealed that tacrolimus treatment in periodontitis-induced rats showed less bone loss associated with periodontitis, through a mechanism involving IL-1β, TNF-α, and IL-6. However, a study by Nivethitha et al. [24] reported a severe case of generalized gingival overgrowth caused by tacrolimus-induced treatment following a renal transplant.
Over the last few years, many chemical substances have been reported to have efects on bacterial cell adhesion. Some, such as chlorhexidine [25], delmopinol [26], and triclosan [27,28] have shown potent inhibitory efects on bioflm development and maturation. Nevertheless, they exhibit several side efects such as tooth and tongue staining, taste alteration, and increased supragingival calculus formation [29]. Terefore, an attempt to explore new materials with minimal side efects to inhibit bioflm formation is necessary.
Chitosan is a natural polysaccharide derived from chitin, composed of 2-amino-2-deoxy-D-glycopyranose and 2acetamide-2-desoxy-D-glycopyranose units linked by -1,4 glycosidic bonds. Chitosan is obtained by the deacetylation of chitin which is particularly abundant in shrimp shells, crab shells, crayfsh, and krill shells [30]. Kumari et al. [31] reported that shrimp shells were found to be the best choice for chitosan production. All the physicochemical properties, such as the degree of deacetylation value, average molecular weight, and solubility data, support this. In addition, the XRD and FTIR patterns of shrimp chitosan were very similar to those of commercial chitosan.
Chitosan is used in biomedical applications due to its high biocompatibility and antimicrobial properties [32]. Several studies have shown that chitosan has antibacterial and antiplaque efects as well as antiadhesive properties against S. mutans and other Streptococci [33,34]. A study by Costa et al. [34] revealed that chitosan was capable of inhibiting S. mutans adhesion and bioflm formation. Another study by Aliasghari et al. [33] demonstrated that chitosan and chitosan nanoparticles at a concentration of 5 mg/ml reduced S. mutans bioflm formation by up to 92.5% and 93.4%, respectively.
Chitosan nanoparticles are bioactive and environmentally friendly materials with unique physicochemical properties. Nano chitosan is a nanoparticle of a chitosan derivative product that has a smaller size than chitosan (10-1,000 nm) [35]. Smaller nano chitosan particle size can increase the solubility and penetration ability of the molecules into the bioflm [33,36].
Te accumulation of oral bioflm is one of the main causes of oral diseases that may lead to systemic diseases; thus, agents with antibioflm properties are required for preventing the diseases. Te search for new antibioflm agents continues due to the lack of truly efective treatment options despite all the currently available products. Since bacteria exist in multispecies bioflms in the oral cavity, this in vitro research attempted to simulate the microecology of an oral Streptococci bioflm by cultivating S. mutans and S. sanguinis together. Te purpose of the present study was to evaluate the efect of giant freshwater prawn shell nano chitosan (PSNC) on S. mutans and S. sanguinis bioflm development in vitro.

Extraction of Chitosan.
Chitosan was extracted from freshwater prawn (Macrobrachium rosenbergii) shells through three major steps, namely, deproteination, demineralization, and deacetylation processes. In brief, the shells of the giant freshwater prawn were washed thoroughly with water to remove any adhering dirt and then dried in an oven at a temperature of 45°C for 3 days. Te dry samples were pulverized and treated with 4% NaOH (Merck, Germany) for 60 minutes at a temperature of 80°C (deproteination). After the protein was depleted, the sample was subsequently washed thoroughly with distilled water and then dried at room temperature for 24 hours. Te deproteinized sample was treated with 1 M HCl (Merck, Germany) for 3 hours to yield chitin (demineralization). Te sample was washed thoroughly with distilled water to remove the excess HCl present in the chitin. Afterward, the demineralized sample was immersed in a 4% NaOCl solution. Te deacetylation process was carried out by treating the sample with 50% NaOH for 3 hours at a temperature of 100°C. After washing thoroughly with distilled water, the sample was subsequently dried for 24 hours to obtain chitosan isolates.

Preparation of Giant Freshwater Prawn Shell Nano
Chitosan (PSNC). Nano chitosan was created by the ionic gelation method. Chitosan was dissolved in 1% acetic acid, and 1% tripolyphosphate (TPP) (Xilong AR, China) was then added drop by drop while stirring vigorously until it reached a ratio of chitosan to TPP � 2 : 1. Te PSNC solution was dried using a spray dryer at a temperature of 120°C.
PSNC was dissolved in acetic acid (Merck, Germany) and kept under magnetic stirring overnight to completely dissolve the particles. Te pH was adjusted with NaOH to a fnal value of 6.0. To obtain the desired volume, distilled water was added.

Microorganisms and Inoculum Preparation.
S. mutans ATCC 25175 and S. sanguinis ATCC10556 were provided by the Integrated Research Laboratory, Faculty of Dentistry, Universitas Gadjah Mada, Indonesia. A single colony of S. mutans or S. sanguinis was cultured in BHI broth medium at 37°C overnight. Te turbidity of the bacterial suspension was then adjusted to 0.5 McFarland (1.5 × 10 8 CFU/mL).

Bioflm Formation Inhibition Assay.
Te efect of PSNC on S. mutans ATCC 25175 and S. sanguinis ATCC10556 bioflm formation was evaluated using a crystal violet assay. A total of 50 μl BHI supplemented with 2% sucrose, 5 μl of S. mutans, 5 μl of S. sanguinis, and 40 μl of various concentrations of PSNC (fnal concentration of PSNC was 5, 2.5, and 1.25 mg/ml), were transferred into a 96-well polystyrene microtiter plate (Iwaki, Japan). Te plate was incubated at 37°C under anaerobic conditions for 24 h and 48 h. Phosphate-bufered saline (PBS) solutions and 0.2% chlorhexidine (CHX) were used as negative and positive controls, respectively.
After rinsing with PBS twice, the bioflms attached to the bottom of the microplates were stained with 125 μl of 0.1% crystal violet for 15 min and washed with PBS to remove the residual dye. Te bounded crystal violet was subsequently released with 200 μl 96% ethanol. Te absorbance of the released crystal violet in ethanol was recorded at OD540 nm by a spectrophotometer (Termo Scientifc, USA). Te experiments were performed in quadruplicate. Te inhibition percentage was calculated using the following formula [37]: The percentage of biofilm inhibition � OD growth control − OD sample OD growth control × 100.

Statistical Analysis.
Te bioflm formation inhibition assay was independently repeated at least four times. Data were expressed as the mean ± standard deviation (SD) from a representative experiment. A one-way analysis of variance (ANOVA) followed by a post hoc LSD test was performed to determine the signifcance of the groups. Statistical analysis was performed using the SPSS software, version 16.0. A p value less than 0.05 was considered signifcant. Figure 1. Experimental data show bioflm formation inhibition percentages of 98.92 ± 0.30, 93.69 ± 1.59, and 38.58 ± 2.40 when the bacteria were exposed to 5, 2.5, and 1.25 mg/ml PSNC for 24 h, respectively. However, the bioflm formation inhibition percentage of 0.2% Chx (positive control) was 97.15 ± 0.55. After being exposed to PSNC at concentrations of 5, 2.5, and 1.25 mg/ml for 48 h, the experimental data of bioflm inhibition percentages were 85.30 ± 0.26, 41.69 ± 0.57, and 33.66 ± 0.99, respectively. Te results show that the bioflm formation inhibition percentage of 0.2% Chx was 84.94 ± 0.16 (Figure 1).

Bioflm Formation Inhibition. Te efect of PSNC on bioflm formation is shown in
Te results revealed that PSNC exposure for 24 h and 48 h inhibited the formation of S. mutans and S. sanguinis bioflms. Te bioflm formation inhibition percentage increased with an increase in the concentration of PSNC (p < 0.05). Furthermore, 5 mg/ml PSNC showed the highest inhibitory activity among the other concentrations of PSNC (p < 0.05). LSD showed no signifcant diferences (p > 0.05) between 5 mg/ml PSNC and 0.2% Chx at 24 h and 48 h exposure, indicating that both materials have the same effectiveness in inhibiting bioflm formation.

Confocal Laser Scanning Microscopy Observation.
Based on the 3D CLSM images (Figure 2), 24 h S. mutans and S. sanguinis bioflms of the negative control group appear thick and densely distributed, most of which are stained green, indicating live cells. Te CLSM images revealed substantial disintegration of the bioflm structure and decreased surface coverage after being treated with 1.25 mg/ml PSNC for 24 h. Te bioflm appeared in clusters, and some blank areas on the surface were observed. Te disruption of the bioflm was obviously increased as the concentration of PSNC increased. Te bacteria were scattered and did not form bioflm in the sample exposed to 2.5 mg/ml and 5 mg/ml PSNC as well as 0.2% Chx (positive control). Te number of bacteria was reduced signifcantly; the remaining bacteria were mostly stained red, indicating that they were dead or dying.
In comparison to the 24 h bioflm, the CLSM images of the 48 h of S. mutans and S. sanguinis bioflms of the negative control group appear thicker and denser. Te bioflm showed green fuorescence, indicating active living cells. Te formation of the bioflm was disrupted after the bacteria were exposed to PSNC. Te CLSM images of samples treated with 1.25 mg/ml and 2.5 mg/ml PSNC for 48 h displayed thinner bioflm-containing cell clusters and decreased surface coverage. Te disruption of the bioflm was most obvious at 5 mg/ml PSNC exposure. Te cell clusters were scattered, and most bacteria were stained red, indicating dead or damaged cells. CLSM results from the group exposed to PSNC 5 mg/ml gave a similar picture to the positive control, indicating the same efectiveness in inhibiting bioflm formation (Figure 2).

Scanning Electron Microscope
Observation. SEM images of S. mutans and S. sanguinis bioflms are shown in Figure 3. In the control group, the 24 h bioflm covering the coverslips showed a typical pattern of bioflm with dense bacterial colonies. Te bacteria overlapped and gathered in clusters and appear to be embedded in EPS. After 24 h of 2.5 mg/ml PSNC exposure, the number of bioflm cells that adhered to the glass coverslips diminished, and the cells showed a scattered distribution. Te bacteria were arranged in the form of aggregates or as individualized cells. After being exposed to 5 mg/ml PSNC, the number of bacteria signifcantly decreases, and most of them are seen as individual cells.
Scanning electron micrographs of 48 h bioflm (Figure 3) appeared thicker and sheathed in a thicker matrix of bioflm than 24 h bioflm. In comparison to the negative control, after 48 h of 2.5 mg/ml PSNC exposure, the number of bioflm cells adhered to glass coverslips decreased, and the bioflm matrix was partially disrupted. Te bioflm diminished after being exposed to 5 mg/ml PSNC. Te remaining bacteria are arranged in the form of aggregates or simply as individualized cells.

Discussion
Te results of the present study evidenced that PSNC inhibited S. mutans and S. sanguinis bioflm formation in vitro. Bioflms are complex microbial communities characterized by cells adhered to substrate surfaces, interfaces, or each other, embedded in cell-generated extracellular polymeric matrices (glycolipids, proteins, 4 International Journal of Dentistry   International Journal of Dentistry glycoproteins, and extracellular DNA) that protect the microbial community [1,38]. Microbial behavior within bioflms difers signifcantly in terms of growth rate and gene transcription from the behavior of the same organisms studied under planktonic conditions. Te bacteria residing within bioflms are more resistant to antibiotics than planktonic bacteria; thus, they play an important role in the development of chronic oral infections, including caries and periodontal diseases [39]. Caries and periodontal diseases are both complex in nature and share a variety of contributing factors that either directly or indirectly link them. Deep pockets of severe chronic periodontitis exhibit low oxygen tension, which may promote the growth of microaerophilic species like S. mutans. Previous studies revealed that untreated periodontitis patients have signifcant S. mutans recovery rates from saliva and subgingival plaque [40].
Bacteria interact with cells in oral tissues and trigger infammatory responses that lead to tissue destruction. Evidence showed downregulated transglutaminase gene expression (TG1 and TG3) in gingival tissues of chronic periodontitis patients. Tis indicates a disruption of the structural integrity of the gingival epithelium, making the gingival epithelium susceptible to bacterial invasion [8].
Furthermore, bacteria negatively infuence host responses to secret high-sensitivity C-reactive protein (hs-CRP), nitric oxide, and various infammatory mediators through mechanisms that are locally regulated by miRNAs [10]. Recent research has revealed that miRNAs have a role in epigenetic processes associated with cardiovascular disease, increased oxidative stress, and periodontitis [9]. Several studies showed that periodontitis is signifcantly linked to cardiovascular diseases [10].
Chitosan is a natural linear polysaccharide produced from chitin by solid-state deacetylation under alkaline conditions or by enzymatic hydrolysis of chitin deacetylase.
Some of chitosan's most notable properties in the medical context are its nontoxicity, biocompatibility, biodegradability, and immune-enhancing activities [36]. Previous studies have shown that nanosized chitosan has excellent activities such as antibacterial efects, drug delivery systems, gene and/or vaccine delivery systems, and antitumor efects [41][42][43]. In the present study, chitosan was extracted from giant freshwater prawn shells. Te degree of deacetylation of chitosan obtained was 77.61%. Te chitosan was further processed by ion gelation with TPP to produce PSNC. Te average particle size of PSNC that formed was 432.9 nm with a polydispersity index � 0.288 and a molecular weight � 34.67 kDa. In addition, the zeta potential test result for PSNC was +51.2 (unpublished data).
Te results of the crystal violet assays as well as CLSM and SEM analysis in the present study demonstrated that PSNC exposure for 24 and 48 h inhibited the development of S. mutans and S. sanguinis (dual species) bioflm. Te antibioflm efect on the dual-species bioflm increased as the concentration of PSNC increased. Te present study revealed that the highest PSNC concentration examined (5 mg/ml) demonstrated the strongest antibioflm activity since the bioflm architecture was strongly disrupted upon treatment. Tese results are consistent with those of previous studies [33,44].
Aliasghari et al. [33] investigated the efects of chitosan and nano chitosan against single cariogenic bacteria (S. mutans, S. salivarius, S. sanguinis, and S. sobrinus) in planktonic and bioflm forms. Te result of their study showed that chitosan and nano chitosan have bacteriostatic or bactericidal activities as well as antiadhesion efects against those bacteria. Furthermore, both materials reduce bioflm/plaque formation in vitro. Similar to our fndings, they also revealed that by increasing the concentration of chitosan and nanochitosan, the antiadhesion activity on tested bacteria increased, and a 5 mg/mL concentration was In untreated control groups, the bioflm is clearly visible with the bacteria embedded in EPS. Te bioflm masses become less after being exposed to PSNC for 24 and 48 h. 6 International Journal of Dentistry found to be the most efective. Another study conducted by Costa et al. [44] found that chitosan-containing mouthwash was capable of hindering the bioflm formation and maturation of S. mutans, L. acidophilus, E. faecium, C. albicans, and P. intermedia. A possible mechanism for PSNC's antibioflm properties is due to its polycationic nature conferred by the functional amino group (NH3+) of the N-acetylglucosamine unit. Te positive charges of PSNC are expected to electrostatically react with negatively charged bioflm components such as EPS, proteins, and DNA, resulting in inhibitory efects on bacterial bioflms [36,45,46]. Interaction between the polycationic nature of nano chitosan and the anionic sites of microbial cell membrane proteins results in the leakage of intracellular components, causing bacterial cell death [47]. In addition, the interaction may also result in bacterial cell focculation, thus preventing the attachment of bacteria to the surface [48]. A study by Strand et al. [49] demonstrated that chitosan focculated Escherichia coli suspensions.
Another possibility of the PSNC mechanism that inhibits bioflm formation is that PSNC interferes with protein synthesis, which afects the bacterial attachment. PSNC produced in this study had a small particle size (about 432.9 nm) and low molecular weight (34.67 kDa). Te small particle size of PSNC may cause the molecules to easily penetrate the nucleus and bind to bacterial DNA [46]. In addition, PSNC with a low molecular weight more easily binds to the DNA of bacterial cells, thus disrupting the synthesis process of certain protein molecules, including proteins that support bacterial attachment [50]. Chávez de Paz et al. [51] demonstrated that chitosan nanoparticles prepared from low molecular weight chitosan induced >95% damage to S. mutans bioflms.
Due to the small size of PSNC particles (432.9 nm), PSNC may penetrate the bacterial cell wall and accumulate in the bacterial cell membrane, which in turn may interfere with bacterial cell metabolism [52]. In addition, electrostatic interactions between nano chitosan molecules and bacterial cell membranes may cause changes in cell membrane permeability. Te alteration of the bacterial cell membrane may then afect their hydrophobicity which in turn prevents the adherence of the bacteria to the surface, thus inhibiting bioflm formation [53].
In conclusion, the results of the present study provide evidence that PSNC inhibits the development of S. mutans and S. sanguinis bioflm in vitro. Te fndings indicated the high potential of PSNC as antiplaque and anticaries agent, suggesting their potential application in dental biomaterials that might be integrated into oral health care products such as mouthwash and toothpaste.

Data Availability
Te data supporting the results of this study are included in the article.

Conflicts of Interest
Te authors declare that there are no conficts of interest.